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.

which stage the disease becomes fatal. In CRPC, existent treatment modalities provide limited effectiveness, and the prognosis is poor. 2 Hence, improved and targeted therapeutic regimens are the need of the hour. On average, DNA in cells of the human body undergo tens of thousands of damages; these lesions can be classified as endogenous (e.g., reactive oxygen species and hydrolytic reactions) or exogenous (e.g., chemicals and radiations), which obstruct DNA replication and transcription, leading to cell cycle arrest or DNA lysis and collapse. 3 Meanwhile, DNA damage response (DDR) is required to participate in DNA repair to protect the cell from the damage. The main executors of DDR are the poly adenosine diphosphate-ribose polymerase (PARP) family of inhibitors consisting of 17 members, of which PARP1 and PARP2 play a major role in the repair of singlestrand breaks (SSBs). 4 The accumulation of SSBs eventually leads to double-strand breaks (DSBs). Disruptive mutations in DNA damage repair genes such as BRCA1/2, ATM, and RAD51 initiate the process of tumourigenesis. BRCA1/2 and ATM are essential in the homologous recombination (HR) pathway, which plays an important role in DSBs.
Tumours carry HR gene mutations that are sensitive to PARP inhibitors, leading to synthetic lethality, which is when deficiencies in the expression of two or more genes led to cell death. [5][6][7] Up to 30% of patients with metastatic CRPC carry genomic alterations in the DDR, including in genes related to homologous recombination repair (HRR) deficiency. Among these genetic changes, the mutations in BRCA2 are the most common. 8 PARP inhibitor olaparib targets cancer cells with defects in the HRR, resulting in synthetic lethality. Given that olaparib is only suitable for mCRPC patients with deleterious or suspected deleterious germline or somatic HRR gene mutations, a study to improve the antitumour effect of drugs and to expand the population of drug applications so as to provide patients with more effective targeted treatment options is needed.
The proto-oncogene mesenchymal-epithelial transition (MET) tyrosine kinase and its ligand hepatocyte growth factor (HGF) play an important role in the migration, proliferation, and invasion of tumour cells. 9 Overexpression of the MET receptor is related to the poor prognosis of patients and drug resistance. 10,11 In addition, AR inhibition can significantly upregulate the expression level of MET, thereby playing a role in the transformation of androgen-dependent PC to androgen-independent PC. [12][13][14] MET overexpression is observed in patients with advanced PC and is related to drug resistance. 15,16 It has been proposed that combined inhibition of MET and AR is more efficacious than using either drug alone. 17 Moreover, MET phosphorylates PARP1 at pTyr907, which increases PARP1 enzyme activity and reduces the binding capacity of PARP inhibitors, resulting in drug resistance; while inhibition of MET enhances the antitumour effect of PARP inhibitors, 18 but whether targeting MET also can improve the antitumour effect of PARP inhibitors in PC is currently unknown. In this study, we found that MET is highly expressed in PC cell lines exposed to olaparib in a concentrationdependent manner, and its expression is closely related to sensitivity to olaparib. We also examined whether the PARP inhibitor olaparib, used either alone or in combination with the MET inhibitor crizotinib, could effectively be used to treat PC.

| Cell culture and inhibitors
Human PC cell lines (DU145, PC3, LNCaP, and 22RV1) were purchased from Procell. PC3, LNCaP, and 22RV1 cells were cultured in RPMI 1640 medium (Sevenbio) with 10% fetal bovine serum (FBS; Gibco), and DU145 cells were cultured in Dulbecco's modified eagle medium (Sevenbio) supplemented with 10% FBS. All the cell lines were cultured in a cell incubator with 5% CO 2 , and the temperature was maintained at 37°C. Olaparib and crizotinib were obtained from Selleck, SC79 (HY-18749) was purchased from MedChemExpress.

| Western blot analysis
Protein was extracted from harvested cells using radioimmunoprecipitation assay buffer (KeyGEN BioTECH) supplemented with the protease inhibitor phenylmethylsulfonyl fluoride (PMSF; KeyGEN BioTECH) and phosphatase inhibitor cocktails (KeyGEN BioTECH). All the protein concentrations were detected using BCA (bicinchoninic acid) assay kit (Thermo Fisher Scientific). The same amounts of the extracted protein samples were supplemented with loading buffer (KeyGEN BioTECH) and separated in 4%-12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (Sevenbio), and then transferred to a nitrocellulose membrane (Millipore, Missouri, USA). After incubation of the membranes with antibodies, the protein on the membranes was detected on an enhanced chemiluminescence (ECL) Western blotting substrate (Tanon, China). Protein membranes were analyzed by Image J software. All the experiments were conducted at least by three independent researchers.

