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. 2014 Oct 30;13(18):2962–2974. doi: 10.4161/15384101.2014.947203

Epithelial cell-derived periostin functions as a tumor suppressor in gastric cancer through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14ARF/Mdm2 signaling pathway

Hongjun Lv 1,†, Rui Liu 2,†, Jiao Fu 1, Qi Yang 1, Jing Shi 1, Pu Chen 1, Meiju Ji 3, Bingyin Shi 1, Peng Hou 1,*
PMCID: PMC4614259  PMID: 25486483

Abstract

Periostin is usually considered as an oncogene in diverse human cancers, including breast, prostate, colon, esophagus, and pancreas cancers, whereas it acts as a tumor suppressor in bladder cancer. In gastric cancer, it has been demonstrated that periglandular periostin expression is decreased whereas stromal periostin expression is significantly increased as compared with normal gastric tissues. Moreover, periostin produced by stromal myofibroblasts markedly promotes gastric cancer cell growth. These observations suggest that periostin derived from different types of cells may play distinct biological roles in gastric tumorigenesis. The aim of this study was to explore the biological functions and related molecular mechanisms of epithelial cell-derived periostin in gastric cancer. Our data showed that periglandular periostin was significantly down-regulated in gastric cancer tissues as compared with matched normal gastric mucosa. In addition, its expression in metastatic lymph nodes was significantly lower than that in their primary cancer tissues. Our data also demonstrated that periglandular periostin expression was negatively associated with tumor stage. More importantly, restoration of periostin expression in gastric cancer cells dramatically suppressed cell growth and invasiveness. Elucidation of the mechanisms involved revealed that periostin restoration enhanced Rb phosphorylation and sequentially activated the transcription of E2F1 target gene p14ARF, leading to Mdm2 inactivation and the stabilization of p53 and E-cadherin proteins. Strikingly, these effects of periostin were abolished upon Rb deletion. Collectively, we have for the first time demonstrated that epithelial cell-derived periostin exerts tumor-suppressor activities in gastric cancer through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14ARF/Mdm2 signaling pathway.

Keywords: E-cadherin, Gastric cancer, Mdm2, p53, periostin, Rb/E2F1 pathway

Abbreviations:

Akt/PKB

serine/threonine kinase/protein kinase B

DMEM

Dulbecco's modified Eagles medium

DMSO

dimethyl sulfoxide

E2F1

E2F transcription factor 1

ECM

extracellular matrix

EDTA

Ethylenediaminetetraacetic acid

EGFR

epidermal growth factor receptor

ELISA

enzyme linked immunosorbent assay

EMT

epithelial-to-mesenchymal transition

ESCC

esophageal squamous cell carcinoma

FAK

focal adhesion kinase

FITC

fluoresceine isothiocyanate

HRP

horseradish peroxidase

HC

immunohistochemistry

Mdm2

mouse double minute 2

MMPs

metalloproteinases

MTT

3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide

NSCLC

non-small-cell lung cancer

PBS

phosphate buffered saline

PI3K

phosphatidylinositol 3-kinase

PVDF

polyvinylidene fluoride

Rb

retinoblastoma

RPMI 1640

Roswell Park Memorial Institute 1640

RT-PCR

Reverse-transcription polymerase chain reaction

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SENP8

Sentrin-specific protease 8

shRNA

short hairpin RNA.

Introduction

Gastric cancer is a devastating malignancy that is highly prevalent in Asia, particularly China, and is the second most common cause of cancer-related death worldwide.1 Common risk factors associated with gastric cancer include H. pylori infection, smoking, high salt intake, and other dietary factors.2 Although diagnostic and therapeutic advances, such as Her2 staining and targeted therapies, have provided pronounced survival benefit, gastric cancer is usually diagnosed at an advanced stage and clinical outcomes remain dismal due to a lack of early symptoms and limited advances in our understanding of the pathogenesis of this disease.2–4 Therefore, there is an urgent need to clarify the molecular events that regulate the aggressive behaviors of gastric cancer, and to identify novel molecular targets for early screening and developing new therapeutic approaches.

