Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance

Significance

Poly(ADP-ribose) polymerase (PARP) inhibitors have produced responses in homologous recombination (HR) repair-deficient cancers, such as those with a mutated breast cancer 1, early onset (BRCA1) gene. We have delineated a two-event mechanism of acquired resistance by using a BRCA1 BRCA C-terminal (BRCT) domain-mutated breast cancer cell line, involving heat shock protein (HSP)90-mediated stabilization of the mutant protein coupled with tumor protein p53 binding protein 1 (TP53BP1) gene mutation, which together restore DNA end resection and RAD51 filament formation, critical steps in HR. Similar events may occur in primary BRCA1-mutated ovarian cancers as cells develop resistance to platinum. The data demonstrate that, even though BRCA1 BRCT domain mutant proteins cannot promote DNA end resection, they retain partial function and can contribute to RAD51 loading and HR. Finally, HSP90 inhibition may prove useful for resensitizing resistant BRCA1-mutant cancer cells to drug treatment.

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. These results provide evidence for a two-event mechanism by which BRCA1-mutant tumors acquire anticancer therapy resistance.

The breast cancer 1, early onset (BRCA1) gene is commonly mutated in hereditary breast and ovarian cancers. The BRCA1 protein has multiple domains that mediate protein interactions; BRCA1 gene mutations may produce truncated proteins that lose the ability to interact with associated proteins. Additionally, mutations in the BRCA C-terminal (BRCT) domain of BRCA1 create protein folding defects that result in protease-mediated degradation (1–3).

Cells that contain dysfunctional BRCA1 proteins are hypersensitive to DNA damaging agents (4). In particular, BRCA1-deficient cell lines are exquisitely sensitive to poly(ADP-ribose) polymerase (PARP) inhibition (5). Despite initial responses of BRCA1-mutant cancers to PARP inhibitor treatment (6), acquired resistance universally develops. Resistance may result from secondary mutations in the BRCA1 gene that restore the reading frame and produce a functional BRCA1 protein (7, 8). In Brca1-mutated mouse mammary tumors, activation of p-glycoprotein or loss of p53 binding protein 1 (53BP1) expression resulting from truncating TP53BP1 mutations confers PARP inhibitor resistance (9). Loss of 53BP1 in BRCA1-deficient cells provides the C-terminal binding protein interacting protein (CtIP) with unrestricted access to DNA breaks, facilitating DNA end resection, an early step in homologous recombination (HR) (9–11).

Following BRCA1-CtIP–mediated activation of DNA end resection, eventual BRCA2-mediated assembly of the RAD51 recombinase in nucleoprotein filaments is a critical step in HR. A role for BRCA1 in RAD51 loading and the mechanisms by which it participates have not been fully clarified. Of note, in PARP inhibitor-resistant BRCA1- and 53BP1-deficient tumors and derived cell lines, RAD51 γ-irradiation–induced foci were detected, although at a lower level than in BRCA1- and 53BP1-proficient cells (9). Previous studies demonstrated that RAD51 foci were partially reduced in BRCA1- or partner and localizer of BRCA2 (PALB2)-deficient cells reconstituted with BRCA1 or PALB2 constructs carrying mutations that disrupt the BRCA1–PALB2 interaction (12, 13), suggesting that BRCA1 may enlist PALB2, which in turn organizes the recruitment of BRCA2 and RAD51.

To date, the described mechanisms of PARP inhibitor resistance occur in only a fraction of the BRCA1 mutant patient population or in PARP inhibitor-resistant Brca1-mutated mouse mammary tumors (8, 10). Here, we used a human breast cancer cell line that contains a BRCT domain BRCA1 mutation to identify additional mechanisms of acquired PARP inhibitor resistance, and demonstrate that stabilization of the mutant BRCA1 protein is critical for the restoration of RAD51 focus formation.

Results

MDA-MB-436 Clones Are Resistant to PARP Inhibitors and Cisplatin.