| Colony formation assay
For the colony formation assay, cells were plated at a uniform concentration of approximately 700 cells/well into six-well plates and then incubated for three weeks in medium containing a single drug or a combination of drugs, and the medium was refreshed every 2 days. To detect colony formation, the cultured cells were then fixed independents with paraformaldehyde, stained with crystal violet, dried, and counted.

| Transwell migration assay
For the cell migration assay, the cells were plated into Transwell chambers (Corning, New York, USA) at a concentration of 2×10 4 per well, incubated with 250 μl of serum-free medium containing one drug or a combination of drugs. After 72 h of incubation, the cells were fixed with paraformaldehyde, stained with crystal violet, dried, and imaged. The results were analyzed using Image J software.  after that, those successfully transfected cells were selected with puromycin (2 μg/ml).

| Drug combination index
The drug combination index (CI) value was measured using CompuSyn software. CI value less than 1 indicates the presence of a synergistic effect between the two drugs, and the smaller the value, the stronger is the synergistic effect; a CI value equal to 1 indicates that there is only an additive effect between the drugs; and a CI value greater than 1 indicates that the drugs are antagonistic. 19
To avoid nonspecific staining, the subcutaneous tumour tissues were probed with antibodies of γH2AX (1:100), Ki67 (1:100), cleaved caspase-3 (1:100), and RAD51 (1:100) at 4°C overnight. Next, the tissue sections were supplemented with biotinylated secondary antibodies for 20 minutes and then stained using a diaminobenzidine kit (Lab Vision). The scores of all the IHC tissue sections were evaluated by two independent researchers. Counterstaining was evaluated using Image J software.

| Statistical analysis
All experimental data were captured using GraphPad Prism 8.0.2 software and the statistical differences were analyzed using SPSS Version 22.0 software. Statistical significance was defined when a p-value was <0.05.

| MET is overexpressed in olaparib-treated PC cells in a concentration-dependent manner
To evaluate the drug sensitivity of olaparib in PC cell lines, the cells (LNCaP, 22RV1, DU145, and PC3) were exposed to olaparib with increasing concentrations (4-64 μM) for 72 h. The sensitivity of the cells to olaparib was detected by CCK-8 after 72 h of treatment ( Figure 1A). The results showed that PC3 and DU145 cells were relatively insensitive to olaparib compared with LNCaP and 22RV1 cells, and the half-maximal inhibitory concentration (IC 50 ) of olaparib in DU145 was approximately 13.5 times that in LNCaP cells ( Table 1).
The proto-oncogene MET, which is highly expressed in CRPC, is known to be associated with cancer occurrence, progression, and treatment resistance. 20 To observe the expression of MET in prostate tissue, we collected human benign prostatic hyperplasia (BPH), hormone-sensitive PC, and CRPC tissues. As indicated, MET protein was overexpressed in CRPC ( Figure 1C). Next, we detected MET expression in the PC cell lines by Western blot analysis, and found that similar to that in CRPC, MET protein was highly expressed in PC3 and DU145 cells ( Figure 1B). Moreover, the levels of MET mRNA and protein in DU145 and PC3 cells treated with increasing concentrations of olaparib showed significant upregulation ( Figure 1D). These results indicate that MET is closely related to sensitivity of PC cell lines to olaparib.

| Targeting MET induces olaparib sensitivity in vitro
To evaluate whether MET mediates tumour sensitivity to olaparib, we used siRNA to silence MET expression and examined the growth of DU145 and PC3 cells in the presence of olaparib. MET silencing enhanced the inhibition of olaparib in DU145 and PC3 cells by decreasing cell viability (Figure 2A). Furthermore, we investigated whether crizotinib could also influence the tumour response to olaparib in PC cells. As indicated, crizotinib rendered the DU145 and PC3 cells more sensitive to olaparib by suppressing cell proliferation ( Figure 2B). In addition, the CI values indicated that treatment with the combination of olaparib and crizotinib could synergistically inhibit the growth of DU145 and PC3 PC cells ( Figure 2C).

