Overexpression Of Survivin Research Articles

Abstract 4447: Targeting Hsp90 with small molecule inhibitors induces the overexpression of the anti-apoptotic molecule, survivin, in human cancer cells

Abstract Survivin is a dual functioning protein. It inhibits the apoptosis of cells by inhibiting caspases, and also promotes cell growth by stabilizing microtubules during mitosis. Furthermore, over-expression of survivin has been shown to induce drug resistance to various chemotherapeutic compounds. Since the molecular chaperone Hsp90 binds and stabilizes survivin, it is widely believed that down-regulation of survivin is one of the important therapeutic functions of Hsp90 inhibitors such as the phase III clinically trialed compound 17-AAG. However, Hsp90 interferes with a number of molecules that positively regulate the intracellular level of survivin, raising the question that clinical use of Hsp90 inhibitors may indirectly induce survivin expression and subsequently promote cancer progression under certain circumstances. Here, we demonstrated that Hsp90 inhibitors, geldanamycin and 17-AAG, induced the over-expression of survivin in three different human cancer cell lines as shown by western blotting. Increased survivin mRNA transcripts were observed in 17-AAG and geldanamycin-treated HT-29 and HONE-1 cancer cells. Interestingly, real-time PCR and translation inhibition studies revealed that survivin was over-expressed partially through the up-regulation of protein translation instead of gene transcription in A549 cancer cells. In addition, 17-AAG-treated A549, HONE-1 and HT-29 cells showed reduced amount of 26S proteasome and decreased proteasomal activity while inhibition of 26S proteasome further increased the amount of survivin protein in cells. At the functional level, down-regulation of survivin by siRNA further increased the drug sensitivity to 17-AAG in the tested cancer cell lines. In conclusion, we showed for the first time that targeting Hsp90 induced the over-expression of survivin through various mechanisms in cancer cells. These data indicate that down-regulation of survivin is not a definite therapeutic function of Hsp90 inhibitors. The current study suggests that dual inhibition of Hsp90 and survivin may be warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4447.

Abstract LB-224: Leptin-induced breast cancer cell migration requires upregulation of antiapoptotic protein, survivin

Abstract Background: Leptin, an adipocytokine secreted by adipose tissue, is responsible for many biological functions including appetite regulation, energy homeostasis, bone formation, and more recently, carcinogenesis. Levels of circulating leptin are typically elevated in obese states and have been associated with increased tumor proliferation, angiogenesis, and increased metastatic behavior. Therefore, obesity has been clearly associated with increased risk of cancer and metastasis. Breast cancer remains as the second leading cause of cancer-related deaths to American women. Many epidemiological studies have shown that obese women are more at risk for developing breast cancer compared to non-obese women. Therefore, it is imperative to understand the contribution of leptin to breast cancer development and progression. Methods and Results: In this study, we used MCF-7 and MDA-MB-231 breast cancer cell lines to examine the relationship between leptin and survivin, a dual-function protein. Survivin has become an interesting target in cancer research because it is highly expressed in cancerous tissues while being nearly undetectable in normal tissues. We observed that leptin increases mRNA and protein expression of survivin in a time-dependent manner. Earlier studies from our lab demonstrated that leptin increases breast cancer cell migration and invasion and underlying mechanisms involved a bidirectional crosstalk between leptin and IGF1 leading to transactivation of EGFR. Examining the underlying mechanisms, we found that blocking EGFR activation decreased leptin-induced survivin expression indicating the involvement of EGFR. To examine the role of survivin in leptin-induced migration of breast cancer cells, we transfected both cell lines with wild-type or p-silencer-survivin and subjected the cells to scratch and electric cell-substrate impedance sensing (ECIS) migration assays. We found that survivin overexpression increased migration potential of breast cancer cells compared to untransfected cells. In addition, leptin treatment further enhanced migration of breast cancer cells with survivin overexpression. Inhibition of survivin inhibited migration of breast cancer cells alone and even in the presence of leptin. These studies showed the importance of survivin in leptin-induced migration of breast cancer cells. For therapeutic purposes, we treated the breast cancer cells with lovastatin, commonly used as an HMG-CoA reducatase inhibitor. Lovastatin has been previously shown to inhibit survivin expression. We treated cells with lovastatin in a dose-dependent manner and observed a complete inhibition of survivin expression. Combination treatment with leptin did not restore survivin protein expression. Importantly, lovastatin treatment efficiently inhibited migration of breast cancer cells even in the presence of leptin. Conclusion: In conclusion, we have established a novel role for survivin in leptin-induced migration of breast cancer cells. Targeting survivin using lovastatin may provide new therapeutic targets for the treatment of metastatic breast carcinogenesis in obese patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-224.

