Breast cancer-associated Abraxas mutation disrupts nuclear localization and DNA damage response functions

Abstract

Breast cancer is the most common cancer in women in developed countries and has a well-established genetic component. Germline mutations in a network of genes encoding BRCA1, BRCA2, and their interacting partners confer hereditary susceptibility to breast cancer. Abraxas directly interacts with the BRCA1 BRCT (BRCA1 carboxyl-terminal) repeats and contributes to BRCA1-dependent DNA damage responses, making Abraxas a candidate for yet unexplained disease susceptibility. Here, we have screened 125 Northern Finnish breast cancer families for coding region and splice-site Abraxas mutations and genotyped three tagging single-nucleotide polymorphisms within the gene from 991 unselected breast cancer cases and 868 female controls for common cancer-associated variants. A novel heterozygous alteration, c.1082G>A (Arg361Gln), that results in abrogated nuclear localization and DNA response activities was identified in three breast cancer families and in one additional familial case from an unselected breast cancer cohort, but not in healthy controls (P = 0.002). On the basis of its exclusive occurrence in familial cancers, disease cosegregation, evolutionary conservation, and disruption of critical BRCA1 functions, the recurrent Abraxas c.1082G>A mutation connects to cancer predisposition. These findings contribute to the concept of a BRCA-centered tumor suppressor network and provide the identity of Abraxas as a new breast cancer susceptibility gene.

Fig. 1

Identification of Abraxas mutation c.1082G>A (Arg361Gln) in breast cancer index cases and cancer families. (A) Schematic diagram of the Abraxas protein and the site of Arg361Gln. Chromatogram of c.1082G>A (Arg361Gln) is displayed directly below the bipartite NLS 358-Lys-Arg-Ser-Arg-361 and 368-Lys-Arg-Ser-Lys-371 shown in yellow. (B) Evolutionary conservation of Abraxas c.1082G and the encoded codon Arg³⁶¹. The conservation scoring was performed by PRALINE. The scoring scheme extends from 0 for the least conserved alignment position up to * for the most conserved alignment position. (C) Pedigrees of two Abraxas c.1082G>A mutation–positive breast cancer families where segregation analysis was possible (BR-0194 and 96-653). Black circles represent breast (Br) cancer cases; other cancer types [brain, colon (Co), endometrial (End), fibrosarcoma (Fsar), head, lip, lung, lymphoma (Ly), neuroblastoma (Nb), ovarian (Ov), pancreatic (Pan), prostate (Pro), sarcoma (Sar), skin, stomach (Sto); Ca, unknown] are marked with gray circles (females) or squares (males). Arrows point to index patients. A slashed symbol indicates a deceased individual. Sample identification codes are provided for all individuals where DNA specimens were available for mutation status analysis. Individuals are marked with a plus sign if mutation-positive. The age at diagnosis is indicated below the patients. The age at monitoring (or age at death), when known, is shown for the healthy individuals.

Fig. 2

Abraxas R361Q disrupts nuclear localization and recruitment to DNA DSBs. (A) IF of HA-tagged Abraxas wild-type (WT) and Abraxas R361Q transiently transfected in U2OS (top) and MCF10A (bottom) cells demonstrates cytoplasmic localization for the Abraxas mutant R361Q. DAPI, 4′,6-diamidino-2-phenylindole. (B) Quantification of cells presenting a predominantly nuclear staining in HeLa, U2OS, and MCF10A stably expressing FLAG-HA Abraxas WT or R361Q. Error bars represent means ± SD. P values were calculated by an unpaired t test. ***P < 0.0001. (C) Immunoblot (IB) of complexes after FLAG immunoprecipitation (IP) of FLAG-HA–tagged Abraxas from whole-cell lysates of 293T cells (-) or 293T cell lines stably expressing either Abraxas WT or R361Q. The location of endogenous and ectopic Abraxas protein is indicated. (D) IB of FLAG-HA–tagged Abraxas complexes after FLAG-IP from cytoplasmic and nuclear fractions of HeLa S3 cell lines that maintain stable expression of either Abraxas WT or Abraxas R361Q.

Fig. 3

Abraxas R361Q impairs BRCA1 and RAP80 DNA damage response functions. (A) IF in U2OS cells at 7 hours after 10 Gy IR demonstrates decreased BRCA1 and RAP80 IRIF in cells expressing Abraxas R361Q. (B) Quantification of the percentage of HeLa, U2OS, and MCF10A cells expressing Abraxas WT or R361Q with greater than five BRCA1 or RAP80 foci per nucleus. The mean was calculated from more than 200 cells for each condition in triplicate. Error bars represent means ± SD. P values were calculated by an unpaired t test. *P = 0.0181; **P = 0.0056; ***P < 0.0001. (C) Clonogenic survival assay of U2OS cells stably expressing Abraxas WT or R361Q after exposure to the indicated doses of γ-radiation. Points represent the average of three independent experiments run in triplicate. Error bars represent means ± SD. (D) Evaluation of the IR-induced G₂-M checkpoint in transiently transfected 293T cells. Mitotic cells were detected by FACS using an antibody against phospho–histone H3. The mean mitotic cell population was calculated from three independent experiments in which, in each sample, 10,000 cells were examined. Error bars represent SD. P values were calculated by Student’s t test assuming unequal variances. ***P < 0.0001. (E) Evaluation of the homology-directed DNA repair of a nuclease-induced DSB after transfection of a DR-GFP reporter cell line with the indicated plasmids. GFP-positive cells indicate the presence of homology-directed DNA repair. Data are representative of four independent experiments. Error bars represent means ± SD. P values were calculated by an unpaired t test. **P = 0.0028; ***P < 0.0001.