Survivin and pancreatic cancer

Abstract

Pancreatic cancer is estimated to be the fourth most common cancer in men and fifth in women in the world and has poor prognosis. In recent years, more and more effort has been put on the relationship between pancreatic cancer and apoptosis. As a newly discovered inhibitor of apoptosis, survivin has drawn more attention. Strong evidence has shown that survivin is expressed in pancreatic cancer cells on frozen sections. Survivin increases in the development of pancreatic ductal adenocarcinoma and its expression can be a marker in evaluating the prognosis of pancreatic cancer patients. Survivin itself may be a new target in the treatment of pancreatic cancer and a survivin DNA vaccine could generate specific antitumor effects in pancreatic carcinoma models.

INTRODUCTION

Pancreatic cancer is estimated to be the fourth most common cancer in men and fifth in women in the world. Despite the development of surgical resection, radiotherapy and chemotherapy, the prognosis is poor since 5-year survival after surgery in patients with resectable disease is approximately 15% to 20%[1]. The effort for new targets in the treatment of pancreatic cancer has drawn more and more attention recently.

Inhibition of apoptosis is very important in the development of cancer. Inhibitor of apoptosis can not only accelerate the development of tumor but also promote the resistance to therapy. Survivin is a new member of inhibitor of apoptosis (IAP) family. It is a 16.5 kDa protein and highly conserved. It contains only one baculovirus IAP repeat and lacks a carboxyl-terminal RING finger, which makes survivin different from other IAP proteins. Survivin protein functions to inhibit caspase activation, thereby leading to negative regulation of programmed cell death (apoptosis). It can also partially inhibit the cell death induced by Fas and Bax[2,3] and now is considered as a mitotic regulator[4,5]. As a newly-discovered IAP, survivin is found to be expressed in many carcinomas, including human cancers of lung, colon, pancreas, prostate and breast[2,3]. Strong expression of survivin can also be found in some apoptosis-regulated fetal tissues, including the stem cell layer of stratified epithelia, endocrine pancreas, and thymic medulla. It is also expressed in human fetal lung, liver, heart, kidney, and gastrointestinal tract, which may contribute to tissue homeostasis and differentiation[6]. However, no survivin is detected in normal terminally differentiated adult tissues[2]. The levels of survivin are low in resting endothelial cells and could be up-regulated on activation to proliferate. Vascular endothelial-cadherin (VE-cadherin) expression is believed to be one of the factors to maintain low levels of survivin in endothelial cells[7]. More studies have focused on the relationship between pancreatic cancer and survivin.

EXPRESSION OF SURVIVIN IN PANCREATIC CANCER

Satoh et al[8] found survivin expressed in 76.9% cases of pancreatic duct cell adenocarcinoma (PDC) and 56.3% intraductal papillary-mucinous tumor (IPMT) lesions. Malignant tumors expressed survivin more frequently than benign tumors. In PDC, the increased expression of survivin was accompanied with the reduction of apoptotic index in tumor cells. Sarela et al[9] also reported that survivin was expressed in majority of pancreatic adenocarcinomas tested and correlated with both cellular proliferation and apoptosis. Yang et al[10] investigated three pancreatic cancer cell lines and found high expression of survivin in these tumor cells.

Jinfeng et al[11] investigated twenty-two lesions from patients with IPMT, including 12 benign ones (IPMT adenoma) and 10 malignant ones (4 IPMT Carcinoma in Situ [CIS] and 6 invasive IPMT lesions), and found the expression of survivin and p53 increased in the development from IPMT adenoma to IPMT CIS with the reduction of apoptosis in tumor cells. The results suggest that survivin and p53 may promote the progress from benign lesions to malignant ones.

Studies indicate that survivin may become a future marker for pancreatic cancer cells in frozen sections. Yang et al[12], synthesized molecular beacons (MBs, short hairpin oligonucleotide probes which can bind to specific oligonucleotide sequences and show fluorescent signals) targeting transcripts of mutant K-ras and survivin, and examined the specificity for detecting the two genes in pancreatic cancer cells using a fluorescence imaging-based technique. Survivin MBs were found binding to survivin gene and a bright fluorescent signal was produced specifically in pancreatic cancer cells. In frozen sections of pancreatic cancer tissues, survivin MBs were also found to have a high specificity in identifying cancer cells. The results provide pathologists a new choice for detecting pancreatic cancer cells in frozen sections, and may be used in clinical practice in the future. However, Jhala et al[13] investigated the biomarkers in diagnosing pancreatic carcinoma using fine needle aspirates and found that survivin expression was not a good marker for separating reactive ductal cells from pancreatic adenocarcinoma.

