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+Model CLINRE-244; No. of Pages 8 ARTICLE IN PRESS Clinics and Research in Hepatology and Gastroenterology (2012) xxx, xxx—xxx Available online at www.sciencedirect.com ORIGINAL ARTICLE Reduced expression of PTEN and increased PTEN phosphorylation at residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation Zhen Yang a,1,2,3,4, Xiao-Gang Yuan a,2, Jiang Chen a,2, Shi-Wen Luo a,b,4, Zhi-Jun Luo c,4, Nong-Hua Lu a,1,4,∗ a Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, 330006 Jiangxi, PR China Center for Laboratory Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, 330006 Jiangxi, PR China c Department of Biochemistry, Boston University School of Medicine, MA 02118 Boston, USA b Summary Aim: PTEN is a tumor suppressor gene in different cancers. This study was to determine the protein expression of PTEN and phosphorylation of PTEN (p-PTEN) at residue Ser380 in different histology specimens of gastric tissues. Methods: A total of 179 tissue specimens of normal gastric mucosa, chronic gastritis, intestinal metaplasia, dysplasia, and gastric cancer were recruited for immunohistochemical analysis of PTEN and p-PTEN expression. Four gastric cancer AGS, MKN-45, MKN-28, and SGC-7901 cell lines and a non-cancerous gastric GES-1 cell line were used to detect expression of PTEN and p-PTEN protein using Western blot. Results: Expression level of PTEN protein was significantly decreased in gastric cancer tissues compared to normal gastric mucosa, chronic gastritis, intestinal metaplasia and dysplasia (P < 0.05). In contrast, p-PTEN protein level was significantly increased in intestinal metaplasia, dysplasia and gastric cancer compared to normal gastric mucosa and chronic gastritis (P < 0.05). However, there was no any association of PTEN and p-PTEN expression with clinicopathological characteristics from gastric cancer patients. Moreover, the ratio of p-PTEN and PTEN was higher in gastric cancer cell lines than that of the non-malignant cells. Conclusions: This study demonstrated that aberrant expression of PTEN and p-PTEN at residue Ser380 was early event that could contribute to gastric carcinogenesis, and that PTEN phosphorylation at residue Ser380 could be a mechanism for PTEN inactivation. © 2012 Elsevier Masson SAS. All rights reserved. ∗ 1 2 3 4 Corresponding author. Tel.: +86 791 88692705; fax: +86 791 88623153. E-mail address: [email protected] (N.-H. Lu). Designed the study. Performed the study. Analyzed the data. Drafted the manuscript. 2210-7401/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.clinre.2012.03.002 Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012), doi:10.1016/j.clinre.2012.03.002 +Model CLINRE-244; No. of Pages 8 ARTICLE IN PRESS 2 Z. Yang et al. Introduction Gastric cancer is the fourth most common cancer worldwide, with an estimated one million new cases in 2007. Gastric cancer is the second leading cause of cancer-related death in men and the fourth among women resulting in approximately 800,000 deaths in 2007, indicating that it is still one of the most significant health burdens in the world, although the incidence and mortality of gastric cancer have declined [1,2]. To date, surgical resection remains the only curative means to improve survival of patients with gastric cancer [3], while chemotherapy has limited effects and is usually served to palliatively relieve patients’ symptoms and increase survival time. Like most other cancers, the underlying mechanism responsible for gastric cancer development and progression is still poorly understood. Therefore, it is necessary to investigate the mechanisms of gastric carcinogenesis and to develop novel strategies to prevent gastric cancer. For this purpose, our research objective focuses on PTEN (phosphatase and tensin homolog deleted on chromosome ten), a tumor suppressor gene identified in 1997 by three independent groups using slightly different strategies [4—6]. PTEN encodes a dually functional phosphatase with lipid and protein phosphatase activities and is involved in regulation of a variety of signaling transduction pathways [7—10], which are critical in cell differentiation, apoptosis, adhesion, and mobility as well as reproduction. PTEN is frequently inactivated in different human cancers [4,5,11]. Recent