Wild-type Survivin Research Articles

Aurora B confers cancer cell resistance to TRAIL-induced apoptosis via phosphorylation of survivin

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. In this study, we examined whether Aurora B, which is frequently overexpressed in cancer cells, is associated with TRAIL resistance. The protein levels of Aurora B were higher in TRAIL-resistant cancer cell lines than in TRAIL-sensitive cancer cell lines. Exogenously expressed Aurora B attenuated TRAIL-induced apoptosis in the tested TRAIL-sensitive cancer cell lines, whereas the small interfering RNA-mediated suppression of Aurora B expression stimulated TRAIL-mediated apoptosis in the tested TRAIL-resistant cancer cell lines. Furthermore, combined treatment with TRAIL and ZM447439, a specific inhibitor of Aurora B, synergistically induced apoptosis in various TRAIL-resistant cancer cells, suggesting that this combined regimen may represent an attractive strategy for effectively treating TRAIL-resistant malignant cancers. Mechanistically, the inhibition of Aurora B activity in various cancer cells commonly downregulated survivin protein levels and potentiated the activation of caspase-3. In addition, Aurora B inhibition induced mitotic catastrophe, which also contributed to the sensitization of cells to TRAIL-mediated apoptosis. Interestingly, forced overexpression of Aurora B increased the protein levels of survivin, but not those of a non-phosphorylatable survivin mutant in which threonine 117 was replaced by alanine, indicating that phosphorylation of survivin is required for this effect. Furthermore, TRAIL-induced apoptosis in MDA-MB-435S cells was attenuated by wild-type survivin but not by the non-phosphorylatable survivin mutant. Collectively, our results demonstrate that Aurora B confers TRAIL resistance to cancer cells via phosphorylation of survivin.

Phosphorylated-Survivin at Ser81 Induced Protein Kinase A (PKA): A Back Loop

BACKGROUND: Survivin, a bifunctional protein, acts as suppressor of apoptosis and has an essential role in mitosis. Survivin is physically phosphorylated on Thr34, and other important sites such as Thr117, Ser20, Thr48 and Ser81. Our previous report has shown that Ser81 of survivin plays role in cytoprotection. In order to investigate the underlying mechanism, all motifs with medium stringency were scanned. We found that site of survivin at Ser81 was correlated to PKA, which is well reported to many cell signal machineries, including cell survival. Therefore, we focused our current investigation in finding possible correlation and interaction between survivin’s Ser81 site and PKA.METHODS: Wild-type survivin (Survivin), antisense survivin (Survivin-AS), mutated-survivin and mutated-survivin Ser81Ala (Survivin-S81A) were constructed. Each retroviral product was produced. Some cell lysates were prepared and immunoprecipitated. For analysis, we performed immunoblotting and PKA’s activity assays.RESULTS: In our current results, phosphorylated-PKA was correlated with survivin. Infection of survivin could lead to acceleration of PKA’s activity in a viral particle dependent manner. This positive back loop induction by survivin was shown to be correlated to Ser81 site, since survivin-mediated PKA activity was not resulted by mutated form of survivin at Ser81 to nonphosphorylatable Ala (S81A).CONCLUSIONS: Our results suggested a possible back loop of survivin to activate PKA, and Ser81 could be an important site to mediate the survivin-PKA back loop signaling. Survivin-induced activation of PKA might be related to cytoprotection.KEYWORDS: survivin, S81A, L929, PKA

Detection of survivin expression in cervical cancer cells using molecular beacon imaging: new strategy for the diagnosis of cervical cancer

ObjectiveDevelopment of novel approaches for quantitative analysis of gene expression in intact tumour cells could provide new methods for the detection of cervical cancer. Molecular beacons (MBs) are single-stranded oligonucleotide probes that form stem-and-loop structures. Survivin, a member of the ‘inhibitor of apoptosis’ family of proteins, is highly expressed in cervical cancer. The aim of this study was to determine the feasibility of MBs targeting wild-type survivin in the diagnosis of cervical cancer. Study designMBs with oligonucleotide sequences complementing survivin mRNA and covalently linked with FITC or Cy3 were designed and synthesized. The specificity and sensitivity of survivin MBs were examined in human cervical cancer cell lines and smears from cervical cancer patients, and confirmed by reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry, Western blotting and the thinprep cytological test (TCT). ResultsBoth survivin MB-FITC and MB-Cy3 produced a strong fluorescent signal in cervical cancer cells, and the intensity was consistent with the results from RT-PCR, Western blotting and immunocytochemical staining. In the initial clinical cohort study, the sensitivity of survivin MBs was 61.37% (27/44) and the specificity was 72.72% (34/44); the sensitivities of Western blotting and TCT were 76.1% (32/42) and 68.19% (30/44), respectively. No significant difference in sensitivity was observed between MBs, Western blotting and TCT for different tumour grades and International Federation of Gynecology and Obstetrics (FIGO) stages. ConclusionsSurvivin MBs are specific and sensitive molecular probes for the detection of cervical cancer cells. They have great potential in the early detection and follow-up of patients with cervical cancer.