Increased expression of survivin is also reported in the development of pancreatic ductal adenocarcinoma (PDA). Bhanot et al[14] used laser capture microdissection, real-time polymerase chain reaction and immunohistochemistry to measure transcriptional levels of survivin and its protein expression in normal pancreatic ducts, pancreatic intraepithelial neoplasia (PanIN) and PDA. A steady increase in mRNA and protein expression of survivin was found from low-grade lesions (PanINs-1) to high-grade lesions (PanINs-2 and 3) and further to PDA. The results indicate that survivin may promote the changing process and could be a signal for malignant lesions.

SURVIVIN EXPRESSION AS A MARKER FOR EVALUATING PROGNOSIS

Specimens from pancreatic cancer patients who accepted surgery with or without postoperative radiation therapy (PORT) were assessed and the relationship between the expression of survivin and the prognosis was evaluated. Patients with positive survivin expression had shorter survival time than those who had no survivin expression, whereas PORT had no impact on survival time in both survivin positive patients and survivin negative patients. The research indicates that survivin may become a prognostic marker for pancreatic cancer[15].

The study of Grabowski et al[16] also showed that nuclear survivin expression was a potent prognostic marker for shorter survival time in gastroenteropancreatic neuroendocrine tumor disease. Determination of nuclear survivin expression may be used to individualize therapeutic strategies. Survivin has been shown to reside in mitochondria, nucleus and cytosol of tumors[17,18]. Tonini et al[19] made the first study on the prognostic relevance of survivin expression in pancreatic cancer in relation with its intracellular distribution. They investigated nuclear and cytoplasmic expression of survivin in 67 patients with pancreatic cancer and reported that patients with high nuclear survivin expression had a longer survival time, while patients with high cytoplasmic survivin expression had a shorter survival time. The median survival time for patients with positive nuclear expression was 27 mo while for patients with negative nuclear expression it was only 10 mo. However, the median survival for patients with positive cytoplasmic expression was 10 months compared with 25 months for patients with negative cytoplasmic expression. In other malignant tumors, such as colorectal cancer, cytoplasmic survivin overexpression is also associated with a poor prognosis, while nuclear survivin overexpression is associated with a better one. The mechanisms for the intracellular distribution of survivin in human cancer cells are still unclear; however, determination of the different expression of survivin may make a new marker for evaluating the prognosis of patients with pancreatic cancer[20].

Theodoropoulos et al[21], investigated survivin gene polymorphisms and the characteristics of pancreatic cancer. The genotypes of the survivin promoter are GG, CC and CG. The frequency of the 31G/C polymorphism was investigated in 80 patients with pancreatic cancer and 160 controls. A significant relationship was found between survivin C carrier and the advanced T stage accompanied with the presence of lymph node metastasis, indicating that the status of survivin C carriage was related to more aggressive features of the tumor.

SIGNIFICANCE OF SURVIVIN EXPRESSION IN THE TREATMENT OF PANCREATIC CANCER

The expression of survivin may be associated with the route of metastasis and the sensitivity to chemotherapy for patients with pancreatic cancer. Lee et al[22] investigated 49 cases of pancreatic cancer and found that 93.9% of them were positive for survivin expression. In patients with positive expression of survivin, perineural invasion was more common; while in patients with negative expression of survivin, venous invasion seemed more common. These findings suggest that survivin may be associated with perineural or venous invasion, which indicate the metastatic route. However, the reason for the relationship between survivin expression and invasion mode is not clear. Among these patients, 14 received epirubicin, cisplatin and 5-FU combination chemotherapy. Patients with lower expression of survivin were more sensitive to the chemotherapeutic protocol. The results suggest that survivin may be used as a potential predictive marker in chemotherapy.

Expression of survivin is also a radioresistance factor in patients with pancreatic cancer. Asanuma et al[23] found an inverse relationship between survivin mRNA expression and radiosensitivity in 5 pancreatic cancer cell lines using a quantitative RT-PCR, indicating that survivin may act as a radioresistance factor in pancreatic cancer cells. They also found that the survivin mRNA increased significantly after X-ray irradiation, implying that survivin was an inducible radioresistance factor in pancreatic cancer cells. Further results of this study suggest that survivin expression directly down-regulates radiosensitivity[24].

Studies also reveal that down-regulation of survivin diminishes radioresistance of pancreatic cancer cells. Kami et al[25] evaluated the effect of short interfering RNA (siRNA) directly against survivin expression in radioresistant cells (AsPC-1). The activity of the survivin promoter and the expression of survivin mRNA were examined in 3 pancreatic cancer cell lines. Various levels of survivin mRNA and the transcriptional activity of the survivin promoter were found in pancreatic cancer cells, and both of them correlated with the radiosensitivity of tumor cells. On the other hand, radiation could increase the activity of the survivin promoter and mRNA expression in these cells. However, siRNA treatment markedly decreased the expression of survivin mRNA in AsPC-1 cells, and diminished the radioresistance of pancreatic cancer cells. These results indicate that combined therapy of a survivin inhibitor and radiation may be effective in the treatment of pancreatic cancer.

