Abstract 5467: BRCA1 N-terminal-deficient proteins provide PARP inhibitor and platinum resistance
Authors: Wang Yifan , Bernhardy Andrea , Nicolas Emmanuelle , Winters Ryan , Cai Kathy , Duncan Kelly , Duncan James , Harrell Maria , Swisher Elizabeth , Johnson Neil 
Published: August 2015

Introduction: Tumors harboring BRCA1 mutations initially respond well to platinum and PARP inhibitor therapy; however, resistance invariably arises and is a major clinical problem. The BRCA1 185delAG allele is a common founder mutation located close to the protein translation start site, thought to produce a short peptide devoid of function.

Experimental procedures: In this study, we utilized the SUM1315MO2 breast cancer cell line that harbors a BRCA1 185delAG mutation to study mechanisms of PARP inhibitor and platinum resistance. SUM1315MO2 cells were cultured in the presence of increasing concentrations of the PARP inhibitor rucaparib or cisplatin until rucaparib resistant (RR) and cisplatin resistant (CR) clones emerged.

Results: DNA sequencing revealed that no BRCA1 gene reversion mutations were present in resistant cells. We next measured BRCA1 protein levels by Western blotting with antibodies specific for the N- and C-terminal domains of BRCA1. As a control, the wild-type BRCA1 protein expressed in MDA-MB-231 cells was detectable with both N- and C-terminal antibodies. In contrast, BRCA1 protein was undetectable in both SUM1315MO2 parental and resistant clones using the N-terminal specific antibody. However, the C-terminal specific antibody identified a more quickly migrating band of low abundance in parental cells, but with elevated expression in both RR and CR clones. We inferred that this protein was devoid of the extreme N-terminal RING domain-containing region that mediates interaction with BARD1. To investigate the functionality of the N-terminal truncated BRCA1 protein (Nt-BRCA1), we measured BRCA1 and RAD51 irradiation-induced focus formation by immunofluorescence. RR and CR clones demonstrated 2.25 - 2.75-fold increase (P = 0.024) in the number of cells with BRCA1 foci compared to parental cells. Similarly, we detected a 2 - 2.85-fold increase (P = 0.0325) in RAD51 foci formation in resistant clones compared to parental cells. Additionally, BRCA1 siRNA treated cells were 12- and 9-fold more sensitive to rucaparib compared to scrambled siRNA-treated control cells (P = 0.0002). Similarly, CR-1 cells treated with BRCA1 siRNAs were 1.56- and 1.7-fold more sensitive to cisplatin (P = 0.0346) compared to scrambled siRNA treated cells. Furthermore, N-terminal truncated BRCA1 proteins were detectable in a primary tumor from a germline BRCA1185delAG mutation carrier.

Conclusions: Taken together, these results provide evidence for a novel, mutation location-dependent mechanism of PARP inhibitor and platinum resistance.

Citation Format: Yifan Wang, Andrea Bernhardy, Emmanuelle Nicolas, Ryan Winters, Kathy Cai, Kelly Duncan, James Duncan, Maria Harrell, Elizabeth Swisher, Neil Johnson. BRCA1 N-terminal-deficient proteins provide PARP inhibitor and platinum resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5467. doi:10.1158/1538-7445.AM2015-5467