A Recurrent ERCC3 Truncating Mutation Confers Moderate Risk for Breast Cancer

Abstract

Known gene mutations account for approximately 50% of the hereditary risk for breast cancer. Moderate and low penetrance variants, discovered by genomic approaches, account for an as-yet-unknown proportion of the remaining heritability. A truncating mutation c.325C>T:p.Arg109* (R109X) in the ATP-dependent helicase ERCC3 was observed recurrently among exomes sequenced in BRCA wild-type, breast cancer-affected individuals of Ashkenazi Jewish ancestry. Modeling of the mutation in ERCC3-deficient or CRISPR/Cas9-edited cell lines showed a consistent pattern of reduced expression of the protein and concomitant hypomorphic functionality when challenged with UVC exposure or treatment with the DNA alkylating agent IlludinS. Overexpressing the mutant protein in ERCC3-deficient cells only partially rescued their DNA repair-deficient phenotype. Comparison of frequency of this recurrent mutation in over 6,500 chromosomes of breast cancer cases and 6,800 Ashkenazi controls showed significant association with breast cancer risk (OR_BC = 1.53, OR_ER+ = 1.73), particularly for the estrogen receptor-positive subset (P < 0.007).

Significance: A functionally significant recurrent ERCC3 mutation increased the risk for breast cancer in a genetic isolate. Mutated cell lines showed lower survival after in vitro exposure to DNA-damaging agents. Thus, similar to tumors arising in the background of homologous repair defects, mutations in nucleotide excision repair genes such as ERCC3 could constitute potential therapeutic targets in a subset of hereditary breast cancers. Cancer Discov; 6(11); 1267-75. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1197.

Figure 1

Identification of germline mutations in ERCC3 in a family with multiple breast cancer cases. Sequencing was performed on 3 individuals affected with breast cancer, confirming identification of ERCC3 R109X in all 3 affected siblings. Pathology reports for individuals II-3 and II-5, show both with well-differentiated (low grade) invasive ductal carcinoma diagnosed at Stage IA. One of the tumors was ER+PR+Her2+ (II-3) the other one was ER+PR-Her2- (II-5).

Figure 2

Functional Evaluation of the ERCC3 mutant via overexpression in an ERCC3 deficient cell line. (A) transcript and (B) protein levels in XPCS2BA parental cell line and cell lines stably overexpressing a lentiviral contruct containing wild-type ERCC3 (WT) or the R109X mutant (R109X) cDNA. Expression from the mutant cDNA produces a truncated protein fragment of about 12kDa. (C-D) Relative cell viablilty of XPCS2BA and ERCC3 wild-type and mutant overexpressing cell lines at 72 hours following treatment with increasing doses of IlludinS (C) or UVC (D). (E) Host cell reactivation assay showing reduced DNA repair ability of the mutant as opposed to wild-type ERCC3 overexpressing cell lines. Data represents the mean of three experiments with error bars representing the SD (C) or SEM (D, E). (F) Phosphorylation of H2AX and Chk1 in response to UVC-induced DNA damage. XPCS2BA, WT or R109X cell lines were harvested at different time points following exposure to 20J/m2 UVC and activation of H2AX and Chk1 was assessed by western blotting.

Figure 3

Modeling of ERCC3 R109X by CRISPR/Cas9 in a mammary epithelial cell line and functional analysis. (A) ERCC3 transcript and (B) protein levels in HMLE and CRISPR/Cas9 edited cells lines modeling the heterozygous R109X mutation. (C) Relative cell viability of HMLE control and CRISPR edited cell lines at 72 hours following treatment with increasing doses of IlludinS. Data represents the mean of three experiments with error bars representing the SEM. (D) Relative cell viability of HMLE control and combined CRISPR edited cell lines (with and without re-expression of the wild-type ERCC3) following treatment with IlludinS. (E) Quantification of phosphorylated H2AX by flow cytometry in HMLE control and CRISPR edited cell lines harvested at different time points after DNA damage.