BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis

Abstract

Tumor cells have unstable genomes relative to non-tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti-cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2-targeting second-generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non-tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency in vivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti-cancer agent.

Keywords: BRCA2; Cisplatin; DNA repair; Drug sensitization; metabolism; metastasis.

Figure 1

Retrospective analysis of patient survival data based on BRCA2 mutation status. The TCGA Uterine Endometrial Carcinoma dataset was queried using the cBioPortal to determine changes in disease‐free (a) and overall survival, (b) in patients with alterations in BRCA2 (BRCA2 WT N = 215 (blue), BRCA2 mut N = 24 (red)). The TCGA Ovarian Serous Cystadenocarcinoma dataset was queried to determine changes in disease‐free and overall survival in patients with alterations in BRCA2 (BRCA2 WT N = 276, BRCA2 mut N = 39) (c and d) or BRCA1 (BRCA2 WT N = 277, BRCA2 mut N = 38) (e and f).

Figure 2

BRCA2 ASO decreases BRCA2 mRNA, protein, and RAD51 foci formation frequency in A549 cells. a) A549 cells were transfected with 20 nM BRCA2 ASO and total cellular RNA was isolated 24 h later. Relative BRCA2 mRNA levels (mean ± SD) were assessed with RT‐qPCR. b) BRCA2 protein levels were assessed 72 h after BRCA2 ASO transfection. c) RAD51 focus formation (mean ± SD) was quantified 48 h after BRCA2 ASO transfection. Statistical significance was determined using a Student's t‐test (p < 0.05).

Figure 3

BRCA2 regulates cisplatin‐induced reduction of lung, ovarian, and breast tumor cell proliferation. A549 cells were treated with varying concentrations of cisplatin (a) or melphalan (b) and control (black circles) or BRCA2 (white circles) ASO. Cells were counted 96 h post‐transfection to assess effects of BRCA2 knockdown. SKOV‐3 cells (c) and MDA‐MB‐231 cells (d) were treated with different concentrations of cisplatin and control or BRCA2 ASO, and counted 96 h post‐transfection. e) Non‐tumor HK‐2 kidney cells were treated with two concentrations of cisplatin and control or BRCA2 ASO, and relative proliferation was quantified using cell counting 96 h post‐transfection. f) BRCA2 deficient CAPAN‐1 cells were exposed to varying concentrations of cisplatin and control or BRCA2 ASO, and counted 96 h post‐transfection. *Different from cells treated with control ASO (p < 0.05).

Figure 4

BRCA2 ASO reverses acquired cisplatin resistance in human head and neck cancer cells. a) HN‐5a, HN‐5a/carbo‐10a, and HN‐5a/carbo‐15a cells were exposed to cisplatin and effects on proliferation were assessed 72 h after drug treatment. HN‐5a cells (b) and HN‐5a/carbo‐15a cells (c) were transfected with control (black circles) or BRCA2 (white circles) ASO, treated with varying concentrations of cisplatin and then counted 96 h post‐transfection. HN‐5a cells (d) and HN‐15a cells (e) were transfected with control or BRCA2 ASO, treated with different concentrations of cisplatin for 6 h, and then re‐plated at a density of 500 cells per well to determine colony forming ability. *Different from cells treated with control ASO using a Student's t‐test (p < 0.05).

Figure 5

BRCA2 modulates tumor cell metabolic response following cisplatin treatment. A549 cells were exposed to cisplatin (6 μM, 24 h) following 6 h of incubation in medium to determine baseline metabolic levels. At 24 h after addition of cisplatin, medium was exchanged for medium without cisplatin. Measurements of oxygen consumption (a), impedance (b), and changes in medium pH (c) were conducted. Magenta = Control ASO, Blue = BRCA2 ASO, Green = Control ASO + Cisplatin, Red = BRCA2 ASO + cisplatin. A549 cells were transfected with control or BRCA2 ASO and then treated with cisplatin (6 μM). Mitotracker staining (d) and glucose uptake (e) were determined using flow cytometry. Unstained control = Red, Control ASO = blue, BRCA2 ASO = orange, Control ASO + Cisplatin = light green, BRCA2 ASO + cisplatin = dark green. *Different from cells treated with control ASO using a Student's t‐test (p < 0.05).

Figure 6

BRCA2 regulates tumor cell metastatic frequency following cisplatin treatment in vivo. A549 cells were transfected with control (black) or BRCA2 (white) ASO and then exposed to varying concentrations of cisplatin (a) or different doses of ionizing radiation (b). Six hours after treatment, the capacity of cells to form colonies in vitro was determined. c) A549‐GFP cells were transfected with control or BRCA2 ASO, treated with 6 μM cisplatin for 6 h, then injected i.v. into the CAM (Left and Center panel). Metastatic foci were counted 7–9 days following injection (Right panel) (N = ≥6). *Different from cells treated with control ASO (p < 0.05).

Figure 7

BRCA2 mRNA expression levels in different human tumors. The TCGA database was queried for BRCA2 mRNA expression levels across different human cancer types.