Enhanced sensitivity to cisplatin and gemcitabine in Brca1-deficient murine mammary epithelial cells

Abstract

Background: Breast cancers due to germline mutations or altered expression of the BRCA1 gene associate with an aggressive clinical course and frequently exhibit a "triple-negative" phenotype, i.e. lack of expression of the estrogen and progesterone hormone receptors and lack of overexpression of the HER2/NEU oncogene, thereby rendering them relatively insensitive to hormonal manipulation and targeted HER2 therapy, respectively. BRCA1 plays a role in multiple DNA repair pathways, and thus, when mutated, results in sensitivity to certain DNA damaging drugs.

Results: Here, we used a Brca1 murine mammary epithelial cell (MMEC) model to examine the effect of loss of Brca1 on cellular sensitivity to various chemotherapy drugs. To explore novel therapeutic strategies, we included DNA damaging and non-DNA damaging drugs whose mechanisms are dependent and independent of DNA repair, respectively, and drugs that are used in standard and non-standard lines of therapy for breast cancer. To understand the cellular mechanism, we also determined the role that DNA repair plays in sensitivity to these drugs. We found that cisplatin and gemcitabine had the greatest specific therapeutic benefit to Brca1-deficient MMECs, and that when used in combination produced a synergistic effect. This sensitivity may be attributed in part to defective NER, which is one of the DNA repair pathways normally responsible for repairing DNA adducts produced by cisplatin and is shown in this study to be defective in Brca1-deficient MMECs. Brca1-deficient MMECs were not differentially sensitive to the standard breast cancer chemotherapy drugs doxorubicin, docetaxel or 5-FU.

Conclusions: Both cisplatin and gemcitabine should be explored in clinical trials for first line regimens for BRCA1-associated and triple-negative breast cancer.

Figure 1

Effect of BRCA1-deficiency on Sensitivity to DNA Damaging Agents. Brca1^+/+ (black circle) and Brca1^-/- (white square) cells were analyzed for cellular sensitivity to (a) doxorubicin (b) cisplatin (c) carboplatin or (d) oxaliplatin by MTT assay. Each data point represents the average of triplicate readings ± S.D. Graphs are representative of at least three independent experiments.

Figure 2

Effect of Brca1-deficiency on Sensitivity to Non-DNA Damaging Agents. Brca1^+/+ (black circle) and Brca1^-/- (white square) cells were analyzed for cellular sensitivity to taxanes, paclitaxel (a) or docetaxel (b), and to antimetabolites, 5-FU (c) or gemcitabine (d) by MTT assay. Each data point represents the average of triplicate readings ± S.D. Graphs are representative of at least three independent experiments.

Figure 3

Sensitivity to the Combination of Cisplatin and Gemcitabine. Brca1^+/+ (a) and Brca1^-/- (b) MMECs were treated with cisplatin and gemcitabine either alone or in combination and analyzed for sensitivity by MTT assay. The IC₅₀ values determined from treatment with cisplatin and gemcitabine alone were plotted as axial points (black circles) on a Cartesian plot to generate a line of additivity. The IC₅₀ values for each combination of cisplatin and gemcitabine were then plotted as data points (black squares). Data points above the line of additivity represent an antagonistic effect, data points on the line of additivity represent an additive effect, and data points below the line additivity represent a synergistic effect. Data are representative of at least three independent experiments.

Figure 4

Effect of Brca1-deficiency on NER and Double-strand DNA Break Repair. In (a), GGR of CPDs in Brca1^+/+ (black circle) and Brca1^-/- (white square) cells was measured using an immunoslot blot assay. Cells were exposed to 10 J/m² UV-irradiation and collected at the indicated times. DNA repair was expressed as a percentage relative to control. Data from triplicate DNA samples from three different biological experiments were expressed as an average ± S.E.M. In (b), sensitivity to UV-irradiation was determined by MTT assay for Brca1^+/+ (black circle) and Brca1^-/- (white square) cells. In (c), damage-induced expression of Xpc mRNA, an NER gene involved in DNA damage recognition, in Brca1^+/+ and Brca1^-/- cells was measured using RT-qPCR. Brca1^+/+ and Brca1^-/- cells were exposed to 10 J/m² of UV and either harvested immediately (control) or incubated in media and harvested 24 h later. In (d), expression of Xpc mRNA following 24 hours of treatment with 0.1 μM cisplatin or 0.01 μM gemcitabine in Brca1^+/+ and Brca1^-/- cells was measured using RT-qPCR. Data were calculated relative to the untreated control and expressed as the average of three experiments ± S.E.M. In (e), DNA strand breaks were measured at 24 hours following treatment with 0.1 μM cisplatin, 0.01 μM gemcitabine, or 0.1 μM doxorubicin in Brca1^+/+ and Brca1^-/- cells using the alkaline comet assay. Comet tails indicate DNA damage. Unless indicated otherwise, data were expressed as an average of triplicate readings ± S.D. **, p < 0.01; *, p < 0.05.