Guan et al[26] also proved that down regulation of survivin expression by small interfering RNA induced apoptosis in pancreatic cancer cells (cell line PC-2) and enhanced its radioactivity. The sequence-specific siRNA markedly decreased survivin mRNA and protein, which resulted in apoptosis in 7.03% of cells treated with siRNA. However, apoptosis was found in 14.58% of cells treated with siRNA combined with radiation, and only 1.66% of cells were found to be apoptotic in the radiation group. These studies give us new hope in the treatment of pancreatic cancer.

In spite of more and more new aggressive therapies, resistance of many tumors to current therapeutic protocols is still a formidable problem. Thus more and more attempts to improve the effects of treatment for cancer patients now depend on methods targeting the resistance of tumor cells[27]. Survivin may be a new target in the treatment of pancreatic cancer. Fulda et al[28] used survivin antisense oligonucleotides down-regulating the expression of survivin, and revealed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis was sensitized in many tumor cells. The cells they used included neuroblastoma, medulloblastoma, glioblastoma, melanoma, pancreatic carcinoma, prostate carcinoma, and breast carcinoma cells. They also found that G1 arrest was associated with less expression of survivin and sensitization for TRAIL-induced apoptosis.

Liang et al[29] tried survivin as a new target for pancreatic cancer gene therapy. A 10’-23’ anti-survivin mRNA DNAzyme was designed, synthesized and delivered into human pancreatic carcinoma cell PANC-1 through liposomes, and the influence on the growth of PANC-1 cells was evaluated. The results showed that with the destruction of the mRNA substrate of survivin by DNAzyme, apoptosis of PANC-1 was increased and cell growth was inhibited. The designed DNAzyme against survivin mRNA is a promising candidate for gene therapy of human pancreatic carcinoma. Liu et al[30] also proved that human pancreatic cancer cell transfected with a siRNA plasmid expression vector against survivin showed decreased cell growth, spontaneous apoptosis, and a specific G0/G1 arrest accompanied with the reduction of survivin mRNA and protein. In addition, the chemosensitivity of pancreatic cancer cells to gemcitabine was increased markedly after suppressing the expression of survivin. Guan et al[31] proved that knockdown of survivin expression by siRNA could suppress proliferation of human pancreatic cancer cell PC-2. The research of Tsuji et al[32] also suggested that survivin-specific siRNA deserved further investigation as a new approach for the treatment of cancer.

Shen et al[33] performed experiments inhibiting the growth of cancer cells by silencing survivin not only in vitro but also in vivo. In their study, they designed and constructed a short hairpin RNAs (shRNAs) specific to survivin, cloned it into a plasmid vector, and transfected the recombinant plasmids into a human pancreatic cancer cell line Patu8988. The proliferation rates of the cancer cells were reduced markedly when transfected with the survivin-shRNA plasmids. When Patu8988 cells with survivin-shRNA were inoculated into BALB/c nude mice, the growth of the tumor was lower and the size of the tumor was smaller compared with the control group. The results showed that vector-based survivin-shRNAs could inhibit the expression of survivin in human pancreatic cancer Patu8988 cells and ultimately inhibit cell proliferation both in vitro and in vivo. Thus, knockdown expression of survivin may be a future way to treat pancreatic cancer.

Encouraging results have also been obtained in humans. Wobser et al[34] used survivin-based peptide vaccinations consisting of a modified HLA-A2 restricted survivin epitope on a 72-year old patient who suffered from pancreatic cancer with liver metastasis. The patient got partial remission of liver metastasis under vaccination with survivin peptides and later a complete remission with a duration of 8 mo. Although the disease relapsed 6 mo after vaccination stopped, it was the first case of a successful use of survivin-based vaccination, which threw light on the gloomy prognosis of advanced pancreatic cancer.

It has been reported that survivin DNA vaccine generates specific antitumor effects in pancreatic carcinoma in mouse models. Human or mouse survivin DNA was given to a murine pancreatic cancer model and the effect of the vaccination was evaluated. Slower tumor growth and longer lifetime were found in mice vaccinated with survivin DNA, no matter whether it was from human or mouse source. Greater infiltration of lymphocytes was found in tumors of the immunized mice. The research showed that survivin DNA vaccination could generate specific antitumor effects with increased lymphocyte infiltration at the tumor sites, and both xenogeneic survivin and congeneric ones had the same immune response[35].

Now more and more attention is being paid to the relationship between apoptosis and pancreatic cancer, including its development and therapy. As a novel IAP member, survivin inhibits caspase activity thereby negatively regulating apoptosis, which draws more attention in the study of cancer. Further studies of survivin should provide a new choice for the marker in diagnosis and the target treatment of the disease.