Targeting Hsp90 with small molecule inhibitors induces the over-expression of the anti-apoptotic molecule, survivin, in human A549, HONE-1 and HT-29 cancer cells

BackgroundSurvivin is a dual functioning protein. It inhibits the apoptosis of cancer cells by inhibiting caspases, and also promotes cancer cell growth by stabilizing microtubules during mitosis. Since the molecular chaperone Hsp90 binds and stabilizes survivin, it is widely believed that down-regulation of survivin is one of the important therapeutic functions of Hsp90 inhibitors such as the phase III clinically trialed compound 17-AAG. However, Hsp90 interferes with a number of molecules that up-regulate the intracellular level of survivin, raising the question that clinical use of Hsp90 inhibitors may indirectly induce survivin expression and subsequently enhance cancer anti-drug responses. The purpose of this study is to determine whether targeting Hsp90 can alter survivin expression differently in different cancer cell lines and to explore possible mechanisms that cause the alteration in survivin expression.ResultsHere, we demonstrated that Hsp90 inhibitors, geldanamycin and 17-AAG, induced the over-expression of survivin in three different human cancer cell lines as shown by Western blotting. Increased survivin mRNA transcripts were observed in 17-AAG and geldanamycin-treated HT-29 and HONE-1 cancer cells. Interestingly, real-time PCR and translation inhibition studies revealed that survivin was over-expressed partially through the up-regulation of protein translation instead of gene transcription in A549 cancer cells. In addition, 17-AAG-treated A549, HONE-1 and HT-29 cells showed reduced proteasomal activity while inhibition of 26S proteasome activity further increased the amount of survivin protein in cells. At the functional level, down-regulation of survivin by siRNA further increased the drug sensitivity to 17-AAG in the tested cancer cell lines.ConclusionsWe showed for the first time that down-regulation of survivin is not a definite therapeutic function of Hsp90 inhibitors. Instead, targeting Hsp90 with small molecule inhibitors will induce the over-expression of survivin in certain cancer cell lines and subsequently enhances the ability of cell survival in drug-treated situations. The current study suggests that dual inhibition of Hsp90 and survivin may be warranted.

Abstract 2547: Caveolin-1 inhibits survivin and increases sensitivity to paclitaxel in breast cancer cells

Abstract Breast cancer is the second leading cause of death due to cancer in women and approximately 194,280 new cases of breast cancer were diagnosed in 2009. The chemotherapeutic drugs known as taxanes disrupt the microtubule function and arrest cell cycle. Paclitaxel (Taxol) is a taxane that is commonly used to treat patients with breast cancer as well as other types of cancer. However, resistance to paclitaxel remains a hindrance to successful treatment. Previously, we have shown that signaling associated with caveolin-1 (CAV1), a structural protein, is an essential determinant of paclitaxel sensitivity in MCF-7 breast cancer cells. CAV1 is part of this family of proteins that has multiple functions in different cancers but it has been proposed to function as a tumor suppressor in breast cancer. Here we show that CAV1 may promote cell death in response to paclitaxel by inhibiting the function of a pro-survival protein called survivin. Survivin is encoded by the BIRC5 gene and is part of the inhibitors of apoptosis (IAP) family. This protein has numerous functions including inhibition of apoptosis and regulation of the cell cycle. Evidence shows that survivin inhibits cell apoptosis by preventing the activation of caspases, which are proteases associated with cell-mediated apoptosis. Up-regulation of survivin is associated with cancer and usually indicates a poor prognosis. We show that while over-expression of CAV1 down-regulates survivin, over-expression of survivin down-regulates CAV1 in MCF-7 cells. Moreover, MCF-7 cells that over-express survivin show decreased inhibition of cell proliferation in response to 10 nM paclitaxel compared to MCF-7 cells expressing the empty vector (EV, control). Thus, our work highlights the role of CAV1 expression in breast cancer cells as an essential determinant of paclitaxel sensitivity. Findings from this research can help researchers and clinicians to evaluate whether CAV1 expression in breast tumors can help predict its responsiveness to taxanes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2547.

