Survivin Expression In Tumors Research Articles

Intensity of Survivin Expression Correlates with Clinical and Biological Markers of Aggressive R/R DLBCL

Introduction: The SPiReL phase II clinical trial evaluated combination immunotherapy with an immunogenic vaccine formulation to the tumor antigen survivin comprised of maveropepimut-S (MVP-S), pembrolizumab and cyclophosphamide in survivin-expressing relapsed or refractory (R/R) diffuse large B cell lymphoma (DLBCL). The efficacy of this survivin-directed T cell educating therapy may be influenced by the survivin antigen burden in the DLBCL. To further explore this potential relationship, we evaluated the heterogeneity of tumor survivin expression in the SPiReL study cohort and its associations with established clinical-pathological DLBLC variables. Methods: Survivin immunohistochemistry (IHC) was performed at NeoGenomics Laboratories (Aliso Viejo, California, USA) on formalin fixed paraffin embedded (FFPE) tumor slides from diagnostic biopsies obtained at trial screening. Inclusion criteria stipulated >10% of tumor cells expressing survivin by IHC. Semi-quantitative survivin expression metrics, as determined by pathologist assessment, included: total percentage of survivin-positive cells; intensity of survivin staining scored as 0 = None, 1+ = Weak, 2+ = Moderate, 3+ = Strong; and H-score, a linear weighted average (% positive cells x staining intensity level). Radiologic tumor burden was measured at up to 4 time points during the trial by computed tomography (CT). Other pathology (e.g. cell of origin) and laboratory biomarkers (e.g. lactate dehydrogenase, LDH) were extracted from clinical results. Results: The mean and median percentage of survivin-positive tumor cells for all enrolled participants (n= 25) was 91% and 99%, respectively. At the per participant level, the range of survivin expression was 50-100% of the cellular content. The heterogeneity of survivin expression intensity among participants is shown in Figure 1. All participants had some proportion of biopsied cells expressing strong (3+) intensity survivin (range: 5-100% of cells, mean: 30% while only 52% of tumors contained non-survivin expressing (0) cells, ranging from 0% to 50% (mean: 9%) Greater proportions of cells that expressed moderate (2+) and/or strong (3+) survivin significantly correlated with clinical and biological markers of aggressive DLBCL. Non-GCB tumours (11/25) showed greater H-scores compared to GCB tumours (212 vs 167, p = 0.0287). Non-GCB tumours contained greater proportions of strong intensity (3+) survivin cells compared to GCB tumours (44% vs 25%, p= 0.045). In addition, participants with LDH above the upper limit of normal at screening (52%, 13/25) had a greater mean proportion of cells expressing moderate and strong intensity survivin (proportion of 2+ and 3+ combined) (72% vs 54%, p = 0.0447) and a lower mean proportion of weak survivin intensity (1+) cells (p = 0.0374) compared to those with normal LDH. A relationship between total tumor area at baseline and survivin intensity levels was explored. Higher proportions of cells expressing weak intensity (1+) survivin were associated with smaller total tumour area at baseline (p=0.0201). In contrast, higher combined proportions of moderate (2+) and high (3+) intensity survivin was associated with larger total tumour area at baseline (p=0.0285). The significant association with the 2+/3+ intensity of survivin with LDH, cell of origin and tumour burden may suggest a mechanistic threshold in DLBCL pathobiology that drives aggressive clinical behavior. Discussion: Here we demonstrate substantial heterogeneity of survivin protein expression in baseline tumour biopsies of R/R DLBCL patients. Given our findings, it may be of clinical and pathobiological significance to assess both the proportion of survivin-expressing cells as well as the intensity of survivin expression. As the known functions of survivin include protection from apoptosis and cell-cycle progression, the associations demonstrated here support the relevance of survivin and in particular high intensity (2+, 3+) survivin as a biomarker linked with aggressive R/R DLBCL features. These results further support the targeting of survivin pathways in interventional trials and development of validated clinical laboratory survivin assays. Further analysis by multiplex immunofluorescence is in progress to further explore how 2+/3+ vs. 0 host immune cells may also influence survivin immunogenicity and clinical response post-MVP-S immunotherapy.