It has been well-known that human carcinogenesis involves multistep genetic and epigenetic alterations, leading to the inactivation of tumor suppressor genes and the overactivation of oncogenes. These abnormalities cause cancer cells to activate adjacent stromal cells and induce the release of cytokines, growth factors, angiogenic factors, proteolytic enzymes and extracellular matrix (ECM) proteins into tumor stroma to create a tumor-supportive microenvironment.5,6 Periostin is an important ECM proteins and its multifaceted role in tumorigenesis has also been well documented.7 It has been reported to be overexpressed and plays an oncogenic role in different cancers by binding with the integrins to promote the recruitment of EGFR and the activation of Akt/PKB and FAK-mediated signaling pathways, including colon, esophagus, pancreas, breast, lung, ovary and prostate cancers.8–14 Conversely, it is frequently downregulated and acts as a tumor suppressor in bladder cancer.15 Periostin has been shown to be down-regulated in majority of gastric cancer tissues compared with matched normal gastric tissues.10 Moreover, a very recent study has demonstrated that periglandular periostin expression is remarkably downregulated in gastric cancer tissues compared with normal gastric tissues. In contrast, stromal periostin expression is significantly up-regulated in cancer tissues.16 Notably, periostin produced by stromal myofibroblasts has been proved to support gastric cancer cell growth.16 However, the role of epithelial cell-derived periostin in gastric tumorigenesis still remains largely unknown.

In this study, using immunohistochemistry (IHC) assay, periglandular periostin expression was demonstrated to be lower in primary gastric cancers than that in adjacent normal gastric mucosa. Moreover, its expression was significantly down-regulated in metastatic lymph nodes compared with matched primary tumor tissues, and was negatively associated with tumor stage. Further functional studies revealed that periostin re-expression in gastric cancer cells dramatically inhibited cell growth and invasiveness by stabilizing p53 and E-cadherin proteins via the retinoblastoma (Rb)/E2F1/p14ARF/Mdm2 signaling.

Results

Down-regulation of periglandular periostin in primary gastric cancers

To clarify the role of periostin played in gastric carcinogenesis, its expression was investigated in a panel of primary gastric cancers and adjacent normal gastric mucosa by IHC assay. As shown in Fig. 1A, most of normal gastric mucosa showed a strong positive staining (++), and periostin was mainly localized in extracellular strand and ring structures surrounding individual glandulous tubules. As compared with normal gastric mucosa, periglandular periostin expression was dramatically downregulated in primary gastric cancers. Similarly to the findings from a previous study,16 stromal periostin staining was significantly increased in gastric cancer tissues compared with normal gastric tissues (Fig. S1). Next, immunohistochemical staining of periostin was performed in 10 pairs of primary tumors and matched metastatic lymph nodes. The results showed that periglandular periostin expression in metastatic lymph nodes was significantly lower than that in their primary tumor tissues (Fig. 1B). Additionally, it was noted that periglandular periostin expression was negatively associated with tumor stage (Fig. 1C). Taken together, these observations suggest that downregulation of periglandular periostin may be involved in the malignant progression of gastric cancer.

Figure 1.

Figure 1.

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Downregulation of periglandular periostin in primary gastric cancers. Immunohistochemical (IHC) analysis in all tissue sections was performed to evaluate periostin expression. The positive staining was shown in a reddish-brown color. All sections were counterstained with Hematoxylin showing a blue color. The insert shows the magnified image of the area indicated by the black square. −, ±, +, + + represents negative, weak, moderate, and strong staining, respectively. Scale bars, 200 μm. (A) Periglandular periostin expression was significantly down-regulated in primary gastric cancers (T) compared with matched normal gastric mucosa (N). Shown are representative images of IHC staining from one pair of tumor and normal samples. (B) Periglandular periostin expression in the metastatic lymph nodes (L) was remarkably lower than that in in their primary gastric cancers (T). Shown are representative images of IHC staining from one of matched primary tumor-metastatic lymph node pairs. (C) Periglandular periostin expression was negatively associated with tumor stage. Representative images of IHC staining with different tumor stage (I, II, III, and IV) were shown as indicated (upper panel). Periostin expression was scored and plotted according to different tumor stage in the lower panel.

