Tamoxifen-resistant breast cancer cells are resistant to DNA-damaging chemotherapy because of upregulated BARD1 and BRCA1

Abstract

Tamoxifen resistance is accountable for relapse in many ER-positive breast cancer patients. Most of these recurrent patients receive chemotherapy, but their chemosensitivity is unknown. Here, we report that tamoxifen-resistant breast cancer cells express significantly more BARD1 and BRCA1, leading to resistance to DNA-damaging chemotherapy including cisplatin and adriamycin, but not to paclitaxel. Silencing BARD1 or BRCA1 expression or inhibition of BRCA1 phosphorylation by Dinaciclib restores the sensitivity to cisplatin in tamoxifen-resistant cells. Furthermore, we show that activated PI3K/AKT pathway is responsible for the upregulation of BARD1 and BRCA1. PI3K inhibitors decrease the expression of BARD1 and BRCA1 in tamoxifen-resistant cells and re-sensitize them to cisplatin both in vitro and in vivo. Higher BARD1 and BRCA1 expression is associated with worse prognosis of early breast cancer patients, especially the ones that received radiotherapy, indicating the potential use of PI3K inhibitors to reverse chemoresistance and radioresistance in ER-positive breast cancer patients.

Fig. 1

BARD1/BRCA1 is upregulated in tamoxifen-resistant breast cancer cells. a The growth curve of MCF7-Pa and MCF7-Re cells under tamoxifen treatment. b mRNA expression profile analysis shows upregulated proliferation-related genes in MCF7-Re cells. c Kaplan–Meier survival curves of breast cancer patients with low (black) and high (red) BARD1 or BRCA1 expression based on Curtis data set. d The mRNA and protein expression of BARD1 and BRCA1 in MCF7-Pa and MCF7-Re breast cancer cells. In a, d, data show means ± s.d. (n = 3) **p < 0.01; ***p < 0.001 compared with MCF7-Pa by Student’s t test

Fig. 2

Tamoxifen-resistant cells are resistant to DNA-damaging chemotherapy. a Cell viability assay showing the effect of tamoxifen on MCF7-Re cells when transfected with negative control siRNA (nc), BARD1, and/or BRCA1 siRNAs. b MCF7-Pa and MCF7-Re cells were treated with different concentrations of cisplatin (cis), adriamycin (ADM), or paclitaxel, and the proportions of viable cells were examined by cell viability assay. c Apoptosis assay by AnnexinV-FITC/PI staining showing the percentages of apoptotic cells when MCF7-Pa and MCF7-Re cells were treated with cisplatin. d Comet assay showing the DNA damage when MCF7-Pa and MCF7-Re cells were treated with cisplatin. Scale bar indicated 100 μm. e Western blot showing the expression of γH2AX when MCF7-Pa and MCF7-Re cells were treated with cisplatin. f Representative immunofluorescence staining of γH2AX, Rad51, and BRCA1 in MCF7-Pa and MCF7-Re cells treated with cisplatin at 0 h, 2 h, 8 h. Scale bar indicated 25 μm. g Percentages of positive nuclei (10 foci or more per nucleus being considered positive) from three fields per sample are indicated. In a–d, g, data show means ± s.d. (n = 3) *p < 0.05; **p < 0.01; compared with control or indicated lines by Student’s t test

Fig. 3

Reduced BARD1 or BRCA1 expression sensitizes tamoxifen-resistant cells to cisplatin. MCF7-Re cells were transfected with negative control siRNA (nc), BARD1, and/or BRCA1 siRNAs. a, b Cell viability assay showing the sensitivity to cisplatin (a) and ADM (b). c Apoptosis assay by AnnexinV-FITC/PI staining showing the effect of cisplatin on the percentages of apoptotic cells. d The expression of γH2AX induced by cisplatin. e Comet assay showing the DNA damage induced by cisplatin. Scale bar indicated 100 μm. In a–c, e, data show means ± s.d. (n = 3) **p < 0.01 compared with control by Student’s t test

Fig. 4

PI3K inhibition decreases BRCA1/BARD1 expression and sensitizes tamoxifen-resistant cells to cisplatin. a, b The protein (a) and mRNA (b) expression of BARD1/BRCA1 in MCF7-Re cells treated with BKM120 or BYL719. c–f MCF7-Re cells were treated with control, BKM120, or BYL719 and then treated with cisplatin. The sensitivity to increasing concentrations of cisplatin (c), apoptotic cells (d), γH2AX expression (e), and DNA damage shown by comet assay (f) were measured. Scale bar indicated 100 μm. g–i MCF7-Re cells were treated with Dinaciclib and cisplatin. The sensitivity to increasing concentrations of cisplain (g), apoptotic cells (h), and γH2AX expression (i) were measured. In b–d, f–h, data show means ± s.d. (n = 3) **p < 0.01 compared with control by Student’s t test

Fig. 5

PI3K inhibitor restores the sensitivity to cisplatin in xenografts of tamoxifen-resistant tumor in vivo. MCF7-Pa and MCF7-Re cells were inoculated into the mammary fat pads of Balb/c nude mice. When tumors became palpable, tumors were treated with control, cisplatin, or cisplatin with BKM120. The picture (a), tumor volume (b), and immunohistochemical staining of BARD1/BRCA1 and Tunel staining (c) of xenografts derived from MCF7-Pa and MCF7-Re cells with indicated treatment. d IHC scores of BRCA1 and BARD1 and Tunel-positive cell number counts in indicated xenografts. In b, d, data show means ± s.d. (n = 6) **p < 0.01 by Student’s t test. Scale bar indicated 400 µm

Fig. 6

The expression of BARD1 and BRCA1 are increased in metastatic breast cancer and associated with poor prognosis in ER+ breast cancer patients received radiotherapy. a, b Representative pictures (a) and quantification (b) of IHC staining of BARD1, BRCA1, and p-AKT in paired primary and metastatic breast cancer tissues. c, d Correlation analysis of p-AKT expression with BARD1 (c) or BRCA1 (d) expression in primary and metastatic breast cancer samples. e Overall survival of breast cancer patients in Curtis data set with or without radiotherapy whose tumors had high or low BARD1/BRCA1 expression. In b, data show means ± s.d. (n = 27) *p < 0.05; **p < 0.01; ***p < 0.001 compared with that in primary breast cancer by Student’s t test. Scale bar indicated 400 µm