Survivin Expression in Metastatic and Nonmetastatic Oral Squamous Cell Carcinoma: A Comparative Study

Aim: Survivin is a multifunctional protein chiefly involved in apoptosis and cell cycle regulation. Increased expression of survivin in tumors and fetal tissue determines its antiapoptotic activity. The aim of the study is to identify the immunoexpression of survivin in metastatic and nonmetastatic oral squamous cell carcinoma (OSCC) and also to evaluate and compare the expression of survivin in metastatic and nonmetastatic OSCC of buccal mucosa. Materials and Methods: In total, 40 histopathologically proven cases of OSCC, including 20 metastatic and 20 nonmetastatic cases, are selected. Among the 20 metastatic and nonmetastatic cases, 10 well-differentiated and 10 moderately differentiated squamous cell carcinoma cases were included and were subjected to immunohistochemical staining for survivin expression. The results were analyzed by SPSS version 11.5 using chi-square test. Results: The expression of survivin in metastatic and nonmetastatic tumors is 15%–70% and 15%–60%, respectively. When comparing the cases of moderately differentiated squamous cell carcinoma in metastatic and nonmetastatic tumors, 70% cases show moderate staining intensity. Conclusion: The survivin expression was comparatively high in metastatic OSCC. Also based on the aforementioned results, survivin expression was high in increasing grades of OSCC.

Survivin in Insulin-Like Growth Factor-Induced Resistance to Lapatinib in Head and Neck Squamous Carcinoma Cells

Epidermal growth factor receptor (EGFR) inhibitors have limited efficacy in head and neck squamous cell carcinoma (HNSCC) due to various resistance mechanisms, such as activation of the insulin-like growth factor-1 receptor (IGF1R), which initiates pro-survival signaling. Survivin, a member of the inhibitor of apoptosis proteins family, is expressed at relatively high levels in malignant tissues and plays a role in cell division. Expression of survivin in tumors has been shown to correlate with poor prognosis due to chemotherapy resistance and anti-apoptotic behavior. We previously demonstrated that activation of the IGF1R reduces sensitivity to EGFR-tyrosine kinase inhibitors (TKIs) via reduced apoptosis suggesting a role of survivin in this process. This study evaluates the role of survivin in IGF1R-mediated lapatinib resistance. Using HNSCC cell lines FaDu and SCC25, survivin expression increased and lapatinib sensitivity decreased with IGF1R activation. Further, these effects were reversed by the survivin inhibitor YM-155. Conversely, survivin expression and lapatinib sensitivity were unchanged with IGF1R activation in UNC10 cells. YM-155 enhanced the inhibitory effect of lapatinib on UNC10 cells, regardless of activation of the IGF1R. These results demonstrate that enhanced survivin expression correlates with IGF1R-mediated lapatinib resistance in HNSCC cells and suggest that regulation of survivin expression may be a key mechanistic element in IGF1R-based therapeutic resistance. Combinatorial treatment with survivin antagonists and EGFR-TKIs warrants further investigation.

ZIC1 acts a tumor suppressor in breast cancer by targeting survivin.

In this study, we aimed to identify the tumor suppressive roles of zinc finger of the cerebellum 1 (ZIC1) in patients with malignant breast neoplasms and to examine the association between ZIC1 and survivin expression. For this purpose, 140 invasive breast cancer specimens, 1,075 RNA breast cancer samples from The Cancer Genome Atlas (TCGA), 6 human breast cancer cell lines and MCF-10A normal breast epithelial cells were selected in order to compare the expression level of ZIC1 with that of survivin via immunohistochemistry and western blot analysis. Subsequently, the MDA-MB-231 and SK-BR3 cells with a lower ZIC1 expression were transfected with rLV-Zic1-PGK-Puro lentivirus or rLV-ZsGreen-PGK-Puro lentivirus in order to observe any alterations in cell proliferation and apoptosis through MTT assay, colony formation assay, mitochondrial membrane potential assay and flow cytometric analysis, and to analyze the modulation of molecular mechanisms by western blot analysis. In addition, xenograft mouse models were constructed to explore the role of ZIC1 in the growth of implanted tumors. The results revealed that ZIC1 negatively correlated with survivin in tumors and cells, and a higher ZIC1 RNA expression indicated a better overall survival in the 1,075 TCGA RNA breast cancer samples. In vitro, the overexpression of ZIC1 inhibited cell proliferation, reduced mitochondrial membrane potential and promoted the apoptosis of the MDA-MB-231 and SK-BR3 breast cancer cells by inactivating the Akt/mTOR/P70S6K pathway, suppressing survivin expression, modulating the cell cycle, releasing cytochrome c (Cyto-c) into the cytosol and activating caspase proteins. In vivo, an elevated ZIC1 expression suppressed the growth of implanted tumors and downregulated survivin expression in tumors. On the whole, the findings of this study demonstrate that ZIC1 plays a tumor suppressive role in breast cancer, by targeting surviving, significantly downregulating its expression.

