Increasing expression of presenilin 1, β-catenin, and p-PTEN and its regulatory roles on cell invasion in gastric cancer

Abstract

BACKGROUND

Presenilin-1 (PS-1), a part of the gamma-secretase complex, has been implicated as a tumor promoter in various cancers. PS-1 binds to β-catenin through a large hydrophilic loop region that could lead to gastric tumorigenesis by the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin pathway, which is known to inhibit phosphatase and tensin homolog deleted on chromosome ten (PTEN). However, little is known about the mechanisms of PS-1, β-catenin, and PTEN in gastric cancer (GC) tumorigenesis.

AIM

To determine the regulatory correlation among PS-1, β-catenin, and phosphorylation of PTEN (p-PTEN) in GC tumorigenesis .

METHODS

Tissue samples from 116 patients with GC were analyzed by immunohistochemistry. Cell lysates from MGC-803 were used to detect protein levels by western blot. Cell invasion ability and metastatic ability were examined in vitro by Transwell invasion and in vivo via tail vein injection, respectively.

RESULTS

The high expression rates of PS-1, β-catenin, and p-PTEN in GC were 60.3% (70/116), 56.9% (66/116), and 47.4% (55/116), respectively, correlating with advanced tumor stages based on tumor invasion, lymph node metastasis, and 5-year survival. PS-1 expression was positively correlated with expression of β-catenin and p-PTEN in patients with GC. PS-1 regulated PTEN phosphorylation and cytoplasmic localization through β-catenin. PS-1 enhanced GC cell invasion via β-catenin.

CONCLUSION

The expression of PS-1 was positively correlated with that of both β-catenin and p-PTEN in GC. The regulation of PTEN phosphorylation and cytoplasmic localization by PS-1 through β-catenin could be considered potential therapeutic targets to prevent GC tumorigenesis.

Key Words: Gastric cancer; Presenilin 1; β-catenin; Phosphorylation of tensin homolog deleted on chromosome ten; Mechanisms of invasion

Core Tip: The high expression of presenilin 1 was correlated with advanced tumor stages, and its expression was positively correlated with expression of β-catenin and phosphorylation of tensin homolog deleted on chromosome ten in patients with gastric cancer (GC). The regulation of phosphatase and tensin homolog deleted on chromosome ten phosphorylation and cytoplasmic localization by presenilin 1 through β-catenin as a novel signaling pathway of GC progression could contribute to GC tumorigenesis.

INTRODUCTION

Gastric cancer (GC) is the second-leading cause of cancer-related deaths worldwide, particularly in East Asia, where the incidence is high with 40 to 60 cases per 100000 inhabitants[1]. The average 5-year survival rate is no more than 20% due to the lack of sensitive biomarkers for early detection. Presenilin 1 (PS-1) is a component of the γ-secretase complex that cleaves a variety of type I membrane proteins, including the β-amyloid precursor protein, vascular endothelial growth factor receptor, and Notch[2]. Mutations in PS-1 cause the majority of cases of familial Alzheimer’s disease[3].

Recent studies have revealed that PS-1 acts as a regulator of β-catenin and plays a significant role in various tumorigenic processes including cell proliferation, apoptosis, cell adhesion, and others. It has been reported that the loss of PS-1 enhances the β-catenin signaling pathway, contributing to skin tumorigenesis in vitro and in vivo[4]. In our previous study we also demonstrated that PS-1 promoted GC cell invasion and metastasis through the β-catenin signaling pathway[5]. However, the downstream targets of β-catenin regulated by PS-1 in GC remain unclear.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) mutations and/or deletions have been implicated in the development of various cancers. As a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway, PTEN is frequently inactivated in GC[6,7]. Recent reports suggest that partial loss of PTEN function due to post-translational modifications, such as phosphorylation, ubiquitination, and acetylation, may contribute to the onset of certain cancers.

In this study we observed that phosphorylation of PTEN (p-PTEN) was readily detectable in the majority of GC cell lines and patients with GC and played a significant role in GC progression via the PI3K/AKT pathway. PS-1 is also known to activate the PI3K/AKT cell survival pathway in a γ-secretase-independent manner[8]. However, the mechanism by which PTEN is inactivated and how PTEN inactivation promotes GC progression and metastasis remains largely elusive.

