Despite the recent decreasing trend in incidence, gastric cancer is still one of the most common carcinomas and the second leading cause of cancer-related death in the world, leading to an estimated 738,000 deaths in 2008 [
]. The majority of gastric cancer patients are diagnosed at an advanced stage and the limited options are available for the treatment. Though many efforts have been made in the management of gastric cancer, most patients still have a poor prognosis, with a 5-year survival rate less than 25% [
]. Studies have shown that the development of gastric cancer is associated with molecular alterations comprising inactivation of tumor suppressors, such as p16, APC and E-cadherin, some of which could be applied as prognostic factors [
]. However, additional biomarkers that could be used as prognostic marker and potential treatment target of gastric cancer are needed to help predict and improve the prognosis of patients.
Opioid binding protein/cell adhesion molecule-like gene (OPCML), also designated as OBCAM, was initially identified from rat brains and found to possess a specific opioid-binding activity [
]. It was later found to be a member of the IgLON family of GPI-anchored cell adhesion molecules, consisting of protein-protein interaction domains, such as three ‘I’ set Ig domains [
]. Studies show that OPCML is normally expressed in brain and ovary, and also expressed in heart, placenta, testis, kidney, liver, pancreas, colon and stomach [
]. Recently, OPCML was found to be a candidate tumor suppressor, which inhibited tumor growth in ovarian cancer and some other cancers including prostate cancer [
]. A recent small-sample study by Wang et al. revealed that OPCML expression was markedly decreased in primary gastric cancer tissues, in comparison with normal stomach tissues [
]. However,the clinical implications and biological functions of OPCML in the progression of gastric cancer remain unknown.
In this study, we for the first time investigated the association between OPCML expression and clinicopathological features as well as prognosis of cancer patients. We also explored the biological functions of OPCML in tumor progression, and the molecular mechanisms underlying its behavior in gastric cancer. The results suggest that decreased OPCML expression might predict poor prognosis and promote disease progression in gastric cancer.
Subjects and immunohistochemical analysis
Tissue samples were obtained from 30 gastric cancer patients who underwent surgical resection at the First Affiliated Hospital of Sun Yat-sen University between 2010 and 2014. These samples contained both the tumor and tumor-free locations and used to assess the differential expression of OPCML using immunohistochemical analysis. In addition, we included 133 patients undergoing gastrectomy in the First Affiliated Hospital of Sun Yat-sen University between 1995 and 2004. Survival data were available from these 133 patients, with a median follow-up time of 24.4 months. None of the patients was treated with radiotherapy or chemotherapy preoperatively. Tumors were classified according to guidelines of the International Union against Cancer [
]. All patients provided informed consent for collection of the tissue samples and publication of the related data. This study was approved by the Ethics Committee of the Sun Yat-sen University.
Immunohistochemical analysis of OPCML protein was conducted as previously described [
]. The degree of OPCML immunostaining was evaluated according to both the proportion of positively stained cancer cells and intensity of staining. The proportion of cancer cells was scored according to the following criteria: 0 (no positive cancer cells), 1 (<10% positive cancer cells), 2 (10–50% positive cancer cells), and 3 (>50% positive cancer cells). Intensity of staining was graded as follows: 0 (no staining); 1 (weak staining = light yellow), 2 (moderate staining = yellow brown), and 3 (strong staining = brown). Staining index was calculated as the proportion score × the staining intensity score. We assessed the expression of OPCML in gastric cancer specimens according to the staining index, which scores as 0, 1, 2, 3, 4, 6, and 9. The staining index score of 4 was applied as the cut-off to differentiate between low and high expression of OPCML. OPCML antibody was purchased from Abcam (Cambridge, UK). The positive and negative controls for immunostaining are applied to assess the specificity of OPCML antibody [Additional file
]. Assessment of the pathological slides was performed by two experienced pathologists who were blinded to clinicopathological characteristics and clinical outcome of the patients.
Semi-quantitative RT-PCR and quantitative PCR analysis
Total RNA was extracted using NucleoSpin RNA Kit (Macherey-Nagel GmbH, Germany) according to the manufacturer’s instructions. Reverse transcription PCR (RT-PCR) and quantitative PCR was carried out as previously described [
]. PCR primers for the human OPCML gene were as follows: sense: 5′-CCTAGGTCCTCTGAGCAACG-3′, antisense: 5′-GGTCAAGGTAGCAGGAGCAG-3′. Primer sequences for S12 were as follows, sense: 5′- GCATTGCTGCTGGAGGTGTAAT-3′, antisense: 5′- CTGCAACCAACCACTTTACGG-3′.
