AACR Annual Meeting 2008

Abstract

The 2008 American Association for Cancer Research (AACR) Annual Meeting was held in San Diego, CA, April 12–16, 2008 (http://www.aacr.org/home/scientists/meetings--workshops/annual-meeting-2008.aspx). More than 17,000 scientists from 60 countries participated in this meeting that was organized by AACR, the oldest and largest organization in the world focused on cancer research. The scientific presentations included more than 6,000 abstracts and 500 invited talks on new and significant discoveries in basic, clinical, and translational cancer research. Autophagy, as pertaining to tumorigenesis and response to anticancer therapies, was undoubtedly a “hot topic” in this meeting. An educational session, a forum, a minisymposium and several other talks dispersed in different sessions had a strong focus on autophagy. All autophagy-related presentations were very well attended and stimulated lively discussions, clearly indicating that the scientific community is greatly interested in this rapidly-progressing area of research.

In the educational session entitled “Cell Death and Autophagy” and organized by Wafik El-Deiry (University of Pennsylvania), Eileen White (Rutgers University) gave an overview on autophagy and emphasized the essential role that autophagy plays in tumor cell survival and resistance to metabolic stress. She concluded by pointing out the need to further investigate the role of autophagy in tumor suppression and treatment responsiveness, and to rigorously test the validity of autophagy modulation for cancer treatment, analogously to what is already being done with apoptosis.

The forum on “The Role of Autophagy in Cancer” was moderated by John Cleveland (The Scripps Research Institute of Florida), who introduced the paradox of autophagy as both a tumor suppressive mechanism and a process that is essential for tumor development and maintenance. He also reviewed recent preclinical studies indicating that autophagy may be exploited for therapeutic benefit in cancer treatment, as autophagy inhibition enhances therapy-induced apoptosis in a myc-driven mouse lymphoma model¹ and prevents myc-induced mouse lymphomagenesis.² The combination of the deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and the autophagy inhibitor chloroquine was also recently shown to be effective against imatinib mesylate-resistant chronic myelogenous leukemia (CML).³

Eileen White and Craig Thompson (University of Pennsylvania) were the two invited forum speakers. Both emphasized the critical function of autophagy in tumor cell survival under stress conditions and in maintenance of protein and organelle homeostasis. Eileen White presented work supporting the hypothesis that damage mitigation and suppression of chronic cell death by autophagy limits tumorigenesis. Tumor suppression by autophagy is mediated by at least two mechanisms:^4,5 a non-cell-autonomous mechanism involving limitation of necrotic tumor cell death and associated inflammation,⁶ and a cell-autonomous mechanism involving prevention of genome damage and instability.^7,8 Exactly how failure to maintain metabolism through autophagy leads to DNA damage and tumorigenesis has not yet been determined. Possibilities under investigation include insufficient ATP production and damaged protein and/or organelle accumulation under defective autophagy conditions. p62/SQSTM1 is a signaling adaptor/scaffold protein implicated in the formation of intracellular ubiquitin-related protein aggregates in hepatocytes and neurons in association with autophagy defects.⁹ Eileen White presented evidence that p62 is induced in tumor cells under metabolic stress and that high p62 levels and p62-related protein aggregates selectively persist in autophagy-defective cells during recovery. The role of autophagy in metabolic stress management was investigated by a proteomic approach, which revealed that autophagy suppresses demand for protein folding during oxygen-and glucose-deprivation. Evidence that autophagy defects promote protein aggregation and endoplasmic reticulum (ER) stress in cancer cells in vitro and in tumors in vivo was also presented. Furthermore, p62 and ER chaperone accumulation are seen in tissues and tumors from beclin1^+/− mice and in a subset of human tumors, indicating that maintenance of protein quality control through autophagy may function as a tumor suppression mechanism. Eileen White briefly discussed the topic of autophagy modulation for cancer therapy and proposed that autophagy inhibitors may be useful in treatment of autophagy-dependent tumors by inhibiting tumor cell survival during metabolic stress induced by anticancer agents, whereas autophagy stimulators may be applicable in cancer chemoprevention by maintaining protein quality control and enhancing abnormal protein clearance.