In the present study we used GC as the experimental model to investigate the role of PS-1 in β-catenin activation and PTEN inactivation and subcellular location that enhance tumor invasion and metastasis. Our results supported a novel functional interaction between the Wnt/β-catenin and PTEN/PI3K pathways that is crucially important for the activity of the protein as a transcription factor in GC.

MATERIALS AND METHODS

Clinical tumor tissues

All the GC samples were from the Affiliated Hospital of Putian University. Among them the 116 paraffin-embedded GC tissues were collected from October 2013 to September 2015. None of the patients in our study received preoperative chemotherapy, radiotherapy, or other biological treatment. All the patients underwent standard D2 lymph node dissection with curative resection (R0). Postoperative adjuvant chemotherapy was performed with 5-fluorouracil-based drugs plus oxaliplatin in advanced cases. The study was approved by the Affiliated Hospital of Putian University institutional review board, and written informed consent was obtained from all participants. The clinical and pathological staging was performed according to the American Joint Committee on Cancer 7^th edition of GC tumor-node-metastasis staging.

Patient follow-up method

All patients were systematically followed up by trained doctors based on institutional follow-up protocol in several ways via outpatient service, letter, telephone, mail, or visiting.

Immunohistochemical analysis

Immunohistochemical staining for PS-1, β-catenin, and p-PTEN was performed on formalin-fixed, paraffin-embedded gastric tissue sections (3 μm thick). Paraffin-embedded tissue sections from GC specimens were given a heat pretreatment of 70 °C for 1 h, then dewaxed in xylene, rehydrated in an ethanol series (100%-50%), and treated in 0.01 mol/L citrate buffer (pH 6.0) for antigen retrieval. After inhibition of endogenous peroxidase activity for 30 min with methanol containing 0.3% H₂O₂, the sections were stained with a rabbit anti-PS-1 monoclonal antibody (1:200, ab76083, Abcam, Cambridge, United Kingdom), β-catenin (1:200, ab22656, Abcam, Cambridge, United Kingdom), or p-PTEN (1:100, ab109454, Abcam, Cambridge, United Kingdom) at 4 °C overnight. The following experimental procedure was on the basis of the manufacturer’s instructions of the LSAB + kit (Dako, CA, United States). The PS-1, β-catenin, and p-PTEN protein expression was immunohistochemically demonstrated as yellowish to brown staining in gastric glandular cells. Two pathologists blinded to the clinical data reviewed the immunoreactivity for PS-1, β-catenin, p-PTEN protein under a light microscope, and the protein expression was scored independently according to the intensity of cellular staining and the proportion of stained tumor cells. The staining intensity was scored as 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellow brown), and 3 (strong staining, brown), and the proportion of stained tumor cells was classified as 0 (≤ 5% positive cells), 1 (6% to 25% positive cells), 2 (26% to 50% positive cells), and 3 (≥ 51% positive cells).

Western blot analysis

The GC cell line MGC-803 was purchased from Shanghai Institute of Cell Biology, China. Cells were homogenized in ristocetin-induced platelet aggregation protein lysis buffer containing protease inhibitors at 4 °C for 30 min before centrifugation at 12000 × g for 10 min at 4 °C. The supernatants, representing whole cell lysates, were prepared for use in subsequent experiments. The protein concentration was measured using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific, MA, United States). A total of 40 μg protein from each sample was denatured and loaded into each well, separated by sodium-dodecyl sulfate gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, United States).

Subsequently, the membrane was blocked with 5% nonfat milk at room temperature for 1 h. The membrane was incubated with anti-PS-1 (1:1000), anti-β-catenin (1:1000), p-PTEN (1:1000), PETN (1:1000, ab32199, Abcam, Cambridge, United Kingdom) or anti-glyceraldehyde-3-phosphate dehydrogenase (1:1000, ab181602, Abcam, Cambridge, United Kingdom) primary antibodies overnight at 4 °C. After washing with wash buffer (10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1% Tween 20), the membrane was further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma, St. Louis, MO, United States) at a 1:2000 dilution for 1 h at room temperature. Subsequently, the membrane was washed for 30 min with wash buffer and detected using enhanced chemiluminescence (Amersham Corporation, Arlington Heights, IL, United States).

