Virtual Library
Start Your Search
N. Leonard
Author of
-
+
P2.03a - Poster Session with Presenters Present (ID 464)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
-
+
P2.03a-062 - Characterisation and Targeting of the DNA Repair Gene, XRCC6BP1, in Cisplatin Resistant NSCLC (ID 5198)
14:30 - 14:30 | Author(s): N. Leonard
- Abstract
Background:
In the absence of specific treatable mutations, platinum-based doublet chemotherapy remains the gold standard treatment for NSCLC patients. However, its clinical efficacy is hindered in many patients due to intrinsic and acquired resistance to these agents, in particular cisplatin. Alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon.
Methods:
DNA Repair Pathway RT[2 ]Profiler Arrays were used to elucidate key DNA repair genes implicated in chemoresistant NSCLC cells using cisplatin resistant (CisR) and corresponding parental (PT) H460 cells previously established in our laboratory. DNA repair genes significantly altered in CisR cells were validated at the mRNA and protein level, using RT-PCR and Western blot analysis, respectively. 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 using Annexin-V/PI staining. Cellular expression and localisation of XRCC6BP1 protein and γH2AX foci in response to cisplatin were examined by immunofluorescence using the Cytell™ Imaging System. To investigate a role for XRCC6BP1 in lung cancer stem cells, Side Population (SP) studies were used to characterise stem-like cells in chemoresistant cells. XRCC6BP1 mRNA analysis was also examined in ALDH1[+] and ALDH1[- ]subpopulations.
Results:
We identified a number of critical DNA repair genes that were differentially regulated between H460 PT and CisR NSCLC cells, where XRCC6BP1 mRNA and protein expression was significantly increased (mRNA; 19.4-fold) 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 CisR cells and reduced the DNA repair capacity of these cells relative to scrambled (negative) controls. Immunofluorescence studies showed an increase in XRCC6BP1 protein expression and γH2AX foci in CisR cells relative to their PT counterparts. SP analysis revealed a significantly higher stem cell population in CisR cells, while XRCC6BP1 mRNA expression was considerably increased in SKMES-1, H460 and H1299 ALDH1[+] CisR cells compared to ALDH1[-] cells.
Conclusion:
We identified XRCC6BP1 as key DNA repair gene implicated in cisplatin resistant NSCLC. Our 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.
