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

Background: Pancreatic 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. Methods: To 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 2ß. Results: The 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 2ß and a 2- to 8-fold increase in the other five SSVs in the GR cells. Knockdown of all SSV or SSV 2ß only, using small inhibitory RNA (siRNA), sensitized the GR cells to Gem, indicating that these SSVs play a role in PDAC chemoresistance. Conclusion: These findings provide evidence for the potential role of SSV 2ß 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.

Overexpression of survivin in pediatric Hodgkin lymphoma tumor cells: Characterization of protein expression and splice-variants transcription profile.

Survivin is abundantly expressed during fetal development but absent in most differentiated adult tissues; an exception being components of the immune system, such as B and T lymphocytes. Beyond acting as a master regulator of the cell cycle, survivin acts as an inhibitor of apoptosis and is overexpressed in almost all carcinoma types; however, its expression in lymphomas is lesser-explored. Survivin's role in carcinogenesis was subjected to its sub-cellular localization and splice transcripts expression, namely wild-type survivin, survivin-∆Ex3 and survivin-2B. To assess survivin's expression and sub-cellular localization in Epstein Barr virus positive and negative biopsies from treatment naïve pediatric patients with Hodgkin lymphoma (HL), samples were stained for survivin protein by immunofluorescence. The proportion of survivin+ cells was calculated, survivin sub-cellular localization assessed and its fluorescence intensity quantified. Transcription profile of survivin mRNA variants was studied by RT-qPCR. Survivin was overexpressed in the nucleus of tumor cells, and also in a greater proportion of tumor cells, in comparison with the non-tumoral infiltrating cells. Although a higher expression of survivin was observed in advanced clinical stages, no correlation was found between the expression level of survivin and a proliferation marker, or event-free survival. Instead, survivin was related to apoptosis inhibition in tumor cells. Additionally, survivin's transcriptional variants displayed similar expression levels. Present results suggest that although survivin is overexpressed in Hodgkin's tumor cells, it may not play a central role in the progression of classic HL, or act as a suitable progression biomarker, as suggested for most carcinomas.

Survivin Splice Variants in Arsenic Trioxide (As2O3)-Induced Deactivation of PI3K and MAPK Cell Signalling Pathways in MCF-7 Cells.

Several pathways are deregulated during carcinogenesis but most notably, tumour cells can lose cell cycle control and acquire resistance to apoptosis by expressing a number of anti-apoptotic proteins such as the Inhibitors of Apoptosis Protein (IAP) family of proteins that include survivin, which is implicated in cancer development. There is no study which had proven that arsenic trioxide (As2O3) has any effect on the splicing machinery of survivin and its splice variants, hence this study was aimed at determining the cytotoxic effect of As2O3 and its effect on the expression pattern of survivin splice variants in MCF-7 cells. As2O3 inhibited the growth of the MCF-7 cells in a concentration-dependent manner. The Muse® Cell Analyser showed that As2O3-induced G2/M cell cycle arrest, promoted caspase-dependent apoptosis without causing any damage to the mitochondrial membrane of MCF-7 cells. As2O3 also deactivated two survival pathways, Mitogen-Activated Protein Kinase (MAPK) and Phosphoinositide 3-Kinase (PI3K) signalling pathways in MCF-7 cells. Deactivation of the two pathways was accompanied by the upregulation of survivin 3α during As2O3-induced G2/M cell cycle arrest and apoptosis. Survivin 2B was found to be upregulated only during As2O3-induced G2/M cell cycle arrest but downregulated during As2O3-induced apoptosis. Survivin wild-type was highly expressed in the untreated MCF-7 cells, the expression was upregulated during As2O3-induced G2/M cell cycle arrest and it was downregulated during As2O3-induced apoptosis. Survivin variant ΔEx3 was undetected in both untreated and treated MCF-7 cells. Survivin proteins were localised in both the nucleus and cytoplasm in MCF-7 cells and highly upregulated during the As2O3-induced G2/M cell cycle arrest, which can be attributed to the upregulation of survivin-2B. This study has provided the first evidence showing that the novel survivin 2B splice variant may be involved in the regulation of As2O3-induced G2/M cell cycle arrest only. This splice variant can therefore, be targeted for therapeutic purposes against Luminal A breast cancer cells.

Validation of a real-time quantitative polymerase chain reaction method for the quantification of 3 survivin transcripts and evaluation in breast cancer tissues.

BACKGROUND: Survivin is a novel antiapoptotic gene, which is a member of the inhibitor of apoptosis protein (IAP) family. Recently, 3 splice variants of this gene were cloned and characterized. This study aimed to validate a sensitive and specific method for the detection of survivin variants in breast cancer. METHODS: Real-time quantitative polymerase chain reaction (qPCR) was performed on the cDNA with a reverse primer specific for each splice variant and a pair of common hybridization probes. RESULTS: The expression of wild-type survivin was significantly correlated with survivin-2b, survivin-ΔEx3, and the ratio of survivin-ΔEx3 to wild-type survivin (P < .001). The ratio of survivin-2b to wild-type survivin was strongly associated with the ratio of survivin-ΔEx3 to wild-type survivin (P < .001). There was a strong positive association between the grade of the tumor and survivin-2b mRNA, survivin-ΔEx3 mRNA, and the ratio of survivin-ΔEx3 to wild-type survivin mRNA (P < .05). The ratio of survivin-2b to wild-type survivin was significantly associated with the presence of estrogen receptors (P = .05). CONCLUSION: Our validated data suggest that survivin isoforms may be related to clinicopathological features and could be used as molecular prognostic tools or as new therapy targets.

