Silencing of BRCA1 and MAD2L1 leads to resistance to docetaxel. A) 24 hours following transfection, cells were treated with docetaxel 20 nM for a further 24 hours and caspase 3 activity measured. Data were normalised to untreated siNT control. MSTO-211H: siNT NT vs siNT DOCE p=0.0003; siBRCA1 NT vs siBRCA1 DOCE p=0.0206; siNT DOCE vs siBRCA1 DOCE p=0.0013. REN: siNT NT vs siNT DOCE p<0.0001; siBRCA1 NT vs siBRCA1 DOCE p=0.0069; siNT DOCE vs siBRCA1 DOCE p<0.0001. B) Cells were treated with docetaxel 20 nM for 24 hours and caspase 3 activity measured. Data were normalised to parental cells. MSTO-211H NT vs MSTO-211H DOCE p<0.0001; MSTO211H DOCE vs MVR DOCE p<0.0001. REN NT vs REN DOCE p<0.0001; REN VNB vs RVR VNB p<0.0001; RVR NT vs RVR DOCE p=0.0069. C) The cells indicated were treated with 20 nM docetaxel before time-lapse imaging using phase contrast microscopy. Cells were imaged every 15 mins for 25 h following addition of drug. Control cells were treated with DMSO. Stills from representative movies are shown. Scale bar 200μm.
ARTICLE ABSTRACT
Mesothelioma is a universally lethal cancer lacking effective therapy. The spindle poison vinorelbine exhibits clinical activity in the relapsed setting, and in preclinical models requires BRCA1 to initiate apoptosis. However, the mechanisms underlying this regulation and the clinical implications have not been explored. Here, we show that BRCA1 silencing abrogated vinorelbine-induced cell-cycle arrest, recruitment of BUBR1 to kinetochores, and apoptosis. BRCA1 silencing led to codepletion of MAD2L1 at the mRNA and protein levels consistent with its status as a transcriptional target of BRCA1. Silencing of MAD2L1 phenocopied BRCA1 and was sufficient to confer resistance to vinorelbine. This was recapitulated in cell lines selected for resistance to vinorelbine, which acquired loss of both BRCA1 and MAD2L1 expression. Following ex vivo vinorelbine in 20 primary tumor explants, apoptotic response rate was 59% in BRCA1/MAD2L1-positive explants compared with 0% in BRCA1/MAD2L1-negative explants. In 48 patients, BRCA1 and/or MAD2L1 loss of expression was not prognostic; however, in a subset of patients treated with vinorelbine, survival was shorter for patients lacking BRCA1/MAD2L1 expression compared with double-positive patients (5.9 vs. 36.7 months, P = 0.03). Our data implicate BRCA1/MAD2L1 loss as a putative predictive marker of resistance to vinorelbine in mesothelioma and warrant prospective clinical evaluation.