Survivin Monomer Plays an Essential Role in Apoptosis Regulation

Survivin was initially described as an inhibitor of apoptosis and attracted growing attention as one of the most tumor-specific genes in the human genome and a promising target for cancer therapy. Lately, it has been shown that survivin is a multifunctional protein that takes part in several crucial cell processes. At first, it was supposed that survivin functions only as a homodimer, but now data indicate that many processes require monomeric survivin. Moreover, recent studies reveal a special mechanism regulating the balance between monomeric and dimeric forms of the protein. In this paper we studied the mutant form of survivin that was unable to dimerize and investigated its role in apoptosis. We showed that survivin monomer interacts with Smac/DIABLO and X-linked inhibitor of apoptosis protein (XIAP) both in vitro and in vivo. Due to this feature, it protects cells from caspase-dependent apoptosis even more efficiently than the wild-type survivin. We also identified that mutant monomeric survivin prevents apoptosis-inducing factor release from the mitochondrial intermembrane space, protecting human fibrosarcoma HT1080 cells from caspase-independent apoptosis. On the other hand, our results indicate that only wild-type survivin, but not the monomer mutant form, enhances tubulin stability in cells. These findings suggest that survivin partly performs its functions as a monomer and partly as a dimer. The mechanism of dimer-monomer balance regulation may also work as a "switcher" between survivin functions and thereby explain remarkable functional diversities of this protein.

The Oncofetal Gene Survivin Promotes Cell Proliferation and Survival in Primary Human Osteoblastic Cells

Survivin, the smallest member of the inhibitor of apoptosis gene family, is critical for the regulation of mitosis and maintenance of cell viability during embryonic development and cancer, while not being detectable in most adult differentiated tissues. We know little about whether survivin plays any physiological or pathophysiological role in the adult musculoskeletal system. We studied the expression of survivin in primary human osteoblastic cells and its biological functions in vitro. Survivin was detected by immunoblotting and real-time PCR. Subcellular localization was analyzed by immunofluorescence. Transfection of siRNA and plasmids coding for wild-type survivin was performed to study survivin function, i.e., proliferation and apoptosis assays. Survivin mRNA and protein are expressed in primary human osteoblastic cells. During interphase survivin localizes predominantly to the cytoplasmic compartment, which is relevant for the organization of the spindle apparatus during mitosis. Survivin knockdown resulted in an arrest of the cell cycle at the G(2)/M phase and increased rates of apoptosis. Elevated levels of survivin in primary human osteoblasts enhanced proliferation and cell viability. Taken together, we demonstrate for the first time that survivin is expressed in primary human osteoblastic cells on the mRNA and protein levels. Our results indicate that survivin is a critical factor for cell division and cell viability in primary human osteoblastic cells. Learning more about survivin's role in human osteoblasts could be an important step toward understanding the complex processes involved in bone homeostasis and remodeling.

Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR

To investigate three isoforms of survivin in colorectal adenocarcinomas. We used the LightCycler Technology (Roche), along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler-Red fluorophore (LC-Red 640). Real time quantitative polymerase chain reaction (PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues. In the patients group, carcinoembryonic antigen (CEA) and CA19-9 tumor markers were also measured immunochemically. Wild type survivin mRNA isoform was expressed in 48% of the 52 tumor samples, survivin-2b in 38% and survivin-ΔΕx3 in 29%, while no expression was found in normal tissues. The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b, survivin-ΔΕx3, survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin (P < 0.001). The mRNA expression of wild-survivin and survivin-ΔΕx3 was related with tumor size and invasion (P = 0.006 and P < 0.005, respectively). A significant difference was found between survivin-2b and morphologic cancer type. Also, the ratio of survivin-ΔEx3/wild-survivin was significantly associated with prognosis. No association was observed between the three isoforms and grade, metastasis, Dukes stage and gender. The three isoforms were not correlated with CEA and CA19-9. Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer.

