RFWD2 Induces Cellular Proliferation and Proteasome Inhibitor Resistance By Mediating p27 Ubiqutinaiton in Multiple Myeloma

Purpose: In recent years, with the emergence of targeted proteasome inhibitors (PIs), the treatment of multiple myeloma (MM) has made great progress and significantly improves the survival rate of patients. However, MM remains an incurable disease, mainly due to the recurrence of drug resistance. The constitutive photomorphogenic 1 (RFWD2, also known as COP1), is closely related to the occurrence and development of tumors, but its role in MM is largely unknown. This study was aimed to explore the mechanism of RFWD2 on cell proliferation and resistance to proteasome inhibitor in MM.

Experimental Design: Using gene expression profiling (GEP) samples, we verified the relation of RFWD2 to MM patients' survival and drug-resistance. The effect of RFWD2 on cell proliferation was confirmed by MTT and cell cycle analysis in RFWD2-overexpressed and RFWD2-knockdown MM cells. MTT and apoptosis experiments were performed to evaluate whether RFWD2 influenced the sensitivity of MM cells to several chemotherapy drugs. MM xenografts were established in immunodeficient NOD/SCID mice by injecting wild-type or RFWD2 over-expression MM cells with drug intervention. The mechanism of drug resistance was elucidated by analyzing the association of RFWD2 with E3 ligase of p27. Bortezomib-resistant RPMI 8226 cells were used to construct RFWD2 knockdown cells, which were injected into NOD/SCID mice to assess the effect of RFWD2 on bortezomib resistance in vivo.

Results: RFWD2 expression was closely related to poor outcome, relapse and bortezomib resistance in MM patients' GEP cohorts. Elevated RFWD2 induced cell proliferation, while decreased RFWD2 inhibited cell proliferation and induced apoptosis in MM cells. RFWD2-overexpression MM cells resulted in PIs resistance, however, no chemotherapy resistance to adriamycin and dexamethasone was observed in vitro. In addition, overexpressing RFWD2 in MM cells led to bortezomib resistance rather than adriamycin resistance in myeloma xenograft mouse model. RFWD2 regulated the ubiquitination degradation of P27 by interacting with RCHY1 ubiquitin ligase. The knockdown of RFWD2 in bortezomib-resistant RPMI 8226 cells overcame bortezomib resistance in vivo.

Conclusions: Our data demonstrate that elevated RFWD2 induces MM cell proliferation and resistance to PIs, but not to adriamycin and dexamethasone both in vitro and in vivo through mediating the ubiquitination of p27. Collectively, RFWD2 is a novel promising therapeutic target in MM.