Craig Thompson reviewed the role of autophagy in cell survival upon growth factor deprivation and bioenergetic stress. Mammalian cells depend on growth factor signaling to maintain adequate nutrient uptake and sustain survival. Upon growth factor withdrawal, the major glucose transporter GLUT1 is downregulated and cells undergo apoptosis, unless this mechanism of programmed cell death is inactivated. In the latter case, cells activate autophagy and remain viable for several weeks by maintaining ATP production from catabolism of intracellular substrates.¹⁰ The possibility that autophagy-dependent cancer cells may have enhanced metastatic potential was discussed, as these cells appear to be “hyper-sensitive” to chemotactic signals, such as stromal cell-derived factor (SDF)-1. Recent studies from the Thompson lab focus on autophagy as a therapeutic target in cancer and show that enhancement of therapy-induced tumor cell death by chloroquine depends on its ability to inhibit autophagy-mediated cell survival,¹ suggesting that combinatorial treatment with autophagy inhibitors may augment the efficacy of autophagy-inducing anticancer drugs. Craig Thompson also discussed the possible role of autophagy in at least two distinct steps of hematopoietic/erythroid cell development, namely elimination of the nucleus and loss of mitochondria. The essential autophagy regulator atg1, which is a kinase involved in autophagosome formation, appears to play a role in autophagic cargo selectivity, as atg1^−/− mice exhibit mitochondria-retaining red blood cells.

The minisymposium on “Autophagy, metabolic stress, and tumorigenesis” was chaired by Shengkan (Victor) Jin (Robert Wood Johnson Medical School) and Junying Yuan (Harvard Medical School), and included six autophagy-related presentations. Robin Mathew from the White lab presented studies exploring the hypothesis that defective autophagy leads to accumulation of toxic protein aggregates and generation of reactive oxygen species (ROS), thus resulting in oxidative stress, DNA damage and enhanced tumorigenesis. Under metabolic stress conditions, autophagy-defective beclin1^+/− and atg5^−/− immortalized baby mouse kidney (iBMK) cells accumulate the oxidative stress and protein aggregation marker p62/SQSTM1. Also, apoptosis-defective beclin1^+/− iBMK cells exhibit higher induction and persistence of ROS during metabolic stress and recovery, respectively. Proteomic analysis of the cellular response of iBMK cells to metabolic stress reveals enhanced upregulation of glucose-related-protein (GRP) chaperones, metabolic enzymes and mitochondrial proteins in autophagy-defective cancer cells, indicating that autophagy plays an essential role in protein quality control and preservation of cellular fitness, which may in turn constitute a novel tumor suppression mechanism. On a similar note, in the minisymposium on “Programmed Cell Death in Tumorigenesis and Therapy” I reported on the role of autophagy in supporting mammary cell survival during stress and I presented a proteomic study on the response of mammary tumor cells to metabolic stress and the impact of autophagy on this process.

Ayesha Alvero from the Mor lab (Yale University) provided evidence for caspase- and autophagy-independent programmed cell death induced by the novel compound NV128 in otherwise chemoresistant ovarian cancer cells. Cell death is characterized by DNA fragmentation and results from activation of an intracellular pathway involving downregulation of AKT, mTOR, S6 kinase and PKCα; mitochondrial translocation of Beclin 1 leading to Bcl-2 inhibition; mitochondrial depolarization; and nuclear translocation of EndoG. Participation of the essential autophagy regulator Beclin 1 in this process, which is not inhibited by the autophagy inhibitor 3-methyladenine (3MA), suggests that Beclin 1 may have additional functional roles to autophagosome formation in cancer cells.

Aluvia Escalante from the Landowski lab (University of Arizona) presented studies investigating the role of autophagy in multiple myeloma cell survival upon treatment with the proteasome inhibitor bortezomib in the presence and absence of Ca²⁺ channel blockers. Bortezomib induces ER stress, mitochondrial calcium flux, cytochrome C release and apoptosis in multiple myeloma cells. Bortezomib also induces autophagy, likely in response to ER stress induction. Calcium deregulation promotes the autophagic response and enhances cancer cell survival, indicating that autophagy may be a defense mechanism in the ER stress response initiated by bortezomib in multiple myeloma cells, and thus may constitute a drug resistance mechanism regulated by mitochondrial Ca²⁺ signaling and potentially amenable to pharmacological inhibition. Similar findings were presented by Manuela Milani from the Bottini (Breast Unit, Cremona, Italy) and Harris labs (Molecular Oncology Laboratories, Oxford, UK) for bortezomib-treated breast cancer cells in the minisymposium focused on “Molecular Mechanisms of Drug Resistance”. In this case, treatment of MCF7 breast cancer cells with bortezomib results in proliferation arrest associated with the unfolded protein response (UPR), eIF2α phosphorylation in a PERK-independent manner, and LC3/autophagy induction through ATF4. Downregulation of ATF4 and LC3 sensitizes cancer cells to bortezomib treatment, indicating that autophagy inhibition may represent a novel approach to overcoming resistance of breast tumors to bortezomib.