Expression of survivin and human telomerase reverse transcriptase in extramammary Paget's disease

Extramammary Paget's disease (EMPD) is a rare neoplasm of apocrine gland-bearing skin. It is known that over-expression of survivin and human telomerase reverse transcriptase (hTERT) correlates with malignancies. We investigated the expression of hTERT and survivin by Paget's cells and their role in the tissue invasion and recurrence of EMPD. Forty-two patients were enrolled into the study. Expression of survivin and hTERT were analyzed by immunohistochemistry and in situ hybridization techniques. The variables including the expression level of survivin and hTERT, gender, age, lesion location, invasion level and number of surgeries were statistically analyzed using Fisher's exact test. Survivin was positively stained in 18 of 22 cases (81.8%), and hTERT in 18 of 29 cases (62.1%). Significantly higher level of survivin expression was detected in patients with multiple surgeries than those with single one (p = 0.0458). Expression of hTERT was significantly higher in the patients with micro-invasive and invasive lesions than those with non-invasive lesions (p = 0.0478). Over-expression of survivin and hTERT correlated strongly with recurrence and local invasion of EMPD lesions. EMPD has male gender predominance in Oriental population.

Survivin Is a Potential Mediator of Prostate Cancer Metastasis

We examined whether Survivin expression is associated with an increased risk of metastasis in prostate cancer. A total of 205 patients with T1 (23%) and T2 (77%) prostate cancer were treated with conventional external beam radiation therapy from 1991 to 1993 at the Massachusetts General Hospital. Of the patients, 62 had adequate and suitable-stained tumor material for Survivin analysis. Median follow-up was 102 months (range, 5-127 months). Distant failure was determined on the basis of clinical criteria. In preclinical studies, replication-deficient adenovirus encoding phosphorylation-defective Survivin Thr34→Ala dominant-negative mutant pAd-S(T34A) or short hairpin RNA (shRNA) was used to inhibit Survivin in prostate cancer models, and the cell motility, morphology, and metastasis were investigated. Our correlative data on men with early-stage (T1/T2) prostate cancers treated at Massachusetts General Hospital by definitive radiotherapy indicated that overexpression of Survivin (positive staining in ≥10% cells) was associated with a significantly increased risk for the subsequent development of distant metastasis (p = 0.016) in the univariate analysis. In the multivariate analysis, overexpression of Survivin remained an independent predictor of distant metastasis (p = 0.008). The inhibition of Survivin dramatically inhibited invasiveness of prostate cancer cells in the in vitro invasion assay and spontaneous metastasis in the Dunning prostate cancer in vivo model. Furthermore, attenuation of Survivin resulted in changes in the microtubule cytoskeleton, loss of cellular polarity, and loss of motility. This study suggests that Survivin may be a potentially important prognostic marker and promising therapeutic target in metastatic prostate cancer.

Survivin overexpression in HCC and liver cirrhosis differentially correlates with p-STAT3 and E-cadherin.

Survivin, a member of the family of inhibitor of apoptosis proteins, functions as a key regulator of apoptosis and cell proliferation. Overexpression of survivin has been implicated in several human cancers, including human hepatocellular carcinoma (HCC). Although several factors have been shown in vitro to upregulate survivin expression in cancer cells, the in vivo regulators of survivin in human hepato-carcinogenesis are largely unknown. We studied by immunohistochemistry the protein expression of survivin in relation to cyclin D1, phosphorylated signal transducer and activator of transcription 3 (p-STAT3), beta-catenin, E-cadherin and phosphorylated-Akt (p-Akt) in 69 cases of HCC and adjacent liver cirrhosis. Survivin was expressed in 63/69 (91.3%) cases of HCC and in 40/47 (85.1%) cases of liver cirrhosis. Survivin localization in HCC was exclusively nuclear, while intense cytoplasmic and low nuclear expression of survivin was observed in cases of cirrhosis. Survivin expression in HCC correlated significantly with low grade tumors, expression of cyclin D1 and p-STAT3. Expression of survivin in liver cirrhosis correlated with downregulation of E-cadherin expression. There was no significant correlation of survivin with beta-catenin or p-Akt in HCC or liver cirrhosis. In conclusion, we showed an association of nuclear survivin with well differentiated HCC, as well as with the expression of the cell cycle regulator cyclin D1. Activation of STAT3 and loss of E-cadherin but not beta-catenin or Akt pathways seem to be implicated in survivin upregulation in HCC and liver cirrhosis.