Periostin inhibits gastric cancer cell growth

Frequent down-regulation of periglandular periostin in primary gastric cancers suggests its role as a putative tumor suppressor. Thus, the growth-suppressive effect was examined through ectopic expression of periostin in BGC823 and SGC7901 cells. Periostin re-expression in the stably transfected cell clones (BGC823/C10 and /E7, and SGC7901/B6 and /B7) was confirmed by qRT-PCR and western blot assays (Fig. 2A and Fig. S2A). Periostin restoration in these cells caused a significant decrease in cell proliferation (Fig. 2B). The inhibitory effect on cell growth was further confirmed by colony formation assay. As shown in Figure 2C, the colonies formed in cell clones stably expressing periostin were significantly fewer than those formed in control clones (V). Moreover, our data showed that periostin inhibited cellular survival under conditions that mimic hypoxia in BGC823 cells (Fig. S2B). To determine periostin as an ECM protein to play a tumor suppressor effect in gastric cancer, the culture supernatant of BGC823/V, /C10 and /E7cells was collected and the secreted periostin in the supernatant was measured by enzyme linked immunosorbent assay (ELISA). The results showed that the concentration of periostin in the supernatant from cell clones (C10 and E7) stably expressing periostin was significantly elevated as compared with control clone (Fig. S3A). The collected medium was then diluted with fresh medium (1:2) and used as the conditional medium for the culture of BGC823 and SGC7901 cells. As expected, the medium from cell clones stably expressing periostin significantly inhibited the proliferation of both BGC823 and SGC7901 cells as compared with control clone (Fig. S3B). Collectively, these observations suggest that epithelial cell-derived periostin can be secreted to extracellular medium and plays a potential tumor suppressor in gastric cancer.

Figure 2.

Figure 2.

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Inhibition of cell growth and induction of cell cycle arrest and apoptosis by periostin in gastric cancer cells. (A) Cell clones stably expressing periostin (BGC823/C10 and /E7, and SGC7901/B6 and /B7) and control clones (BGC823/V and SGC7901/V) were isolated and periostin re-expression in these clones was confirmed by protein gel blot. GAPDH was used as loading control. (B) Periostin restoration significantly inhibited cell viability in gastric cancer cells. *, P < 0.05; **, P < 0.01. (C) The effect of periostin on cell growth was further confirmed by colony formation assay. Left panel shows the representative images of colony formation in periostin-overexpressing and control clones. Quantitative analysis of colony numbers is shown in the right panel. Data were presented as mean ± SE. *, P < 0.05; **, P < 0.01. (D) Cells were transiently transfected with periostin-expressing plasmid and empty vector. After 48 h or 72 h post-transfection, DNA content was measured by flow cytometry to determine cell cycle fractions. Representative flow cytometric histograms of the cells transfected with different plasmids are shown in the left. The distribution and percentage of these 2 cell lines in each cell cycle phase is indicated in the right. Data were presented as mean ± SE of values from 3 different assays *, P < 0.05. (E) Cells were transiently transfected with different plasmids. Cell apoptosis was then measured 72 h after transfection in cells by flow cytometry using Annexin V/PI Detection Kit. Flow cytometry histograms represent the cells with Annexin V-FITC and PtdIns staining in the left. Relative percentage of apoptotic cells in periostin- and empty vector-transfected cells was presented in the right. Data were presented as mean ± SE of values from 3 independent assays. *, P < 0.05; **, P < 0.01.

