3rd International Cancer Research Symposium 2012: Defining & Translating Science for Disease Prevention and Therapy, Kolkata, West Bengal, India, December 18–21, 2012

Chairs: Sushanta K. Banerjee, Ph.D. and Dhrubajyoti Chattopadhyay, Ph.D.

Co-Chairs: Rajesh Agarwal, Ph.D., C.V. Rao, Ph.D., Shivendra Singh, Ph.D., Gopal Kundu, Ph.D. and Bhudev C. Das, Ph.D.

CLINICAL AGENT DEVELOPMENT FOR COLORECTAL CANCER PREVENTION

Ernest Hawk

Division of Cancer Prevention & Population Sciences, University of Texas MD Anderson Cancer Center; And Duncan Family Institute for Cancer Prevention and Risk Assessment, Division of Cancer Prevention & Population Sciences, University of Texas MD Anderson Cancer Center.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in both men and women in the U.S. Survival rates for late-stage disease remain unacceptably low. In conjunction with screening, effective chemoprevention efforts can also reduce CRC incidence and mortality. New pathophysiologic insights are providing new preventive targets. Many randomized controlled trials have been conducted testing various agents with regard to adenoma recurrence, or more rarely, CRC incidence or mortality, establishing proof-of-principle for CRC chemoprevention. When evaluated in the context of the cardinal elements of clinical trial design—agent, cohort and endpoint—prior trials offer important lessons that can inform and enhance the design and conduct of future CRC chemoprevention trials. These lessons will be briefly reviewed along with current challenges facing the field of CRC preventive agent development. While the process of developing molecular preventive agents for CRC is similar to that for other cancers, the long transformation time of adenomatous polyps into cancer and current standards of care which mandate removal of polyps for histopathologic characterization challenge efforts to demonstrate the effectiveness of molecular preventive agents on the basis of CRC incidence. The rate-limiting step in the identification of novel CRC chemopreventive agents that are both efficacious and safe is the pace of clinical research which is driven by choices of cohorts and endpoints. Various strategies addressing this challenge will be discussed, including a focus on high-risk cohorts, the development of early-stage surrogate end-point biomarkers, nesting prevention endpoints as secondary goals in therapeutic trials, and combining agents with modest efficacy or an unfavorable toxicity profile as individual agents to improve their preventive impact.

IN SEARCH OF THE DRIVERS: EXCAVATING THE ORAL CANCER GENOME

Partha P. Majumder

National Institute of Biomedical Genomics, Kalyani, India

India is a member—through the Department of Biotechnology, Government of India—of the International Cancer Genome Consortium [International network of cancer genome projects. Nature 464, 993–998 (15 April 2010)]. To identify genomic alterations, both germline and somatic, we have been carrying out whole-exomeresequencing of paired blood and tumor DNA of patients with gingivobuccal cancer. This project is being conducted jointly by the Advanced Centre for Treatment, Research and Education on Cancer (ACTREC), Mumbai, and the National Institute of Biomedical Genomics (NIBMG), Kalyani. In this presentation, I shall provide a summary of the results—clinical and genomic—of the work carried out on the first 50 patients. While a very large number of genomic alterations, both germline and somatic, are observed in patients with gingivobuccal oral cancer, a systematic statistical and bioinformatic analysis has revealed consistent alterations in some genes that have earlier been implicated in cancers of various sites, but not gingivobuccal cancer.

IMPACT OF MOLECULAR GENETICS IN THE MANAGEMENT OF CANCER

Mammen Chandy

Director, Tata Medical Center, Kolkata

The discovery of the Philadelphia chromosome by David Hungerford and Peter Knowles in 1960 was the first example of a non-random genetic change which was consistently present in patients with chronic myeloid leukaemia. Janet Rowley subsequently showed that this was a reciprocal translocation between the bcr gene on chromosome 22 and the abl gene on chromosome 9. We now know that this results in up-regulation of a tyrosine kinase which is the key to the increased proliferation of myeloid cells in this neoplasm. Brian Drucker showed that this kinase could be specifically inhibited by the small molecule imatinib and this has been the most successful example of “targeted” therapy for cancer. These discoveries have radically changes the diagnosis and management of this disease: when a patient present with a classical blood picture of CML, the diagnosis is confirmed by Fluorescence in situ hybridization (FISH) to document the presence of the 9:22 translocation and the breakpoint is confirmed by RT-PCR. The RQ_PCR is then used to document the response to imatinib and serially monitored to document early relapse which may be due to a mutation in the Bcr-abl gene which is confirmed by sequencing. If the mutation is T315I then the patient will not respond to the second generation tyrosine kinase inhibitors and will require a drug called pomatinib. This illustrates how molecular genetics has transformed the way we manage CML today. In many other cancers also precision in diagnosis with molecular genetic tools is essential to choose the appropriate management and tailor the therapy. New biomarkers will also help us to track the progression of cancer. Knowledge of the genetic pathways which drive malignant proliferation are providing new targets for therapy. Next generation sequencing is providing the technology to sequence the whole genome in different cancers and this information will be pivotal in devising new strategies for prevention, accurate diagnosis, monitoring and therapy of cancer.

MOLECULAR ANALYSIS OF CELLULAR PATHWAYS ASSOCIATED WITH THE DEVELOPMENT OF UTERINE CERVICAL CARCINOMA

Dipanjana Majumdar, Sraboni Mitra, Partha S. Basu, Ranajit Mondal, Jaydip Biswas, Susanta Roychoudhury^a and Chinmay Kumar Panda

Chittaranjan National Cancer Institute, 37S. P. Mukherjee Road, Kolkata, India. E.Mail: ckpanda.cnci@ gmail.com. ^a Indian Institute of Chemical Biology, CSIR, 4 Raja S. C. Mullick Road, Kolkata, India.

Abstract:

Cancer of uterine cervix (CaCx) is second most common cancer amongst women worldwide. The epidemiological data strongly associates high risk HPVs as a necessary cause of cervical cancer development, though the exact role of HPV in development of the disease is not fully clear. Molecular studies have confirmed complex karyotypic patterns with non-random cytogenetic alterations in several chromosomes like 1, 3, 4, 5, 6, 7, 11, etc. in this tumor. Identification of the candidate genes in the frequently altered chromosomal regions and their associated cellular pathways will help to understand the molecular pathogenesis of the disease.

In our analysis, approximately 90 % of the cervical lesions showed HPV16/18 positivity. Our data suggested that expression of two HPV oncoprotein E6 and E7 was modulated by the genetic and epigenetic status of HPV during progression of CaCx. Next, copy number variation (CNV) of the chromosomal regions showing frequent cytogenetic aberrations has been analyzed by microsatellite and STS markers in the primary cervical lesions. From these chromosomal regions several candidate genes have been identified based on CNV analysis followed by promoter hypermethylation, mutation and expression analyses. These genes were seen to control different cellular pathways like 1) RBSP3-RB associated G1-S cell cycle, 2) RASSF1A, CACNA2D2, EI24 associated apoptotic pathways, 3) SLIT2-ROBO1/2, ITGA9 and CADM1 mediated signaling, 4) MLH1, ATM, CHEK1 associated DNA damage response pathway. Our data showed that alterations of these genes were differentially associated with development of CaCx. Now, systemic approach has been taken to characterize the molecular pathways associated with CACX and to find out the key regulatory steps for efficient management of the disease.

TUMOR NECROSIS FACTOR REGULATES RADIORESISTANCE IN GLIOBLASTOMA

Ellora Sen

National Brain Research Centre, Manesar, Haryana 122 050, India

Gliomas are resistant to radiation therapy as well as to TNFα induced killing. Radiation induced TNFα triggers NF-ĸB mediated radioresistance. As inhibition of NF-ĸB activation sensitizes glioma cells to TNFα induced apoptosis, we investigated whether TNFα modulates the responsiveness of glioma cells to ionizing radiation-mimetic Neocarzinostatin (NCS). TNFα enhanced the ability of NCS to induce glioma cell apoptosis. NCS mediated death involved caspase-9 activation, reduction of mitochondrial copy number and lactate production. Interestingly, death was concurrent with NF-ĸB, Akt and Erk activation. Abrogation of Akt and NF-ĸB activation further potentiated the death inducing ability of NCS in TNFα co-treated cells. NCS induced p53 expression was accompanied by increase in ATM phosphorylation. While NCS induced ATM phosphorylation in a NF-ĸB independent manner, ATM inhibition abrogated NF-ĸB activation. Metabolic gene profiling indicated that TNFα affects NCS mediated regulation of several genes associated with glycolysis. The existence of ATM-NFĸB axis that regulate glycolysis to overcome pro-survival response in NCS and TNFα co-treated cells, suggests mechanisms through which inflammation could affect resistance and adaptation to radiation despite concurrent induction of death.

BETA-ENDORPHIN NEURONAL REGULATION OF CANCER GROWTH, PROGRESSION AND METASTASIS

Dipak K. Sarkar

Distinguished Professor and Director, Rutgers Endocrine Program, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901

It is becoming increasingly clear that stressful life events can impact cancer growth and metastasis by modulating nervous, endocrine and immune system of body. Under the physiological condition a neuroendocrine hormone that plays a critical role in bringing the stress axis homeostasis is the opioid peptide beta-endorphin (BEP). We have recently shown that promotion of endogenous levels of BEP in the hypothalamus via BEP neuron transplantation reduces stress response, promotes immune function. Hence, we have tested the effects of BEP cell transplantation in rat models of prostate, breast, liver and colon cancers. We found that BEP neuron transplantation inhibits the growth and progression of all cancers tested. We also found preventive effect of BEP neuron transplantation on mammary cancer cell metastasis in lung tissue. The cancer preventive role of BEP appear to be caused by the suppression of sympathetic neuronal function and resulting in an increased peripheral NK cell and macrophage activities, elevated levels of anti-inflammatory cytokines and reduced levels of inflammatory cytokines. BEP inhibition of tumor progression also involves alteration in tumor microenvironment possibly due to suppression of catecholamines and inflammatory cytokines production that are known to alter cell–matrix attachments, angiogenic mechanisms, DNA repair and epithelial-mesenchymal transition. Thus, possible therapeutic approaches exist that take advantage of BEP cell therapy to prevent cancer growth and progression. (Supported by NIH grants R37 AA08757 and F31CA132620)

SPDEF, THE PROSTATE-DERIVED ETS-FACTOR, IS A TUMOR METASTASIS SUPPRESSOR

Hari K Koul^{1, 2, 3} and Sweaty Koul¹

¹Signal transduction and Molecular Urology Laboratory-Program in Urosciences, Division of Urology- Department of Surgery, School of Medicine, ²The Denver Veterans Administration Medical Center, and ³The University of Colorado Comprehensive Cancer Center; University of Colorado -School of Medicine, Building P15 or RC2, 12700 E 19th Avenue, Room number 6430D, Aurora, CO 80045, USA

The prostate-derived ETS factor (SPDEF, a.k.a PDEF) is the latest family member of the ETS transcription factor family, although it is unique in many aspects. SPDEF was first discovered as an mRNA transcript highly expressed in prostate tumor cells where it regulates prostate-specific antigen (PSA) gene expression and is an androgen receptor co-regulator. SPDEF expression is highly restricted to epithelial cells and has only been found in prostate, breast, colon, ovary, stomach, and airway epithelium. Our studies for the first time demonstrated that SPDEF is lost during prostate cancer progression. Strong preclinical evidence is emerging that SPDEF is a negative regulator of tumor progression and metastasis. The induction of tumor aggressiveness in response to the loss of SPDEF is thought to be due to the plethora of PDEF-regulated gene targets, many of which are known players in tumor progression including tumor cell invasion and metastasis. Our studies demonstrate that SPDEF inhibited tumor cell invasion in part by transcriptional repression of MMP9. These data lead us to the hypothesis that PDEF is a tumor metastasis suppressor protein. In deed we have recently provided, the first direct demonstration of the function of SPDEF as a tumor metastasis suppressor in vivo. Here we will summarize what is known about SPDEF since its discovery a decade ago and share data from our experiments with suggesting a role for SPDEF as a tumor metastasis suppressor. Studies supported in part by Chair Commitment (H. Koul) and the Department of Surgery, School of Medicine Academic Enrichment Funds (H. Koul). H. Koul is supported in part by VA Merit Award-01BX001258, NIH R01 DK54084, and NIH/NCI R01CA161880).

THE MATRICELLULAR PROTEIN CCN1 IN INFLAMMATORY DISEASES AND CANCER

Lester F. Lau

Department of Biochemistry and Molecular Genetics, University of Illinois College of Medicine

BACKGROUND: The CCN family of matricellular proteins is critical for embryonic development and plays important roles in inflammation, wound healing and tissue repair in adulthood. Deregulation of CCN proteins contributes to the pathobiology of various diseases, many of which may arise when inflammation or tissue injury becomes chronic, including cancer (Jun and Lau, Nat. Rev. Drug Discov. 10:945–963, 2011). CCN1, the first member of the CCN family identified, has been shown to promote the growth of certain cancers through its potent angiogenic activity mediated by its direct binding to integrin αvβ3 in endothelial cells. However, CCN1 can also induce apoptosis and senescence in some cell types through integrin α6β1, suggesting a tumor suppressing function. We have examined the potential tumor-promoting and tumor-suppressing effects of CCN1 linked to its angiogenesis vs. apoptosis/senescence-inducing activities. Experimental Design: We have constructed CCN1 mutants that are specifically disrupted in its binding sites for integrin αvβ3 or α6β1 and therefore unable to induce angiogenesis or apoptosis/senescence, respectively. Using these mutants, we have tested the effects of CCN1 expression in tumor growth that are associated with αvβ3 or α6β1-mediated functions. Results and Conclusions: Through analysis of site-specific mutants, we found that CCN1 has both tumor promoting and suppressing functions in various tumor cells. The implications of these findings in therapeutic approaches will be discussed.

TARGETED DRUG DEVELOPMENT: SYNTHETIC THIADIAZOLE NMK-TD-100, A NOVEL MICROTUBULE MODULATING AGENT, INDUCES APOPTOSIS AND AUTOPHAGY IN CANCER CELLS

Gopal Chakrabarti

Department of Biotechnology and Dr. B.C Guha Centre For Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, India.

Recent drug discovery efforts are highly focused towards design and synthesis of small molecules as anticancer agents. Thiadiazoles are one of the privileged structural fragments in medicinal chemistry having broad spectrum of pharmacological activities. As the microtubule cytoskeleton is one of the major targets in cancer chemotherapy, we have screened for growth inhibitory effect of synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles on A549 cell line, human lung cancer cell line, and identified NMK-TD-100, as the most potent microtubule-depolymerizing agent. We have examined the effects of NMK-TD-100 on cellular microtubules in cells as well as its binding with purified tubulin and microtubules in cell-free system. Cell viability experiments using A549 indicated that the IC₅₀ value is 5.5 ± 0.26 μM in A549. NMK-TD-100 induced delay in exit from mitosis, reduced in mitochondrial membrane potential and induced apoptosis in A549 cells. Again treatment with NMK-TD-100 also induces autophagy in A549 cells and pretreatment with early and late autophagy inhibitors viz. 3MA and chloroquine potentiates the cytotoxicity of NMK-TD-100 in A549 cells. Preservation of mitochondrial integrity by cyclosporin A pretreatment prevented autophagy induction in A549 cells, showing induction of autophagy is downstream of mitochondrial membrane potential disruption. Immunofluorescence studies using an anti-α-tubulin antibody showed a significant depolymerization of the interphase microtubule network and spindle microtubule in a dose-dependent manner. In vitro polymerization of purified tubulin into microtubules was inhibited by NMK-TD-100 with an IC50 value of 17.5 ± 0.35 μM. The binding of NMK-TD-100 with tubulin was studied using NMK-TD-100 fluorescence and intrinsic tryptophan fluorescence of tubulin. The stoichiometry of NMK-TD-100 binding to tubulin was 1:1 (molar ratio) with a dissociation constant of 0.996 ± 0.015 μM. Together these data suggest that NMK-TD-100, a novel microtubule depolymerizer could be as a lead compound for development of potential chemotherapeutic agent.

CANCER PREVENTION BY DIETARY PHYTOCHEMICALS: FROM NRF2-MEDIATED ANTI-OXIDATIVE STRESS, EPIGENETICS TO IN VIVO CANCER MODELS

Ah-Ng Tony Kong

Professor II and Glaxo Endowed Professor of Pharmaceutics, Director of Graduate Program in Pharmaceutical Sciences Leader, Carcinogenesis and Cancer Prevention, Cancer Institute of New Jersey Ernest Mario School of Pharmacy Rutgers, The State University of New Jersey Piscataway, NJ 08854, USA, Email: KongT@pharmacy.Rutgers.Edu

Diverse nutritional phytochemicals are powerful medicinal products in promoting human health and diseases prevention including cancer. These dietary “antioxidants” can trap reactive species RONS, trigger cellular signaling events including “proteins thiol modifications” leading to expression of cellular defense and other genes. Our lab study dietary phenolic-antioxidants, isothiocyanates, tocopherols, omega-3 fatty acids and herbal medicines, which are effective against many animal carcinogenesis models. These compounds modulate kinases, activate Nrf2-mediated antioxidative stress/anti-inflammatory pathways and induce cellular defense genes HO-1, GST, and NQO1. Integrating Nrf2-/- mice with microarray bioinformatics, other genes including apoptosis, cell adhesion, cell growth, kinases, electron transport, transcription factors, and ubiquitination, are also Nrf2 targets, leading to the overall cellular protective effects against oxidative/carcinogenic damages, particularly during carcinogenesis initiation. The Nrf2-/- mice are more prone to carcinogen-induced skin, colon and other cancers and are more susceptible to DSS-induced colon inflammation and DSS-AOM-induced colon carcinogenesis. Inhibition of LPS-induced inflammation in mouse macrophages by sulforaphane (SFN) would require Nrf2. Epigenetically, in the prostate TRAMP tumors, as cancer progresses, a shut-down of Nrf2 via CpG methylation of the promoter region, attenuating Nrf2-mediated genes, which were reversed by dietary PEITC, curcumin and tocopherols. Curcumin could demethylate CpGs of Nrf2 promoter in TRAMP C1 cells and Neurog1 promoter in LNCaP cells, which would impact early carcinogenesis and later stages of cancer development. Hence, nutritional phytochemicals can confer early chemopreventive epigenetic effects resulting in blocking initiation/promotion of carcinogenesis, as well as late stage of epigenetic effects inhibiting progression of carcinogenesis. Supported by NIH grants.

TRANSCRIPTOMIC APPROACHES: PREVENTION AND TREATMENT OF GI CANCERS

C. V. Rao

Center for Cancer Prevention and Drug Development; University of Oklahoma Health Sciences Center, Oklahoma City, OK

Colorectal, stomach, liver, pancreas, esophageal, and oral cavity cancers are major gastrointestinal neoplastic diseases and responsible for more than four million new cases in each year globally. With exception of stomach cancer remaining gastrointestinal cancers incidence rates are increasing each year, more so in developing countries. In spite of many developments in diagnosis and treatments, still most of the stage IV GI cancer patients 5-year survival rates are <10 %. Among these, pancreatic cancer has the worst prognosis and it is lethal to almost all of the patients diagnosed. Lack of early detection and effective interventions are major factors contributing to the poor prognosis and dismal survival rates of pancreatic cancer patients for more than 60 years. As with many epithelial cancers, pre-invasive precursors in the GI tract progress slowly over many years to decades to development of invasive cancers in humans. Particularly, with detection of pancreatic cancer at an early stage might permit life-saving interventions. Use of tumor xenografts and/or orthotopic models to predict valid targets and for design and development of drugs for pancreatic cancer patients has been disappointing. Animal models that recapitulate the molecular pathology of human pancreatic intraepithelial neoplasia (PanINs) and their progression to pancreatic ductal adenocarcinoma (PDAC) are ideal models for establishing pancreatic cancer prevention and treatment strategies. Recent advent in genetically engineered mouse (GEM) models, such as development of transgenic Kras^G12D-dependent mouse models of PDAC that mimic human PDAC offer opportunities to identify putative targets of pancreatic lesion development and targeted drug strategies for PDAC prevention. We will present recent advances on i) selection of optimal Kras mouse models for prevention and early diagnosis, and ii) application of Whole Transcriptome (SOLiD) approaches to identify druggable targets for PanINs and PDAC. Then, iii) we will provide a few examples of development of molecularly targeted chemopreventive drugs using the kras^G12D mouse model. This work was supported by NCI-R01-94962 and N01-CN-53300.

MOLECULAR MECHANISM OF CANCER CHEMOPREVENTION BY RESVERATROL

Robert A. Sclafani, Rajesh Agarwal, Alpna Tyagi, Sunitha Siriwardana, Takenori Takahata, and Barbara Frederick.

University of Colorado Schools of Medicine and Pharmacy and the Cancer Center, Aurora, Colorado, USA.

BACKGROUND: The nutraceutical resveratrol (trans-3,4′,5 Trihydroxystilbene) is a phytoalexin that is found in many plants including red grape skins and peanuts. Resveratrol has no toxic effect on normal cells or animals, but inhibits the growth of wide-array of cancer cells and tumor growth in animal models as a potent chemopreventive agent. Our previous studies have shown that resveratrol selectively arrested head and neck squamous cancer cells (HNSCC) and ovarian cancer cells, but not primary epithelial and fibroblast normal cells, in the S phase of the cell cycle by damaging DNA and activating the DNA damage checkpoint and apoptosis (1,2). Our current studies are focused on HNSCC (Head and Neck squamous cell carcinoma) because of the importance of DNA damage in HNSCC etiology and the possibility of using oral administration of resveratrol. In HNSCC xenografts, resveratrol exerts similar effects and can produce both DNA damage and apoptosis in vivo. Our current model is that resveratrol partially inhibits or slows DNA replication by inhibition of DNA polymerase μM(ki = 3μM) (3,4) resulting in DNA damage and apoptosis in cancer cells because they lack important DNA repair pathways (1,2). Our current studies are focused on HNSCC (Head and Neck squamous cell carcinoma) because of the importance of DNA damage in HNSCC etiology and the possibility of using oral administration of resveratrol. METHODS: In order to test our hypothesis, eliminate other known possibilities, HNSCC cells were treated with cis-resveratrol (cis-3,4′,5 Trihydroxystilbene), which cannot inhibit DNA polymerase (Stivala et al., 2001). Rolipram was used as an cAMP phosphodiesterase (PDE4) inhibitor, which has been shown to mimic the effects of resveratrol and to increase SIRT1 activity in model aging systems (5). DNA damage checkpoint activation and cell cycle phases were measured as previously described (1,2). In order to identify other genes important for resveratrol sensitivity, we performed a chemical synthetic lethal screen (6) in which FaDu HNSCC cells infected with genome-wide lentiviral shRNAs were screened for increased sensitivity to resveratrol by deep-sequencing and bioinformatic analysis of the survivor populations. We also tested if resveratrol could prevent HNSCC in vivo using the 4NQO (4-Nitroquinoline-1-oxide) HNSCC carcinogenic mouse model as a true chemoprevention model (7). RESULTS: In support of our hypothesis, both cis-resveratrol and rolipram were completely ineffective in activating the DNA damage checkpoint even at high concentrations (100μM). We identified a number of important DNA repair genes in our chemical synthetic lethal screen. One of the genes encodes the RECQ1 DNA helicase, which is important for suppressing “illegitimate” recombination events, regulating DNA replication and preventing genomic instability. RECQ1 shRNA knockdowns in HNSCC cells increase the amount of spontaneous DNA damage as has been seen by others (8). In vivo, resveratrol feeding inhibited the progression of 4NQO-induced tongue carcinogenesis by decreasing the total number of tongue lesions, including hyperplastic and dysplastic lesions on the epithelial surface and papilloma. CONCLUSION: We have made significant progress in determining the mechanism of action of resveratrol in HNSCC chemoprevention. Because cis-resveratrol has significant anti-oxidant activity, the anti-oxidant activity of resveratrol is not sufficient for its anti-cancer effect. Furthermore, we think it is unlikely that either PDE4 or SIRT1 are targets of resveratrol in HNSCC. Our hypothesis that DNA polymerase μM is the true target is supported by our data. Our chemical synthetic lethal screen has identified genes important for resveratrol sensitivity. One of these genes is RECQ1, which represents a therapeutic target In HNSCC as it is known to be is overexpressed in both HNSCC cell lines and tumors and is protective against DNA damage by chemotherapeutic agents and radiation that damage DNA (9). We have also begun studies of HNSCC from Fanconi Anemia patients. Fanconi Anemia is a familial, recessive genetic disorder in which the patients are defective in DNA repair by Trans-lesion Synthesis and are at high risk for HNSCC, which makes them an ideal group for chemoprevention by resveratrol.

EPIGENETIC REGULATION OF THE RXRΑ/VDR IN THE PREVENTION OF COLON CANCER AND COLITIS BY NATURAL PRODUCTS.

Michael J. Wargovich¹, Jay Morris¹, Dina Moseley², and Rebecca Knackstedt².

¹University of Texas Health Sciences Center, San Antonio, TX, and ²Medical University of South Carolina, Charleston, SC, USA.

Our laboratory is actively engaged in understanding the mechanism by which chronic inflammation enhances risk for colon cancer through pre-cancerous syndromes, such as chronic colitis. In studies in human colon cancer cell lines, we have found that RXRα, a transcription factor that partners other key nuclear transcription factors, notably the VDR, is silenced epigenetically. The implication is that silencing of RXRα would impair the ability of VDR to control inflammation. In methylation sensitive human colon cancer cell lines, the green tea polyphenol EGCG inhibits specific methylation of the RXRα promoter thus allowing re-expression of down-stream genes involved in inflammatory control. EGCG has no effect on methylation insensitive colon cancer cell lines, nor is RXRα silenced in them. We postulated that silencing of RXRα and/or VDR may occur early in risk for colon cancer and turned our attention to animal models for colitis. The expression of these transcription factors is variable in acute colitis, but in chronic colitis, both RXRα and VDR expression is downregulated. We investigated whether this was due to epigenetic mechanisms or if other mechanisms were involved. Early studies indicate that, at least in the mouse DSS colitis model, epigenetic silencing is not involved, but to investigate a possible mechanism through which VDR was being silenced in acute colitis an in the chronic colitis cancer model, the expression of SNAIL and SLUG and their upstream regulators, TNFa and NfKB were examined. In acute colitis, NFkB expression was not upregulated but TNFa and Snail were significantly upregulated in mice with acute colitis with Slug just short of significance). In the colitis cancer model, the upregulation of TNFa was still present but Slug and Snail were no longer upregulated. The control of inflammation is complex in these models and with transit to cancer, epigenetic silencing assumes a larger role, and is a target for intervention with natural products. Supported by grant R01CA96694 from the National Cancer Institute.

NEW EXPERIMENTAL APPROACHES FOR IDENTIFYING COLON CANCER CHEMOPREVENTIVE AGENTS

Charles Giardina

Department of Molecular and Cell Biology, University of Connecticut, USA

Within the pre-neoplastic colonic mucosa, mutated intestinal stem cells interact with a range of tissue and dietary factors, as well as immune and inflammatory signals. The balance and nature of these interactions determines whether a mutated cell will progress to malignant disease, or will regress and ultimately be eliminated from the tissue. Studies are underway to develop ESC-derived intestinal organoids to rapidly evaluate agents that can inhibit the survival and expansion of mutated intestinal stem cells. In addition, screening studies are being pursued to identify agents that can accentuate the apoptotic activity of death ligands present within the inflammatory micro-environment of an early neoplastic lesion (such as TNF and Fas ligand). A number of agents have been identified with such activity and are presently being studied for their mechanism of action. Together, these efforts are aimed at identifying new agents to study important aspects of cancer prevention signaling, and to potentially identify novel approaches for colon cancer prevention. An update on these efforts will be presented.

SILIBININ ‘THE DOUBLE EDGED SWORD’: TARGETING BOTH THE INITIATORS AND THE INITIATED ONES IN COLON CANCER

Rajesh Agarwal

Professor, Department of Pharmaceutical Sciences, Co-Leader, Cancer Prevention and Control Program, University of Colorado Cancer Center, University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences, 12850 E. Montview Blvd, C238, Room V20-2118, Aurora, CO 80045, Phone: 303-724-4055, Fax: 303-724-7266, Email: Rajesh.Agarwal@UCDenver.edu

Colorectal cancer (CRC) is the second leading cause (both genders combined) of cancer-related deaths in US. Statistical estimates for 2012 indicate ∼103,170 new CRC cases and 51,690 associated deaths in US alone. The current anti-cancer therapy fails to generate significant anti-tumor effect due to a defective apoptotic machinery or resistance of the CRC cells to the specific death mechanisms induced by the drugs. Furthermore, cancer stem cells (CSC), now recognized as the main cause for initiation, promotion and progression of CRC, are inherently resistant to chemo- and radio-therapies and that has been the major reason for failure of most of current therapies. Thus, discovery and development of agents, especially chemopreventive agents, which target both ‘initiated’ stem cells and different death mechanisms might provide opportunities to intervene and prevent the disease in either early stage or at a late stage in cancer therapy as it would eradicate CSC pool, which otherwise escapes the therapeutic insult resulting in cancer relapse. We recently reported the strong preventive and therapeutic efficacy of silibinin (flavonolignan isolated from the seeds of milk thistle), in different CRC pre-clinical models. From a broader view point, the limitation of these studies was that it did not discuss the efficacy of the silibinin treatment in terms of the metabolic profile, energy state and CSC population of the colonic tumors. In this regard, our recent studies, utilizing quantitative high-resolution nuclear magnetic resonance spectroscopy (¹H-, ¹³C- and ³¹P-NMR) to assess the metabolic profile and energy state of the silibinin treated CRC cells, indicated that the cellular damage to tumor cells by silibinin is severe and irreparable due to sustained interference in essential cellular processes such as mitochondrial metabolism, phospholipids, and protein synthesis. In addition, we also found that silibinin strongly decreases the percentage of sphere formation of CRC cells (a stem cell characteristic). Further studies were then aimed to provide an understanding of the mechanisms involved in the inhibitory effect of silibinin on the CSC population in CRC. Specifically, we carried out studies on how silibinin modulates the dynamics of CSC spheroids under in vitro conditions, by examining its effect on the kinetics of CSC spheroids generated from three CRC cell lines, SW480, HT-29 and LoVo, and subjected them to sphere cluster formation assays. Both single and multi-treatment approaches with silibinin were employed to determine the effect, for 1-2 weeks, on the cycling properties of CSC which were enriched in the individual spheroids. Comparative analysis of different cell lines in terms of sphere formation kinetics indicated that the CSC enriched spheroids varied significantly in their growth kinetics, measured as a function of time and related to the individual diameter/area of the spheroids. Effect of silibinin on the growth kinetics and cycling properties of these CSC enriched spheres indicated a significant but differential modulatory effect of silibinin on these properties across cell lines. Furthermore, differentiation assays of these spheroids carried under serum conditions in the presence and absence of silibinin, indicated towards the formation of more differentiated clones by treatment. Subsequent in vitro studies, employing real time RT-PCR, showed silibinin effect to be mediated in part by significant down regulation of the expression of the CSC marker CD44 and Notch mediated signaling in these cell lines. Further studies showed that silibinin also decreased protein levels of cleaved Notch and its transcriptional target Hes-1. The implications of such an effect are tremendous, since the disruption of homeostasis (regulated in part by Notch signaling) among stem cells and progenitor cells in intestinal region results in the expansion of CSC pool together with an increase in proliferative cell populations. These are extremely important findings as they clearly show that silibinin interferes with kinetics of CSC pool expansion via targeting various regulatory signals associated with the survival and multiplication of colon CSC pool leading to effective CRC prevention. This study, inarguably, not only provides us with a complete scenario of the anti-cancer effect of silibinin against CRC growth but in terms of practical and translational aspects, further validates the clinical usefulness of this drug. Supported by NCI R01 grant CA112304.

MULTIFUNCTIONAL NON-HISTONE CHROMATIN PROTEIN PC4 IN GENOME ORGANIZATION AND BREAST CANCER MANIFESTATION

Tapas K. Kundu

Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.

Human Positive transcriptional co activator 4, PC4, is a highly abundant multifunctional nuclear protein involved in several cellular functions which include chromatin compaction/organization, transcriptional co-activation and DNA repair. We had earlier reported that PC4 possesses a histone interaction ability which is essential for its chromatin compaction function. Silencing of PC4 by means of siRNA as well as lentivirus mediated stable knockdown, dramatically alters the transcription activation related epigenetic marks and thereby a huge array of genes. PC4 knockdown cells show remarkable defects in cell cycle, genome size, and also in nuclear architecture, which resembles highly progressive cancerous cells. Significantly, we have observed that PC4 is down regulated in all types of Breast Cancer samples collected across the globe. The cause and functional consequences of this down regulation of PC4 is being investigated.