(-)-Epigallocatechin-3-gallate Modulates the Differential Expression of Survivin Splice Variants and Protects Spermatogenesis During Testicular Torsion.

The anti-apoptotic effect of (-)-epigallocatechin-3-gallate (EGCG) during unilateral testicular torsion and detorsion (TT/D) was established in our previous study. In mice, the smallest inhibitor of apoptosis, survivin, is alternatively spliced into three variants, each suggested to have a unique function. Here, we assessed how EGCG exerts its protective effect through the expression of the different survivin splice variants and determined its effect on the morphology of the seminiferous tubules during TT/D. Three mouse groups were used: sham, TT/D+vehicle and TT/D treated with EGCG. The expression of the survivin variants (140 and 40) and other apoptosis genes (p53, Bax and Bcl-2) was measured with semi-quantitative RT-PCR. Histological analysis was performed to assess DNA fragmentation, damage to spermatogenesis and morphometric changes in the seminiferous tubules. In the TT/D+vehicle group, survivin 140 expression was markedly decreased, whereas survivin 40 expression was not significantly different. In parallel, there was an increase in the mRNA level of p53 and the Bax to Bcl-2 ratio in support of apoptosis induction. Histological analyses revealed increased DNA fragmentation and increased damage to spermatogenesis associated with decreased seminiferous tubular diameter and decreased germinal epithelial cell thickness in the TT/D+vehicle group. These changes were reversed to almost sham levels upon EGCG treatment. Our data indicate that EGCG protects the testis from TT/D-induced damage by protecting the morphology of the seminiferous tubules and modulating survivin 140 expression.

(-)-Epigallocatechin-3-gallate Modulates the Differential Expression of Survivin Splice Variants and Protects Spermatogenesis During Testicular Torsion

The anti-apoptotic effect of (-)-epigallocatechin-3-gallate (EGCG) during unilateral testicular torsion and detorsion (TT/D) was established in our previous study. In mice, the smallest inhibitor of apoptosis, survivin, is alternatively spliced into three variants, each suggested to have a unique function. Here, we assessed how EGCG exerts its protective effect through the expression of the different survivin splice variants and determined its effect on the morphology of the seminiferous tubules during TT/D. Three mouse groups were used: sham, TT/D+vehicle and TT/D treated with EGCG. The expression of the survivin variants (140 and 40) and other apoptosis genes (p53, Bax and Bcl-2) was measured with semi-quantitative RT-PCR. Histological analysis was performed to assess DNA fragmentation, damage to spermatogenesis and morphometric changes in the seminiferous tubules. In the TT/D+vehicle group, survivin 140 expression was markedly decreased, whereas survivin 40 expression was not significantly different. In parallel, there was an increase in the mRNA level of p53 and the Bax to Bcl-2 ratio in support of apoptosis induction. Histological analyses revealed increased DNA fragmentation and increased damage to spermatogenesis associated with decreased seminiferous tubular diameter and decreased germinal epithelial cell thickness in the TT/D+vehicle group. These changes were reversed to almost sham levels upon EGCG treatment. Our data indicate that EGCG protects the testis from TT/D-induced damage by protecting the morphology of the seminiferous tubules and modulating survivin 140 expression.

Survivin splice variants are not essential for mitotic progression or inhibition of apoptosis induced by doxorubicin and radiation.

Survivin is a critical regulator of mitosis, and an inhibitor of apoptosis which is overexpressed in almost all cancers. In the current study, cell cycle profiles of normal proliferating human umbilical vein endothelial cells, prostate cancer, and lung cancer cell lines expressing varying levels of survivin and its splice variants were compared using a novel functional complementation assay. Defects in chromosome segregation and cytokinesis that were observed after depletion of endogenous survivin were not complemented by any of the survivin splice variants: survivin-2B, survivin-3B, survivin-ΔEx3, or survivin-2A when expressed exogenously at a level comparable to endogenous full-length survivin. Survivin variants were not detectable at the endogenous protein level. Cancer cells with higher levels of full-length survivin and survivin-2B expression, exhibited reduced caspase-3 activation following doxorubicin treatment and radiation. Whereas earlier studies focused on function and expression levels of survivin specific to cancer cells, the current study brings forward the essential role of survivin in normal dividing cells. Full-length survivin was found to be associated with Aurora-B kinase in the chromosomal passenger complex and depletion of survivin mimics mitotic phenotypes observed after Aurora-B kinase inhibition, in cancer as well as normal proliferating cells. Thus, our study establishes survivin as a marker of proliferation, rather than a cancer specific marker. Therefore, systemic therapeutic interventions targeting survivin will affect cancer as well as normal proliferating cells.