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. 2015 Mar 1;29(5):489-94.
doi: 10.1101/gad.256214.114.

Resistance to therapy in BRCA2 mutant cells due to loss of the nucleosome remodeling factor CHD4

Shawna Guillemette  1 , Ryan W Serra  1 , Min Peng  1 , Janelle A Hayes  1 , Panagiotis A Konstantinopoulos  2 , Michael R Green  3 , Sharon B Cantor  4
Affiliations

Resistance to therapy in BRCA2 mutant cells due to loss of the nucleosome remodeling factor CHD4

Shawna Guillemette et al. Genes Dev. .

Abstract

Hereditary cancers derive from gene defects that often compromise DNA repair. Thus, BRCA-associated cancers are sensitive to DNA-damaging agents such as cisplatin. The efficacy of cisplatin is limited, however, by the development of resistance. One cisplatin resistance mechanism is restoration of homologous recombination (HR), which can result from BRCA reversion mutations. However, in BRCA2 mutant cancers, cisplatin resistance can occur independently of restored HR by a mechanism that remains unknown. Here we performed a genome-wide shRNA screen and found that loss of the nucleosome remodeling factor CHD4 confers cisplatin resistance. Restoration of cisplatin resistance is independent of HR but correlates with restored cell cycle progression, reduced chromosomal aberrations, and enhanced DNA damage tolerance. Suggesting clinical relevance, cisplatin-resistant clones lacking genetic reversion of BRCA2 show de novo loss of CHD4 expression in vitro. Moreover, BRCA2 mutant ovarian cancers with reduced CHD4 expression significantly correlate with shorter progression-free survival and shorter overall survival. Collectively, our findings indicate that CHD4 modulates therapeutic response in BRCA2 mutant cancer cells.

Keywords: BRCA2; CHD4; DNA repair; hereditary cancer.

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Figures

Figure 1.
Figure 1.
Genome-wide screen identifies CHD4 as a mediator of cisplatin response in BRCA2 mutant cells. (A) Schematic of BRCA2 species found in BRCA2 revertant C4-2 cells or PEO1 cells. (B) Nuclear extracts were analyzed by immunoblot with the indicated antibodies. Truncated BRCA2 in PEO1 cells migrates (∼220 kDa), while the revertant BRCA2 protein in C4-2 cells migrates (∼390 kDa). (C) Cells were left untreated or treated with cisplatin and analyzed for colony survival. (D) Schematic of viral infection with shRNA pools and screening conditions. (E) Colony survival of PEO1 cells containing four unique shRNA vectors targeting CHD4 or NSC as compared with BRCA2 revertant C4-2 cells (SF50 [in micromolar]: PEO1+shNSC, 0.21; shCHD4 (1–4), 0.55–0.56; and C4-2+shNSC, 0.7). PEO1 cells (F,G), FA-D1 cells (H,I), CAPAN1 cells (J,K), and BRCA2 revertant C4-2 cells (L,M) containing two unique pGIPZ shRNA vectors (one or two) targeting CHD4 or NSC were analyzed by immunoblot, left untreated or cisplatin-treated, and analyzed for colony survival. Where shown, error bars represent the standard deviation of the mean of three independent experiments.
Figure 2.
Figure 2.
CHD4 depletion corrects cisplatin-induced cell cycle progression defects and chromosomal aberrations in PEO1 cells. (A) Cells were left untreated or cisplatin-treated and analyzed for cell cycle with propidium iodide. (B,C) Cells were treated with cisplatin (B), and metaphase spreads were analyzed for percentage of radials per metaphase spread (C). At least 75 metaphase spreads were analyzed for each variable (25 per variable in triplicate). Where shown, error bars represent the standard deviation of the mean of three independent experiments. Asterisks denote significance from Student’s two-tailed, unpaired t-test: (***) P ≤ 0.005.
Figure 3.
Figure 3.
CHD4 depletion in BRCA2 mutant cells does not restore HR. (A) Cells were left untreated or treated with cisplatin and coimmunostained with the indicated antibodies. A representative image of cisplatin-treated cells is shown. (B) Depletion of NSC verses CHD4 was confirmed by immunoblot with the indicated antibodies, and the percentage of cells with RAD51 foci or γH2AX foci was quantified. (C) Cells were left untreated or cisplatin-treated, and nuclear extracts were analyzed with the indicated antibodies. (D) VC8 hamster cells were analyzed as in B and quantified for the percentage of GFP-positive cells by flow cytometry using the DR-GFP HR reporter assay.
Figure 4.
Figure 4.
CHD4 depletion in BRCA2 mutant cells enhances DNA damage tolerance. (A–C) BRCA2 mutant PEO1 cells (A), BRCA2 mutant FA-D1 cells (B), and BRCA2 revertant C4-2 cells (C) expressing pLKO.1 vectors targeting NSC or CHD4 were left untreated or cisplatin-treated, and nuclear extracts were analyzed with the indicated antibodies. Arrows indicate molecular weight of ∼37 kDa. (D) Cells were left untreated or cisplatin-treated and coimmunostained with the indicated antibodies. A representative image of cisplatin-treated cells is shown. (E) Quantification of the average fold increase in cells with RAD18 foci. (F) Schematic of HPRT mutagenesis assay. (G) Quantitation of HPRT mutagenesis assay. (H) PEO1 cells expressing pLKO.1 shRNAs targeting NSC or CHD4 were codepleted of RAD18 with two unique vectors versus NSC and analyzed by immunoblot blot with the indicated antibodies. (I) Cells were left untreated or cisplatin-treated and quantified for colony survival. Where shown, error bars represent the standard deviation of the mean of three independent experiments.
Figure 5.
Figure 5.
In BRCA2 mutant cancer cells, reduced CHD4 expression correlates with cisplatin resistance and poor patient response to chemotherapy. (A) Nuclear extracts of C4-2 cells, PEO1 cells, and derived cisplatin-resistant clones C4-4, C4-11, and C4-13 were analyzed by immunoblot with the indicated antibodies. (B) Cells were treated with cisplatin and immunostained with the indicated antibodies. A representative image is shown at 16 h. (C) Cells were left untreated or treated with cisplatin and analyzed for colony survival. (D) Cells were analyzed by immunoblot with the indicated antibodies. (E) Empty vector or HA CHD4 were ectopically expressed and analyzed by immunoblot with the indicated antibodies. (F) Quantification of average fold increase in apoptotic cells from three experiments. (G) Cells expressing pGIPZ shRNAs targeting NSC or RAD18 were analyzed by immunoblot with the indicated antibodies. (H) Cells were left untreated or cisplatin-treated and quantified for colony survival. (I,J) Above median or below median CHD4 mRNA levels were correlated with PFS and OS in sporadic ovarian cancers (I) and BRCA2 mutant ovarian cancers (J). P-values were determined by two-tailed log rank tests. (K) Model depicting the response to cisplatin-induced cross-links in BRCA2 mutant cells expressing or depleted of CHD4.
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