To study PARP inhibitor resistance, we cultured the triple-negative breast cancer cell line MDA-MB-436 in the presence of the PARP inhibitor rucaparib. MDA-MB-436 cells contain a BRCA1 5396 + 1G>A mutation in the splice donor site of exon 20 that results in a BRCT domain-truncated protein (14). Drug-resistant clones, labeled rucaparib-resistant (RR) 1 through 6, emerged ∼2 to 4 mo after initial exposure. Clones were highly resistant to rucaparib, and cross-resistant to olaparib, as well as cisplatin (Fig. 1A). Concentrations required to reduce colony formation by 50% (lethal concentration 50, LC₅₀) were 482- to 590-fold (P < 0.0001), 254- to 492-fold (P < 0.0001), 150- to 173-fold (P < 0.0001), and 27- to 59-fold (P = 0.0056) greater than those for parental cells for rucaparib, rucaparib after a 6-mo holiday from rucaparib selection, olaparib, and cisplatin, respectively. Additionally, MDA-MB-436–resistant clones had a marked decrease in the number of aberrant chromosome structures after treatment with rucaparib compared with the parental cell line, with 10- to 20-fold (P < 0.0001) and 7- to 15-fold (P < 0.0001) fewer aberrations and radials per cell, respectively (Fig. 1B).

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.

To rule out drug efflux as a mechanism of PARP inhibitor resistance, we measured the ability of rucaparib to inhibit the PARP enzyme by assessing cellular poly(ADP-ribose) (PAR) levels by Western blot in the absence of activated DNA. Rucaparib reduced the levels of PAR to a similar degree in MDA-MB-436 parental cells and in all the resistant clones except for RR-1 (Fig. S1A). Of note, clones RR-5 and RR-6 had reduced basal PAR levels. To assess if the lack of PARP inhibition in RR-1 cells accounted for drug resistance, we used siRNA to deplete PARP-1 and PARP-2 levels. PAR levels were reduced after siRNA treatment (Fig. S1B); however, the colony forming potential of RR-1 cells was not significantly impacted (Fig. S1C). We conclude that, although rucaparib did not inhibit PARP as effectively in RR-1 cells, additional events may have contributed to rucaparib resistance.

Increased Mutant BRCA1 Protein in Resistant Clones.

We next measured BRCA1 and RAD51 protein levels by Western blot. MCF7 cells express WT BRCA1 protein and were used as a positive control. Mutant BRCA1 protein was undetectable in MDA-MB-436 parental cells, but was abundant in resistant clones. RAD51 protein levels were similar in parental cells and resistant clones (Fig. 2A). To determine whether BRCA1 reversion mutation had occurred, we sequenced BRCA1 gene introns and exons. MDA-MB-436–resistant clones retained the original 5396+1G>A mutation, and did not harbor any additional mutations in BRCA1 (Fig. S2A). Furthermore, the BRCA1 mRNA sequences of parental cells and resistant clones were identical (Fig. S2 B and C). The BRCA1 protein detected in resistant clones by an N-terminal BRCA1 antibody was C-terminal truncated and consequently not recognized by a C-terminal–specific antibody (Fig. 2B). The BRCA1 5396+1G>A mutation produces two splice variants (14). We used siRNAs specific to each isoform to determine which variant accounted for the reexpressed protein. MDA-MB-436 parental cells stably expressing an exogenous WT BRCA1 protein (MDA-MB-436+WT) were used as a control for nonspecific BRCA1 protein knockdown. We demonstrated that siRNA specifically targeting the exon 20 deletion variant resulted in knockdown of mutant but not WT BRCA1 protein. Therefore, it is likely that the exon 20 deletion variant accounted for the reexpressed protein in resistant clones (Fig. S2 D and E).

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.

The mutant BRCA1 protein could be detected in association with chromatin (Fig. S3). As expected, γ-irradiation–induced BRCA1 foci were not detectable in MDA-MB-436 parental cells; in contrast, BRCA1 foci were readily detectable in resistant clones. Similarly, RAD51 foci were not detected in parental cells, despite the abundance of RAD51 protein; however, resistant clones readily formed RAD51 foci following irradiation. Formation of γ-H2AX foci, a marker of DNA damage, was present to the same degree in parental and resistant cells (Fig. 2C).

Protein Stability Accounts for Increased Mutant BRCA1 Protein.

