PO-013 BRCA1 and BRCA2 next generation sequencing in high-grade serous ovarian cancer

Abstract

IntroductionProtein arginine methyltransferases (PRMTs) are composed of nine family members that catalyse the methylation of the guanidine group of arginine residues. PRMT1, the main asymmetric dimethyltransferase, is responsible for 85% of all cellular arginine methylation, and has been increasingly associated with cancer pathogenesis. This is particularly true within the breast: PRMT1 levels are increased in breast cancer compare to the normal mammary gland and its expression correlates with poor patient survival. Proteomic approaches were used to identify novel PRMT1 substrates, enabling a better understanding into how PRMT1 promotes malignant progression.Material and methodsTAP-tag purification and mass spectrometry has identified the deubiquitinating enzyme (DUB) USP11 as a novel substrate of PRMT1. Methyl acceptor site was established by mass spectrometry and in vivo and in vitro methylation assays were performed to confirm PRMT1-mediated methylation. Generation of USP11 knockdown MCF7 cells reconstituted with wild type or methyl-deficient USP11 were used to probe biological significance.Results and discussionsWe confirmed that USP11 is a PRMT1 interacting protein by co-immunoprecipitation, but more importantly demonstrated that USP11 is a substrate of PRMT1 using recombinant hUSP11 and [3H]-S-adenosyl methionine in an in vitro methylation assay. Critically, methylation of USP11 in MCF7 cells, was reduced upon PRMT1 depletion. We identified R433 as the main PRMT1-mediated methylated arginine residue of USP11. R433 is located in the catalytic core domain of USP11, a residue that is conserved in different species and in its paralogues, USP4 and USP15. USP11 is a known regulator of the DNA damage response. Interestingly, methyl-deficient USP11-R433K showed a reduction in its catalytic activity in vitro whilst expression of USP11-R433K resulted in 53 BP1 retention and in a decrease in RAD51 foci formation, indicative of defective homologous recombination-mediated double strand break repair. Finally, we observed a crosstalk between arginine methylation and ubiquitination because overexpression of USP11 promotes the deubiquitination of PRMT1.ConclusionOur studies identify for the first time that DUBs are modified by arginine methylation, and that this post-translational modification has the potential to control the enzymatic activity of DUBs involved in double strand break repair. Understanding how methylation of USP11 contributes to genome stability will be important for understanding drug resistance in breast cancer.