Expression Of Survivin Splice Variants Research Articles

Survivin Splice Variant 2β Enhances Pancreatic Ductal Adenocarcinoma Resistance to Gemcitabine.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with poor prognosis, as it is difficult to predict or circumvent, and it develops chemoresistance quickly. One cellular mechanism associated with chemoresistance is alternative splicing dysfunction, a process through which nascent mRNA is spliced into different isoforms. Survivin (Baculoviral IAP Repeat-Containing Protein 5 (BIRC5)), a member of the inhibitor of apoptosis (IAP) protein family and a cell cycle-associated oncoprotein, is overexpressed in most cancers and undergoes alternative splicing (AS) to generate six different splicing isoforms.MethodsTo determine if survivin splice variants (SSV) could be involved in PDAC chemoresistance, a Gemcitabine (Gem) resistant (GR) cell line, MIA PaCa-2 GR, was created and assessed for its SSV levels and their potential association with GR. Cross-resistance was assessed in MIA-PaCa-2 GR cells to FIRINOX (5-fluorouracil (5-FU), irinotecan, and oxaliplatin). Once chemoresistance was confirmed, RT-qPCR was used to assess the expression of survivin splice variants (SSVs) in PDAC cell lines. To confirm the effect of SSVs on chemoresistance, we used siRNA to knockdown all SSVs or SSV 2β.ResultsThe MIA PaCa-2 GR cell line was 40 times more resistant to Gem and revealed increased resistance to FIRINOX (5-fluorouracil (5-FU), irinotecan, and oxaliplatin); when compared to the parental MIA-PaCa-2 cells. RT-qPCR studies revealed an 8-fold relative expression increase in SSV 2β and a 2- to 8-fold increase in the other five SSVs in the GR cells. Knockdown of all SSV or SSV 2β only, using small inhibitory RNA (siRNA), sensitized the GR cells to Gem, indicating that these SSVs play a role in PDAC chemoresistance.ConclusionThese findings provide evidence for the potential role of SSV 2β and other SSVs in innate and acquired PDAC chemoresistance. We also show that the expression of SSVs is not affected by the type of chemoresistance, therefore targeting survivin splice variants in combination with chemotherapy could benefit a wide range of patients.

Altered expression of survivin and its splice variants ∆Ex3 and 2B contributes to disease development in breast cancer

AimSurvivin is a unique member of inhibitor of apoptotic protein family present in embryonic development but absent in normal tissues. Splicing of the survivin gene give rise to five different splice variants and differential expression of these splice variants has been associated with disease progression and development in many human cancers. The aim of our study was to investigate the expression level of survivin (survivin-wild type) and two of its splice variants (∆Ex3 & 2B) in human breast cancer and normal adjacent. MethodFifty breast cancer patients were included in this study. The mRNA expression level of survivin and its variants was checked by SYBR Green based real-time PCR (qRT-PCR) method. ResultThe expression of surviving WT, ∆Ex3 and survivin-2B mRNA was 88% (44/50), 76% (38/50), and 72% (36/50) respectively. Further correlation of expression of survivin splice variants with clinico-pathologic parameters in breast cancer patients indicated a stage and grade specific increase in survivin-wt mRNA, and stage and grade dependent change in expression for variants ∆Ex3 and 2B. ConclusionOur data demonstrates that differential expression of survivin splice variants is a key feature of breast cancer. Apart from survivin, targeting survivin splice variants can also be an important diagnostic and therapeutic target in cancer. Our findings may provide novel avenues for development of both diagnostics and biotheraepeutics in cancer.

