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 6

Rdd-BRCA1 proteins are expressed in tumors.

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Rdd-BRCA1 proteins are expressed in tumors. (A) Mice carrying SUM1315MO2...
(A) Mice carrying SUM1315MO2 parental tumors were treated with rucaparib or cisplatin and tumor growth monitored. When tumors reached approximately 1,000 mm3 in size, they were harvested and reimplanted. Red and black lines represent individual tumors from each set of treatments; asterisks represent the days of drug administration. n = 2 mice per treatment group. (B) Assessment of BRCA1 protein levels from tumors described in A by Western blot analysis. MDA-MB-231 cells were used as BRCA1 WT controls. (C) Bar graph shows quantification of staining intensities using C-terminal Ab of at least 2 individual tumors for each treatment. MDA-MB-231 xenografts were used as BRCA1 WT controls; SUM1315MO2 tumors were treated with vehicle or rucaparib, and cisplatin-resistant tumors from A were assessed by IHC for BRCA1 expression using N- and C-terminal–specific Abs. Scale bar: 20 μm. Two tumors for each treatment group were assessed. *P < 0.05, 2-tailed Student’s t test. Bar graph shows quantification of staining intensities using C-terminal Ab of at least 2 individual tumors for each treatment. (D) MDA-MB-231, PDX WO-24 (#24), and PDX WO-21 (#21) were assessed by Western blot analysis for BRCA1 protein expression using a C-terminal–specific Ab. Three individual tumors were assessed for each PDX model. Samples were run on parallel gels, and the tubulin blot was derived from duplicate samples run on a parallel gel. (E) Primary tumors from patients with WT or BRCA1185delAG germline mutations were stained with BRCA1 N- and C-terminal–specific Abs by IHC. Scale bar: 20 μm. See Supplemental Table 2 for more details.