PROSTATE CANCER: POTENTIAL FOR PREVENTION AND THERAPY BY BITTER MELON

Ratna Ray, Peng Ru, Robert Steele, Nancy Phillips and Susan Crawford

Department of Pathology, Saint Louis University, St. Louis, Missouri, USA

BACKGROUND: Prostate cancer remains the second leading cause of cancer deaths among American men. Earlier diagnosis increases survival rate in patients. However, treatments for advanced disease are limited to hormone ablation techniques and palliative care. Thus, new methods of treatment and prevention are necessary for inhibiting progression of the disease to a hormone refractory state. For centuries, Ayurveda has recommended the use of bitter melon (Momordica charantia) as a functional food to prevent and treat human health related issues. METHODS: Prostate cancer cells were treated with bitter melon extract (BME) at different time points. Cell lysates were analyzed for the expression different signaling molecules using specific antibodies. BME was gavaged in TRAMP and nude mice and prostate tumor was monitored. RESULTS: We have observed an anti-proliferative effect of bitter melon extract (BME) in a number of cancer cell lines, including prostate, without affecting normal cell growth. We observed that prostate cancer cells treated with BME accumulate during the S phase of the cell cycle and modulate cyclin D1, cyclin E, and p21 expression. Treatment of prostate cancer cells with BME enhanced Bax expression and induced PARP cleavage. Oral gavage of BME, as a dietary compound, delayed the progression to high-grade prostatic intraepithelial neoplasia in TRAMP (transgenic adenocarcinoma of mouse prostate) mice (31 %). Prostate tissue from BME-fed mice displayed approximately 51 % reduction of proliferating cell nuclear antigen expression. Further therapeutic study suggested that oral gavage of BME regresses prostate tumor growth in xenograft model. CONCLUSION: Together our results strongly suggested a chemotherapeutic role of bitter melon in prostate cancer preclinical models.

ENERGY CRISIS IN MAMMARY CANCER PREVENTION BY WITHAFERIN A IN A CLINICALLY-RELEVANT MOUSE MODEL

Shivendra V. Singh

University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

BACKGROUND: Despite screening efforts and continuous evolution of targeted therapies, breast cancer is a major health concern for women worldwide. Novel approaches for prevention of breast cancer are desirable not only because many risk factors associated with this disease are not easily modifiable but also due to the fact that currently available preventive options are sub-optimal. The present study was undertaken to determine the efficacy of Ayurvedic medicine constituent withaferin A (WA) for prevention of mammary cancer using MMTV-neu transgenic model. METHODS: Incidence and burden of mammary cancer and pulmonary metastasis were scored in female MMTV-neu mice after 28 weeks of intraperitoneal administration with 100 μg WA (three times/week) (n = 32) or vehicle (n = 29). Mechanisms underlying mammary cancer prevention by WA were investigated by determination of tumor cell proliferation, apoptosis, metabolomics, and proteomics using plasma and/or tumor tissues. Spectrophotometric assays were performed to determine activities of complex III and complex IV. RESULTS: WA administration resulted in a statistically significant decrease in macroscopic mammary tumor size, microscopic mammary tumor area, and the incidence of pulmonary metastasis. For example, the mean area of invasive cancer was lower by 95.14 % in WA treatment group compared with control group (P = .05). Mammary cancer prevention by WA treatment was associated with increased apoptosis, inhibition of complex III activity, and reduced levels of glycolysis intermediates. Proteomics confirmed downregulation of many glycolysis-related proteins in the tumor of WA-treated mice compared with control, including M2-type pyruvate kinase, phosphoglycerate kinase, and fructose-bisphosphate aldolase A isoform 2. CONCLUSION: The present study reveals suppression of glycolysis in WA-mediated mammary cancer prevention in a clinically-relevant mouse model. This investigation was supported by the grant RO1 CA142694-03 awarded by the National Cancer Institute.

RECIPROCAL REGULATION OF HER-2 AND ANNEXIN A2 IN BREAST CANCER

Jamboor K. Vishwanatha, Praveenkumar K. Shetty and Sanjay I. Thamake

Department of Molecular Biology and Immunology and Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, Texas.

Her-2 (ErbB-2), Estrogen Receptor (ER) and Progesterone Receptor (PR) are the most commonly used biomarkers and therapeutic targets in breast cancer patients. However, these biomarkers are not expressed in 17–30 % of women with breast cancer which limits the use of existing therapies. Patients under hormone deprivation and Herceptin therapy, a most common therapeutic option, tend to acquire resistance to such therapies over time. Alternative survival pathways are commonly seen to be upregulated upon inhibition of receptor tyrosine kinases (RTK), including Her-2. It is established that treatment with Herceptin leads to selective overexpression and activation of epidermal growth factor receptor (EGFR) and Src which further contributes to oncogenesis in Herceptin resistant and triple negative breast cancer (TNBC) patients. Here, we show a co-regulated upregulation in the expression of Annexin A2 (AnxA2), a known substrate of Src and one of the regulators of EGFR receptor endocytosis, in Herceptin resistant and Her-2 negative breast cancer. Immunohistochemical expression analysis revealed a reciprocal regulation between Her-2 and AnxA2 in breast cancer clinical samples as well as in cell lines as confirmed by protein and RNA analysis. The siRNA and Herceptin mediated downregulation/inhibition of Her-2 in Her-2 amplified cells induced AnxA2 expression and membrane translocation. In this study we report a possible involvement of AnxA2 in maintaining constitutively activated EGFR downstream signaling intermediates and hence in cell proliferation, migration and viability. This effect was consistent in Herceptin resistant JIMT-1 cells as well as in Her-2 negative breast cancer. The siRNA mediated AnxA2 downregulation leads to increased apoptosis, decreased cell viability and migration. Our studies further indicate the role of AnxA2 in EGFR-Src membrane bound signaling complex and ligand induced activation of downstream signaling pathways. Targeting this AnxA2 dependent positive regulation of EGFR signaling cascade may be of therapeutic value in Her-2 negative breast cancer.

ACTIVATED MMP-2 POTENTIAL MARKER FOR BREAST CANCER

Amitava Chatterjee

Assistant Director (Retired), Chittaranjan National Cancer Inst, 37, S P Mukherjee Road. Kolkta, India

BACKGROUND: The present study aimed to detect expression, activity and activation status of matrix metalloproteinase-2 (MMP-2) and also the correlation with prognostic parameters like ER, PR and HER2 in 81 malignant breast tumor and adjacent normal tissues of TNM stage-II and III. METHODS: The main experimental methods performed in the study were immunohistochemistry, ELISA, immunoblot, zymography, and semi quantitative RT-PCR. RESULTS: Pro-MMP-2 was highly expressed in node positive tumors more than adjacent normal breast tissue. The mature forms of MMP-2 (68kD and 64kD) were observed in T2–T4 stages. We observed positive expression of TIMP-2 and Induction of MT-1-MMP in tumors. Increased expression of EMPRIN and translocation of NFқB were also observed in tumors, compared to adjacent normal breast tissue. CONCLUSION: Activation of MMP-2 in tumors, compared to adjacent normal breast tissue of both TNM stage-II and III suggests its role in advanced stage of breast cancer. Appreciable expression of TIMP-2 and induction of MT-1-MMP and EMPRIN in tumors indicate their involvement in activation of pro-MMP-2 in tumors and transcription factor NFκB translocation to nucleus suggest their possible role as key regulators of MMP-2 activation in HER2 positive breast cancer. Our observations clearly suggest MMP-2 as a biological marker of breast cancer in different clinical stages evaluating valuable information in breast cancer treatment.

ASPIRIN IN BREAST CARCINOGENESIS: PREVENTION AND RISK

Gargi, Maity, Archana De, Snigdha Banerjee, Amlan Das and Sushanta K. Banerjee

Division of Hematology and Oncology, Department of Medicine, and Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas. USA. Sbanerjee2@kumc.edu

Background: Aspirin (ASA), a classical non-steroidal anti-inflammatory drug (NSAID) is widely used to reduce pains and fever. Epidemiological and experimental studies suggested that Aspirin use reduces the risk of different cancers including breast cancer. These studies have raised the tempting possibility that Aspirin could serve as a preventive medicine for breast cancer. However lack of in-depth knowledge of the mechanism of action(s) of ASA reshapes the debate of risk and benefit of Aspirin in prevention of breast cancer. Aims: Our aim is to investigate effects of ASA on pathophysiological events like epithelial to mesenchymal transition (EMT), migration, and stemness of cell in breast cancer cells. Methods: Both in vitro and in vivo xenograft model were used to test the objectives. Results: Our studies using in vitro and in vivo tumor xenograft model show a strong beneficial effects of Aspirin in prevention of breast carcinogenesis. We find Aspirin not only prevents breast tumor cell proliferation in vitro and tumor growth in xenograft mouse model, it also significantly inhibits other pathophysiological events in breast cancer such as EMT, cell migration as well as reprogramming of stemness in breast cancer cells. Conclusions: Collectively, the studies suggest that intake of an ASPIRIN a day might offer additional avenues for breast cancer prevention and treatment.

CAN DEGUELIN BE DEVELOPED FOR BREAST CANCER TREATMENT?

Rajendra G. Mehta

Cancer Biology Division, IIT Research Institute, Chicago, IL 60616, USA

BACKGROUND: Deguelin was initially isolated from Mundulea sericea by activity guided fractionation and now chemically synthesized. Earlier we reported chemopreventive efficacy of deguelin both in vitro and in vivo. Deguelin at non-toxic concentrations was chemopreventive in chemically-induced skin, mammary and colon carcinogenesis models. Previously, we also reported that deguelin inhibited proliferation of all breast cancer cell-types: ER⁺, PR⁺, HER2⁺, ER⁻, PR⁻, HER2⁺ and triple negative breast cancer (TNBC) cells. METHODS: Human TNBC MDA-MB-231 and murine metastasizing TNBC 4T1 cells were used. Cell proliferation, invasion, immunostaining, western blot analyses and real-time PCR assays were used for the studies. For in vivo experiments cells were injected subcutaneously for evaluating deguelin efficacy and intravenously to determine its effects on metastasis. RESULTS: Deguelin inhibited proliferation of TNBC cells by 50–75 % and confirmed by immunostaining of Ki67 and PCNA. Deguelin treatment of 4T1 cells resulted in downregulation of nuclear c-Met, and its downstream signaling molecules p-ERK and p-AKT. The suppression of ERK-mediated signaling by deguelin was confirmed by using appropriate ERK and AKT inhibitors. Growth of 4T1 cells injected subcutaneously in Balb/c mice was suppressed by deguelin at 2 mg/kg body weight in vivo. Deguelin also suppressed lung metastasis of 4T1 cells injected intravenously. Deguelin also inhibited growth of MDA-MB-231 cells in vitro and in vivo. CONCLUSION: Unlike patients with hormone receptor positive or ER⁻, PR⁻, Her2⁺ tumors, there is no well-established targeted therapeutic strategy for TNBC patients. The current study provides a very good rationale to develop deguelin for the therapy of breast cancer patients.

ARE CATECHOLAMINES REGULATOR OF TUMOR ANGIOGENESIS?

Sujit Basu^{1, 2}, Debanjan Chakroborty¹, Chandrani Sarkar¹, and Partha Sarathi Dasgupta³

¹Department of Pathology, ²Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA and ³Chittaranjan National Cancer Institute, Kolkata, India

BACKGROUND: The roles of catecholamine neurotransmitters (dopamine, norepinephrine and epinephrine) in the regulation of several physiological functions are well established. Interestingly, recent reports also indicate unique correlations between these neurotransmitters and cancer. Angiogenesis or formation of new blood vessels is a critical process limiting growth of tumors. This process in turn is tightly regulated by a critical balance between pro and antiangiogenic factors. RESULTS: We have recently demonstrated that dopamine (DA) by acting through its D2 receptors can inhibit angiogenesis (microvessel permeability, adult endothelial cell migration and proliferation) and vasculogenesis (mobilization of bone-marrow derived endothelial progenitor cells to tumor microenvironment) by suppressing VEGF receptor 2 phosphorylation (VEGFR-2) in tumor endothelial cells (TEC) and endothelial progenitor cells (EPC) and thereby, inhibit growth of several malignant tumors in preclinical animal models. In contrast, norepinephrine and epinephrine induces angiogenesis and thereby, promote tumor growth. CONCLUSIONS: Because these drugs are being currently used in the clinics for the treatment of other disorders, therefore our results can be translated from the bench to the clinics for the treatment of cancer patients.

STRATEGIES TO TAME THE ACTIVITIES OF ANDROGEN RECEPTOR FOR TREATING ANDROGEN-INDEPENDENT PROSTATE CANCER

Dr. Mohammad Saleem

Molecular Chemoprevention and Therapeutics, The Hormel Institute, University of Minnesota, MN, USA

Androgen Receptor (AR) is a member of the steroid hormone receptor family that shares sequence homology with other members such as progesterone (PR), glucocorticoid (GR), mineralocorticoid (MR) and estrogen (ER) receptors. Aberrant AR expression and activation promoted by mutations, and binding partner mis-regulation is presented in several clinical manifestations including androgen insensitivity syndrome, acne vulgaris, androgenetic alopecia, benign prostate hyperplasia (BPH), and different type of cancers in humans. Despite many decades of investigation, the role of AR in gene regulation of cells and tissues remains only partially characterized. The initial stage of prostate cancer (CaP) is dependent on androgen and can be managed by a series of therapies that are antagonist to AR or suppress AR signaling. However, after a short remission period, tumors reappear as castration-resistant prostate cancer (CRPC). Recently, it has been observed that overexpression of AR is the most common event associated with CRPC. Multiple mechanisms activate AR in CaP cells during CRPC emergence. These include ligand-independent activation of AR in an androgen depleted environment, AR gene amplification and overexpression of AR co-activators. The detection of AR splice variants in CRPC disease has given another important dimension to the significance of AR during this disease. AR splice variants have also been observed in different CaP cell lines. Though LBD is absent in AR splice variants, yet exhibit higher AR transcriptional activity in

CaP cells. It has been reported that splicing of exon within AR intron 2 introduces a stop codon upstream of exon 3 in the AR transcript that would encode an AR protein (lacking the second zinc finger of the DBD and LBD) if translated. In our ongoing efforts to characterize AR and its splice variants, and devise strategies to target these for treating human CaP, we provide evidence that ARv7 or AR3 is a major splice variant detected in human CaP cell lines, xenografts and human malignant prostate-tissues. We provide evidence that ARv7 levels are generally higher in CRPC tumor of humans. We provide a novel mechanism that we believe regulates the expression of ARv7 in CRPC cells. We also provide a novel agent that we show effectively targets ARv7 function in CRPC cells. We show that ARv7 sustains PSA generations and growth of cells in a low-androgen environment. We suggest that AR plays a critical role in the CaP and CRPC pathogenesis, thus is an ideal molecule for targeted therapies. Several approaches have been utilized to tame the AR function and majority of these points towards competitive inhibitors (for androgen-binding to LBD) and nuclear translocation inhibitors. Based on our studies and published reports, we suggest that in addition to targeting AR directly, blocking co-activators (needed as a prerequisite for AR function), should form a part of therapeutic strategy.

N-GLYCAN TARGETS ANGIOGENESIS AND IMPACTS THE PROGRESSION/REGRESSION OF BREAST TUMOR GROWTH: TWO SIDES OF A SINGLE TARGET

Dipak K. Banerjee

Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical sciences Campus, San Juan

BACKGROUND: Breast cancer is a highly proliferative disorder with a complex etiology, and continues to be a major health problem worldwide. Irrespective of mutations in tumor cells, neovascularization remains a major event in tumor progression. Aberrant glycosylation has been observed in essentially all types of experimental and human cancers. Inhibition of hybrid and complex-type N-glycans synthesis inhibits the formation of capillary tubes. But, inhibition of complex-type N-glycans, and not hybrid-type N-glycans is the blocker of the tube formation. Our focus is the interrelationship between the dynamic process of asparagine-linked (N-linked) protein glycosylation and angiogenesis. METHODS: Evaluation of the significance of a cross-talk between mannosylphospho dolichol synthase (DPMS) and N-acetylglucosaminyl 1-phosphate transferase (GPT) and its dynamic relationship to angiogenesis in vitro (a non-transformed capillary endothelial cell line) and in vivo (nude mice with or without breast tumor). RESULTS: Increased synthesis and turnover of lipid-linked oligosaccharide (LLO, Glc₃Man₉GlcNAc₂-PP-Dol) due to phosphorylation activation of DPMS increased protein N-glycosylation, which resulted in accelerated cell cycle and increased angiogenesis. DPMS overexpression can mimic it. Tunicamycin, a GPT inhibitor reduces the LLO level, down-regulates the VEGF signaling, induces ER stress, and affect 577 genes leading to cell cycle arrest and apoptosis. Raman Spectroscopy detects protein denaturation and consequently the unfolded protein response. When tested in a double-negative (ER⁻/PR⁻?HER-2⁺) breast cancer, tunicamycin prevented the tumor progression by ∼55 % in 3 weeks. Tumor microvasculature exhibited ER stress. CONCLUSION: N-glycan is an excellent molecular target for developing novel anti-angiogenic glycotherapy treating breast cancer progression/elimination.

OSTEOPONTIN AND RELATED PROTEINS: ROLE IN INFLAMMATION, CANCER AND PATHOLOGICAL ANGIOGENESIS

Gopal C. Kundu

National Centre for Cell Science, NCCS Complex, Pune 411 007, India

Background: Wound healing, inflammation and cancer are pathological events associated with rapid neovascularization in adults. Earlier reports have suggested that inflammatory cells play an important role in pathological angiogenesis. The regulation of cancer progression towards its malignancy needs the interplay among various cytokines, transcription factors and the effector molecules. Osteopontin (OPN), a cytokine like, calcified ECM associated SIBLING family of protein plays an important role in determining the oncogenic potential of various cancers. However the molecular mechanism by which stroma- and tumor-derived OPN that regulate tumor growth and angiogenesis are not well defined. Methods and Results: We have reported that OPN triggers VEGF dependent breast tumor growth and angiogenesis by activating various signaling cascades. Our results also revealed that both stroma and tumor-derived OPN controls tumor progression and angiogenesis through activation of various kinases and downstream target molecules. Previous reports indicated that OPN is a hypoxia inducible gene. Our recent reports highlight the emerging role of OPN in the hypoxic adaptation of breast cancer cells that ultimately leads to HIF-1alpha dependent VEGF expression and breast tumor angiogenesis. Moreover, the role of CD20 and CD133 positive cancer stem cells and how that correlate OPN expression and regulate melanoma growth and angiogenesis are in progress. Furthermore, OPN derived RGD conjugated chitosan nanoparticle mediated drug delivery in breast tumor model is the subject of intense investigation. Conclusion: Thus, targeting OPN and its associated genes could be novel therapeutic approach for the control of tumor growth and angiogenesis.

ANTI-ANGIOGENIC THERAPY ON IN VIVO TUMOR BEARING MOUSE MODEL SYSTEM

Samarendra Nath Banerjee and Srabantika Mallick

Department of Zoology, Rammohan College, 102/1, Raja Rammohan Sarani, Kolkata – 700009, India Email: samban2kcal@yahoo.com, samar_banerjee@rediffmail.com

BACKGROUND: The process of angiogenesis plays a critical role in both normal physiological events as well as in many pathological processes such as tumor growth and metastasis. Different in vitro and in vivo techniques are used to elucidate the mechanisms controlling angiogenesis and to investigate how cancer therapies in particular anti-angiogenesis therapy may interfere with the angiogenic process to induce tumor regression with less toxicity. 2-Methoxyestradiol (2ME) has emerged as a promising agent for cancer treatment. A dose-dependent biphasic effect of 2ME on VEGF mRNA expression and cell proliferation was found in some cell lines. But it is necessary to investigate the effect of 2ME on in vivo system. Till now little has been done on the effect of 2ME on somatic chromosome of the tumor bearing mouse, particularly during the period of tumor regression. The present study was undertaken to evaluate the efficacy and safety of 2ME, either alone or in combination with Cyclophosphamide (CP) on in vivo mouse tumor model considering tumor growth rate and bone marrow toxicity. METHODS: Tumor regression in response to 2ME, either alone or in combination with CP was studied at different time intervals by morphometric analysis of tumor size and BrdU labeling cell proliferation study. Bone marrow toxicity was measured by chromosomal aberration analysis. RESULTS: Analysis of tumor cell and bone marrow metaphases clearly points out that 2ME in combination with CP can protect bone marrow as it inhibits different types of chromosomal aberrations. The regression of the tumor during the course of therapy was also determined and correlated with the gradual increase of dead cell and decrease of living cell frequency in the treatment series of S-180 ascitic tumor bearing cells. CONCLUSION: The results indicate that 2ME in combination with CP not only gave protection to bone marrow chromosomes of mouse but also induced solid tumor regression.

TRANSLATIONAL STUDIES WITHPHELLODENDRON AMURENSEBARK EXTRACT: MYTH OR REALITY?

Addanki P. Kumar, Paul Rivas, Craig H. Robson, Guiming Li, Roble Bedolla, Joseph E Basler, Gregory P Swanson, William E Jones, Nikolas Papanikolaou, I-Tien Yeh, Robert M Reddick and Rita Ghosh.

Department of Urology, School of Medicine, South Texas Veterans Health Care System and The University of Texas Health Science Center at San Antonio, Texas.

Therapeutic resistance is one of the limitations for successful management of prostate cancer (PCA). 15–30 % of men with localized PCA treated with radiotherapy will be diagnosed with recurrent disease. In addition these treatments are already delivered at a dose intensity that results in toxicity to normal tissue. One approach to circumvent these problems is to combine radiation with Nexrutine^R. Such a combination has the potential to (i) increase the sensitivity of tumor cells to lower doses of radiation; (ii) reduce the cytotoxic effects of radiation on normal cells and (iii) improve therapeutic index of the treatment modality. We investigated if intervention with Nexrutine^R in vivo would potentiate cytotoxic effects of radiotherapy. Twenty-week-old TRAMP mice (at which time they display invasive carcinoma) were fed pelleted diet containing 600 mg/kg Phellodendron Amurense bark extract 6 weeks prior to undergoing radiation therapy. These animals were compared to a group that received radiation alone. Efficacy was evaluated by histological analysis of prostate tissue at the termination of the experiment at 34 weeks. Preliminary data demonstrate that a majority of animals that received Phellodendron amurense bark extract for 6 weeks prior to radiation therapy had no overt cancer but exhibited features consistent with high grade prostatic intraepithelial neoplasia. In contrast animals that received radiation alone exhibited features consistent with well to poorly differentiated adenocarcinoma. These differences indicate a dramatic improvement in response. Investigation of molecular pathways associated with Nexrutine^R-mediated inhibition of cell proliferation showed the inhibition of Akt/CREB/Cyclin D1 network and induction of apoptosis. This led to a phase 0/I clinical trial in men undergoing radiotherapy/surgery for prostate cancer. These results will be discussed. Supported by NIH and pilot funds through CTRC (APK).

TARGETING INTRA-TUMOR ANDROGEN SIGNALING VIA STEROID SULFATION: A VITAMIN D AND STEROL-REGULATED MECHANISM FOR PROSTATE CANCER INHIBITION

Bandana Chatterjee

University of Texas Health Science Center at San Antonio, USA

BACKGROUND: Reactivated androgen receptor, fueled by elevated tumor tissue 5α-dihydrotestosterone (DHT), plays a key role in the reemergence of prostate cancer. Primary prostate cancer almost always regresses in response to androgen deprivation therapy. The disease, however, recurs and progresses to therapy resistance and death in ∼30 % patients. New-generation drugs that inhibit androgen receptor (MDV3100, TAK-700□, Dutasteride□) extend survival of post-chemotherapy patients for a limited period following which therapy resistance emerges—hence the challenge to develop alternate means for reducing intra-tumor androgen signaling. METHODS and RESULTS: 3□-sulfation of dehydroepiandrosterone (DHEA), mediated by the prostate-expressed sulfotransferase SULT2B1b (aka SULT2B), is likely to reduce intra-prostate androgen levels since sulfated DHEA cannot be converted to androstenedione and thus to testosterone and DHT. We tested whether enzyme-catalyzed sulfation of DHEA, the obligate precursor steroid for androgen synthesis, is targetable to inhibit prostate cancer. Results show that SULT2B silencing increased prostate cancer cell proliferation. Clinical prostate cancer specimens showed markedly reduced SULT2B expression, revealed by immunohistochemistry, western blotting and mRNA quantification. 1,25-dihydroxy vitamin D₃ (D₃) and sterols enhanced SULT2B expression due to induction of the SULT2B-encoding gene by D₃-activated vitamin D receptor (VDR) and sterol-activated liver X receptor (LXR□). CONCLUSION: Tumor SULT2B levels may be a marker for predicting prostate cancer recurrence and progression. We also anticipate that enhancement of SULT2B activity in tumors by activating VDR and LXR□ through combined therapeutic intervention with D₃ and sterols would limit the tumor androgen pool and inhibit recurrent, therapy-resistant prostate cancer.

ADA3, A NOVEL MOLECULAR TARGET FOR CANCER THERAPY

Vaibhav Chand, Rince John, Neha Jaiswal and Alo Nag

Dept. of Biochemistry, Univ. of Delhi South Campus, New Delhi, India.

Cancer is one of the most identifiable human disease caused by cells displaying uncontrolled growth and invasion properties leading to metastasis and poor patient survival. Many viral oncoproteins cause cancer by abrogating the function of key cellular proteins involved in the maintenance of normal physiology. Our study focuses on human ADA3 (hADA3), a recently identified cellular target of Human Papilloma Virus (HPV) 16 E6. ADA3 is an essential component of the ADA transcriptional coactivator complex. High risk HPV-E6 has been shown to bind and abrogate coactivator function of hADA3. This implies a crucial contribution of E6 induced hADA3 inactivation towards HPV mediated oncogenesis. Till date, very little is known about the mechanism of degradation of hADA3 by E6. Here, we show that HPV positive cervical cancer cell lines exhibit reduced level of hADA3. Half life assay with overexpressed E6 confirmed its role in destabilization of hADA3. HPV-E6 mediated degradation of hADA3 was shown to be mediated through ubiquitin proteasome pathway with an active participation of E6-AP. HPV-E6 was also shown to induce other posttranslational modifications of hADA3 which may account for its destabilization. Interestingly, depletion of hADA3 was found to induce oncogenic EMT like phenotype in cervical cancer cells. In summary, present investigation provides novel insight into the mechanistic role of hADA3 in tumor suppression. Thus, our findings show promise in development of novel therapies against HPV related malignancies.

THE POTENTIAL OF CELECOXIB-LOADED HYDROXYAPATITE-CHITOSAN NANOCOMPOSITE FOR THE TREATMENT OF COLON CANCER

Mahitosh Mandal

School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302, India.

Celecoxib has shown potential anticancer activity against most carcinomas, especially in patients with familial adenomatous polyposis and precancerous disease of the colon. However, serious side effects of celecoxib restrict its generalized use for cancer therapy. In order to resolve these issues and develop an alternative strategy/preliminary approach, chitosan modified hydroxyapatite nanocarriers-mediated celecoxib delivery represents a viable strategy. We characterized the nanoparticle for morphology, particle size, zeta potential, crystalinity, functional group analysis, entrapment efficiency, drug release and hemocompatibility. The effects of celecoxib-loaded nanoparticles on colon cancer cell proliferation, morphology, cytoskeleton, cellular uptake and apoptosis were analysed in vitro. Further, we evaluated the antiproliferative, apoptotic and tumor inhibitory efficacy of celecoxib-loaded nanocarriers in a nude mouse human xenograft model. Nanoparticles exhibited small, narrow hydrodynamic size distributions, hemocompatibility, high entrapment efficiencies and sustained release profiles. In vitro studies showed significant antiproliferation, apoptosis and time-dependent cytoplasmic uptake of celecoxib-loaded Hap-Cht nanoparticles in HCT 15 and HT 29 colon cancer cells. Additional in vivo studies demonstrated significantly greater inhibition of tumor growth following treatment with this modified nanoparticle system. The present study indicates a promising, effective and safe means of using celecoxib, and potentially other therapeutic agents for colon cancer therapy.

ELUCIDATING THE MOLECULAR SIGNALING ADAPTED BY ARTEMISININ IN CERVICAL CANCER CELLS

Urmi Chatterji

Associate Professor, Department of Zoology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata – 700 019, India. Tel: +91-93393 76535; Email: urmichatterji@gmail.com

Background: In an attempt to identify a compound that can be used for effective prognosis of cervical cancer, we investigated the effects of artemisinin (ART) on human cervical cancer cells.

ART has anti-cancer activities both in vitro and in vivo. The present study focuses on the molecular alterations induced by artemisinin in human cervical cancer cells, with special emphasis to the estrogen receptor and β-catenin pathways, which regulate cell proliferation, angiogenesis and apoptosis. Methods: Effects of artemisinin on proliferation of ME-180 cells were measured by flow cytometry. Expression of genes and proteins related to cell proliferation, angiogenesis and apoptosis were quantified both at the transcriptional and translational levels by semi-quantitative RT-PCR, western blot analysis and immunocytochemistry, respectively. Results: Dose and time-dependent action of artemisinin indicated that artemisinin is capable of down regulating the expression of ERα and β-catenin, both at the transcriptional and translational level. The downstream genes of the canonical Wnt/β-catenin signaling pathway related to cell proliferation, like cyclin D1, CDK4 were down regulated, whereas p21^Waf1/Cip1 was up regulated. Concomitant down regulation of VEGF, hTR, hTERT, E6 and E7 oncogenes was also observed in response to artemisinin. Artemisinin-induced apoptosis was confirmed by nuclear chromatin condensation, annexin V staining and p53 up regulation. Conclusion: Our results clearly indicate that artemisinin induces anti-proliferative, anti-angiogenic and pro-apoptotic effects in HPV-39 infected ME-180 cells, mainly by targeting the ER and canonical Wnt/β-catenin.

CANCER STEM CELLS: IMPLICATIONS FOR COLON CARCINOGENESIS AND DEVELOPMENT OF THERAPEUTIC STRATEGIES WITH NATURAL AGENTS

Adhip P. N. Majumdar

Veterans Administration Medical Center, Karmanos Cancer Institute, Department of Medicine, Wayne State University School of Medicine, Detroit, Michigan, USA

BACKGROUND: Recent evidence supports the contention that epithelial cancers, including sporadic colorectal cancer (CRC), the incidence of which increases with aging, are diseases driven by self-renewing cancer stem cells (CSCs). We reported an age-related increase in colon CSCs. One of the characteristics of CSCs is their resistance to 5-FU based chemotherapy, the mainstay of colon cancer therapeutic with significant toxicities, resulting in high disease recurrence underscoring the need for improved CSC-targeted non-toxic therapeutics for CRC. Investigations were carried out to examine the role of CSCs in the development of CRC during aging and to develop therapeutic strategies using natural agents to combat this malignancy. METHODS: Both in vitro and in vivo studies were conducted. Immunohistochemistry and quantitative real-time PCR as well as Western-blot analysis were used to determine the relative concentration of markers of colon CSCs. Flow cytometry was utilized to examine the proportion of colon CSCs. RESULTS: We found that curcumin, the major active ingredient of turmeric (Curcuma longa), used in South Asian cuisine, with no discernible toxicity synergizes with FOLFOX (5-FU+Oxaliplatin) and attenuates EGFR signaling resulting in inhibition of colon cancer cell growth. In subsequent studies, we observed that difluorinated curcumin (CDF), a novel analog of curcumin, either alone or together with FOLFOX markedly stimulates apoptosis of FOLFOX-resistant colon cancer cells and disintegrates colonospheres (surrogate tumors) that are highly enriched in CSCs. Interestingly, eicosapentaenoic acid (EPA), one of the components of omega-3 polyunsaturated fatty acids, together with FOLFOX has also been found to be highly effective in inhibiting the growth of FOLFOX-resistant colon cancer cells, accompanied by a marked reduction in expression of ALDH1, one of markers of colon CSCs. Another approach we utilized was to induce differentiation of colon CSCs by down-regulating miroRNA-21 (upregulated in CRC) and subjecting the differentiated/differentiating cancer cells to CDF either alone or together with 5-FU based chemotherapy. This approach resulted in a marked attenuation of EGFR signaling, β-catenin/TCF driven gene transcription and reduction in colon CSCs and cellular growth. CONCLUSION: The conventional 5-FU based chemotherapy together with one of the following agents (a) curcumin, (b) CDF or (c) omega-3 fatty acids that eliminates colon CSCs could be effectively utilized for treatment of colorectal cancer. Differentiation of colon CSCs also renders the chemo-resistant cells highly susceptible to the combination of 5-FU based chemotherapy and CDF.