We next investigated factors that could contribute to changes in BRCA1 protein levels in PARP inhibitor-resistant clones. There were no changes in BRCA1 gene copy number (Fig. S4A); additionally, resistant clones demonstrated only a 1.5- to 2.7-fold (P = 0.0061) increase in BRCA1 mRNA by quantitative RT-PCR analyses (Fig. S4B). To determine if increased BRCA1 protein expression was dependent on transcription or translation, we treated parental and resistant clones with cycloheximide to inhibit protein translation. We could detect a faint BRCA1 band in MDA-MB-436 parental cells when we increased protein loading and film exposure time; however, BRCA1 protein was undetectable at 6 h after cycloheximide treatment. In contrast, BRCA1 protein levels were maintained for as long as 24 h after cycloheximide treatment in RR clones (Fig. S4C). These data suggest that the increase in mutant BRCA1 protein in resistant cells was likely a result of protein stabilization rather than hyperactivation of BRCA1 transcription or translation.

BRCT domain mutations often result in an inability of the mutant protein to fold correctly; consequently, the unfolded protein is more susceptible to protease-mediated degradation (1–3). It is therefore possible that the mutant BRCA1 protein in MDA-MB-436 parent cells is undetectable as a result of an inability to correctly fold, with subsequent degradation by the proteasome. Consistent with this hypothesis, MDA-MB-436 parental cells treated with the proteasome inhibitors MG132 or bortezomib had detectable levels of mutant BRCA1 protein, suggesting protein was being generated but rapidly degraded due to folding defects (Fig. S4D).

HSP90 Stabilizes Mutant BRCA1 Protein.

Because HSP90 has been implicated in the folding of cancer-related mutant proteins (15), we investigated the dependency of BRCA1 mutant protein levels on HSP90 activity. First, we assessed the association of BRCA1 proteins with HSP90 by determining levels of BRCA1 protein in HSP90 immunoprecipitates from MDA-MB-436+WT cells or PARP inhibitor-resistant clones. Mutant and ectopically expressed WT BRCA1 protein from the parental cell line were absent or weakly in complex with HSP90. In contrast, mutant BRCA1 protein from resistant clones was readily found in association with HSP90 (Fig. 3A). Similarly, when we immunoprecipitated BRCA1 from MDA-MB-436+WT cells or RR cells, HSP90 could only be found in association with the mutant BRCA1 proteins (Fig. 3B). Next, we treated MDA-MB-436+WT BRCA1 cells, as well as RR cells, with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). WT BRCA1 protein remained at levels comparable to untreated cells at 72 h posttreatment. In contrast, RR cells had reduced mutant BRCA1 protein levels by 48 h posttreatment. HSP70 levels increased in response to 17-DMAG, indicating HSP90 was inhibited to an equal degree in all cell lines (Fig. 3C). Furthermore, 17-DMAG treatment of resistant clones restored sensitivity to rucaparib; compared with DMSO/rucaparib, clonal survival of RR-1, RR-5, and RR-6 in 17-DMAG/rucaparib was reduced 4.7-fold (P < 0.0001), 13.1-fold (P = 0.0007), and 4.9-fold (P = 0.0023), respectively (Fig. 3D). Together, these data suggest that HSP90 promotes mutant BRCA1 protein folding and conformational stability in RR cells. Of note, 17-DMAG treatment also sensitized MDA-MB-436+WT cells to rucaparib treatment (Fig. S5A), likely mediated through a reduction in BRCA2 and RAD51 protein levels (16) (Fig. S5B).

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).

Reduced 53BP1 Facilitates BRCA1-Independent DNA End Resection.

We next analyzed the contribution of stabilized mutant BRCA1 in RR cells to two critical steps of HR, DNA end resection and RAD51 loading. First, we investigated the ability of exogenous WT BRCA1 in parental cells and the C-terminal truncated mutant BRCA1 protein in resistant clones to interact with proteins known to complex with BRCA1. Analyses of immunoprecipitated exogenous WT BRCA1 protein from MDA-MB-436+WT cells demonstrated that BARD1, PALB2, BRCA2, RAD51, CtIP, and RAP80 could all be detected in association with WT BRCA1. Similarly, BARD1, PALB2, BRCA2, and RAD51 associated with endogenous mutant BRCA1 protein immunoprecipitated from resistant clones. However, the BRCT domain interacting proteins CtIP and RAP80 were not found to interact with the C-terminal truncated BRCA1 protein from MDA-MB-436 resistant cells (Fig. S6A).