On a different note, Bulent Ozpolat from the Lopez-Berestein lab (UT M.D. Anderson Cancer Center) examined the role of autophagy as a caspase-independent form of cell death in breast cancer cells. Bcl-2 downregulation (alone or in combination with doxorubicin) in MCF7 breast cancer cells induces non-apoptotic cell death associated with autophagy induction, as demonstrated by autophagosome formation, LC3 membrane-translocation, and upregulation of ATG5 and Beclin 1. ATG5 knockdown inhibits autophagy and cell death induced by Bcl-2 downregulation, indicating that targeted silencing of Bcl-2 induces ATG5-mediated cell death in breast cancer cells and may be used as a therapeutic strategy for breast tumors expressing Bcl-2.

The role of autophagy as a cell survival mechanism in response to starvation has been well established. However, more than one pathway is likely involved in maintaining cell viability under starvation. James Phang (National Cancer Institute) presented work on the role of extracellular matrix-derived proline as a stress responder to nutrient deprivation, in a process possibly overlapping with autophagy. In addition to downregulating mTOR-mediated phosphorylation of S6 kinase, glucose deprivation upregulates proline oxidase (POX) that can possibly be involved in ATP production and maintenance of bioenergetics by donating proline-derived electrons to the electron transport chain.

Maureen Murphy (Fox Chase Cancer Center) presented studies exploring the relationship between the Alternative Reading Frame (ARF) product of the INK4a/ARF locus, autophagy and tumorigenesis. ARF is overexpressed in a subset of human tumors, despite its well-recognized p53-dependent and -independent growth suppressive functions. An explanation as to why human tumors may benefit from retaining ARF expression comes from the findings that ARF is markedly upregulated in myc-driven lymphomas and during nutrient deprivation, and results in autophagy induction, which may enable tumor cell survival under stress.

Autophagy is negatively regulated by the Class I PI3K/AKT/mTOR pathway, whereas mTOR inhibition induces autophagy and growth arrest in traditional monolayer culture conditions. In the minisymposium focused on “Novel Approaches to Drug Discovery”, Amy Howes from the Vuori lab (Burnham Institute for Medical Research) presented data indicating that treatment of human cancer cells cultured as three-dimensional (3-D) spheroids with rapamycin and low dose irradiation induces autophagy and cell death with apoptotic and/or necrotic features, particularly in the center of the treated spheroids.

In the minisymposium on “Novel Mechanisms of Drug Action”, Margaret Park from the Dent lab (VCU Massey Cancer Center) presented evidence that treatment with OSU-03012 (OSU), a derivative of the COX2 inhibitor celecoxib lacking COX2 inhibitory activity, results in PERK-dependent Beclin 1 and ATG5 upregulation and subsequent autophagosome formation, followed by pro-caspase 4 cleavage and caspase 4-dependent cathepsin B activation, likely contributing to cell death. Thus, OSU promotes both cell survival and cell death processes. This finding constitutes a recurrent theme in cancer therapy, as autophagy is emerging as a survival, and possibly drug resistance, mechanism activated in response to the antiproliferative and/or cytotoxic effects of many anticancer agents.

Finally, autophagy was a topic of interest in the Seventh Annual Dorothy P. Landon-AACR Prize for Translational Cancer Research Lecture by John Mendelsohn (UT M.D. Anderson Cancer Center) and in the Presidential Address by William Hait (Ortho Biotech Oncology Research and Development). John Mendelsohn gave an overview of the development of epidermal growth factor receptor (EGFR) inhibitors for cancer therapy and summarized recent studies indicating that EGFR is essential for cancer cell survival independent of its tyrosine kinase activity, as downregulation of EGFR by siRNA induces autophagy under regular culture conditions and results in cell death, indicating that autophagy may be a therapeutic target for potentiating the antitumor effects of EGFR inhibition. William Hait, the 2007–2008 AACR president, presented his personal scientific journey in translational cancer research, including work on the eukaryotic elongation factor 2 kinase (eEF-2K), which contributes to the starvation-mediated decrease in protein synthesis, and plays a role in induction of autophagy and maintenance of cell viability during nutrient depletion. Downregulation of eEF-2K by siRNA blunts starvation-induced autophagy and compromises cancer cell survival, a finding currently under NCI-sponsored clinical investigation.

Thus, interest in autophagy is clearly increasing within the cancer research community due to the role of this pathway in tumor suppression and its common deregulation in human cancer, but many questions have not been answered yet. We need to better understand the role of autophagy in the stress response in tumors, and we need to rigorously explore the possibility of autophagy modulation to augment cancer prevention and therapy. There is no doubt that next year’s annual AACR meeting will include even more interesting studies and presentations focused on autophagy.