Periostin induces cell cycle arrest and apoptosis in gastric cancer cells

Given that inhibition of cell growth is usually bound up with concomitant cell cycle arrest and the activation of apoptosis pathway, the contribution of cell cycle arrest and apoptosis to the growth inhibitory role of periostin was investigated in this study. As shown in Fig. 2D, compared with empty vector-transfected cells, cell cycle was arrest at the G2/M phage in periostin-transfected BGC823 cells. The percentage of G2/M phage was increased from 14.8 ± 0.6% to 25.0 ± 2.1% (P < 0.05). However, periostin restoration mainly caused S phase cell cycle arrest in SGC7901 cells compared with control (Fig. 2D). The percentage of S phage was increased from 35.5 ± 0.8% to 43.7 ± 2.3% (P < 0.05). Next, the impact of periostin restoration on apoptosis in these cells was also been assessed. As shown in Fig. 2E, periostin transfection caused an increase of the amount of apoptotic cells compared to empty vector transfection (12.1 ± 0.4% vs. 19.8 ± 1.3% in BGC823 cells, P < 0.01; 10.3 ± 0.4% vs. 15.5 ± 1.6% in SGC7901 cells, P < 0.05). These findings further support the growth inhibitory effect of epithelial cell-derived periostin in gastric cancer.

Periostin inhibits gastric cancer cell migration and invasion

Periglandular periostin expression was significantly down-regulated in metastatic lymph nodes as compared with their primary cancer tissues, suggesting that periostin may affect the metastatic ability of gastric cancer cells. As expected, periostin restoration strongly inhibited cell migration. There was a dramatically lower number of migrated cells in cell clones stably expressing periostin than control clones (P < 0.05) (Fig. 3A, upper panel). Moreover, the invasion assay also demonstrated that periostin significantly inhibited the invasive potential of gastric cancer cells. The number of cells that passed through Matrigel-coated membrane into the lower chamber was significantly lower in periostin-overexpressing cell clones than in control clones (Fig. 3A, lower panel). To determine whether the effect of periostin restoration on cell metastasis was associated with matrix metalloproteinases (MMPs), qRT-PCR was used to evaluate the expression of MMP-9 and -14 genes, 2 representative MMPs involved in cancer cell invasion.17 As shown in Fig. 3B, periostin restoration significantly inhibited the expression of these 2 genes in these cells, suggesting that the decrease in the metastasis-associated phenotypes may be link to the inhibition of MMP-9 and -14.

Figure 3.

Figure 3.

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Inhibition of gastric cancer cell migration and invasion by periostin. (A) Periostin-overexpressing and control clones were starved overnight and then seeded in the Transwell chambers without Matrigel for migration assay, and coated with Matrigel for invasion assay, respectively. After a 24 h or 48 h-culture, non-migrating or non-invading cells in the upper chamber were removed and migrating or invading cells were stained and calculated in 5 microscopic fields per sample. Shown are representative images of migrating or invading cells (left panels). The bar graphs (right panels), corresponding to left panels, show means ± SE of the numbers of migrating or invading cells from 3 independent assays. *, P < 0.05. (B) qRT-PCR assay was used to investigate the effect of periostin restoration on the expression of metastasis-related genes MMP-9 and -14 in gastric cancer cells. Expression levels of these genes were normalized with 18s rRNA level. Data were presented as mean ± SE. **, P < 0.01.

Downregulation of periostin promotes gastric cancer cell growth and invasiveness

To further confirm tumor-suppressor function of periostin in gastric cancer, shRNA approach was employed to down-regulate periostin in SGC7901 cells. After infection with lentivirus expressing specific periostin shRNA followed by selection, one SGC7901 cell clone (F10) was obtained in which periostin expression was stably knock-downed. Specifically, as shown in Fig. 4A, periostin shRNA dramatically inhibited periostin expression as compared with control clone (SC) stably expressing non-specific shRNA containing scrambled nucleotides. As shown in Fig. 4B and C, periostin shRNA strongly promoted cell proliferation and transformation as compared with control shRNA. Moreover, our data showed that specific down-regulation of periostin enhanced cell migratory ability (Fig. 4D), and increased the expression of MMP-9 and -14 (Fig. 4E). These observations further confirm tumor-suppressor effect of epithelial cell-derived periostin in gastric cancer.

Figure 4.

Figure 4.