DNA end resection is dependent on the activities of BRCA1 and CtIP proteins (17, 18). We investigated the role of the mutant BRCA1 protein in DNA end resection by measuring the formation of RPA32 foci after γ-irradiation (Fig. S6 B and C). MCF7 and MDA-MB-436+WT cells express WT BRCA1 protein and were used for comparison with mutant BRCA1 proteins. Depletion of WT BRCA1 by using three individual siRNAs resulted in a fourfold (P = 0.0001) and 3- to 15-fold (P = 0.0011) decrease in the formation of RPA32 foci compared with scrambled siRNA control-treated MCF7 and MDA-MB-436+WT cells, respectively. In contrast, depletion of mutant BRCA1 protein from RR clones RR-1 and RR-5 did not affect the formation of RPA32 foci. Depletion of CtIP by using three individual siRNAs resulted in a three- to fivefold (P < 0.0001), 3- to 15-fold (P = 0.0042), 5- to 21-fold (P < 0.0001), and 13- to 25-fold (P < 0.0001) decrease in the formation of RPA32 foci compared with scrambled siRNA control-treated MCF7, MDA-MB-436+WT, RR-1, and RR-5 cells, respectively (Fig. S6C). These data indicate that the truncated C-terminal BRCA1 protein that did not interact with CtIP did not influence DNA end resection, and that CtIP activates DNA end resection independently of BRCA1 interaction in MDA-MB-436 resistant clones.

TP53BP1 mutation and loss of function have been demonstrated to confer PARP inhibitor resistance (9–11). We sequenced TP53BP1 gene introns and exons in parental cells and resistant clones. MDA-MB-436 parental cells contained homozygous WT TP53BP1 gene sequences. In contrast, all resistant clones contained a heterozygous 3708 del 11 mutation located in exon 18 (Fig. S7A). This was a microhomology-mediated deletion (Fig. S7B), a mechanism of deletion common to BRCA1/2-mutant cancers (19). The mutation creates a frameshift and an early stop codon predicted to produce a truncated protein (p.P1235PfsX37). Consistent with a heterozygous TP53BP1 gene loss-of-function mutation, PARP inhibitor-resistant clones had lower levels of 53BP1 protein compared with parental cells (Fig. S7C). 53BP1 protein was reduced four- to sevenfold compared with parental cells (by densitometry), a greater reduction than expected from loss of one allele, suggesting possible transcriptional silencing of the remaining WT allele.

To investigate if loss of function of 53BP1 accounted for PARP inhibitor resistance, we depleted 53BP1 from parental MDA-MB-436 cells by using siRNA and measured PARP inhibitor sensitivity. Consistent with a previous report (20), siRNA-mediated depletion of 53BP1 conferred only a slight degree of resistance to PARP inhibitor treatment in MDA-MB-436 cells (3.4-fold increase in rucaparib LC₅₀ value vs. scrambled siRNA treatment; P = 0.1295, unpaired t test; Fig. S7D). In contrast, ectopic expression of WT BRCA1 (MDA-MB-436+WT) conferred substantial rucaparib resistance (426-fold increase in rucaparib LC₅₀ value compared with GFP control cells; P < 0.0001, unpaired t test; Fig. S7E), similar to that seen in our RR clones (Fig. 1A). These data indicate that disruption of 53BP1 function alone could not fully account for the resistance acquired by the MDA-MB-436 clones derived under rucaparib selection pressure.

Because 53BP1 deletion was previously shown to provide PARP inhibitor resistance in mouse Brca1 mutant cell lines, we further investigated the effect of 53BP1 depletion on additional human BRCA1 mutated cancer cell lines, including SUM1315 (185delAG) and HCC1395 (5251C>T). Consistent with the data in MDA-MB-436 cells, siRNA mediated-depletion of 53BP1 in SUM1315 and HCC1395 cells conferred a 5.1-fold and 5.7-fold increase in rucaparib LC₅₀ value compared with scrambled siRNA treatment (P = 0.145 and P = 0.083, unpaired t test), respectively (Fig. S7 F and G).