studies have indicated that loss or reduced expression of PTEN commonly occurs in gastric cancers due to genetic or epigenetic changes, such as mutation, loss of heterozygosity (LOH), and promoter hypermethylation [12—16]. However, it remains to be defined whether other mechanisms account for inactivation of PTEN and how PTEN inactivation promotes carcinogenesis. Indeed, several recent studies have demonstrated that PTEN protein can be regulated by a variety of other mechanisms, including phosphorylation, oxidation, lipid ligands and protein binding partners. Among these, phosphorylation is the most important mechanism of post-translational modification of PTEN. To date, there are multiple phosphorylation sites in PTEN protein, such as Ser380, Thr382, and Thr383, which leads to a loss of phosphatase activities or a gain of PTEN stability [17,18], and in turn results in loss of tumor suppressor function and increased cancer susceptibility. Among the phosphorylation sites of PTEN protein, Ser380 is more critical for regulation of PTEN function [17]. Development of gastric cancer is a multistep process from normal gastric mucosa to chronic gastritis, intestinal metaplasia, dysplasia, and to invasive cancer [19]. In this study, we aimed to determine expression of PTEN vs. phosphorylated PTEN (pPTEN) at residue Ser380 in different stages of gastric lesions and to gain an insight into the role of PTEN protein and most importantly, p-PTEN in gastric cancer development. Patients and methods Patients Gastric tissue samples were collected from patients who underwent a gastroduodenoscopy at the First Affiliated Hospital of Nanchang University from January 2007 to September 2009. A total of 179 patients were enrolled in this study, which included 20 cases of normal gastric mucosa, 21 chronic gastritis, 40 intestinal metaplasia, 48 dysplasia (including 33 low-grade and 15 high-grade intraepithelial neoplasia), and 50 gastric cancers. The clinical characteristics of these patients are summarized in Table 1 and there was no significant difference in the age and gender distribution among these groups. The clinicopathological characteristics were also obtained from 43 of 50 patients with gastric carcinoma who underwent gastrectomy. This study was approved by the ethics committee of the First Affiliated Hospital of Nanchang University. Histological examinations All biopsies or surgical specimens from patients with different gastric lesions were taken from the gastric antrum and individual lesions from patients. The tissues used for histological analysis were fixed in 10% formaldehyde in Ca2+ and Mg2+ -free phosphate buffered saline (PBS) overnight at 4 ◦ C before paraffin embedding. Paraffin sections of 4 m were cut with a microtome and stored at room temperature. Pathologic diagnosis and classification were made according to the criteria of the World Health Organization [20] and the updated Sydney system [21]. Immunohistochemistry Paraffin sections were prepared form these gastric tissues or biopsy specimens. For immunohistochemistry, these tissue sections were first dewaxed in xylene and sequentially dehydrated in 100%, 95%, and 85% ethanol. The sections were immunostained using the PV-9000 Polymer Detection System (Zhongshan Golden-bridge, Beijing, China) according to the standard staining protocol. Briefly, the sections were first washed in PBS and endogenous peroxidase was blocked using 3% H2 O2 . After the specimens were incubated with the primary antibody overnight at 4 o C (rabbit polyclonal anti-human PTEN [ab31392] and monoclonal antihuman p-PTEN [Ser 380] [ab76431], diluted to 1:150, Abcam, Cambridge, UK), they were washed with PBS again, followed by incubation with polymer helper for 30 min and polyperoxidase-anti-rabbit IgG for 30 min. After washing three times with PBS, the sections were further incubated with 3,3-diaminobenzidin (DAB, Zhongshan Golden-bridge) for developing positive color. The negative control sections were just incubated with PBS without the primary antibodies. After that, the sections were counterstained with hematoxylin and mounted with coverslips. Review and scoring of immunostained sections The stained sections were reviewed and scored under a light microscope by two pathologists without knowing the clinicopathological