Tissue samples were collected from 70 patients at the Wenzhou People's Hospital between July 2018 and July 2019. All patients were male and the mean age was 61.52±8.31 (range, 51–82) years. The distance between the healthy and the cancer tissue was <5 cm. The inclusion criteria were as follows: Patients with pathologically confirmed primary PCa with different metastasis. The exclusion criteria were as follows: Recurrent PCa, treatment with androgen-deprivation, chemotherapy or radiotherapy prior to sampling, other infectious disease, malignant tumor, severe liver and kidney disease, pulmonary fibrosis, bone metabolic disease, secondary renal hypertension, systemic immune disease or malignant tumor complications. Adjacent tissues in the same patients were also collected as the normal control. The Sixth Edition of the TNM Classification of Malignant Tumors (Union for International Cancer Control) (20) was used for TNM staging. This study was approved by the Ethics Committee of the Wenzhou People's Hospital [Ethics Review (2018) No. (11)], and oral and written informed consent was obtained from all patients according to the committee's regulations. All tissue samples and data were collected and shared in accordance with good medical practices and local laws. The clinical characteristics of all patients are shown in Table I.

Full-length CMTM5 cDNA (Sino Biological, Inc.) was used to construct the overexpressing CMTM5 lentivirus vector, which was packed using 1.5 µg CMTM5 pLVX–IRES-ZsGreen1 plasmid (YouBio), according to manufacturer's protocol (pMDL:VSV-G:REV=5:3:2, multiplicity of infection=20). A 3rd generation system and 293 cell line (Shenzhen Haodi Huatuo Biotechnology Co., Ltd.) were used. The PCa cell line, DU145 (Shenzhen Haodi Huatuo Biotechnology Co., Ltd.), was transfected with the overexpressing CMTM5 lentiviral vector (CMTM5-overexpressing cells) and empty vector (vector control cells) to construct stable transfection. The duration of transduction into cells of interest was 12 h. A flow cytometer (cat. no. 175487; Beckman Coulter, Inc.) was used to detect the expression of CMTM5. Cells were then cultured in the mixture of RPMI-1640 medium (HyClone; Cytiva) containing 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 U/ml) at 37°C with 5% CO₂. Subsequent experiments were performed at 24 h post-transfection.

Immunohistochemical analysis was performed to detect the cellular localization and expression of CMTM5 and EGFR. Tissue samples were fixed in 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin, sectioned to 4–5 µm thickness and analyzed via immunohistochemistry. Samples were deparaffinized with xylene, dehydrated with alcohol and subjected to antigen retrieval by boiling samples in decreasing concentrations of EDTA buffer (pH 9.0) at 120°C for 2 min, followed by cooling to room temperature for 30 min. Then, the sections were soaked in 3% peroxide solution for 15 min to quench endogenous peroxidase. After washing in PBS buffer, the sections were blocked with 10% normal goat serum (Sigma-Aldrich; Merck KGaA) for 10 min (pH 7.5) at room temperature and incubated with rabbit polyclonal to human CMTM5 antibody (1:100; cat. no. ab187980; Abcam) and primary antibodies against EGFR (1:50; cat. no. MA5-13070; Thermo Fisher Scientific, Inc.) at 4°C overnight. On the following day, the sections were washed with PBS for 5 min and incubated with biotin-labeled goat anti-rabbit IgG (cat. no. ab205718; 1:5,000; Abcam) at 37°C for 30 min. The slides were washed and visualized with substrate diaminobenzidine tetrahydrochloride, and then counterstained with hematoxylin at 37°C for 1 min. The stained samples were imaged under a light microscope (×20, ×50 and ×100 magnification; Olympus CX21; Olympus Corporation). According to the percentage of positive cells (0, <10%; 1, 10–24%; 2, 25–50%; and 3, >50%), the grades of staining intensity were 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). The CMTM5 score was calculated by the percentage of positive cells × staining intensity, and the range was 0–9. For all cases, a total score of ≥4 was high expression, a score of 0–3 was low expression.