Survivin S81A Enhanced TRAIL's Activity in Inducing Apoptosis

BACKGROUND: Survivin is rarely expressed in normal healthy adult tissues, however, it is upregulated in the majority of cancers. Survivin, which belongs to IAPs family, has been widely reported to protect cells from apoptosis by inhibiting caspases pathway. Survivin’s mitotic activity is modulated by many kinases, and its phosphor status can also influence its ability to inhibit apoptosis. There are several important survivin’s phosphorylation sites, such as S20 and T34. We have continued our investigation on other potential survivin’s phosphorylation sites that could be important site for regulating survivin’s cyto-protection.METHODS: By assuming that S81 could be a potential target to modify activity of survivin, wild-type survivin (Survivin), antisense survivin (Survivin-AS), mutated-survivin Thr34Ala (Survivin-T34A) and mutated-survivin Ser81Ala (Survivin-S81A) were constructed and inserted into pMSCV-IRES-GFP vector with cytomegalovirus (CMV) promoter. Each retroviral product was produced in BOSC23 cells. LY294002 pretreatment and TRAIL treatment along with infection of retroviral products were performed in murine fibrosarcoma L929 cells. For analysis, flow cytometric apoptosis assay and western blot were performed.RESULTS: In our present study, survivin for providing cytoprotection was regulated by PI3K. The results showed that LY294002, an inhibitor of PI3K, effectively suppressed survivin-modulated cytoprotection in a TRAIL-induced apoptotic model. In addition, mutated survivin S81A showed marked suppression on survivin’s cytoprotection. Along with that, TRAIL’s apoptotic activity was enhanced for inducing apoptosis.CONCLUSION: We suggested that survivin could inhibit apoptosis through PI3K and S81A could be another potential target in order to inhibit Survivin-modulated cytoprotection as well as to sensitize efficacy of TRAIL or other related apoptotic inducers.KEYWORDS: apoptosis, survivin, TRAIL, S81A, L929, LY294002

Abstract 2685: The intratumoral expression of survivin associated with Wnt1 expression, not Wnt5a expression in non-small cell lung cancer

Abstract Introduction: Survivin is a member of the inhibitor of apoptosis protein (IAP) family. Recent studies have revealed that survivin is associated not only with apoptosis but also with cell proliferation. As a result, the survivin expression is considered to be involved in tumorigenesis, including non-small cell lung cancer (NSCLC). On the other hand, the Wnt genes are involved in embryogenesis and tumorigenesis. Among them, Wnt1 stimulates the canonical Wnt signaling pathway, which regulates the transcription of various tumor-associated Wnt-target genes with TCF/LEF1 motif. We have reported that Wnt1 overexpression promotes tumorigenesis in NSCLC through the induction of c-Myc, cyclin D1, VEGF-A (Huang C, et al. Eur J Cancer 2008). Recently, survivin is also reported to be a target gene of the canonical Wnt signaling pathway. Therefore, we performed a clinical study on survivin expression in relation to the expression of Wnt1 (canonical Wnt) and Wnt5a (non-canonical Wnt) in NSCLC. Method: One hundred and ten NSCLC patients were investigated. The expression of wild-type survivin (survivin) was evaluated by semi-quantitative RT-PCR because real-time RT-PCR is not an appropriate method to discriminate between wild-type survivin and other splice variants, such as survivin-2B and survivin-deltaEx3. The intratumoral expressions of survivin, Wnt1 and Wnt5a were evaluated by immunohistochemistry. The Ki-67 proliferation index was also investigated by immunohistochemistry, and the apoptotic index was evaluated by TUNEL method. Results: (1) Sixty carcinomas (54.5%) had an overexpression of survivin. The apoptotic index was significantly lower in survivin-high tumors than in survivin-low tumors (P=0.003). In addition, the Ki-67 proliferation index was significantly higher in survivin-high tumors than in survivin-low tumors (P=0.014). (2) The percentage of Wnt1-positive tumor cells significantly correlated with the survivin gene expression (P&amp;lt;0.001). The survivin gene expression was significantly higher in Wnt1-positive tumors than in Wnt1-negative tumors (P=0.002). (3) The percentage of Wnt5a-positive tumor cells did not correlate with the survivin gene expression (P=0.155). There was no difference in the survivin gene expression according to the Wnt5a expression. (4) As a result, the apoptotic index was significantly lower in Wnt1-high tumors than in Wnt1-low tumors (P=0.032). In addition, the Ki-67 proliferation index was significantly higher in Wnt1-high tumors than in Wnt1-low tumors (P=0.001). Conclusions: The intratumoral Wnt1 expression affects the expression of wild-type survivin in NSCLC to produce more aggressive tumors with low tumor apoptosis and high tumor proliferation rate. 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 2685.

