To investigate whether ATM is elevated in prostate cancer tissues from patients with CRPC who are prone to develop metastasis. We collected surgical specimens of prostate cancer from April 2006 to April 2016 in our hospital. All patients were male, aged 67–84 years, of whom 62 were HDPC and 50 were CRPC. 112 surgical specimens isolated from patients with prostate cancer were obtained. All patients provided informed consent. The present study was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University (Suzhou, China). Among the total patients included in the present study, 62 patients with HDPC but without local and distant metastases were included, all of which underwent radical prostatectomy. The remaining 50 patients with CRPC had developed varying local or distant metastases and underwent palliative surgery to alleviate urinary tract symptoms.

CRPC cell lines C4-2 and CWR22Rv1 were used in the present study. C4-2 cells were obtained from the China Center for Type Culture Collection (Wuhan, China). CWR22Rv1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% charcoal stripped fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.). CP466722 (Selleck Chemicals, Houston, TX, USA), JAK inhibitor 1 (EMD Millipore, Billerica, MA, USA) and Stattic (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) inhibitors were added to cultured tumor cells (1:1,000 for all inhibitors) at 37°C, at 6, 12 and 24 h time intervals to inhibit the expression of ATM, JAK and STAT3, respectively. RT-qPCR, western blot and cell migration assay were performed at 6, 12 and 24 h respectively.

Tumor tissues were fixed in 10% (v/v) formaldehyde in PBS, embedded in paraffin, and cut into 5-µm thick sections. The sections were air-dried, then cleared and dehydrated. Antigen retrieval was performed by boiling sections in sodium citrate buffer. Subsequently, sections were incubated for 1 h at room temperature in blocking solution 5% BSA (AR0004, Boster Biological Technology, Pleasanton, CA, USA). Incubation with primary antibody Phosphorylated (p)-ATM (MA1-46069, Thermo Fisher Scientific, Inc. USA) was at 4°C overnight. Following washes with PBS three times, incubation with secondary rabbit anti-mouse antibody (61–0200, Thermo Fisher Scientific, Inc. USA) at room temperature for 20 min followed by three additional washes with PBS. Following staining, tissues were counterstained with hematoxylin. Error bars and significance of values were determined by counting positively stained cells in three randomly selected areas of each slide. Tumor tissue sections were immunostained using a mouse and rabbit specific HRP/DAB detection IHC kit (ab80436, Abcam, Cambridge, UK). Following this, tissues were counterstained by hematoxylin. A light microscope was used (Olympus Corporation, Tokyo, Japan), at magnification, ×40. Error bars and significance values were obtained by manually counting positively stained cells from one randomly chosen area of slides of three different stains.

ATM knockout cells and corresponding control cells were established via lentivirus infection. The lentiviral pLenti-II vector (Addgene, Inc., Cambridge, MA, USA) carried ATM small interfering (si)RNA (ATMSi) or scrambled (Sc) gene sequences as follows: ATM scramble forward, 5′-UUCUCCGAACGUGUCACGUdTdT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAAdTdT-3′ and ATM siRNA forward, 5′-CAUACUACUCAAAGACAUUdTdT-3′ and reverse, 5′-AAUGUCUUUGAGUAGUAUGdTdT-3′. pLent-II-ATMSi or Sc, psPAX2 (virus-packaging plasmid) and pMD2G (envelope plasmid; All from Qiagen, Inc., Valencia, CA, USA) were transfected into 293T cells at a ratio of 4:3:2 using PolyFect transfection reagent (Qiagen, Inc.). Following C4-2 and CWR22Rv1 cells being virally infected overnight, the culture media containing the virus were replaced with normal culture media and maintained under normal cell culture conditions. After subculturing cells, the ATM-knockdown cells were selected by culturing cells in the presence of puromycin (2 µg^xml⁻¹; 540411; Millipore, Billerica, MA, USA) and maintained in media containing 0.1 µg^xml⁻¹ puromycin.

Tumor cells (1×10⁴) were seeded into the upper chamber of a Transwell support (Corning Incorporated, Corning, NY, USA) with serum-free media, whereas medium containing 10% FBS was added to the lower chamber. Following incubation for 24 h at 37°C, cells were fixed in methanol and then stained with crystal violet. Positively stained cells in three randomly selected visual fields were counted under a light microscope (Olympus Corporation), magnification, ×20 (inlet, ×100). Each assay was repeated three times. Antibodies used were: JAK inhibitor 1 (420099; EMD Millipore), CP466722 (S2245; Selleck Chemicals), PD-L1 Antibody (62-5982-80, Thermo Fisher Scientific, Inc.).

Tumor cells (1×10³) were seeded in a 96-well plate, the ATM inhibitor CP466722 was added and a control group was set up without CP466722. The growth of cells in MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA) was assessed daily, DMSO was used to dissolve formazan in cells and the optical density of each well was determined at 570 nm relative to the control group.

Total RNA from cells (1 µg) was subjected to reverse transcription using Superscript III transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) as follows: 25°C for 10 min, 42°C for 50 min, 70°C for 15 min and 4°C hold. Quantitative PCR was conducted using the appropriate primers and a Bio-Rad CFX96 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with SYBR green to determine the mRNA expression levels of genes of interest. Enzyme activation and DNA initial denaturation occurred at 95°C for 3 min, followed by for 40 cycles of: DNA denaturation at 95°C for 15 sec and annealing/extension at 60°C for 45 sec. A melting curve analysis was performed. GraphPad Prism 5 was used to analyze the results. The 2^−ΔΔCq method was used to calculate the relative expression level of each gene. Expression levels were normalized to GAPDH.