data. The concordance rates were generally high and data with any grading discrepancies were rereviewed and discussed by them to make a final score. Epithelial cells stained positively with yellow or brown color in the nuclear and/or cytoplasm were defined as positive Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012), doi:10.1016/j.clinre.2012.03.002 +Model CLINRE-244; No. of Pages 8 ARTICLE IN PRESS Reduced expression of PTEN and increased PTEN phosphorylation in gastric cancer tissues 1 1 1 1 2 2 2 3 3 4 0 0 0 0 0 0 0 0 6 2 1 9 5.0 9.5 82.5 60.4 44.0 48.6 Cell lines and culture 1 2 27 27 21 78 +++ 19 19 7 19 28 92 The immortalized gastric epithelial mucosa cell line GES-1, established by Beijing Institute for Cancer Research (Beijing, China), was a kind gift from Prof. Y Ke of Beijing Institute for Cancer Research. Human gastric cancer SGC7901 (moderately differentiated), MKN28 (moderately differentiated), MKN45 (poorly differentiated) and AGS (poorly differentiated) cell lines were kind gifts from Prof. DM Fan of Xijing Hospital (Xi’an, China). These cell lines were cultured at 37 o C in an atmosphere of 5% CO2 in Dulbecco modified Eagle’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum, 100 unit penicillin, and 100 g/mL streptomycin (Gibco BRL, Life Technologies, New York, USA). 1 1 1 1 2 2 2 3 3 4 85.0 95.2 67.5 72.9 40.0 66.5 4 2 4 4 1 15 4 8 8 15 9 44 9 10 15 16 10 60 20 21 40 48 50 179 Normal gastric mucosa1 Chronic gastritis2 Intestinal metaplasia3 Dysplasia4 Gastric cancer5 Overall PR: positive rate. a P < 0.05 was considered as statistically significant. 3 1 13 13 30 60 12/8 10/11 21/19 26/22 31/19 100/79 ± ± ± ± ± ± n 50.1 54.2 54.9 57.4 54.0 54.7 14.5 14.7 12.3 12.5 12.3 13.8 (M/F) (mean ± SD) − Sex Age PTEN + ++ +++ PR (%) P vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. 2: 3: 4: 5: 3: 4: 5: 4: 5: 5: 0.685 0.167 0.410 0.001a 0.048a 0.185 0.000a 0.458 0.017a 0.001a − p-PTEN + ++ PR (%) P vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. 2: 3: 4: 5: 3: 4: 5: 4: 5: 5: 0.583 0.000a 0.000a 0.002a 0.000a 0.000a 0.005a 0.010a 0.000a 0.098 staining. Each section was chosen, reviewed, and scored from five randomly selected fields (magnification of × 200). In each microscopic field from the representative areas, 100 immunoreactive cells were assessed and quantified as the percentage and then averaged from the five fields for % of immunostaining, i.e. 0, < 5.0%; 1, 5.1—25.0%; 2, 25.1—50.0%; 3, 50.1—75.0%; and 4 > 75.0%. Moreover, the staining intensity was also semi-quantitatively assessed as 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. After that, the integrals of the ‘‘area × intensity’’ were calculated, based on which the overall expression levels of the proteins in the section as follows: negative (−), score 0—2; weak positive (+), score 3—5; moderate positive (++), score 6—8; and strong positive (+++), score 9—12. Protein extraction and Western blotting Group Differential expression of PTEN and p-PTEN at different histology of gastric tissue specimens. Table 1 3 Approximately 5 × 106 cells at log growth phase were collected and lysed in a buffer containing 0.5% Lubrol-PX, 50 mM KCl, 2 mM CaCl2 , 20% glycerol, 50 mM Tris-HCl (pH 7.4), and 0.1% protease and 1% phosphatase inhibitors (Sigma-Aldrich, Saint-Louis, MO, USA). The concentrations of protein sample were measured based on the BCA’s method (Pierce Biotechnology, Rockford, IL, USA). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4 ×) sample buffer (250 mM Tris-HCl, pH6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% mercaptoethanol) was added to the protein samples and in turn denatured by boiling for 5 min in a water bath. Samples containing 50 g of protein were analyzed on 10% SDS-PAGE gels and electroblotted onto nitrocellulose membranes (Whatman GmbH, Dassel, Germany). After blocking with 5% nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST), the blots were incubated overnight with the primary antibody at 4 o C (i.e., a rabbit monoclonal anti-human PTEN [#9559] at a dilution of 1:1,000 from Cell Signaling Biotechnology [Danvers, MA, USA] or a monoclonal anti-human p-PTEN [Ser 380] [ab76431] antibody at a dilution of 1:1,000 from Abcam [Cambridge, UK], or a rabbit polyclonal anti-human -actin antibody [sc-1615-R] at 1:1,000 dilution