Survivin, a novel target of the Hedgehog/GLI signaling pathway in human tumor cells.

Survivin, an important antiapoptotic protein, is expressed in tumors, whereas in normal tissues the expression of this protein is extremely low, defining a role for survivin as a cancer gene. Survivin exhibits multifunctional activity in tumor cells. However, why survivin expression is sharply and invariably restricted to tumor tissue remains unclear. Here, we identified 11 putative consensus binding sites for GLI transcription factors in the survivin promoter and characterized the promoter activity. Inhibitors of the Hedgehog/GLI pathway, cyclopamine and GANT61, decreased the promoter activity in reporter assays. ΔNGLI2 (which lacks the repressor domain) was the most potent vector in activating the survivin promoter–reporter. Moreover, GANT61, a GLI1/2 inhibitor, repressed endogenous survivin protein and mRNA expression in most cells across a large panel of tumor cell lines. Chromatin immunoprecipitation showed GLI2 binding to the survivin promoter. The ectopic GLI2-evoked expression of endogenous survivin was observed in normal human fibroblasts. GANT61 decreased survivin level in nude mice tumors, mimicking the activity of GANT61 in cultured cells. The immunohistochemistry and double immunofluorescence of human tumors revealed a correlation between the tissue regions showing high GLI2 and survivin positivity. Thus, these results demonstrated that survivin is a classical transcriptional target of GLI2, a Hedgehog pathway signaling effector. This potentially reflects the high expression of survivin in human tumor cells. As the Hedgehog pathway is upregulated in virtually all types of cancer cells, these findings substantially contribute to the explanation of uniform survivin expression in tumors as a potential target for the development of a more effective treatment of cancers through the inhibition of GLI2 to restrain survivin activity.

Insights into the Regulation of Survivin Expression in Tumors

Survivin, an anti-apoptotic protein was found to be expressed in tumors, whereas in normal tissues the expression of this protein was shown to be absent or extremely low. Survivin exhibits multifunctional activity in tumor cells. Survivin is the smallest member of the inhibitor of apoptosis protein family and was demonstrated to have key roles in regulating cell division and inhibiting apoptosis. The cancer protein survivin was shown to be associated with tumor cell progression, invasiveness, resistance to therapeutics and poor prognosis. Its level in tumors is associated with deregulation of several oncogenic pathways. In this review, a delineation of survivin expression and progress in understanding the survivin transcriptional regulation with the emphasis of augmented apoptosis in cancer and its link to the Hedgehog pathway and cancer stem cells is discussed.

Correlation of local and systemic expression of survivin with histopathological parameters of cutaneous melanoma.

Survivin is a multifunctional protein abundantly expressed in tumors of various types, including melanoma. There are still sparse data regarding relationship of melanoma cell survivin expression with accepted histopathological characteristics as well as serum concentration. The aim of this study was to investigate the association of local tumor survivin expression (primary tumor and metastatic lesions) and serum concentration with clinical and histopathological parameters in melanoma patients. The level of survivin expression was determined immunocytochemically in tumor tissue and with ELISA test in the serum of 84 melanoma patients diagnosed from 2009 to 2013 at the Institute for Pathology and Forensic Medicine and Institute for Medical Research at Military Medical Academy, Belgrade, Serbia. The intensity of survivin expression was significantly higher in the patients whose tumor had ulceration, higher mitotic index, higher Clark and Breslow stage, that made vascular invasion or spread through lymphatic vessels in primary tumor, and was significantly higher in the patients with metastatic disease. Survivin expression and the number of survivin positive cells in metastatic lesions were significantly associated with the duration of disease free interval (DFI). The patients with high expression score had almost double shorter DFI comparing to those with weak local survivin expression and a small number of survivin+cells (9 ± 7 vs 19 ± 13 months, respectively). The degree of tumor infiltrating lymphocytes presence in tumor tissue was significantly associated with serum survivin concentration, with lowest average level detected in samples of patients with the highest degree of infiltration. Serum survivin concentrations were highest in samples of melanoma patients with IA American Joint Commission on Cancer (AJCC) clinical stage, pT1a histological stage, patients whose tumors were still in horizontal growth phase, without signs of lympho-hematological disease spreading, with the highest number of mitoses and the smallest Clark index. Survivin expression in tumor tissue and its serum concetration significantly correlate with clinical and histopathological parameters. Serum levels could be important in initial follow-up as indicators of those patients that would have aggressive local tumor growth and spreading. Survivin determination in tumor tissue is of great significance in estimation of DFI.

