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. 2014 Oct;34(20):3828-42.
doi: 10.1128/MCB.01646-13. Epub 2014 Aug 4.

BRCA1 pathway function in basal-like breast cancer cells

Sarah J Hill  1 , Allison P Clark  2 , Daniel P Silver  3 , David M Livingston  4
Affiliations

BRCA1 pathway function in basal-like breast cancer cells

Sarah J Hill et al. Mol Cell Biol. 2014 Oct.

Abstract

Sporadic basal-like cancers (BLCs) are a common subtype of breast cancer that share multiple biological properties with BRCA1-mutated breast tumors. Despite being BRCA1(+/+), sporadic BLCs are widely viewed as phenocopies of BRCA1-mutated breast cancers, because they are hypothesized to manifest a BRCA1 functional defect or breakdown of a pathway(s) in which BRCA1 plays a major role. The role of BRCA1 in the repair of double-strand DNA breaks by homologous recombination (HR) is its best understood function and the function most often implicated in BRCA1 breast cancer suppression. Therefore, it is suspected that sporadic BLCs exhibit a defect in HR. To test this hypothesis, multiple DNA damage repair assays focused on several types of repair were performed on a group of cell lines classified as sporadic BLCs and on controls. The sporadic BLC cell lines failed to exhibit an overt HR defect. Rather, they exhibited defects in the repair of stalled replication forks, another BRCA1 function. These results provide insight into why clinical trials of poly(ADP-ribose) polymerase (PARP) inhibitors, which require an HR defect for efficacy, have been unsuccessful in sporadic BLCs, unlike cisplatin, which elicits DNA damage that requires stalled fork repair and has shown efficacy in sporadic BLCs.

