RING domain–deficient BRCA1 promotes PARP inhibitor and platinum resistance
Yifan Wang, … , Fiona Simpkins, Neil Johnson
Yifan Wang, … , Fiona Simpkins, Neil Johnson
Published July 25, 2016
Citation Information: J Clin Invest. 2016;126(8):3145-3157. https://doi.org/10.1172/JCI87033.
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Research Article Oncology Therapeutics

RING domain–deficient BRCA1 promotes PARP inhibitor and platinum resistance

Abstract

Patients with cancers that harbor breast cancer 1 (BRCA1) mutations initially respond well to platinum and poly(ADP-ribose) polymerase inhibitor (PARPi) therapy; however, resistance invariably arises in these patients and is a major clinical problem. The BRCA1185delAG allele is a common inherited mutation located close to the protein translation start site that is thought to produce a shortened, nonfunctional peptide. In this study, we investigated the mechanisms that lead to PARPi and platinum resistance in the SUM1315MO2 breast cancer cell line, which harbors a hemizygous BRCA1185delAG mutation. SUM1315MO2 cells were initially sensitive to PARPi and cisplatin but readily acquired resistance. PARPi- and cisplatin-resistant clones did not harbor secondary reversion mutations; rather, PARPi and platinum resistance required increased expression of a really interesting gene (RING) domain–deficient BRCA1 protein (Rdd-BRCA1). Initiation of translation occurred downstream of the frameshift mutation, probably at the BRCA1-Met-297 codon. In contrast to full-length BRCA1, Rdd-BRCA1 did not require BRCA1-associated RING domain 1 (BARD1) interaction for stability. Functionally, Rdd-BRCA1 formed irradiation-induced foci and supported RAD51 foci formation. Ectopic overexpression of Rdd-BRCA1 promoted partial PARPi and cisplatin resistance. Furthermore, Rdd-BRCA1 protein expression was detected in recurrent carcinomas from patients who carried germline BRCA1185delAG mutations. Taken together, these results indicate that RING-deficient BRCA1 proteins are hypomorphic and capable of contributing to PARPi and platinum resistance when expressed at high levels.

Authors

Yifan Wang, John J. Krais, Andrea J. Bernhardy, Emmanuelle Nicolas, Kathy Q. Cai, Maria I. Harrell, Hyoung H. Kim, Erin George, Elizabeth M. Swisher, Fiona Simpkins, Neil Johnson

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Figure 2

SUM1315MO2 cells express a RING domain–deficient BRCA1 protein.

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SUM1315MO2 cells express a RING domain–deficient BRCA1 protein. (A) Cell...
(A) Cell lysates were generated from MDA-MB-231 (abbreviated as 231 in the figure), SUM1315MO2 parental (Par), RR1–3, and CR1–3 cells, and BRCA1 protein was detected by Western blot analysis using C-terminal–specific (C-term) and N-terminal–specific (N-term) BRCA1 Abs. Molecular weight markers are indicated. Three independent experiments were performed. (B) SUM1315MO2 parental, RR1–3, and CR1–3 cells were treated with IR, and BRCA1, RAD51, γ-H2AX, and DAPI were detected by immunofluorescence (representative images are shown). Scale bar: 10 μm. Bar graphs show the mean and SEM percentage of cells containing more than 5 foci (n = 3 independent experiments. *P < 0.05, 2-tailed Student’s t test). (C) In-frame methionine translation start sites are depicted, along with the location of the stop codon generated from the BRCA1185delAG mutation (above). BRCA1 was immunoprecipitated from MDA-MB-231 and SUM1315MO2 RR1 cells and subjected to mass spectrometry. BRCA1 peptides detected from MDA-MB-231 cells are highlighted in yellow and SUM1315MO2 RR1 cells in green, Met-297 is highlighted in red (below). See Supplemental Figure 1 for complete information on peptide coverage. (D) Cell lysates were collected from MDA-MB-231 and SUM1315MO2 RR1 and CR1 cells, as well as from MDA-MB-436 cells that expressed exogenous mCherry (mCh), BRCA1 full-length (FL), BRCA1-Met128 (M128), or BRCA1-Met-297 (M297). BRCA1 gel migration was detected by Western blot analysis using N- and C-terminal–specific Abs; HSP90 was measured as a loading control. (E) CRISPR/Cas9 targeting sg_GFP, sg_exon 2 (E2), or sg_exon 11 (E11) in MDA-MB-231 and SUM1315MO2 RR1 cells. Western blot analyses of BRCA1 levels using a C-terminal–specific Ab (see Supplemental Figure 2 for additional information). Three independent experiments were performed.