SUBSEQUENT HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT) ASSOCIATED WITH LONGER SURVIVAL IN PATIENTS WITH RELAPSED/REFRACTORY (R/R) ACUTE MYELOGENOUS LEUKEMIA (AML) AFTER CLO+ARA-C OR ARA-C ALONE: A LANDMARK ANALYSIS FROM THE CLASSIC I TRIAL

Siddhartha Ganguly¹, Kantarjian, H.M.², Wetzler, M.³, Rizzieri, D.⁴, Schiller, G.⁵, Jagasia, M.⁶, Stuart, R.⁷, Avigan, D.⁸, Craig, M.⁹, Collins, R.¹⁰, Maris, M.¹¹, Kovacsovics, T.¹², Golberg, S.¹³, Seiter, K.¹⁴, Hari, P.¹⁵, Greiner, J.¹⁶, Vey, N.¹⁷, Recher, C.¹⁸, Ravandi, F.², Wang, E.³, Eckert, S.¹⁹, Partisano, A.¹⁹, Faderl, S.²

¹University of Kansas Medical Center, Kansas City, KS; ²University of Texas MD Anderson Cancer Center, Houston, TX; ³Roswell Park Cancer Institute, Buffalo, NY; ⁴Duke University Medical Center, Durham, NC; ⁵University of California Los Angeles, Los Angeles, CA; ⁶Vanderbilt University Medical Center, Nashville, TN; ⁷Medical University of South Carolina, Charleston, SC; ⁸Beth Israel Deaconess Medical Center, Boston, MA; ⁹West Virginia University, Morgantown, WV; ¹⁰University of Texas Southwestern Medical Center at Dallas, Dallas, TX; ¹¹Rocky Mountain Cancer Centers, Denver-Midtown, Denver, CO; ¹²Oregon Health and Science University Center for Hematological Malignancies, Portland, OR; ¹³Hackensack University Medical Center, Hackensack, NJ; ¹⁴New York Medical College, Valhalla, NY; ¹⁵Medical College of Wisconsin, Milwaukee, WI; ¹⁶Universitatsklinikum Ulm, Ulm, Germany; ¹⁷Service d’H_ematologie, Institut Paoli Calmettes, Marseille, France; ¹⁸Service d’H_ematologie, Hopital Purpan, Toulouse, France; ¹⁹Sanofi Oncology, Cambridge, MA

BACKGROUND: If feasible, hematopoietic stem cell transplantation (HSCT) is the only option for patients with relapsed or refractory (R/R) acute myeloid leukemia (AML) to achieve long-term survival. We previously reported the efficacy/safety outcomes of the CLASSIC I study in older patients (pts) with R/R AML (Faderl et al., JCO 2012 Jul 10; 30: 2492–9). In this report, we evaluate the outcomes of HSCT after Clo+Ara-C or Ara-C alone in CLASSIC I. METHODS: CLASSIC I was a randomized, double-blind trial that compared Clo+Ara-C to Ara-C in pts 55 years or older with R/R AML. Pts were randomized to either Clo 40 mg/m2 or placebo followed by Ara-C 1 g/m2 each for 5 consecutive days. While the primary endpoint of OS did not differ between arms, there were statistically significant improvements in the secondary endpoints (ORR, CR & EFS) in the Clo+Ara-C arm. In this post-hoc analysis, patient characteristics and safety were analyzed for pts who underwent HSCT. Since the median time to HSCT was 3.8 months from the initiation of the study drug, a 4-month landmark analysis was conducted to evaluate OS following HSCT. To minimize bias, we conducted post-hoc landmark analysis to assess the extent of association between HSCT and OS at 4 and 8 months. RESULTS: Among 320 pts with centrally confirmed AML (median age, 67 years), 66 (21 %; median age 63.5 years) underwent HSCT [Clo+Ara-C: n = 34; Ara-C: n = 32]. Of these, 47 pts (71 %) went to HSCT in CR; 36 in CR from study drug [Clo + Ara-C: n = 22 vs. Ara-C: n = 14] and 11 in CR from alternative therapy. 64 pts received an allogeneic transplant [sibling donor: n = 46 (70 %), unrelated donor: n = 18 (27 %)] and 2 (3 %) were autologous transplants. Preparative regimens included myeloablative in 22 pts (33 %) and reduced intensity conditioning/non-myeloablative in 43 pts (65 %). Multiple analyses were consistent with a survival benefit of HSCT: a naive estimate comparing OS in pts with and without HSCT; a 4-month landmark analysis comparing OS in pts with and without HSCT; and landmark analyses assessing the impact of HSCT on OS for patients who achieved CR. Although there was evidence that HSCT was associated with longer survival, there was little evidence of a differential effect across the treatment arms (interaction p = 0.32 [not shown]). Post HSCT safety findings were similar across treatment arms and raised no new concerns. CONCLUSION: This post-hoc analysis of the CLASSIC I study indicates that subsequent HSCT in elderly patients with R/R AML is feasible and appears to be associated with longer survival; though there is little evidence of a differential effect across treatment arms. A prospective randomized study is needed to determine the validity and clinical applicability of these findings.

MODULATING AKT INDUCED EPITHELIAL MESENCHYMAL TRANSITION FOR PREVENTION AND TREATMENT OF COLON CANCER

Chendil Damodaran

Texas Tech University Health Science Center, El Paso, TX-79912

Activation of serine/threonine kinase AKT has emerged as central feature of epithelial to mesenchymal transition (EMT) which is initial step for metastasis leading to aggressiveness of the disease in many cancer models including colorectal cancer (CRC). The focus of current study is to dissect the role of AKT and its molecular signaling in regulation of EMT in CRC. HCT-116 colon cancer cells stably over expressing AKT (AKT/HCT-116) showed significantly higher cell proliferation when compared to the vector transfected cells (pCMV/HCT-116). An elevated expression of important EMT transcription factors and genes like Snail, Slug, β-catenin, MMP-2, MMP-9 correlated with increased migration and invasive phenotypes of AKT/HCT-116 cells. Further, in vivo studies confirmed that AKT/HCT-116 xenografts are highly aggressive and angiogenic in nature when compared with the mice injected with pCMV/HCT-116 cells. Molecular analysis from tumor samples, revealed a transcriptional regulation of snail, slug, β-catenin, MMP-2, MMP-9 in AKT/HCT-116 when compared with control tumors. Further these results were supported with immunohistochemistry analysis. Low levels of E-cadherin expression with concomitant increase and nuclear localization of β-catenin was evident in AKT/HCT-116 tumors as compared to controls. Increase in micro-vessel formation with high expression of Factor VIII and RETIC confirms the angiogenic property of the tumors. Altogether, our results confirms the potential role of AKT signaling in regulating EMT and angiogenesis in colon cancer and inhibiting AKT can serve as an important therapeutic strategy in modulating EMT in colon cancer growth and metastasis.

TARGETING CANCER STEM CELLS: AN APPROACH TOWARDS ‘NEXT GENERATION’ CANCER THERAPY

Tanya Das

Division of Molecular Medicine, Bose Institute, Kolkata

Introduction: Increasing evidence suggests that cancer development is due to a rare population of cells, termed cancer stem cells (CSCs) that uniquely initiates and sustains disease. However, the role of CSCs in multistage cancer progression, particularly with respect to drug-resistance and metastasis, has not been well-defined. This study, thus, aims to discern the contribution of these CSCs to cancer drug-resistance and metastasis, to eventually identify novel treatment modalities to fight cancer recurrence and progression. Methodology/Results: Exploiting various new-generation technologies we observed that a small population of tumor cells always fails to surrender even at lethal doses of chemotherapeutic drugs. These drug-spared cells (DSCs) not only displayed self-renewal, tumorigenic and differentiation properties, but also exhibited ‘stemness signature’—thereby confirming their identity as cancer stem cells (CSCs). An effort to unveil the cause underneath such resistance property demonstrated that the presence of drug efflux pumps, slow-cycling character, and the anti-oxidant phenotype of CSCs contributed in the escape of these cells from drug-insult. In fact, chemotherapy augmented CSC population which displayed low ROS even at high dose of the genotoxic drugs. Under this condition, ATM encouraged NFκB-dependent chemo-resistance while down-regulating ATM-JNK pro-death circuitry in CSCs. Interestingly, additional genotoxic insults triggered self-renewal in these cells thereby increasing their numbers in the total population. Consistent with this in vitro experimental data, increased percentage of CSCs was observed in human breast tumors after neo-adjuvant chemotherapy when compared with un-treated ones. Furthermore, enrichment of CSCs was observed in breast tumors of patients undertaking increasing cycles of anti-cancer drugs. Additional investigation exposed that in a cancer tissue or cell population, only CSCs acquire high migratory potential thereby indicating their possibility of being exclusively responsible for invasion and metastasis. Detail search discovered highly aggravated migration property, higher levels of pro-migratory proteins such as MMPs, integrins, slug and Twist, as well as mesenchymal phenotype rather than epithelial morphology of these CSCs as compared to the parental cancer cells. Importantly, intervening at any molecular step, defined so far, restored chemosensitivity and down-regulated Oct-4, resulting in significant perturbation of ‘spheroid’ formation and ‘stemness’ signature. Conclusion: On the basis of these findings we propose a unified model of cancer progression in which CSCs play a central role in both tumorigenesis and metastasis. An approach towards targeting these ‘root of all evils’ may, therefore, be the endeavor of next generation therapy for complete regression of drug-resistant and metastatic cancers.

DEVELOPMENT OF NOTCH INHIBITORS IN BREAST CANCER: TARGETING STEM CELL PATHWAYS

Albain KS¹, Czerlanis C¹, Rajan P¹, Zlobin A¹, Godellas C¹, Bova D¹, Lo SS¹, Robinson P¹, Sarker S¹, Gaynor ER¹, Cooper R¹, Aranha G¹, Czaplicki K¹, Busby B¹, Rizzo P¹, Chisamore M², Demuth T², Blackman S², Watters J², Stiff P¹, Fuqua SAW³, Covington, KR³, Antonio Pannuti⁴, Ingrid Espinoza⁴, Miele Lucio⁴

¹Loyola University Chicago Cardinal Bernardin Cancer Center, Maywood, IL; ²Merck Oncology, North Wales, PA; ³Baylor Breast Center, Houston, TX; ⁴University of Mississippi Cancer Institute, Jackson, MS

BACKGROUND: Breast tumor initiating cells (TIC) are thought to use Notch signaling with other pathways for self renewal. This is suggested to be a major cause of tumor recurrence and progression. Notch inhibitors are being tested in breast cancer clinical trials. However, successful development of these agents in oncology will require a mechanistic understanding of how Notch cross-talks with other therapeutically relevant pathways. This is has been the focus of our lab for the last 15 years. In ERα-positive breast cancer models, we showed that estrogen decreases Notch activity. Estrogen deprivation or tamoxifen (tam) re-activate Notch signaling and are more sensitive to pharmacological Notch inhibitors. Treatment with γ-secretase inhibitors (GSIs) potentiates the effects of tam in T47D xenografts and overcomes tam-resistance in a well-characterized in vivo model. Additionally, we have shown that Notch-1 can activate ERα-dependent transcription in the absence of estrogen, potentially leading to endocrine resistance. Both Notch-1 and Notch-4 render ER+ cells estrogen-independent and tam-resistance, through different pathways. It was unknown whether GSIs plus endocrine therapy result in modulation of Notch and other proliferation and TIC markers in human breast cancer. We conducted a pilot clinical trial to add short exposure of the GSI MK-0752 to ongoing tam or letrozole (letr) during the presurgical window to determine 1) feasibility, 2) safety/tolerance, and 3) impact on biomarkers. The trial was completed in December, 2011. We report data from this pilot study, also shown at SABCS (ClinTrials.gov NCT00756717). METHODS: Patients (pts) with early stage ERα+ breast cancer were treated with 25 days of tam or letr. On day 15 MK-0752 was added to endocrine therapy (350 mg orally 3 days on, 4 days off, 3 days on), with definitive surgery day 25. Formalin-fixed, paraffin embedded biopsies were obtained at baseline, day 14 and final surgery, with histologic confirmation of tumor content (>50 %) and RNA extraction by standard methods. Q-PCR was done for Notch-1, Notch-3, Notch-4, Deltex, Jagged-1, c-myc, HEY1, HEY2, HES1, PS2, C-Myc, Cyclin A2, NOXA, Ki67, Dicer-1, RPL13 (internal control). Ct averages for 3 replicates were used and mRNA levels were calculated by the 2ΔΔCt method. Baseline gene expression levels were used as comparators for days 14 and 25 levels in each pt. RESULTS: 22 patients were enrolled to reach the planned sample size of 20 participants who received all therapy. 11 patients received tamoxifen, 9 letrozole. 2 withdrew before receiving study drug. All biopsies and definitive surgeries were performed on schedule. Toxicity was minimal (1 grade 1 periorbital edema and cough, 4 grade 1 acneiform facial rash, no diarrhea or surgical complications). Microarray analysis (in progress) indicated modulation of multiple TIC pathways, including Notch, Wnt, insulin, MAPK, chemokine and ERB. Expression of Notch-1, Notch-4, NOXA, Ki-67, RUNX1 and ADAM19 was significantly modulated by GSI in 75 % or more (≥15/20) of the patients. “Canonical” Notch targets HES1, HEYL and HES5were significantly modulated in 13/20, 12/20 and 6/20 patients respectively. Cyclin D1 and Notch-3 were modulated in 11/20 and 10/20 patients respectively. 100 % of patients showed either a decrease in Ki67 (17/20), an increase in NOXA (15/20) or both. This was a molecular indication of anti-proliferative and pro-apoptotic effects of MK0752 plus endocrine therapy compared to endocrine therapy alone. CONCLUSIONS: 1) Addition of the gamma secretase inhibitor MK-0752 to ongoing endocrine therapy was feasible, safe and well-tolerated; 2) Despite short treatment at a dose much lower than phase I MTD, all tumors showed significant modulation of Notch pathway genes and other genes relevant to breast cancer TIC biology; 3) MK-0752 favorably affected targets related to proliferation, apoptosis, stem cells, endocrine resistance and metastasis; and impacted critical cancer pathways. A phase 2 trial is warranted and is being planned. Funding: Swim Across America, Inc. (clinical trial costs); Merck (drug supply, profiling)

PREGNANCY, HORMONES, AND BREAST CANCER

Rajkumar Lakshmanaswamy

Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, TX, USA

Breast cancer is the most common cancer in women worldwide and attempts at prevention of breast cancer are important areas of clinical and experimental investigations. Women who undergo a full-term pregnancy before the age of 20, with or without lactation, have about one half the risk of developing breast cancer compared to women who undergo their first pregnancy after the age of 35 or women who have never undergone a full term pregnancy. This universal protective effect of pregnancy serves as a major clue and starting point in the search for new strategies and novel biomarkers related to the prevention of breast cancer. For the current study, women were recruited into one of the following categories; 1) women who had their first child ≤25 years of age [early parous], 2) women who had their first child ≥35 years of age [late parous], and 3) a control group of women the same age as those in groups 1 and 2 but who have never given birth [nulliparous]. Breast tissue and blood was collected from the volunteers. High throughput genomics and proteomics analysis were performed. Based on data we have identified early pregnancy associated signature genes and proteins that could serve as biomarkers for breast cancer prevention.

OMEGA 3-FATTY ACIDS DIFFERENTIALLY TARGET ONCOGENIC AND TUMOR SUPPRESSOR MICRO-RNAS TO PREVENT BREAST CANCER METASTASIS

Nandini Ghosh-Choudhury

South Texas Veterans Health Care System and Department of Pathology, University of Health Science Center at San Antonio, Texas, USA

We have shown that docosahexaenoic acid (DHA), the main ω3-fatty acid component of fish oil (FO), targets breast cancer stem cells to prevent tumor growth and osteolytic metastasis in mouse models. More recently we documented suppression of the oncogenic microRNA-21 (miR-21) expression by DHA as one of the mechanisms for preventing osteolytic metastasis of breast cancer. Breast cancer cells undergo epithelial to mesenchymal transition (EMT) characterized by transcriptional repression of E-cadherin to initiate tumor metastasis. We investigated the mechanism of fish oil action on EMT. Breast tumors from fish oil-fed mice showed significantly increased expression of E-cadherin mRNA. Incubation of bone metastatic MDA-MB-231 (MDA) human breast cancer cells with DHA markedly enhanced E-cadherin mRNA and protein expression, indicating reversal of EMT. Breast cancer metastasis is associated with increased expression of Zeb1, a repressor for E-cadherin transcription. DHA inhibited the expression of Zeb1 protein and mRNA. Importantly, tumors from FO fed mice showed significant reduction in Zeb1 mRNA expression. The mechanism by which FO regulates Zeb1 expression is not known. We considered both transcriptional mechanism and post-transcriptional regulation by microRNA. Transient transfection assays using a Zeb1 promoter reporter construct in MDA cells showed significant decrease in transcription of Zeb1 by DHA. Using ChIP assay we identified involvement of NFkB transcription factor in Zeb1 transcription, which was targeted by DHA. 3′UTR of Zeb1 mRNA contains recognition element for miR-200c. FO-fed mice tumors showed increased levels of pre-miR-200c and mature miR-200c. Similarly, DHA markedly enhanced the expression of both pre and mature miR-200c in MDA cells. Finally, reporter assays using miR-200c promoter in MDA cells showed significant increase in miR-200c transcription in response to DHA. Together our results demonstrate a salutary effect of FO to prevent osteolytic metastasis by downregulating miR-21 expression and to reverse EMT of breast cancer cells by increased miR-200c, which dampens the expression of Zeb1 to upregulate E-cadherin.

PROTEIN KINASE C SIGNALING IN TRIPLE NEGATIVE BREAST CANCER METASTASIS

Soumen Paul

Department of Pathology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA

BACKGROUND: Triple negative breast cancer (TNBC) represents ∼12–17 % of all breast cancers and lacks expression of estrogen receptor, progesterone receptor, and epidermal growth factor receptor type 2. TNBC often found to be associated with aggressive pathologic features with higher rate of metastasis and recurrence leading to limited clinical outcome. Till date, no targeted therapy is available to prevent metastatic progression of TNBC. This is due to the lack of our poor understanding about signaling networks that influence TNBC metastasis. Thus, delineation of such signaling pathways could lead to the development of targeted therapies for TNBC recurrence and metastasis. METHODS: To understand signaling mechanisms that could regulate TNBC metastasis, we focused on specific protein kinase C (PKC) isoforms, as they are abundantly expressed in TNBC. We used TNBC cell lines and spontaneous models of TNBC metastasis and inhibited or depleted PKC isoforms to test TNBC cell invasion and metastasis. RESULTS: We discovered that PKC-isoform signaling is prometastatic and inhibition of PKC signaling inhibits TNBC cell invasion and metastasis in the lung. CONCLUSION: Our work indicates that PKC signaling can be targeted as a therapeutic modality to treat TNBC metastasis and recurrence.

PROBABLE NEW THERAPEUTIC DRUGS FOR BREAST AND COLON CANCERS

Subhash Basu¹, Rui Ma², Joseph R. Moskal ³, Manju Basu¹, and Sipra Banerjee⁴

¹Department of Chemistry and Biochemistry and Cancer Drug Delivery Research Foundation, University of Notre Dame, Notre Dame, IN 46556,; ² Siemens, P.O. Box 2297,Elkhart IN 46515; ³ The Falk Center for Molecular Therapeutics, Northwestern University, Evanston, IL 60201; ⁴ Cleveland Clinic Foundation, Department of Cancer Biology, Cleveland, OH, 44129;USA

Almost 10 % (50, 000) of total breast cancer-diagnosed patients die every year, in the United States. Luminal A- and luminal B-type breast cancers are characterized by a low chance of metastasis and relatively good medical prognoses. On the other hand basal-like breast cancers (BLBC) are highly invasive and migrate aggressively to distal organs, and are untreatable medically either by available chemotherapy or through combined chemo-radiation treatments. Evidence indicates that blood group-related Lewis (Le) antigens (glycosphingolipids; GSLs and Glycoproteins; GPs) are tumor-associated cell surface molecules. The Le^a, Le^b, Le^X, Le^Y, and their sialosyl-derivatives, all N-acetylglucosaminyl-containing glycoconjugate antigens, are over-expressed on the surfaces of breast, colon, and ovarian cancer cells during metastasis. Our goal is to find first a few potent anti-cancer agents which would kill the metastatic cancer cells when injected into the blood stream. All these chemicals: L-PPMP, D-PDMP, cis-Platin, GD3, GD1b, Tamoxifen, and Betulinic Acid (a herbal drug used for cancer treatment in China) induced the apoptotic pathways (intrinsic or extrinsic) in the metastatic breast (SKBR-3, MDA-468, and MCF-7) and colon (Colo-205) cancer cells. All of these apoptotic agents activated Caspass-3, -8, and -9 and also modulated a few specific genes for glycosyltransferases (studied by DNA-Microarray). Use of the specific antibodies against cell surface-specific glyco-antigens could be one of the targeted drug deliveries and will be the answer to effective cancer chemotherapy by apoptotic agents.

TARGETING INTEGRATORS OF LIFE AND DEATH SIGNALING IN BREAST CANCER

Savitha Sridharan, Kirti Jain, Deepanwita Pal and Alakananda Basu

University of North Texas Health Science Center and Institute for Cancer Research, Department of Molecular Biology & Immunology, 3500 Camp Bowie Blvd., Fort Worth, Texas - 76107, USA.

BACKGROUND: The 40S ribosomal protein S6 kinase (S6K) acts downstream of the mammalian target of rapamycin (mTOR), an important target for cancer therapy. mTOR inhibitors are, however, of limited success, due to feedback activation of the survival pathways. S6K exists as two homologs, S6K1 and S6K2. While both homologs are overexpressed in breast cancer, little is known about the function of S6K2. The objective of the present study is to discern the function of S6K homologs in breast cancer. METHODS: S6Ks and their potential downstream targets or upstream regulators were manipulated by siRNA silencing or ectopic expression. The levels of various proteins were determined by Western blotting. Cell death was determined by the PARP cleavage and staining of cells with Annexin V/Yo-pro and propidium iodide following treatment with various apoptotic stimuli. RESULTS: Depletion of S6K2 potentiated cell death by apoptotic stimuli in several breast cancer cells whereas depletion of S6K1 promoted cell survival. In contrast to S6K1, depletion of S6K2 decreased Akt phosphorylation in MCF-7 and T47D cells. Knockdown of S6K2 but not S6K1 caused a decrease in anti-apoptotic and an increase in proapoptotic Bcl-2 family proteins and ectopic expression of Akt partly restored cell survival in S6K2-depleted cells. CONCLUSION: Our study demonstrates that the two S6K homologs have opposite effects on Akt activation and breast cancer cell survival. Thus, targeting S6K2 rather than mTOR or S6K1 is expected to be an effective therapeutic strategy to treat breast cancers.

Session III, Hall C

USING MASSAGE THERAPY TO TREAT CHEMOTHERAPY-INDUCED PERIPHERAL NEUROPATHY (CIPN)

Joan E. Cunningham¹, Teresa Kelechi¹, Steve Chin¹, Pierre Giglio¹, Katherine Sterba¹, Viswanathan Ramakrishnan¹, David Stickler¹, Nikki Barthelemy², and Paul Falkowski².

¹Medical University of South Carolina, Charleston, SC ²Integrative Cancer Care, Inc., Charleston, SC

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is a common, miserable side-effect of many standard anti-cancer drugs and may be very long-lasting. Effective, tolerable treatment is lacking and underlying physiologic mechanisms poorly understood. Anecdotal data suggest massage therapy has potential to alleviate CIPN symptoms, and may promote nerve healing through improved circulation. This presentation highlights design and operational challenges for clinical investigation of massage therapy, and presents preliminary data from an on-going feasibility/pilot study in patients with solid tumors who have Grade 2 CIPN secondary to taxane and/or platin chemotherapy. METHODS: This non-randomized controlled trial includes a structured intervention, delivered by licensed practitioners in 12 treatments over 5 weeks, plus several months follow-up. Main outcomes measures are compliance; pre-post changes in CIPN, quality of life (QoL) and superficial blood circulation; and comparison to observation-only control patients. Assessment instruments include TNSr-SF, NPS-CIN and CIPNAT (for CIPN), EORTC-QLQ and -CIPN20 (for QoL), plus infra-red thermistry and laser-doppler flowmetry (for blood circulation). Patients are asked for feedback about study design elements. RESULTS: To date, 5 of 15 intervention patients have received treatment, with 100 % compliance in treatment schedule and no adverse events. None of 8 controls has been recruited. Patients varied in qualitative and quantitative symptoms. Preliminary analyses indicate every patient experienced reduction in various pain indices, each index highly significant in aggregate. Measured changes in temperature and perfusion were variable. Durability of response is being investigated. CONCLUSION: Preliminary results suggest massage therapy may be an excellent alternative to pharmacologic symptom control in CIPN patients. Supported by award UL1RR029882 from the National Center for Research Resources, and by the Massage Therapy Foundation.

THE BANERJI PROTOCOLS: THE REGRESSION OF MALIGNANT TUMORS BY A NON-TOXIC AND NON-INVASIVE ORAL MEDICAL APPROACH

Prasanta Banerji and Pratip Banerji

PBH Research Foundation, 10/3/1 Elgin Road, Kolkata -700020, India, Tel: +913330582815 Fax: +913322877275, Email: info@pbhrfindia.org, Website: www.pbhrfindia.org

Dr. Christian Friedrich Samuel Hahnemann postulated the most important principles of Homeopathy. In 1810, he published the “The Organon of the Rational Art of Healing”, his greatest book, wherein was elucidated systematically, the method and principles of a medical treatment to which he had given the name of Homeopathy. In comparison to the conceptual changes in allopathy, homoeopathic concepts are still based on symptoms only and not based on modern development of science but so far all important clinical trials in homoeopathy have not been based on the Theory of Individualization. Thus, even 200 years after Hahnemann’s discoveries, homoeopathy still lacks a firm and “modern day acceptable” scientific foundation.

This oral presentation is meant to introduce the methodology of “The Banerji Protocols” and to present retrospective data on our outcomes in using disease-specific homeopathic medicines for treatment of cancers and other critical intractable diseases. Although many studies have been conducted on the role of alternative medicine in the treatment of cancer, only a few reports have been published presenting the total regression of malignant tumors. A series of cancer cases cured using “The Banerji Protocols” was previously evaluated and validated by The National Cancer Institute (NCI), USA, Best Case Series (BCS) Program. We will present a brief description of how the “The Banerji Protocols” differ from the methods of classical homeopathy, and then will present treatment data and different cases of cancers and other serious illnesses. “The Banerji Protocols” provide standardized disease-specific protocols that can be consistently applied to all cases of specific cancers as well as other serious diseases, as primary treatment using homeopathic medicines without the use of any “conventional therapies”. The Banerji Protocols provide a viable treatment option for cancer patients who are unwilling or unable to receive conventional therapy. The survival rates in this study were comparable to or better than to those reported in SEER 1973–2004. We have a higher cure rate for some cancers that are not typically curable through conventional methods, such as lung and esophagus tumors and glioblastoma multiforme. In this conference we will talk about homeopathy itself and the remarkable contribution of Hahnemann to science. We will show the results of the treatment of cancer, the dreaded disease threatening mankind with no recourse to a solution offered by any system of medicine or surgery. We will also speak about our efforts to bring about scientific acceptance to this remarkable system of medicine. We will talk about the treatment of cancer by the use of homeopathic medicines as specific medicines for specific diseases in specific potencies in preset protocols. The objective being to establish homeopathy on scientific grounds elevating it to its rightful position as the medicine of the masses and to simplify the practice of this marvelous treatment system for benefit of suffering humanity.

CALCAREA CARB INDUCES APOPTOSIS IN CANCER CELLS VIA AN IMMUNO-MODULATORY CIRCUIT

Rathin Chakraborty

Bholanath Chakrabarty Trust, 5, Subol Koley Lane, Howrah 711101, India. E-mail: billroop27@ yahoo. co.in

BACKGROUND: Homeopathic remedies are reported to have healing potential for various diseases including cancer although the mechanism underneath their anticancer effect is still not known. To this end we attempted to evaluate the anti-cancer effects of the homeopathic remedy, calcarea carb, and simultaneously investigated the molecular mechanism underlying this drug-induced tumor regression. METHODS: Various biochemical, genetic alteration, flowcytometric, confocal-imaging, as well as immunological methods were employed to unveil the mechanisms of Calcarea carb induced regression of cancer both in in vitro and in vivo models. RESULTS: Although calcarea carb administration to ascites carcinoma-bearing mice resulted in 30–35 % tumor cell apoptosis, it failed to induce any significant cell death in ex vivo conditions. These results prompted us to examine whether calcarea carb employs the immuno-modulatory circuit in asserting its anti-tumor effects. In tumor-bearing mice, there was profound depletion of CD4⁺ and CD8⁺ cells in peripheral blood, dominance of T helper cell type-2 (Th2) that dampened T cytotoxic cell type-1 immune responses, and inhibition of T cell proliferation. Calcarea carb in turn prevented such loss of effector T cell repertoire, reversed type-2 cytokine bias and attenuated tumor-induced inhibition of T cell proliferation in tumor-bearing host. To confirm the role of immune system in calcarea carb-induced cancer cell death, a battery of cancer cells were co-cultured with calcarea carb-primed T cells. Our results indicated a “two-step” mechanism of the induction of apoptosis in tumor cells by calcarea carb i.e., (1) activation of the immune system of the host; and (2) induction of cancer cell apoptosis via immuno-modulatory circuit. CONCLUSION: These observations delineate the significance of immuno-modulatory circuit during calcarea carb-mediated tumor apoptosis. The molecular mechanism identified may serve as a platform for involving homeopathic inclusions into immunotherapeutic strategies for effective tumor regression.

UPDATE OF CLINICAL TRIALS IN BREAST CANCER

Barbara Dunn

National Cancer Institute, Bethesda, Maryland

Breast cancer is the most common cancer among women in the US, with an estimated 226,870 new cases and 39,510 deaths due to this disease estimated for 2012. Despite advances in treatment, the persisting high incidence of disease has contributed to an increasing emphasis on prevention. Seventy percent of breast cancers are estrogen receptor (ER)-positive, and are therefore presumed to be hormone-responsive and potentially treatable or preventable by anti-estrogenic agents. To date, all the large phase 3 randomized controlled breast cancer prevention trials have tested or are currently testing only hormonal drugs designed to antagonize the carcinogenic effect of endogenous estrogen. These agents are thus expected to prevent only ER-positive breast cancers; they are either selective estrogen receptor modulators (SERMs) or aromatase inhibitors (AIs). The SERMs, tamoxifen and raloxifene, have been shown in such large trials to reduce the risk of ER-positive breast cancers, leading to US FDA (Food and Drug Administration) approval of these drugs for risk reduction of breast cancer in high-risk women. A phase 3 prevention trial of the AI exemestane has been completed and shows a 65 % relative reduction in annual incidence of invasive breast cancer in women taking the AI compared to placebo. The risk-reducing properties of a second AI, anastrozole, are currently being investigated in the International Breast Cancer Intervention Study (IBIS)-II. The balance between the benefits (efficacy) and risks (toxicities) of these ER-positive breast cancer-directed agents will be discussed. Interest is now focusing on developing agents with a broader spectrum of preventive activity, particularly with regard to ER-negative subtypes of breast cancer, including the challenging triple-negative (ER-, PR-, Her2-negative) subset. A number of phase 1 and 2 trials using tissue-derived surrogate-endpoint biomarkers (SEBs) as outcomes have been implemented. These smaller trials address prevention not only of ER-negative but also ER-positive breast cancers, since approximately 50 % of the latter have been shown to be resistant to the estrogen-targeting drugs used in the large trials. Issues of importance in these smaller trials include choice of agent, selection of appropriate trial participants, trial design, method of access to breast tissue in women without cancer, selection and monitoring of SEBs, and monitoring of drug toxicity. Among the agents which have recently garnered interest for their potential activity in preventing ER-negative cancers is metformin. The application of this anti-diabetic drug to breast cancer, both for treatment and prevention, offers an example of the repurposing of an agent in widespread use for one disease to another medical condition. The search for anti-cancer signals in large non-cancer trials is increasingly being used as a tool for discovering agents with potential for prevention of cancers in general and breast cancer in particular.

PROSTATE CANCER TREATMENT AND PREVENTION: CONVENTIONAL VS. TRADITIONAL APPROACH

Shailesh Singh

Morehouse School of Medicine, Atlanta, GA USA

Despite recent advance and state of art tools available in the clinics, prostate cancer is still a second-leading cause of cancer-related deaths among men. Conventional cancer treatments are the first choice of modality offered in the clinics. This harms the immune system, which is required to fight against the disease and makes it difficult to deliver a cancer cure. Recently, chemokines/chemokine receptors have been shown to play a crucial role in prostate cancer progression and metastasis. This presentation will shed light on the potential role of CC Chemokine receptor-9 (CCR9) and its natural ligand (CCL25) directed therapy in improving chemotherapeutic efficacy. Furthermore, this presentation will cover the potentials of immune system in cancer progression and strategies currently in use to boot immunity against cancer. As a preventive strategy the role of natural product in potentiating immunity against cancer will be discussed.

ROLE OF MYELOID TRANSCRIPTION FACTORS AND LEUKEMIC FUSION PROTEINS IN THE PATHOGENESIS OF LEUKEMIC CANCERS

Sheo Mohan Singh

Director, International Centre for Stem Cells, Cancer and Biotechnology (ICSCCB), Pune, India. Email: director@icsccb.org or sheomohan@yahoo.com, Mobile Tel: +91-9545089202 Website: www.icsccb.org

The rational design of targeted therapies for acute myeloid leukemia (AML) requires the discovery of novel protein pathways in the systems biology of a specific AML-subtype. We have shown that in the AML-subtype with translocation t(8;21), the leukemic fusion protein AML1-ETO inhibits the function of transcription factors PU.1 and C/EBPalpha via direct protein-protein interaction. In addition, using proteomics we have also shown that the AML subtypes differ in their proteome, interactome and post translational modifications (PTMs). We, therefore, hypothesized that the systematic identification of target proteins of AML1-ETO on a global proteome-wide level will lead to novel insights into the systems biology of t(8;21)-AML on a post-genomic functional level. Thus, 6 h after inducible expression of AML1-ETO, protein expression changes were identified by 2D-gel electrophoresis, and 28 target proteins of AML1-ETO including Prohibitin, NM23, HSP27 and Annexin1 were identified by MALDI-TOF mass spectrometry. AML1-ETO upregulated the differentiation inhibitory factor NM23 protein expression after 6 h and the NM23 mRNA expression was also elevated in t(8;21)-AML patient samples in comparison to normal bone marrow. AML1-ETO inhibited the ability of C/EBP transcription factors to downregulate the NM23 promoter. These data suggest a model in which AML1-ETO inhibits the C/EBP-induced downregulation of the NM23 promoter, and thereby increases the protein level of differentiation inhibitory factor NM23. Proteomic pathway discovery can identify novel functional pathways in AML, such as the AML1-ETO-C/EBP-NM23 pathway, as main step towards a systems biology and therapy of AML.