from Santa Cruz Biotechnology [Santa Cruz, CA, USA]). In the next day, HRP-conjugated goat anti-rabbit IgG (diluted 1:10,000, Zhongshan Golden-bridge) was used as a secondary antibody and incubated with the membrane Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012), doi:10.1016/j.clinre.2012.03.002 +Model CLINRE-244; No. of Pages 8 4 ARTICLE IN PRESS Z. Yang et al. Figure 1 Immunohistochemical staining of PTEN and p-PTEN protein in different histology of gastric tissues. Magnification at × 200. A—E. The expression of PTEN in normal gastric mucosa (A), chronic gastritis (B), intestinal metaplasia (C), dysplasia (D), and gastric cancer (E). F—J. The expression of p-PTEN in normal gastric mucosa (F), chronic gastritis (G), intestinal metaplasia (H), dysplasia (I), and gastric cancer (J). Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012), doi:10.1016/j.clinre.2012.03.002 +Model CLINRE-244; No. of Pages 8 ARTICLE IN PRESS Reduced expression of PTEN and increased PTEN phosphorylation in gastric cancer tissues 5 at 4 o C for 4 h. The reactions were subjected to incubation with the enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology, Rockford, IL, USA) and then exposed to X-ray film for visualizing the positive bands. Statistical analysis Data are summarized as mean ± standard deviation (SD) or percentage of control. The Chi2 test (SPSS v. 16.0 for windows; SPSS, Inc., Chicago, IL, USA) was performed to evaluate difference in categorical variables, such as gender among different defined groups. The one-way ANOVA (SPSS v.16.0) was used to determine the differences, such as patients’ ages in numerical variables among the groups. Kruskal-Wallis test or Mann-Whitney test (SPSS v.16.0) was used to determine the differences in numerical variables between differently defined groups. A P value of less than 0.05 was considered statistically significant. Results Differential expression of PTEN and p-PTEN in different gastric lesions Immunohistochemically, PTEN protein was primarily expressed in the cytoplasm of epithelial cells, with occasionally expression in the nuclei. The immunohistochemical data of PTEN expression was then analyzed semi-quantitatively. The data showed that weak to strong expression of PTEN was observed in 85.0%, 95.2%, 67.5%, 72.9%, and 40.0% of normal gastric mucosa, chronic gastritis, intestinal metaplasia, dysplasia, and gastric cancer, respectively. PTEN expression level was significantly decreased in gastric cancer compared to the normal gastric mucosa, chronic gastritis, intestinal metaplasia, or dysplasia (P < 0.05; Fig. 1 and Table 1). In addition, there was significant difference in PTEN expression between the tissue specimens of chronic gastritis and intestinal metaplasia (P < 0.05; Fig. 1 and Table 1). Moreover, p-PTEN was expressed in the cytoplasm of positive cells (Fig. 1). Weak to strong expression of p-PTEN was observed in 5.0%, 9.5%, 82.5%, 60.4%, and 44.0% of normal gastric mucosa, chronic gastritis, intestinal metaplasia, dysplasia, and gastric cancer, respectively, indicating that p-PTEN expression level was significantly increased in intestinal metaplasia, dysplasia, and gastric cancer compared to the normal gastric mucosa and chronic gastritis (P < 0.05; Fig. 1 and Table 1). Again, there was significant difference in p-PTEN expression between intestinal metaplasia and dysplasia or gastric cancer (P < 0.05; Fig. 1 and Table 1). Nevertheless, our data obtained from 43 cases of gastric cancer do not show an obvious association between PTEN or p-PTEN levels and clinicopathological grades (Table 2). Figure 2 Expression of p-PTEN and PTEN in non-cancerous and cancerous gastric cell lines by Western blot. A. Western blot. These cell lines were grown for two days and then subjected to total cellular protein isolation and Western blot analysis of PTEN and p-PTEN expression. B. Quantitative data of A. Immunoblots of PTEN and p-PTEN were scanned and the ratio of p-PTEN to PTEN densitometric units was expressed as % of -actin. * P < 0.05 compared with GES-1. levels of both PTEN and p-PTEN protein were higher in gastric cancer cell lines (i.e., SGC-7901, MKN-28, MKN-45, and AGS) than in non-cancerous GES-1 cells (Fig. 2A). Moreover, further analysis of data on Western blots revealed that the PTEN phosphorylation