Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin

Abstract

Evasion of apoptosis allows many cancers to resist chemotherapy. Apoptosis is mediated by the serial activation of caspase family proteins. These proteases are often activated upon the release of cytochrome c from the mitochondria, which is promoted by the proapoptotic Bcl-2 family protein, Bax. This function of Bax is enhanced by the MOAP-1 (modulator of apoptosis protein 1) protein in response to DNA damage. Previously, we reported that MOAP-1 is targeted for ubiquitylation and degradation by the APC/CCdh1 ubiquitin ligase. In this study, we identify the HECT (homologous to the E6-AP carboxyl terminus) family E3 ubiquitin ligase, UBR5, as a novel ubiquitin ligase for MOAP-1. We demonstrate that UBR5 interacts physically with MOAP-1, ubiquitylates MOAP-1 in vitro and inhibits MOAP-1 stability in cultured cells. In addition, we show that Dyrk2 kinase, a reported UBR5 interactor, cooperates with UBR5 in mediating MOAP-1 ubiquitylation. Importantly, we found that cisplatin-resistant ovarian cancer cell lines exhibit lower levels of MOAP-1 accumulation than their sensitive counterparts upon cisplatin treatment, consistent with the previously reported role of MOAP-1 in modulating cisplatin-induced apoptosis. Accordingly, UBR5 knockdown increased MOAP-1 expression, enhanced Bax activation and sensitized otherwise resistant cells to cisplatin-induced apoptosis. Furthermore, UBR5 expression was higher in ovarian cancers from cisplatin-resistant patients than from cisplatin-responsive patients. These results show that UBR5 downregulates proapoptotic MOAP-1 and suggest that UBR5 can confer cisplatin resistance in ovarian cancer. Thus UBR5 may be an attractive therapeutic target for ovarian cancer treatment.

MOAP-1, UBR5 and cisplatin resistance in ovarian cancer

“…Matsuura et al . confirmed the sensitization to cisplatin upon UBR5 knockdown and showed that co-knockdown of MOAP-1 reduced the cisplatin sensitization effect of UBR5 knockdown, suggesting that UBR5 uses MOAP-1 in part to induce cisplatin resistance (3). Moreover, our lab showed that ectopic overexpression of UBR5, but not a UBR5 point mutant in which the critical cysteine required for its ubiquitin ligase activity was mutated, enhanced cisplatin resistance in cells (15), showing that overexpression of UBR5 alone was sufficient to induce cisplatin resistance, dependent upon its ability to ubiquitinate its substrates.…”

“…have discovered a novel mechanism of cisplatin resistance in ovarian cancer that works through destabilization of MOAP-1 (modulator of apoptosis 1), a protein with a BH3-like motif that stimulates activation of the pro-apoptotic Bcl-2 family protein Bax (2,3). MOAP-1 is an effector of the RASSF1A (Ras association domain family 1A) tumor suppressor that regulates extrinsic apoptosis through the TNFα (tumor necrosis factor α) and TRAIL (TNF-related apoptosis inducing ligand) pathways (4).…”

“…now show that in S and G2 phases, MOAP-1 is targeted for degradation by the HECT (Homologous to the E6-AP Carboxyl Terminus) domain-containing E3 ubiquitin ligase UBR5 (Ubiquitin Protein Ligase E3 Component N-Recognin 5) (3). UBR5 is a 309 kDa nuclear phosphoprotein that has reported roles in the DNA damage response and G2 checkpoint control (10–12).…”

“…UBR5 is also upregulated in several ovarian cancer cell lines (15). Kornbluth’s lab identified UBR5 as a MOAP-1 binding partner in a co-immunoprecipitation after etoposide treatment and speculated that it might regulate MOAP-1 ubiquitination and degradation (3). Indeed, they showed that UBR5 knockdown prolonged MOAP-1 protein half-life and decreased MOAP-1 poly-ubiquitination in cells.…”

“…UBR5 is a part of the EDVP E3 ubiquitin ligase complex, which is composed of UBR5, DDB1, VPRBP and the protein kinase DYRK2, with some substrates being targeted for phosphorylation by DYRK2 prior to ubiquitination by UBR5 (16). While it is unknown if MOAP-1 is a DYRK2 substrate, MOAP-1 associated with each component of the complex in co-immunoprecipitations, the addition of purified components of the complex enhanced MOAP-1 ubiquitination in vitro and DYRK2 knockdown enhanced MOAP-1 half-life in cells (3). Interestingly, UBR5 specifically regulated MOAP-1 protein levels in a cell cycle-dependent manner.…”