SIGNALING CROSSTALK BETWEEN Β-CATENIN AND EGFR IN GSK3Β INACTIVATED PROSTATE CANCER CELLS THAT LEAD TO ENHANCED PROLIFERATION AND SURVIVAL

Mrinal K. Ghosh

Indian Institute of Chemical Biology (CSIR), Kolkata

Background: Wnt/β-catenin and EGFR pathways are important in cancer development and often aberrantly activated in multiple human cancers. However, it is very important to understand the mechanism responsible for this activation and the relation between them. Expressions of β-catenin and EGFR are elevated in various cancers specifically in prostate cancer (PCa) cells, DU145. Methods: To undertake the study a wide variety of cell biology and molecular biology approaches were employed. Normal and cancer cell lines were maintained regularly and were the source material for most experiments. Physiological drugs/inhibitors were used to specifically block or activate cellular machineries. Transient transfections were done to over-express proteins and RNAi approach was taken for knock-down experiments. Cells were subsequently harvested for the preparation of WCLs or cytoplasmic-nuclear extracts or extraction of total RNA. Lysates were used for western blotting or co-immunoprecipitation experiments. RNA was used for qPCR analysis. Genes were cloned and sub-cloned into various vectors as and when required. Chromatin immunoprecipitation and luciferase assays were done to evaluate EGFR promoter occupancy by transcriptionally active β-catenin. Immunocytochemical and immunohistochemical techniques were performed on tissue sections and used to visualize proteins inside cells. Results: When GSK3β is inactivated in these cells, β-catenin gets stabilized, phosphorylated at Ser⁵⁵² by PKA, accumulates in the nucleus and regulates the expression of its target genes that include EGFR. Chromatin Immunoprecipitation (ChIP) and promoter analysis revealed that the EGFR promoter gets occupied by transcriptionally active β-catenin when elevated in GSK3β inactivated cells. This phenomenon not only leads to increased expression of EGFR but also initiates the activation of its downstream molecules such as ERK1/2 and Stat3, ultimately resulting in upregulation of multiple genes involved in cell proliferation and survival. We recently established the mechanism of EGFR expression by transcriptionally active β-catenin that eventually leads to enhanced proliferation and survival of prostate cancer cells. Conclusion: The study provided a strong correlation between inactivating phosphorylation of GSK3β with the up-regulated expressions of both β-catenin and EGFR in PCa compared with PHp. In a nutshell the present study describes three major findings: (i) GSK3β inhibition promotes β-catenin stabilization and its PKA-dependent phosphorylation of Ser⁵⁵² and Ser⁶⁷⁵ residues in DU145 cells. (ii) Phosphorylation of β-catenin at Ser⁵⁵² by PKA promotes its transcriptional activity, including EGFR gene expression. (iii) Increased expression of EGFR through phospho-β-catenin-Ser⁵⁵² may facilitate the activation of ERK1/2 and Stat3, which provide a molecular mechanism how β-catenin promotes survival and proliferation of these cells.*This work was supported by grants provided by DST (SR/SO/HS-0150/2010) and CSIR (EMPOWER: OLP-2) to Dr. M. K. Ghosh.

CANCER IMMUNOEDITING: INTEGRATING THE ROLE OF IMMUNITY IN CANCER SUPPRESSION AND PROMOTION

Gaurisankar Sa

Professor of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata 700054, India.

E-mail: gauri@boseinst.ernet.in

Introduction: The regulatory T (T_reg) cell lineage is indispensable for induction of T cell tolerance, which happens to be one of the mechanisms of cancer immune evasion in tumor condition. FoxP3, a lineage-specification factor required for T_reg cell differentiation and function, executes its multiple activities mostly through transcriptional regulation of its target genes. The molecular bases underlying the phenotypic and functional diversity of FoxP3⁺ T_reg cells remain obscure. Experimental Procedures: Various biochemical, genetic alteration, proteomic, structural biology, flowcytometric, micro-imaging, ChIP/re-ChIP, as well as immunological methods were employed to unveil the mechanisms of il10 gene transcription in T_reg cells in breast cancer patients as well as in tumor-implanted mice model. Results: Adding to the knowledge of abundant T cell plasticity in terms of cytokine production our study identified a population of FoxP3-expressing IL10-producing adaptive T_reg cells, a subtype distinct from FoxP3-negative IL10-producing class-1 T_reg cells that contributes to IL10-dependent type-2 cytokine bias in breast cancer patients. An in-depth analysis reveals that FoxP3, in association with HAT1 modify il10 promoter epigenetically, making a space for pocketing STAT3-FoxP3 complex. As soon as HAT1 modify il10 promoter the FoxP3 dimer forms complex with dimeric STAT3. The interaction between FoxP3 and STAT3 induces certain conformational change which drives them to bind with chromatin DNA. A high-throughput docking module with target-receptor specificity and exon deletion mutation study showed that STAT3 dimer binds specifically to the exon-2 β-sheet region of FoxP3 through its N-terminal floppy domain to form STAT3-FoxP3 complex. Such activity of FoxP3 is extended to other STAT3 target genes, e.g., il6, vegf, c-myc, bclxl, ccnd1, that lack FoxP3-binding site. Conclusion: These results suggest a novel function of FoxP3 where failing to achieve direct promoter-occupancy, FoxP3 promotes transcription in association with a locus-specific transcription factor, STAT3, in T_reg cells.

PRIMING EFFECT OF 2-(5-SELENOCYANATO-PENTYL)-6-AMINO-BENZO [DE]ISOQUINOLINE-1, 3-DIONE TO AUGMENT CYTOTOXIC ACTION OF CYCLOPHOSPHAMIDE AGAINST TUMOR CELLS WITH REDUCED SIDE EFFECTS: IMPLICATION OF ALTERED ROS LEVEL, ANTIOXIDANT ENZYMES ACTIVITY, APOPTOSIS AND DNA DAMAGE

S. Singha Roy, P. Chakraborty, J. Biswas & S. Bhattacharya

Dept. of Cancer Chemoprevention, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Kolkata 700 026, India, E-mail: sudinb19572004@yahoo.co.in

BACKGROUND: The discovery of chemotherapy has opened a novel avenue in cancer treatment. But the general limitation of these chemotherapeutic drugs is their toxicity towards normal cells. Induction of oxidative stress is one of the primary reasons for the toxicity. METHODS: In the course of our anticarcinogenesis—drug development programme, a series of novel organoselenocyanate compounds are developed. These compounds were tested for their toxicity in normal Swiss albino mice. The selenium compound was administered orally as a suspension in aqueous propylene glycol and cyclophosphamide was given intraperitoneally. We also evaluated the antioxidant property of these compounds against a reference mutagen as well as a standard chemotherapeutic drug, cyclophosphamide. Further, one of the synthetic compound, 2-(5-selenocyanato-pentyl)-6-amino-benzo[de]isoquinoline-1,3-dione (most active in the series) was used as an adjuvant with cyclophosphamide in tumor bearing mice to see the interference of the organoselenium compound in the therapeutic efficacy of the standard antineoplastic drug. RESULTS: The compound is much less toxic than dietary sodium selenite. The non-toxic organoselenium compound showed the ability to intensify the antioxidative defense system. The compound showed the potential to inhibit the CP induced cellular toxicity through its action, in part, by enhancing the antioxidant enzyme levels and by neutralizing the reactive oxygen species whereas in a therapeutic point of view the selenium compound showed enhancement of antineoplastic effect on tumor cells by inducing oxidative stress and apoptosis. It exerted synergistic effect with CP by enhancing the life span of the tumour bearing mice. CONCLUSION: The compound showed its potential to act both as a chemoprotector and as a chemoenhnacer when used as an adjuvant with the standard antineoplastic drug, cyclophosphamide.

MOLECULAR CHARACTERIZATION OF HPV16 RELATED CERVICAL CANCERS HIGHLIGHTS SIGNIFICANT VARIATIONS BETWEEN CASES HARBOUR EPISOMAL AND INTEGRATED HPV16 GENOMES

Sharmila Sengupta

National Institute of Biomedical Genomics, Netaji Subhas Sanatorium, 2nd Floor, P.O.: N.S.S., Kalyani 741251, West Bengal, India. E-mail: ssg1@nibmg.ac.in/sharmilasg@gmail.com

In India, among the HPV positive cervical cancer cases (CaCx), 50 % are HPV16 DNA positive, and this is also the most prevalent type within normal populations. Of these, 14 % are Asian American variants (AA) and 86 % are European variants (E). However, not all HPV infected individuals develop the disease in the long run. Sixty percent of the CaCx cases in India harbor intact E2 portraying the presence of episomal HPV16, despite the fact that HPV-16 E2 protein negatively regulates transcription of the E6 and E7 genes. We focused on interrogating the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, and (iv) viral load, in HPV16 related CaCx pathogenesis. High E7 expression in CaCx cases having episomal (pure episomes or concomitant) viral genomes with intact E2 could be attributable to loss of E2 repressor activity due to E2BS-I/II methylations. High viral load among CaCx cases having episomal (pure and concomitant) viral genomes as compared to those with purely integrated viral genomes could be attributable to the potential of E2 in maintenance of replication and segregation of viral genomes in such cases. This prompted us to speculate that HPV16 positive CaCx cases that harbour episomal viral genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis. Analysis of candidate host miRNA expression revealed significant upregulation of 3 miRNAs and down regulation of 5 miRNAs among CaCx cases in contrast to controls, HPV16 positive or HPV negative. Differential downregulation was recorded for miRNA-200a in HPV16 positive normal samples compared to HPV negative controls, and miRNA-181c in CaCx cases harboring episomal as opposed to integrated HPV16. Our studies thus appear to be instrumental in providing insights into host-virus interaction in cervical cancer pathogenesis. Such studies could be of translational relevance providing directions towards identification of novel strategies for combating HPV infections and cervical carcinogenesis.

EPIGENETIC CHEMOPREVENTION OF TOBACCO-RELATED URINARY BLADDER CARCINOGENESIS BY KAWAIN

Xiaolin Zi

Associate Professor of Urology, Pharmacology and Pharmaceutical Sciences, University of California, Irvine, USA

BACKGROUND: To test the hypothesis that epigenetic enhancement of H3K4 methylation by Kawain (a principal active compound in traditional kava drink) may represent a new avenue for bladder cancer (BCa) prevention. METHODS: Normal Human Bladder Epithelial Cells (NHBEC) and BCa cell lines (5637, T24 and RT4) were treated with tobacco carcinogen 4-aminobiphenyl (4-ABP) and/or kawain, and methylation levels of H3K4 were examined. In addition, mice were gavaged with hydroxybutyl(butyl)nitrosamine (OH-BBN) to induce BCa and 0.6 % kawain or vehicle in the food was administered as a dietary supplement. Experimental endpoints (tumor weight, the survival of the mice, and the expression of biomarkers, etc.) were evaluated by pathological, immunohistochemistry and statistical analyses. RESULTS: In the OH-BBN model, more than 95 % of mice with 0.6 % kawain in their diet survived throughout the period of experimentation while only about 65 % of mice fed with vehicle diet survived. Dietary feeding of 0.6 % kawain significantly decreased the mean bladder wet weight of the mice by 72.4 %. Compared to untreated normal urothelium, decreased amounts of H3K4 mono-methylation, up-regulation of potential H3K4 target genes (i.e. HOX C8, 11, 13 and D1), and down-regulation of sFRP1 gene were seen in BCa tissues of mice treated with OH-BBN; these effects of OH-BBN were all reversed by kawain treatment. Treatment of NHBEC with 4-ABP also decreased the levels of H3K4 methylation, whereas kawain induced H3K4 mono- and di-methylation in BCa cells. CONCLUSION: Kawain has the potential to reverse tobacco carcinogen-induced H3K4 demethylation, leading to inhibition of bladder carcinogenesis.

FUNCTIONAL CONTRIBUTION OF TRANSCRIPTION FACTOR IN MIRNA MEDIATED TRANSCRIPTIONAL REGULATION OF HPV IN CANCER STEM CELLS

Abhishek Tyagi¹, Gouri SIsodia^{1, 2}, Alok Bharti² and B.C. Das¹

¹ Dr. B. R. Ambedkar Center for Biomedical Research (ACBR), University of Delhi, Delhi-110 007, ² Institute of Cytology and Preventive Oncology (ICPO), ICMR, 1-7, Sector - 39, Noida-201301

Cancer stem cells (CSC) have been reported in many human tumors and are thought to be responsible for tumor initiation, therapy resistance, progression, relapse, and metastasis. Like many other solid tumors, cervical cancer caused by Human Papillomavirus (HPV), may harbor a heterogeneous population of cancer cells known as “Cervical Cancer Stem-like Cells” having the capacity to self renew as well as differentiate into multiple lineages whose identification and characterization may facilitate the development of novel strategies for the treatment of advanced and metastatic cervical cancer. However, despite of their potential clinical importance, how intrinsic CSCs properties are regulated at the molecular level is poorly understood. Recent discoveries of microRNAs (miRNA) have provided a new avenue in understanding the regulatory mechanisms in CSCs. Therefore, miRNA based therapeutics that specifically target CSCs may provide novel firepower to the anticancer arsenal by targeting distinctively and concertedly regulated key and interconnected biological properties of CSCs responsible for tumor initiation, maintenance and malignant transformation. Hence, elucidation of these networks is of clinical importance as it allow for the identification of novel therapeutic targets and the development of strategies to target putative cervical cancer stem-like cell (CxCSC). Present study is designed in order to investigate the role of Human Papillomavirus (HPV) in enriched populations of human cervical cancer stem-like cells that were isolated according to their differential expression of CD49, CD71, ABCG2 along with other putative stem cell markers and their correlation with expression of miR29a, miR21 and Let-7a from HPV+ve (HeLa, SiHa) and HPV-ve (C33a) cervical cancer cell lines. Also, their tumorigenic potential and sensitivity to specific herbal drugs will be presented.

GENETIC AND EPIGENETIC REGULATION OF CARCINOGENESIS IN HUMAN PAPILLOMA VIRUS ASSOCIATED EPITHELIAL CANCERS

Mausumi Bharadwaj*, Showket Hussain, Nisha Thakur

Division of Molecular Genetics & Biochemistry, Institute of Cytology and Preventive Oncology (ICMR), Noida, India. E-mail. mausumi.bharadwj@gmail.com/bharadwajm@icmr.org.in

Human papillomavirus (HPV) is the major causative agent for the development of cancer of uterine cervix, the most common gynecological cancer in developing countries including India. It involves a series of events, in which normal cervical cells gradually undergo changes leading to the development of precancerous lesions spanning 10–15 years. These lesions can either regress to normal condition or they may progress to higher lesions and then have the potential to develop into invasive cancer if left untreated. The oncogenic potential of HPV is attributed to its E6 and E7 genes. The products of these two genes stimulate cell proliferation by activating the cell-cycle-specific proteins and interfere with the functions of cellular growth regulatory proteins, p53 and Rb. Various robust molecular approaches are being used to study gene expression profile, SNPs, promoter methylation, microRNAs etc. in order to develop cancer biomarkers. Therefore, we sought to identify host encoded microRNAs and their target candidate genes involved in epithelial cancers in conjunction with HPV infection. Thus, our group showed comparative analysis of HPV infection in different organ sites provides an interesting clue(s) for possible interaction of HPV infection with host genes. We demonstrated SNPs in immunomodulatory genes as well as genes in cell cycle regulatory pathways may play a significant role in HPV associated cancers and their regulations through microRNAs. In addition, the promoter methylation of tumor suppressor genes FHIT and RASSF1 shows a significant association in cervical cancer. Therefore, our group demonstrated the genetic/epigenetic predisposition to cervical carcinogenesis in addition to HPV infection.

DECREASED EXPRESSION OF MIR-125B ALTERS CELL CYCLE PROGRESSION THROUGH MITOSIS BY TARGETING SPINDLE ASSEMBLY CHECKPOINT GENE MAD1 IN HEAD AND NECK CANCER

Sumana Bhattacharjya¹, Somsubhra Nath¹, Jayeeta Ghose², Guru Prasad Maiti³, Nabendu Biswas¹, Santu Bandyopadhyay¹, Chinmay K. Panda³, Susanta Roychoudhury¹and Nitai P. Bhattacharyya²

¹Cancer Biology and Inflammatory Disorder Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4, Raja S.C. Mullick Road, Kolkata 700 032, India, ²Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700 064, India, ³Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata-700 026, India

Altered expressions of micro RNA (miRNA), a negative regulator of coding genes, have been implicated in many cancers. It is known that miRNAs target many genes involved in the regulation of cell cycle, one of the hallmarks of cancers. To find out whether miRNA targets genes in the spindle assembly checkpoint (SAC) and alters in head and neck cancer, we first used the published literatures and various databases. Such in silico analysis revealed an inverse correlation in the expressions of miR-125b and MAD1L1 (encodes Mad1, a core SAC protein). MAD1L1 gene has recognition site of miR-125b. We experimentally confirmed using various methods that MAD1L1 gene is a target of miR-125b. We showed that the ectopic expression of miR-125b in UPCI: SCC084 cells downregulates Mad1. Further, we observed that this Mad1 downregulation results in a transient activation of the SAC which delays cells in mitosis. Over time, this delay leads to an accumulation of chromosomal abnormalities within the cells and they ultimately proceed towards apoptotic death. On the other hand, it was observed that HCT116 cells progressed through mitosis faster when the endogenous Mad1 levels were increased by inhibiting the expression of miR-125b. Moreover, anti-miR-125b-mediated Mad1 upregulation was able to maintain the viability of the cells. Finally, we have also verified the expression of miR-125b and Mad1 in head and neck tumours to obtain the in vivo relevance of the cell line observations.

PLANT FLAVONOIDS AS EPIGENETIC MODULATORS IN CANCER: IMPLICATIONS IN CANCER CHEMOPREVENTION

Sanjay Gupta

Department of Urology, Case Western Reserve University, University Hospitals Case Medical Center, 10900 Euclid Avenue, Cleveland, Ohio-44106 USA.

Background: Diet and lifestyle factors contribute to cancer development by inducing both epigenetic and genetic changes that, in combination with genetic make-up, result in the disruption of key cellular processes leading to neoplastic transformation. In prostatic epithelium, it has been shown that the vast majority of high-grade PIN lesions and adenocarcinomas exhibit early loss of GSTP1 expression, associated with hypermethylation of the CpG islands encompassing the GSTP1 promoter. It has been proposed that GSTP1 is a caretaker gene, protecting the cells against genomic damage mediated by oxidants and electrophiles from inflammation or dietary exposures. Our studies demonstrate a significant increase in the levels of 8-oxo-2′-deoxogunosine (8-OHdG), an oxidative DNA damage marker, in prostate adenocarcinomas, compared to benign tissue from same individuals, which positively correlated with the loss of GSTP1 activity and correlates with GSTP1 promoter hypermethylation [Kanwal et al. Mol Carcinog. 2012 (Epub ahead of print)]. Dietary flavonoids have been reported to demonstrate many interesting biological activities, including induction of epigenetic changes and cancer prevention. We have previously demonstrated that green tea polyphenols cause re-expression of glutathione-S-transferase pi, epigenetically silenced during cancer progression, whose signatures are observed in human prostate cancer LNCaP cells [Int J Cancer. 126:2520–33, 2010].Methods: We used human prostate cancer LNCaP cells as an in vitro model and green tea polyphenols and other plant flavonoids for our studies. Results: In search of the mechanism(s) of the anticancer action of green tea polyphenols and its major constituent (–)–epigallocatechin-3-gallate, we have observed that these promising compounds could modulate epigenetic mechanisms at various levels. The presentation will discuss these epigenetic modifications elicited by green tea polyphenols and other plant flavonoids which could lead to the re-expression of GSTP1. Conclusions/Summary: Understanding the mechanism(s) of epigenetic regulation and its reversibility by plant flavonoids will result in the development of new strategies for the prevention and/or treatment of cancer

TARGETED THERAPEUTICS REQUIRE TUMOR MICROENVIRONMENT CONSIDERATIONS

Neil A. Bhowmick

Department of Medicine. Cedars-Sinai Medical Center, Samuel Oschin Comprehensive Cancer Institute, and Greater Los Angeles Veterans Administration, Los Angeles CA. USA.

BACKGROUND: Recent advances in chemotherapeutics and targeted drugs have improved outcomes for patients with advanced cancer. Yet therapeutic resistance of prostate cancer makes it the second leading cause of cancer-related death in USA. The primary target of current prostate cancer therapy, androgen ablation and taxol-based therapies, is toward the cancer epithelia. Work in our laboratory and others indicate cancer-associated stromal fibroblasts (CAF) contribute to the progression of carcinomas. The paracrine factors generated by CAF impact cancer proliferation, metastatic potential, and therapeutic resistance. RESULTS: We find that the loss of transforming growth factor-beta receptor type II in the prostatic CAF is associated with the up regulation of the anti-apoptotic gene Mcl-1, a member of Bcl2 family of genes. Mcl1 is frequently associated with advanced human prostate cancer and castrate resistant growth. Through the use of novel transgenic and xenograft mouse models we find that antagonizing Mcl1, by a well-tolerated drug named Sabutoclax, can mediate prostate tumor sensitivity to androgen ablation and taxol therapy. Another important component of the tumor microenvironment is its vasculature. Thus, renal cancer therapy often involves VEGF antagonism; yet acquisition of therapeutic resistance has required another tact. To this effort we have targeted both the tumor and its microenvironment through the taxol-conjugation of an EphA2 binding domain. EphA2, is a transmembrane receptor found in many tumors and associated vasculature. CONCLUSION: We have found that such dual targeting approaches improve therapeutic efficacy and delay resistance development.

TRANSLATING SKIN SCIENCE RESEARCH INTO APPLICABLE HUMAN PRODUCTS.

Theresa R. Pacheco

Associate Professor, Department of Dermatology, University of Colorado, School of Medicine, Anschutz Medical Campus, Aurora, CO

Skin science research and development occurring in academic institutions provides insight into mechanism of skin health and disease. Research looking at skin diseases caused by cutaneous DNA damage (solar radiation, ionizing radiation and other insults) is promising, but application of this research to meet the mission and priorities is lacking. It is necessary to establish long-term collaborative goals of researchers studying any relevant skin disease so that the clinical relevance of routine basic skin science experiments and study design of these studies will have outcomes that translate into clinic human disease therapeutic development. Technology Readiness Levels (TRLS) are well established and provide a firm roadmap toward this effort. Using a human disease model highlighting DNA damage, a review of TRLS levels 1–9 will be reviewed. Specially, highlights of the development of appropriate and relevant animal model(s) and appropriate and relevant assays will be reviewed and basic manufacturing guidelines for concurrent product development.

POLYCOMB GENES, CHEMOPREVENTION AND SKIN CANCER CELL RESPONSE TO CELL STRESS

Richard L. Eckert, Sivaprakasam Balasubramanian, Santosh Kanade, and Bingshe Han

Departments of Biochemistry and Molecular Biology, Dermatology, and Obstetrics and Gynecology³, University of Maryland School of Medicine, Baltimore, Maryland, USA

The polycomb group (PcG) proteins, Bmi-1 and Ezh2, are important epigenetic regulators that enhance skin cancer cell survival. We recently showed that Bmi-1 and Ezh2 protein level is reduced by treatment with the dietary chemopreventive agents, sulforaphane and green tea polyphenol, and that this reduction involves ubuiquitination of Bmi-1 and Ezh2, suggesting a key role of the proteasome. In the present study we observe a surprising outcome that Bmi-1 and Ezh2 levels are reduced by treatment with the proteasome inhibitor, MG132. We show that this is associated with a compensatory increase in the level of mRNA encoding proteasome protein subunits in response to MG132 treatment and an increase in proteasome activity. The increase in proteasome subunit level is associated with increased Nrf1 and Nrf2 level. Moreover, knockdown of Nrf1 attenuates the MG132-dependent increase in proteasome subunit expression and restores Bmi-1 and Ezh2 expression. The MG132-dependent loss of Bmi-1 and Ezh2 is associated with reduced cell proliferation, accumulation of cells in G2, and increased apoptosis. These effects are attenuated by forced expression of Bmi-1, suggesting that PcG proteins, consistent with a pro-survival action, may antagonize the action of MG132. These studies describe a compensatory Nrf1-dependent, and to a lesser extent Nrf2-dependent, increase in proteasome subunit level in proteasome inhibitor-treated cells, and confirm that PcG protein levels are regulated by proteasome activity

NOVEL PROTOONCOGENE RBM3 PROMOTES TUMOR ANGIOGENESIS

Satish Ramalingam^1,3, Dharmalingam Subramaniam^1,3, David Standing¹, Shahid Umar^1,3, Animesh Dhar^2,3, Shrikant Anant^1,3.

Departments of ¹Molecular and Integrative Physiology, ²Cancer Biology and ³KU Cancer Center, University of Kansas Medical Center, Kansas City, KS

Angiogenesis is critical to the growth, invasion, and metastasis of tumors. Because targeting angiogenesis has emerged as a promising avenue of cancer treatment, understanding the regulation of the pro-angiogenic factors in endothelial cells has become increasingly important. RBM3 belongs to the evolutionarily conserved RNA binding protein family that interacts with 60S ribosomal subunit to globally regulate translation. We have recently demonstrated that RBM3 is a protooncogene whose expression is significantly upregulated in a variety of cancers. In addition, we demonstrated that RBM3 induces the translation of cyclooxygenase-2, Vascular endothelial growth factor (VEGF)-A and IL-8 mRNA. Given the significance of angiogenesis, we determined whether there are any differences in the number of microvessels in the primary xenograft tissues. Immunohistochemistry analyses of the tumors demonstrated increased number of microvessels that were lined by either CD31+ or LYVE1+ cells suggesting the increase in heme- and lymphangiogenesis, respectively. On the other hand, inhibition of RBM3 expression using specific siRNA resulted in significant reduction in both CD31+ and LYVE1+ microvessels suggesting that RBM3 expression is essential for angiogenesis. To directly demonstrate the role of RBM3 in angiogenesis, in vitro tube formation assays were performed with human umbilical vein endothelial (HUVEC) and human lymphatic endothelial (HLEC) cells. In both cases, RBM3 overexpression increased while RBM3 knockdown inhibited tube formation, again suggesting that RBM3 expression regulates heme- and lymphangiogenesis. Moreover, treatment of HUVEC and HLEC cells with conditioned media from RBM3 overexpressing HCT116 cells resulted in increased tube formation in both cell lines. We next determined the mechanism by which RBM3 induces angiogenesis. VEGF-A binds to VEGFR-1 (Flt1) and VEGFR-2 (Flk1/KDR), VEGF-B binds to only VEGFR-1, while VEGF-C and VEGF-D bind to VEGFR-2 and VEGFR-3 (Flt4). VEGFR-2 and VEGFR-3 are expressed in heme- and lymphatic endothelial cells, respectively. RBM3 overexpression increased the steady state transcript levels of all three VEGF isoforms in HCT-116 colon cancer cells when compared to controls. On the other hand, knockdown of RBM3 resulted in significant reduction in the expression of the three VEGF genes. Moreover, actinomycin D-stability assays demonstrated that there was increased the half-life of all three VEGF mRNAs in HCT116-RBM3 cells when compared to HCT116 control cells. To further demonstrate that VEGFs are essential for RBM3-induced angiogenesis, HUVEC and HLEC tube formation assays were performed with conditioned media from HCT116-RBM3 overexpressing cells and controls in the setting of VEGF-A, -C and -D downregulation using specific siRNAs. Knockdown of either VEGF species resulted in a significant reduction in tube formation in both HUVEC and HLEC cells, even in the presence of increased RBM3 expression demonstrating that RBM3-induced angiogenesis is mediated by increased VEGF expression. Taken together these data suggests that RBM3 is a key regulator of angiogenesis, and could be a potential target for inhibiting tumor angiogenesis.

APIGENIN INDUCES APOPTOSIS IN HUMAN LEUKEMIA CELLS AND EXHIBITS ANTI-LEUKEMIC ACTIVITY IN VIVO

Amit Budhraja and Xianglin Shi,

Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky, USA

In this study, we investigated the functional role of Akt and c-jun-NH2-kinase (JNK) signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 downregulation, cytochrome c release from mitochondria, and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspase activation, and apoptosis. Conversely, LY294002 and a dominant-negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of the JNK pathway showed marked reduction in apigenin-induced caspase activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumor growth in U937 xenografts accompanied by inactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens.

ACTIN CYTOSKELETON MODULATORY PROTEIN LIM KINASE 1 AND FUNCTIONAL REGULATION OF MT1-MMP: IMPLICATION IN INVASION OF PROSTATE CANCER CELLS

Ratna Chakrabarti

Department of Molecular Biology and Microbiology, Burnettt School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL.

BACKGROUND: LIM kinase 1 (LIMK1) is a downstream effector of Rho signaling pathway, which modulates actin dynamics through inactivation of the ADF family member cofilin by phosphorylation. LIMK1, a unique serine/threonine kinase containing two N-terminal LIM domains in tandem and a PDZ domain, is a newly identified candidate that promotes prostate and breast cancer metastasis. Rho family protein Rac 1 activates LIMK1 for its kinase activity through PAK1/PAK4 mediated phosphorylation at T⁵⁰⁸, which results in cofilin inactivation and accumulation of F actin. Membrane type matrix metalloproteinase 1 (MT1-MMP) is a critical modulator of extracellular matrix (ECM) turnover and thus plays crucial roles in neoplastic cell invasion and metastasis. MT1-MMP and its substrates pro-MMP-2 and pro-MMP-9 are often overexpressed in a variety of cancers including prostate cancer and the expression levels correlate with the grade of malignancy in prostate cancer cells. The abundant expression of these enzymes contributes to the changes in the tumor microenvironment, which facilitates degradation of the surrounding collagen matrix and migration of cells through the matrix defects. Tumor cell migration also requires reorganization of actin cytoskeleton that is regulated by Rho family of small GTPases. Specifically, Rac1 has been implicated in induction of formation of lamellipodia/membrane ruffles and invasion of otherwise non-invasive epithelial cells. In this study, we determined a functional relationship between LIMK1 and MT1-MMP and its implication in cell invasion. METHODS: We used prostate cell culture models for in vitro invasion assays, gelatin zymography, immunoblotting and immunoprecipitation, flow cytometry, confocal microscopy and luciferase reporter assays. We used prostate tissue microarray for immunohistochemistry and xenograft models for prostate tumor development. RESULTS: Our results showed that overexpression of LIMK1 increased collagenolytic activities of MMP-2 and secretion of pro-MMP-2 and pro-MMP-9. LIMK1 expression facilitates expression, transcriptional activation and plasma membrane targeting of MT1-MMP. Physical association between LIMK1 and MT1-MMP and their colocalization to the golgi vesicles were also noted. A positive correlation of expression of LIMK1 and MT-MMP in prostate tumor specimens and in xenograft models shows the clinical relevance of the functional relationship between these two proteins. CONCLUSION: This study provides evidence for a novel association between MT1-MMP and LIMK1, which may contribute to the invasiveness of prostate cancer cells.

CHANGES IN INNATE IMMUNE SIGNALING PATHWAYS DURING TUMOR METASTASIS AND THEIR MODULATION BY NATURALLY OCCURRING ISOTHIOCYANATES

Saumendra N. Sarkar

University of Pittsburgh Cancer Institute, Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA

Tumor metastasis is responsible for >90 % of cancer fatality. Despite its clinical importance, the genetic and biochemical determinants of metastasis has remained elusive. Here, we have used paired cell lines, as well as fresh tumor specimens, derived from autologous primary and metastatic head and neck squamous cell carcinoma to investigate the changes in innate immune signaling pathways. Toll-like receptor 3 (TLR3) exhibits a potent pro-apoptotic activity in cancer cells, when activated by its cognate ligand, double stranded (ds) RNA. We show that compared to primary tumor cells, metastatic tumor cells are highly sensitive to TLR3 mediated apoptosis after dsRNA treatment. The enhanced apoptosis of metastatic cells is dependent on dsRNA, its receptor TLR3, and its adaptor TRIF. We show that a defective NF-kB response in metastatic cells causes increased. These results established a novel mechanism of TLR3 mediated apoptosis in metastatic cells (Cancer Res; 72; 45–55.). To exploit this phenomenon in targeting metastatic tumor cells, we have used several naturally occurring isothiocyanates (ITC) from cruciferous vegetables and studied their effects on TLR3 signaling. We find that two most promising ITCs, phenethyl isothiocyanate (PEITC) and D,L-sulforaphane (SFN), have differential effects on dsRNA mediated innate immune signaling through TLR3. PEITC preferentially inhibited TLR3 mediated IRF3 signaling and downstream gene expression in vivo and in vitro, whereas SFN caused inhibition of TLR3 mediated NF-kB signaling and downstream gene expression. Mechanistically, PEITC inhibited ligand (dsRNA) dependent dimerization of TLR3 resulting in inhibition of signaling through IRF3. Our results indicate biologically relevant functional differences between two structurally similar ITC may provide important insights in therapeutic development of these compounds targeted to metastatic tumor.

