RAD51-Mediated DNA Homologous Recombination Is Independent of Mutational Status.

UNLABELLED

PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of -mutated and other HR-deficient (HRD) tumors.

METHODS

Primary -deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and -null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and γH2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. -deficient MEFs served as an HR-deficient control, while the stable knockdown of () served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and -expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of -null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of and its paralogs were examined as a function of the status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs.

RESULTS

knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking . Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, -mutated; U251 and U373, -null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against () display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of -inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of and its paralogs (, and ), regardless of the mutational status.

CONCLUSIONS

Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural -null cells, PTEN-deficient tumor cell lines, and primary -mutant GBM patient-derived tissue specimens and BTICs.