Extracellular, cell-permeable survivin inhibits apoptosis while promoting proliferative and metastatic potential.

The tumour microenvironment is believed to be involved in development, growth, metastasis, and therapy resistance of many cancers. Here we show survivin, a member of the inhibitor of apoptosis protein (IAP) family, implicated in apoptosis inhibition and the regulation of mitosis in cancer cells, exists in a novel extracellular pool in tumour cells. Furthermore, we have constructed stable cell lines that provide the extracellular pool with either wild-type survivin (Surv-WT) or the previously described dominant-negative mutant survivin (Surv-T34A), which has proven pro-apoptotic effects in cancer cells but not in normal proliferating cells. Cancer cells grown in conditioned medium (CM) taken from Surv-WT cells absorbed survivin and experienced enhanced protection against genotoxic stresses. These cells also exhibited an increased replicative and metastatic potential, suggesting that survivin in the tumour microenvironment may be directly associated with malignant progression, further supporting survivin's function in tumourigenesis. Alternatively, cancer cells grown in CM taken from the Surv-T34A cells began to apoptose through a caspase-2- and caspase-9-dependent pathway that was further enhanced by the addition of other chemo- and radiotherapeutic modalities. Together our findings suggest a novel microenvironmental function for survivin in the control of cancer aggressiveness and spread, and should result in the genesis of additional cancer treatment modalities.

Comparative study on the immunogenicity between an HLA-A24-restricted cytotoxic T-cell epitope derived from survivin and that from its splice variant survivin-2B in oral cancer patients

BackgroundWe previously reported an HLA-A24-restricted cytotoxic T-cell epitope, Survivin-2B80-88, derived from a splice variant of survivin, survivin-2B. In this report, we show a novel HLA-A24-restricted T-cell epitope, Survivin-C58, derived from a wild type survivin, and compared their immunogenicity in oral cancer patients.MethodsBy stimulating peripheral blood lymphocytes of HLA-A24-positive cancer patients with Survivin-C58 peptide in vitro, the peptide-specific CTLs were induced. In order to compare the immunogenic potential between C58 peptide and 2B80-88 peptide, peripheral blood T-cells from thirteen HLA-A24-positive oral cancer patients were stimulated with either or both of these two peptides.ResultsSurvivin-2B80-88 peptide-specific CTLs were induced from four patients, and C58 peptide-specific CTLs were induced from three out of eight patients with over stage II progression. The CTLs exerted cytotoxicity against HLA-A24-positive tumor cells. In contrast, CTL induction failed from a healthy volunteer and all four patients with cancer stage I.ConclusionIt was indicated that a splicing variant-derived peptide and wild type survivin-derived peptide might have a comparable potency of CTL induction, and survivin targeting immunotherapy using survivin-2B80-88 and C58 peptide cocktail should be suitable for HLA-A24+ oral cancer patients.

Identification and Characterization of Sur3γ and Sur3γV, Two Novel Splice Variants of Survivin in HSPC