Total RNA was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) from tissue samples. RNA purity was measured via ultraviolet spectrophotometry, and RNA samples were stored at −80°C for later use. Subsequently, cDNA was synthesized from 1 µg RNA using a PrimeScript RT Master Mix kit (Takara Bio, Inc.) in accordance with manufacturer's protocols. The mRNA expression levels of CMTM5, EGFR, AKT, PI3K, Bcl-2 and Bax were quantitatively analyzed using SYBR Green qRT PCR Master mix (Thermo Fisher Scientific, Inc.) on ABI 7300 Plus real-time PCR system (Thermo Fisher Scientific, Inc.). Thermocycling conditions were as follows: Initial hold at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The mRNA expression level was normalized to β-actin expression in the same sample. The primers were as follows: CMTM5 forward, 5′-CTTCCTCACCTCCCACAAG-3′ and reverse, 5′-AGATGGAAACCAGGATGATG-3′; EGFR forward, 5′-TTGCCGCAAAGTGTGTAACG-3′ and reverse, 5′-GTCACCCCTAAATGCCACCG-3′; AKT forward, 5′-AGCGACGTGGCTATTGTGAAG-3′ and reverse, 5′-GCCATCATTCTTGAGGAGGAAGT-3′; PI3K forward, 5′-TATTTGGACTTTGCGACAAGACT-3′ and reverse, 5′-TCGAACGTACTGGTCTGGATAG-3′; Bcl-2 forward, 5′-GGTGGGGTCATGTGTGTGG-3′ and reverse, 5′-CGGTTCAGGTACTCAGTCATCC-3′; Bax forward, 5′-CCCGAGAGGTCTTTTTCCGAG-3′ and reverse, 5′-CCAGCCCATGATGGTTCTGAT-3′; and β-actin (as an internal control) forward, 5′-GGCACTCTTCCAGCCTTCC-3′ and reverse, 5′-GAGCCGCCGATCCACAC-3′. The 2^−ΔΔCq method was used to calculate relative expression of CLCA2 mRNA (21).

Total protein was extracted from the tissue samples using RIPA buffer (cat. no. BYL40825; JRDUN Biotechnology) containing protease inhibitor and phosphatase inhibitors, followed by centrifugation at 16,000 g for 30 min at 4°C to collect the supernatant. Then, samples were separated via 10% SDS-PAGE and transferred onto a PVDF membrane, and 40 µg protein/lane was separated via 8–12% SDS-PAGE. The membrane was blocked with 5% non-fat milk in TBS at 37°C for 2 h, and then incubated with antibodies against CMTM5 (1:500; cat. no. ab187980; Abcam), EGFR (1:500; cat. no. MA5-13070; Thermo Fisher Scientific, Inc.), p-EGFR (1:200; cat. no. 44–788G; Thermo Fisher Scientific, Inc.) and β-actin (1:1,000; cat. no. ab8226; Abcam) at 4°C overnight. After three washes in 0.05% TBS-Tween-20, the membrane was incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. sc2030; Santa Cruz Biotechnology, Inc.) and HRP-conjugated Affinipure Goat Anti-Mouse IgG (1:2,500, cat. no. SA00001-1; ProteinTech Group, Inc.), at room temperature for 1 h. According to the manufacturer's instructions, subsequent detection was performed using the ECL western blotting system (Amersham; Cytiva).

Western blotting was also used to detect the relative protein expression level of CMTM5 in CMTM5-overexpressing cells and vector control cells. The procedure was repeated as aforementioned.

Moreover, primary antibodies against β-actin (1:1,000; Abcam; cat. no. ab8226), AKT (1:800; cat. no. 4691; Cell Signaling Technology, Inc.), phosphorylated (p)-AKT (1:800; cat. no. 4051; Cell Signaling Technology, Inc.), PI3K (1:800; cat. no. ab86714; Abcam), Bcl-2 (1:200; cat. no. ab117115; Abcam) and Bax (1:200; cat. no. ab32503, Abcam) were used detect cell apoptosis. The procedure was repeated as described previously.

ImageJ software (version 1.46; National Institutes of Health) was used to measure the grey values of protein bands.

A wound healing assay was performed to assess the effect of CMTM5 on cell lateral migration. CMTM5-overexpressing cells and vector control cells were placed in 6-well plates (500 l/well). A scratch was created using a 100 µl-sterile micropipette tip. Cells were then washed twice with PBS to remove cellular debris and cultured at 37°C in serum-free DMEM for 24 h. Cells were imaged using an inverted light microscope (×20 magnification, DM IL; Leica Microsystems GmbH) at 0 and 48 h, and the wound width was analyzed using ImageJ software 1.46 (National Institutes of Health).