Plasmid construction and transfection

Lentivirus overexpressing PS-1, short hairpin RNA (shRNA), and control shRNA were purchased from Gene Chem Corporation (GCPL45123, Shanghai, China). The PS-1 complementary DNA open reading frame was cloned into the p-HBLV-IRES-ZsGreen-PGK-Puro plasmid (HanbioTM, Shanghai, China) for lentiviral production. β-catenin small interfering RNA (siRNA) (#6225) and control siRNA (#6568) were purchased from Cell Signaling Technology, MA, United States. Stable cell lines were screened with puromycin and identified by western blot.

Subcellular fraction assay

The assays were performed according to the manufacturer’s instructions for the Qproteome Cell Compartment Kit (Qiagen, Germany). A total of 40 μg protein from each fraction was denatured and loaded into each well, and sodium-dodecyl sulfate gel electrophoresis and western blot were conducted as described above. Antibodies against lamin A/C was purchased from Santa Cruz Biotechnology.

Cell invasion assay

Cell invasion was measured using a Transwell chamber (8 μm, 24-well format; Corning, Lowell, MA, United States). To measure invasion diluted Matrigel (BD Biosciences, CA, United States) was used to coat the insert chamber membrane. Then 8 × 10⁴ cells were resuspended in 0.3 mL of serum-free medium and added to the upper chamber while 0.8 mL of medium containing 10% fetal bovine serum was added to the lower chamber. Finally, cells that invaded into the lower chamber were fixed with methanol, stained with crystal violet, and counted in five random fields.

Immunofluorescence assay

GC cell line MGC-803 with γ-secretase inhibitor (GSI) treated and control were plated in a 24-well plate chamber for 24 h. Cells were washed with 1 × PBS and fixed with an acetone/methanol (1:1) mixture for 15 min at 4 °C. Cells were then washed twice with 1 × PBS, followed by blocking with 10% normal goat serum blocking solution (ZSGB Biotech, China) for 1 h at room temperature. Cells were hybridized with the specific primary antibodies against β-catenin or PTEN diluted in 1 × PBS (1:400) overnight at 4 °C. Cells were washed three times in 1 × PBS and hybridized with fluorescently conjugated secondary antibody (1:200) for 45 min at room temperature. β-catenin and PTEN were detected by fluorescein isothiocyanate red fluorescence. Cells were washed in 1 × PBS, mounted with Vectashield/DAPI (Vector Laboratories, Burlingame, CA, United States), and examined by a laser scanning confocal microscope (Leica, Germany). At least 200 cells were counted from each experiment.

In vivo tumorigenesis

Specific pathogen-free-grade male BALB/c nude mice were purchased from the Institute of Zoology, Chinese Academy of Sciences. For the metastasis experiment in vivo, 5 × 10⁶ MGC-803 cells and GSI-treated MGC-803 cells were resuspended in 0.3 mL of Dulbecco’s modified Eagle’s medium and injected intravenously into 4-week-old male nude mice (n = 4 per group). The mice were anesthetized after 8 weeks, and metastatic nodules were counted throughout the liver and lungs.

Statistical analysis

All measurement data are presented as the means ± standard error and were analyzed using SPSS 17.0 for Windows (SPSS, Chicago, IL, United States) and Prism 5.0 software (GraphPad). The relationships between the PS-1 expression level and clinicopathologic parameters were calculated with the Pearson χ² test. Survival curves were explored by the Kaplan-Meier method, and differences between two groups were evaluated by the log-rank test. Comparisons were performed using the Student’s t test (two groups) or one-way analysis of variance (multiple groups). The statistical significance threshold was set as P < 0.05.

