Overexpression of BLM promotes DNA damage and increased sensitivity to platinum salts in triple-negative breast and serous ovarian cancers

Abstract

Background: Platinum-based therapy is an effective treatment for a subset of triple-negative breast cancer and ovarian cancer patients. In order to increase response rate and decrease unnecessary use, robust biomarkers that predict response to therapy are needed.

Patients and methods: We performed an integrated genomic approach combining differential analysis of gene expression and DNA copy number in sensitive compared with resistant triple-negative breast cancers in two independent neoadjuvant cisplatin-treated cohorts. Functional relevance of significant hits was investigated in vitro by overexpression, knockdown and targeted inhibitor treatment.

Results: We identified two genes, the Bloom helicase (BLM) and Fanconi anemia complementation group I (FANCI), that have both increased DNA copy number and gene expression in the platinum-sensitive cases. Increased level of expression of these two genes was also associated with platinum but not with taxane response in ovarian cancer. As a functional validation, we found that overexpression of BLM promotes DNA damage and induces sensitivity to cisplatin but has no effect on paclitaxel sensitivity.

Conclusions: A biomarker based on the expression levels of the BLM and FANCI genes is a potential predictor of platinum sensitivity in triple-negative breast cancer and ovarian cancer.

Figure 1.

BLM and FANCI are significantly associated with response to cisplatin chemotherapy. Scatter-plots of LOO analysis in cisplatin-1 and cisplatin-2 cohorts identifies BLM and FANCI as the only two genes that show significant association with response in both clinical trials, based on both gene expression data (A) and copy number aberration data (B). Red dashed lines indicate significance thresholds based on permutation testing. Color intensity indicates overlapping genes. Association between array expression of BLM, FANCI, and the ratio of BLM+FANCI expression to BRCA1, and response to cisplatin chemotherapy in cisplatin-1 (C, E, G), cisplatin-2 (D, F, H) and OV01 carboplatin-treated (I, J, K) and OV01 paclitaxel-treated (L, M, N) cohorts. Red dots indicate the BRCA1 mutant cases.

Figure 2.

Relationships of BLM, FANCI, and BRCA1 expression levels in breast cancer cell lines and association with therapy sensitivities. (A) Western blot analysis of BLM, BRCA1, and Actin protein abundance in a panel of breast cancer cell lines. BT549, HCC1143, HCC38, MDA231, and MDA453 are BRCA1 wild-type genotype. HCC1937 and MDA436 are BRCA1 mutated (B) Densitometry of Western blot in A for quantification of BLM/Actin and (C) BRCA1/BLM ratio. (D–G) Bar plots of IC50 to treatments in panel of cell lines. Breast cancer cells lines were irradiated with increasing doses of UV-C or subjected to cisplatin, olaparib or paclitaxel treatment, and 4 weeks later assayed for colony formation. Error bars represent the standard deviation between three independent experiments. (D) IC50 to cisplatin (E) IC50 to UV-C (F) IC50 to PARP-inhibitory olaparib (G) IC50 to paclitaxel. (H) Western blot showing the effect of shRNA BRCA1 or shLuciferase control (shLuc) on expression of BLM (left side) and FANCI (right side) in U2OS cells. GAPDH is shown as a loading control. (I) Western blot demonstrating gene-specific siRNA knockdown of BLM or FANCI expression in BT549 breast tumor cells. (J) Bar plots indicate the ratio of IC₅₀ for the cisplatin (black bars) and paclitaxel (gray bars) in gene-specific siRNA-treated cells, relative to scramble control siRNA-treated cells. (K) Western blot for endogenous BLM and HA-tag in MDA231 cells transfected with control vector (lane 1), HA-tag BLM cDNA (BLM, lane2), HA-tag BLM co-treated with BLM helicase small molecule inhibitory (BLM + BLMi, lane 3), and HA-tag BLM co-treated with 50 nM siRNA BLM (BLM + siBLM, lane 4). (L) Bar plots indicate the ratio of IC₅₀ for the cisplatin in MDA231 cells treated with control vector (black bar), HA-tag BLM cDNA (BLM, medium gray bar), HA-tag BLM co-treated with BLM helicase small molecule inhibitory (BLM + BLMi, dark gray bar), and HA-tag BLM co-treated with 50 nM siRNA BLM (BLM + siBLM, light gray bar).

Figure 3.

Increased DNA damage upon BLM overexpression in MDA231 cells. MDA231 cells were infected with control vector or HA-tag BLM cDNA (BLM) and co-treated with 20 µM BLM small molecule inhibitory (Bi) or 100 nM siBLM (si). Cells were mock treated (cisplatin: 0 h, A) or treated with 10 µM cisplatin for 4-h (cisplatin: 4 h, panel B) and released for 24 h. Immunofluorescence for phospho-H2Ax and phospho-53BP1 was carried out and nuclei counterstained with DAPI. (A) Representative immunofluorescent images for indicated markers in mock treated cells (cisplatin: 0 h) indicating spontaneous DNA damage foci. (B) Representative immunofluorescent image in cells treated with 10 µM cisplatin for 4 h (cisplatin: 4 h) indicating drug induced damage foci. All images were obtained at the same magnification and exposure time. All images were analyzed in parallel for each experiment. (C and D) Cells containing foci recognized by relevant antibodies in immunofluorescence assays, were identified and counted. At least 100 cells were counted for each category of foci at each time point. Bar plots indicate percentages of cells, noted above, that contain γH2AX-p foci (C) and PB53-p foci (D) in mock treated cells (cisplatin: 0 h, black bars) and 10 µM cisplatin treated cells (cisplatin: 4 h, gray bars).