The Transwell assay was performed to assess the efficacy of CMTM5 on cell longitudinal migration and invasion. CMTM5-overexpressing cells and vector control cells were placed in common Transwell plate (Corning, Inc.) chambers to evaluate the migratory ability. Cells were resuspended in 200 µl serum-free DMEM, and the cell suspension was then added into the upper chambers (1×10⁵ cells/well) for 24 h, and DMEM containing 10% FBS was added to the lower chambers (600 µl/well). After culturing at 37°C in 5% CO₂ for 48 h, the membrane was washed with PBS, fixed in 4% paraformaldehyde at room temperature for 30 min and stained with 0.1% crystal violet at room temperature for 10 min. Cells that did not pass through the polycarbonate membrane were scraped away gently with a cotton swab, and the number of migrated cells was counted using an inverted light microscope (MSHOT/MC30 Digital imaging system; Guangzhou Micro-shot Technology Co., Ltd.) under a ×200 magnification.

The invasive ability of cells was determined in the same approach, but the cells were added into upper chambers precoated with Matrigel (MilliporeSigma) at room temperature for 15 min.

CMTM5-overexpressing cells and vector control cells were inoculated in 96-well plates in triplicate at the density of 2×10³ cells/well, and cultured at 37°C. Then, 10 µl CCK-8 solution (Tomos Biotools Shanghai Co., Ltd.) was added to each well at the indicated time points according to the manufacturer's instructions following transfection for 2 h at 37°C. Cells were incubated for 2 h in a 5% CO₂ incubator at 37°C. The number of viable cells was calculated via absorbance measurements at 570 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

CMTM5-overexpressing cells and vector control cells in the logarithmic phase were selected and digested with 0.05% trypsin into single cells. Cells were suspended in RPMI-1640 medium containing 10% FBS, and the cell concentration was adjusted to 1×10³ cells/ml. In total, 200 µl CMTM5-overexpressing cell and vector control cell suspensions were seeded into culture dishes (60-mm; 200 cells per dish) and complete medium was added to 10 ml. The plates were incubated in a cell incubator with 5% CO₂ at 37°C for 2–3 weeks. Then, cells were fixed in 5 ml methanol for 15 min at room temperature and dyed with Giemsa stain for 20 min at room temperature. Then, 0.1% crystal violet staining solution was used for staining for 30 min at room temperature, and then the stain was washed away with double distilled water and samples were allowed to dry naturally. A transparent film with grids was placed under a culture dish and the number of colonies (>50 cells) was counted using a light microscope (magnification, ×100; Olympus Corporation).

CMTM5-overexpressing cells and vector control cells were subjected grown on glass slides, washed with PBS and fixed in using 4% buffered paraformaldehyde dissolved in 0.2 M phosphate buffer (pH 7.3) for 15 min at room temperature. The samples were then blocked with 2% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature and incubated overnight with p-EGFR (1:200; cat. no. 44–788G; Thermo Fisher Scientific, Inc.) or E-cadherin primary antibodies (1:200; cat. no. 14–3249-82; Thermo Fisher Scientific, Inc.). After washing three times with PBS, the samples were incubated in a dark chamber with fluorochrome-conjugated secondary antibody (1:400; cat. no. 4408S; Cell Signaling Technology, Inc.) for 2 h at room temperature. The samples were washed with PBS and cover slips were mounted with 90% glycerol in PBS. DAPI (Roche Diagnostics) was used for nuclear staining for 10 min at room temperature. The fluorescent images were observed under a fluorescence microscope (magnification, ×100, Nikon TE-2000U; Nikon Corporation). The total area of target marker-positive cells was then evaluated using ImageJ software (version 1.8.0, National Institutes of Health).

CMTM5-overexpressing cells and vector control cells were digested with trypsin and both were either stimulated with or without the AKT inhibitor MK-2206 (50 nmol/l; MedChem Express) for 2 h at room temperature. On this basis, the four samples were plated in 96-well plates at a density of 1×10⁴ cells/well in triplicate. The apoptosis rate of cells was evaluated via Annexin V FITC/PI staining using corresponding apoptosis detection kits (BD Biosciences). The cells were stained with 5 µl Annexin V FITC and 5 µl PI and incubated at room temperature in the dark for 15 min. The extent of apoptosis was analyzed using a flow cytometer (FACScan; BD Biosciences). Data were analyzed using FlowJo software V10.7.1 (FlowJo, LLC).