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Enhanced gastric cancer cell growth and invasiveness by down-regulation of periostin. (A) Cell clone (SGC7901/F10) stably expressing shRNA targeting periostin was isolated, and downregulation of periostin was verified by western blot and qRT-PCR assays. 18s rRNA was used as a normalized control. GAPDH was used as loading control for protein gel blot assay. **, P < 0.01. (B) The stable cell clone (F10) exhibited a significant acceleration in cell proliferation. *, P < 0.05; **, P < 0.01. (C) Down-regulation of periostin significantly inhibited colony formation in gastric cancer cells. Representative images of colony formation in F10 and control (SC) clones are shown in the left panel. Quantitative analysis of colony numbers is shown in the right panel. Data were presented as mean ± SE. *, P < 0.05. (D) Downregulation of periostin dramatically inhibited cancer cell migration. Shown are the representative images of the migration of F10 and SC clones in scratch wound-healing assay. The bar graphs show means ± SE. of gap width of wounds at the indicated times from 3 independent assays. *, P < 0.05. (E) qRT-PCR assay was performed to evaluate the expression of MMP-9 and -14 in F10 and SC clones. 18s rRNA was used as a normalized control. Data were presented as mean ± SE. *, P < 0.05; **, P < 0.01.

Contribution of p53 and E-cadherin to tumor-suppressor activities of periostin

Given that p53 signaling plays a key role in inducing cell-cycle arrest, apoptosis and senescence in tumorigenesis,18 the effect of periostin restoration on the expression of p53 and its downstream targets was investigated in gastric cancer cells. As shown in Fig. 5A, periostin restoration enhanced the accumulation of p53 protein in these cells. Moreover, Smac, an apoptogenic factor and participates in p53-mediated mitochondrial apoptosis, was strikingly upregulated in BGC823 cell clones stably expressing periostin. Similarly, 2 other p53 target genes Gadd45a and p21 were significantly up-regulated in cell clones stably expressing periostin (Fig. 5B).

Figure 5.

Figure 5.

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Periostin stabilizes p53 and E-cadherin proteins by down-regulating Mdm2 expression. (A) Periostin-overexpressing and control clones were lysed. Supernatants were then collected and subjected to western blot analysis. The antibodies against p53 and its downstream target Smac were used to determine the effect of periostin re-expression on the p53 signaling. GAPDH was used as loading control. (B) qRT-PCR assay was used to test the effect of periostin restoration on the expression of 2 p53 targets Gadd45a and p21 in these clones. 18s rRNA was used as an internal control. Data were presented as mean ± SE. *, P < 0.05; **, P < 0.01. (C) Western blot analysis was used to test the effect of periostin restoration on E-cadherin (E-cad) expression in these clones. GAPDH was used as loading control. (D) Periostin re-expression in BGC823 cells enhanced cell-cell adhesion and cells exhibit an epithelial morphology. (E) qRT-PCR assay was performed to investigate the effect of periostin restoration on mRNA expression of E-cadherin (E-cad) and its transcription suppressors Twist, Snail-1 and -2 in gastric cancer cells. 18s rRNA was used as an internal control. Data were presented as mean ± SE. *,P < 0.05; **, P < 0.01. (F) Cell clones stably expressing periostin C10 and E7 and control clone were treated with 50 μM proteasome inhibitor MG132 for 4 h and then lysed in ubiquitin-preserving buffer. Western blotting assay was performed to detect the amount of ligated ubiquitin. p53 and E-cadherin (E-cad) proteins were used as loading controls. Ub-p53, ubiquitinated p53; Ub-E-cadherin, ubiquitinated E-cadherin. (G) Anti-Mdm2 antibody was used to determine the effect of periostin restoration on Mdm2 expression in gastric cancer cells. GAPDH was used as loading control.