Tumor cells were collected following centrifugation at 300 × g for 5 min at 4°C) and supernatant removal. Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 0.2 mM PMSF). Proteins (20–40 µg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (EMD Millipore). The PVDF membrane was placed in blocking buffer and put on shaker for 30 min at room temperature and wash three times with PBS for 10 min. Subsequently, membranes were incubated with primary antibodies (1:1,000), for 1 h at room temperature on a shaker. Following 3 washes for 10 min each, with PBS with Tween 20, HRP-conjugated secondary antibodies (1:5,000; ab97030; Abcam) incubated for 1 h at room temperature on a shaker. Finally, band intensities were visualized in Imager (Bio-Rad Laboratories) using ECL system (34095; Thermo Fisher Scientific, Inc.). Primary antibodies used in the assay included: Phosphorylated (p)-ATM (MA1-46069; Thermo Fisher Scientific, Inc.), PD-L1, p-JAK1, p-JAK2 and p-STAT3 and GAPDH (14-5983-82, 44–422G, 710928, 44–384G, MA5-15738; Thermo Fisher Scientific, Inc.).

All statistical analyzes were performed using SPSS 19.0 (IBM Corp., Armonk, NY, USA) statistical software. All experiments were performed three times which are presented as the mean ± standard deviation and differences between two groups were analyzed using a two-tailed Student's t-test. One-way analysis of variance followed by Fisher's least significant difference post hoc test was used for comparisons among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

ATM expression in prostate cancer specimens was investigated via RT-qPCR and IHC. IHC was performed to determine the levels of p-ATM, since ATM is predominantly activated by phosphorylation. Based on clinical pathological findings of prostate cancer specimens, the RT-qPCR results revealed that the mRNA expression levels of p-ATM were significantly enhanced in prostate cancer tissues obtained from patients with CRPC compared with from patients with HDPC (P<0.01; Fig. 1A). The results of IHC analysis demonstrated that the expression levels of p-ATM were significantly enhanced in prostate cancer tissues obtained from patients with CRPC compared with from patients with HDPC (P<0.01; Fig. 1B and C).

Using the CRPC cell lines C4-2 and CWR22Rv1 as model systems, the successful establishment of ATM knockout cell lines C4-2-ATMSi and CWR22Rv1-ATMSi, and control lines C4-2-Sc and CWR22Rv1-Sc, was revealed (Fig. 2A). The migration of ATM knockout cells was significantly decreased 24 h post-transfection in C4-2-ATMSi and CWR22Rv1-ATMSi cells compared with in the control cells (Fig. 2B). The expression levels of EMT-associated markers were investigated in ATM knockout cells by RT-qPCR; the results demonstrated that the expression levels of N-Cad, vimentin and vascular endothelial growth factor were significantly decreased compared with in the control cells, whereas E-Cad expression was significantly increased in ATM knockout cells compared with in the control cells (Fig. 2C). These results suggested that ATM knockout suppresses EMT in tumors.

To validate the aforementioned experimental results, an ATM inhibitor (CP466722) was incubated with C4-2, CWR22Rv1, C4-2-Sc and CWR22Rv1-Sc cells. Following 6 h of incubation, a significant decrease in p-ATM expression was revealed by RT-qPCR analysis (Fig. 3A). Furthermore, the proliferative ability of the cells was significantly decreased 3 and 4 days post incubation with CP466722 compared with in the control group (Fig. 3B). In addition, the migratory ability of ATM knockout cells and cells incubated with CP466722 were significantly decreased, thus suggesting that ATM inhibition may suppress the growth and metastasis of CRPC cells (Fig. 3C).

The aforementioned results demonstrated that ATM has an important role in CRPC invasion and metastasis via mechanisms that are not yet fully understood. Therefore, the present study aimed to investigate the expression of PD-L1, which is closely associated with EMT (14–16,25). Firstly, the expression of PD-L1 in C4-2-ATMSi and CWR22Rv1-ATMSi cells was investigated, and the results demonstrated that compared with in the control group, the expression of PD-L1 was significantly suppressed in ATM knockout cells (Fig. 4A). In order to determine the mechanism underlying the alteration of PD-L1 levels, ATM downstream regulatory factors were examined, particularly the extensively studied JAK/STAT3 signaling pathway, which is involved in PD-L1 regulation (26,27). The expression levels of p-JAK1, p-JAK2 and p-STAT3 in ATM knockout cells were decreased compared with in the control cells (Fig. 4B). Following 6 h of co-culture with JAK inhibitor 1 or Stattic, changes in PD-L1 mRNA expression were investigated by RT-qPCR, the results of which revealed that incubation with JAK inhibitor 1 resulted in a significant decrease in the expression levels of PD-L1, whereas incubation with Stattic did not exert a significant effect (Fig. 4C). These results suggested that downregulation of the JAK signaling pathway may inhibit the expression of PD-L1.

To investigate the roles of PD-L1 expression and the JAK signaling pathway in ATM-associated regulation of CRPC metastasis, PD-L1 antibodies and JAK inhibitor 1 were incubated with C4-2-Sc and CWR22Rv1-Sc cells. The results demonstrated that PD-L1 antibodies and JAK inhibitor 1 significantly decreased the migration of cells (Fig. 5A). Furthermore, the results demonstrated that following incubation with either PD-L1 antibodies or JAK inhibitor 1, the overexpression of EMT-associated marker genes was effectively normalized (Fig. 5B). These findings suggested that downregulation of the JAK-PD-L1 signaling pathway may inhibit metastasis and EMT in CRPC.