Each experiment was repeated three times. SPSS 20.0 statistical software (IBM Corp.) and GraphPad Prism software (version 8.0.2; GraphPad Software, Inc.) were used for data analysis. ImageJ software (v1.53a, National Institutes of Health) was used to analyze the image results of cell experiments. All data are presented as the mean ± SD. The results were accessed using a χ² test, paired t-test, unpaired t-test and one-way ANOVA followed by Bonferroni correction comparisons. P<0.05 was considered to indicate a statistically significant difference.

CMTM5 was predominately localized in the cytoplasm (Fig. 1A). The negative expression, weak expression and medium or strong expression of CMTM5 in prostate tumor tissue samples were 47.1% (33/70), 35.7% (25/70) and 17.1% (12/70), respectively. By contrast, CMTM5 in the 70 normal prostate tissue samples was either strongly (37/70; 52.9%) or moderately (30/70; 42.9%) expressed, with only a few with weakly positive staining (3/70; 4.3%). This indicated that the high expression of CMTM5 was significantly lower in PCa tissues compared with that in normal prostate tissues (P<0.001; Fig. 1B). Western blotting and RT-qPCR results suggested that the protein and mRNA expression levels of CMTM5 were significantly downregulated in PCa tissues compared with adjacent tissues (both P<0.001; Fig. 1C-E).

The immunohistochemistry results identified that EGFR was mainly localized on the cell biofilm system, such as plasma membrane, endoplasmic reticulum and vacuolar, as well as a small amount on the nuclear and cytoplasmic matrix (Fig. 2A). In the 70 PCa tissues, 64 samples (91.4%) showed high EGFR expression and only 6 samples (8.6%) showed low EGFR expression. However, in the 70 adjacent prostate tissues, low EGFR expression was found in 39 samples (55.7%) and high EGFR expression in 31 samples (44.3%) (P<0.01; Fig. 2B). The RT-qPCR results demonstrated that EGFR mRNA expression was upregulated in PCa tissues (P<0.001; Fig. 2C). To further confirm this finding, EGFR and p-EGFR expression was detected using western blotting, and found that the protein expression levels of EGFR and p-EGFR were upregulated in PCa tissues (both P<0.05; Fig. 2D and E).

To determine the biological function of CMTM5 in pathogenesis of PCa, CMTM5 overexpression was induced in DU145 cells. As shown in Fig. 3A, the lentivirus-mediated overexpression was established, and a higher expression of CMTM5 protein was detected in CMTM5-overexpressing cells (P<0.001). CCK-8 and clone formation assays indicated that the proliferation and colony number of cells were significantly decreased in CMTM5 overexpression group (P<0.05 and P<0.001, respectively; Fig. 3B and C). Moreover, the wound healing assay revealed that CMTM5-overexpressing cells exhibited a lower scratch closure rate compared with that of vector control cells (P<0.001; Fig. 3D). Similarly, the results of the Transwell migration assay indicated that CMTM5 overexpression significantly reduced the number of migrated cells (P<0.001; Fig. 3E). Furthermore, the Transwell invasion assay was conducted to evaluate the effect of CMTM5 overexpression on the invasive ability of DU145 cells, and it was found that CMTM5 overexpression inhibited cell invasion (P<0.001; Fig. 3E). Taken together, these results suggested that CMTM5 inhibited cell proliferation, migration and invasion in vitro.

As detected via immunofluorescence, CMTM5 overexpression led to a significant upregulation of E-cadherin expression when compared with control group (Fig. 4A). On the contrary, p-EGFR was negatively expressed in CMTM5-overexpressing cells, but positively expressed in vector control cells (Fig. 4B). Both the results indicated that CMTM5 may inhibit metastasis of PCa according to the expression of the analyzed marker proteins.

As shown in Fig. 5A, CMTM5 overexpression significantly downregulated the protein expression levels of p-AKT, PI3K and Bcl-2 and upregulated Bax protein expression (P<0.001) when compared with the control group. Vector control cells treated with the AKT inhibitor MK-2206 had higher early apoptosis, late apoptosis and necrosis rates compared with those without MK-2206 (P<0.001; Fig. 5B). The late apoptosis and necrosis rates of CMTM5-overexpressing cells treated with MK-2206 were significantly higher compared with those without MK-2206 (P<0.001; Fig. 5B). These findings indicated CMTM5 could promote apoptosis by downregulating the PI3K/AKT signaling pathway components in PCa cells.