Abstract
BackgroundSurvivin, belonging to the inhibitor of apoptosis (IAP) gene family, is abundantly expressed in tumors. It has been hypothesized that Survivin facilitates carcinogenesis by inhibition of apoptosis resulting in improved survival of tumorigenic progeny. Additionally, Survivin plays an essential role during mitosis. Together with its molecular partners Aurora B, Borealin and inner centromere protein it secures bipolar chromosome segregation. However, whether increased Survivin levels contribute to progression of tumors by inducing chromosomal instability remains unclear.MethodsWe overexpressed Survivin in U251-MG, SVGp12, U87-MG, HCT116 and p53-deficient U87-MGshp53 and HCT116p53−/− cells. The resulting phenotype was investigated by FACS-assisted cell cycle analysis, Western Blot analysis, confocal laser scan microscopy, proliferation assays, spectral karyotyping and in a U251-MG xenograft model using immune-deficient mice.ResultsOverexpression of Survivin affected cells with knockdown of p53, cells harboring mutant p53 and SV40 large T antigen, respectively, resulting in the increase of cell fractions harboring 4n and >4n DNA contents. Increased γH2AX levels, indicative of DNA damage were monitored in all Survivin-transduced cell lines, but only in p53 wild type cells this was accompanied by an attenuated S-phase entry and activation of p21waf/cip. Overexpression of Survivin caused a DNA damage response characterized by increased appearance pDNA-PKcs foci in cell nuclei and elevated levels of pATM S1981 and pCHK2 T68. Additionally, evolving structural chromosomal aberrations in U251-MG cells transduced with Survivin indicated a DNA-repair by non-homologous end joining recombination. Subcutaneous transplantation of U251-MG cells overexpressing Survivin and mycN instead of mycN oncogene alone generated tumors with shortened latency and decreased apoptosis. Subsequent SKY-analysis of Survivin/mycN-tumors revealed an increase in structural chromosomal aberrations in cells when compared to mycN-tumors.ConclusionsOur data suggest that increased Survivin levels promote adaptive evolution of tumors through combining induction of genetic heterogeneity with inhibition of apoptosis.
Highlights
Survivin, belonging to the inhibitor of apoptosis (IAP) gene family, is abundantly expressed in tumors
Our results show that overexpression of Survivin lead to a significant increase in the fraction of cells experiencing mitotic defects and DNA damage in p53 wild type cells (U87-MG, HCT116), Simian virus 40 (SV40) large T antigen (SV40 TAg) expressing cells (SVGp12), mutant p53 cells (U251-MG) and cells with knockdown of p53 (U87-MGshp53, HCT116p53−/−)
Overexpression of Survivin leads to an increase of cells with mitotic defects In order to corroborate whether increased levels of Survivin affects mitosis and eventually leads to chromosomal instability, we generated lentiviral vectors for expression of the 142 amino acid coding sequence of Survivin fused to c-myc and HA-tags and used the empty vector as control (Fig. 1a)
Summary
Survivin, belonging to the inhibitor of apoptosis (IAP) gene family, is abundantly expressed in tumors. Whether increased Survivin levels contribute to progression of tumors by inducing chromosomal instability remains unclear. In particular chromosomal instability (CIN) contributes to the development of genetic heterogeneity in tumors and allows the outgrowth of tumorigenic cells with advantageous karyotypes [2]. Survivin, which is found overexpressed in cancer tissues [3], might play a pivotal role in this kind of adaptive evolution of tumors since it exerts dual function in apoptosis and mitosis [4,5,6]. BIRC5, the gene encoding Survivin is transcriptionally repressed by p53 [8, 9]. That loss of functional p53 either due to mutations or its increased mdm2-mediated degradation caused by deregulated signaling pathways in glioblastoma (i.e. loss of PTEN, enhanced PI3K-signaling, for reviewing see [10]) increases a tumorpromoting activity of Survivin
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