To clarify the molecular mechanism of periostin contributing to cell migration and invasion, the effect of periostin restoration on E-cadherin expression was tested, whose loss function ostensibly promotes metastatic dissemination by inducing wide-ranging transcriptional and functional alterations.19 As shown in Fig. 5C, periostin restoration substantially promoted E-cadherin expression in these cell lines. This finding was supported by the observation that periostin restoration enhanced cell-cell adhesion and the individual cells displayed an epithelial morphology (Fig. 5D). Next, the effect of periostin restoration on mRNA expression of E-cadherin and its transcription suppressors Twist, Snail-1 and -2 was investigated in these cell clones. Surprisingly, mRNA expression of E-cadherin was significantly reduced in cell clones stably expressing periostin as compared with that in control clones (Fig. 5E). Accordingly, its transcription suppressors, particularly Twist was dramatically upregulated in periostin-overexpressing cell clones compared with control clones (Fig. 5E). There was an opposite relationship between protein and mRNA expression, indicating that periostin regulates E-cadherin expression probably by a post-transcriptional mechanism rather than transcriptional mechanism. It has been well documented that the stability of E-cadherin and p53 proteins can be regulated by ubiquitin-proteasome system.18,20 As expected, periostin restoration remarkably decreased ubiquitination of p53 and E-cadherin proteins in BGC823-C10 and -E7 cell clones (Fig. 5F). Considering the important role of Mdm2 played in the process of ubiquitination and subsequent proteasomal degradation of p53 and E-cadherin proteins, the effect of periostin restoration on Mdm2 expression in gastric cancer cells was evaluated. As shown in Fig. 5G, Mdm2 expression was strongly reduced in cell clones stably expressing periostin compared with control clones. These data suggest that epithelial cell-derived periostin enhanced the activity and stability of p53 and E-cadherin through inhibiting Mdm2 mediated ubiquitin-proteasome pathway.

Periostin stabilizes p53 and E-cadherin proteins by modulating the Rb/E2F1/p14ARF/Mdm2 signaling pathway

Next, the mechanism underlying downregulation of Mdm2 mediated by periostin was clarified. It is clear that Mdm2 can be regulated by several major signaling pathways, particularly the PI3K/Akt pathway.21 Thus, the effect of periostin restoration on the activity of PI3K/Akt pathway was investigated. As shown in Fig. 6A, restoring periostin expression did not affect phosphorylation of Akt in these cells. However, surprisingly, the expression of phosphorylated Rb (p-Rb) was significantly elevated in cell clones stably expressing periostin as compared with control clones (Fig. 6A). In line with this finding, our data showed a positive relationship between protein expression of periostin and p-Rb in primary gastric cancer tissues (Fig. 6B). Similar to periostin expression pattern, the expression of p-Rb protein was also dramatically reduced in metastatic lymph nodes as compared with their primary cancer tissues (Fig. S4). Accumulated evidence has demonstrated that E2F1 will be released and allowed to activate its downstream target genes when Rb is phosphorylated.18,22 Thus, the effect of periostin restoration on the expression of 2 key E2F1 target genes p14ARF and p73 in these cell lines was tested. The former can not only accelerate Mdm2 degradation,23,24 but also accumulate p53 protein by physical interaction of p53 with Mdm2.25,26 The latter can induce apoptosis by a p53-independent pathway.18 As expected, the expression of p14ARF and p73 was significantly up-regulated in periostin-overexpressing cell clones compared with control clones (Fig. 6C). These findings suggest that epithelial cell-derived periostin stabilizes p53 and E-cadherin proteins, contributing to inhibition of gastric cancer cell growth and invasiveness probably by modulating the Rb/E2F1/p14ARF/Mdm2 pathway.

Figure 6.

Figure 6.