| MET suppression downregulates ATM/ATR pathway and enhances sensitivity to PARP inhibition
HRR-deficient cells that are treated with PARP inhibitors trigger synthetic lethality, causing irreversible disruption of chromosomal construction, cell cycle arrest, and ultimately cell death. 21

| Cotargeting PARP and MET induces apoptosis of PC cells by inhibiting the PI3K/ AKT pathway
The PI3K/AKT pathway is a crucial sensor of genomic integrity. PI3K signaling promotes DNA double-strand repair by interacting with the HRR complex, and PI3K suppression enhances the antitumor effect of PARP inhibitors. [25][26][27] In addition, MET/HGF signaling protects tumour cells from DNA damage by activating the PI3K/AKT pathway. 28 Therefore, we tested whether the inhibition of MET could downregulate the expression of PI3K pathway. As expected, crizotinib suppressed the phosphorylation of PI3K and AKT, which resulted in HRR deficiency, and finally enhanced the antineoplastic effects in PC cells ( Figure 4A). Given that cleaved PARP and cleaved caspase 3 are the cleaved version of PARP and caspase 3 proteins, respectively, their activation plays an important role in apoptosis and can be used as apoptosis markers. 29 Therefore, we examined the expression levels 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 over-activation. We used SC79, an AKT activator, after the combined treatment of crizotinib and olaparib in PC cells. As indicated, SC79 could suppress the expression of cleaved PARP and cleaved caspase 3 and decreased cell apoptosis ( Figure 4C). Furthermore, we conducted the overexpression of exogenous MET in prostate cancer cell lines LNCaP and 22RV1 to reach olaparib-resistance states, and following treatment with olaparib or crizotinib alone or a combination of the two, then detected the expression of PI3K/AKT by western blot. As indicated, the combined treatment in LNCaP and 22RV1 could suppress the PI3K/AKT pathway ( Figure 4D). Our results showed that the combined inhibition of PARP and MET induces apoptosis of DU145 and PC3 cells by inhibiting the PI3K/AKT pathway.

| Olaparib and crizotinib synergistically inhibit the growth of subcutaneous tumours in vivo
To further confirm our in vitro experimental results, we established DU145 subcutaneous tumour models in vivo. As indicated, the combined treatment of olaparib and crizotinib significantly slowed down the growth of subcutaneous tumours in mice compared with those treated with olaparib or crizotinib alone ( Figure 5A-C). In addition, we removed the subcutaneous tumours from the nude mice and weighed it after the completion of the experiment. The tumours weighed significantly lower in mice treated with the combination therapy compared with those in mice in the monotherapy and control groups ( Figure 5D). An analysis of the subcutaneous tumours showed substantially reduced Ki67 staining and RAD51 protein abundance but increased cleaved caspase 3 and γH2AX protein levels in mice in the combination treatment group compared with mice in the monotherapy and control groups ( Figure 5E). In addition, the phosphorylation expression levels of PI3K, AKT, ATR and ATM decreased in the combination therapy compared with monotherapy or vehicle in subcutaneous tumours ( Figure 5F). Therefore, our results suggested that the combination of olaparib and crizotinib could synergistically inhibit the growth of subcutaneous PC tumours in vivo. sensor of genomic integrity and the PI3K downstream gene AKT is activated when exposed to PARP inhibitors, and these changes limit the efficacy of PARP inhibitors in the treatment of cancers. In addition, some studies found that the aberrant expression and activation of MET result in the activation of the ERBB3/PI3K/AKT pathway, which is associated with the resistance of EGFR inhibitors in lung cancer, and this resistance can be reversed by combining MET inhibitors and EGFR inhibitors. In the current study, we observed that MET inhibition downregulated the PI3K/AKT signaling pathway, induced apoptosis, and enhanced the drug sensitivity of olaparib in PC. In conclusion, our study demonstrates that MET inhibition enhances the efficacy of PARP inhibitors in PC and provides a novel, targeted, therapy regimen for the management of advanced PC.

ACK N OWLED G EM ENTS
This study was supported by National Natural Science Foundation of

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.