Combined analysis of survivin autoantibody and carcinoembryonic antigen biomarkers for improved detection of colorectal cancer

Survivin is a member of the family of inhibitor of apoptosis proteins that is overexpressed in several human tumors. Previous studies have found that overexpression of survivin in cancer cells induces an antibody response. We compared 232 serum samples from colorectal cancer (CRC) patients and 365 samples from healthy volunteers using an in vitro enzyme-linked immunosorbent assay to evaluate the survivin autoantibody response in patients. The sensitivity of the anti-survivin response from patients with CRC was 56.9%, and the specificity was 64.1%. When a cut-off value of 5.0 ng/mL was chosen for carcinoembryonic antigen (CEA) in these same serum samples, the values for sensitivity and specificity were 40.9% and 86.6%, respectively. Combined detection using survivin autoantibodies and CEA produced better sensitivity (51.3%) and specificity (89.9%) compared to the sensitivity of CEA (40.9%) and the specificities of the individual markers (64.1% and 86.6%, respectively). The area under a receiver operating characteristic curve was 0.617 for survivin autoantibodies, 0.630 for CEA and 0.694 for both markers together. A positive association between autoantibodies against survivin and preoperative CEA concentrations in sera of patients with CRCs was established. Our results suggest that analysis of both parameters would assist in screening patients with CRC.

Induction of apoptosis of non-small cell lung cancer by a methylated oligonucleotide targeting survivin gene

Survivin overexpression is closely linked to lung oncogenesis. Silencing of survivin gene by molecular antagonists has shown promise as novel anticancer strategies. DNA methylation is a critical epigenetic modification that silences gene transcription. In this study, we used a new methylated oligonucleotide, which mediates sequence-specific DNA methylation in cell, as a strategic alternative to survivin-targeting treatment, and investigated its efficacy in suppressing survivin expression and the consequent apoptotic induction potential in NCI-H460 cells. SurKex1, a methylated sense oligonucleotide, was shown to reduce specifically survivin mRNA expression and induce cell apoptosis. In addition, it has been supposed that the methylated oligonucleotide exerts its effect of gene silencing through the activation of DNA methyltransferase (DNMT1), but the exact mechanism is still unknown. Our data suggest for the first time that the SurKex1 exerts its down-regulatory effects on survivin expression through the activation of DNMT1.

Detection of Autoantibodies against Survivin in Sera from Cancer Dogs

Survivin overexpression has been reported in relation to tumor malignancy, suggesting that it is an unfavorable prognostic marker, and antibody responses to this protein have been confirmed in human cancer patients. In this study, we investigated antibody responses to survivin in canine cancer cases, and examined the prevalence of such responses by enzyme-linked immunosorbent assay (ELISA) using recombinant canine survivin protein as the antigen. The cut-off value for positivity in the anti-survivin ELISA was 0.35, as determined using the mean absorbance +2 S.D. of samples from healthy dogs. Sera from 16 of 59 (27.1%) cancer and 3 of 25 (12%) non-cancer disease dogs were positive on ELISA. The highest positivity rates (>50%) among the cancer cases were seen in dogs with mammary tumor, squamous cell carcinoma and melanoma.

The neem limonoids azadirachtin and nimbolide induce cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells

Limonoids from the neem tree (Azadirachta indica) have attracted considerable research attention in recent years owing to their potent antioxidant and anti-proliferative effects. The present study was designed to investigate the cellular and molecular mechanisms by which azadirachtin and nimbolide exert cytotoxic effects in the human cervical cancer (HeLa) cell line. Both azadirachtin and nimbolide significantly suppressed the viability of HeLa cells in a dose-dependent manner by inducing cell cycle arrest at G0/G1 phase accompanied by p53-dependent p21 accumulation and down-regulation of the cell cycle regulatory proteins cyclin B, cyclin D1 and PCNA. Characteristic changes in nuclear morphology, presence of a subdiploid peak and annexin-V staining pointed to apoptosis as the mode of cell death. Increased generation of reactive oxygen species with decline in the mitochondrial transmembrane potential and release of cytochrome c confirmed that the neem limonoids transduced the apoptotic signal via the mitochondrial pathway. Altered expression of the Bcl-2 family of proteins, inhibition of NF-κB activation and over-expression of caspases and survivin provide compelling evidence that azadirachtin and nimbolide induce a shift of balance toward a pro-apoptotic phenotype. Antioxidants such as azadirachtin and nimbolide that can simultaneously arrest the cell cycle and target multiple molecules involved in mitochondrial apoptosis offer immense potential as anti-cancer therapeutic drugs.