RLIP76: A VALID TARGET FOR CHEMOTHERAPY

Yogesh C. Awasthi¹, Sharad S. Singhal² and Sanjay Awasthi²

¹Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, USA; ²Department of Diabetes and Metabolic Diseases Research, City of Hope National Medical Center, Duarte, California, USA.

BACKGROUND: RLIP76 (syn, RalBP1) is a multifunctional protein that mediates the ATP-dependent transport of glutathione conjugates of electrophilic xeno-and endo-biotics. Some of the other functions of RLIP76 include its role as a Ral effector protein linking Ral GTPase to Rho pathway, role in signal termination through clathrin coated pit-mediated endocytosis, and mitotic spindle formation. Our previous studies have shown that RLIP 76 is over expressed in most of the cancer cells studied so far and that the inhibition of its expression or functions leads to apoptosis of cancer cells. Using previous published studies and recent data, this presentation is aimed towards providing evidence that RLIP76 plays a central role in the mechanisms of carcinogenesis. METHODS: Using state of the art biochemical, molecular, immunological, and biophysical techniques, we have studied the role of RLIP76 in the mechanisms of carcinogenesis in vivo animal models and in vitro cell culture models. RESULTS: We have previously shown that RLIP76 is the major transporter of the glutathione-conjugates of electrophilic compounds such as 4-hydroxynonenal (HNE). Cells transfected with RLIP76 acquire resistance to apoptosis and many cancer cell lines develop resistance to chemotherapy by over expressing RLIP76 to accelerate the efflux of drugs. Antibodies against RLIP76 and RLIP76 siRNA, or RLIP76 antisense (R508) induce apoptosis in cancer cells in vitro and also cause nearly complete remission of the xenografts of human lung, colon, kidney, and pancreas cancer in nude mice without any apparent toxicity. The ATP-dependent transport of GSH-conjugates is coupled with the clathrin coated pit-mediated endocytosis (CDE). In cells lacking RLIP76 expression and also in RLIP76 knock-out mice in vivo, CDE is attenuated. In RLIP76 knock-out mice, polycyclic hydrocarbon e.g. benzo (a) pyrene (BaP)-induced skin and lung carcinogenesis is inhibited. Phorbol ester-induced activation of PKCα and neoplastic transformation caused by the topical application of polycyclic hydrocarbons in skin is also inhibited in RLIP76 knock-out mice. CONCLUSION: Together, these results strongly suggest that RLIP76 plays a central role in the mechanisms involved in the initiation and progression of cancer and is a valid target for chemotherapy. (This work was supported in part by NIH Grants ES012171, EY004396 (Y.C.A.), and CA77495 (S.A.).

TARGETING APOPTOSIS PATHWAYS IN LEUKEMIA

Varsha Gandhi

Ruby E. Rutherford Distinguished Professor, MD Anderson Cancer Center, Houston, TX, USA

BACKGROUND: Caspase-mediated apoptosis pathways are blocked by over-expression of Bcl-2 as well as IAP antiapoptotic proteins in primary leukemia cells. This inherent blockage in apoptosis is further enhanced by bone marrow microenvironment. Corollary to these observations is the fact that these proteins could be targeted by small molecule inhibitors resulting in caspase-dependent apoptosis. METHODS: Using primary leukemia cells from patients with chronic lymphocytic leukemia (CLL) we evaluated small molecule Bcl-2 antagonists such as gossypol, AT-101, apogossypol analogues, and ABT-737. To evaluate microenvironment-induced drug-resistance, we investigated impact of these molecules on leukemia cells when they are co-cultured with microenvironment. Normal lymphocytes from healthy donors were used to determine therapeutic index. Efficacy of this strategy was tested in a clinical trial. RESULTS: Bcl-2 antagonists induced apoptosis in leukemia cells after 24 h treatment with 3 and 10 micromolar of gossypol, AT-101, and apogossypol analogues. This biological effect was not blocked when cells were in microenvironment milieu. Bone marrow stromal cells induced Mcl-1 antiapoptotic protein. ABT-737 was most potent inhibitor with activity at nanomolar level. During therapy, ABT-263 (an oral ABT-737) sensitized cells to apoptosis by Bax or Bak oligomerization. Cells lacking Bax and Bak pro-apoptotic proteins were resistant to Bcl-2 antagonists suggesting intrinsic cell death pathway. CONCLUSION: Bcl-2 antagonists represent a novel strategy to treat hematological and other malignancies where survival of cancer cells depends on Bcl-2 antiapoptotic proteins. Clinical activity of single agent ABT-263 provides a proof-of-concept in CLL. These agents could be combined with established chemotherapeutics or targeted novel drugs.

EXPRESSION OF MITOTIC CHECKPOINT GENES AND ITS IMPLICATION IN ESOPHAGEAL CANCERS IN RAW BETEL-NUT CHEWERS

A Banerjee, S Kurkalang, P Yadav, GM Rangad², M Islam², H Dakhar², A Chatterjee

Molecular Genetics Laboratory, Department of Biotechnology & Bioinformatics, North-Eastern Hill University, Shillong-793022, India., ² Surgical Division, Nazareth Hospital, Laitumkhrah, Shillong-793003. Ph: 913642722403 Email: chatterjeeanupam@hotmail.com; anupamchatterjee@nehu.ac.in

Introduction-Raw betel-nut (RBN) chewing is an important contributing factor for esophageal cancers. Genetic instability is a hallmark of carcinogenesis and frequently presents as dynamic changes in chromosome copy number, referred to as chromosome instability (CIN). Defects in components of the mitotic spindle checkpoint have been associated with CIN in human cancer cells, but understanding of their role is still limited. The aim of this study was to examine the expression of mitotic checkpoint genes and their association with premature sister-chromatid separation in the presence of spindle-inhibitors in the samples collected from RBN-chewers.

Materials and methods—RBN-extract was administered ad libitum in the drinking water with and without lime and it was estimated that each mouse consumed 2 mg of RBN-extract in a day for 15 to 180 days. In another set of experiments, loss of heterozygosity (LOH) mapping was performed in 81 esophageal tumors using 6 polymorphic loci covered the area from 4q25 to 4q28 of chromosome 4. The expression levels of aurora A (AuA), aurora B (AuB), MAD2 and Bub1 genes were analyzed in bone marrow cells (BMC) and esophageal samples in mice and in human cancer samples. Results—Deletions at 4q27 (D4S2975) exhibited highest frequency (∼44 %), suggesting the site of a candidate tumor suppressor gene in esophageal cancer samples. The frequency of premature sister chromatid separation was increased consistently from 15 to 180 days treatment in mouse-BMC and particularly such precocious anaphase was seen higher in the samples treated with RBN-extract and lime. The overall expression level of four mitotic checkpoint genes showed a tendency to deviate from their normal expression level in mouse BMC and esophageal cells as well as human esophageal cancer samples. Conclusion—Previous LOH studies around 4q27, to which MAD2 has been mapped, has been identified in several different tumor types. We report that deletion of one MAD2 allele results in a defective mitotic checkpoint in both human cancer cells and murine primary embryonic fibroblasts and that leads to develop aneuploid cells. Checkpoint-defective cells show premature sister-chromatid separation in the presence of spindle inhibitors which could contribute for the elevation of chromosome mis-segregation events, implicating defects in the mitotic checkpoint in tumorigenesis.

TELOMERE DYNAMICS AS DETERMINANTS OF CANCER DEVELOPMENT, TREATMENT AND PREVENTION

Sen Pathak

Department of Genetics, The University of Texas M.D. Anderson Cancer Center at Houston, Texas 77030, USA

Genetic instability is the hallmark of cancer cells. Telomeres, the nucleoprotein structures present at both ends of linear chromosomes, serve as guardian and ensure normal chromosome morphology as well as proper segregation of genetic material during cell division. Telomere length is determined by the telomerase complex consisting of two subunits, an enzyme subunit, TERT and a RNA component, Terc. Telomere attrition has been implicated in aging and cancer which is considered the disease of aged cells. Since cancer originates in the organ- or tissue-specific stem cells which are known to have longer telomeres, it is not unreasonable to state that attrition of telomeres is the root cause of genetic instability. We have studied the in vivo and in vitro biology of the Terc knockout mice from early to late generations (G1 to G6). G4 and higher generation mice showed dysfunctional telomeres resulting into the phenotypes of aging, reduced fertility, genetic instability and initiation of epithelial malignancies. This has also been observed in different human syndromes and has been implicated in various birth defects as well as in chronic diseases including cancer (Pathak, 2004). Cancer stem (CS) cells, responsible for cancer recurrence and resistance to chemo- and radio-therapy, have provided additional complexity in the successful treatment of metastatic disease. Metastatic cancer cells have amplification of telomeric DNA which helps generate clones of different biological characteristics. Since most cancer death occurs due to metastatic disease, it is vital to discover drugs that will target telomeres in CS cells. “Life style” is the most important factor for the causation of cancer. We hypothesize that chromosomes with dysfunctional telomeres tend to undergo translocation, inversion and mitotic anomalies. Cells with specific chromosome anomalies may be present in the peripheral blood of cancer patients and even in some asymptomatic family members. Such precancerous cells with minimal genetic defects are not the circulating tumor cells (CTC), as known today. In conclusion—telomere dynamics as determinant of cancer development, treatment and prevention needs further exploration by both clinicians and basic scientists together in order to eliminate this dreadful disease called—Cancer.

A RECIPROCAL RELATIONSHIP BETWEEN MICRORNA-21 AND TUMOR SUPPRESSOR PTEN FORCES A FEEDBACK LOOP INVOLVING NF-ĸB IN RENAL CANCER CELLS

Goutam Ghosh-Choudhury

South Texas Veterans Health Care System and University of Texas Health Science Center at San Antonio, Texas, USA.

BACKGROUND: About 30 % of patients with clear cell renal carcinoma (RCC) develop invasive disease with chemoresistance. Aberrant expression of microRNAs has been linked to human cancers. Profiling studies revealed differential expression of miR-21 among 31 solid tumors including RCC. METHODS: Using qRT-PCR, we found increased expression of miR-21 in grade 2 and grade 3 RCC samples. RESULTS: Enhanced levels of mature, pre- and pri-miR-21 were detected in cultured renal cancer cells compared to normal proximal tubular epithelial cells. Neither the underlying mechanism of its expression nor its downstream targets those force renal carcinogenesis are known. Expression of miR-21 Sponge to quench endogenous mature miR-21 significantly inhibited proliferation and invasion of renal cancer cells concomitant with attenuation of cyclin D1 mRNA and protein expression. Although mutation of PTEN has been detected in various cancers, reduced expression of this tumor suppressor predominantly contributes to tumorigenesis. In fact 25 % of patients with RCC show reduced levels of PTEN. siRNA-mediated downregulation of PTEN or overexpression of constitutively active Akt reversed miR-21 Sponge-induced inhibition of renal cancer cell proliferation and migration. mTORC1 plays an important role in renal carcinogenesis. miR-21 Sponge arrested the inactivating phosphorylation of the tumor suppressor protein TSC2 (tuberin), resulting in suppression of mTORC1 activity. Expression of constitutively active mTORC1 prevented miR-21 Sponge-suppressed proliferation and migration of renal cancer cells. Besides these data, a positive feedback loop involving activation of NFkB in the context of miR-21 regulation as a driver of renal carcinogenesis will be discussed. CONCLUSION: Increased miR-21 levels in renal cancer cells contribute to their proliferation and invasion by increasing mTORC1 activity.

PREVENTION OF OVARIAN CARCINOGENESIS BY AMLA

Alok De, Archana De, Snigdha Banerjee, Inamul Haque, and Sushanta K. Banerjee

Department of OB/GYN, School of Medicine, University of Missouri Kansas City, 2411 Holmes Street, Kansas City, Missouri, Division of Hematology and Oncology, Department of Medicine, University of Kansas Medical Center, Kansas City, Kansas

BACKGROUND: Ovarian cancers are commonly treated by surgery, chemotherapy and radiation therapy. However, none of these strategies are effective enough. Several plant-based natural products/supplements, including extracts from Emblica officinalis (Amla) are known to be potent anticancer agents. METHODS: For in vitro study OVCAR3 and SW626 cells and for in vivo study mouse xenograft model were treated with Amla extracts (AE). Cell proliferation, autophagy and angiogenesis were studied both in vitro and in vivo conditions. RESULTS: AE reduced cell proliferation in ovarian cancer cells in vitro. AE did not induce apoptotic cell death, but did significantly increase the expression of the autophagic proteins beclin1 and lc3b under in vitro conditions. AE also significantly inhibited in vitro capillary branch formation in human umbilical vein endothelial cells, and reduced the expression of several angiogenic genes, including hypoxia-inducible factor 1α in OVCAR3 cells. AE also showed anti proliferative effects and induced the expression of autophagic proteins beclin 1 and lc3b in mouse xenograft tumors. Additionally, AE reduced endothelial cells antigen—CD31 positive blood vessels and Hif-1α expression in mouse xenograft tumors. CONCLUSION: These studies suggest AE reduce cell proliferation and tumor growth possibly by blocking angiogenesis and activating autophagy in ovarian cancer. AE may prove useful as an alternative therapeutic approach in the fight against ovarian cancer.

CELL-NANOMATERIAL INTERACTION: IMPLICATIONS IN CELL SIGNALING AND TARGETED DELIVERY

Priyabrata Mukherjee

Associate Professor, Senior Associate Consultant, College of Medicine, Mayo Clinic, Rochester, MN-55905, USA. Email: mukherjee.priyabrata@mayo.edu, Phone: 507-284-8563, Fax: 507-293-1058, mukherjee.priyabrata@mayo.edu, Lab page:http://mayoresearch.mayo.edu/mayo/research/mukherjee-lab

The application of nanotechnology in biology and medicine, called biomedical nanotechnology, is a burgeoning field that brings with it a myriad of opportunities and possibilities for advancing medical science and disease treatment. This multidisciplinary field spans the disciplines of biology, chemistry, materials science, engineering and medicine. At the nanoscale, the physico-chemical properties of materials (e.g., metals, semiconductors) differ fundamentally from their corresponding bulk counterpart because of the quantum size effect. While metallic gold is the well-known golden yellow, gold nanoparticles (AuNPs) can change their characteristic from a wine red color to pink, violet, or blue simply by altering their size and shape. With this flexibility, nanoparticles also have a large surface area capable for the loading of multiple diagnostic (such as optical, radioisotope, magnetic) and therapeutic (such as drugs) agents. These two fundamental characteristics of gold nanoparticles render them an attractive research tool. Therefore, understanding the basic molecular interactions between nanoparticles and biomolecules is important and essential to successfully apply nanotechnology to biomedical applications. To achieve this goal, our group is pursuing three major areas; Protein-nanoparticle interactions and their implication in angiogenesis-dependent disorder; Cell-nanoparticle interactions and their implication in targeted therapy; Synthesis of differently shaped nanoparticles based on their protein structure and application in biology. In this particular talk we will discuss how investigating the basic principles of cell-nanomaterial interaction may lead to the discovery new molecular targets and signaling events in various malignancies.

TARGETING TRANSFORMING GROWTH FACTOR-ß SIGNALING IN ADVANCED COLORECTAL CANCER

Pran K. Datta

Professor and Director of Research, Hematology/Oncology Division, Department of Medicine, UAB Comprehensive Cancer Center, The University of Alabama at Birmingham; Research Scientist, Veterans Affairs Medical Center; 1824 6th Ave South, WTI 520C, Birmingham, AL 35294, Phone: 205-975-6039, Fax: 205-934-9573.

BACKGROUND: Although TGF-ß/Smad signaling pathway plays an important tumor suppressor role, it also exhibits pro-metastatic functions in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer (CRC) is directly correlated to poor survival. However, the functional role of Smad signaling in CRC metastasis to liver and the therapeutic potential of TGF-β receptor kinase inhibitors (TRKI) in CRC progression and metastasis have not been elucidated. METHODS: We have investigated the role of TGF-β/Smad signaling on cell proliferation, migration/invasion, tumorigenicity and metastasis (by imaging) in Smad4-null colon carcinoma cell lines, MC38 and SW620 after stably expressing Smad4. We also determined the mechanism of function of the TRKI, LY2109761 in the inhibition of colon cancer metastasis in mice. RESULTS: TGF-β induces migration/invasion, tumorigenicity and metastasis of Smad4-null MC38 and SW620 cells, and treatment of mice with LY2109761 efficiently inhibits these effects. In addition, LY2109761 blocks liver metastasis by inducing the expression of E-cadherin and reducing the expression of tumor promoting proteins like MMP-9, nm23, uPA and COX-2. Transgenic expression of Smad4 significantly reduces the oncogenic potential of MC38 and SW620 cells and reversed the role of TGF-β from a tumor promoter to tumor suppressor. CONCLUSION: These data demonstrate an important anti-metastatic role of TGF-β/Smad signaling in colon cancer metastases. In colorectal cancer patients, loss of Smad4 can explain the functional shift of TGF-β from tumor suppressor to a tumor promoter, and inhibition of TGF-β signaling in these patients can be an effective therapeutic strategy.

UNDERSTANDING CERVICAL CANCER IN INDIAN WOMEN: HPV VIRAL LOAD, PHYSICAL STATUS AND ONCOGENE EXPRESSION

Rita Mulherkar

ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai, 410210

Global cancer rates could increase by 50 % to 15 million by 2020, according to World Cancer Report (WHO), with the majority of the cancer burden being in developing countries. Early detection can bring down the incidence of two of the major cancers in India—oral cancer and cervical cancer. In developed countries early cytological detection of cervical cancer has led to a significant reduction of mortality while in the developing countries including India, incidence and mortality rates are still very high. Besides focusing on screening, diet and tobacco habit to bring down the incidence of cancer, it is important to curb infections which cause cancer. Human Papillomavirus (HPV) plays a major role in the etiology of cervical cancer. Early detection of the high risk HPV types has been shown to reduce the cervical cancer burden.

In order to understand the cervical cancer scenario in India, it is important to study the types of HPV, viral load and their integration status in Indian cervical cancer samples. Using high throughput HPV genotyping and PCR with SPF1/2 degenerate primers, we have identified different HPV types in our cohort of cervical cancer patients (n = 270). The association of HPV with carcinoma of the cervix in this cohort of patients was found to be 95 %, with HPV 16 (66 %), 18 (2 %) and dual infection by 16 + 18 (6 %), being most common. Other HR-HPV types infecting the cohort included 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 73. Association with clinical outcome showed that HPV16 positive cases had better prognosis in terms of response to radiation as compared to the HPV18 positive cases. The site of integration of the virus has also been studied using APOT (Amplification of Papilloma virus Oncogene Transcript) assay in 86 cases with advanced cervical carcinoma. Viral integration was found in 79 % cases and was more frequent at chromosomal loci 1p, 3q, 6q, 11q, 13q and 20q. Also, regions within or near fragile sites and known genes were identified as integration ‘hotspots’. Association with the clinical outcome revealed that both physical status and site of integration of the virus could have important bearing on disease prognosis. It was observed that estimation of the viral copy number and levels of viral transcripts could further complement the information and together with physical status of the virus provide useful insights into the pathophysiology of HPV infection and its relationship to disease prognosis.

EPIGENETIC MODULATION IN CANCER THERAPY

Pamela Munster

University of California, San Francisco, CA, USA

The escape of cancer cells under therapy requires the ability for rapid molecular adjustment and inheritability of resistance features. The development of genomic mutations is slow and a cellular change in protein and receptor expression does not allow inheritability. In contrast, epigenetic modifications may account for both. Novel approaches to cancer therapy include a better understanding of the epigenome in tumor progression, the development of treatment resistance and the introduction of novel therapeutics that interfere with the epigenome. These include histone deacetylase inhibitors and DNA methylation inhibitors. Focusing on breast cancer, we will demonstrate how therapy resistance develops and how the integration of HDAC inhibitors and DNMT inhibitors may restore treatment sensitivity. An update on the preclinical and clinical studies on combining HDAC inhibitors with anti-hormonal therapy will be presented.

COX-2 AND COX-2 INDUCED MIRNAS IN BREAST CANCER PROGRESSION: SUSTENANCE OF STEM-LIKE CELLS

Mousumi Mazumder¹, Leanna Dunn¹, Erin Landman¹, Xiping Xin¹ and Peeyush K Lala^1,2

Departments of ¹Anatomy and Cell Biology and ²Oncology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A5C1

BACKGROUND: We established that high COX-2 expression promotes breast cancer progression via multiple mechanisms including inactivation of immune cells and promotion of cancer cell migration, invasiveness, angiogenesis and lymphangiogenesis, largely via activation of the prostaglandin receptor EP4, which was validated as an effective therapeutic target in a COX-2 expressing murine breast cancer model. To define the roles of COX-2 and COX-2 -induced miRNAs in the induction and sustenance of Stem-Like Cells (SLC) in breast cancer, we stably transfected COX-2 cDNA into COX-2-ve, HER-2-ve MCF-7 and COX-2-ve, HER-2 +ve SK-BR-3 human breast cancer cell lines. Experimental design and Results: MCF-7-COX-2 and SK-BR-3-COX-2 cells exhibited SLC properties including EMT, increased tumorsphere forming ability for successive generations and ALDH activity. Tumorspheres produced by several COX-2-disparate cell lines revealed dramatically higher COX-2 expression than monolayer-grown cells, indicating a key role of COX-2 in SLC growth. In vivo SLC properties of MCF-7-COX-2 cells was confirmed by a dramatic increase in their lung colony forming capacity after IV injection in NOD/SCID/GUSB null mice (tumor cells identified by the GUSB marker), and orthotopic tumoriginity for successive generations in NOD/SCID/IL2R γ null mice. Using differential gene and miRNA microarrays we identified two COX-2 upregulated miRNAs (miR655 and 526b) and their 13 target tumor suppressor-like genes in MCF-7-COX-2 cells. There was a positive correlation between COX-2 and expression of both miRNAs in numerous COX-2 disparate cell lines; the miRNA expression was blocked with COX-2 inhibitor and EP4 antagonist, affirming the roles of COX-2/EP4 in miRNA upregulation. Treatment with EP4 antagonist or knockdown of EP4 or the miRNAs in MCF-7-COX-2 cells abrogated their SLC properties establishing their roles in SLC functions. CONCLUSION: These miRNAs can be used as SLC biomarkers to personalize breast cancer therapy with EP4 antagonists. Grant Support: Supported by Grants from the Ontario Institute of Cancer Research and the Canadian Breast Cancer Foundation, Ontario Chapter to PKL. MM is a TBCRU Scholar

CANCER VACCINES: EVOLVING PARADIGMS

Robert Suriano, Shilpi Rajoria, Andrea George, Elyse Hanly, Jan Geliebter, Abraham Mittelman, Raj K. Tiwari.

Microbiology and Immunology, New York Medical College, NY, USA

The major challenge in cancer immunotherapy is related to the generation of cancer specific multivalent antigenic response that can break immune-tolerance and generate a sustained humoral and cell mediated cancer targeted immune response. We have tried to address each of these challenges in our attempts to generate a therapeutic synthetic peptide based cancer vaccine in a prostate cancer model system. These synthetic peptides are designed to re-educate the immune to break tolerance to cancer associated antigens and has tremendous clinical utility. We identified tumor associated heat shock proteins (HSPs) as the antigens of choice and using differential panning over phage display single chain antibody (scFv) libraries generated the tumor associated antibodies. Heat shock proteins are conserved antigens and are not immunogenic per se rather the immunogenicity is conferred by the peptides associated with HSPS. The scFv reactive against multiple HSP-peptide are against multivalent tumor epitopes. Using these scFvs we identified synthetic peptide mimotopes from two synthetic phage display peptide libraries, LX-8 and X-15 by panning the libraries against different antibodies that tumor specific HSPs-peptide complexes. Several different peptides were identified and sequenced. The immunogenicity of these peptides was tested. The peptides were injected in mice that resulted in peptide specific immune response. Cross reactivity and specificity and tumor rejection was examined and metastatic ablative peptides defined in a prostate cancer model system. These were deemed to be candidates for immunotherapeutic vaccine(s). In conclusion we have used the phage display technology to define immunogenic synthetic peptides that can possibly generate both humoral and cell mediated immune response in mediating tumor rejection. Combinations of these tolerance breaking peptides are new generation immuno-therapeutics that has promise to bypass tumor induced immune evasion mechanisms and overcome some of the major challenges of immunotherapy clinically.

THE GREEN TEA POLYPHENOL EGCG INDUCES MESENCHYMAL TO EPITHELIAL TRANSITION (MET) AND TUMOR REGRESSION IN TRIPLE NEGATIVE BREAST CANCER (TNBC) CELLS AND MOUSE XENOGRAFT MODEL: INVOLVEMENT OF CCN5

Amlan Das^1,3, Snigdha Banerjee^1,2, Archana De¹, Inamul Haque¹, Gargi Maity¹, Matt McEwen^1,,and Sushanta K. Banerjee^1,2,3

¹Cancer Research Unit, VA Medical Center, Kansas City, MO 64128 and ²Division of Hematology and Oncology, ³Department of Medicine and Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66205, USA.

BACKGROUND: Epithelial to Mesenchymal transition (EMT) is an important and coordinated series of events associated with tumor metastasis and invasion. Recent studies had shown the importance of CCN5 (also known as WISP-2, Wnt-1-induced signaling protein-2) in the regulation of various carcinomas including the breast cancer. Recent studies had showed that ectopic expression of CCN5 can reverse Epithelial-Mesenchymal transition (EMT) and inhibit cancer metastasis. Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, has been extensively studied as a bioactive dietary component against various types of carcinomas through multiple mechanisms such as anti-oxidation, induction of apoptosis, inhibition of angiogenesis and metastasis. However, the mechanism of action of EGCG in breast carcinoma is uncertain. OBJECTIVE: The objective of the present study is to determine whether CCN5 plays any significant role in EGCG-mediated cytotoxicity in triple negative breast cancer cells. RESULTS: In our study we have observed that exposure of triple negative human breast cancer cell (TNBC), MDA-MB-231 to recombinant CCN5, resulted in a dose-dependent inhibition of proliferation after 48 h and the IC50 was observed around 500 ng/mL concentrations, while the effect was undetected in MCF-7 cells. Interestingly, at the sub-lethal dose (100 ng/mL) of rec. CCN5 protein, a distinct morphological alteration (i.e., Mesenchymal type to epithelial type) of MDA-MB-231 cells was observed as confirmed by the staining of actin cytoskeleton and differential regulation of EMT markers. Interestingly it was also observed that exposure of TNBC cells, MDA-MB-231 and HCC70 to EGCG resulted in a dose-dependent inhibition of proliferation after 72 h and the IC₅₀ was observed around 75 μM for MDA-MB-231 cells and 50 μM for HCC70 respectively. We found EGCG-treatment effectively induces MET and inhibits the in vitro migration parallel with the induction of CCN5 expression in TNBC cells in a dose-dependent fashion. Furthermore, consistent with in vitro findings, tumor progression was drastically inhibited in EGCG-treated MDA-MB-231-tumor xenograft in nude mouse model. CONCLUSION: EGCG imparts its anti-cancer activity in both TNBC cells as well as MB-231-tumor xenografts via induction of CCN5.

INHIBITION OF EPITHELIAL-MESENCHYMAL TRANSITION BY BENZYL ISOTHIOCYANATE, A CRUCIFEROUS VEGETABLE-DERIVED CANCER CHEMOPREVENTIVE AGENT

Anuradha Sehrawat and Shivendra V. Singh

University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

BACKGROUND: Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g., garden cress), inhibits chemically-induced cancer in experimental rodents. Previous studies from our own laboratory have revealed that BITC not only inhibits growth of cultured (MDA-MB-231 and MCF-7) and xenografted (MDA-MB-231) human breast cancer cells but also suppresses mammary cancer development in a transgenic mouse model (MMTV-neu). This presentation extends these observations and summarizes the results of our more recent published and unpublished studies demonstrating novel actions of BITC [i.e., inhibition of epithelial-mesenchymal transition (EMT)]. METHODS: Effect of BITC treatment on protein and mRNA expression of epithelial and mesenchymal markers in cells was determined by Western blotting and reverse transcription or real time PCR, respectively. Immunohistochemistry or Western blotting was performed to determine the in vivo effect of BITC administration on E-cadherin, vimentin, fibronectin, uPAR and FoxQ1 protein expression. Overexpression or knockdown of desired protein was achieved by transfection. Transwell chamber assay was used to determine cell migration. RESULTS: EMT inhibition by BITC in representative breast cancer cells (MDA-MB-231, SUM159, MCF-7) was characterized by up-regulation of epithelial markers (e.g., E-cadherin and/or occludin) with concomitant suppression of mesenchymal markers (vimentin, fibronectin, and snail). Experimental EMT induced by exposure to TGFβ and TNFα in a spontaneously immortalized and non-tumorigenic human mammary epithelial cell line (MCF-10A) was also largely reversible in the presence of BITC. Inhibition of MDA-MB-231 xenograft growth in vivo in female athymic mice and breast cancer prevention in MMTV-neu mice by BITC administration was accompanied by induction of E-cadherin and/or suppression of vimentin protein expression. BITC-mediated inhibition of EMT was independent of urokinase-type plasminogen activator receptor (uPAR), but partly linked to suppression of FoxQ1. CONCLUSION: In conclusion BITC mediated inhibition of EMT in breast cancer is mediated by induction of E-cadherin and downregulation of mesenchymal markers in breast cancer. In addition, partial suppression of FoxQ1 plays important role in inhibitory effect of BITC against EMT. These studies were supported by the National Cancer Institute grant RO1 CA129347-06.

STRATEGIC MANAGEMENT OF BREAST CANCER CELL MIGRATION BY REGULATION OF E-CADHERIN THROUGH NUCLEAR MATRIX PROTEIN SMAR1

Arghya Adhikary¹, Samik Chakraborty¹, Shravanti Mukherjee¹, Minakshi Mazumdar¹, Argha Manna¹, Samit Chattopadhyay² and Tanya Das^1,*

¹Division of Molecular Medicine, Bose Institute, Kolkata, India. ²National Centre for Cell Science, Pune, India.

INTRODUCTION: The evolution of the cancer cell into a metastatic entity is the major cause of death in patients with cancer. It has been acknowledged that aberrant activation of a latent embryonic program—known as the epithelial-mesenchymal transition (EMT)—can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence. A key initial step in EMT involves the down-regulation of the adhesion protein, E-cadherin. Dissecting the molecular mechanisms that regulate E-cadherin expression has, therefore, become pivotal for understanding tumor invasiveness and metastasis. METHODS: Experiments like Western blotting, Immunoprecipitation, Reverse transcription, Chip assay, Unidirectional wound healing assay, Transwell migration assay were done to solve the mystery. RESULTS: In the present study we demonstrated that the tumor suppressor SMAR1, a matrix attachment region-binding protein, enhanced E-cadherin expression in breast carcinoma cells in two ways. In one hand SMAR1 represses Slug, a negative regulator of E-cadherin. In fact, our gene manipulation studies and CHIP analysis strongly proved that binding of SMAR1 to the matrix attachment region site present in Slug promoter and recruitment of co-repressor complex to the Slug promoter resulted in its transcriptional repression. On the other, SMAR1 disrupted E-cadherin-MDM2 crosstalk, required for E-cadherin degradation, thereby resulting in E-cadherin protein stabilization. The resultant outcome of these molecular interactions declined the migratory potential of breast carcinoma cells. CONCLUSIONS: Cumulatively, our in-depth analysis strongly suggests that SMAR1 reverts epithelial-mesenchymal transition by up-regulating E-cadherin expression in a two-way manner, thereby dampening the metastatic potential of breast carcinoma cells. SMAR1, therefore, may offer its candidature as a crucial target for therapeutic intervention of metastatic breast cancer. *To correspond: tanya@bic.boseinst.ernet.in

CHOLESTEROL-LOWERING DRUG TARGETS OSTEOCLASTOGENIC FACTORS TO PREVENT OSTEOLYTIC BONE METASTASIS OF BREAST CANCER

Chandi C. Mandal^1,#,*, Goutam Ghosh-Choudhury² and Nandini Ghosh-Choudhury¹

¹Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, ² Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

BACKGROUND: Breast cancer metastasis is the main cause of morbidity and mortality in patients. Once in bone microenvironment, tumor cells release cytokines which increase osteoclast activity and bone microenvironment provides a fertile soil to breast tumor cells for their metastatic growth. We have recently documented that simvastatin (SIM), a member of statins group, a cholesterol-lowering drug, blocks osteolytic metastasis of breast cancer cells by targeting p53-mediated CD44 gene expression. Intracardiac administration of human breast cancer cell (MDA-MB-231) in immune compromised mice increases osteolytic activity which is inhibited by SIM treatment. SIM-mediated inhibition of osteolytic bone metastasis could be at least in part due to inhibition of osteoclast activity. Expressions of osteoclastogenic factor, colony stimulating factor 1 (CSF-1) and dickkopf-1 (DKK-1), a known Wnt inhibitor, positively correlate to cancer metastasis and are shown to be essential for osteoclast activity. In bone microenvironment tumor cells and osteoblast cells both secrete CSF-1 and DKK-1 which either directly or indirectly activate ostecolast activity. METHODS: q-RTPCR, ELISA and immunoblotting were performed to determine expression of CSF-1 mRNA, CSF-1 protein and DKK-1 protein respectively. Osteoblast-supported osteoclast activity was examined by co-culture experiment using osteoblast 2 T3 and osteoclast precursor spleen cells. Luciferase assays were carried out to test promoter activity using DKK-1 and CSF-1 promoter driven luciferase plasmid constructs. RESULTS: We have noticed that bone specific metastatic breast cancer cell shows significant increased expressions of CSF-1 and DKK-1 in compare to its parental cell (MDA-MB-231). We found that SIM treatment significantly decreased the expressions of CSF-1 and DKK-1 in MDA-MB-231 and in osteoblast 2T3 cells. We examined the efficacy of SIM on osteoclast formation in a co-culture assay using 2T3 and mouse spleen cells as osteoclast precursor cells. SIM dramatically blocked osteoblast-induced formation of TRAP positive multinucleated (MNC) osteoclasts. We tested the involvement of DKK-1 in CSF-1 expression to understand the molecular mechanism underlying in SIM-mediated inhibition of osteoclast activity. We found that SIM treatment was unable to inhibit DKK-1-induced CSF-1 expression in 2T3 cells. CONCLUSION: This observation suggested that SIM-mediated inhibition of CSF-1 is regulated by DKK-1. This observation for the first time shows that simvastatin targets DKK-1 to block osteoclastogenic factor CSF-1, expression of which is essential for osteolytic activity in metastatic bone microenvironment.