and PTEN ratio was higher in gastric cancer cell lines than that of non-malignant GES1 cells, e.g., 2.00 ± 0.37 (P < 0.05), 5.60 ± 0.88 (P < 0.05), 6.15 ± 0.72 (P < 0.05), and 3.20 ± 0.29 (P < 0.05) folds of MKN-28, MKN-45, SGC-7901, and AGS, respectively to the ratio of GES-1 (Fig. 2B). Discussion Expression of PTEN and p-PTEN in non-malignant and malignant gastric cell lines Next, we examined PTEN and p-PTEN expression in noncancerous and cancerous gastric cell lines and found that In the present study, expression of PTEN protein was shown to be progressively lost during gastric carcinogenesis from normal gastric mucosa via chronic gastritis, intestinal metaplasia, and dysplasia to gastric cancer, whereas p-PTEN Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012), doi:10.1016/j.clinre.2012.03.002 +Model ARTICLE IN PRESS CLINRE-244; No. of Pages 8 6 Z. Yang et al. Table 2 Association between PTEN and p-PTEN expression and clinicopathological data from gastric cancer patients. Characteristics n Overall score of the protein expression PTEN p-PTEN − + ++ +++ PR (%) Gender Male Female 23 20 15 11 6 4 2 4 0 1 34.8 45.0 Age (years) ≥ 55 < 55 22 21 14 12 4 6 3 3 1 0 36.4 42.9 Location Antrum Body and cardia 24 19 14 12 7 3 2 4 1 0 41.7 36.8 Gross type (Borrmann) I + II III + IV Differentiation Well and moderately Poorly and undifferentiatedly Invasive depth Above submucosa Muscularis propria Below subserosa TNM I + II III + IV Lymph node metastasis + − P − + ++ 13 15 7 4 3 1 0 0 43.5 25.0 13 15 5 6 4 0 0 0 40.9 28.6 15 13 5 6 4 0 0 0 37.5 31.6 +++ PR (%) 0.323 0.192 0.835 0.242 0.922 0.458 0.659 6 37 3 23 2 8 1 5 0 1 50.0 37.8 23 16 4 3 0 20 10 6 3 7 4 32 5 3 18 1 0 9 12 31 9 17 1 9 0.381 3 25 2 9 1 3 0 0 50.0 32.4 30.4 17 4 2 0 26.1 1 50.0 11 7 2 0 45.0 1 1 4 0 0 1 28.6 25.0 43.8 5 2 21 1 1 9 1 1 2 0 0 0 28.6 50.0 34.4 2 4 0 1 25.0 45.2 8 20 2 9 2 2 0 0 33.3 35.5 0.205 1 vs. 2: 1.000 1 vs. 3: 0.520 2 vs. 3: 0.649 0.241 0.322 17 9 8 0 6 2 0 1 45.2 25.0 1 vs. 2: 0.509 1 vs. 3: 0.895 2 vs. 3: 0.422 0.923 0.487 31 12 P 0.161 18 10 10 1 3 1 0 0 41.9 16.7 PR: positive rate. protein was progressively increased from normal gastric mucosa to intestinal metaplasia, and taking into account total PTEN reduction, was sustained high expression in dysplasia and gastric cancer. Moreover, these data were also confirmed in gastric cancer cell lines compared to the non-cancerous gastric cells. Nevertheless, the aberrant expression of PTEN and p-PTEN protein was not associated with any of the clinicopathological data we collected, such as gender, tumor differentiation, stages, and metastasis of gastric cancer. The current data demonstrates that the altered PTEN protein expression is an early event and may contribute to gastric cancer development. Detection of altered PTEN and p-PTEN at residue Ser380 expression could serve as a biomarker for diagnosis or risk prediction of gastric cancer. Our current findings confirm a previous observation reported by Yang et al. [22] that expression of PTEN protein was progressively decreased from normal gastric mucosa to gastric cancer. In their study, expression of PTEN protein was lower in the diffuse-type of gastric cancer than that of the intestinal-type gastric cancer and was conversely associated with lymph node metastasis of gastric cancer and poor and undifferentiated cancer. The reason why we were unable to show any association of PTEN expression with clinicopathological data might be because of the small sample size. However, in accordance with our study, two previous studies showed that the loss of PTEN expression was not associated with clinicopathological data, such as tumor size, differentiation, and lymph node metastasis of gastric cancers [16,23]. The function of PTEN protein is to dephosphorylate phosphatidylinositol second messengers, leading to blocking PI3 K signaling [18]. Phosphorylation of the three residues (Ser380, Thr382, and Thr383) within the tail of PTEN protein increases protein stability and inhibits PTEN phosphatase activity [17]. Thus, previous studies have reported that phosphorylation of PTEN protein at Ser380, Thr382, and Please cite this article in press as: Yang Z, et al. Reduced expression of PTEN and increased PTEN phosphorylation at residue Ser380 in gastric cancer tissues: A novel mechanism