Survivin multifaceted activity in head and neck carcinoma: Current evidence and future therapeutic challenges

Conclusions:Survivin expression should be studied as a potential hallmark of higher risk oral, oropharyngeal and laryngeal squamous cell carcinomas (SCCs) to develop loco-regional recurrences. These outcomes could have a significant impact on both the treatment modalities and the intensity of post-treatment follow-up. Further investigation is necessary before considering elective neck dissection in patients with laryngeal SCC with high survivin expression. Objectives: Functioning simultaneously at cell division and apoptosis inhibition, survivin, a member of the inhibitor of apoptosis proteins family, plays a pivotal role in determining cell survival. Significant over-expression of survivin has been demonstrated in most human malignancies and correlated with more aggressive forms. This review focuses on the attempts to translate survivin biologic properties toward both a diagnostic/prognostic tool and a novel therapeutic target in head and neck SCC (HNSCC). Materials and methods: An exhaustive review of literature was performed to investigate available evidence about survivin expression, biological role and therapeutic potential in HNSCC. Results: Multiple evidence indicates that, in HNSCC cell lines, survivin inhibition by gene therapy and by small molecule inhibitors significantly increases the anti-tumour activity of several cytotoxic and other targeted therapies.

Abstract A99: EZN-3042 (E), a novel survivin messenger ribonucleic acid (mRNA) antagonist, administered as a single agent (SA) or with docetaxel (D): results of a Phase 1, first-in-human, pharmacokinetic (PK), combination after single agent (CASA) dose-escalation study

Abstract Background: Survivin, a member of the inhibitors of apoptosis protein gene family, is a compelling molecular target in cancer research. In addition to modulating apoptosis, survivin acts as an essential regulator of mitosis. Overexpression of survivin in many cancers is correlated with advanced disease and reduced survival. EZN-3042 is a potent locked nucleic acid antisense oligonucleotide that down-modulates survivin mRNA and consequently protein in vitro (IC50 <5 nM). Preclinically, EZN-3042 enhances apoptotic and antitumor effects of taxanes. Methods: This study was designed to determine the safety and tolerability, maximum tolerated/recommended dose, PK, pharmacodynamics (PD), and preliminary evidence of antitumor activity of EZN-3042 as a SA and in combination (E+D). Using a novel dose-escalation design, patients with advanced tumors first received SA EZN-3042 as a 2-h IV infusion qwk (2 doses Wk 1). After disease progression following SA therapy, patients could receive EZN-3042 (qwk) + docetaxel (75 mg/m2 q3wk). Dose escalation (3+3) for SA and for the combination of E+D was independently based on toxicities observed during Cycle 1 of SA or E+D, respectively (CASA modified Fibonacci dose escalation). Results: Ten patients (8 men; median age = 64 y [46–70 y]) received SA doses of 2.5 mg/kg (n=3), 5 mg/kg (n=3), and 8 mg/kg (n=4). Tumor types included colorectal (CRC) (n=5), prostate (PC) (n=4), and unknown primary (n=1) cancers. Nine patients had received multiple prior chemotherapies (median number of prior regimens = 2; range = 1–6). Five patients (all men) received E (2.5 mg/kg) + D. Dose-limiting toxicity (DLT) was observed in 1 patient in Cohort 3 who had received 8 mg/kg of SA EZN-3042 (DLT = Grade 3 increased aspartate aminotransferase [AST]) and in 1 patient who had received E+D (DLT = Grade 3 neutropenic fever). The most common drug-related adverse events (AEs) (in >2 patients) for SA EZN-3042 were fatigue (70%); neutropenia (40%); and alopecia, anorexia, AST increase, diarrhea, leukopenia, and nausea (30% each). The most common drug-related AEs (in >2 patients) for E+D were neutropenia and fatigue (80% each); and alopecia, anorexia, and leukopenia (60% each). Overall, most AEs were Grade 1 or 2. All 10 patients who received SA treatment provided data for PK analysis. A qualitative assessment of the PK data indicated that the EZN-3042 concentration appears to increase in a dose-proportional manner at the doses evaluated. Preliminary assessment of antitumor activity indicates that the best response for SA EZN-3042 was stable disease in 1 patient with CRC (8 mg/kg, 6 weeks); the best response for E+D was a confirmed partial response for E+D in 1 patient with PC (21 wks). Conclusions: EZN-3042 was well tolerated in previously treated patients with advanced tumors. Dose escalation is ongoing; updated results will be presented at the meeting. The CASA dose-escalation design is an innovative approach that allows one to follow the safety and efficacy of SA and combination therapy in the same patient. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A99.