Western blot analysis
Total protein of cells was extracted by lysis buffer and concentration determination was conducted using the DC protein assay method of Bradford (Bio-Rad, Hercules, CA). Twenty mg of protein was loaded and separated in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA). The antibodies used for the analysis are as follows, OPCML (Abcam, Cambridge, UK), cyclin-dependent kinase inhibitor 1B (p27), cleaved caspase 3, cleaved caspase 9, poly ADP-ribose polymerase (PARP), phospho-Protein Kinase B (AKT) (Ser473) and phosphor-glycogen synthesis kinase 3β (GSK3β) (Cell Signaling Technology, Inc., Danvers, MA), and GAPDH (Good Here Biotechnology, Hangzhou, P.R. China).
Gastric cancer cell lines and 5-aza-2′-deoxycytidine treatment
Gastric cancer cell lines (SGC7901, BGC823, AGS, MKN28, NCI-N87, SNU1 and MKN45) were obtained from American Type Culture Collection (Manassas, VA) in 2011. The cell lines were last tested and authenticated via cell line STR genotyping assay in 6 months before the experiment ended. The cell lines were cultured in RPMI-1640 media. Treatment of cell lines (SGC7901, BGC823 and AGS) with 5-aza-2′-deoxycytidine was performed as previously described [
DNA construct and stable transfection
The cDNA of human OPCML was inserted into pcDNA3.1 (Invitrogen, Carlsbad, CA), as previously described [
]. SGC7901 and BGC823 cells were transfected with OPCML-pcDNA3.1 and the empty vector pcDNA3.1 using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA), and stable clones were then generated and confirmed as previously described [
Bisulfite treatment of DNA and methylation-specific PCR (MSP)
Genomic DNA was extracted using NucleoSpin DNA Kit (Macherey-Nagel GmbH, Germany) according to the manufacturer’s instructions, and was analyzed by the methylation-specific PCR (MSP) posterior to bisulfite conversion, as previously reported [
]. MSP primers used for OPCML promoter was as follows, M, sense: 5′-CGTTTAGTTTTTCGTGCGTTC-3′, antisense: 5′-CGAAAACGCGCAACCGACG-3′; U, sense: 5′-TTTGTTTAGTTTTTTGTGTGTTTG-3′, antisense: 5′-CAAAACAAAAACACACAACAACA-3′.
Cell viability was examined using Cell Counting Assay Kit-8 (CCK-8) (YiYuan Biotech, Guangzhou, China), according to the manufacturer’s instructions. Anchorage-dependent and -independent growth was respectively assessed by colony formation assay and colony formation in soft agar, as previously described [
]. By flow cytometry, cell cycle assay was performed using propidium iodide staining, and apoptosis assay was conducted using annexin V-FITC/PI staining (BD Biosciences, Erembodegem, Belgium), as previously reported [
Tumor xenograft formation and in vivo experiment
All the experiments on mice were approved by the Sun Yat-Sen University Committee for Animal Research and conducted in accordance with the highest international standards of humane care in animal experimentation. Four-week-old male athymic BALB/c nude mice were purchased from Vital River Laboratories (Beijing, China). All mice had free access to sterilized food and autoclaved water. In vivo experiments were initiated posterior to 1 week of acclimatization. 1 × 10
suspended SGC-7901 cells (in 0.1 ml phosphate-buffered saline) transfected with OPCML or empty vector were injected subcutaneously into the dorsal left flank of each mouse. Tumor size was measured at each third day for 4 weeks. Tumor volume (mm
) and tumor weight were determined as previously described [
t test, One-way ANOVA, Mann-Whitney U test and X
2 test were used to determine the statistical difference. The survival curves were plotted by the Kaplan–Meier method, and comparison of survival times was conducted using the log-rank test. All tests were two-sided, and a
P < 0.05 was considered to be statistically significant. Statistical analysis was carried out using SPSS 16.0 and Graphpad Prism V4.0.
In the current study, we showed that OPCML was expressed in the normal stomach while markedly down-regulated or lost in gastric cancer. We first used immunohistochemical assay to directly compare the expression of OPCML in gastric cancer tissues and their adjacent normal tissues in 30 gastric cancer patients. OPCML protein was shown to be significantly reduced in gastric cancer tissues, while readily expressed in adjacent normal stomach tissues. Next, we assessed the differential expression of OPCML in tumor samples from 133 patients with gastric cancer. The results revealed that the expression of OPCML was reduced in tumor samples from 96/133(72.2%) patients with gastric cancer and was completely lost in tumor tissues from 45/133 (33.8%) gastric cancers. A small-sample study by Wang et al. demonstrated a reduced expression of OPCML in primary gastric cancer tissues, compared with normal stomach tissues, similar to the finding in our study [
Moreover, we evaluated the association of OPCML expression and clinicopathological characteristics and prognosis in gastric cancer. Notably, the decrease of OPCML expression was significantly more frequent in the advanced stages of gastric cancer, in comparison to the early stages. In addition, poorly differentiated cancers exhibited a significantly lower expression of OPCML protein than well and moderately differentiated ones. Interestingly, gastric cancer patients with decreased OPCML expression possessed a significantly shorter survival as compared to cancers with preserved OPCML expression. Further multivariate regression analysis revealed that reduced OPCML expression is an independent predictor for poor outcome of gastric cancer patients. Taken together, these data revealed that reduction of OPCML expression might have the important clinical significance in the progression of gastric cancer.