Differential localization and high expression of SURVIVIN splice variants in human embryonic stem cells but not in differentiated cells implicate a role for SURVIVIN in pluripotency

The BIRC5 gene encodes the oncofetal protein SURVIVIN, as well as four additional splice variants (ΔEx3, 2B, 3B and 2α). SURVIVIN, an inhibitor of apoptosis, is also a chromosomal passenger protein (CPP). Previous results have demonstrated that SURVIVIN is expressed at high levels in embryonic stem cells and inhibition of SURVIVIN function results in apoptosis, however these studies have not investigated the other four splice variants. In this study, we demonstrate that all variants are expressed at significantly higher levels in human embryonic stem (hES) cells than in differentiated cells. We examined the subcellular localization of the three most highly expressed variants. SURVIVIN displayed canonical CPP localization in mitotic cells and cytoplasmic localization in interphase cells. In contrast, SURVIVIN–ΔEx3 and SURVIVIN–2B did not localize as a CPP; SURVIVIN–ΔEx3 was found constitutively in the nucleus while SURVIVIN–2B was distributed along the chromosomes during mitosis and also to the mitotic spindle poles. We used inducible shRNA against SURVIVIN to inhibit expression in a titratable fashion. Using this system, we reduced the mRNA levels of these three variants to approx. 40%, resulting in a concomitant reduction of OCT4 and NANOG mRNA, suggesting a role for the SURVIVIN variants in pluripotency.

BIRC5 (survivin) splice variant expression correlates with refractory disease and poor outcome in pediatric acute myeloid leukemia: A report from the Children's Oncology Group

The inhibitor-of-apoptosis protein survivin, encoded by BIRC5, regulates apoptosis, cell division and proliferation. Several survivin splice variants have been described however, the prognostic significance of their expression has not been well defined in pediatric acute myeloid leukemia (AML). Quantitative expression analyses of BIRC5 mRNA (n = 306) and survivin transcript splice variants (n = 90) were performed on diagnostic bone marrow samples from children with de novo AML treated on the clinical trials CCG-2961 and AAML03P1, then correlated with disease characteristics and clinical outcome. Total BIRC5 expression did not correlate with clinical outcome. Fragment length analysis and sequencing of the entire BIRC5 transcript demonstrated three splice variants. The most prominent product, wild-type survivin, was expressed in all samples tested. Two minor transcripts were present in 90 patients treated on CCG-2961; survivin-2B and a novel variant, survivin-ΔEx2, characterized by deletion of BIRC5 exon II. A high 2B/ΔEx2 expression ratio (≥1) correlated with increased diagnostic WBC count, monocytic phenotype, +8 cytogenetics, lower complete remission (45% [n = 10] vs. 88% [n = 59], P < 0.001) and higher induction failure rates (23% [n = 5] vs. 3% [n = 2], P = 0.009). Consistent with this poor induction response, patients with a 2B/ΔEx2 ratio ≥1 had inferior 5-year survival rates (OS 36% vs. 60%, P = 0.011; EFS 23% vs. 53% at 5 years, P = 0.001) and appear to have increased relapse risk (P = 0.056). Subset analyses suggest that relative over-expression of 2B, rather than under-expression of ΔEx2 determines clinical response. High survivin-2B/ΔEx2 ratios are associated with refractory disease and inferior survival in childhood AML. Survivin splice variant expression warrants prospective evaluation in clinical trials.

Clinico-pathologic relevance of Survivin splice variant expression in cancer

Survivin is a member of the inhibitor of apoptosis (IAP) family and has multifunctional properties that include aspects of proliferation, invasion and cell survival control. Survivin is a promising candidate for targeted cancer therapy as its expression is associated with poor clinical outcome, more aggressive clinico-pathologic features, and resistance to radiation and chemotherapy. In the present review the different properties of the Survivin splice variants are discussed and their activities correlated with different aspects of cancer cell biology, to include subcellular location. Special emphasis is placed on our current understanding of these Survivin splice variants influence on each other and on the phenotypic responses to therapy that they may control.

