Regulation of Heat-Induced Apoptosis by Loss of miR-23a-Dependent Control of Noxa in Human Cells and its Suppression by HSP70

Protein damaging stress such as hyperthermia can activate a process of cellular destruction known as apoptosis. Exposure to hyperthermia can also induce the synthesis of heat shock proteins including HSP70, which can protect cells from stress-induced apoptosis. Apoptosis is controlled by pro- and anti-apoptotic members of the Bcl-2 family. The pro-apoptotic BH3-only protein NOXA activates this death pathway by inhibiting the anti-apoptotic Bcl-2 family protein MCL-1. In this thesis we sought to investigate (i) whether Noxa mRNA levels are regulated post-transcriptionally by miRNA-mediated suppression, (ii) if expression of this miRNA is altered in heat stressed cells and (iii) if the effect of hyperthermia on expression of this miRNA is affected by HSP70 levels. We found that the miRNA miR-23a binds the Noxa mRNA 3’ UTR and suppresses translation. miR-23a has a short half-life, which is reduced in heat shocked cells leading to Noxa mRNA stabilization and increased NOXA protein expression. Cells overexpressing HSP70 contain elevated levels of miR-23a due to a reduced turnover rate. miR-23a levels remain elevated in HSP70 expressing cells exposed to hyperthermia and consequently NOXA protein levels are not increased and apoptosis is suppressed. This thesis also provides evidence that CDK5, an atypical cyclin-dependent kinase, regulates the expression of miR-23a. Hyperthermic treatment led to a loss of CDK5 tyrosine-15 phosphorylation, which was prevented by HSP70 overexpression. Treatment with the CDK5 inhibitor roscovitine caused an increase in NOXA protein levels and sensitized cells to heat-induced apoptosis. The accumulation of NOXA was not due to an effect of CDK5 on the stability of NOXA since its half-life was not affected in roscovitine-treated cells or in cells expressing a dominant-negative CDK5. Instead, CDK5 inhibition resulted in a decrease in miR-23a levels causing Noxa mRNA levels to accumulate. Cells overexpressing miR-23a were found to be resistant to roscovitine induced cell death. All together, these results indicate that HSP70 inhibits stress-induced apoptosis by maintaining miR-23a levels and thereby preventing NOXA accumulation. We suggest that HSP70 could be beneficial to tumor cells by helping to maintain the expression of oncogenic miRNAs under conditions of cellular stress.