Survivin-specific CD4+ T cells are decreased in patients with survivin-positive myeloma.

BackgroundSurvivin is a small protein inhibitor of apoptosis and a tumor associated antigen. Survivin expression in multiple myeloma is associated with poor prognosis, disease progression, and drug resistance. The CD4+ response against survivin remains uncharacterized.MethodsIn order to better understand the anti-tumor immune response to survivin, and optimize vaccination strategies, we characterized the spontaneous CD4+CD25- T cell response against survivin in healthy donors and myeloma patients using survivin derived peptide pools.ResultsHealthy donors and myeloma patients’ CD4+CD25- T cells exhibited a proliferative and IFN-gamma response against survivin peptides loaded onto autologous dendritic cells. We employed limiting dilution analysis to quantify the precursor frequency of survivin reactive CD4+CD25- T cells. Multiple myeloma patients (range 0% to 2.2x10-3%, n=12) had fewer survivin reactive CD4+CD25- T cells than healthy blood donors (range 1.1x10-3 to 8.4x10-3%, n=10), p = 0.021. The survivin reactive CD4+CD25- T cell precursor frequency was inversely associated with tumor survivin mRNA expression (p = 0.0028, r = −1.0, n = 6), and survivin tumor protein expression by IHC (p = 0.0295, r = −0.67, n = 10). A full length mutant survivin protein-pulsed dendritic cell vaccine expanded survivin reactive CD4+CD25- T cells after 12 days of in vitro culture (range 0-540x,median = 42x), and expansion was achieved even in patients with low baseline survivin reactive CD4+ precursors.ConclusionsWe have, for the first time, quantified the circulating CD4+CD25- precursor frequency against survivin and demonstrated this is lower in myeloma patients than healthy donors. The number of survivin reactive CD4+CD25- T cells is inversely associated with tumor survivin expression suggesting suppression of survivin responsive CD4+CD25- T cells. Further exploration of a full length mutant survivin protein vaccine which expands survivin reactive CD4+ cells independent of the survivin reactive precursor frequency is warranted.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-015-0065-1) contains supplementary material, which is available to authorized users.

Abstract B35: Survivin inhibition via EZN-3042 in canine models of lymphoma and osteosarcoma

Abstract Canine lymphoma (LSA) and osteosarcoma (OS) have high mortality rates and remain in need of more effective therapeutic approaches. Due to the strong similarities between canine and human LSA and OS, canine LSA and OS are excellent models for the human disease. Survivin, an IAP family member protein that inhibits apoptosis and drives cell proliferation, is commonly elevated in human and canine cancer. Survivin expression is a negative prognostic factor in dogs and humans with LSA and OS. The objective of our research was to determine the effects of survivin inhibition using a locked nucleic acid antisense oligonucleotide (EZN-3042) on canine LSA and canine OS cell lines, with respect to cell proliferation, apoptosis, and chemosensitivity in vitro. Furthermore, we sought to determine the efficacy of EZN-3042 on inhibition of survivin transcription, survivin protein production, and tumor growth in subcutaneous and orthotopic canine OS xenografts. We inhibited survivin using EZN-3042 in two canine LSA and two canine OS cell lines. Survivin inhibition was confirmed by qRT-PCR and western blot. Percent dead and total cell number was assessed by manual cell counting with trypan blue. Growth inhibition was confirmed with a bioreductive fluorometric assay. A caspase-3/7 assay was used to determine levels of apoptosis in EZN-3042 treated cells compared to controls. Cells were then treated with antineoplastic drug doxorubicin (DOX) +/- EZN-3042 and assays repeated. In vivo, nude mice with subcutaneous and orthotopic OS xenografts were given 100 mg/kg EZN3042 intraperitoneally. Survivin inhibition was confirmed with Immunohistochemistry and qRT-PCR analysis. Survivin inhibition in canine LSA and OS cells via EZN-3042 resulted in 34-72% decrease in survivin protein expression and a 1.3-3.4 fold decrease in endogenous survivin mRNA expression. When EZN-3042 treated cells were compared to controls, total and viable cell numbers were decreased, and apoptosis was increased. Survivin inhibition via EZN-3042 enhanced canine LSA and OS cell lines sensitivity to DOX. IHC and qRT-PCR analysis of subcutaneous and orthotopic canine OS xenografts confirmed decreased tumor survivin expression in EZN-3042 treated mice. Mice treated with EZN-3042 in combination with DOX had significantly decreased tumor growth when compared to single agent treatment and control groups. These results demonstrate that survivin inhibition via EZN-3042 decreased LSA and OS cell proliferation, and increased cellular apoptosis and chemosensitivity to DOX, and that EZN-3042 treatment inhibited tumor survivin expression in vivo and significantly decreased tumor growth when combined with DOX. Survivin-directed therapies may be highly effective in treatment of both canine and human LSA and OS, and spontaneous canine cancer may be a valuable model for the evaluation of survivin-targeted treatment. Citation Format: Jenette K. Shoeneman, Eugene J. Ehrhart, III, Joseph B. Charles, Douglas H. Thamm. Survivin inhibition via EZN-3042 in canine models of lymphoma and osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B35.