Role of Survivin in Drug Resistant B-Cell Acute Lymphoblastic Leukemia.

Abstract 10Survivin/BIRC5, an inhibitor of apoptosis (IAP) protein,is critical for the survival and proliferation of cancerous cells and has become the target of an increasing number of preclinical novel therapies against primarily solid tumors. Survivin is expressed in AML and ALL cells and has been implicted in leukemia relapse. Here we test the hypothesis that Survivin is critical to chemotherapeutic drug resistance in ALL. To test this hypothesis, we initially compared survivin mRNA levels in both patient-derived B-ALL cells and B-ALL cell lines versus normal B-cells in various stages of development using real-time PCR. ALL cells encompassing various cytogenetic subgroups showed significantly greater mRNA levels of Survivin versus normal B cell precursors ranging from 2 to greater than 20-fold versus controls. To determine whether Survivin contributes to drug resistance, we lentivirally overexpressed Survivin in primary B-ALL and B-ALL cell lines. Overexpression of Survivin attenuated the effect of Vincristine on ALL cell proliferation when compared to ALL cells transduced with empty vector controls. Vincristine IC50 value determination for the control was 0.1 nM, whereas Survivin overexpression resulted an IC50 value of 10 nM (p<0.01). Similarly, significantly higher concentrations of L-Asparaginase (>0.01 U/l vs ab >0.1; p<0.05) and Dexamethasone (0.1 nmol/l vs >10 nmol/l; p<0.013) were needed to achieve drug cytotoxicity. We conclude that Survivin overexpression decreases sensitivity of ALL cells to standard chemotherapy. Conversely, lentivirally-mediated survivin shRNA knockdown of the same cell type significantly sensitized a greater percentage of the leukemia to 10nM vincristine versus non-silencing controls. Survivin shRNA targeting resulted in a 30.2% greater affected population than controls, assayed using MTT (p<0.003). Overexpression or knockdown of Survivin was confirmed by Western Blot and real-time PCR. Of note, expression levels of Survivin is heterogeneous within the same ALL sample: Transduction of patient-derived ALL cells with a Survivin-GFP reporter construct revealed that a small subpopulation of Survivin-GFP high ALL exhibiting approximately 7-fold higher levels of endogenous Survivin than bulk leukemia cells. Consistent with cell cycle-dependent regulation of Survivin, Survivin-GFP high ALL were enriched in G2/M phase of the cell cycle. In addition, endogenous variations of Survivin protein levels were in the same range as changes in Survivin protein levels in overexpression and knockdown experiments, which indicates that these experiments tested physiologically occurring levels of Survivin. Next, we determined whether Survivin contributes to self-renewal of drug resistant ALL cells. Survivin overexpression of primary samples yielded three times more colonies, than controls in a primary plating CFU assay (p<0.019). Taken together, our data suggests that targeting Survivin in B-ALL may sensitize to chemotherapy and highlights the role of Survivin in drug resistant B-ALL as a target for novel therapies. Disclosures:No relevant conflicts of interest to declare.

Gene Expression Profiling Reveals Activation of Multiple Oncogenic Pathways and Over-Expression of Survivin in the Pathogenesis of Extranodal Nasal-Type NK/T Cell Lymphoma.