We hypothesized that the reduction in 53BP1 protein levels in PARP inhibitor-resistant clones enabled CtIP to activate DNA end resection and RPA32 loading in the absence of CtIP–BRCA1 protein interaction. We demonstrated that a twofold increase (by densitometry) in 53BP1 protein levels in RR-1 cells engineered to express ectopic WT 53BP1 resulted in a 1.5-fold (P = 0.005, unpaired t test) and 1.3-fold (P = 0.025, unpaired t test) reduction in the percentage of RPA32 and RAD51 foci-positive cells compared with control RR-1 cells, respectively (Fig. S8A). Furthermore, reexpression of 53BP1 increased RR-1 sensitivity to PARP inhibitor treatment with a twofold decrease (P = 0.049, unpaired t test) in the LC₅₀ value of rucaparib compared with control cells (Fig. S8B).

RAD51 Focus Formation Is Dependent on Mutant BRCA1.

To determine why decreased 53BP1 protein levels conferred only modest PARP inhibitor resistance in MDA-MB-436 cells, we studied RAD51 assembly following DNA damage in these cells. Of note, RNF8 and RNF168 have been implicated in RAD51 loading during HR in the absence of BRCA1 and 53BP1 (21). However, levels of these proteins remained unchanged in resistant clones (Fig. S8C).

We measured the effect of 53BP1 depletion on RPA32 and RAD51 foci after γ-irradiation treatment in MDA-MB-436 cells engineered to express GFP control or exogenous WT BRCA1 (Fig. 4A). Depletion of 53BP1 increased RPA32 foci 3.4-fold (P < 0.001) and 4.9-fold (P < 0.001) in MDA-MB-436 control (+GFP) and MDA-MB-436+WT cells, respectively. Therefore, the presence or absence of BRCA1 protein did not affect the increase in formation of RPA32 foci after 53BP1 depletion. In contrast, depletion of 53BP1 resulted in a 3.3-fold increase (P < 0.001) in RAD51 foci in MDA-MB-436+WT cells that contained WT BRCA1 protein; however, RAD51 foci remained completely absent in MDA-MB-436 control cells. Similar to MDA-MB-436+WT cells, depletion of 53BP1 in MCF7 cells expressing endogenous WT BRCA1 also resulted in a 2.2-fold (P < 0.005) and 2.4-fold (P < 0.001) increase in RPA32 and RAD51 focus formation, respectively (Fig. S8 D and E). Therefore, in MDA-MB-436 cells that lack BRCA1 protein, reducing 53BP1 protein levels enabled CtIP to activate DNA end resection and enhance RPA32 loading, but did not facilitate efficient RAD51 recruitment. Consequently, 53BP1 depletion did not afford dramatic PARP inhibitor resistance in MDA-MB-436 parental cells, in contrast to the more substantial effects of 53BP1 depletion in other model systems (10, 11).

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).

MDA-MB-436 resistant clones readily form RAD51 foci (Fig. 2C). We therefore investigated the role of the mutant BRCA1 protein in restoring RAD51 focus formation. Depletion of BRCA1 by using three individual siRNAs abolished formation of RAD51 foci in MDA-MB-436 resistant clones, indicating that recruitment of RAD51 following DNA damage was dependent on mutant BRCA1 protein. siRNA-mediated depletion of BRCA1 had no effect on the formation of γ-H2AX foci or RAD51 protein levels (Fig. 4B). Additionally, siRNA-mediated BRCA1 depletion dramatically resensitized resistant clones, but not already-sensitive parental cells, to PARP inhibitor treatment. Following expression of BRCA1 siRNA, rucaparib LC₅₀ values were reduced 1- to 3.6-fold (P = 0.3), 132- to 175-fold (P < 0.0001), 69- to 153-fold (P < 0.0001), and 33- to 115-fold (P < 0.0001) compared with scrambled siRNA treatment in MDA-MB-436 parental, RR-1, RR-5, and RR-6 cells, respectively (Fig. 4C).

Increased Mutant BRCA1 Protein in Human Cancers.

