Abstract
BRCA1, a multifunctional protein with an important role in DNA double-strand break repair by homologous recombination (HR), is subjected to ubiquitin-dependent degradation. To date, several E3 ubiquitin ligases have been identified to govern BRCA1 stability, but the deubiquitinase that counteracts its turnover remains undefined. In this study, we report that the ubiquitin-specific protease 9X (USP9X) is a bona fide deubiquitinase for BRCA1 in human cancer cells. Reciprocal immunoprecipitation assays demonstrated that USP9X interacted with BRCA1. Depletion of USP9X by short interfering RNAs or inhibition of USP9X by the small-molecular inhibitor WP1130 significantly reduced BRCA1 protein abundance, without affecting its mRNA levels. In contrast, overexpression of wild-type USP9X, but not its deubiquitinase activity-defective mutant (C1566S), resulted in an upregulation of BRCA1 protein levels. Moreover, USP9X depletion reduced the half-life of BRCA1, accompanied by an increase in its ubiquitination. HR assays showed that knockdown of USP9X significantly reduced HR efficiency, which was partially rescued by reintroduction of BRCA1 into USP9X-depleted cells. In support of these findings, USP9X knockdown significantly enhanced sensitivity to PARP inhibitor Olaparib and methyl methanesulfonate (MMS). Collectively, these results establish USP9X as a deubiquitinase for BRCA1 and reveal a previously unrecognized role of USP9X in the regulation of HR repair and the sensitivity of cancer cells to DNA-damaging agents.