Survivin is a member of the inhibitor-of-apoptosis (IAP) family of endogenous caspase inhibitors. We have previously shown that Survivin is expressed in primitive hematopoietic stem and progenitor (HSPC) cells and required for HSC maintenance and for entry into cell cycle. Several alternative Survivin splice variants have been described that are associated with differential prognosis in patients with cancer and leukemia, however, the function of alternative splice variants in normal and cancer cells is not fully understood. Using rapid amplification of cDNA ends (RACE) with mRNA from HL-60 leukemia cells we have identified two novel alternative splice variants, Survivin-3γ (Sur3γ) and Survivin-3γ-Variant (Sur3γV) that differ by only 4 bases, however due to changes in the reading frame, they result in proteins with very different C-termini, with Sur3γV being the predominant variant. We have characterized the expression of Sur3γV and Sur3γ in normal HSPC, leukemia and solid cancer cells and evaluated their anti-apoptotic function in cancer cell lines and primary hematopoietic cells. In contrast to cell lines and most cancers, where SurWT is the predominant transcript, Sur3γV is expressed at higher levels than SurWT in a number of normal human tissues including bone marrow, pericardium, placenta, seminal vesicles, testis and uterus. In placenta and bone marrow, Sur3γV mRNA is expressed at 25-fold and 65-fold higher levels respectively, than wild-type Survivin (SurWT). Sur3γV is expressed at ~16-fold higher levels in CD34+ HSPC and at ~80-fold higher level in primary CD34+ CD38− HSPC. In CD34+ cells, stimulation with SCF, TPO, FLT3L was associated with 30 – 55 fold increase in expression of SurWT, however expression of Sur3γ and Sur3γV were not altered, suggesting that these variants may be associated with HSPC maintenance/quiescence. Sur3γV and SurWT are expressed at equivalent levels in AML however, in blast crisis CML, Sur3γV is expressed at 5-fold higher level than SurWT. Over expression of Sur3γ and Sur3γV in Yac1, a mouse lymphoma cell line, confers ~ 2-fold increased resistance to Taxol similar to that observed using Survivin-WT. Similarly, Yac-1 cells over expressing Sur3γV or Sur3γ were equally resistant to Etoposide as cells over expressing SurWT. Over-expression of Sur3γ and Sur3γV using the bi-cistronic MIEG vector in primary murine bone marrow mononuclear cells results in 1.7-fold increase in total CFU-GM, similar to SurWT. In addition, over-expression of Sur3γV and Sur3γ protected CFU-GM from apoptosis induced by delayed growth factor addition resulting in a 1.6 fold increase in the number of CFU-GM colonies detected. These studies clearly indicate that both Sur3γ and Sur3γV possess anti-apoptotic function at least equivalent to SurWT. In summary, we have identified 2 novel splice variants of Survivin that differ from one another by 4 bases. Unlike other Survivin splice variants that are expressed at low levels, Sur3γV is expressed at a higher level than SurWT in primitive HSPC and in some primary leukemia samples. Expression of Sur3γ and Sur3γV is not altered by hematopoietic growth factors that stimulate HSC proliferation. Over-expression of Sur3γ and Sur3γV protects cells from chemotherapeutic drugs and ectopic expression of Sur3γ and 3γV stimulates HPC proliferation and protects primary mouse HSPC from apoptosis. These studies also suggest that differential expression of Survivin splice variants may play a critical role in HSC function and differentiation.

Overlapping but Distinct Role of p21WAF1/CDKN1 and Survivin in Hematopoietic Progenitor Cell Proliferation.

