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. 2013 Oct 15;110(42):17041-6.
doi: 10.1073/pnas.1305170110. Epub 2013 Oct 1.

Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance (V体育ios版)

Neil Johnson  1 , Shawn F Johnson, Wei Yao, Yu-Chen Li, Young-Eun Choi, Andrea J Bernhardy, Yifan Wang, Marzia Capelletti, Kristopher A Sarosiek, Lisa A Moreau, Dipanjan Chowdhury, Anneka Wickramanayake, Maria I Harrell, Joyce F Liu, Alan D D'Andrea, Alexander Miron, Elizabeth M Swisher, Geoffrey I Shapiro
Affiliations

Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance

Neil Johnson et al. Proc Natl Acad Sci U S A. .

Abstract

Breast Cancer Type 1 Susceptibility Protein (BRCA1)-deficient cells have compromised DNA repair and are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Despite initial responses, the development of resistance limits clinical efficacy. Mutations in the BRCA C-terminal (BRCT) domain of BRCA1 frequently create protein products unable to fold that are subject to protease-mediated degradation. Here, we show HSP90-mediated stabilization of a BRCT domain mutant BRCA1 protein under PARP inhibitor selection pressure. The stabilized mutant BRCA1 protein interacted with PALB2-BRCA2-RAD51, was essential for RAD51 focus formation, and conferred PARP inhibitor as well as cisplatin resistance. Treatment of resistant cells with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibition. Resistant cells also acquired a TP53BP1 mutation that facilitated DNA end resection in the absence of a BRCA1 protein capable of binding CtIP. Finally, concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occur in clinical samples of BRCA1-mutated recurrent ovarian carcinomas that have developed resistance to platinum VSports手机版. These results provide evidence for a two-event mechanism by which BRCA1-mutant tumors acquire anticancer therapy resistance. .

Keywords: cancer therapy; homologous recombination. V体育安卓版.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
MDA-MB-436 clones are resistant to PARP inhibitors and cisplatin. (A) MDA-MB-436 clones RR-1 to RR-6 were significantly more resistant to rucaparib than parental cells (red curve). Cells cultured in the absence of drug for 6 mo remained resistant to rucaparib (+6 mo). Cells were also cross-resistant to olaparib and cisplatin, as measured by colony formation assay (n = 3, mean ± SEM of colonies formed relative to DMSO-treated cells). (B) Metaphase spread analyses of chromosome aberrations and radial formations after treatment with rucaparib (1 µM) for 24 h (n = 3, mean ± SEM). (Inset) Representative metaphase spreads.
Fig. 2.
Fig. 2.
Mutant BRCA1 protein is abundant in MDA-MB-436 resistant clones. (A) BRCA1, RAD51, histone H3, and tubulin levels were measured in cytoplasmic (marked as “c”) and nuclear (marked as “n”) extracts from MCF7 cells, MDA-MB-436 parental cells and resistant clones RR-1 to RR-6 by Western blot. (B) MCF7 cells, MDA-MB-436 parental cells and resistant clones RR-1, RR-5, and RR-6 were treated with DMSO (−) or 1 µM rucaparib (+) for 24 h, and BRCA1 protein levels were assessed by using BRCA1 N- or C-terminal–specific antibodies by Western blot. (C) Detection of BRCA1, RAD51, γ-H2AX, and DAPI by immunofluorescence in MDA-MB-436 parental and resistant cells (n = 3, mean ± SEM percentage of cells containing more than five foci). (Inset) Representative cells.
Fig. 3.
Fig. 3.
HSP90 stabilizes mutant BRCA1. (A) HSP90 was immunoprecipitated from MDA-MB-436 control (GFP) cells, MDA-MB-436+WT cells, and RR clones 1 to 6, and HSP90 and BRCA1 protein levels were analyzed by Western blot (WCE, whole cell extract). (B) BRCA1 was immunoprecipitated from MDA-MB-436 control (GFP) cells, MDA-MB-436+WT cells, and RR clones 1 to 3, and BRCA1 and HSP90 protein levels were analyzed by Western blot. (C) MDA-MB-436+WT, RR-1, RR-5, and RR-6 were treated with 100 nM 17-DMAG for the indicated times, and BRCA1, HSP70, and tubulin protein levels were measured by Western blot. (D) RR-1, RR-5, and RR-6 were treated with vehicle (marked as “V”) or 50 nM 17-DMAG (marked as “D”) in the presence of vehicle (marked as “V”) or 100 nM rucaparib (marked as “R”), and colony formation was assessed (n = 3, mean ± SEM of colonies formed relative to vehicle + vehicle-treated cells).
Fig. 4.
Fig. 4.
Mutant BRCA1 protein promotes RAD51 focus formation. (A) MDA-MB-436 control (GFP) and MDA-MB-436+WT BRCA1 cells (WT) were treated with scrambled (Sc) or 53BP1 siRNA and fixed 6 h after γ-irradiation. RPA32 and RAD51 foci were detected by immunofluorescence. (Left) Western blot demonstrating 53BP1 knockdown and images of representative DAPI-stained cells. (Right) Quantification of RPA32 and RAD51 foci (n = 3, mean ± SEM percentage of cells containing more than five foci). (B) MCF7 and MDA-MB-436 resistant clones RR-1, RR-5, and RR-6 were left untreated (−) or treated with scrambled (Sc) control or three individual BRCA1 siRNAs. After 72 h, cells were fixed 6 h after γ-irradiation treatment. (Left) BRCA1 and RAD51 protein knockdown measured by Western blot and images of representative RR-1 cells after detection of BRCA1, RAD51, γ-H2AX and DAPI by immunofluorescence. (Right) Quantification of foci positive cells (n = 3, mean ± SEM percentage of cells containing more than five foci). (C) MDA-MB-436 parental cells or resistant clones were treated with scrambled (Sc) or three individual BRCA1 siRNAs, exposed to increasing concentrations of rucaparib for 72 h and replated for colony formation. Colony formation was calculated as in Fig. 1A (n = 3, mean ± SEM of colonies formed relative to DMSO-treated cells).
Fig. 5.
Fig. 5.
Increased mutant BRCA1 protein in a platinum-resistant ovarian carcinoma. BRCA1 and 53BP1 protein levels measured by immunohistochemistry from patient 149101. Representative stains of biopsies taken from the platinum-sensitive primary ovarian tumor and the recurrent resistant tumor.
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References

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