DOPAMINE AS A MARKER AND THERAPEUTIC AGENT IN CANCER

Chandrani Sarkar, Debanjan Chakroborty, Partha Sarathi Dasgupta and Sujit Basu

Department of Pathology and James Comprehensive Cancer center, The Ohio State University Medical Center, USA

BACKGROUND: A substantial amount of dopamine (DA) is present in peripheral tissues including the gastrointestinal tract (GI) tract. This peripheral DA has been implicated in the regulation of the several functions of GI tract. Also blood vessels supplying the organs of the GI tract are innervated by sympathetic nerves containing DA, and DA regulates functions of normal blood vessels through its receptors present in these vessels. We therefore investigated the role, if any, of this neurotransmitter in the growth and progression of gastrointestinal tract cancers. METHODS: Initially, the status of DA and tyrosine hydroxylase, the rate-limiting enzyme required for DA synthesis, were determined in normal stomach and colon tissues and mouse and human gastric and colon cancer tissues. The status of DA in blood vessels of normal colon and in blood vessels of malignant colon tissues was also determined. On the basis of our observation of inverse correlation between stomach and colon DA and cancer growth, we determined the effect of pharmacological dose of DA on the angiogenesis and growth of Hs746T gastric cancer and HT 29 colon cancer in nude mice. RESULTS: DA and tyrosine hydroxylase were absent in both human and mouse gastric cancer and colon cancer tissues. On the contrary, exogenous administration of low nontoxic dose of DA significantly retarded tumor angiogenesis by inhibiting VEGFR-2 phosphorylation in tumor endothelial cells, which expressed DA D2 receptors. This action of DA was associated with the inhibition of gastric and colon cancer growth in mice. Furthermore, in combination with anticancer drug (5-fluorouracil), DA significantly inhibited tumor growth and increased the life span of HT29 colon cancer bearing mice when compared with treatment with DA or 5-flurouracil alone. This was possible as DA significantly increases the concentration of 5-flurouracil in tumor tissues. CONCLUSION: Our study demonstrates that there is an inverse correlation between endogenous stomach and colon DA and of gastric and colon cancer and indicates that DA can significantly enhance the concentration and hence the efficacy of commonly used anticancer drugs and also indicates that an inexpensive drug like DA or its D2 receptor agonist, which are being extensively used in the clinics, can be an option for the treatment of gastric and colon cancer.

HLA CLASS I GENETIC AND EXPRESSIONAL ALTERATIONS ASSOCIATED WITH HPV16 RELATED CERVICAL CANCER PATHOGENESIS

Damayanti Das Ghosh¹, Indranil Mukhopadhyay ², Sharmila Sengupta ³

¹Present Address: Indian Institute Of Chemical Biology, Kolkata; ²Human Genetics Unit, Indian Statistical Institute, Kolkata; ³National Institute of Biomedical Genomics, Kalyani. E-mail: damayantidas@gmail.com, indranilm100@gmail.com, sharmilasg@gmail.com

BACKGROUND: HLA class I molecules present endogenous antigens to the immune system and are relevant for HPV-infections and cervical cancer (CaCx) pathogenesis. OBJECTIVES: We tested whether (i) certain HLA class I alleles/haplotypes predispose towards or protect against HPV16 infections and CaCx, and (ii) HLA class I expression is significantly downregulated in CaCx and is affected by promoter-hypermethylation. METHODS: Polymorphic HLA class I (A, B, C) exons 2 and 3 were resequenced in 75 HPV16 (+)ve cases, 70 HPV16 (+)ve normals and 45 HPV (−)ve controls. Different softwares like Phredphrap, PHASE, and PLINK were used for sequence-alignment, haplotype-construction and association-testing, respectively. HLA class I expression was determined by quantitative PCR among HPV16 (+)ve cases (n = 50), and normals (n = 18) and HPV (−)ve controls (n = 25) and promoter-methylation was determined by quantitative PCR. RESULTS: HLA B*40060101 significantly increased disease risk. Six SNPs in HLA-B were found to be significantly associated with disease risk/protection. HLA-A, HLA-B and HLA-C were significantly downregulated among CaCx cases both in contrast to HPV16 (+)ve normals (4.78, 6.037 and 2.99 folds, respectively) and HPV (−)ve controls (8.38, 8.7 and 3.52 folds, respectively). HLA-A and HLA-C showed higher repression (3.68 and 5.06 fold, respectively) among integrated than episomal cases. HLA class I promoter methylation was not found to be associated with CaCx pathogenesis. CONCLUSION: Genetic diversity of HLA class I B alleles and downregulated expression of HLA A, B and C, significantly influence HPV16 related CaCx pathogenesis. Differential expression of HLA A and C between episomal and integrated types of CaCx indicates that they are likely to be distinct biologically.

ROLE OF DOPAMINE IN TUMOR VASCULAR REMODELING

Debanjan Chakroborty, Chandrani Sarkar, Hongmei Yu, Jiang Wang, Zhongfa Liu, Partha Sarathi Dasgupta and Sujit Basu.

Department of Pathology and James Comprehensive Cancer center, The Ohio State University Medical Center, USA.

BACKGROUND: Tumor progression is associated with an excessive increase in the number of highly disorganized, dysregulated and dysfunctional blood vessels that pose a serious problem for therapies due to incompetent and inadequate blood flow that leads to major loss and restricted delivery of antineoplastic agents to the tumor. Hence restoration of proper blood flow is crucial to ensure proper delivery of therapeutic agents in cancer. Since the neurotransmitter dopamine (DA) is effective in inhibiting tumor angiogenesis and blood vessels are innervated by sympathetic nerves that contain DA, we investigated the role of DA if any in regulating the morphology and hence function of tumor blood vessels. METHODS: The effect of administration of low nontoxic dose of DA to tumor bearing mice on the morphology and function of tumor blood vessels was determined. DA was also administered in combination with conventional antineoplastic agents and the concentration of the drugs in tumor tissue and the effect on this combination treatment on tumor progression was determined. RESULTS: Dopamine restores normal blood flow in tumors, inhibits leakiness of tumor blood vessels, and reduces hypoxia in tumor tissues leading to an increase in the concentration and efficacy of anticancer drugs in tumor tissues which results in inhibition of tumor growth and development. CONCLUSION: Our results demonstrate that dopamine in addition to inhibiting angiogenesis can also restore normal vasculature in tumors and thus may be important as an effective therapeutic agent not only in the treatment of cancer but also in other diseases or disorders where aberrant and dysfunctional blood vessels is an important feature of the disease progression.

DELINEATING THE MOLECULAR MECHANISM OF THE BREAST CANCER-INDUCED BRAIN METASTASIS AND A ROLE OF A NOVEL PAN-TGF-Β INHIBITOR AS A POTENTIAL THERAPY FOR BRAIN METASTASIS

Keya De Mukhopadhyay and LuZhe Sun

Department of Cellular & Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229

Breast cancer (BCa) is the most common malignant disease in women in the U.S. Nearly 20 % of patients with advanced BCa are eventually diagnosed with brain lesions, which is a devastating complication in patients with BCa over-expressing EGF receptor family members including Her2 positive and triple negative breast cancer. It is the most feared complication of BCa in part because are not capable of significantly treating the BCa-induced brain metastases due to the inability of the available treatment regimens to effectively penetrate the blood brain barrier (BBB) and also due to our limited knowledge on cellular and molecular mechanisms that drive the homing to and growth in the brain of BCa cells. Therefore, there is a need of efficient model system that can significantly contribute towards our understanding of different factors from both host and tumor leading to brain metastasis. We have recently isolated a novel BCa cell line B6TC that was generated through fusion between human BCa, MDA-MB-231 and ZR-75-1 cells in mouse bone marrow. This B6TC cell line showed higher propensity to metastasize to brain than its parental cells when inoculated through intracardiac injection in female athymic nude mice. In order to generate a highly brain metastatic breast cancer model for mechanistic research, we subjected the B6TC cells through four rounds of selection for cells that were capable of trans-endothelial cell invasion to obtain cells that could invade through BBB. This in vitro selected cell line was further subjected through three rounds of in vivo selection for cells that were capable of metastasizing to the brain and the cells after third round selection was named N3LR, which has the highest potential to cause brain metastasis. In searching for genes and pathways that may contribute to the increased brain metastasis of N3-LR cell with microarray analysis, we found that the transforming growth factor-beta (TGFβ) signaling pathway is up-regulated in N3-LR cell in comparison with B6TC cell, in addition to the EGF and prostaglandin signaling pathways that have been reported to be associated with brain metastatic breast cancer cells. Functional comparison also showed that N3-LR cell was more migratory than B6TC cell and more responsive to TGFβ-induced phosphorylation of Smad3 as well as migration, suggesting that TGFβ signaling may contribute to the increased brain metastatic potential. We next investigated whether metastatic tumor growth in the brain microenvironment can be inhibited by systemic administration of a potent pan-TGFβ inhibitor, BGERII- a recombinant fusion protein containing the endoglin domain of betaglycan (BGE) and the extracellular domain of RII. The animals were inoculated intracardiacally with N3LR, the most potent sub-line of highly metastatic B6TC cells, and were then treated with vehicle or BGERII systemically via i.p. injection right after the inoculation. After 3 weeks, the BGERII treated group showed lower brain metastasis incidence and tumor burden as detected by whole mouse bioluminescence and GFP imaging. Further analyses to understand the underlying molecular and regulatory mechanism of brain metastasis and its intervention in our mouse model is underway for the discovery of novel molecularly targeted drugs to prevent and eradicate BCa metastasis initiation, progression and recurrence.

DIFFERENTIAL EXPRESSION PATTERN OF ONCOMIRNA—222 IN HEPATOCELLULAR CARCINOMA CELLS EXPRESSING HBV AND WITHOUT EXPRESSING HBV

Manikankana Bandopadhyay¹, Neelakshi Sarkar¹, Ananya Pal¹, Rajesh Panigrahi¹, Shivram P. Singh², Arup Banerjee ¹, Avik Biswas¹, Shekhar Chakrabarti^{1, 3}, Runu Chakravarty¹

¹ICMR Virus Unit, Kolkata, Indian Council of Medical Research, Kolkata, ²Department of Gastroenterology, SCB Medical College, Cuttack, ¹National Institute of Cholera and Enteric Diseases, Kolkata, India. Telephone No. 09674382354, E-mail address: manikankana.c@gmail.com

INTRODUCTION: Hepatocellular carcinoma (HCC) is the one of most common malignancies worldwide and chronic Hepatitis B Virus (HBV) infection is one of the major risk factors in development of hepatocellular carcinoma. Information about miRNA mediated regulation of viral infections is just emerging. During HBV infection, perturbations of cell cycle regulating miRNA expression might have significant correlation with HCC development. miRNA-222 is often over expressed in HCC and targets p27^Kip1—a tumor suppressor gene. In this context of the background information, our aim is to assess the role of miRNA-222 in different stages of HBV infection leading to HCC. METHODS: In the study HepG2, hepatocellular carcinoma cell without HBV infection and hepatocellular carcinoma cell HepG2.2.15 that constitutively produce HBV were used. HepG2 cells transiently transfected with HBV plasmid and HepG2.2.15 were used for studying miRNA-222, its target mRNA and target protein. RNA and the protein were extracted following time kinetics. Expression of miRNA-222 was measured by real time PCR and target protein p²⁷ by western blot. miR-222 expression in healthy individuals was also compared with samples from liver cirrhosis and HCC patients. RESULTS: At 24 h post transfection the expression of miR-222 was increased significantly followed by reduced expression at 48, 72 and 96 h in HBV transfected HepG2 cells. This decreased expression was reflected in HepG2.2.15 cell line at 48 h. Target mRNA and protein p27 were also down regulated and later up regulated, following the same trend. Reduced expression of miR-222 was found in liver cirrhosis and HCC patients compared with healthy controls. CONCLUSION: Differential expression of miRNA-222 during different stages of HBV infection may suggest using miR-222 as biomarker for progression of serious liver diseases like liver cirrhosis and HCC in HBV carriers.

STEROID RECEPTOR COACTIVATOR IN ONCOGENIC REPROGRAMMING OF CANCER CELL METABOLISM

Subhamoy Dasgupta, Bin Zhang, Nagireddy Putluri, Arun Sreekumar, Bert W. O’Malley

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030

BACKGROUND: Steroid receptor coactivators/p160 (SRC) are frequently amplified and overexpressed in various human cancers which mediate transcriptional functions of nuclear receptors and other transcription factors, providing survival advantage to cancer cells. Recent findings from genomic profiling of human prostate tumors have identified SRC-2 (also known as NCOA2/TIF2), one of the SRC family member as the major oncogene. SRC-2 was found to be significantly enhanced in 8 % of primary tumors and 37 % of metastatic tumors, and its expression positively correlated with disease recurrence. This suggests SRC-2 may play crucial role in the progression of prostate cancer. The current study is aimed to understand the molecular function of SRC-2 in the pathogenesis of prostate cancer. METHODS: Loss and gain of functional studies were performed by lentiviral and adenoviral transduction methods followed by metabolic profiling using LC-MS. Metabolic phenotyping of cancer cells were analyzed using kinetic assays using Biolog-Spectra. Gene expression studies and chromatin immune-precipitation were performed to determine transcriptome complex. RESULTS: Knockdown of SRC-2 altered the metabolic profile of prostate cancer cells. Total lipid content of the tumor cells particularly palmitate and oleate were significantly reduced due to loss of SRC-2. We found SRC-2 transcriptionally modulates the fatty acid biosynthesis pathway, as SRC-2 ablation specifically reduced fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) both at the mRNA and protein level. Using metabolite-tracers, we identified SRC-2 modulates glutamine dependent fat synthesis and loss of SRC-2 substantially decreased glutamine metabolism, and prostate cancer cell growth. ChIP analysis identified direct association of SRC-2 on FASN and SCD promoter. CONCLUSION: SRC-2 acts as metabolic coactivator and promotes prostate cancer cell growth by modulating transcriptional responses.

TETRANDRINE (A NOVEL BIS-BENZYLISOQUINOLINE ALKALOID) INHIBITS ANDROGEN RECEPTOR SIGNALING AND INDUCES APOPTOSIS OF ANDROGEN RESPONSIVE AS WELL AS CASTRATE RESISTANT PROSTATE CANCER CELLS: IMPLICATIONS FOR PROSTATE CANCER TREATMENT

Hari K Koul and Sweaty Koul

¹Signal transduction and Molecular Urology Laboratory-Program in Urosciences, Division of Urology- Department of Surgery, School of Medicine, ²The Denver Veterans Administration Medical Center, and ³The University of Colorado Comprehensive Cancer Center; University of Colorado -School of Medicine, Building P15 or RC2, 12700 E 19th Avenue, Room number 6430D, Aurora, CO 80045, USA

Castrate resistant prostate cancer is the second leading cause of cancer deaths in men. Reduction in serum prostate-specific antigen (PSA) levels has been proposed as an endpoint biomarker for human prostate cancer intervention Androgen receptor signaling remains active even during castration resistance, as such is an attractive therapeutic target in prostate cancer. We investigated the effects of Tetrandrine, a novel derivative of Tetrandrine (an active constituent of Chinese herb S. tetrandra) on PSA synthesis and secretion and cell growth and proliferation in LNCaP cells (hormone sensitive prostate cancer cells) and LNCaP C4 and C-4B (castrate resistant prostate cancer cells). Treatment of LNCaP cells with Tetrandrine resulted in significant decrease in PSA synthesis and secretion. Tetrandrine treatment inhibited androgen receptor activity as well as PSA secretion and down regulation of androgen receptor levels. Tetrandrine also suppressed the growth and proliferation of LNCaP cells. These effects of Tetrandrine on inhibition of growth and proliferation were time (24–96 h) and concentration (5–20 mM) dependent. Cell Cycle analysis following 24 h treatment revealed that Tetrandrine treatments increased the number of cells in G₀/G₁-phase of the cell cycle, consistent with G₀/G₁-phase cell cycle arrest. Long-term treatment (48 h or greater) of the cells in vitro as well as in tumor xenografts (in vivo) with Tetrandrine resulted in accumulation of the cells Sub-G₀/G₁-phase, and cell death. Taken together these studies demonstrate that Tetrandrine targets androgen receptor signaling in both androgen responsive as well as castrate resistant prostate cancer cells. These results demonstrate that Tetrandrine as a potential agent for intervention of prostate cancer. Studies supported in part by VA Merit Award-01BX001258, and NIH/NCI R01CA161880; H. Koul is supported in part by NIH R01 DK54084, and Chair Commitment (H. Koul) and the Department of Surgery, School of Medicine Academic Enrichment Funds (H. Koul).

FOXP3 ACT AS A CO-TRANSCRIPTION FACTOR OF STAT-3 IN TREGULATORY CELLS

Abir Kumar Panda, Dewan Md Sakib Hossain, Sreeparna Chakraborty, Kirti Kajal and Gaurisankar Sa

Division of Molecular Medicine, Bose Institute, P1/12, CIT Scheme VIIM, Kolkata, India.

Introduction: Immunological self-tolerance against self-reactive T cells is maintained at least in part by regulatory T (T_reg) cells.T_reg cells operate primarily at the site of inflammation where they modulate the immune reaction through a) direct killing of cytotoxic cells through cell-to-cell contact, b) inhibition of cytokine production by cytotoxic cells by interleukin-2, c) direct secretion of immunomodulatory cytokines, in particular TGF-beta and interleukin-10.The X-chromosome encoded FoxP3 is a lineage-specifying factor responsible for the differentiation and function of T-regulatory cells which are indispensible for controlling autoimmunity and excessive inflammation. Continued expression of FoxP3 in mature T_reg cells is essential to maintain the gene expression programme enabling suppressive function of T_reg cells. Materials and Methods: Flow cytometry, Real-Time PCR, Western Blotting, Chromatin Immunoprecipitation, MALDI-2D gel electrophoresis. Results: In our recent study we identify a population of IL-10-producing T_reg cells, essentially FoxP3-positive, which contributes to IL-10-dependent type-2 cytokine bias in breast cancer patients. Although FoxP3 knock-out inhibited il10 transcription in these cells, genome-wide analysis of FoxP3-target genes rules out the role of FoxP3 as a transcription factor for il10. An in-depth analysis reveals that FoxP3, in association with HAT1, docks on STAT3 to epigenetically modify il10 promoter through histone acetylation thereby inducing transcription. Conclusion: These results suggest a ‘conditional’ gain-of-function of FoxP3, even in absence of mutation, where failing to achieve direct promoter-occupancy FoxP3 promotes transcription in association with a locus-specific transcription factor, STAT3, in T_reg cells in the periphery.

IMPACT OF SYNONYMOUS VARIATIONS WITHIN HPV16 L2 GENE AND GENE EXPRESSION PROFILING IN CERVICAL CANCER

Paramita Mandal, Saroj K. Mahapatra, Ankur Mukherjee, Samsiddhi Bhattacharjee, Analabha Basu and Sharmila Sengupta

National Institute of Biomedical Genomics, Netaji Subhas Sanatorium (T.B. Hospital), 2^nd Floor, P.O.:N.S.S., Kalyani-741251,West Bengal,India. Contact no.:+918420304333, Email address:pm1@nibmg.ac.in

Introduction: HPV16 E2 gene disruption causes loss of negative feedback control of viral oncogene expression causing cervical cancer (CaCx). Presence of intact E2 genes in a large number of CaCx cases led towards exploring new paradigms of cervical carcinogenesis. Objective: (i) Determining the association of synonymous variations within L2 and the non-coding region (NCR2), with E2 intact CaCx pathogenesis and (ii) identifying whether such cases are similar or different from those with disrupted E2, in terms of host gene expression. Materials and Methods: We resequenced intact HPV16 isolates (∼8 kb) from 70 cases and 25 nonmalignant DNA samples. L2 expression (mRNA/protein) was determined by quantitative PCR and western blotting. Gene expression profiling was performed on 4 HPV16 positive CaCx cases (intact E2=2, disrupted E2=2) and 2 HPV negative controls. Results: Synonymous variations were significantly higher within the E6 (p = 0.016), E5 (p = 0.005) and L2 (p = 0.0003) of cases with intact E2 and the percentage of such cases harboring humanized codons was significantly (p < 0.01) higher only within the L2 gene (100 %) of cases compared to the HPV16 positive non-tumors (32 %). In contrast to cases with E2 disruption, we recorded the expression of L2 gene (mRNA and protein) only among the E2 intact cases. Further analysis of such samples revealed that the percentage of humanized codons are significantly higher (p value < 0.001) in Asian Americans (4–6) compared to Europeans cases which, harbored either no or few (1–2) humanized codons. Majority of E variant cases (16/17; 94 %) harbored SNPs within the short non-coding region (NCR2) between E5 and L2 genes. These resulted in loss of two miRNA binding sites within NCR2. Pathway analysis (KEGG) of microarray data considering both significantly (p < 0.001) up or downregulated genes among the cases in contrast to HPV negative controls, revealed significant association of (i) JAK-STAT and cytokine signaling pathways in E2 disrupted CaCx cases and (ii) DNA replication and cancer related pathways in E2 intact CaCx cases. The association of cell cycle and p53 signaling pathways with HPV16 positive CaCx cases was common to both E2 intact and disrupted. Conclusion: The findings highlight (i) involvement of multiple pathways in HPV16 related CaCx causation, and (ii) CaCx cases that harbour HPV16 genomes with intact E2 are likely to be distinct biologically, from those with disrupted E2.

EXPRESSION OF SELECTED MIRNA AND MIRNA PROCESSING GENES IN ORAL PRECANCER AND CANCER TISSUES

Roshni Roy, Navonil De Sarkar and Bidyut Roy

Human Genetics Unit, Indian Statistical Institute, 203 B. T. Road, Kolkata 700108. Email: roshniroy16@gmail.com

Introduction: Oral leukoplakia is a premalignant lesion that long has been considered to confer increased risk for the development of Oral Cancer which is highly prevalent in India. Oral lichen planus (OLP) on the other hand is a chronic inflammatory disease whose malignant potential is debatable. Since not all oral precancerous lesions progress to oral cancer, molecular markers like miRNA expression could be used to diagnose and predict progression of disease that cannot be detected histopathologically. Objective: Study of intra- and inter-variation in the expression of a few microRNAs (26a1, 423, 34b, 29a, 193a, 137 and 205) and microRNA processing (XPO5, GEMIN3 and RAN) genes in the three disease types, namely, oral cancer, leukoplakia and lichen planus. Methodology: Total RNA was extracted from paired disease and normal tissues from 15 oral cancer, 19 leukoplakia and 19 OLP patients. Expression of microRNAs and microRNA processing genes were studied using TaqMan assay. Subsequent statistical analyses were performed for interpretation of expression data. Results: Expression of mir 26a-1 and mir 137 genes was dysregulated >2 folds in 70 % of the cancer samples while expression of remaining microRNAs and mir-processing genes were deregulated in a few samples. In leukoplakia, expression of mir-26a-1 was dysregulated >2 folds in 50 % of the samples while dysregulation in expression of the remaining miRNAs was observed in 1/3^rd of the patients. In OLP, there was an interesting trend of unidirectional up or down regulation in the expression of mir26a-1, mir29a, mir-34b and mir-137 genes. We also observed that the average expression of miRNA genes differed among the three patient groups. Conclusion: Deregulation in microRNA expression could not be correlated with individual’s tobacco habits. Variation in expression of microRNA as well as miRNA processing genes was observed within individuals of the same patient group (i.e. cancer or leukoplakia or lichen planus) and between any two patients groups. There was correlation in expression of microRNAs which probably explains the unidirectional increase or decrease of miR expression in OLP. Finally, the mean expression level in the three types of disease tissues differed significantly. But expression of more miRNA genes in more samples would be necessary for molecular markers.

NOVEL INTERACTING PARTNERS OF THE DEUBIQUITINATING ENZYME HAUSP

Seemana Bhattacharya, Mrinal Kanti Ghosh

Division of Cancer Biology and Inflammatory Disorders, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata-700032, India. Phone: 91-33-24995882, Mobile: 9903567493. Email: seemana.bhattacharya@gmail.com.

Introduction: HAUSP, Herpesvirus-Associated Ubiquitin-Specific Protease, a deubiquitinating enzyme, has varied roles in a number of biological processes ranging from genome stability, epigenetic regulation, cell cycle and apoptosis, to viral infection, immunity and stem cell maintenance and hence, emerges to be a very important candidate with implications in cancer as well as other pathologies. The dual, context-specific role of HAUSP, both as an oncogene and a tumor suppressor, makes its role versatile. Materials and Methods: HAUSP was cloned in pGZ vector and transfected in HEK293 cells. Whole cell lysates from vector and HAUSP transfected cells were separated by 2D-gel electrophoresis. Gels were coomassie stained and spots were identified using PDQuest software. Spots were excised and tryptic digests of proteins isolated using In-gel trypsinization kit. MALDI-TOF/TOF MS/MS and GPS analysis were performed for identification of proteins. Immunoprecipitates from HAUSP overexpressed cells were analyzed similarly for identification of putative interacting partners of HAUSP. Results and Conclusion: In this study, we have shown the role of exogenously expressed HAUSP on the overall proteome. Of the 38 proteins identified, 16 were upregulated, 8 down-regulated, 7 unique spots and 3 spots were absent when compared to the control gel. The proteins identified indicate a probable involvement of HAUSP in tumor suppression, transport, metabolism, cytoskeletal organization, gene expression, chaperones, apoptosis etc. Enriched pool of interacting proteins by immunoprecipitation has elucidated a number of probable and yet unknown partners. Of the 36 interacting proteins, some of the proteins like TRRAP, FBF-1, etc. with changed profiles also interact with HAUSP. Gene ontology based classification of the proteins reveals several enzymes, proteins involved in transportation, apoptosis, gene expression, etc. Five Zinc Finger Proteins were identified with the same C2H2 domain. Also a few uncharacterized proteins have been identified. The overall aim of this study is to explore the global picture of the changes in the protein profile and putative interacting partners of HAUSP by mass spectrometric analysis, hence attributing newer functions to the protein.

*This work was supported by grants provided by DST (SR/SO/HS-0150/2010).

CANCER STEM CELLS: UNLOCKING THE SECRETS OF DRUG RESISTANCE AND CANCER RECURRENCE

Shilpi Saha, Sanhita Mukherjee and Tanya Das

Division of Molecular Medicine, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata, India.

Introduction: Cancer represents a major health challenge for the 21st century. The failure to eradicate cancer may be as fundamental as mis-identification of the target. Current anti-cancer therapies succeed at eliminating bulk disease but often fail to root out tumor reservoir. Accumulating evidences suggest that this resting cellular subpopulation with stem cell-like features referred to as cancer stem cells (CSCs) is the source of disease recurrence and secondary tumor development. This study, thus, aims to discern the ripostes of these cells to chemotherapy, to eventually identify novel treatment modalities to fight cancer recurrence. Methodology/Results: Herein we report that treatment of different cancer cell lines with doxorubicin, cisplatin, or etoposide resulted in the selection of drug surviving cells (DSCs). These DSCs displayed self-renewal, tumorigenic and differentiation property of putative CSCs. It was observed that presence of drug efflux pumps, slow-cycling and the anti-oxidant phenotype of CSCs contributed in the rare escape of these cells from drug-induced cell death. Interestingly, our study unveiled that, additional genotoxic hits to DSCs triggered self-renewal in CSC-like cells trailed by induction of differentiation resulting in TA progenitors and differentiated cells. Consistent with this in vitro experimental data, we observed increased percentage of CSCs in breast tumors obtained after neo-adjuvant chemotherapy compared with samples that did not receive preoperative chemotherapy. Furthermore, enrichment of CSCs was observed in breast tumors of patients undertaking increasing cycles of chemotherapy. A search for the underlying mechanisms revealed that drugs-induced NFκB fuelled efflux pumps on one hand and increased Oct-4 on the other that contributes to the transition of CSCs from a quiescent state to a proliferating stage. Intervening NFκB restored chemosensitivity and down-regulated Oct-4, resulting in significant perturbation of ‘spheroid’ formation and ‘stemness’ signature, manifesting the role of NFκB in imparting and sustaining chemoresistant and self-renewal property of CSCs post-chemotherapy. Conclusion: In conclusion this study for the first time assigns NF-κB as an attractive therapeutic tool to potentially stop the “seeds” of the tumor before they have a chance to germinate and spread.

ANTI-CARCINOGENIC EFFECTS OF IL-21 AND CURCUMIN IN ALBINO MICE

Souvarish Sarkar, Pijush Kumar Maiti, Mohit Jain, Richa Agarwal, Shubhanjana Sikdar, Riddhi Goswami

Department of Biotechnology, Heritage Institute of Technology, Chowbaga Road, Anandapur, Kolkata – 700107. Email: riddhi.goswami@heritageit.edu

IL-21 is a member of a large family of cytokines (IL-2, IL-4, IL-7, IL-9 and IL-15) whose receptors share a common receptor c chain (cc).IL-21 is made by a range of activated CD4+ Th cells, including Th1 and Th17 cells, activated NKT cells, and T follicular helper cells. Data emerging from pre-clinical and clinical studies indicates that forced over expression of IL-21 in tumour cells suppresses their growth, through enhanced antitumor immunity. Nonetheless, studies in models of colitis-associated colon cancer strongly support the tumour-promoting role of IL-21, raising the possibility that IL-21 could have opposing functions on the growth of tumors. Curcumin (diferuloylmethane) is a polyphenol derived from the plant Curcuma longa, commonly called turmeric. Extensive research over the last 50 years has indicated this polyphenol can both prevent and treat cancer. The anticancer potential of curcumin stems from its ability to suppress proliferation of a wide variety of tumour cells, down-regulate transcription factors NF-κB, AP-1 and Egr-1; down-regulate the expression of COX2, LOX, NOS, MMP-9, uPA, TNF, chemokines, cell surface adhesion molecules and cyclin D1; down-regulate growth factor receptors (such as EGFR and HER2); and inhibit the activity of c-Jun N-terminal kinase, protein tyrosine kinases and protein serine/threonine kinases. In several systems, curcumin has been described as a potent anti-cancer agent. The main aim of our study is to see the combined effect of IL21 and Curcumin in tumour induced albino mice. In the present study tumourogenic condition was induced in Swiss Albino mice (Mus musculus) with selected doses of Carbon tetrachloride. Then they were injected with different doses of IL-21, Curcumin separately and a combination of both over varying exposure periods. Standard anti-oxidant parameters and chromosomal aberrations were assessed in different exposure groups to ascertain the anti-carcinogenic effects of the therapeutic compounds.

DIFFERENTIAL TRANSCRIPTIONAL REGULATION OF SMAD3/E2F/CMYC AXIS OF TGFΒ SIGNALING PATHWAY DURING PROSTATE CARCINOGENESIS

Subhadipa Majumder^#, Arnab Gupta⁺, Urmi Chatterji* and Sanghamitra Sengupta^#.

Departments of Biochemistry^# and Zoology*, University of Calcutta, 35 Ballygunge Circular Road, Kolkata-700019, India. Saroj Gupta Cancer Centre and Research Institute⁺, Mahatma Gandhi Road, Thakurpukur, Kolkata -700063, India. Email ID: sanghamitrasg@yahoo.com

Introduction: TGFβ is a pleiotropic growth factor that has been implicated in multiple, and often diametrically opposed functions. In cancer cells, TGFβ acts as a growth promoter, whereas in normal cells it appears to inhibit cell growth. In the present study, we conducted a quantitative expression based analysis of components of TGFβ signaling pathway to identify the molecular discriminators that lead to carcinogenesis of the prostate gland. Materials and methods: In our study, a TaqMan based differential gene expression profiling of components of TGFβ pathway was conducted using tissue samples representing human prostate cancer and benign prostatic hyperplasia (BPH). The coexpression status of E2F proteins, its coactivators and the relevant upstream and downstream members belonging to TGFβ signaling pathway was examined by qRT-PCR, western blot and immune-staining analysis. Results: E2F5 was found to be ranked top among the ten upregulated genes in TaqMan assay. E2F5 expression level was correlated with the Gleason Score of prostate cancer patients. The genes that were upregulated in cancer tissues compare to BPH samples include: SMAD3, p300, CyclinD1, c-Myc and Ki-67. Among the different phospho-isoforms of SMAD3, the one with phosphorylation at linker region (Ser208) was found to be prevalent in cancer samples. Of the three MAPKs, ERK and p38 were significantly phosphorylated too. We detected 1.5 fold higher expression of k-ras while TGFβRI was significantly downregulated in cancer samples. Conclusion: Taken together our data suggests that E2F5 is a potent transcriptional regulator in prostate cancer. Differential phosphorylation of SMAD3 is also an important feature in prostate carcinogenesis. Studies are in progress to reveal if the different phospho-isoforms of SMAD3 in collaboration with E2F5 is responsible for uncontrolled cell proliferation during prostate tumorigenesis.

