Expression of survivin and its splice variants survivin-2B and survivin-DeltaEx3 in breast cancer

Alternative splicing of survivin mRNA gives rise to multiple isoforms, that is, survivin and 3 splice variants, survivin-2B, survivin-3B and survivin-DeltaEx3. The aim of this study was to compare the expression of survivin, survivin-2B and survivin-DeltaEx3 in normal breast tissue, fibroadenomas, primary breast cancer and axillary nodal metastases. Survivin, survivin-2B and survivin-DeltaEx3 mRNA were measured using semiquantitative RT-PCR. In the primary carcinomas, we related mRNA for each form of survivin to both survivin protein and apoptosis. For each type of breast tissue, survivin was the predominant form detected, being present in 146 out of 156 (93.6%) primary breast carcinomas, 11 out of 11 (100%) axillary nodal metastases, 21 out of 31 (67.7%) fibroadenomas and five out of 22 (22.7%) specimens of normal breast tissue. Levels of the three forms of survivin were significantly higher in the carcinomas compared to normal breast tissue (P < 0.0001). Levels of both survivin-2B and survivin-DeltaEx3 but not survivin were significantly higher in nodal metastases than primary carcinomas. Survivin mRNA levels correlated significantly with survivin protein. Finally, both survivin and survivin-DeltaEx3 but not survivin-2B correlated positively with apoptosis. Although survivin, survivin-2B and survivin-DeltaEx3 were all detected in both malignant and nonmalignant breast tissue, the predominant form was survivin. Our results suggest that the different forms of survivin may have different roles in apoptosis in breast cancer.     

Role of survivin and its splice variants in tumorigenesis

Survivin, a unique member of the inhibitor of apoptosis (IAP) protein family, is highly expressed in cancer but is undetectable in nonproliferating normal adult tissues, suggesting a potential role in tumorigenesis. Differential splicing of survivin pre-mRNA results in three new survivin variants, survivin-DeltaEx3, survivin-2B, and survivin-3B. Loss of survivin-2B expression was found in the later stage of cancer development, while survivin and survivin-DeltaEx3 are not, suggesting a differential role of them in tumour development. In this minireview, the author intends to summarise and discuss the current data relevant to the role of survivin and its splicing variants in tumorigenesis, which may facilitate further investigation in this interesting area.     

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.     

Survivin及其异构体survivin-2B和survivin-△Ex3在膀胱癌中的表达及临床意义

背景与目的：研究发现survivin基因参与了包括膀胱癌在内的人类多种肿瘤的发生。近期研究表明smwivin的异构体同样具有独特的亚分子定位和功能。本研究通过检测survivin及其异构体smwivin-2B和survivin-△Ex3 mRNA在膀胱癌组织中的表达,探讨这两个异构体潜存的临床意义。方法：选取手术切除的原发膀胱移行细胞癌（bladder transitional cell carcinoma,BTCC）新鲜标本60例,膀胱良性疾病患者病变膀胱组织标本12例作为对照。应用实时定量PCR方法检测survivin、survivin-2B和survivin-△Ex3 mRNA的表达量,分析其表达与不同病理分级和临床分期膀胱癌的关系。结果：Survivin、Survivin-2B和sunivin—△Ex3 mRNA在BTCC组织中均存在表达,表达量分别为0．333±0．163、0．056±0．017、0．124±0．096;对照组12例均表达survivin-2B mRNA,其中分别有3例千兀4例标本存在suwivin和survivin—△Ex3 mRNA的微量表达。survivin和survivin-△Ex3 mRNA的表达与病理分级和临床分期呈正相关（0〈r＇s〈1,P〈0．05）,survivin-2B呈负相关（-1〈r＇s〈0,P〈0．05）。3例复发患者原膀胱肿瘤组织中survivin mRNA的表达较相同病理级别者明显高,而survivin-2B却低。结论：检测survivin及其异构体survivin-2B和survivin-△Ex3 mRNA在BTCC组织中的表达,对判断膀胱肿瘤的恶性程度以及术后复发具有潜在的临床应用价值。     

Implication of alternative splice transcripts of caspase-3 and survivin in chemoresistance