Abstract 3931Poster Board III-867Extranodal nasal-type Natural Killer/T-cell lymphoma (NKTCL) is a distinct clinicopathologic entity most commonly affecting Asians and Central and South Americans, and characterized by a clonal proliferation of NK or T cells with a cytotoxic phenotype. There is a strong association with Epstein-Barr Virus. The tumor is aggressive with patient usually surviving short duration even with chemotherapy. We performed the first comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type Natural Killer/T-cell lymphoma (NKTCL) using formalin-fixed paraffin embedded (FFPE) tissue (n=25) and NK cell lines (n=5) and compared the results to the GEP of normal NK cells using the Illumina DASL whole genome array, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTCL and to identify potential therapeutic targets. Quantitative-PCR validation of the GEP findings revealed over-expression of candidate genes BIRC5 (survivin), EZH2 and STMN1 in NK cell lines compared to normal NK cells, consistent with the GEP data. We then extracted a list of genes that are differentially expressed between NKTCL and normal NK cells and tissue controls. We than subjected this list of genes to pathway and network analysis using Metacore. This revealed a significant enrichment for cell cycle related genes and pathways. Furthermore, the network analysis results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTCL characterized by activation of Myc and nuclear factor kappa B (NF-KB), and deregulation of p53. This was further corroborated using immunohistochemistry on tissue microarray of NKTCL (n=33, including all cases with GEP performed). We observed a significant percentage of NKTCL showing overexpression for c-Myc (45.4%), p53 (87.9%) and NF-KB p50 (67.7%) on immunohistochemistry. Notably, overexpression of survivin was observed in 97% of the cases. Based on our findings, we propose a model of NKTCL pathogenesis where deregulation of p53 together with activation of MYC and NF-KB, possibly driven by EBV LMP-1, result in the cumulative upregulation of survivin. When KHYG and NKYS cell lines were treated with a compound IDR E804 which inhibited survivin, there is significant inhibition of cell growth as assess by MTS assay and induction of apoptosis as measured using Annexin V staining by flow cytometry. This suggests that compounds inhibiting survivin may be a potentially useful novel therapeutic approach in NKTCL. Disclosures:No relevant conflicts of interest to declare.

Involvement of mTOR and survivin inhibition in tamoxifen-induced apoptosis in human hepatoblastoma cell line HepG2

Patients with advanced hepatocellular carcinoma (HCC) have shown to benefit from tamoxifen treatment. The mechanisms of tamoxifen effects in HCC, however, are not yet clearly understood. The PI3K/Akt/mTOR signal pathway is involved in cell proliferation, tumorigenesis, and apoptosis. Over-expression of survivin has played an important role in leading to antiapoptosis. The current study investigated changes in mTOR pathway and survivin expression in hepatocarcinoma cell line HepG2 treated with tamoxifen. We detected apoptosis of hepatocarcinoma cells by flow cytometry assay. Survivin transcription level and p70S6k was demonstrated by PCR, dual-luciferase reporter assay and western blot analysis respectively. Our results are showed as follows: tamoxifen leads to apoptosis of the cells, and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6 kinase. Twenty micromoles tamoxifen treatment signifcantly depresses transcription of survivin mRNA. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduce survivin protein level but did not affect survivin transcription, which indicated that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells. Our results suggest that tamoxifen down-regulate survivin expression in HepG2 cells is mediated by transcriptional and posttranscriptional level via PI3K/Akt/mTOR pathway to induce apoptosis.

Perturbation of survivin expression affects chromosome alignment and spindle checkpoint in mouse oocyte meiotic maturation

Survivin is a member of inhibitors of apoptosis proteins (IAPs), which have multiple regulatory functions in mitosis, but its roles in meiosis remain unknown. Here, we report its expression, localization and functions in mouse oocyte meiosis. Survivin displayed maximal expression levels in GV stages, and then gradually decreased from Pro-MI to MII stages. Immunofluorescent staining showed that survivin was restricted to the germinal vesicle, associated with centromeres from pro-metaphase I to metaphase I stages, distributed at the midzone and midbody of anaphase and telophase spindles, and located to centromeres at metaphase II stages. Depletion of survivin by antibody injection and morpholino injection resulted in severe chromosome misalignment, precocious polar body extrusion, and larger-than-normal polar bodies. Overexpression of survivin resulted in severe chromosome misalignment and prometaphase I or metaphase I arrest in a large proportion of oocytes. Our data suggest that survivin is required for chromosome alignment and that it may regulate spindle checkpoint activity during mouse oocyte meiosis.

Increased expression of inhibitor of apoptosis proteins, survivin and XIAP, in non-small cell lung carcinoma