INHIBITORY STUDIES OF ANDROGRAPHOLIDE AND NANO-ANDROGRAPHOLIDE ON B16F10 MELANOMA GROWTH BY INHIBITING PI3K/AKT/NF-κB PATHWAY

Sumit Kumar Dey, Abhishek Nandy, Rudra Narayan Munda, Nabanita Chatterjee, Subhadip Das, Debasree Ghosh, Sujata Das & Krishna Das Saha^Ψ .

Cancer Biology & Inflammatory Disorder Division; CSIR- Indian Institute of Chemical Biology; 4, Raja S.C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. ^Ψ Corresponding author: Phone: +91-33-24995810; Email: krishna@iicb.res.in

Introduction: Cancer treatment using compounds isolated from medicinal plants is becoming popular. Present study is directed to compare the anticancer activity of both the Andrographolide (AN) isolated from Andrographis paniculata and its nano formulation (NA) on a murine melanoma model and its effect on the PI3K/AKT/NF-κB signaling pathway involved in the survival of melanoma cancer cells. Material methods: The apoptotic effect of NA towards the B16F10 cell line (in vitro) and the murine (BALB/c) melanoma model (in vivo) was evaluated by observing the expression of apoptotic mediators like BCl-2 family, PARP, NF-κB, Matrix metalloproteinase (MMP)-2,-9, Cyclooxygenase-2 (COX-2), Extracellular signal-regulated kinase-2 (ERK-2) and TGF-β by western blotting, ELISA and immunocytochemistry. Results: In vitro experiments showed that NA reduced B16F10 cell viability more effectively than AN in a concentration and time-dependent manner. NA induced death of B16F10 was associated with characteristic apoptotic changes with DNA fragmentation, and annexin V binding. Also, alteration of caspase-3, - 9, Bcl-2 and PARP levels, loss of mitochondrial membrane potential, and enhanced level of cytosolic cytochrome C were observed in treated B16F10 cells. NA treatment raised the survival of B16F10 tumor bearing mice than AN significantly (P < 0.01) by apoptotic mediators in the B16F10 tumor cells. NA treatment modulated PI3-kinase activity with inhibition of NF-κB p65 translocation and phosphorylation of AKT in tumor cells more efficiently than AN. NA decreased the elevated expression of Matrix metalloproteinase (MMP) -2, -9, Cyclooxygenase-2 (COX-2), Extracellular signal-regulated kinase-2 (ERK-2) and increased TGF- β in melanoma tissues than AN. The expressions of VEGF and CD 34 in the tumor tissue were decreased by NA treatment in greater extent. Conclusion: The present investigation highly suggests that nano-andrographolide (NA) has the potential use as a therapy for melanoma.

NMK-TD-100, A NOVEL MICROTUBULE MODULATING AGENT INDUCES APOPTOSIS AND AUTOPHAGY IN A549 LUNG CANCER CELLS

Surela Bhattacharya¹, Maruthi Kumar Narayanam², Arnab Ganguli¹, Dalip Kumar² and Gopal Chakrabarti¹*

¹ Department of Biotechnology And Dr. B.C Guha Centre For Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019. ²Department of Chemistry, Birla Institute of Technology and Science, Pilani, Rajasthan. *Correspondence to gcbcg@caluniv.ac.in

Introduction: The essential role of microtubules in cell division makes them important target for anticancer drugs. However, the toxicity and resistance to the anti microtubule agents in clinical use prompt for rigorous screening of new agents. We have screened for growth inhibitory effect of synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles on A549 cell line and identified NMK-TD-100 as the most potent microtubule-depolymerizing agent. Material and Methods: The cell viability and colony formation assays were performed with MTT and crystal violet, respectively. Immunofluorescence, flow cytometry and Western blot analysis were used to study the cellular microtubule and apoptotic and autophagic mechanisms. Tubulin was purified from goat brain and the fluorescence and absorption measurements were performed in a fluorescence spectrophotometer (PTI, USA) and JASCO (Japan) V-530 UV-visible spectrophotometer, respectively. Results: We have examined the effects of NMK-TD-100 on microtubules in cells as well as in cell free system. NMK-TD-100 effectively reduced the cell viability, with an IC₅₀ value of 5.5 ± 0.26 μM in A549, inhibited the colony-formation ability, and induced delay in exit from mitosis, reduced mitochondrial membrane potential (MMP) and induced apoptosis in A549 cells. NMK-TD-100 also induced protective autophagy in A549 cells and pretreatment with autophagy inhibitors potentiated its cytotoxicity. Time-dependent study of change in MMP and preservation of mitochondrial integrity in A549 cells strongly suggested that induction of autophagy is downstream of mitochondrial membrane potential disruption by NMK-TD-100. Immunofluorescence studies showed a significant depolymerization of the interphase microtubule network in a dose-dependent manner. In cell free system, polymerization of purified tubulin into microtubules was inhibited by NMK-TD-100 with an IC₅₀ value of 17.5 ± 0.35 μM. The fluorescence studies showed that the stoichiometry of NMK-TD-100 binding to tubulin is 1:1 (molar ratio) with a dissociation constant of 0.996 ± 0.015 μM and its binding site is near colchicine binding site on tubulin. Conclusion: Together these data suggest that NMK-TD-100, a novel microtubule depolymerizing agent could be used for development of a potential chemotherapeutic agent.

TARGETING THE SERCA PUMP WITH A SYNTHETIC DIHYDROPYRIMIDONE, NIFETEPIMINE: A MECHANISTIC APPROACH TOWARDS IMMUNE REJUVENATION FROM TUMOR-INDUCED APOPTOSIS

Swatilekha Ghosh, Arghya Adhikary, Supriya Chakraborty, Gaurisankar Sa, Tanya Das and Parimal.C.Sen

Division of Molecular Medicine, Bose Institute, P1/12 CIT Scheme VIIM Kolkata, India. Email: swati_gh6933@yahoo.co.in

Introduction: Multiple mechanisms have been proposed by which tumors induce T cell apoptosis to circumvent tumor immune-surveillance. Although Sarco/endoplasmic reticulum (SR/ER) Ca²⁺-ATPase (SERCA) have long been known to regulate intracellular Ca²⁺ homeostasis, few studies have examined the role of SERCA in processes of T lymphocyte survival and activation. It remains largely unexplored as to how tumors jeopardize SERCA function to disable T cell-mediated anti-tumor immunity. Materials and Methods: Human CD4⁺ T cells were cultured with cell free spent media of human breast carcinoma cell, MCF-7, to evaluate the changes in T cell signaling cascade. Studies were also extended in tumor-bearing mice model. Results: Here, we have seen that human CD4⁺ T cells in presence of tumor conditions manifested an up-regulation of SERCA3 expression which resulted in development of ER stress leading to CD4⁺ T cell apoptosis. Prostaglandin E₂ produced by the tumor cell plays a critical role in up-regulating SERCA3 by enhancing the binding of its transcription factor Sp1. Gene manipulation and pharmacological approaches further established that increase in SERCA expression also resulted in subsequent inhibition of PKCα and θ and retention of NFκB in the cytosol. However, down-modulation of SERCA3 expression by a dihydropyrimidone derivative, ethyl-4-(3-nitro)-phenyl-6-methyl-2-oxo-1, 2, 3, 4-tetrahydropyrimidine-5 carboxylate (Nifetepimine) protected the CD4⁺ T cells from tumor-induced apoptosis. In fact nifetepimine-mediated restoration of PKC activity resulted in nuclear translocation of p65NFκB thereby ensuring its survival. Studies further undertaken in mice model re-validated the its immunoprotective role. Conclusion: Our present study thus strongly suggest that imbalance in cellular calcium homeostasis is an important factor leading to CD4⁺ T cell death during cancer and holds promise that nifetepimine may have the potential to be used as an immunorestoring agent in cancer bearers.

ACCUMULATED NUCLEOTIDE SUBSTITUTIONS IN THE HEPATITIS B VIRUS GENOME CAN SERVE AS EARLY DETERMINANT OF HEPATOCELLULAR CARCINOMA

Alip Ghosh, Somenath Datta, Gaurav Roy, Kausik Das, Simanti Datta, Abhijit Chowdhury and Soma Banerjee*

Center for Liver Research, SDLD, I.P.G.M.E & R, Kolkata-20. *Email: somabanerjee70@gmail.com

Backgrounds: Chronic Hepatitis B virus (HBV) infection induces the progression of liver diseases to cirrhosis with increasing potential to develop liver failure or Hepatocellular carcinoma (HCC). During natural course of infection the virus accumulated several nucleotide substitutions (NSs) in the genome. These accumulated NSs induce the progression of the liver disease towards HCC. Aim: The aim of the study was to find out NSs or combination of NSs in D genotype of HBV genome associated with the progression of HBV mediated liver disease towards HCC in eastern Indian population. Methods: Blood samples were collected from 17 patients with no fibrosis (NB),9 patients with early fibrosis (EB), 20 patients with liver cirrhosis (LC) and 22 HBV related HCC patients. NSs were detected by DNA sequencing. Data were analysed using SPSS16.0 and R project. Results: 6 single substitutions and 3 double substitutions in different ORF of HBV genome were significantly associated with the progression of the liver diseases through fibrosis to HCC. In the Basal core promoter (BCP) A1762T/G1764A (K130M+V131I in HBx) were found with 35.29 % in NB, 44.4 % EB, 65 % in LC & 77.27 % in HCC groups respectively. T12S (17.65 % >33.33 % >35.00 % >50.00 %), S35T (0.00 % >11.11 % > 15.00 % >27.27 %), T67N (23.53 % >33.33 % >35.00 % >40.91 %) and T147C (0.00 % >11.11 % >35.00 % >40.91 %) in the core region, T1050G/A (17.65 % >22.22 % >35.00 % >40.91 %) and T1050G+A1053G (0.00 % >11.11 % >25.00 % >31.82 %) in the Enhancer-I and HBX promoter region and S98T (0.00 % >11.11 % >20.00 % >31.82 %) in the PreS1 region of HBV were the other substitutions associated with the progression towards HCC. Conclusion: From this study we can conclude that accumulation of nucleotide substitution at critical locus such as A1762T/G1764A, K130M+V131I, T12S, S35T, T67N, T147C, T1050G/A, S98T and T1050G+A1053G during the early phase of disease in HBV infected patients increases the risk for HCC development and they may be used as early marker of HCC development.

CYTOGENETICS CHANGES AMONG BETEL QUID CHEWERS FROM DIFFERENT REGION OF WEST BENGAL AND NORTH EASTERN STATES

Adhikari Aniket, De Auley, Podder Gargi, Halder Ajanta, Roychowdhury Ranjan, Banerjee Jayashree, De Manas, De Madhusnata

Department of Genetics, Ramakrishna Mission Seva Pratishthan, Vivekananda Institute of Medical Sciences, 99 Sarat Bose Road, Kolkata – 700026. E-mail address: aniket_adhikari@rediffmail.com

Introduction: Oral cancer is most common cancer in males and third most common in females, the main causative agent being use of chewing betel quid (BQ). Areca nut, Cathechu, Slaked lime are major components of Betel quid. Nitrosamines formed from alkaloids in betel nut during betel quid chewing may be implicated in the etiology of oral cancer. Reactive oxygen species are generated due to slaked lime which is also present in betel quid. Micronuclei (MN) have been proposed as a good biomarker to assess cytogenetic damage. Methods: In this present study total 209 subjects were screened from different camps and Department of E.N.T. & Oral and Maxillofacial surgery of RKMSP hospital, Kolkata. Peripheral blood leukocyte culture were analyzed for mitotic index (MI) and exfoliated cell from the buccal mucosa were examined for micronuclei (MN). Results: It has been found that micronuclei percentage was higher and mitotic index higher in oral cancer cases than normal. Most of the subjects had betel quid chewing habit. Conclusion: Betel quid has an immense role in changing the oral pathology and developing oral cancer.

PRO-EMT AND NEO-ANGIOGENIC ATTRIBUTES OF ORAL SUB-MUCOUS FIBROSIS AND VISUALIZATION OF STAGE PROGRESSION PANORAMA THROUGH AUTOMATED IMAGE STITCHING

Anji Anura*¹, Raunak Kumar Das¹, Mousumi Pal², Swarnendu Bag¹, Sailesh Conjeti¹, Debdoot Sheet¹ Ranjan Rashmi Paul², Sanghamitra Sengupta³, Ajoy K. Ray⁴ Chandan Chakraborty¹, Jyotirmoy Chatterjee¹

^1. School of Medical Science and Technology, IIT Kharagpur-721302, India; ^2.Gurunanak Institute of Dental Science and Research, Kolkata 700114, India; ^3.Dept. of Biochemistry, University of Calcutta, Kolkata 700019, India; ⁴.Bengal Engineering and Science University, Shibpur, Howrah 711103, India

*Correspondence: anji.anura7@gmail.com

Introduction: Oral sub-mucous fibrosis (OSF) a high-risk pre-cancer, prevalent in the population of Indian sub-continent having deleterious oral habits. If untreated, OSF progresses into oral cancer. Pathologically, it shows fibrosis of lamina propria and atrophic epithelium with or without dysplasia and chronic inflammation. Being primary site of neoplastic initiation, oral epithelium needs attention in assessing malignant potentiality. Here analysis of molecular features for epithelial alteration related to progressive maturation, cell–cell adhesion, as well as proliferative potential and neo-angiogenic attributes could be crucial. Hence related prime candidate genes are explored in different stages of OSF. Materials & Methods: In histopathologically confirmed normal oral mucosa, dysplastic and non-dysplastic OSF, epithelial regulators like p63, E-cadherin, beta-catenin, PARD-3, c-Myc and angiogenic regulators like VEGF, VEGFRII and CD105 are explored at proteomic and trancriptomic levels by immuno-histochemistry and qPCR. To have a panoramic view of alteration in molecular expression through stage-progression from normal to pre-cancer, microscopic images were stitched by an computational algorithm that takes in overlapping images. Results: A significant down regulation of E-cadherin, beta-catenin, TAp63 and PARD3 with an up-regulation of delta N p63 and c-Myc at proteomic and transcritomic levels indicated pro-EMT changes . Decreased expressions of VEGF and VEGFRII in non-dysplastic OSF supported its avasculrity but upregulation of VEGF in epithelium as well as VEGFRII⁺ and CD105⁺ microvasculature in dysplasia showed its neo-angiogenigic potential. Image stitching algorithm visualized p63 expression, through pathosis progression by overcoming constraints of limited field of view. Conclusion: The data established progression of malignant potentiality of OSF through dysplastic conditions with pro-EMT and neo-angiogenesis associated molecular changes.

IDENTIFYING DIFFERENTIALLY EXPRESSED GENES IN BENIGN PROSTATIC HYPERPLASIA AND PROSTATE CANCER AS PROSPECTIVE CANCER MARKERS

Ankur Bhowal*, Sanghamitra Sengupta^#, Urmi Chatterji*

Departments of *Zoology and ^#Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Kolkata-700019, India. *Email: urmichatterji@gmail.com

Introduction: Benign prostatic hyperplasia (BPH) and prostate cancer have become major medical problem in elderly men in India. Prostate cancer is the second most common adenocarcinoma amongst men whereas BPH is the non-malignant overgrowth of the periurethral and transitional region of prostate gland. Both these proliferative diseases found to be highly dependent on androgen and androgen receptor (AR)-related signaling pathways. However, with time, adenocarcinoma of prostate often becomes androgen independent, which makes it non-responsive to conventional androgen ablation therapies. Hence, there is great necessity for identifying precise markers during cancer progression. Material and Methods: TaqMan® gene expression array was used to narrow down our search for differentially expressed genes between BPH and cancer tissue samples. Differentially expressed genes were selected for construction of probable hypothetical pathway. Selected candidate gene expressions were validated in a larger cohort of patient samples with the help of real time PCR and Western blot analysis. Protein localization was analyzed by immunohistochemistry for candidate genes. Results: From the microarray data, significant over expression of an immunophilin molecule, FKBP4 was identified in prostate cancer. cMyc protein along with its co-regulator p300 was also found to be over expressed in prostate cancer samples. Conclusion: Our study proposes a probable molecular mechanism that involves cMyc and FKBP4 to enhance the androgen receptor signaling efficacy. The enhanced AR signaling pathway can also be correlated with the over expressed FGF8 and KLK3 gene in prostate cancer tissues, and the above genes may serve as probable prognostic markers for prostate cancer progression and detection.

DYNAMIC PATTERN OF INDIVIDUAL INTEGRIN BETA-1 CLUSTERS DETERMINE CANCER CELL ADHESION AND ORCHESTRATES MIGRATORY SIGNAL GENERATION

Argha Manna, Arghya Adhikary, Samik Chakraborty, Anirban Roy, Tanya Das*

Division of Molecular Medicine, Bose Institute

Introduction: Integrin beta-1 is a crucial adhesion receptors at tumor cell surface involved in adhesion invasion and migration. A central, yet unresolved question regarding the function of integrins is how these receptors regulate both their conformation and dynamic nanoscale organization on the membrane to generate adhesion-competent clusters upon ligand binding. Here we exploit the spatial accuracy and temporal resolution of single particle tracking (SPT) to dissect the relationship, lateral mobility, and micro clustering of the integrin beta-1 receptor. Materils and Methods: Surface immunolabelling of live tumor cells, single particle tracking (SPT) and data processing with own built computer program, western blotting and co-immunoprecipitation. Results: We found that during early phase of cancer cell adhesion integrin beta-1 show random motility and free diffusion. But interestingly after stable adhesion to fibronectin (FN) extra cellular matrix integrin beta-1 is confined to adhesive platforms beneath the cell membrane. After ligand(FN)-receptor (integrin beta-1) anchorage motility of integrin clusters is restricted by the membrane lipid rafts. We also found that although having the ability to bind to their ligands, these active clusters failed to support firm adhesion and secondary outside-in migratory signalling cascade generation in static conditions after cholesterol depletion, because mobility and clustering capacity were highly compromised. Conclusion: Altogether, our work demonstrates an intricate coupling between membrane lipid raft domains and lateral diffusion of integrin beta-1 and further underscores the crucial role of mobility for the onset of integrin beta-1 mediated cancer cell adhesion and migratory signals. *Corresponds to: tanya@bic.boseinst.ernet.in

STRUCTURAL BASIS OF BRCA1/2 AND GENETIC COUNSELING

Ashok K Varma

Tata Memorial Centre, Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi -Mumbai - 410 210, INDIA, Email: avarma@actrec.gov.in

BRCA1/2 genes are most studies genes among the breast and ovarian cancer patient. BRCA1 and 2 are located in chromosome 17 and 13 respectively. BRCA1 comprises several functional domains including N-terminal RING-finger motif, DNA binding domain at the central region and repeats of BRCTs at C-terminus. BRCA2 has only few reported binding domains like RAD51, DSS1. It is well reported that carriers of BRCA1/2 mutations are at risk to Breast and Ovarian cancer. Several mutations have been discovered across the full length of BRCA1 gene, and categorizing their grade of pathogenicity is a major challenge. The Breast Cancer Information Core (BIC) is a repository for genetic mutations/variants observed in the BRCA1/2 genes. Scientists/clinicians are looking diagnostic/prognostic marker for cancer. However, there is no great success. Structural component of cancer genomics, can provide novel information regarding the pathogenicity of mutants at the atomic level. This also helps to visualize the disease related components and design the small molecule drug leads. Structural studies of cancer causing mutations discovered in Indian families using multi-disciplinary molecular, biophysical, structural and functional assays may help in clinical management.

EXOME SEQUENCING REVEALS COMPREHENSIVE GENOMIC ALTERATIONS IN HUMAN CANCER CELL LINE

Atreyee Saha, Neha Sanghi, Pinaki Mondal and Susanta Roychoudhury

Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata. susanta@iicb.res.in

Introduction: It is well established that genomic alterations play an essential role in oncogenesis, tumor progression, and response of tumor to therapeutic intervention. For decades the human immortal cancer cell lines are used as a convenient model to investigate cancer biology and to explore potential efficacy of anticancer drugs. The advancement of next-generation sequencing technologies (NGS) provide immense advantage to scan genomic changes such as mutations, deletions, and alterations of chromosomal copy numbers. However, the cost of the whole genome sequencing still prevents the routine application of NGS, alternately capturing and sequencing the coding exons of genes (the “exome”) can be a cost-effective approach for identifying changes that result in alteration of protein sequences. We embarked in a project to sequence all human coding genes of a few cell lines of the three selected cancer types (oral cancer, lung adenocarcinoma and glioblastoma). As a proof of concept of this study, we sequenced one lung adenocarcinoma (A549) cell line to identify the sequence variation and mutations in A549 cell line. Materials and Methods: The whole exome from A549 genomic DNA were captured using Agilent Superselect 50 Mb kit and sequenced in SOLiD platform (SOLiD 5500xl). Lifescope Software (Life Technologies, http://www.lifetechnologies.com/in/en/ home.html) v 2.5 was utilized for both secondary (mapping) and tertiary analysis of the sequence reads. Results and conclusion: Out of 71 million raw reads generated, 59 million could be successfully mapped back to the hg19 reference assembly (http://www.ncbi.nlm.nih.gov). 86 % of the target bases were covered with at least one sequencing read, and the average sequencing read depth was 41x in target regions. We detected 32,060 sequence variants (differences from the human reference genome) in A549 cell line. The majority of these differences (94 %) are known polymorphisms in normal human population (i.e. recorded in NCBI dbSNP database, build 130). And the rest of the variants that we found (which are not present in dbSNP database) represent novel sequence variations and/or somatic mutations. Out of those novel variants, non-synonymous ones are more likely to change protein functions and impact cellular phenotypes. Efforts are being made to construct altered cellular pathways in A549 cell line based on this exonic mutation profile.

PACLITAXEL LOADED SOLID LIPID NANOPARTICLE SHOWS ENHANCED POTENCY AND ANTICANCER EFFICACY TO A549 CELL LINE

Debabrata Ghosh Dastidar, Arnab Ganguli, Amlan Das and Gopal Chakrabarti

Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 19, WB, India.

E-mail: gcbcg@caluniv.ac.in

Introduction: Paclitaxel is a potent mitotic inhibitor. It is an effective broad spectrum anti-cancer drug mainly used for lung, ovarian and breast cancer. But poor aqueous solubility, non-targeted delivery and dose-dependent side effects are the problems associated with the proper clinical use of it. Therefore, development of a delivery system that can target the cancer tissue, enhance the potency and anticancer efficacy is of great interest. Methods: Paclitaxel loaded Solid Lipid Nanoparticle was synthesized from microemulsion template. Particle size and zeta potential were measured by Zetasizer. The entrapment efficient was determined by High Performance Liquid Chromatography (HPLC). Morphological studies were done with AFM, TEM, and EFM. Cell viability (MTT assay), cell cycle analysis and drug uptake studies were done in A549 cell line. The uptake of drug by A549 cell line was quantitatively determined by HPLC. The tubulin network and nucleus of A549 cells after treatment were studied by confocal and fluorescence microscope. Expression level of different marker proteins was studied by Western blotting. Results and Conclusion: Nanoparticles of 78 nm diameter and having 98 % entrapment efficiency were synthesised. The particles were spherical in shape and the drug was entrapped into the core of the particles. The entrapment of paclitaxel in SLN decreased the IC₅₀ value and thus increased the potency by six times and caused arrest of 25 % of cells at G2/M phase more than that of aqueous solution of paclitaxel, at 100 nM dose in 24 h. The uptake of paclitaxel was nearly double (1.8 times) when delivered as entrapped into SLN. Treatment with paclitaxel loaded SLN caused the over expression of different apoptotic marker proteins like p53, BAX more than that of aqueous solution of paclitaxel. All these experimental results lead us to conclude that paclitaxel loaded solid lipid nanoparticle has enhanced the potency and anticancer efficacy to A549 cell line.

THE UBIQUITIN LIGASE CHIP REGULATES C-MYC STABILITY AND TRANSCRIPTIONAL ACTIVITY

Indranil Paul, Syed Feroj Ahmed, Arijit Bhowmik, Satamita Deb and Mrinal K Ghosh

Laboratory of Signal Transduction in Cancer and Stem cells, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, West Bengal 700032, India. E-mail: indranil.res@gmail.com

Background. c-Myc is a proto-oncogenic transcription factor and its rapid turnover mediated by the ubiquitin-proteasome system (UPS) is critical for maintaining normal cellular homeostasis. Multiple ubiquitin ligases have been assigned for c-Myc regulation till date. However, the available data suggest for the possible existence of additional E3 ligase(s). Materials and methods. Physiological drugs/inhibitors were used to specifically block or activate cellular specific machineries. Transient transfections were done to over-express proteins and RNAi approach was taken for knock-down experiments. Cells were subsequently harvested for the preparation of WCLs or cytoplasmic-nuclear extracts or extraction of total RNA. Lysates were used for western blotting or coimmunoprecipitation experiments. RNA was used for qPCR analysis. Genes were cloned and sub-cloned into various vectors as and when required. Immunocytochemical and immunohistochemical techniques were used to visualize proteins inside cells. Results. CHIP interacts and ubiquitinates c-Myc thus targeting it for proteasome-mediated degradation. Over-expression of CHIP could accelerate the turnover rate of c-Myc protein. Conversely, knockdown of CHIP by RNAi stabilizes endogenous c-Myc. The interaction between CHIP and c-Myc depends on the N-terminally located tetratricopeptide repeats (TPR) of CHIP which has been implicated as a chaperone binding motif. Inhibition of Hsp90 chaperone activity by 17-AAG reduces c-Myc protein level. We found that the association between CHIP and c-Myc is dependent on the chaperones; particularly Hsp70. CHIP antagonizes the transcriptional activity of c-Myc and decreases the abundance of the transcripts of its target genes. Conclusions. We report CHIP as a new E3 ligase for c-Myc. CHIP is a chaperone-associated Ubox containing E3 ligase. To the best of our knowledge, this is the first report of c-Myc being regulated by a bona-fide chaperone associated E3 ligase. Because CHIP has been reported earlier to be negatively regulating Akt1, BCR-ABL and hTERT, and now c-Myc, the present study may strengthen the view that CHIP acts as a tumour suppressor.

Β-CATENIN/TCF4 COMPLEX INDUCES THE EXPRESSION OF RNA HELICASE P68 PROMOTING EPITHELIAL TO MESENCHYMAL TRANSITION(EMT)

Kiran Kumar Naidu Guturi, Nilanjana Das, Moumita Sarkar, Mrinal K Ghosh*

*Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB), 4 Raja S C Mullick Road, Kolkata, 700032, West Bengal, India. kirankumarnd@yahoo.com, Ph no-9088275277

Introduction: Among evolutionary conserved signaling pathways the pleiotropic effects of Wnt/β-catenin signaling functions are well established in biological process including embryogenesis, tumorigenesis, stem cell biology and cell polarity. Deregulation of the Wnt/β-catenin signaling pathway has been linked to various human cancer including breast, prostate, colon, leukemia, and glioma. Again acetylation of β-catenin is important for its stability as well as transcriptional coactivation of TCF4 target genes. Similarly RNA helicase P68 is found to be over expressed in cancer which has been implicated in a variety of processes, including miRNA processing, RNA secondary structures rearrangement, RNA splicing, gene transcription and tumor development. Material and Methods: Immunoblotting and Immunoprecipitation, Chromatin Immunoprecipation (ChIP), Luciferase reporter assay (Promoter analysis),Q-PCR, Immunofluorescence microscopy. Results: It is very important to understand the mechanism responsible for activation of β-catenin and the relation with P68 expression. Here, we report the mechanism of P68 expression by transcriptionally active β-catenin in breast cancer cells that eventually leads to epithelial to mesenchymal transition. We show β-catenin as well as c-Myc (β-catenin target gene) are the crucial mediators of P68 expression in cancer. The p68 promoter contains three putative consensus of TCF-binding sites, one of which is crucial for β-catenin dependent activation when analyzed by luciferase assay. We further observed that β-catenin occupies the P68 promoter and directly induce transcription of P68 or indirectly through c-Myc. This regulation promotes EMT by upregulating Mesenchymal markers like N-Cadherin and vimetin and down regulating E-Cadherin in breast cancer cells. Conclusion: Wnt/β-catenin signaling plays an important role in cancer progression by upregulating P68 expression which consequently leads to EMT.

NOVEL CHITOSAN BASED NON VIRAL VECTOR FOR EFFICIENT GENE DELIVERY TO CERVICAL CANCER CELL

Kishor Sarkar¹, Manish Debnath¹, A. Chatterjee², G. Chakraborti², P. P. Kundu^1*

¹Department of Polymer Science & Technology, University of Calcutta, 92 A. P. C. Road, Kolkata-700009, India. ²Department of Biotechnology & Dr. B. C. Guha Centre for genetic Engineering, University of Calcutta, 35, Ballygunje Circular Road, Kolkata – 700019, India.

Introduction: Gene therapy is a promising technique for curing various genetic diseases like AIDS, Parkinson’s disease and cancer. Currently, development of safe and efficient DNA carrier is the main hurdle to the success of gene therapy. Chitosan (CTS) is a natural, nontoxic, biodegradable, biocompatible linear cationic polymer and it is intensively studied as a non viral gene delivery vector. However, the transfection efficiency of chitosan is still too low for clinical trials. Materials and Methods: To improve the transfection efficiency of CTS, we prepared dendronized chitosan (DCTS) by Michael addition reaction between N-maleyl chitosan and polyamidoamine (PAMAM) dendrimer. Self assembled DCTS/pDNA complexes were prepared by complex coacervation method at different N/P (nitrogen to phosphate ratio) ratios. The in vitro transfection efficiency of the copolymer was investigated on HeLa cell line. Results: The dendronized chitosan copolymer showed good DNA binding ability and formed spherical shaped nanoscale sized particle (100–200 nm) with homogeneous size distribution with pDNA. The copolymer also showed very low toxicity compared to that of polyethyleneimine (PEI), which is most widely used nonviral vector. The transfection efficiency of the copolymer into HeLa cell increased significantly compared to that of chitosan and reached up to 29 ± 2 % at very low N/P ratio (N/P = 3.0) almost equal to that of PEI (31 ± 4 %, at N/P = 10). Conclusion: Therefore, dendronized chitosan copolymer may be a strong and efficient alternative candidate as a novel non viral vector for therapeutic gene delivery into cancerous cell instead of most widely used PEI.

SYNTHESIS OF MAGNETITE (FE3O4) HOLLOW SPHERES FOR HYPERTHERMIA THERAPY IN CANCER TREATMENT

Madhuri Mandal*, Debasish Sarkar

Dept. of Condensed Matter Physics and Material Science, SN Bose National Centre for Basic Sciences, Block JD, sector III, Salt lake, Kolkata 700098, India. Email: madhuri@bose.res.in

Solvo-thermal technique has been used to synthesize hollow like nanospheres of magnetite. PVP, ethylene glycol, urea, ferric chloride etc. have been used to synthesize the particles. We have shown that PVP plays an important role to take the shape of the particles as hollow spheres and also helps to control the size of the particles. Structural analysis was done by XRD measurement and morphological measurements like SEM and TEM was performed to confirm the structure, shape and size of hollow sphere particles. We have fabricated an instrument for hyperthermia therapy. The detail ac-dc magnetic measurements from this instrument give an idea about the application of these hollow-nanospheres for hyperthermia therapy in cancer treatment. Being hollow in structure and magnetic in nature such materials will also be useful in other application fields like in drug delivery, arsenic and heavy metal removal by adsorption technique, magnetic separation etc.

CORRELATION BETWEEN NUCLEAR SURVIVIN EXPRESSION AND STEMNESS-RETENTION OF BCSCS: ROLE OF THE PLEURIPOTENCY FACTOR, OCT-4

Minakshi Mazumdar, Shravanti Mukherjee, Argha Manna and Tanya Das

Introduction: Disease recurrence originates from residual treatment-resistant cells, which regenerate the initial breast cancer phenotype. The discovery of the normal breast stem cell has re-ignited interest in the identity and properties of breast cancer stem-like cells (BCSC) and the relationship of these cells to the tumorigenic ability of treatment-resistant cells. While the origin and identity of BCSC is contentious, resistant cells survive and propagate only because of the recruitment of aberrant and potentially druggable signaling pathways. Survivin, a bifunctional “Inhibitor of Apoptosis” (IAP), that has been implicated in protection from apoptosis and regulation of mitosis is highly expressed in BCSC as compared to the non-stem cell tumor population. This indicates that survivin might be an essential factor for CSC survival and maintenance. Oct-4, expressed in undifferentiated embryonic stem cells (ESC) and embryonal carcinoma cells, is a well known ESC marker and recently, fore fronted for its presence in a number of solid tumor stem cells. Moreover, in ESC it has been shown that Oct-4 activates the survival gene survivin by phosphorylation and activation of its transcriptional activator STAT3. Materials and Methods: Present study deals with isolation, characterization and propagation of BCSCs as mammospheres in vitro using flowcytomerty and confocal microscopy followed by western blot analysis, along with affirmation of presence of survivin and Oct-4 in different passages of mammospheres. Results: Present study showed that the expression of survivin was overall found to be increased through passage/degree of mammosphere (bearing BCSC) formation along with pleuripotency marker-Oct-4 both of which were turned down upon re-differentiation. Interestingly, survivin expression was increased in nuclear compartment when compared to its cytoplasmic expression. This altered distribution of survivin pointed towards a different functional significance of survivin in BCSCs. Moreover, when survivin expression was downregulated by silencing its transcriptional regulator, STAT-3 or by DN-Akt-cDNA, it effectively lowered stemness as well as Oct-4 expression. Furthermore, from bioinformatic analysis a binding sequence of Oct-4 was predicted on the survivin promoter that projects towards a transcriptional regulation of survivin by Oct-4. Further investigation for the underlying mechanisms of regulation of survivin protein by Oct-4 is needed. Conclusions: All these findings suggest that survivin may pose a relationship with Oct-4 in maintaining stemness of BCSC.