Evaluation of Antitumor Activity Using Change in Tumor Size of the Survivin Antisense Oligonucleotide LY2181308 in Combination with Docetaxel for Second-Line Treatment of Patients with Non–Small-Cell Lung Cancer: A Randomized Open-Label Phase II Study

Chemoresistance is mediated, in part, by the inhibition of apoptosis in tumor cells. Survivin is an antiapoptotic protein that blocks chemotherapy-induced apoptosis. To investigate whether blocking survivin expression enhances docetaxel-induced apoptosis in patients with non-small-cell lung cancer (NSCLC), we compared the antitumor activity of the survivin inhibitor LY2181308 plus docetaxel with docetaxel alone. We used change in tumor size (CTS) as a primary endpoint to assess its use in early decision-making for this and future studies of novel agents in NSCLC. Patients (N = 162) eligible for second-line NSCLC treatment (stage IIIB/IV) with an Eastern Cooperative Oncology Group performance status of 0 to 1 were randomized 2:1 to receive LY2181308 (750 mg intravenously, weekly) and docetaxel (75 mg/m intravenously, day 1) or docetaxel alone every 21 days. CTS from baseline to the end of cycle 2 was compared between the two treatment arms. The mean (SD) tumor size ratio for LY2181308/docetaxel and docetaxel was 1.05 (0.21) and 1.00 (0.15) (p = 0.200), respectively, suggesting no significant improvement in antitumor activity between the arms. Because there was also no significant difference between the two arms for progression-free survival (PFS) (2.83 months with LY2181308/docetaxel and 3.35 months with docetaxel [p = 0.191]), both arms were combined. Using the combined arms, CTS correlated with PFS (PFS = 4.63 months in patients with decreased CTS compared with 2.66 months in patients with increased CTS), supporting its use in early decision-making in phase II studies.

Tumor priming enhances siRNA delivery and transfection in intraperitoneal tumors

Cancers originating from the digestive system account for 290,000 or ~20% of all new cancer cases annually in the US. We previously developed paclitaxel-loaded tumor-penetrating microparticles (TPM) for intraperitoneal (IP) treatment of peritoneal tumors (Lu et al., 2008; Tsai et al., 2007; Tsai et al., 2013). TPM is undergoing NIH-supported IND-enabling studies for clinical evaluation. The present study evaluated the hypothesis that TPM, via inducing apoptosis and expanding the interstitial space, promotes the delivery and transfection of lipid vectors containing siRNA. The in vivo model was the metastatic human Hs766T pancreatic tumor that, upon IP injection, produced widely distributed solid tumors and ascites in the peritoneal cavity in 100% of animals. The target gene was survivin, an anti-apoptotic protein induced by chemotherapy and associated with metastases and poor prognosis of patients with gastric and colorectal cancers. The siRNA carrier was pegylated liposomes comprising cationic and neutral lipids plus a fusogenic lipid (PCat). PCat-loaded with survivin siRNA (PCat–siSurvivin) was active in cultured cells (decreased survivin mRNA and protein levels, reduced cell clonogenicity, enhanced paclitaxel activity), but lost its activity in vivo; this difference is consistent with the well-known problem of inadequate delivery and transfection of siRNA in vivo. In comparison, single agent TPM prolonged animal survival and, as expected, induced survivin expression in tumors. Addition of PCat–siSurvivin reversed the TPM-induced survivin expression and enhanced the antitumor activity of TPM. The finding that in vivo survivin knockdown by PCat–siSurvivin was successful only when it was given in combination with TPM provides the proof-of-concept that tumor priming promotes the delivery and transfection of liposomal siRNA. The data further suggest the TPM/PCat–siSurvivin combination as a potentially useful chemo-gene therapy for peritoneal cancer.