of PTEN inactivation. Clin Res Hepatol Gastroenterol (2012), doi:10.1016/j.clinre.2012.03.002 +Model CLINRE-244; No. of Pages 8 ARTICLE IN PRESS Reduced expression of PTEN and increased PTEN phosphorylation in gastric cancer tissues Thr383 results in the loss of tumor suppressor function of PTEN [17,18], and Ser380 phosphorylation is more critical in inactivation of PTEN protein [17]. In keeping with this, we showed both reduction of PTEN abundance and increases in its phosphorylation at Ser380, which might contribute to the loss of PTEN function in cells, although other phosphorylation sites of PTEN protein are also worthwhile studying and future development of antibodies will aid in these studies. Our investigation of PTEN expression revealed that the expression levels in cancer cell lines were higher than that of normal gastric cells, which is in contrast to cancer specimens. The reason of this discrepancy remains to be determined, but it might be attributed to the following; first, we did not analyze enough normal gastric cell lines and second, it is possible that cultured cancer cell lines somehow overcome the suppressive mechanism of expression. Nevertheless, after the ratio of phosphorylated to total PTEN was measured, we found that ratio was increased, which is consistent with the data obtained from human cancer tissue specimens. Hence, this suggests that detection of the increased PTEN phosphorylation is more reliable than that of total of PTEN in determining malignancy of gastric cells. Based on the data observed in the present study, we concluded that alterations in the expression of PTEN and p-PTEN at residue Ser380 during gastric carcinogenesis are an early event. It is true that their alterations first occur in lesions of chronic gastritis, intestinal metaplasia, and dysplasia. Thus, evaluation of changes in expression of PTEN and p-PTEN proteins at residue Ser380 may be a useful biomarker for prediction of the malignant transformation of non-cancerous lesions. In a future study, we will expand the sample size to confirm our findings. However, there is a discrepancy in this study, i.e., PTEN phosphorylation at residue Ser380 could increase PTEN protein stability, but our data showed reduced expression of total PTEN protein. The reason for this discrepancy is unknown. However, previous studies suggested that loss or reduced expression of PTEN protein commonly occurs in gastric cancers due to genetic or epigenetic changes, such as mutation, LOH, and gene promoter hypermethylation [12—16]. We speculate that a combination of these mechanisms may lead to reduction in PTEN expression, while increased activity of some kinases is able to phosphorylate the remaining PTEN protein and keep PTEN protein more stable in the early stage of gastric carcinogenesis (such as chronic gastritis and intestinal metaplasia). Reduction of PTEN expression mainly occurs in intestinal metaplasia, whereas PTEN phosphorylation mainly starts to occur in chronic gastritis. Once the alterations have occurred, the altered expression of PTEN protein is sustained, which is why expression of PTEN was shown to be progressively lost during gastric carcinogenesis, whereas p-PTEN protein progressively increased from normal gastric mucosa to intestinal metaplasia. However, further studies incorporating these independent mechanisms are needed. In conclusion, this study demonstrated that PTEN expression is progressively reduced from normal gastric mucosa to gastric cancer, whereas p-PTEN protein increases in the transition from normal gastric mucosa to intestinal metaplasia, and is sustained at high levels in dysplasia and gastric cancer, indicating that alteration of PTEN and p-PTEN at residue Ser380 protein may contribute to gastric caner 7 development. This study also supports the notion that phosphorylation of PTEN at Ser380 is a mechanism by which PTEN inactivated during gastric carcinogenesis. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported in part by grants from the National Natural Science Foundation of China (Grant Number: 81060038) and Graduate Innovative Foundation of Jiangxi Province, China (Grant Number: YC10A020). We thank Medjaden Bioscience Limited for assisting in the preparation of this manuscript. 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