BRCA1185delAG tumors may acquire therapy resistance through expression of RING-less BRCA1
Rinske Drost, … , Peter Bouwman, Jos Jonkers
Rinske Drost, … , Peter Bouwman, Jos Jonkers
Published August 1, 2016; First published July 25, 2016
Citation Information: J Clin Invest. 2016;126(8):2903-2918. https://doi.org/10.1172/JCI70196.
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Categories: Research Article Cell biology Oncology Therapeutics

BRCA1185delAG tumors may acquire therapy resistance through expression of RING-less BRCA1

Abstract

Heterozygous germline mutations in breast cancer 1 (BRCA1) strongly predispose women to breast cancer. BRCA1 plays an important role in DNA double-strand break (DSB) repair via homologous recombination (HR), which is important for tumor suppression. Although BRCA1-deficient cells are highly sensitive to treatment with DSB-inducing agents through their HR deficiency (HRD), BRCA1-associated tumors display heterogeneous responses to platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors in clinical trials. It is unclear whether all pathogenic BRCA1 mutations have similar effects on the response to therapy. Here, we have investigated mammary tumorigenesis and therapy sensitivity in mice carrying the Brca1185stop and Brca15382stop alleles, which respectively mimic the 2 most common BRCA1 founder mutations, BRCA1185delAG and BRCA15382insC. Both the Brca1185stop and Brca15382stop mutations predisposed animals to mammary tumors, but Brca1185stop tumors responded markedly worse to HRD-targeted therapy than did Brca15382stop tumors. Mice expressing Brca1185stop mutations also developed therapy resistance more rapidly than did mice expressing Brca15382stop. We determined that both murine Brca1185stop tumors and human BRCA1185delAG breast cancer cells expressed a really interesting new gene domain–less (RING-less) BRCA1 protein that mediated resistance to HRD-targeted therapies. Together, these results suggest that expression of RING-less BRCA1 may serve as a marker to predict poor response to DSB-inducing therapy in human cancer patients.

Authors

Rinske Drost, Kiranjit K. Dhillon, Hanneke van der Gulden, Ingrid van der Heijden, Inger Brandsma, Cristina Cruz, Dafni Chondronasiou, Marta Castroviejo-Bermejo, Ute Boon, Eva Schut, Eline van der Burg, Ellen Wientjens, Mark Pieterse, Christiaan Klijn, Sjoerd Klarenbeek, Fabricio Loayza-Puch, Ran Elkon, Liesbeth van Deemter, Sven Rottenberg, Marieke van de Ven, Dick H.W. Dekkers, Jeroen A.A. Demmers, Dik C. van Gent, Reuven Agami, Judith Balmaña, Violeta Serra, Toshiyasu Taniguchi, Peter Bouwman, Jos Jonkers

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Figure 7

BRCA1185delAG tumor cells are dependent on RING-less BRCA1 for proliferation, DNA damage signaling, and treatment response.

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BRCA1185delAG tumor cells are dependent on RING-less BRCA1 for prolifer...
(A) BRCA1 protein levels after BRCA1 knockdown in BRCA1185delAG-mutant SUM1315MO2 tumor cells. –, no shRNA. Expression of POLII was used as a loading control, and protein size markers are indicated. (B) Quantification of RAD51 IRIF–positive SUM1315MO2 cells after BRCA1 knockdown as in A. The percentage of cells with 10 or more RAD51 foci was evaluated for at least 300 cells per condition. Statistical significance was calculated using Fisher’s exact test, and results are representative of 2 independent experiments. (C) Complementation of SUM1315MO2 cells by stably transfected cDNA constructs expressing WT BRCA1 (WT) or BRCA1 N-terminal truncation variants (M48, M128, or M297). NTF, nontransfected control. Cells were assayed for clonal growth after transduction with the BRCA1 3′-UTR–targeting shRNA (sh1) or the NT shRNA (shNT) control. P = 0.0005 by 2-tailed, unpaired t test for NTF control shNT versus sh1. Significant complementation of proliferation is indicated by **P < 0.01 or #P < 0.0001. Results shown are representative of 2 independent experiments. (D) RAD51 IRIF formation in Brca1 WT (KP) cells, the KB1(185stop)P (288) cell line, and its Brca1 deficient subclone C3 (288-C3). Cells were irradiated with 10 Gy, and RAD51 foci formation in S and G2 phases was compared with a nonirradiated control (no IR). Red bars indicate the mean number of foci in at least 116 EdU-positive cells. P value was determined using a 2-tailed, unpaired t test, and data are representative of 2 independent experiments. (E and F) IC50 values of KB1(185stop)P cell line 288 and subclones C1-3 for olaparib (E) and cisplatin (F). Error bars indicate the SD for 3 independent experiments. (G and H) OS of mice transplanted with KB1(185stop)P cell lines 288 and 288-C3 and treated with 100 mg/kg AZD2461 daily for 28 consecutive days (G) or with 6 mg/kg cisplatin on days 0 and 14 (H), or untreated (G). P values were calculated using the log-rank test.