Tumor survivin expression in locally advanced non-small cell lung cancer (NSCLC) patients treated with platinum-based chemoradiation followed by surgical resection

7595 Background: Recently tumor molecular markers have shown promise as prognostic and predictive indicators for survival in early and advanced stage NSCLC patients (pts.) treated with chemotherapy. The objective of this study was to correlate immunohistochemistry (IHC) markers of pre-treatment biopsies in locally advanced NSCLC patients treated with concurrent platinum based chemoradiation followed by surgical resection. Methods: This is a retrospective study that included stage III NSCLC pts who had adequate pre-treatment tumor specimens and were treated with platinum based chemotherapy regimens and concurrent thoracic radiation (40 Gy). Thirty three pts had sufficient pre-treatment tissue for IHC and were identified from a surgical database. Cells were stained by IHC for frequency (0–4) and intensity (0–4) of ERCC1, PTEN, and survivin and analyzed by log-rank and multivariate Cox PH regression for potential relationships to pathologic complete response (pCR), time to recurrence (TTR), and overall survival (OS). Results: Characteristics of 33 pts: 15 females; median age 61; 17 adenocarcinoma, 10 squamous(sq), 5 undifferentiated, 1 adeno-sq. Median OS was 23 months (mo) (5.9–140), and median TTR was 14.7 mo (3.5–121). Following chemoradiation, 9 patients had pCR. pCR was associated with improved TTR, p < .027. ERCC1 and PTEN were not significantly related to OS, TTR, or pCR. High nuclear survivin frequency (>2) was associated with worse OS, HR 0.4, p< .045 and lower nuclear survivin intensity (<4) was marginally associated with pCR, p< .10. Conclusions: In this exploratory analysis, higher survivin expression was associated with worse prognosis in locally advanced NSCLC patients treated with chemoradiation followed by surgery. These results suggest that additional studies of survivin are warranted in NSCLC and that adding survivin inhibitors to chemoradiation is a reasonable strategy for locally advanced NSCLC with high survivin expression. No significant financial relationships to disclose.

Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Upregulates Survivin Expression in KSHV-Associated B-Lymphoma Cells and Contributes to Their Proliferation

Survivin is a master regulator of cell proliferation and cell viability and is highly expressed in most human tumors. The molecular network linked to survivin expression in tumors has not been completely elucidated. In this study, we show that latency-associated nuclear antigen (LANA), a multifunctional protein of Kaposi's sarcoma-associated herpesvirus (KSHV) that is found in Kaposi's sarcoma tumors, upregulates survivin expression and increases the proliferation of KSHV-infected B cells. Analysis of pathway-specific gene arrays showed that survivin expression was highly upregulated in BJAB cells expressing LANA. The mRNA levels of survivin were also upregulated in HEK 293 and BJAB cells expressing LANA. Similarly, protein levels of survivin were significantly higher in LANA-expressing, as well as KSHV-infected, cells. Survivin promoter activity assays identified GC/Sp1 and p53 cis-acting elements within the core promoter region as being important for LANA activity. Gel mobility shift assays revealed that LANA forms a complex with Sp1 or Sp1-like proteins bound to the GC/Sp1 box of the survivin promoter. In addition, a LANA/p53 complex bound to the p53 cis-acting element within the survivin promoter, indicating that upregulation of survivin expression can also occur through suppression of p53 function. Furthermore, immunohistochemistry analyses revealed that survivin expression was upregulated in KSHV-associated Kaposi's sarcoma tissue, suggesting that LANA plays an important role in the upregulation of survivin expression in KSHV-infected endothelial cells. Knockdown of survivin expression by lentivirus-delivered small hairpin RNA resulted in loss of cell proliferation in KSHV-infected cells. Therefore, upregulation of survivin expression in KSHV-associated human cells contributes to their proliferation.