RESULTS

Prognostic value and relationship of PS-1, β-catenin and p-PTEN in GC tissues

To investigate the associations among PS-1, β-catenin, and p-PTEN, we detected the expression of PS-1, β-catenin, and p-PTEN in the 116 GC samples using immunohistochemistry (Figure 1A). Based on the protein expressions in the GC tissues, we classified samples into two groups: High group and low group. The high expression rates of PS-1, β-catenin, and p-PTEN were 60.3% (70/116), 56.9% (66/116), and 47.4% (55/116), respectively. According to the χ² test (Table 1), both tumor invasion and lymphatic metastasis were simultaneously associated with PS-1, β-catenin, and p-PTEN, and it was consistent with the results that patients with high expression of PS-1, β-catenin, and p-PTEN have significantly more metastatic lymph nodes than those with low expression (Figure 1B).

Figure 1 Prognostic values of presenilin-1, β-catenin, and phosphorylation of tensin homolog deleted on chromosome ten in gastric cancer tissues. A: Representative immunohistochemical staining of presenilin-1 (PS-1), β-catenin, and phosphorylation of tensin homolog deleted on chromosome ten (p-PTEN) in continuous tissue sections from gastric tumor tissues. Original magnification, × 40; B: Significant differences of metastatic lymph nodes between low and high expression of PS-1 (left), β-catenin (middle), and p-PTEN (right); C: The overall survival rate of patients with high PS-1 (left), β-catenin (middle), and p-PTEN (right) expression were significantly lower than that of patients with low protein expression. PS-1: Presenilin-1; p-PTEN: Phosphorylation of tensin homolog deleted on chromosome ten.

Table 1 Characteristics of 116 patients with gastric cancer among presenilin-1, β-catenin, and phosphorylation of tensin homolog deleted on chromosome ten expressions.

Clinicopathological features	PS-1		β-catenin		p-PTEN		P value
	Total (n)	High expression	P value	High expression	P value	High expression
Age (years)			0.056		0.068		0.028
≤ 60	63	33		31		24
> 60	53	37		35		31
Gender			0.673		0.919		0.604
Male	91	54		52		42
Female	25	16		14		13
Tumor site			0.147		0.272		0.405
Upper	40	19		23		18
Middle	22	15		16		13
Lower	46	32		24		22
Mixed	8	4		3		2
Tumor size (cm)			0.004		0.005		0.269
≤ 5	59	28		26		25
> 5	57	42		40		30
Grade of differentiation			0.405		0.389		0.439
Well and moderate	55	31		29		24
Poor and not	61	39		37		31
Neurovascular invasion			0.433		0.031		0.014
Negative	54	29		25		19
Positive	62	41		41		36
Depth of invasion			< 0.001		0.001		0.002
T1	4	1		0		1
T2	11	2		2		0
T3	48	25		26		22
T4	53	42		38		32
Lymphatic metastasis			< 0.001		< 0.001		0.003
N0	15	3		3		2
N1	20	7		7		7
N2	35	21		20		16
N3	46	39		36		30
Distal metastasis			0.041		0.112		0.004
M0	109	63		60		48
M1	7	7		6		7

p-PTEN: Phosphorylation of tensin homolog deleted on chromosome ten; PS-1: Presenilin-1; T: Tumor; N: Node; M: Metastasis.

Moreover, we compared the overall survival of patients categorized by PS-1, β-catenin, and p-PTEN expression. The overall survival rate of patients with high PS-1 expression was significantly lower than that of patients with low PS-1 expression. Similar results were observed in β-catenin and p-PTEN (Figure 1C). Interestingly, using the Spearman rank correlation test, we found that PS-1 expression levels were positively correlated with both β-catenin and p-PTEN levels in patients with GC. In the samples with high PS-1 expression, both β-catenin and p-PTEN protein expression tended to be significantly higher than that in the low PS-1 expression samples (r values were 0.362 and 0.471, respectively, Table 2). In the samples with high β-catenin expression, p-PTEN protein expression tended to be significantly higher than that in the low β-catenin expression samples (r value was 0.338, Table 2). These results suggested that the functional effects of PS-1 in GC could probably be conducted by β-catenin and p-PTEN.

Table 2 Correlation among expressions of presenilin 1, β-catenin and phosphorylation of tensin homolog deleted on chromosome ten.