Survivin is a member of the inhibitor of apoptosis protein family that has been implicated in cell cycle control, anti-apoptosis and cell division. Our previous studies and others have shown that Survivin and the cyclin dependent kinase inhibitor p21WAF1/CDKN1 (p21) are functionally associated and are involved in cell cycle, anti-apoptosis and cytokinesis in cancer cells and in normal hematopoietic progenitor cells (HPC). P21 is highly expressed in quiescent hematopoietic stem cells (HSC) in steady state, but the proportion of quiescent HSCs in G0 phase is reduced in p21−/− mice. In contrast, p21 has been shown as positive regulator on cell cycle of normal HPC since p21 deficiency results in fewer total CFU in mouse bone marrow (BM) cells with fewer CFU in S-phase and retrovirus transduction of p21 in p21 deficient bone marrow cells restores total and cycling CFU. We have previously reported that Survivin increases the proliferation of mouse primary HPC and that this enhancing effect is on HPC proliferation is absent when p21 is functionally deleted, suggesting that p21 is required for Survivin to enhance HPC proliferation. In addition, ITD-Flt3 mutations that are normally expressed in patients with acute myeloid leukemia and associate poor prognosis increase expression of both Survivin and p21, implicating their involvement in aberrant proliferation of HPC expressing ITD-Flt3. Herein we have characterized the functional association between p21 and Survivin in normal and transformed cell proliferation. Antagonizing wild-type Survivin in mouse BaF3 cells by retrovirus transduction of a T34A dominant negative mutant Survivin or anti-sense increased p21 expression, even though Survivin requires p21 to enhance HPC proliferation. Ectopic p21 in Survivin+/+ primary mouse bone marrow cells increased the number of immunophenotypically defined c-kit+, lin− (KL) cells, which is consistent with a positive role of p21 in HPC proliferation, however; ectopic expression of p21 failed to increase HPC proliferation in Survivin deficient primary bone marrow cells, suggesting that p21 alone is not sufficient to substitute for Survivin's enhancing function on normal HPC proliferation. Over-expression of ITD-Flt3 enhanced growth factor independent proliferation of primary mouse marrow c-kit+, Sca-1+, lin− (KSL) cell number; however, co-expression of p21 with ITD-Flt3 dramatically decreased the number of growth factor independent KSL cells (80±6% reduction: P<0.01). Furthermore, the inhibitory effect of p21 on KLS proliferation was further enhanced by Survivin knockout bone marrow cells (64±5% reduction compared with presence of Survivin: P<0.05). These findings indicate that Survivin and p21 have a overlapping but distinct roles in regulating normal HPC proliferation and that manipulating p21 and Survivin may represent a potential therapeutic target for acute leukemia cells expressing ITD-Flt3.

Nuclear Survivin Abrogates Multiple Cell Cycle Checkpoints and Enhances Viral Oncolysis

Survivin (BIRC5) promotes cell division and survival with roles as chromosomal passenger protein and inhibitor of apoptosis protein (IAP). It is overexpressed in many cancers and is associated with resistance to chemotherapy and radiation. Previously, we showed that expression of survivin within the nucleus of HeLa cells accelerates its degradation and blocks apoptosis inhibition without affecting localization during mitosis. Here, we have investigated the effects of survivin on cell cycle control and potential therapeutic consequences using HeLa and IGROV1 cells expressing wild-type and nuclear-targeted survivin. We show that overexpression of survivin, especially within the nucleus, increases control over G(1)-S checkpoint via increased nuclear accumulation of cyclin D and cyclin-dependent kinase 4 and subsequent pRb phosphorylation. We investigated the influence of survivin on the activity of the E1A CR2-deleted oncolytic adenovirus dl922-947, which depends critically on an aberrant G(1)-S checkpoint. Nuclear expression of survivin augments virus-induced S-phase induction and increases viral protein expression and overall viral replication. There is a consequent increase in antitumor activity both in vitro and in vivo. The increased dl922-947 activity is restricted to malignant cells and is not associated with induction of apoptosis, nor does it rely on the role of survivin as an IAP. In addition, we observe the appearance of a large >or=4N population coincident with multiple mitotic defects in dl922-947-infected cells, both of which are significantly increased by nuclear survivin. This indicates that adenoviral activity is facilitated by abrogation of multiple cell cycle checkpoints and can be enhanced by expression of survivin within the nucleus.

A study into the potential role of Survivin localization in resistance to drug-induced apoptosis

The aim of this study was to test the hypothesis that diverting the cytoplasmic subcellular localization of the anti-apoptotic form of Survivin to the nucleus would sensitize cancer cells to chemotherapeutics. Apoptosis is a morphologically and biochemically distinctive form of cell death, critical in the maintenance of tissue homeostasis. Caspases are a family of cysteine proteases that have a vital role in the implementation of apoptosis, and their activity is regulated by Inhibitors of Apoptosis Proteins. Recent studies indicate that one such inhibitor, Survivin, may have dual functions that are specific to its cellular location, including suppression of apoptotis (cytosolic) and regulation of cell division (nuclear). Since both apoptosis and proliferation are altered in cancer, identifying whether these roles for Survivin are dependent on its subcellular localization will inform future approaches to treat chemotherapeutically resistant tumour cells. After initially confirming the specificity of several Survivin antibodies, the distribution of Survivin was examined by immunofluorescence microscopy and sub-cellular fractionation in breast cancer cell lines. The threshold of drug-induced apoptosis was compared in cells over-expressing either wildtype Survivin or a form of Survivin unable to exit the nucleus due to a mutation in its nuclear export sequence. Endogenous Survivin localized to both nucleus and cytoplasm of breast cancer cell lines. Over-expressed Survivin had an anti-apoptotic, protective function. In contrast, cells expressing Survivin with the mutated nuclear export sequence had a lower apoptotic threshold to chemotherapeutic drugs. These results demonstrate for the first time that Survivin is localized to both the nucleus and cytoplasm of breast cancer cell lines. Importantly, the sensitivity of cells to chemotherapeutic drugs was increased when Survivin's localization was restricted to the nucleus, consistent with cytoplasmic Survivin having the anti-apoptotic role. Since clinical studies have shown that nuclear Survivin is a positive prognostic factor in breast cancer patients, the data suggest that strategies to alter Survivin distribution may be useful in the fight against cancer.

