PO-510 XRCC6BP1: a novel role in the DNA repair of platinum resistant NSCLC cells

Abstract

IntroductionAlterations in the DNA repair capacity of damaged cells are now recognised as an important factor in mediating resistance to chemotherapeutic agents.Material and methodsDNA Repair Pathway RT2 Profiler Arrays were used to elucidate key DNA repair genes implicated in chemoresistant NSCLC cells using cisplatin resistant (CisR) and corresponding parental (PT) NSCLC cell lines generated in our laboratory. DNA repair genes significantly altered in CisR cells were validated at the mRNA and protein level. The translational relevance of differentially expressed genes was examined in a cohort of chemo-naïve matched normal and tumour lung tissues from NSCLC patients. Loss of function studies were carried out using siRNA technology. The effect of XRCC6BP1 gene knockdown on apoptosis was assessed by FACS. Cellular expression and localization of XRCC6BP1 protein and gH2AX foci in response to cisplatin were examined by immunofluorescence (Cytell). To investigate a role for XRCC6BP1 in lung cancer stem cells, Side Population (SP) studies were used to characterise stem-like subpopulations within chemoresistant cells. XRCC6BP1 mRNA analysis was also examined in ALDH1+ and ALDH1- subpopulations. Immunohistochemistry analysis was carried out in resected lung tumour tissues and XRCC6BP1 expression was correlated with survival and other clinicopathological parameters.Results and discussionsWe identified a number of critical DNA repair genes that were differentially expressed between PT and CisR NSCLC cells. XRCC6BP1 mRNA and protein expression was significantly increased in H460 CisR cells relative to their PT counterparts. Relative to matched normal lung tissues, XRCC6BP1 mRNA was significantly increased in lung adenocarcinoma patients. Gene silencing of XRCC6BP1 induced significant apoptosis of chemoresistant cells and reduced their DNA repair capacity. Immunofluorescence studies showed an increase in XRCC6BP1 protein expression and gH2AX foci in CisR cells. SP analysis revealed a significantly higher stem cell population in resistant cells, while XRCC6BP1 mRNA expression was considerably increased in SKMES-1, H460 and H1299 ALDH1+ CisR cells compared to ALDH1- cells. IHC scoring of XRCC6BP1 showed that lung cancer patients with high expression had worse survival outcomes.ConclusionOur data highlight the potential of targeting components of the DNA repair pathway in chemoresistant lung cancer, in particular XRCC6BP1, either alone or in combination with conventional cytotoxic therapies such as cisplatin.