Protein	β-catenin expression			p-PTEN expression
	Low	High	r value	Low	High	r value
Presenilin-1 1 expression			0.362a			0.417a
Low	30	16		36	10
High	20	50		25	45
p-PTEN expression			0.338a
Low	36	25
High	14	41

^aP < 0.05. p-PTEN: Phosphorylation of tensin homolog deleted on chromosome ten.

PS-1 enhanced the expression of p-PTEN in GC cells

MGC-803 GC cells were successfully infected with PS-1 overexpression (vector-PS-1) and PS-1 knockdown (ctrl-shPS-1) lentivirus. We examined protein levels of PS-1, β-catenin, p-PTEN, and PTEN by western blot analysis. We found that the protein levels of p-PTEN and β-catenin were noticeably decreased with downregulation of PS-1 (Figure 2A) while the level of p-PTEN and β-catenin were increased when PS-1 was upregulated (Figure 2B). However, PTEN protein expression was not significantly changed in MGC-803 cells either in the overexpression group or in the knockdown group. These results suggested that PS-1 might affect phosphorylation of PTEN as well as β-catenin expression but did not affect total PTEN protein expression.

Figure 2 Presenilin-1 enhanced the expression of phosphorylation of tensin homolog deleted on chromosome ten in gastric cancer cells. A and B: Western blot analysis testing the expression of presenilin-1 (PS-1), β-catenin, phosphorylation of tensin homolog deleted on chromosome ten (PTEN), and PTEN by upregulating and downregulating PS-1. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. The quantified analysis of the expression of PS-1 and phosphorylation of PTEN is presented. The values were shown as mean ± SD of three independent experiments. ^bP < 0.01. PS-1: Presenilin-1; p-PTEN: Phosphorylation of tensin homolog deleted on chromosome ten; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; Ctrl: Control; PTEN: Phosphorylation of tensin homolog deleted on chromosome ten.

PS-1 regulated PTEN phosphorylation and cytoplasmic localization through β-catenin

We performed a subcellular fraction assay to measure subcellular localization of β-catenin, PTEN, and p-PTEN by GSI treatment or lentivirus PS-1. GSI obviously decreased expression of β-catenin (in both cytoplasm and nucleus) and p-PTEN (just in cytoplasm) without effecting the total amount of PTEN. Interestingly, nuclear localization of PTEN upon GSI treatment was also consistently observed in GC cells (Figure 3A-C). Overexpression of PS-1 induced β-catenin expression in both the cytoplasm and nucleus with an increase in p-PTEN only in the cytoplasm (Figure 3D). We further investigated whether PS-1 regulated PTEN phosphorylation through β-catenin. As expected cotransfection of β-catenin siRNA and lentivirus PS-1 could rescue the phosphorylation of PTEN by PS-1 (Figure 3E). These data indicated that PS-1 regulated PTEN phosphorylation and cytoplasmic localization through activating β-catenin in GC cells.

Figure 3 Presenilin-1 regulated tensin homolog deleted on chromosome ten phosphorylation and subcellular localization through activating β-catenin. A: MGC-803 cells were treated with 1 μM γ-secretase inhibitor (GSI) for 12 h, 24 h, and 36 h. Time-dependent protein expressions of β-catenin, phosphorylation of tensin homolog deleted on chromosome ten (PTEN), and PTEN with or without GSI treatment were detected via western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control; B: Localization of β-catenin and PTEN after GSI treatment. Cells were treated with 1 μM GSI for 24 h, and immunocytochemical analysis was performed with different antibodies. Nuclei were detected by DAPI staining; C-E: Subcellular localization of phosphorylation of PTEN, PTEN, and β-catenin after treatment of GSI, lentivirus presenilin-1, and both lentivirus presenilin-1 and β-catenin small interfering RNA. Cytoplasmic and nuclear fractions were isolated via the subcellular fraction assay. Glyceraldehyde-3-phosphate dehydrogenase was used as a control in the cytoplasm and lamin A/C in the nucleus. GSI: Γ-secretase inhibitor; p-PTEN: Phosphorylation of tensin homolog deleted on chromosome ten; PTEN: Tensin homolog deleted on chromosome ten; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