Members of the inhibitor of apoptosis protein (IAP) family, survivin and X-chromosome-linked IAP (XIAP), contribute to apoptosis resistance of cancer cells, and an increase in their expression may elevate the apoptotic threshold of malignant tumours during their growth and progression. In the present study, we investigated the expression status of survivin and its interactants hepatitis B X-interacting protein (HBXIP) and XIAP in non-small cell lung carcinoma (NSCLC) cell lines and NSCLC tumours and matched lungs from surgically treated patients in relation to their clinicopathological data. The expression of survivin, HBXIP and XIAP mRNAs was quantitated by real-time RT-PCR. The expression of survivin and XIAP proteins was analysed by Western blotting and ELISA. Survivin mRNA and protein levels were highly upregulated in NSCLC cells and tissues as compared to the lungs. In fact, the levels of survivin mRNA and protein in the tumours were more than 10-fold higher in 96 (64%) and 72 (82%) of the 150 and 88 examined NSCLC patients, respectively. The expression of survivin mRNA was higher in squamous cell lung carcinomas than in lung adenocarcinomas (LACs; P=0.003) and in less-differentiated tumours than in well-differentiated ones (P=0.007). The level of survivin protein was higher in stage IB and stage II+III tumours (P=0.049 and P=0.044), than in stage IA tumours. The BIRC5 promoter polymorphism at nucleotide -31 did not influence the expression of survivin mRNA and protein in NSCLC cells and tumours. HBXIP mRNA was abundantly expressed in NSCLC cell lines and NSCLC tumours and lungs, while its level was comparable in the tumours and lungs. The expression of XIAP mRNA in NSCLC cell lines and NSCLC tumours and lungs was not significantly different. However, the expression of XIAP protein was higher in NSCLC tumours, particularly in LACs, as compared to the lungs (P=0.017 and P=0.004). In conclusion, the overexpression of survivin in the majority of NSCLCs together with the abundant or upregulated expression of HBXIP and XIAP suggest that tumours are endowed with resistance against a variety of apoptosis-inducing conditions.

Deletion of caveolin-1 protects hyperoxia-induced apoptosis via survivin-mediated pathways

Hyperoxia-induced lung injury is an established model that mimics human acute respiratory distress syndrome. Cell death is a prominent feature in lungs following prolonged hyperoxia. Caveolae are omega-shaped invaginations of the plasma membrane. Caveolin-1 (cav-1), a 22-kDa transmembrane scaffolding protein, is the principal structural component of caveolae. We have recently shown that deletion of cav-1 (cav-1-/-) protected against hyperoxia-induced cell death and lung injury both in vitro and in vivo; however, the mechanisms remain unclear. Survivin, a member of the inhibitor of apoptosis protein family, inhibits apoptosis in tumor cells. Although emerging evidence suggests that survivin is involved in wound healing, especially in vascular injuries, its role in hyperoxia-induced lung injury has not been investigated. Our current data demonstrated that hyperoxia induced apoptosis via suppressing survivin expression. Deletion of cav-1 abolished this suppression and subsequently protected against hyperoxia-induced apoptosis. Using "gain" and "loss" of function assays, we determined that survivin protected lung cells from hyperoxia-induced apoptosis via the inhibition of apoptosis executor caspase-3. Overexpression of survivin by deletion of cav-1 was regulated by Egr-1. Egr-1 functioned as a negative regulator of survivin expression. Deletion of cav-1 upregulated survivin via decreased Egr-1 binding of the survivin promoter region. Together, this study illustrates the effect of hyperoxia on survivin expression and the role of survivin in hyperoxia-induced apoptosis. We also demonstrate that deletion of cav-1 protects hyperoxia-induced apoptosis via modulation of survivin expression.

Adenovirus-mediated transfer of siRNA against survivin enhances the radiosensitivity of human non-small cell lung cancer cells

Expression of survivin has been reported to be correlated with shorter survival in patients with non-small cell lung cancer (NSCLC), and overexpression of survivin may lead to radioresistance in various human cancers. In this study, we inhibited survivin expression by using an adenoviral vector (AdsiSurvivin)-mediated RNA interference to elucidate the combined effect of survivin-targeting gene therapy and radiotherapy on the NSCLC cells. Our data showed that AdsiSurvivin exerted survivin gene silencing, induced apoptosis, and significantly attenuated the growth potential in NSCLC cells within 72 h after infection. The combined treatment modalities with AdsiSurvivin infection and radiation were significantly more potent on cell-growth inhibition than monotherapy. In H1650, H460, A549, and H1975 human NSCLC cells, the survival ratios of AdsiSurvivin-treated groups at multiplicity of infection of 25 and 50 were significantly lower than those of control groups at varying radiation dose (0-8 Gy; three-way analysis of variance, P<0.05). The cytotoxicity of combined AdsiSurvivin infection and irradiation increased in a dose-dependent manner in both the virus and the irradiation treatment. Knockdown of the survivin gene expression seems to be a promising treatment strategy for NSCLC. Our data warrant the need for further effort to develop survivin-targeted radiosensitizer for lung cancer treatment.