A survivin double point mutant has potent inhibitory effect on the growth of hepatocellular cancer cells

Survivin is an attractive target in cancer therapy. Previous studies have demonstrated that survivin dominant-negative mutants T34A and C84A were able to induce apoptosis in cancer cells. Given that they had different mechanisms in inducing apoptosis, our study was undertaken to determine whether a survivin double point mutant (TC34,84AA) could achieve more potent inhibitory effect on the growth of hepatocellular cancer cells. Adenoviruses expressing survivin mutants were constructed and transduced into hepatocellular cancer cells. The inhibitory effect of the survivin mutants on cancer cell growth was measured. Transduction of cancer cells with all three survivin mutants resulted in significant apoptosis. Compared with survivin mutants T34A or C84A alone, the cancer killing effect of survivin TC34,84AA was much stronger. In addition, the survivin mutants were more sensitive than wild type survivin to the degradation in the ubiquitin-proteasome pathway. Our results suggest that adenovirus-delivered dominant-negative survivin TC34,84AA promotes apoptosis-mediated hepatocellular carcinoma suppression, and could potentially be a promising candidate for cancer therapies.

The clinical significance of splice variants and subcellular localisation of survivin in non-small cell lung cancers

Survivin is a member of the inhibitor of apoptosis protein family. Survivin has splice variants with different biological functions associated with tumorigenesis. We investigated 134 non-small cell lung cancers (NSCLCs) to study the clinical significance of wild-type survivin, survivin-2B, and survivin-deltaEx3. Real-time PCR analyses were performed for their gene expressions. The subcellular localisation of survivin proteins was evaluated by immunohistochemistry. The Ki-67 proliferation index and the apoptotic index were also evaluated. The survivin-deltaEx3 gene expression was significantly higher in stage II–III than in stage I (P=0.0174), and significantly correlated with the nuclear pan-survivin expression (P<0.0001). The Ki-67 index was significantly higher in wild-type survivin-positive tumours (P<0.0001), survivin-deltaEx3-positive tumours (P<0.0001), and tumours with positive expression of the nuclear pan-survivin (P=0.0047). In contrast, the apoptotic index was significantly lower only in wild-type survivin-positive tumours (P<0.0001). Thus, the wild-type survivin gene expression was associated with apoptotic inhibition and tumour proliferation. Furthermore, the survivin-deltaEx3 gene expression was strongly associated with tumour proliferation, especially in advanced stage NSCLCs. In contrast, the survivin-2B gene expression did not correlate with tumour proliferation or tumour apoptosis.

Alternative Splice Variants of Survivin as Potential Targets in Cancer

Survivin, a member of the IAP family is an attractive target for cancer treatment due to it's over expression in most cancers and low or no expression in most differentiated adult tissues. Survivin expression is a poor prognostic marker in a number of cancers. Clinical trials are currently underway evaluating anti-sense oligonucleotides against Survivin, immunotherapy using Survivin primed dentritic cells and peptide mimics that block interaction of Survivin with Hsp90 resulting in loss of Survivin protein stability. Additional approaches using ribozymes against Survivin mRNA, or dominant-negative cDNA to block Survivin function are in pre-clinical stages. Like many genes, Survivin is alternately spliced and a number of new splice variants have recently been identified. Expression of some of these splice variants correlates with loss of steroid receptors as well as the tumor suppressor p53, in some cancers, suggesting that like wild-type Survivin, at least some of these splice variants may also have prognostic relevance. This review will focus on the current understanding of the function of Survivin splice variants and their expression and sub-cellular localization in normal and neoplastic tissues as well as critically evaluating the potential toxicity of the Survivin directed therapies and their predicted effect on the alternatively spliced Survivin isoforms.