PS-1 promoted GC cells invasion via β-catenin

To investigate the functional significance of the variation of β-catenin regulated by PS-1, we first employed Transwell invasion assay to assess the effect of PS-1 overexpression on GC cell migration. The results showed that MGC-803 cells stably infected with lentivirus PS-1 exhibited promotion of cell invasion compared with the control cells. However, cotransfection of β-catenin siRNA and lentivirus PS-1 could rescue the promotion of cell invasion by PS-1. Furthermore, we noticed that PTEN phosphorylation was noticeably decreased and was accompanied by decreasing β-catenin (Figure 4A and B). In contrast, shRNA-mediated knockdown of PS-1 in MGC-803 cells led to significantly reducing cell invasion. Overexpression of β-catenin completely reversed the effect of PS-1 knockdown in invasion. Importantly, the protein level of p-PTEN was positively correlated with the protein level of PS-1 and β-catenin (Figure 4C and D). These data provided evidence that PS-1 promoted the invasion of GC cells via enhancing β-catenin expression that might increase PTEN phosphorylation.

Figure 4 Presenilin-1 promoted gastric cancer cell invasion via β-catenin. A: The expression of total phosphorylation of tensin homolog deleted on chromosome ten, tensin homolog deleted on chromosome ten, and β-catenin were measured by western blot after transfection with β-catenin small interfering RNA and lentiviral presenilin-1 (PS-1); B: Decreasing expression of β-catenin reversed the enhanced invasion induced by upregulating PS-1. The quantifications were presented as mean ± SD on the right; C: The above proteins were measured after overexpression of β-catenin in stable knockdown PS-1 cell line MGC-803; D: Β-catenin increased the invasion ability of MGC-803 cells when PS-1 was downregulated. The quantifications were presented as mean ± SD on the right. ^bP < 0.01. NS: No significance; PS-1: Presenilin-1; p-PTEN: Phosphorylation of tensin homolog deleted on chromosome ten; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; Ctrl: Control; PTEN: Phosphorylation of tensin homolog deleted on chromosome ten.

GSI inhibited GC cell metastases in nude mice

To demonstrate that PS-1, a critical component of γ-secretase, is highly relevant for GC tumorigenesis, we conducted a preclinical assay to investigate the impact of GSI on immunocompromised mice. We randomly divided the nude mice into two groups and injected the same amount of MGC-803 cells into the lateral tail veins. One group was treated with normal saline (control group), and the other group was treated with 10 mg/kg GSI (GSI group). Three months after injection, the mice were sacrificed to evaluate metastatic nodules in the lungs (Figure 5). We found mice injected with saline harbored more metastatic foci throughout their lungs compared with those injected with the GSI. The result showed the potential therapeutic efficacy of GSI for the treatment of human GC.

Figure 5 γ-secretase inhibitor inhibited gastric cancer cell metastases in nude mice. A: Metastases (black arrows) in the lungs at 40 days after inoculation; B: The corresponding quantifications were presented as mean ± SD. Metastases were seldom detected in the γ-secretase inhibitor treatment group. ^bP < 0.01. GSI: γ-secretase inhibitor; Ctrl: Control.

DISCUSSION

PS-1, a multipass transmembrane protein important in development, is tightly linked to many cases of early-onset familial Alzheimer’s disease indicating a causal relationship between these mutants and Alzheimer’s disease pathology[9-11]. Recently, an increasing number of reports have shown that PS-1 plays an exclusive and significant role in various tumorigenic processes including cell proliferation, apoptosis, cell adhesion and others[12,13]. In prostate cancer PS-1 was demonstrated to induce intramembrane proteolysis of transforming growth factor-β receptor I, promoting transforming growth factor-β-induced invasion of cancer cells in vitro[14]. Enhanced expression of proteolytically active PS-1 is associated with E-cadherin proteolysis and nuclear translocation, which promotes peritoneal metastasis in colorectal cancer[15].