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Epithelial cell-derived periostin acts as a tumor suppressor by stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14ARF/Mdm2 signaling pathway. (A) Cell clones were lysed and lysates were subjected to protein gel blot assays. The antibodies against phospho-AktSer473 (p-Akt), phospho-RbSer811 (p-Rb) and Rb were used to determine the effect of periostin restoration on the activity of the PI3K/Akt pathway and Rb. GAPDH was used as a loading control. (B) IHC assay was used to investigate protein expression of periostin and p-Rb in a panel of primary gastric cancer tissues. Shown are representative images of IHC staining from 2 tumor samples. Scale bars, 200 μm. (C) qRT-PCR assay was used to evaluate the expression of E2F1 target genes p14ARF and p73 in periostin-overexpressing and control clones. 18s rRNA was used as a normalized control. Data were presented as mean ± SE. *, P < 0.05. (D) Western blotting assay was performed to test the effect of Rb deletion on the expression of p-Rb, Rb, Mdm2, p53 and E-cadherin (E-cad) proteins. GAPDH was used loading control. (E) qRT-PCR assay was used to test the effect of Rb deletion in BGC823 cells on the expression of p53 target genes p21 and Gadd45a, and E2F1 target genes p73 and p14ARF, respectively. 18s rRNA was used as a normalized control. Data were presented as mean ± SE. *, P < 0.05. (F) Anti-proliferative effect of periostin was inhibited when Rb was knock-downed in gastric cancer cells. Data were presented as mean ± SE. *, P < 0.05. (G) Schematic model of molecular mechanisms underlying tumor-suppressive role of epithelial cell-derived periostin in gastric cancer. Periostin restoration in gastric cancer cells promotes Rb phosphorylation and sequentially activates the transcription of E2F1 target genes p73 and p14ARF. The former induces cell apoptosis by a p53-independent pathway. The latter inhibits the expression and activity of Mdm2, leading to the stabilization of p53 and E-cadherin proteins, ultimately contributing to induction of cell cycle arrest and p53-dependent apoptosis, as well as inhibition of cancer cell invasiveness. Moreover, periostin restoration can up-regulate SENP8 expression, promoting transactivation activity of E2F1 at certain oncosuppressor genes by E2F1 deNEDDylation, such as p73 and p14ARF, further contributing to inhibition of cancer cell growth and invasiveness.

To further determine that periostin plays a tumor-suppressor role by regulating the Rb/E2F1/p14ARF/Mdm2 pathway in gastric cancer, specific siRNA was used to inhibit Rb or p73 expression in BGC823-cell clones stably expressing periostin and control clone. As shown in Fig. 6D and Fig. S5, specific Rb siRNA strongly inhibited the expression of Rb and p-Rb proteins compared with control siRNA. Expectedly, Mdm2 expression was restored when Rb was downregulated in periostin-overexpressing cell clones. Accordingly, Rb deletion attenuated the accumulation of p53 and E-cadherin proteins in cell clones stably expressing periostin (Fig. 6D). As shown in Fig. 6E, the effect of periostin on the expression of p53 target genes p21 and Gadd45a, and E2F1 target genes p73 and p14ARF was abolished when Rb was deleted in these clones. Most importantly, anti-proliferative effect of periostin was attenuated upon Rb or p73 deletion in these clones (Fig. 6F & Fig. S6). These data further support that epithelial cell-derived periostin functions as a tumor suppressor in gastric cancer by modulating the Rb/E2F1/p14ARF signaling pathway.

It has been reported that NEDDylation as a molecular modification on E2F1 can inhibit its proapoptotic transcriptional activity.27 Sentrin-specific protease 8 (SENP8), a cysteine protease, can cause deNEDDylation of E2F1 and promote its transactivation activity at the proapoptosis-related genes, such as p73 gene, but not at the cell cycle progression-related genes, such as E2F2 gene.27 To be consistent with this, our data showed that SENP8 was significantly upregulated in periostin-overexpressing cell clones compared with control clones (Fig. S7), implicating a possible mechanism underlying periostin-induced apoptosis in gastric cancer.

Discussion

In this study, we found that periglandular periostin was frequently down-regulated in primary gastric cancer tissues compared with adjacent non-tumor tissues in vivo, as supported by a previous study that all normal gastric tissues showed strong periostin immunoreactivity, however, only 58% of gastric cancer tissues showed positive staining.10 Moreover, mRNA expression of periostin was found in the cytoplasm of gastric cancer cells using in situ mRNA hybridization, suggesting that periostin can be produced by gastric epithelial cells.10 Most recently, a study demonstrated that periglandular periostin expression was downregulated in gastric cancer tissues compared with normal gastric tissues.16 Similar to the findings from the present study, down-regulation of periglandular periostin was strongly correlated with higher tumor stage.16 However, stromal periostin expression was significantly up-regulated in gastric cancer tissues compared with normal gastric tissues and periostin produced by cancer-associated fibroblasts significantly promoted cancer cell growth.16 These findings suggest that the exact roles of periostin in tumorigenesis depend on what types of cells it derived from.