Survivin and laryngeal carcinoma prognosis: nuclear localization and expression of splice variants

Aberrant survivin expression in cancer cells has been associated with tumour progression, radiation/drug resistance and shorter patient survival. The aim of the present study was to investigate survivin expression in laryngeal carcinoma (LSCC) tissue and - for the first time at this site - the expression of survivin splice variants. P53 was also studied. Survivin and p53 expression was determined immunohistochemically in 86 consecutive patients operated for LSCC. Survivin mRNA expression was assessed by quantitative real-time polymerase chain reaction (PCR). Hot-spot mutations in exons 5, 6, 7 and 8 of the TP53 gene were studied by sequencing analysis. A nuclear localization for survivin predominated. There was a significant association between a higher nuclear survivin expression and LSCC recurrence (P = 0.046). Disease-free survival (DFS) for LSCC patients with a nuclear survivin expression >7.0% was shorter than in cases whose expression was ≤7.0% (P = 0.05). Wild-type survivin correlated significantly with nuclear survivin expression (P = 0.02). p53 expression was associated with the co-expression of wild-type survivin and survivin-2B (P = 0.01). Nuclear expression of survivin appears to influence LSCC aggressiveness, a higher nuclear survivin expression correlating with a higher recurrence rate and a shorter DFS. Wild-type survivin was the most frequently detected splice variant in LSCC tissues.

The survivin -31 snp in human colorectal cancer correlates with survivin splice variant expression and improved overall survival

Survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC) have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386 C/T and -31 G/C polymorphisms were investigated. Quantitative RT-PCR was used to assess mRNA levels in fresh/frozen tissue samples. Protein levels were immunohistochemically evaluated on formalin-fixed paraffin-embedded tissue sections. Individuals were genotyped using real time PCR. Expression of all 5 survivin splice variants as well as survivin protein was elevated in colorectal carcinomas compared to normal tissue. Specific splice variant expression differentially correlated with clinicopathological parameters. Furthermore, both snps correlated with splice variant levels or their ratios in colorectal carcinomas while the -31 G/C snp may be related to CRC development and improved overall survival. Our results support a role of survivin in colorectal carcinogenesis while the -31 G/C snp may constitute a marker of survival.

The Survivin −31 Snp in Human Colorectal Cancer Correlates with Survivin Splice Variant Expression and Improved Overall Survival

Background: Survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC) have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386C/T and −31G/C polymorphisms were investigated.Methods: Quantitative RT-PCR was used to assess mRNA levels in fresh/frozen tissue samples. Protein levels were immunohistochemically evaluated on formalin-fixed paraffin-embedded tissue sections. Individuals were genotyped using real time PCR.Results: Expression of all 5 survivin splice variants as well as survivin protein was elevated in colorectal carcinomas compared to normal tissue. Specific splice variant expression differentially correlated with clinicopathological parameters. Furthermore, both snps correlated with splice variant levels or their ratios in colorectal carcinomas while the −31G/C snp may be related to CRC development and improved overall survival.Conclusion: Our results support a role of survivin in colorectal carcinogenesis while the −31G/C snp may constitute a marker of survival.

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.

Association of p53 gene alterations with the expression of antiapoptotic survivin splice variants in breast cancer

Survivin, a member of the inhibitory apoptosis protein family, gives rise, by an alternative splicing, to four variants with different functions. Many experimental studies indicate that p53 can regulate the expression of survivin and some of its splice variants. Although both the expression of survivin splice variants and the p53 gene were frequently altered in human cancers, nothing is known about their interactions in in vivo tumour samples. Here, we report that, in 162 breast carcinomas, p53 mutations are significantly associated with an increased expression of survivin and, in particular, its antiapoptotic splice variants (survivin-DeltaEx3 and survivin-3B). The upregulation of these variant expressions is particularly related to p53 mutations occurring in the residues belonging to the tetramerization domain. The loss of heterozygosity in the p53 gene is also associated with an increased expression of the survivin-DeltaEx3 variant. The expression of the proapoptotic variants (survivin-2B and survivin-2alpha) is not affected by any of these alterations. Our results provide for the first time in vivo evidence that, in human breast cancer, the survivin expression as well as its splicing depends on the p53 status. The results also suggest that the upregulation of antiapoptotic survivin variant expression by the mutant p53 may increase breast cancer cells survival and resistance to therapy.

