Inactivation of BRCA2 in human cancer cells identifies a subset of tumors with enhanced sensitivity towards death receptor-mediated apoptosis

Abstract

Purpose: DNA repair defects due to detrimental BRCA2-mutations confer increased susceptibility towards DNA interstrand-crosslinking (ICL) agents and define patient subpopulations for individualized genotype-based cancer therapy. However, due to the side effects of these drugs, there is a need to identify additional agents, which could be used alone or in combination with ICL-agents. Therefore, we investigated whether BRCA2-mutations might also increase the sensitivity towards TRAIL-receptors (TRAIL-R)-targeting compounds.

Experimental design: Two independent model systems were applied: a BRCA2 gene knockout and a BRCA2 gene complementation model. The effects of TRAIL-R-targeting compounds and ICL-agents on cell viability, apoptosis and cell cycle distribution were compared in BRCA2-proficient versus-deficient cancer cells in vitro. In addition, the effects of the TRAIL-R2-targeting antibody LBY135 were assessed in vivo using a murine tumor xenograft model.

Results: BRCA2-deficient cancer cells displayed an increased sensitivity towards TRAIL-R-targeting agents. These effects exceeded and were mechanistically distinguishable from the well-established effects of ICL-agents. In vitro, ICL-agents expectedly induced an early cell cycle arrest followed by delayed apoptosis, whereas TRAIL-R-targeting compounds caused early apoptosis without prior cell cycle arrest. In vivo, treatment with LBY135 significantly reduced the tumor growth of BRCA2-deficient cancer cells in a xenograft model.

Conclusions: BRCA2 mutations strongly increase the in vitro- and in vivo-sensitivity of cancer cells towards TRAIL-R-mediated apoptosis. This effect is mechanistically distinguishable from the well-established ICL-hypersensitivity of BRCA2-deficient cells. Our study thus defines a new genetic subpopulation of cancers susceptible towards TRAIL-R-targeting compounds, which could facilitate novel therapeutic approaches for patients with BRCA2-deficient tumors.

Keywords: BRCA2; TRAIL; apoptosis; gene targeting; targeted therapy.

Figure 1. Genetic BRCA2 inactivation enhances the sensitivity of cancer cells towards TRAIL-R-mediated apoptosis

(A) Proliferation assays of BRCA2-proficient DLD1 and corresponding BRCA2^+/− A9A2 cells versus two corresponding homozygously BRCA2-deleted clones (A10-1 BRCA2^−/− and A10-3 BRCA2^−/−) after treatment with the indicated agents. (B) Confirmatory proliferation assays of BRCA2-deficient parental (CAPAN1) and empty vector-transfected (CAPAN1/NEO) pancreatic cancer cells versus two BRCA2-complemented cell clones CAPAN1/CIN and CAPAN1/236 after treatment with the indicated agents. All experiments were performed in triplicate with error bars representing SEM from three independent experiments. * = extrapolated value. (C, D) Effects of BRCA2-status on cell viability upon administration of the clinically viable TRAIL-R2-targeting agonistic antibodies LBY-135 and tigatuzumab in BRCA2-proficient and BRCA2-deficient DLD1 cells (C) and in BRCA2-proficient and BRCA2-deficient CAPAN1 cells (D).

Figure 2. siRNA-mediated BRCA2 knockdown enhances the sensitivity of cancer cells towards TRAIL-R-mediated apoptosis

Western blot analysis of BRCA2 expression levels upon siRNA-mediated knockdown of untreated, control- and BRCA2-siRNA-transfected DLD1 cells (upper panel). Corresponding proliferation assays after treatment with LBY135 (lower panel). Experiments were performed in triplicate with error bars representing SEM from three independent experiments.

Figure 3. TRAIL-R stimulation causes early onset of apoptosis without concomitant cell cycle arrest in BRCA2^−/− cells

(A) Cell cycle profiles (left panels) and subG1 fraction (right panels) of parental DLD1 cells (upper panels) versus BRCA2^−/− A10-1 cells (lower panels), treated with MMC at 50 nM or left untreated and assessed at the indicated time points. Arrows indicate samples displaying a significant G2/M arrest. (B) Cell cycle distribution (left panel) and subG1 fraction (right panel) of parental DLD1 cells versus BRCA2^−/− A10-1 cells, treated with LBY135 at the indicated concentrations or left untreated, assessed at 24 h after treatment. (C) Cell cycle profiles displaying representative results from one of at least three experiments derived from (B).

Figure 4. Apoptosis and CASPASE8 recruitment in BRCA2^−/− cells are not dependent on the mitochondrial pathway or on the regulation of TRAIL-receptors

(A) Typical features of apoptosis (chromatin condensation and nuclear fragmentation) in DLD1 BRCA2^−/− A10-1 cells 24 h after treatment with LBY135 at 125 ng/ml, as assessed by fluorescence microscopy after Hoechst staining. (B + C) Western blotting to detect caspase 8 and caspase 3 cleavage upon treatment with LBY at 125 ng/ml at the indicated time points in parental DLD1 versus BRCA2^−/− A10-1 cells (B) and in BRCA2-deficient parental (CAPAN1) and empty vector-transfected (CAPAN1/NEO) cancer cells versus two BRCA2-complemented cell clones (CAPAN1/CIN and CAPAN1/236) (C). (D) Western blotting to assess the baseline expression levels of the indicated regulators of the mitochondrial pathway in BRCA2-proficient versus BRCA2-deficient DLD1 cells. BCL-2 was detectable in control cell lines (not shown) but not in DLD1 cells. (E) FACS analysis of surface receptor staining of TRAIL-R1 and TRAIL-R2 in BRCA2-proficient versus BRCA2-deficient DLD1 cells.

Figure 5. BRCA2-deficiency delays tumor growth upon administration of LBY135 in a murine tumor xenograft model in vivo

(A) Time course (21d) of tumor growth as assessed by repeated measurements of xenograft tumors in mice subcutaneously injected with parental DLD1 cells or A10-3 BRCA2^−/− cell clones, respectively, which were consecutively treated intraperitoneally thrice a week with either LBY135 at 5 mg/kg or vehicle. Values are expressed as mean and standard error of the size of tumors at the indicated time points expressed as percentage to the baseline dimensions. t-test: *p < 0.01, #p < 0.005, §p < 0.001. (B) Representative pictures from excised tumors derived from parental and BRCA2^−/− A10-3 DLD1 cells.