Recent studies have shown that resistance to apoptosis may contribute to chemoresistance. Alteration of caspases, such as caspase-3, results on decreased apoptosis. Genes of IAP (inhibitor of apoptosis proteins) family, such as survivin, were also implicated in tumor development where they are mutated or have deregulated expression. Initial studies revealed strong survivin expression in several fetal tissues and some proliferating adult tissues, whereas no survivin expression was detected in a range of adult tissues. Although the factors at the origins on survivin re-expression in tumors are still unknown, the anti-apoptotic function of survivin is mediated in part by inhibiting caspase-3 activity. Recently, functionally divergent splice variants resulting from alternative splicing, with apoptotic (for caspase-3) or anti-apoptotic (for survivin) opposite activities have been described. The alternative splice variant, caspase-3s results from exon 6 deletion and shows antagonist of apoptotic property of caspase-3. Three alternative splice variants of survivin (survivin-DeltaEx3, survivin-2B and survivin-3B) differing in their anti-apoptotic properties were reported. While the anti-apoptotic effect of survivin-DeltaEx3 is preserved, survivin-2B has lost its anti-apoptotic potential and may act as a naturally occurring antagonist of survivin and survivin-DeltaEx3. At present, little is known about properties of survivin-3B. Several evidences indicate that in several cancers, the ratio of splice variants is significantly altered, and modifications of splicing pathways have been developed for cancer treatment. Recent investigations have shown that expression of alternative splice variants of caspase-3 and of survivin were also altered in many human cancers, and that variations in their expression were associated with tumor progression and chemoresistance. In this article, we describe recent data concerning alternative splice variants of these two proteins.     

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.     

An alternatively spliced survivin variant is positively regulated by p53 and sensitizes leukemia cells to chemotherapy

Survivin is a unique member of the inhibitor of apoptosis protein family, and its expression is regulated by p53. Recent identification of several functionally divergent survivin variants augments the complexity of survivin action as well as its regulation. Here we report that survivin-2B (retaining a part of intron 2 as a cryptic exon) is positively regulated by p53, and its overexpression plays a role in sensitizing leukemia cells to chemotherapeutic drug doxorubicin. Doxorubicin treatment activated p53, downregulated survivin and survivin-DeltaEx3 but upregulated survivin-2B in EU-3, an acute lymphocytic leukemia (ALL) cell line with wild-type (wt)-p53 phenotype. In contrast, doxorubicin treatment failed to induce these alterations in EU-6 cells, a mutant-p53 ALL cell line. To specify the role of wt-p53 in regulating survivin and its variants, a temperature-sensitive p53 mutant plasmid p53-143 was transfected into EU-4, a p53-null ALL cell line, to establish a subline EU-4/p53-143. When EU-4/p53-143 cell culture was shifted from 37.5 degrees C to the wt-p53-permissive temperature (32.5 degrees C), the expression of survivin and survivin-DeltaEx3 was decreased whereas survivin-2B expression was increased, confirming the distinct regulatory effect of p53 on survivin and its variants. To clarify the role of survivin-2B in the process of apoptosis, survivin-2B cDNA was cloned into pcDNA3HA vector and transfected into EU-4 cells. Enforced expression of survivin-2B in EU-4 cells inhibited cell growth and sensitized these cells to doxorubicin-induced apoptosis. These results suggest that survivin-2B variant is a proapoptotic factor and its expression is upregulated by p53.     

Differential expression of survivin-2B and survivin-DeltaEx3 is inversely associated with disease relapse and patient survival in non-small-cell lung cancer (NSCLC)

Although it was observed that inhibition of the antiapoptotic protein survivin expression in lung cancer cells induces apoptosis, the expression and role of survivin variants (survivin-2B and survivin-DeltaEx3) in lung cancer have not yet been characterized. We analyzed 24 non-small-cell lung cancer (NSCLC) samples by semi-quantitative RT-PCR. Surprisingly, our results revealed that high-level expression of survivin-2B is significantly associated with the patient category of "no relapse and alive" (p-value<0.0001). In contrast, high-level expression of survivin-DeltaEx3 is highly associated with the patient category of "relapse and dead" (p-value<0.0001). Consistent with this observation, exogenous expression of survivin-2B in A549 lung cancer cells inhibited cell growth, disrupted the mitochondria potential, and induced apoptotic cell death, while expression of survivin-DeltaEx3 protected the mitochondria potential and facilitated cell survival. These findings provide evidence that survivin-2B and survivin-DeltaEx3 play opposite roles in disease relapse and NSCLC cell survival, which is likely through the differential modulation of mitochondrial potential. Thus, controlling the differential expression of survivin-2B and survivin-DeltaEx3 may represent novel approaches for cancer therapeutics in NSCLC.     