INVOLVEMENT OF P68 RNA HELICASE IN THE REGULATION OF TUMOR-SUPPRESSOR FOXO3A

Moumita Sarkar, G. Kiran Kumar Naidu and Mrinal K. Ghosh

Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S C Mullick Road, Kolkata, 700032, West Bengal, India. E-mail: moumitasarkar.biology@gmail.com

Introduction: RNA helicase p68 belongs to the DEAD box family and plays essential roles in RNA metabolism. Recent reports implicate it to be overexpressed in several cancers. It act as a transcriptional co-activator of several highly oncogenic transcription factors, one such being β-catenin. Forkhead box class O 3a (FOXO3a) is a bona fide tumor suppressor and an important transcription factor that modulate the expression of genes involved in apoptosis, cell cycle, DNA damage repair, oxidative stress and cellular differentiation. FOXO3a is phosphorylated by AKT followed by nuclear exclusion and degradation via skp2 in the presence of growth factor signaling. Our study aims in the investigation of p68 mediated regulation of FOXO3a in cancer. Material and Methods: Human colon cancer cells HCT-116 and HCT-15 were transiently transfected with p68 and were harvested after 24 h. Whole cells lysates were prepared and protein expressions were analyzed using immunoblotting. For analysis of mRNA expression, total RNA was quantified using real time PCR. AKT promoter was cloned into pGL3 vector and luciferase assay was performed to check promoter activity. Results: Dose dependent exogenous expression of p68 in colon cancer cells showed a marked reduction of FOXO3a along with increased expression and activation of AKT. Also, qRT-PCR analysis indicates that over-expression of p68 led to increase in AKT and decrease in FOXO3a target genes expression. But expression of FOXO3a remains unaltered implying post-translational regulation of FOXO3a due to increased AKT as a possible mechanism of downregulation of FOXO3a by p68 in cancer. In the presence of proteasomal inhibitor MG132, FOXO3a level was not reduced thus suggesting increased degradation of FOXO3a in p68 overexpressed cells. Our studies also showed that p68 induced AKT promoter activity. Conclusion: Our study presents a novel role of p68 in regulating AKT-FOXO3a signaling axis. Continuing work will assess the dependence of p68 on AKT expression to control FOXO3a. Recent reports states the presence of several TCF4 sites in the AKT promoter and thus regulated by β-catenin. Therefore it would be intriguing to investigate whether p68 in conjunction with β-catenin regulates AKT expression. This work was supported by grants provided by DST (SR/SO/HS-0150/2010).

DUAL ROLE OF BLACK TEA EXTRACT AS ANTIOXIDANT AND PRO-OXIDANT: A NEW STRATEGY IN CANCER THERAPIES

Debjani Ghosh, Chabita Saha^* and Subrata Kumar Dey

School of Biotechnology and Biological Sciences, West Bengal University of Technology, BF-142, Sector I, Salt Lake, Kolkata 700064, India. *Corresponding author: k.chabita@gmail.com

Introduction: Dietary antioxidant supplements (DAS) have long been used during conventional chemo and radiation therapy to reduce oxidative damage, though the efficacy and safety of this complementary treatment is still controversial. Results on combining antioxidant supplement and anticancer therapy are meagre, making room for more in vitro studies as well as clinical trials to promote DAS during cancer therapy. In the present study attempts have been made to optimise the dose of black tea extract (BTE) to be used as a DAS in vitro, during radiation and drug (Daunomycin) induced oxidative damage in normal lymphocytes and leukemic K562 cells.

Materials and Methods: Presence of different polyphenols in BTE and their uptake were assessed by HPLC studies. Effect of BTE in drug or radiation induced oxidative stress-mediated ROS generation and detection of apoptosis was studied by flow cytometry. Further investigation on apoptotic pathway was followed by detection of caspase 3 and comet assay. Results: Catechins, caffeine and few other flavanols like Myricetin, Quercetin and Kaempferol were identified in the BTE. HPLC studies showed differential uptake of tea catechins in normal lymphocytes and K562 cells. Further drug or radiation induced genotoxicity was evaluated by comet assay. These studies suggested that DNA damage was efficiently minimised in normal cells compared to cancerous cells by pre-treating them with BTE. These findings are supported by increase in ROS, casapase 3 levels and evaluation of apoptosis in both types of cells. Conclusion: A comprehensive understanding about the role of antioxidant supplements in the oxidative injury to noncancerous as well as cancerous cells will be essential for the improvement of alternative strategies in cancer treatment modalities and our study will contribute to improve these strategies.

COMBINATORIAL THERAPY OF CANCER STEM CELLS: A MECHANISTIC APPROACH

Poulami Khan, Shilpi Saha, Minakshi Majumder and Tanya Das*

Division of Molecular Medicine, Bose Institute, Kolkata, India

Introduction: In spite of the ongoing researches for decades cancer is still incurable and to investigate the root of that cause, a recent concept introduces the term ‘cancer stem cells’ or ‘cancer stem-like cells’ (CSCs). CSCs are the identifiable subpopulations of tumor cells with unlimited capacity for self-renewal and are highly resistant to chemotherapy or radiotherapy. In a clinical setting, cytotoxic drugs like etoposide, doxorubicin, cisplatin, tamoxifan, etc., are now given in various combinations and schedules to try to avoid the emergence of resistant cells and to kill pre-existing cells that already are drug resistant. But, although chemotherapy or radiotherapy kills most of the cells in tumor, it is believed to leave cancer stem cells behind. Since the dietary phytochemical, curcumin is now well established as potent inhibitor of different cancers, without any side effects—it could be an interesting approach to study its effect, if any, in combination with these well-known anti-cancer drugs. In our study we aimed at determining whether the combinatorial therapy of chemotherapeutic drugs with curcuminin could be a remedy to target and sensitize the otherwise chemoresistant CSCs, to regress cancer from its origin. Materials and methods: Flowcytometry was used to isolate and purify putative cancer stem cells, which were then characterized by CD44⁺/CD24 ⁻subpopulations. Apoptosis was studied using AnnexinV binding assay and analysed by flowcytomatry. Intracellular p53 level was checked by western blot analysis. Results: Our work established the apoptotic dose of curcumin and doxorubicin combination that spared normal cells. A search for the underlying mechanism revealed the involvement of the tumor suppressor protein p53. In fact, this combinatorial therapy modulated the expression of pro-apoptotic and anti-stemness factor p53 in spheroids derived from highly resistant non-small cell lung carcinoma (NSCLC) cells. Interestingly, direct treatment of spheroids with curcumin took longer time for sensitization than pre-treatment of the parental adherent cancer cells with curcumin. Moreover, CSC spheres presented more differentiated population of cells up on combinatorial therapy than the control ones. Conclusions: Curcumin sensitizes highly resistant cancer stem cell populations towards doxorubicin. This combinatorial therapy further induces differentiation in these mostly undifferentiated CSC populations and reduces stemness in them. Such combinatorial therapy may in future lead to the development of effective remedy of highly resistant cancers.

ONCOGENIC RAS-INDUCED PROLIFERATION CHANNELIZES TOWARDS APOPTOSIS ON SENSITIZING MEK/ERK SIGNALING

Pushpak Bhattacharjee, Shuvomoy Banerjee, Juny Chakrabarty, Deblina Guha, Shilpa Saha, Gaurisankar Sa*

Division of Molecular Medicine, Bose Institute, Kolkata 700 054, India

Introduction: Active-mutant or oncogenic Ras is best known for its ability to induce malignant transformation through the stimulation of uncontrolled cell proliferation. Interestingly, targeting down-stream Raf/MEK/ERK pathway sensitizes active-mutant Ras-expressing cells although the mechanism as to how oncogenic transformation promotes apoptosis is not well elucidated. Materials & Methods: Flow cytometry & immuno-blotting techniques are used to evaluate down-stream MEK/ERK pathway in active-mutant Ras-expressing cells. Gene manipulation and pharmacological intervention studies were performed to confirm the mechanism underneath the apoptogenicity of oncogenic Ras. The mechanism of action was further been validated in RAS mutated mice model. Results: We found under normal condition, oncogenic Ras signals through Raf/MEK/ERK pathway to resist apoptosis and favour proliferation, this protection is antagonized by intervening in this signal transduction cascade. In fact, targeting Ras-downstream factors of this proliferative pathway channelizes oncogenic Ras-signaling towards p38MAPK/JNK1 pro-death circuitry that phosphorylate p53 at Ser³³/Ser⁴⁶ and Thr⁸¹, respectively, of its transactivation domain required for optimal activation of p53. In contrast, in mitogen-stimulated wild-type Ras-expressing cells, Raf/MEK/ERK perturbation fails to induce p38MAPK-/JNK-mediated p53 activation as well as apoptosis. These results establish a hitherto unexplored role of oncogenic Ras as a molecular switch between growth-promoting and growth-inhibitory cascades in which the ultimate balance between these pathways defines cellular homeostasis, leading to survival or induction of programmed cell death. Conclusion: Since, oncogenic Ras is responsible for >30 % of cancers, this pathway describing a novel approach towards anti-tumorigenesis through differential regulation of different MAP kinase family members by oncogenic Ras, is of potential therapeutic significance. *To communicate: gauri@bic.boseinst.ernet.in

KNOWLEDGE, ATTITUDE, BELIEF AND PRACTICE (KABP) OF THE WOMEN IN KOLKATA TOWARDS UTERINE CERVICAL CANCER AND ITS RISK FACTORS

Dr. Ramdas Chatterjee*, Dr. Aditi Nag Chaudhury

Lady Brabourne College, Kolkata. meramdas@yahoo.co.in; ramdas372@gmail.com

Introduction: Despite being a well-preventable disease majority of the cervical cancer cases are presented at an advanced stage to the hospitals in India. By 2020 India will be contributing to an estimated 29 % and 30 % respectively of the global burden of the cases and mortality. The key to reducing cervical cancer morbidity and mortality is early detection and treatment of cervical precancerous lesions. Hence, it is imperative to enhance KABP of the women towards the disease. Methods: Knowledge level and attitude of the sexually active women (aged 20–60 years) in Kolkata towards cervical cancer were studied through a schedule based survey work. Following the survey work women were imparted the relevant knowledge on all aspects of the disease through group meetings and audio-visuals. Results: Results of this ongoing study, mostly among the women within 31–50 years age, will be presented. Preliminary results indicated that about 50 % could not identify any of the risk factors for the cervical cancer, 90 % never heard of the human papillomavirus (HPV), a major etiologic agent for the cancer. Similarly a large portion (68 %) was unaware about Pap test, a well-known diagnostic tool. The disease was thought to be a curse by no less than 24 % of the participants. The ignorance level was almost similar among the women who had education up to primary level or up to secondary/higher secondary level. A large majority (90 % approximately) of the women never heard or attended a similar kind of awareness program. Although a large proportions of the community female health workers were aware of the risk factors for cervical cancer, their knowledge about HPV and Pap test were very poor. Conclusion: Any such in-depth study would recommend for a developing country to go all-out for (i) more awareness and education programs among women about cervical cancer and Pap smear screening and (ii) motivate women to alleviate their fears regarding medical procedures and treatment.

A CROSS-TALK BETWEEN P53 AND SMAR1 ENSURES SUPPRESSION OF TUMOR-ANGIOGENESIS: ANTI-ANGIOGENIC ROLE OF CAPSAICIN

Samik Chakraborty, Arghya Adhikary, Deblina Guha, Samit Chattopadhyay and Tanya Das*

Division of Molecular Medicine, Bose Institute, Kolkata, India

Introduction: Anti-angiogenic approach is a new means to target invasive cancers. However, conventional anti-angiogenic drugs have several side affects including impairment of normal physiological angiogenesis. On the contrary, dietary components like natural phytochemicals are fairly proficient as anti-tumor and anti-angiogenic agents and are devoid of any significant side effects. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), the active component of chili peppers, is known to induce apoptosis in different tumors. However, the mechanism underneath the anti-angiogenic effect of capsaicin, if any, is still the Cinderella of investigation. Materials and Methods: Present study deals with characterization and quantification of pro-angiogenic factors like VEGF, HIF-1α and Cox-2 along with anti-angiogenic factors like p53 and SMAR1 using flowcytomerty and confocal microscopy followed by western blot analysis, along with assertion of cancer cell-induced endothelial cell (EC) migration by the wound healing assay. Results: Here we show that although mammary epithelial carcinoma cells (MECs) encouraged EC migration and sprout formation, such cross-talk between MEC and EC did not require cell–cell contact. These results suggested the involvement of MEC-shed soluble bio-modulators. Interestingly, cell-free media of MECs, pre-treated with capsaicin, significantly halted the migration and sprout formation of endothelial cells. An in depth analysis into the underlying mechanisms revealed that capsaicin ameliorated hypoxia-mediated VEGF up-regulation at both mRNA and protein levels in MECs. Gene manipulation as well as biochemical and pharmacological studies further unveiled that capsaicin targeted hypoxia inducible factor, HIF-1α, adopting a bi-faceted approach, i.e., (a) p53-dependent HIF-1α degradation and (b) SMAR1-dependent decrease in the nuclear localization and transcriptional aptitude of HIF-1α due to suppression of pro-angiogenic factor Cox-2. Our results hint towards the presence of a feed-back loop between p53 and SMAR1 in down-modulating the angiogenic potency of HIF-1α. Conclusion: These novel findings not only disclose the detail mechanism underlying cancer cell-endothelial cell cross-talk in ensuring sprout formation but also bring to light capsaicin as a crucial player in regulating neo-angiogenesis and hence progression of breast cancer. *Correspond to: tanya@bic.boseinst.ernet.in

NEW COMBINATORIAL THERAPY FOR BREAST CANCER

Sankalpa Chakraborty¹, Anuska Das²

¹2nd year postgraduate student, ²1st year postgraduate student, Department of Biophysics and Molecular Biology, University of Calcutta. Email-chakrabortysankalpa@gmail.com

Introduction: Discovery of the existing equilibrium between non-cancer stem cells (non-CSC) & cancer stem cells (CSC) in tumor made cancer therapy more complicated. The de-novo generation of CSCs has implication for the effectiveness of anticancer therapies that exclusively target CSCs because non-CSCs would regenerate CSCs after cessation of therapy. So, in order to be effective, cancer therapies will need to combine agents that are selectively toxic to CSCs & that will inhibit transition from non-CSC to CSC state. The transitions between these states occur through Epithelial Mesenchymal Transition(EMT).Here we propose a combinatorial therapy for the management of Breast cancer(BCa) through two strategies: A. INHIBITING CSCs: Using the idea of ‘oncogene addiction’, as BCa cells are known to be addicted to cmyc oncogene in mice, we may inhibit CSCs using a protein called NUCLEOLIN, reported to inhibit transcription of c-myc gene by stabilizing c-myc DNA quadruplex. Methodology: cDNA clone expressing NUCLEOLIN protein will be delivered in a breast tumor of animal model. For specific delivery into CSCs, antibody against GD2 may be used. Expected Result: Inhibition of c-myc transcription is expected to inhibit CSCs. B. BLOCKING TRANSITION FROM NON-CSC TO CSC STATE: Various reports have demonstrated that TGF-β & RAS signaling pathways are the most important determinants of EMT & by blocking NF-kB, a common downstream messenger of both pathways, a complete inhibition of EMT can be achieved. Methodology: The downregulation of NF-kB and corresponding blockage of EMT in BCa cell line will be examined by treating cells with DITHIOLETHIONONE, a known inhibitor of NF-kB. Once we are successful, we would try to investigate the similar kind of inhibition in animal model. Expected Result: Inhibition of NF-kB expression in epithelial cells is expected to block EMT in cell line & animal model. Conclusion: A combination of proposed therapies may be applied to follow the tumor regression in animal model.

INTERPLAY BETWEEN GENETIC AND EPIGENETIC MACHINERY IN THE ETIOPATHOGENESIS OF PROSTATE CANCER

Sanmitra Basu¹, Subhadipa Majumder¹, Ankur Bhowal¹, Arnab Gupta², Sanghamitra Sengupta¹

¹Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Kolkata-700019. ²Cancer Centre Welfare Home and Research Institute, MG Road, Thakurpukur, Kolkata-700063. Email Id: sanmitrabasu@gmail.com

Introduction: The DNA repair system is integral to the maintenance of genomic stability and suppression of tumorigenesis in many cancers. We have hypothesized that deficiency in the activities of DNA repair enzymes might be one of the major determinants in the etiology of prostate carcinogenesis. Materials and methods: We conducted a population-based case–control study using 12 SNPs located on MSH6, MSH2, MLH1, XRCC1 and OGG1genes among 100 prostate cancer (CaP) cases and 166 age-matched Benign prostatic hyperplasia (BPH) patients as control. Genotypic proportions of each locus were determined using PCR-based RFLP and sequencing. To decode functionality of the polymorphisms with CaP risk, expression of DNA repair genes in tissues was compared between CaP and BPH at mRNA level by relative quantification that was further quantified at the protein level densitometrically by Western blot. To determine CpG methylation pattern of the downregulated genes in CaP tissues compared to benign tissues, we have conducted methylation-specific-PCR on bisulfite-modified genomic DNA extracted from tissues. Result: Twelve SNPs located on the candidate DNA repair genes were genotyped. Only MSH2_rs6753135 was found to be monomorphic. Of the 11 polymorphic markers, OGG1_rs1052133 deviated from Hardy-Weinberg’s equilibrium and was excluded from this study. The genotypic (OR = 1.95; CI = 1.16–3.26; P value = 0.03 and OR = 3.73; CI = 1.43–9.75; P value = 0.001) and allelic (P value = 0.01 and P value = 0.004) proportions of two MSH6 exonic SNPs namely rs1042821 and rs1800932 were found to be significantly different between the two groups. Comparison of gene expression demonstrated a significant downregulation of MSH6, MLH1 and MSH2 genes in CaP tissues compared to BPH which was validated at protein level also. To inquire the role of CpG island methylation for the observed repression of gene expression we quantitated the methylation status of the MLH1 and MSH2 promoters. Our primary observations indicate that aberrant promoter methylation may be responsible for down-regulation of MLH1 in CaP. Conclusion: Taken together our study indicates that CaP has a complex etiology arising presumably because of interplay between genetic and epigenetic abnormalities escorting the normal cells to transform into their malignant derivatives.

HESPERITIN INDUCES APOPTOSIS IN HUMAN BREAST CARCINOMA MCF-7 CELLS BY TRIGGERING ACCUMULATION OF ROS AND ACTIVATION OF ASK 1/JNK SIGNALING PATHWAY

Shreyasi Palit, Susanta Kar, Gunjan Sharma, Dr. Pijush K. Das

Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India. E-mail address: mail2shreyasi@gmail.com

Introduction: As currently used anti-cancer drugs lead to undesirable side effects, phytochemicals are considered as attractive alternatives and flavonoids especially are considered as potentially important tumor suppressive agents. We, therefore, evaluated the anti-cancer activities of hesperitin, a naturally occurring flavanone glycoside predominant in citrus fruits and attempted to decipher the underlying molecular mechanisms. Materials and Methods: The growth inhibitory effect of hesperitin on MCF-7 cells was assessed by MTT assay. The induction of apoptosis was evaluated and validated by Annexin V-PI flow cytometry and TUNEL assay. Mitochondrial membrane potential and the accumulation of ROS were measured by JC-1 and DCFDA flow-cytometry respectively. Activation of caspases, expression of Bax, Bcl-2, release of cytochrome-c and activation of ASK1/JNK pathway in MCF-7 cells was validated mainly by immunoblotting and immuno-precipitation. Results: Hesperitin exhibited significant cytotoxic effect in MCF-7 cells in a concentration- and time-dependent manner, which was due to the induction of apoptosis. Apoptosis was associated with the activation of caspase-9, loss of mitochondrial membrane potential, release of cytochrome c and increase in Bax:Bcl-2 ratio. Further, hesperitin-mediated apoptosis involved generation of ROS along with the activation of ASK 1/JNK signalling pathway. Conclusion: Hesperitin induced cytotoxicity and intrinsic mitochondrial apoptotic cascade in MCF-7 cells by triggering generation of cytosolic ROS and activating downstream ASK1/JNK stress activated signaling pathway.

HPV16 RELATED CERVICAL CANCER PATHOGENESIS: SOME INSIGHTS FROM ANALYSIS OF METHYLATION STATUS OF VIRAL AND HOST GENOMES

Shrinka Sen, Paramita Mandal and Sharmila Sengupta

National Institute of Biomedical Genomics (NIBMG), Netaji Subhas Sanatorium (T.B. Hospital), 2nd Floor. P.O: N.S.S., Kalyani-741251, West Bengal, India. Email ID: shrinka_sen@yahoo.co.in, ss3@nibmg.ac.in

Introduction: Alterations of CpG methylation in both HPV and host genomes are likely to influence cervical cancer (CaCx) pathogenesis. Objectives: We tested whether (i) CpG methylation within the LCR (early promoter, P97), and late promoter (P670) and in immunostimulatory motifs of E6 and E7 of HPV16 and (ii) global DNA hypomethylation of the host genome, are altered in HPV16 positive CaCx cases (E2 intact/disrupted) compared to HPV16 negative/positive controls. Materials & Methods: Methylation was determined by bisulphite sequencing of LCR (nt 7465-115), late promoter region and immunostimulatory motifs of E6 and E7 of HPV16. DNA hypomethylation was estimated by Taqman assay of ALU repeats/Beta-ACTIN and expressed as percent of methylated reference (PMR). TLR9 expression was estimated by quantitative PCR. Results: Methylation was most prominent at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted with lack of methylation at replication origin in case of both. Methylation of CpGs within immunostimulatory motifs of E6 and E7, were significantly higher among cases (24/43, [p = 0.011] and 38/48, [p = 0.004], respectively) compared to HPV16-positive controls (3/16, and 6/15, respectively). TLR9 mRNA expression was lower among cases portraying methylated but not unmethylated E6/E7 motifs, compared to HPV16 positive controls. Analysis of 7 CpGs in late promoter region showed that median methylation was significantly higher among E2-disrupted CaCx cases samples compared to E2-intact samples (Mann–Whitney U test; p = .017), with significantly higher methylation (p = 0.006) specifically at nt 494 and nt 502. Median PMR values reflected significant hypomethylation of CpGs in ALU repeats among HPV16-positive CaCx cases irrespective of E2-status, compared to HPV negative/positive controls. Conclusions: The findings reflect that both viral and host genome methylation are significantly associated with cervical cancer pathogenesis and might be biologically relevant.

CARCINOGENIC AND MUTAGENIC EFFECT OF BETEL NUT EXTRACT—IS IT ANGIOGENIC

Srabantika Mallick, Sudipta Chowdhury, and Samarendra Nath Banerjee

Department of Zoology, Rammohan College 102/1, Raja Rammohan Sarani, Kolkata- 700009, INDIA

samban2kcal@yahoo.com; samar_banerjee@rediffmail.com

Background: Chewing of betel nut (also known as ‘supari’) plays a vital role in the induction of oral and oesophageal cancer in the people of India and neighboring countries. Earlier studies were performed to know the genotoxic potential of Betel Nut Extract (BNE) on in vivo mammalian cells which indicates different types of chromosomal aberrations, sperm head abnormalities and sister chromatid exchanges. The carcinogenic potentiality as well as its cell transformation ability has also been reported. Therefore, the present study was undertaken to determine the genotoxic potentiality of betel nut extract on in vivo S-180 tumour bearing mouse considering tumour cell proliferation rate and bone marrow toxicity. Moreover, our aim was to determine whether BNE alone can induce angiogenesis during solid tumour growth. Methods: Ascites form of S-180 cells was injected subcutaneously in the leg of normal mouse ventrally for induction of tumour. Tumour growth rate in response to BNE was studied at different time intervals by morphometric analysis of tumour size. Bone marrow toxicity was measured by chromosomal aberration analysis. Results: Analysis of tumour cell and bone marrow metaphases clearly points out that BNE can induce different types of chromosomal aberrations. The enlargement of the tumour during the course of treatment was also determined and correlated with the gradual decrease of dead cell and increase of living cell frequency of BNE treated S-180 ascitic tumour bearing mouse. Conclusion: The results indicated that betel nut extracts not only induce chromosomal abnormalities of mouse but also enhance solid tumour growth as well as angiogenesis.

INFLAMMATION INDUCED PANCREATIC DYSFUNCTION: A MOLECULAR APPROACH

Sreetama Choudhury^a#, Sudeshna Mukherjee^a#, Sayan Ghosh^b# and Sreya Chattopadhyay^a*

^a Department of Physiology, University of Calcutta, 92 APC Road, Kolkata 700 009. ^b S.N. Pradhan Centre for Neurosciences, University of Calcutta, 35 BC Road, Kolkata 700 019. *Corresponding author. ^# First three authors have equal credit.

Type 2 Diabetes mellitus (T2DM) represents a significant global health problem and it is estimated that this disease would become one of the world’s most prevalent causes of preventable mortality. Over the past decades it has been hypothesised that pathogenesis of T2DM is related to a state of preclinical inflammation. Activation of inflammatory cascade releases several cytokines (IL-6, TNF-α, IFN-γ, IL-1β, etc.) which contribute to initiating a state of impaired glucose resistance and a subsequent elevated risk of T2DM. To correlate the link between inflammation and diabetes we treated pancreatic cells with different doses of lipopolysaccharide in vitro to assess whether it leads to inflammatory damage to pancreatic cells. It was observed the LPS treatment on pancreatic cells decreased the number of viable cells in the pancreas in a dose dependent manner. LPS also down-regulated survival proteins (pAkt, Bcl2) while it upregulated the pro-apoptotic proteins like Bax and caspase-3. LPS also increased phosphorylation of IĸB thereby indicating the nuclear translocation of NFĸB in the pancreatic cells. Thus LPS causes stress in the pancreatic cells which triggers apoptotic and thus causes pancreatic cells death. The present study supports the hypothesis that there is a strong association between inflammation and T2DM. The establishment of the link between T2DM and inflammation would open up new vistas in combating the disease.

MOLECULAR ALTERATIONS OF SFRP1/2 IN UTERINE CERVICAL CARCINOMA OF INDIAN PATIENTS

Sudip Samadder^1#, Chandraditya Chakraborty¹, Nupur Mukherjee¹, Sankhadeep Dutta¹, Partha S. Basu¹, Ranajit Mondal¹, Jaydip Biswas¹, Susanta Roychoudhury² and Chinmay Kumar Panda^1*

¹Chittaranjan National Cancer Institute, 37 S. P. Mukherjee Road, Kolkata. ²Indian Institute of Chemical Biology, CSIR, 4 Raja S. C. Mullick Road, Kolkata, India

Cancer of uterine cervix (CaCx) is the second most frequent malignancy (18 % of total cancer) amongst female in India where the infection of human papillomavirus (HPV 16, 18 etc.) is considered as the most important epidemiological factors along with other cofactors like high parity, tobacco smoking, oral contraceptives etc. During progression of tumor small portion of the cells, cancer stem cells (CSCs), have self-renewal capacity and are resistant to chemotherapy/radiotherapy. Thus molecular analysis of the self-renewal pathways will help to understand the molecular pathogenesis of tumor development and to develop proper therapeutic intervention. Among the different self-renewal pathways, alteration of Wnt signaling pathway has been reported in different tumors. To understand the association of Wnt pathway in development of CaCx we have analyzed copy number variation (CNV) of SFRP1 and SFRP2, the extracellular Wnt signaling antagonists, in 114 primary cervical lesions of Indian patients using different microsatellite markers. Comparable frequency of CNV was seen in SFRP1 locus (23 %) and SFRP2 locus (18 %). Similar trend has also been seen in microsatellite size alteration frequencies in the loci. Co-alterations have also been observed in some samples. In a case–control study of (CA) repeat length polymorphism of rs11269473 in SFPR2 locus, it was evident that (CA)₂₀ and (CA)₂₁ repeats were suggested to be risk allele for the development of CaCx.

EFFECT OF BLEOMYCIN ON IN VIVO TUMOUR ANGIOGENESIS USING S-180 TUMOUR BEARING MOUSE

Sudipta Chowdhury, Srabantika Mallick, Rusa Basu Roy, Priyanka Ghosh, and Samarendra Nath Banerjee

Department of Zoology, Rammohan College, 102/1, Raja Rammohan Sarani, Kolkata- 700009, INDIA

samban2kcal@yahoo.com; samar_banerjee@rediffmail.com

Background: The majority of anti-neoplastic agents used in cancer treatments are ‘S-dependent’. In this respect, they differ from ionizing radiation whose lesions are not dependent upon S-phase for conversion to aberrations with the consequence that all stages of the cell cycle are vulnerable. Bleomycin is an important compound in some forms of cancer therapy because it has an action very similar to radiation. Such chemical is known as radiomimetic. Recent studies indicated that angiogenesis not only provide nutrients to the tumor cells but also help the tumor cells to metastasize to the different organs of the body. Thus, targeting angiogenesis is considered as the most important approach for cancer therapy. Therefore, the present study was undertaken to determine the anti-angiogenic effect and genotoxic potentiality of Bleomycin in vivo S-180 tumour bearing mouse considering tumour cell proliferation rate and bone marrow toxicity. Moreover, our aim was to determine whether Bleomycin alone can induce angiogenesis during solid tumour growth. Methods: Tumour growth rate in response to Bleomycin was studied at different time intervals by morphometric analysis of tumour size. Bone marrow toxicity was measured by chromosomal aberration analysis. Results: A steady decrease in tumour volume was noted after the administration of Bleomycin at therapeutic dose in S-180 ascitic tumour bearing mouse which was correlated with the gradual increase of dead cells and decrease of living cells frequency at 24 h and 48 h of treatment. Conclusion: In the present study, we have demonstrated that Bleomycin acts as an important anti-angiogenic agent that controls solid tumour growth through regulation of angiogenesis.

AN EXPLORATORY ANALYSIS OF THE INVOLVEMENT OF HOXA11-AS1, HOTAIR AND HOXD10 EXPRESSION IN HPV16 RELATED CERVICAL CANCER PATHOGENESIS

Sweta Sharma, Damayanti Das Ghosh* and Sharmila Sengupta

National Institute of Biomedical Genomics, Netaji Subhas Sanatorium, 2nd Floor, P.O.: N.S.S., Kalyani-741251, West Bengal, India. Email: ss4@nibmg.ac.in; *Present Address: Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700 032, West Bengal, India

Introduction: Long non-coding RNAs (lncRNAs) from the HOX cluster regulate expression of the HOX cluster genes which might influence cervical cancer (CaCx) pathogenesis. Objectives: To test the hypothesis that lncRNAs HOXA11-AS1 and HOTAIR, and HOXD10 (a gene regulated by HOTAIR) are deregulated in HPV16 positive CaCx cases with intact/disrupted E2 genes, as compared to HPV negative controls and this alteration is associated with HPV E7 protein. Methodologies: Expression analysis of noncoding RNAs was done by quantitative PCR (SYBR Green) on (i) 17 E2-intact and 12 E2-disrupted cases and 16 HPV negative controls for HOXA11-AS1, (ii) 9 E2-intact and 10 E2-disrupted cases and 16 HPV negative controls for HOTAIR and (iii) 5 E2-intact and 3 E2-disrupted cases and 5 HPV negative controls for HOXD10 with GAPDH as normalization control. Statistical significance of the fold change differences (2^−ΔΔC_T) were determined based on Mann–Whitney U test. Results: HOXA11-AS1 was upregulated among cases compared to HPV negative controls, which was more prominent among E2-intact cases (14.92-fold; p = 0.002) compared to E2-disrupted cases (13.45-fold; p = 0.009). HOTAIR was significantly downregulated among E2-intact cases (16.56-fold, p = 0.017) but not in E2-disrupted cases (13.93-fold; p = 0.16) compared to HPV negative controls. E2-intact cases harbouring episomal and concomitant HPV do not differ in E7 expression, and showed no difference in expression of both HOTAIR and HOXA11-AS1. However, E2-disrupted/integrated cases harboring high E7 expression, revealed significant downregulation of HOTAIR (59.7-fold; p = 0.026) in contrast to low E7 expressing cases, indicating an E7 dependent role, and no change in HOXA11-AS1 expression. However, HOXD10 showed a statistically non-significant downregulation in both E2-intact and E2-disrupted cases (12.13-fold; p = 0.2 and 96.34; p = 0.18 respectively) as compared to HPV negative controls but no association with E7 expression and viral load. Conclusion: The expression pattern of HOTAIR in CaCx pathogenesis appears to differ from that in other cancers and such deregulation influences HPV16 related CaCx pathogenesis in an E7 dependent manner.