PO-012 Ferulic acid increases ABT-888 sensitivity in breast cancer cells

Abstract

IntroductionThe MRE11-RAD50-NBS1 (MRN) protein complex mediates DNA damage signalling and double-strand break repair. MRE11 can be regulated by histone modification, resulting in variable expression levels. We previously found that high MRE11 expression was associated with improved survival following radiotherapy, contrary to our expectations in terms of DNA DSB repair efficiency. We also discovered a C-terminally truncated form of MRE11 (TR-MRE11) in patients’ bladder tumour samples. To understand these findings further, we looked for the cleavage site of TR MRE11, and its functional impact on DNA damage repair.Material and methodsRT112 bladder carcinoma cells were lysed for immunoprecipitation with MRE11 antibody. After Coomassie staining, bands corresponding to full-length (FL) or TR-MRE11 were excised and in-gel digestion carried out with trypsin or elastase. Samples were analysed on an Orbitrap Fusion Lumos Mass Spectrometer. We then inserted point mutations into the DNA sequence of MRE11 corresponding to exons 15 to 20, which resulted in a premature stop codon, in an attempt to investigate the core functional MRE11 sequences which contribute to cell survival after radiation. Then various types of DNA damage by several methods such as overgrowth, starvation, irradiation or drug treatments to assess the effects on TR-MRE11 expression.Results and discussionsFL and TR MRE11 mass spectrometry narrowed down the cleavage site position to between αα 538 and 592. Cells transfected with MRE11 plasmids containing different lengths of c-terminal truncation showed two major patterns: low and high survival after irradiation. The c-terminal truncations occurring before exon 16 showed higher sensitivity to irradiation than c-terminal truncations after exon 16. These two patterns suggest that an important role in DNA damage repair exists in MRE11 after exon 16. Among various cells subjected to DNA damage, the cells overgrown consistently showed significantly increased TR-MRE11. This work is ongoing, and we plan to use molecules related to overgrowth in functional assays to determine the exact cleavage site.ConclusionIf lack of the c-terminal has functional impact in terms of DNA repair efficiency, this could explain our ‘paradoxical’ MRE11 IHC results seen in primary bladder cancer samples. Furthermore, further investigation of TR-MRE11 induction and cleavage sites will enable us to use this data for clinical benefit.