An HLA-A24-restricted cytotoxic T lymphocyte epitope of a tumor-associated protein, survivin.

To date an increasing number of T-cell epitopes derived from various tumor-associated antigens have been reported, and they proved to play significant roles for tumor rejection both in vivo and in vitro. Survivin was originally identified as a member of the inhibitor of apoptosis protein family. Expression of this gene is developmentally regulated. Although survivin is expressed during normal fetal development, the expression is barely detected in terminally differentiated adult tissues except for testis, thymus, and placenta. In contrast, it is abundantly expressed in a wide variety of malignant tissues. We examined the expression of survivin and the two splicing variants survivin-2B and survivin-DeltaEx3 in various cancer cells, immortalized cells, and normal adult tissues. It was demonstrated that two splicing variants were detected in various types of cancer cells as well as survivin, and their expression was more restricted to cancer cells as compared with survivin expression. To identify HLA-A24-restricted T-cell epitopes from survivin and the variant proteins, three peptides were selected from amino acid sequence of these proteins, based on the HLA-A24-binding motif. Peptide binding assay to HLA-A24 revealed that only one peptide designated as survivin-2B80-88 (AYACNTSTL) was capable of binding to HLA-A24. By stimulating peripheral blood lymphocytes with the peptide-pulsed antigen-presenting cells, CTLs were successfully induced in vitro from five of five HLA-A24-positive cancer patients. The CTLs showed significant cytotoxicity against HLA-A24-positive survivin-2B-positive cancer cells. These data suggest that survivin-2B80-88 may be a potent T-cell epitope eliciting CTL response against a splicing variant survivin-2B, which is specifically expressed in many kinds of cancer cells.     

High expression levels of x-linked inhibitor of apoptosis protein and survivin correlate with poor overall survival in childhood de novo acute myeloid leukemia.

PURPOSE: Apoptosis-related proteins are important molecules for predicting chemotherapy response and prognosis in adult acute myeloid leukemia (AML). However, data on the expression and prognostic impact of these molecules in childhood AML are rare. EXPERIMENTAL DESIGN: Using flow cytometry and Western blot analysis, we, therefore, investigated 45 leukemic cell samples from children with de novo AML enrolled and treated within the German AML-BFM93 study for the expression of apoptosis-regulating proteins [CD95, Bcl-2, Bax, Bcl-xL, procaspase-3, X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein-1 (cIAP-1), survivin]. RESULTS: XIAP (P < 0.002) but no other apoptosis regulators showed maturation-dependent expression differences as determined by French-American-British (FAB) morphology with the highest expression levels observed within the immature M0/1 subtypes. XIAP (P < 0.01) and Bcl-xL (P < 0.01) expression was lower in patients with favorable rather than intermediate/poor cytogenetics. After a mean follow-up of 34 months, a shorter overall survival was associated with high expression levels of XIAP [30 (n = 10) versus 41 months (n = 34); P < 0.05] and survivin [27 (n = 10) versus 41 months (n = 34); P < 0.05]. CONCLUSIONS: We conclude that apoptosis-related molecules are associated with maturation stage, cytogenetic risk groups, and therapy outcome in childhood de novo AML. The observed association of XIAP with immature FAB types, intermediate/poor cytogenetics, and poor overall survival should be confirmed within prospective pediatric AML trials.     

Chk2 phosphorylation of survivin-DeltaEx3 contributes to a DNA damage-sensing checkpoint in cancer

Survivin is an oncogene that functions in cancer cell cytoprotection and mitosis. Here we report that differential expression in cancer cells of a C-terminal splice variant of survivin, termed survivin-ΔEx3, is tightly associated with aggressive disease and markers of unfavorable prognosis. In contrast to other survivin variants, survivin-ΔEx3 localized exclusively to nuclei in tumor cells and was phosphorylated at multiple residues by the checkpoint kinase Chk2 during DNA damage. Mutagenesis of the Chk2 phosphorylation sites enhanced the stability of survivin-ΔEx3 in tumor cells, inhibited the expression of phosphorylated H2AX (γH2AX) in response to double-strand DNA breaks, and impaired growth after DNA damage. DNA damage induced Chk2 phosphorylation, stabilization of p53, induction of the cyclin-dependent kinase inhibitor p21, and homologous recombination-induced repair were not affected. In vivo, active Chk2 was detected at the earliest stages of the colorectal adenoma-to-carcinoma transition, persisted in advanced tumors, and correlated with increased survivin expression. Together, our findings suggest that Chk2-mediated phosphorylation of survivin-ΔEx3 contributes to a DNA damage-sensing checkpoint that may affect cancer cell sensitivity to genotoxic therapies.     

