AbstractResistance to radiotherapy is a significant cause of treatment failure and tumour relapse. Therefore, strategies to improve the efficiency of cell death induction by ionizing radiation (IR) are required to improve therapeutic outcomes. FLIP, anendogenous caspase-8 inhibitor is a critical regulator of cell death that is frequently overexpressed in various cancers. FLIP overexpression in cancer contributes to resistance to death receptor- and chemotherapy- induced cell death. Thus, this Thesis investigated the role of FLIP in regulating resistance to IR. FLIP protein levels were down regulated by RNA interference or stably overexpressed by retroviral methods, and resulted in enhanced radio protection and radio resistance respectively. The effect of FLIP modulation on IR-induced cell death was assessed by caspase activity assays, short-term cell death and clonogenic survival assays. Overexpression of a FLIP mutant which cannot interact with its binding partner FADD, and thus cannot inhibit caspase-8 activation, did not protect cells from IR induced cell death, indicating that the FLIP-FADD protein-protein interaction and consequential inhibition of caspase-8 activity is mainly responsible for radio protection conferred by FLIP overexpression. This study provides convincing evidence indicating that FLIP should be investigated in clinical samples as a predictive biomarker of resistance to radiotherapy.
Clinically approved HDAC inhibitors were used to down regulate FLIP protein levels, and the effect on IR-induced cell death was assessed. The results presented in this study reveal that F1DAC inhibitors sensitize non-small cell lung carcinoma (NSCLC) cell lines to IR. Furthermore, the radiosensitizing properties of HDAC inhibitors were highly dependent on their ability to suppress FLIP expression.Therefore. FLIP down-regulation induced by HDAC inhibitors is a potential clinical strategy to radiosensitize and improve control of non-small cell lung carcinoma tumours, and thereby increase the poor survival rates of patients.
The levels of FLIP protein localized within the nucleus increased following IR.suggesting that FLIP has a nuclear role in the response to DNA damage, independent from caspase-inhibition at the membrane-bound death-inducing signalling complex, FLIP overexpression resulted in aberrant constitutive activation of the DNA damage response and increased the levels of endogenous DNA damage in untreated cells.Thus. FLIP overexpressing cells may have increased levels of genomic instability,which fuels cancer development and may also contribute to resistance to radio- and chemotherapy. Loss of FLIP resulted in spontaneous and enhanced IR-induced DN Adamage, which was partially independent of caspase activity; thus, agents which downregulate FLIP expression may also sensitize NSCLC cells to IR by enhancing DNA damage.
Overall, this study provides key evidence that pharmacological inhibition of FLIPmay improve the response of NSCLC to IR and enable successful control of moreradiation-resistant subpopulations of tumour cells.
|Date of Award||Jul 2016|
|Supervisor||Daniel Longley (Supervisor) & Kevin Prise (Supervisor)|