The effects of knockdown of wild-type survivin, survivin-2B or survivin-Δ3 on the radiosensitization in a soft tissue sarcoma cells in vitro under different oxygen conditions

The inhibitor of apoptosis wild-type survivin is a multifunctional protein that suppresses apoptosis and regulates cell cycle progression. An association between wild-type survivin expression and radiosensitivity has been described in different tumor cells. The effects of siRNA-induced knockdown of wild-type survivin and survivin-splice variants survivin-2B and survivin-Delta3 were investigated under normoxic and hypoxic conditions in the human sarcoma cell line US 8-93 (mutant p53). Inhibition of the survivin isoforms by siRNA resulted in a decrease of target mRNA down to 14-70% compared to cells treated with control siRNA independent of the oxygen level. The mRNA expression of survivin isoforms was decreased by the factor of 1-12 when the cells were cultivated under hypoxic conditions. Moreover, the knockdown of wild-type survivin reduced colony formation independent of oxygen concentration down to 70% and induced formation of polyploid cells. Less reduction of plating efficiency was observed after specific knockdown of survivin-2B and survivin-Delta3 under hypoxic or normoxic conditions. A knockdown of wild-type survivin, survivin-Delta3 and survivin-2B isoforms in combination with irradiation caused no radiosensitization in cell line US 8-93, neither under hypoxic nor under normoxic conditions tested in the colony-forming assay. However, knockdown of wild-type survivin caused radiosensitization in the megacolony assay.

P2-076: The expression of anti-apoptotic protein, survivin, in non-small cell lung cancer

Survivin is a member of Inhibitor of Apoptosis (IAP) family and considered to play a significant role of tumorigenesis and proliferation. Over expression of survivin was reported in several cancers including non-small cell lung cancer (NSCLC), and these reports suggested there is correlation between survivin expression and patient's prognosis. The purpose of this report is to clarify this relationship in NSCLC. We examined 100 NSCLC tissues resected from 2000 Oct. to 2004 Dec. in Tohoku University Hospital. The 100 patients consisted of 71 men and 29 women. The median age was 66.2 (range 30-80). The histology and pathological staging were following: adenocarcinoma 71, squamous cell carcinoma 29, IA 35, IB 24, IIA 5, IIB 10, IIIA 16, IIIB 9, IV 1. We assessed the expression of survivin, p53, NF kappa B, and Bcl-2 by immunohistochemistry. These proteins were considered to have correlation with survivin. Moreover mRNA expression of survivin variants was also examined by real time polymerase chain reaction (PCR). PCR primers were set for wild-type survivin, survivin-deltaEx3, survivin-2B, survivin-3B, and survivin-2alpha. The number of cytoplasmic survivin-positive tissue was 88, and nuclear survivin-positive tissue was 40 (both positive was 36). We have 57 p53 positive, 67 NF kappa B positive, and 12 Bcl-2 positive tissues. The results for real time PCR will be reported. We will analyse these data and show the relationship between expression of apoptosis related proteins including survivin and prognosis or stage.

Expression of survivin and its splice variants in gastric cancer

Survivin variants specific real time quantitative RT-PCR was developed to analyze their expression in 53 paired cancer and para-cancerous tissues, and the expression of the wild-type survivin protein was detected by immunohistochemistry. The results showed that survivin mRNA and protein were expressed in gastric cancer and para-cancerous tissues. The survivin-2B was dominantly expressed in para-cancerous tissues, whereas the survivin-DeltaEx3 was more frequently detected in cancer tissues. The positive rate of survivin-2a was 100% in both cancer and para-cancerous tissues, but its relative transcript expression level was not significantly increased in cancer tissues in comparison with para-cancerous tissues. The correlation analysis revealed that the expression of survivin-2a mRNA was significantly associated with that of total survivin (r (s)=0.4178, P=0.0018), whereas inversely to that of survivin-DeltaEX3 (r (s)=-0.4506, P=0.0007). It was suggested that survivin-2a may act as an antagonist of survivin-DeltaEX3. The balance between antiapoptotic survivin iso-forms and nonantiapoptotic ones may play an important role in tumorigenesis and tumor progression. Promising value is hinted to analyze survivin and its variants in tumor early diagnosis and distinguishing malignant tumors from benign ones.