Tolfenamic acid enhances pancreatic cancer cell and tumor response to radiation therapy by inhibiting survivin protein expression

Survivin is overexpressed in most human cancers, including pancreatic adenocarcinoma. Expression of survivin is regulated by specificity protein (Sp) proteins and related to resistance to radiation therapy. Tolfenamic acid induces Sp protein degradation in several cancer cell lines. The purpose of this study is to investigate whether tolfenamic acid inhibits survivin expression and sensitizes pancreatic cancer cells/tumor to radiotherapy. Panc1 and L3.6pl cells have been used to study the effect of radiation on survivin expression and to investigate the efficacy of tolfenamic acid in enhancing the response to radiation therapy. In addition, an orthotopic model for human pancreatic cancer has been used to confirm the efficacy of tolfenamic acid to enhance tumor response to radiation in vivo. Pancreatic cancer cell lines express variable levels of survivin mRNA/protein, which correlate with their radiosensitivity. Radiation increased survivin promoter activity and protein expression in Panc1 and L3.6pl cells and tolfenamic acid inhibited both constitutive and radiation-induced survivin protein expression and enhanced the response of pancreatic cancer cells to radiation therapy. In vivo studies show that tolfenamic acid enhanced the radiation-induced apoptosis associated with decreased survivin expression in tumors and this correlates with the enhanced response of these tumors to the radiation. Thus, tolfenamic acid significantly enhances pancreatic cancer cells/tumor response to radiation therapy. The underlying mechanism includes tolfenamic acid-induced degradation of Sp proteins, which in tumor decreases expression of the Sp-dependent antiapoptotic protein survivin. These preclinical data suggest that tolfenamic acid has the potential to increase the response of pancreatic adenocarcinoma to radiation therapy.

Survivin expression in esophageal cancer: correlation with p53 mutations and promoter polymorphism

Survivin is an inhibitor of apoptosis protein, which is selectively up-regulated in various cancers including esophageal cancer. The underlying mechanism of survivin overexpression in cancers is still unclear. We investigated resected tumor specimens from 100 esophageal cancer patients. Reverse transcription polymerase chain reaction was performed to evaluate survivin gene expression. Polymerase chain reaction-single strand conformation polymorphism was performed to investigate mutations of p53. We found that the survivin expression in tumors with mutant p53 is higher than that in tumors with wild type p53. Furthermore, the distribution of three polymorphisms in survivin promoter region in esophageal cancer patients was studied. The result indicated that the survivin expression was caused by a C allele in the survivin promoter polymorphism -625G/C in some degree. The methylation profile of survivin exon1 was also evaluated using bisulfite sequencing PCR. Our result indicated that survivin mRNA overexpression in cancer was not caused by its dysmethylation status. Therefore, our results suggested that the survivin expression depended on the p53 status and the C allele in the survivin promoter polymorphism -625G/C might increase the possibility of the survivin overexpression in esophageal cancer patients.

A novel combination: ranpirnase and rosiglitazone induce a synergistic apoptotic effect by down-regulating Fra-1 and Survivin in cancer cells

Accumulating evidence supports the idea that two known phosphatidylinositol 3'-kinase (PI3K) downstream proteins, Fra-1 and Survivin, are potential targets for cancer therapy. Increased expression of Fra-1, a Fos family member of the transcription factor activator protein-1, has been implicated in both the maintenance and the progression of the transformed state of several cancer cells. In addition, high Survivin expression in tumors correlates with more aggressive behavior, lower response to chemotherapeutic drugs, and shortened survival time. Previously, we reported that, in malignant mesothelioma cells with increased PI3K activity, small-molecule inhibitors of the PI3K/AKT pathway acted cooperatively with the amphibian RNase chemotherapeutic drug ranpirnase to inhibit cell growth. Because the thiazolidinedione antidiabetic drug rosiglitazone targets the PI3K/AKT pathway, we investigated the effect of the combination of these two drugs in cell survival in several cancer cell lines. We show here that the combination of ranpirnase and rosiglitazone synergistically decreases cell viability and increases cell apoptosis in several cancer cell lines. Cell killing is associated with decreased Fra-1 and Survivin expression and knockdown of Fra-1 increases cell killing by ranpirnase in a dose-dependent manner but not by rosiglitazone. The drug combination does not have a synergistic effect on killing in Fra-1 knockdown cells, showing that Fra-1 modulation accounts in part for the synergism. The novel drug combination of ranpirnase and rosiglitazone is a promising combination to treat cancers with increased PI3K-dependent Fra-1 expression or Survivin.