However, little is known regarding the downstream targets of PS-1 in the development of GC. We therefore aimed to evaluate the involvement of PS-1 in GC. In the current study we found that the expression of PS-1 in patients with GC was higher in tumor tissues than in adjacent nontumor tissues, and these patients exhibited more lymph node metastases and a poorer prognosis. In addition, our study was the first to verify the positive correlation between PS-1 and β-catenin levels in GC, providing scientific clinical experimental evidence for further in vitro research. In the in vitro cell model, overexpression of PS-1 markedly promoted cell invasiveness, whereas PS-1 knockdown significantly inhibited GC cell invasion. These effects could be reversed by regulation of β-catenin levels. These results were consistent with those obtained for gastric tumor tissues, indicating that PS-1 likely acts as a tumor promoter to enhance tumor cell invasion by β-catenin.

Several studies focusing on the role of PS-1 and β-catenin have been published in a broad variety of cancer types. A study reported that myoinositol-induced downregulation of PS-1 could reduce β-catenin translocation, which interfered with the epithelial-mesenchymal transition and led to anticancer activity[16]. In addition, in our previous study we also presented that PS-1 promoted cell invasion and metastasis through β-catenin in GC[5]. However, it has also been reported that PS-1 deficiency resulted in increased β-catenin stability in tumorigenesis in vitro and in vivo[4,17,18]. The diverse and controversial impact of PS-1 on β-catenin in the above-mentioned tumors probably resulted from the activation of a variety of downstream signaling pathways. Overall, our findings identified a PS-1/β-catenin signaling pathway as a novel component of the carcinogenic machinery in GC.

PTEN was independently discovered by three laboratories in 1997 as a tumor suppressor that was often lost in tumorigenesis[19-21]. Later studies established that PTEN played a critical role in growth and survival in various cancer types[22,23]. Recent studies indicated that regulation of PTEN in GC included mutation, promoter hypermethylation, etc. However, it remains to be investigated whether other regulatory mechanisms account for the inactivation of PTEN and the induction of tumorigenesis in GC. Indeed, several studies demonstrated that phosphorylation of the PTEN protein (including Ser380, Thr382, and Thr383) led to a loss of phosphatase activities and in turn resulted in a loss of tumor suppressor function. This was the most important mechanism of modification of PTEN in some types of tumors[24,25].

In the current study we explored whether the phosphorylation of PTEN occurred and how it was regulated in GC. Our results indicated that the high expression rate of p-PTEN in gastric tumor tissues was higher than that in nontumor tissues. Cytoplasmic p-PTEN expression levels were positively correlated with both PS-1 and β-catenin levels in patients with GC, and this was consistent with in vitro results that PS-1 regulated PTEN phosphorylation and subcellular localization through activating β-catenin. These results are consistent with those of previous studies showing phosphorylation of PTEN inducing its nuclear exclusion and membrane location[26,27].

Interestingly, although p-PTEN was upregulated by PS-1, the expression of total PTEN remained unchanged. This was inconsistent with previous studies showing a loss of PTEN activity through a decrease in total PTEN expression level and phosphorylation of its C-terminus in lung cancer cells[28]. It is possible that this was just the decreasing unphosphorylated PTEN that resulted in abrogation of the tumor suppressive effect of PTEN in GC. However, it still remains unclear whether PTEN is phosphorylated by the PS-1/β-catenin complex directly or indirectly. In the future it will be interesting to investigate how PTEN is phosphorylated by the PS-1/β-catenin complex and whether p-PTEN performs biological functions in GC.

CONCLUSION

Our current study represented the first investigation into the functional relationship among PS-1, β-catenin, and p-PTEN in GC progression through a survey of tumor tissues with well-documented clinical pathology and history. We also have demonstrated that PS-1 regulated PTEN phosphorylation and cytoplasmic localization through β-catenin. This work identified a novel PS-1/β-catenin/PTEN signaling axis contributing to GC pathogenesis. It mechanistically explained how high PS-1 expression promoted GC aggressiveness by inactivating the key tumor suppressor PTEN via β-catenin-dependent phosphorylation and mislocalization. Components of this pathway (PS-1, β-catenin, specific PTEN kinases) represent potential therapeutic targets for GC. Taken together, our findings indicate that PS-1, β-catenin, and p-PTEN are important for GC progression and could be considered potential therapeutic targets to prevent GC tumorigenesis.