Distinct expression of Survivin splice variants in breast carcinomas

Survivin, a member of the inhibitor apoptosis family, is expressed in several human tumours, and its expression is regulated by p53. Recently, three alternative splice variants (Survivin-2B, Survivin-deltaEx3 and Survivin-3B), differing in their antiapoptotic properties, were identified. To date, little is known about the expression of all Survivin splice variants in breast cancer, particularly the recently identified Survivin-3B variant. In this study, we show that all Survivin transcripts were expressed in breast tumour cell lines and breast carcinomas, with a very weak expression detected in adjacent normal tissue. Frequency of proapoptotic Survivin-2B was significantly higher in small tumour size (p=0.026) and was inversely associated with axillary node positive carcinomas (p=0.004). In contrast, Survivin-3B was more frequent in high-grade carcinomas (p=0.004). Correlation with p53 status revealed that Survivin-3B was significantly more frequent in carcinomas with p53 gene mutation (p=0.036). After neoadjuvant chemotherapy, a significant reduction in the percentage of expressing Survivin (p=0.016) and Survivin-2B (p=0.027) was observed, while no change was found for Survivin-deltaEx3 and Survivin-3B variants. These results demonstrate for the first time that Survivin variants are differentially expressed in breast cancer according to tumour progression and treatment and suggest that Survivin-3B might act as an antiapoptotic factor in this lesion, with its expression regulated by p53.

Elevated expression of survivin-splice variants predicts a poor outcome for soft-tissue sarcomas patients

The aim of this study was to investigate the level and the prognostic value of the expression of different survivin transcript variants--survivin, survivin-DeltaEx3 and survivin-2B--in tumours of 76 soft tissue sarcoma (STS) patients. The expression of survivin transcript variants in STS tissue samples and in 12 nonmalignant control tissues was analysed by quantitative RT-PCRs. Expression levels of all survivin transcript variants were strongly elevated in STS compared to normal tissues. A positive correlation between expression of splice variants and tumour stage was found (P=0.02; chi2 test). The multivariate Cox's proportional hazards regression model revealed a 7.3-fold increased risk of tumour-related death for patients with survivin-DeltaEx3 overexpressing tumours (P=0.007). The effect of surivivin (wildtype variant) and survivin-2B was less pronounced but still significant (2.2- and 1.9-fold, resp., P<0.05 each). Our results show for the first time that mRNA expression of survivin-variants is significantly correlated to a poor prognosis for STS patients, and we suggest expression of survivin splice variants together with tumour stage as independent predictor of survival.

Survivin Overexpression in Hepatocellular Carcinoma Is Associated With p53 Dysregulation

Survivin is a recently described anti-apoptotic protein that is suppressed by wild-type p53 and is overexpressed in 41-70% of hepatocellular carcinomas from Asia. Two alternatively spliced transcripts have also been described: anti-apoptotic survivin-DeltaEx3 and non-anti-apoptotic survivin-2B. Survivin splice variant expression has not been studied in HCC, and little is known about survivin expression in hepatocellular carcinomas arising in other parts of the world, where risk factors are often different than they are in Asia. We studied survivin mRNA and protein expression in a United States cohort of hepatocellular carcinomas and correlated the findings with p53 immunopositivity. RT-PCR was performed for survivin, survivin-2B, and survivin-DeltaEx3 in 20 HCCs and one intrahepatic cholangiocarcinoma. Expression levels of total survivin were evaluated with real-time PCR. Protein expression was examined by immunohistochemistry. Survivin was the major transcript, and all transcripts were present in all normal and neoplastic tissues; 11/20 (55%) HCCs and the one cholangiocarcinoma showed twofold or greater overexpression of survivin. Next, we examined survivin and p53 protein expression by immunohistochemistry on a separate series of 79 HCC, 13 fibrolamellar carcinomas, and 15 hepatic adenomas; 14/79 (17%) HCC, but none of the fibrolamellar carcinomas or hepatic adenomas, showed survivin protein overexpression, and 25/79 HCC (32%) showed abnormal nuclear accumulation of p53, which correlated with increased survivin expression. All three survivin transcripts are present in normal liver and HCC. Survivin is the dominant transcript in HCC and is overexpressed in 55% of cases. Survivin protein overexpression is associated with aberrant p53 nuclear positivity.