Inhibition of BRCT(BRCA1)-phosphoprotein interaction enhances the cytotoxic effect of olaparib in breast cancer cells: a proof of concept study for synthetic lethal therapeutic option

Abstract

Synthetic lethal therapeutic strategy using poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitor olaparib in carriers of BRCA1 or BRCA2 mutation has shown promise in clinical settings. Since <5 % of patients are BRCA1 or BRCA2 mutation carriers, small molecules that functionally mimic BRCA1 or BRCA2 mutations will extend the synthetic lethal therapeutic option for non-mutation carriers. Here we provide proof of principle for this strategy using a BRCA1 inhibitor peptide 2 that targets the BRCT(BRCA1)-phosphoprotein interaction and mimics the M177R/K BRCA1 mutation. Reciprocal immunoprecipitation and immunoblotting of BRCA1 and Abraxas was used to demonstrate inhibitor 2 targets BRCT(BRCA1)-Abraxas interface. Immunostaining of γH2AX, cell cycle analysis and homologous recombination (HR) assays were conducted to confirm that inhibitor 2 functionally mimics a chemosensitizing BRCA1 mutation. The concept of synthetic lethal therapeutic strategy with the BRCA1 inhibitor 2 and the PARP inhibitor Olaparib was explored in HeLa, MDA-MB-231, and HCC1937 cell lines. The results show that inhibition of BRCA1 by 2 sensitizes HeLa and MDA-MB-231 cells but not HCC1937 to Olaparib mediated growth inhibition and apoptosis. These results provide the basis for developing high affinity BRCT(BRCA1) inhibitors as adjuvants to treat sporadic breast and ovarian cancers.

Fig. 1

(A) Proposed hypothesis: Small molecule inhibitors of BRCT(BRCA1)-phosphoprotein interaction will increase the sensitivity of cancer cells to DNA damage based therapeutics. The inhibitor is represented as a red X. (B) Competitive inhibition curves for BRCT(BRCA1) inhibitor peptides 1 (Ac-pSPTF-CO₂H) and 2 (Ac-R₁₀G-pSPTF-CO₂H). (C) Immunoprecipitation (IP) and Immunoblotting (IB) studies with and without the BRCT inhibitor 2 in the presence and absence of IR-induced DNA damage.

Fig. 1

(A) Proposed hypothesis: Small molecule inhibitors of BRCT(BRCA1)-phosphoprotein interaction will increase the sensitivity of cancer cells to DNA damage based therapeutics. The inhibitor is represented as a red X. (B) Competitive inhibition curves for BRCT(BRCA1) inhibitor peptides 1 (Ac-pSPTF-CO₂H) and 2 (Ac-R₁₀G-pSPTF-CO₂H). (C) Immunoprecipitation (IP) and Immunoblotting (IB) studies with and without the BRCT inhibitor 2 in the presence and absence of IR-induced DNA damage.

Fig. 1

(A) Proposed hypothesis: Small molecule inhibitors of BRCT(BRCA1)-phosphoprotein interaction will increase the sensitivity of cancer cells to DNA damage based therapeutics. The inhibitor is represented as a red X. (B) Competitive inhibition curves for BRCT(BRCA1) inhibitor peptides 1 (Ac-pSPTF-CO₂H) and 2 (Ac-R₁₀G-pSPTF-CO₂H). (C) Immunoprecipitation (IP) and Immunoblotting (IB) studies with and without the BRCT inhibitor 2 in the presence and absence of IR-induced DNA damage.

Fig. 2

(A) Left Cell-based double strand break repair assay. Right U2OS-DR cells with an integrated reporter construct for HR-mediated repair of GFP was subjected to inhibitor 2. (B) Inhibition of BRCT(BRCA1) by inhibitor 2 sensitizes cells to DNA damage. DNA damage was induced by increasing doses of IR in the presence (10 μM) and absence of inhibitor 2. DNA damage was assessed by γH2AX staining and DAPI was used to stain the nucleus. The bar chart on the right shows quantification.

Fig. 2

(A) Left Cell-based double strand break repair assay. Right U2OS-DR cells with an integrated reporter construct for HR-mediated repair of GFP was subjected to inhibitor 2. (B) Inhibition of BRCT(BRCA1) by inhibitor 2 sensitizes cells to DNA damage. DNA damage was induced by increasing doses of IR in the presence (10 μM) and absence of inhibitor 2. DNA damage was assessed by γH2AX staining and DAPI was used to stain the nucleus. The bar chart on the right shows quantification.

Fig. 3

BRCT(BRCA1) inhibitor 2 releases IR induced G2/M arrest. The cells were subjected to the inhibitor (100 μM) in the presence and or absence of IR (15 Gy). After 24 h the cells were analyzed by flow cytometry.

Fig. 4

(A) Viability of cells with wild type BRCA1 (HeLa and MDA-MB-231) and truncated BRCA1 (HCC1937) in the presence of inhibitor 2, Olaparib (PARP inhibitor) and the combination was determined. (B) Induction of apoptosis was measured in a Caspase 3/7 assay. Cells were treated with inhibitor 2 at 50 μM, Olaparib at 25 μM and combination at these concentrations for 48 h and caspase 3/7 activity was measured. * P < 0.05 and ** P < 0.005

Fig. 4

(A) Viability of cells with wild type BRCA1 (HeLa and MDA-MB-231) and truncated BRCA1 (HCC1937) in the presence of inhibitor 2, Olaparib (PARP inhibitor) and the combination was determined. (B) Induction of apoptosis was measured in a Caspase 3/7 assay. Cells were treated with inhibitor 2 at 50 μM, Olaparib at 25 μM and combination at these concentrations for 48 h and caspase 3/7 activity was measured. * P < 0.05 and ** P < 0.005