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Figures

FIG 1
FIG 1
Sporadic BLC cell lines express p220 and Δ11b. Full-length BRCA1 and the Δ11b BRCA1 isoform were immunoprecipitated from lysates of the sporadic BLC cell lines, normal controls, and BRCA1 mutant lines, using a C-terminal epitope BRCA1 antibody (sc6954). Blots were performed with an N-terminal epitope BRCA1 antibody (MS110). p220 migrates between the 148- and 250-kDa markers and is marked by a red arrow in the molecular mass marker lane. Δ11b migrates just above the 98-kDa marker and is marked by a green arrow in the molecular mass marker lane. Different colored circles mark the type of line that was tested in each blot, and the key for the circles is shown. Several BLC cell lines were blotted more than once to confirm the results.
FIG 2
FIG 2
Postdamage Rad51 foci in sporadic BLC cells and controls. (A) MDA MB 231 cells treated or not treated with 5 Gy irradiation and stained with γH2AX and Rad51 antibodies 8 h after treatment. An arrow has been placed next to a representative cell in which there is colocalization. The brightness was increased by 20% and the contrast was increased by 20% using PowerPoint in every panel to alleviate difficulties with the conversion of the images to PDF. DAPI, 4′,6-diamidino-2-phenylindole. (B) All sporadic BLC cell lines, normal controls, BRCA1 mutant lines, and non-BLC cell lines were treated with 5 Gy or mock treated (0 Gy), allowed to recover for 8 h, and then fixed and stained for Rad51. The percentage of cells containing three or more Rad51 foci was calculated for each cell line under each condition. This experiment was repeated 2 to 3 times for each cell line, and the bars in the bar graph represent the average percentage of cells containing 3 or more Rad51 foci, as determined in these experiments. The error bars represent the standard deviations between the experiments.
FIG 3
FIG 3
Representative immunofluorescence results after exposure to different DNA-damaging agents. (A) MDA MB 231 cells treated or not treated with 5 Gy irradiation and stained with γH2AX and BRCA1 antibodies 8 h after treatment. (B) MDA MB 231 cells treated or not treated with 5 Gy irradiation and stained with γH2AX and 53BP1 antibodies 8 h after treatment. (C and D) MDA MB 231 cells 4 h after 30-J UV irradiation through micropores stained with BRCA1 and CPD antibodies (C) or BRCA1 and γH2AX antibodies (D). CPDs are known to mark sites of UV damage. Arrows are placed next to a few representative cells in which there is colocalization. The brightness was been increased by 20% and the contrast was increased by 20% using PowerPoint in every panel to alleviate difficulties with the conversion of the images to PDF.
FIG 4
FIG 4
Sensitivity of cell lines to various DNA-damaging agents. Cells of each line were plated in triplicate at a density suitable for colony formation, allowed to settle, treated with various doses of different DNA-damaging agents, and then allowed to recover and grow at 37°C until colonies became visible. After colonies of the appropriate size had grown, the cells were stained with crystal violet staining solution and counted with a Microbiology International ProtoCOL colony counter. IC50s for each cell line for each treatment were calculated on the basis of the dose-response curves generated from these counts. The bars in the bar graphs represent the average IC50s for each cell line from 2 to 3 experiments after the following: gamma irradiation (A), UV irradiation (B), MMC treatment (C), and MMS treatment (D). The error bars represent the standard deviations between experiments. The color of the brackets grouping certain cell lines represents the cell line type, as depicted in the key at the bottom.
FIG 5
FIG 5
BLC cell lines are HR proficient regardless of gamma irradiation sensitivity status. (A) Southern blots were performed on multiple clones of the three cell lines used in the HR reporter experiments in this figure to identify a clone from each line that carries a single, integrated copy of the I-SceI–GFP HR reporter. Separate blots for the three clones studied in the HR reporter experiments are shown here. Based on the sequence of the reporter, we digested clonal genomic DNA separately with two different enzymes to assess the reporter copy number. We digested with HindIII, which should give rise to a band of 812 bp and another single band of various sizes in clones bearing a single copy of the reporter. We also digested with StuI, which should give rise to a band of 2,017 bp and another single band of various sizes in clones bearing a single copy of the reporter. (B) Map of the target sites of the four BRCA1 siRNAs used in the HR experiments on p220 and Δ11b. (C) (Left) The average percentage of GFP-positive cells in the HR reporter assay for each cell line with each siRNA is represented in the bar graph. The error bars represent the standard deviations from four experiments. (Right) The HR reporter results have been normalized to those for the siGL2 control for each respective day in each respective cell line, and the bars in the graph represent the average percentage of GFP-positive cells relative to the number of GFP-positive siGL2-transfected control cells for that cell line, with the error bars representing the standard deviations from the four experiments. (D) IP-Western blotting was performed on extracts of each cell line transfected with each siRNA to demonstrate the efficacy of depletion of the specific isoform by each siRNA. IPs were performed with a C-terminal BRCA1 antibody, and the blots were developed with an N-terminal BRCA1 antibody so that both the p220 and Δ11b isoforms could be detected. Green arrow, Δ11b; red arrow, p220. (E) Western blotting (WB) assays were performed on half of the cells from one round of the HR reporter assay to demonstrate that the I-SceI protein levels were the same in each cell line after each siRNA treatment. The I-SceI that was transfected was tagged with HA. After loading of equivalent amounts of protein on the gel from each cell line transfected with each siRNA, the blots were stained with HA antibody to detect I-SceI. Tubulin blotting was performed to show that equivalent amounts of protein were loaded in each lane. B1, BRCA1; ex, exon.
FIG 6
FIG 6
HR capacity of HS578T and MDA MB 157 cell lines. The HR capacity of the sporadic BLC cell lines HS578T and MDA MB157 was tested by transfecting these lines with a control siRNA or two different BRCA1 siRNAs, seeding the cells at a density suitable for colony formation, and exposing the cells to various concentrations of the PARP inhibitor olaparib. The cells were grown in PARP inhibitor-containing medium until colonies of suitable size had formed. Colonies were stained and counted, and IC50s were estimated from the dose response curves generated by these colony counts. (A and C) The average IC50 for each siRNA from at least three separate experiments is shown in the bar graphs for HS578T (A) and MDA MB 157 (C) cells. Since the siBRCA1 IC50s are so low compared to those for the controls, a table with the relevant values is introduced below each bar graph. The error bars in the bar graphs represent the standard deviations between the averages for multiple experiments. The P values comparing each set of siBRCA1 results to the siGL2 results were calculated using a two-tailed t test in GraphPad Prism software. (B and D) BRCA1 IP-Western blots of HS578T and MDA MB 157 cells transfected with the siRNAs used for the assays whose results are presented in panels A and C demonstrate the ability of these siRNAs to deplete BRCA1 and are shown for HS578T (B) and MDA MB 157 (D) cells. In these blots, the p220 and Δ11b isoforms are indicated by arrows of different colors. UTR, untranslated region.
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