| PurposeThe purpose of this thesis is to study the role of Aurora A kinase (AurA) and survivin in gastric cancer drug resistance particularly DNA damaging agent and to study underlying mechanism of AurA mediated survivin protein stabilization.BackgroundGastric cancer is a malignant tumor arising from the epithelial lining of stomach. Although its incidence have seen a decline in West, it is still more prevalent in Asia and remains the fourth major cause of cancer related mortalities. It is an aggressive disease with major health burden as it responds poorly to existing remedies if diagnosed in advanced stage. Prognosis of patients with advanced gastric cancer remains poor with 5-year survival rate <10% and median overall survival of less than one year.Cancer treatment using existing therapies has a little potential to cure the disease due to intrinsic or acquired drug resistance of tumor cells. Cancer cells aberrantly express a broad array of genes that makes them self-sufficient in growth factor signaling and help them bypass drug-damage induced cell cycle checkpoints.Mitotic kinase AurA belongs to a conserved serine/thereonine kinase family and has been frequently amplified and/or overexpressed in different cancers including gastric cancer. It is a putative oncogene that has the capacity to transform certain cell lines when overexpressed. Human survivin is the smallest member of inhibitor of apoptosis protein family with elevated expression in most of the solid and hematological malignancies. Both AurA and cytoplasmic survivin expression has been associated with poor clinical outcome, drug resistance. Moreover, AurA can be targeted by small molecule inhibitors whereas survivin still remains non-druggable due to its structural features.Experimental ProceduresThis thesis is mainly comprised of four parts. In the first part, gastric cancer tissue samples were analyzed for AurA expression and its association with clinicopathological features. This was achieved by recruiting a retrospective cohort of 240 gastric patients analyzing their tissues for AurA protein level. Clinical data of relevant patients was collected and analyzed for correlation between AurA and gastric cancer clinicopathological features.The second part of this study is focused on role of AurA in gastric cancer DNA damage. DNA damage was induced in gastric cells by doxorubicin followed by determination of DNA-damage induced yH2AX foci formation and AurA activation.The third part of this thesis comprises of experiments unraveling survivin as a downstream target of AurA. Moreover, effect of AurA and survivin on gastric cancer cells drug sensitivity as well as effect of AurA depletion, overexpression, and kinase inhibition were evaluated on survivin protein level.Final part of this study dissects the mechanism for AurA mediated survivin protein stabilization. A series of experiments were performed including stable knockdown of AurA in gastric cancer cells followed by analyzing survivin mRNA and protein level, cell-cycle synchronization followed by AurA and survivin protein analysis, survivin mRNA interaction with cytoplasmic polyadenylation element binding protein 1 (CPEB1), inhibition of mammalian target of rapamycin and survivin protein level, effect of AurA on survivin post-translational modifications, AurA mediated inhibition of survivin ubiquitylation, unraveling a putative E3 ligase for survivin degradation and revealing signaling cascade beginning from AurA to survivin protein stabilization.ResultsIn an immunohistochemistry screen of 240 gastric cancer tissue specimens, we found 71.7% cases had high AurA expression. These results were corroborated with Western blot analysis of AurA expression in a randomly selected set of tumor tissues specimen compared to adjacent normal tissues. The AurA positivity was correlated with tumor stage (p=0.006), clinical stage (p=0.002), lymph node metastasis (p=0.007) and distant metastasis (p<0.0001). High AurA expressing gastric cancer patients showed significantly shorter overall survival (DFS, p=0.001; OS, p<0.001) compared to low AurA expressing gastric cancer patients.Furthermore, we show for the first time that AurA protected gastric cancer cells from genotoxic effects of doxorubicin. AurA overexpressing cells showed less sensitivity to doxorubicin whereas AurA depleted cells showed an enhanced sensitivity to doxorubicin underscoring pivotal role of AurA in overriding DNA damage checkpoints and sustaining cells survival even in the presence of doxorubicin.Interestingly, AurA kinase inhibition decreased survivin protein expression in a dose dependent manner. In addition, AurA depletion led to decreased survivin protein level whereas AurA overexpression enhanced survivin protein level. These results were corroborated with immunohistochemistry analysis of 62 pairs of gastric cancer specimens that found AurA expression positively correlated with survivin expression (p=0.001). Moreover, elevated survivin expression was correlated with reduced sensitivity to DNA damaging agent while survivin depletion exacerbated gastric cancer cell death compared with control cells suggesting its potential role in cancer drug resistance. AurA enhance survivin T34 phosphorylation which is required for proper survivin function. In addition, small molecule Aura kinase inhibitor and doxorubicin synergistically diminished survivin protein suggesting potential use of combination therapy for targeting survivin in gastric cancer patients.While studying the mechanism of AurA mediated survivin protein stabilization, we found that both AurA and survivin were expressed in gastric cancer cell lines in a cell cycle dependent manner. AurA stabilized survivin protein level by activating ERK1/2 kinase and inhibiting ubiquitin mediated survivin proteosomal degradation. Finally, we found that NEDD4L was downstream mediator of AurA and it acted as novel E3 ligase for survivin ubiquitylation in AurA depleted experimental settings.ConclusionOur data suggested that AurA stabilized survivin by upregulating ERK1/2 signaling and downregulating E3 ubiquitin ligase, NEDD4L in gastric cancer cells. These findings pave way for including small molecule AurA inhibitor to target both AurA and survivin hence promoting the efficacy of DNA damaging agents for gastric cancer management. |
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