We assessed a panel of carboplatin-treated recurrent BRCA1 mutant ovarian carcinomas for platinum sensitivity, secondary BRCA1 reversion mutations, and increased BRCA1 and decreased 53BP1 staining (Table S1). Increased BRCA1 protein expression in the absence of BRCA1 reversion mutation occurred in two of four tumors carrying BRCT domain mutations (5382insC to 5622C>T). Similar to RR MDA-MB-436 cells, the recurrent carcinoma from patient 149101 had an increase in BRCA1 staining combined with a reduction in 53BP1 staining, and was resistant to platinum (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.

Discussion

Mutations in the BRCT domains of BRCA1 often prevent proper protein folding, and misfolded proteins are subject to protease-mediated degradation (1–3). In the present study, we show that, under PARP inhibitor selection pressure, HSP90 interacts with and stabilizes mutant BRCA1 proteins. The stabilized C-terminal truncated protein is semifunctional, as it is unable to interact with CtIP, but retains the protein domains necessary to mediate interactions with PALB2-BRCA2-RAD51 (12, 22). Importantly, the mutant BRCA1 protein is capable of promoting RAD51 loading onto DNA following DNA damage.

Because the BRCT domain-deficient BRCA1 protein is incapable of interacting with CtIP, cells require further genetic adaptations to survive selection pressure. In our model, PARP inhibitor-resistant MDA-MB-436 cells had reduced 53BP1 protein levels as a result of a heterozygous loss-of-function mutation, an event that provided CtIP with unrestricted access to DNA ends and facilitated BRCA1-independent DNA end resection (10, 11). Consistent with other studies, 53BP1 depletion alone contributed to PARP inhibitor resistance (10, 11), but conferred only a slight degree of resistance in this BRCA1 mutant human cancer cell line model (20). Although 53BP1 depletion hyperactivated DNA end resection and RPA32 loading, without stabilization and increased expression of the mutant BRCA1 protein, RAD51 assembly could not occur following DNA damage.

The present study shows inherent partial function of a BRCT domain-mutated BRCA1 protein that can contribute to HR. Other studies have demonstrated functionality of N-terminal missense mutations, and knock-in Brca1-deficient mouse models expressing these mutants responded poorly to platinum drugs, mitomycin C, or PARP inhibition (23, 24). Additionally, although a reduction in 53BP1 expression may facilitate DNA end resection irrespective of the primary BRCA1 mutation, this event may be obligatory in BRCT domain-mutated cells so that end-resection can occur in the absence of a BRCA1 BRCT domain–CtIP interaction. Finally, our data suggest that HSP90 inhibition may reverse PARP inhibitor resistance and may be a rational strategy particularly germane in BRCA1 BRCT domain mutant cancers.

Materials and Methods

Cell Culture.

MDA-MB-436 cells were cultured in the presence of gradually increasing concentrations of rucaparib until resistant clones emerged that grew in 1 µM drug. Colonies were labeled RR 1 through 6. Cells were cultured in the absence of rucaparib for at least 1 mo before they were used for experiments. Cells were routinely analyzed 6 h after 10 Gy γ-irradiation treatment.

Genomic Manipulations and Immunoprecipitation.

Lentiviral generation and infections and siRNA transfections were carried out according to standard protocols. Protein knockdown or reexpression was routinely assessed 72 h after transfection or 96 h after infection. BRCA1 or HSP90 complexes were recovered from 2 mg of nuclear extract by using the Pierce Classic IP Kit (Thermo Scientific) according to the manufacturer’s instructions.

Preparation of DNA.

Genomic DNA was isolated from cells using the DNeasy tissue kit (Qiagen). BRCA1 gene sequencing was carried out as previously described (7). SNP chip array was carried out using the human SNP 6.0 array according to the manufacturer’s instructions (Affymetrix). Total RNA was isolated from cell lines by using TRIzol (Invitrogen) and purified by using an RNeasy cleanup kit (Qiagen).

Statistics.

Mean and SE values were compared by using unpaired t tests. For multiple comparisons we used one-way ANOVA. P < 0.05 was considered statistically significant.

Further Details.

Further details are provided in SI Materials and Methods.