MET inhibition enhances PARP inhibitor efficacy in castration-resistant prostate cancer by suppressing the ATM/ATR and PI3K/AKT pathways

Abstract

Up to 30% of patients with metastatic castration-resistant prostate cancer (CRPC) patients carry altered DNA damage response genes, enabling the use of poly adenosine diphosphate-ribose polymerase (PARP) inhibitors in advanced CRPC. The proto-oncogene mesenchymal-epithelial transition (MET) is crucial in the migration, proliferation, and invasion of tumour cells. Aberrant expression of MET and its ligand hepatocyte growth factor is associated with drug resistance in cancer therapy. Here, we found that MET was highly expressed in human CRPC tissues and overexpressed in DU145 and PC3 cells in a drug concentration-dependent manner and is closely related to sensitivity to PARP inhibitors. Combining the PARP inhibitor olaparib with the MET inhibitor crizotinib synergistically inhibited CRPC cell growth both in vivo and in vitro. Further analysis of the underlying molecular mechanism underlying the MET suppression-induced drug sensitivity revealed that olaparib and crizotinib could together downregulate the ATM/ATR signaling pathway, inducing apoptosis by inhibiting the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, enhancing the olaparib-induced antitumour effect in DU145 and PC3 cells. In conclusion, we demonstrated that MET inhibition enhances sensitivity of CRPC to PARP inhibitors by suppressing the ATM/ATR and PI3K/AKT pathways and provides a novel, targeted therapy regimen for CRPC.

Keywords: ATM/ATR pathway; CRPC; DNA damage response; MET inhibitor; PARP inhibitor; PI3K/AKT pathway.

FIGURE 1

MET is overexpressed in a concentration‐dependent manner in olaparib‐treated prostate cancer (PC) cells. (A) PC cells (LNCaP, 22RV1, DU145, and PC3) were treated with increasing concentrations (4–64 μM) of olaparib or dimethyl sulphoxide for 72 h. (B) Representative immunohistochemical analysis of MET protein expression in human BPH, hormone‐sensitive prostate cancer, and CRPC tissues. (C) Western blot analysis of the expression of MET in PC cells (LNCaP, 22RV1, DU145, and PC3). (D) MET mRNA and protein expression increased in olaparib‐treated PC cells (DU145 and PC3) in a concentration‐dependent manner. Statistically significant differences were assessed by Student's t test in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. ns = no statistical difference

FIGURE 2

MET suppression induces olaparib sensitivity in vitro. (A) Western blot shows siRNAs effectively silencing MET in PC cells. Viability of DU145 and PC3 cells transfected with control or MET siRNAs and treated with dimethyl sulphoxide or olaparib for 3 days. (B) Cell viability of the combination treatment of Cri (MET inhibitor crizotinib) and Ola (PARP inhibitor olaparib) or monotherapy in DU145 and PC3 cells for different durations (0–96 h). (C) Synergistic effects (combination index, CI) of Cri and Ola in PC cells (DU145 and PC3) was measured using CompuSyn software. (D) Clonogenicity of DU145 and PC3 cells were performed by clonogenic formation assay after treatment with Cri (2 μM) and Ola (64 μM), monotherapy or dimethyl sulphoxide for 14 days. E, Migration assays were performed in the presence of Cri (2 μM) and Ola (40 μM), Cri or Ola monotherapy, or dimethyl sulphoxide in DU145 and PC3 cells for 3 days. F, Western blot shows the overexpression of exogenous MET in prostate cancer cell lines LNCaP and 22RV1, then examining the drug resistance following in the presence of different drug concentrations. Statistically significant differences were assessed by Student's t test in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. ns =no statistical difference

FIGURE 3

γH2AX staining and ATM/ATR pathway downregulation following MET suppression and enhancing sensitivity to olaparib. (A) Representative immunofluorescent staining of γH2AX (red) and DAPI (blue) in DU145 and PC3 cells in the presence of Cri ((MET inhibitor crizotinib; 4 μM) and Ola (PARP inhibitor olaparib; 64 μM), Cri or Ola monotherapy, or dimethyl sulphoxide for 3 days. Scale bar, 50 μm. More than five foci per nucleus were considered as positive cells. (B) Western blot analysis of γH2AX expression in DU145 and PC3 cells. (C‐D) Western blot analysis of p‐ATM/ATM, p‐ATR/ATR, and RAD51 expression in DU145 and PC3 cells. Statistically significant differences were assessed by Student's t test in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. ns = no statistical difference

FIGURE 4

Combined treatment of olaparib with crizotinib induces apoptosis of the PC cells by inhibiting the PI3K/AKT pathway. (A) Western blot analysis of the expression of p‐PI3K/PI3K and p‐AKT/AKT in DU145 and PC3 cells. (B) Western blot analysis of the expression of BCL‐2, BAX, cleaved PARP, and cleaved caspase 3 in DU145 and PC3 cells. (C) Cells were pretreated with or without 2 μg/ml SC79 and then treated with Cri (4 μM) and Ola (64 μM), Cri or Ola monotherapy, or dimethyl sulphoxide for 3 days, then Western blot analysis of the expression of p‐AKT, cleaved PARP, and cleaved caspase 3. (D) The OE‐MET of LNCaP and 22RV1 in the presence of Cri (4 μM) and Ola (64 μM), Cri or Ola monotherapy, or dimethyl sulphoxide for 3 days, then Western blot analysis of the expression of PI3K/AKT pathway. Statistically significant differences were assayed by Student's t test in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. ns = no statistical difference

FIGURE 5

Olaparib and crizotinib synergistically inhibit the growth of subcutaneous tumours in vivo. Once DU145 subcutaneous tumour reached 50 mm³, mice were injected intraperitoneally with the olaparib (Ola, 40 mg/kg) and crizotinib (Cri, 5 mg/kg), either alone or in combination for 4 weeks (5 day per week). (A) Brief experiment process diagram. (B–D) Curves of tumour volume growth, representative gross images of tumour sizes, and tumour weight after treating with different groups as indicated. (E‐F) The representative immunohistochemical analysis of the expression of Ki67, RAD51, cleaved caspase 3, γH2AX, p‐PI3K, p‐AKT, p‐ATR and p‐ATM proteins in DU145 xenografted tumour cells after treating with different groups as indicated. Statistically significant differences were assessed by one‐way analysis of variance and Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001. ns = no statistical difference