In addition, our study showed that OPCML protein and mRNA were significantly decreased or lost in all 7 gastric cancer cell lines, as compared to the normal stomach tissues. Given the significant association between reduction of OPCML expression and gene methylation [
], we analyzed the methylation status of OCPML promoter in these cell lines. The results showed that the silencing of OPCML gene was significantly correlated with promoter methylation. Furthermore, we also assessed the expression of OPCML mRNA after incubation with the methylation inhibitor 5-aza-2′-deoxycytidine in three cell lines. OPCML mRNA was found to be restored in all three cell lines, hence further confirming the link between the loss of OPCML mRNA expression and gene methylation.
Next, our study explored the effect of OPCML on the growth of gastric cancer cells in vitro and in vivo. The results revealed that ectopic expression of OPCML led to the inhibition of cell viability, and the suppression of both anchorage-dependent and -independent growth colony formation of gastric cells. Besides, the suppressive effect of OPCML on the growth of gastric cancer cells was confirmed in the subsequent experiments on nude mice. Sellar et al. reported that OPCML exhibited growth inhibitory activity in epithelial ovarian cancer, similar to the tumor-suppressive effect of OPCML indicated in our study [
]. Consistently, the in vitro study by Cui et al. also revealed that ectopic expression of OPCML suppressed tumor cell clonogenicity of prostate cancer and colon cancer cell lines [
]. Further analysis by flow cytometry showed that OPCML arrested gastric cells in the G0/G1 phase of the cell cycle. Also, OPCML was demonstrated to increase both early and late apoptosis in these cell lines. To our knowledge, no evidence has so far been found regarding the impact of OPCML on cell cycle and apoptosis of cancer cells. Taken together, these data thus suggest the role of OPCML as a candidate tumor suppressor in gastric cancer.
The possible molecular mechanism underlying OPCML arresting cell cycle in G0/G1 phase and inducing cell apoptosis was also investigated. In this study, p27 was found to be up-regulated in both SGC7901 and BGC823 cell lines by ectopic expression of OPCML. The transitions of cell cycle are strictly controlled by a series of cyclins, CDKs and inhibitory proteins. p27 possesses the inhibitory activity of CDK4 and CDK6, which phosphorylate Rb to promote the transition of G1 to S phase [
]. In addition, ectopic OPCML expression led to the increase of activated form of caspase 9 and caspase 3. Caspase 9 is the crucial regulator of mitochondria-dependent apoptotic pathway, the activation of which launch the intrinsic apoptosis process via activating downstream apoptosis-associated molecules including caspase-3 and PARP.
This study revealed that AKT and its downstream target GSK3β were constitutively phosphorylated in the two gastric cancer cell lines and their phosphorylation levels were markedly suppressed by ectopic expression of OPCML. AKT has been shown to play an important role in the development and progression of numerous cancers through promoting cell proliferation and suppressing apoptosis [
]. Constitutively phosphorylated AKT has been reported in some cancers, and activated AKT promotes cell proliferation and induces apoptosis via phosphorylating a number of transcription factors including GSK3β [
]. The findings in this study suggested that AKT/GSK3β signaling pathway might be implicated in the tumor-suppressing function of OPCML in gastric cancer.
This study was supported by National Natural Science Foundation of China (81,000,887, 81,201,932) (the design of the study, collection of the data and writing the manuscript), Natural Science Foundation of Guangdong Province (2015A030313116) (collection and analysis of the data), Science and Technology Planning Project of Guangdong Province (2016A020215041) (analysis and interpretation of the data), Medical Scientific Research Foundation of Guangdong Province (A2015283) (analysis of the data and writing the manuscript) and the Junior Teacher Cultivation Project of Sun Yat-sen University (15ykpy18) (collection and analysis of the data).
Availability of data and materials
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
XX, CW, MS and CM designed the project and prepared the manuscript. SH, LM, JJ, YY and LL conducted the experiments and statistical analysis. WY, LM, JJ and ZX collected the tissue samples and clinical data, fulfilled ethical authorizations. All authors approved the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All patients provided written informed consent for collection of the tissue samples and published the related data. This study was approved by the Ethics Committee of the Sun Yat-sen University.
All the experiments on mice were approved by the Sun Yat-Sen University Committee for Animal Research and conducted in accordance with the highest international standards of humane care in animal experimentation.
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