Regulation of survivin expression by IGF-1/mTOR signaling

Survivin is a dual regulator of cell proliferation and cell viability overexpressed in most human tumors. Although strategies to lower survivin levels have been pursued for rational cancer therapy, the molecular circuitries controlling survivin expression in tumors have not been completely elucidated. Here, we show that stimulation with insulin-like growth factor-1 (IGF-1) results in increased survivin expression in prostate cancer cells. This response is independent of de novo gene transcription, changes in mRNA expression or modifications of survivin protein stability. Instead, IGF-1 induced persistence and translation of a pool of survivin mRNA, in a reaction abolished by the mTOR (mammalian target of rapamycin) inhibitor, rapamycin. Forced expression of the mTOR target p70S6K1 reproduced the increase in survivin expression in prostate cancer cells, whereas acute ablation of endogenous p70S6K1 by small interfering RNA downregulated survivin levels. Rapamycin, alone or in combination with suboptimal concentrations of taxol reduced survivin protein levels, and decreased viability of prostate cancer cells. Therefore, IGF-1/mTOR signaling elevates survivin in prostate cancer cells via rapid changes in mRNA translation. Antagonists of this pathway may be beneficial to lower an antiapoptotic threshold maintained by survivin in prostate cancer.

Survivin in esophageal cancer: An accurate prognostic marker for squamous cell carcinoma but not adenocarcinoma

We quantified the expression of survivin, both as mRNA in real-time PCR and protein in immunohistochemistry, in tumor samples of 112 patients with esophageal cancer (56 squamous cell carcinomas and 56 adenocarcinomas). Overall survival of squamous cell carcinoma patients with high survivin mRNA levels was significantly less than that of patients with low survivin mRNA levels (p = 0.0033). Distribution pattern of survivin (nuclear vs. cytoplasmic or mixed) was not correlated to survival, while the extent of immunostaining was significantly correlated to survivin mRNA values (p = 0.016) and had prognostic relevance in univariate analysis (p = 0.0012). Cox's proportional-hazard regression model showed that tumor survivin expression in esophageal squamous cell carcinoma was the most important prognostic factor, independent of tumor stage and other histopathological factors, both as mRNA relative value (p = 0.0259) and protein immunostaining (p = 0.0147). In esophageal adenocarcinoma, survivin expression and pattern of distribution had no prognostic relevance. Thus, quantifying survivin expression provides a prognostic marker only for esophageal squamous tumors.

Survivin gene expression is negatively regulated by the p53 tumor suppressor gene in non-small cell lung cancer

Survivin is considered to be associated with tumorigenesis by regulating apoptosis and cell proliferation. Recent experimental studies reported survivin gene expression to be negatively regulated by wild-type p53. We investigated resected tumor specimens from 140 non-small cell lung cancer (NSCLC) patients. Quantitative reverse-transcription PCR analysis was performed to evaluate survivin gene expression. PCR-single strand conformation polymorphism following sequencing was performed to investigate mutations of p53. The apoptotic index and the Ki-67 proliferation index were also evaluated according to the survivin expression. The survivin expression was low in normal lung tissue. In contrast, the survivin expression varied greatly among tumor tissues. The survivin expression in squamous cell carcinomas was significantly higher than that in adenocarcinomas (P=0.0109). The survivin expression in moderately or poorly differentiated tumors was significantly higher than that in well-differentiated tumors (P=0.0334). Furthermore, the survivin expression in tumors with mutant p53 was significantly higher than that in tumors with wild-type p53 (P=0.0026). In addition, the apoptotic index was significantly lower in high-survivin tumors than in low-survivin tumors (P<0.0001). The Ki-67 proliferation index was significantly higher in high-survivin tumors than in low-survivin tumors (P<0.0047). This study indicated survivin gene expression to be negatively regulated by p53 in NSCLC, and that survivin expression could inhibit apoptosis and accelerate tumor cell proliferation to produce more aggressive carcinomas.