乳腺癌中survivin表达的预后价值

目的：研究凋亡抑制基因survivin在乳腺癌中表达的预后价值。方法：采用免疫组化SP法检测180例乳腺癌石蜡标本中survivin表达情况,并对survivin表达与乳腺癌的临床分期、雌激素受体表达、月经状况、腋淋巴结转移、病理类型、无病生存时间以及总生存时间的关系进行了分析。结果：Survivin在乳腺癌中的阳性表达率为66.1%,survivin的表达与乳腺癌的临床分期、雌激素受体、月经状况、腋淋巴结是否转移及病理类型无关（P〉0.05）;单因素分析结果表明survivin表达与乳腺癌病人的无病生存时间和总生存时间显著相关,survivin表达阳性病人的无病生存时间和总生存时间均显著低于survivin表达阴性的病人;多因素Cox回归分析结果表明survivin的表达状况是决定乳腺癌病人无病生存时间的独立因素,而与总生存时间无关。结论：Survivin是判断乳腺癌预后的一个独立生物学指标,阳性表达病人的预后较阴性表达病人差。     

Nuclear survivin expression is associated with HPV-independent carcinogenesis and is an indicator of poor prognosis in oropharyngeal cancer

The relationship between expression of the inhibitor of apoptosis protein survivin and the presence of high-risk human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OSCC) remains unclear. This also accounts for its role as a predictor of survival. Therefore, we conducted a multicentre retrospective study on 106 consecutive oropharyngeal cancer patients. Human papillomavirus sequences were detected by nested PCR protocols. Survivin and p16 expression as a surrogate marker for HPV status were analysed by immunohistochemistry. Sequences of high-risk HPV were detected in 29% of cases. Prominent cytoplasmatic expression of survivin was found in 58% of cases and nuclear expression of survivin was found in 19% of the survivin-positive tumours. Nuclear expression of survivin was significantly correlated with HPV-negative tumours (P=0.023) and with a poor disease-free survival rate with an estimated 3-year disease-free survival probability of 35% for tumours with nuclear expression of survivin vs 78% for tumours with non-nuclear expression of survivin (hazard ratio=8.264; 95% confidence interval (95% CI)=2.510-27.210; P<0.001). In multivariate analysis, p16 expression status as well as nuclear expression of survivin were strong independent and opposing prognostic indicators of disease-free survival (hazard ratio=0.068; 95% CI=0.005-0.892; P=0.041 and hazard ratio=15.975; 95% CI=2.377-107.360; P=0.004, respectively). Our data show that nuclear accumulation of survivin correlates with HPV-independent carcinogenesis and is an independent predictor of poor survival in patients with OSCC.     

Survivin-deltaEx3 and survivin-2B: two novel splice variants of the apoptosis inhibitor survivin with different antiapoptotic properties

Recently, a novel antiapoptosis gene, i.e., survivin, was identified as a structurally unique member of the inhibitor of apoptosis protein family. Survivin expression is turned off during fetal development and not found in non-neoplastic adult human tissues but is again turned on in the most common human cancers. The antiapoptotic properties of survivin might provide a significant growth advantage in tumors and possibly also contribute to chemoresistance of cancer. Therefore, we analyzed the expression of survivin in human renal cell carcinomas (RCCs), known to be largely resistant to chemotherapy. Northern blot analysis and RT-PCR revealed survivin expression in newly established RCC cell lines (n = 11) of all major histological types. Moreover, we identified two novel splice variants of survivin, lacking exon 3 (survivin-deltaEx3) or retaining a part of intron 2 as a cryptic exon (survivin-2B). Both sequence alterations cause marked changes in the structure of the corresponding proteins, including structural modifications of the baculovirus inhibitor of apoptosis protein repeat domain. The role of the novel isoforms in the regulation of apoptosis was assessed in transfection experiments, showing conservation of antiapoptotic properties for survivin-deltaEx3 and a markedly reduced antiapoptotic potential for survivin-2B. In conclusion, our observations suggest a complex regulatory balance between the different isoforms of survivin, which might determine the response to proapoptotic stimuli, not only in human RCCs but also in fetal tissues and other types of cancer.