Given that periglandular periostin expression was dramatically decreased in gastric cancer tissues compared with normal gastric tissues and negatively associated with tumor stage, we speculate that epithelial cell-derived periostin may be a potential tumor suppressor in gastric cancer. As expected, periostin restoration in gastric cancer cells showed significant growth-inhibitory effect by inhibition of cell proliferation and colony formation as well as induction of cell cycle arrest and apoptosis. Strikingly, the tumors with reduced periostin expression exhibited a high metastatic propensity, as supported by our findings that periostin restoration significantly inhibited gastric cancer cell migration and invasion. These findings suggest that epithelial cell-derived periostin may be a putative tumor suppressor gene in gastric cancer.

The exploration of the mechanism behind periostin contributing to inhibition of cell growth and invasiveness revealed that periostin restoration increased the accumulation of p53 and E-cadherin proteins at the posttranslational level. This finding was supported by a recent study that inducible knock-down of periostin in esophageal squamous cell carcinoma xenograft tumors displayed decreased p53 expression.28 E-cadherin is crucial in preventing the epithelial cells undergoing epithelial-to-mesenchymal transitions, which denotes a cell reprogramming course during which epithelial cells acquire fibroblast-like properties and show reduced intercellular adhesion and enhanced migratory potential of tumor cells.29

The PI3K/Akt pathway plays a fundamental role in the development of gastric cancer,30 and accumulating evidences have indicated that periostin can promote cancer cell growth and inhibit apoptosis by activating the PI3K/Akt pathway in human cancers, such as colon cancer, cholangiocarcinoma, prostate cancer, and non-small-cell lung cancer (NSCLC).8,31–33 Conversely, periostin plays a tumor-suppressor role in bladder cancer by repressing its activity.32 Moreover, periostin has also been revealed to exhibit a biphasic effect on Akt activity in pancreatic cancer cells with an increase of Akt activity at the high concentration but a decrease at the low concentration.34 It has long been known that active Akt binds to and phosphorylates Mdm2 to enhance protein stability, leading to enhanced p53 or E-cadherin degradation through ubiquitin-proteasome system.18,20 Unexpectedly, the activity of Akt kinase was not influenced by periostin restoration in gastric cancer cells in this study. However, surprisingly, our data revealed that periostin restoration significantly elevated the expression of phosphorylated Rb protein in these cells, implicating that this molecular event may play a vital role in tumor-suppressor activities of periostin.

The Rb is now considered as a transcriptional co-factor that can bind to and either antagonize or potentiate the function of numerous transcription factors, among which E2Fs appears to be predominance.22,35 When Rb is phosphorylated, it loses the ability to bind E2Fs and triggers the transcription of E2Fs targeted genes.22,35 As the first discovered tumor suppressor, oncosuppressor role of Rb is originally thought to be largely due to its capacity to arrest cells in G1 phase by inhibiting the activity of E2Fs transcription.22 However, under certain conditions, Rb presents anti-apoptotic functions that seems paradoxical to its role as a tumor suppressor. This probably occurs in large part due to its dual capability to negatively regulate both E2Fs-induced cell cycle entry and E2F1-induced apoptosis.36 It is clear that E2F1 has a key role in both cell proliferation and apoptosis.36,37 Under proliferative conditions, E2F1 induces the expression of cell cycle progression-related genes, such as E2F2 gene, whereas under proapoptotic conditions E2F1 induces the expression of proapoptosis-related genes, such as p73 gene.38,39 Moreover, a recent study has demonstrated that SENP8, a NEDD8-specific cysteine protease, can deneddylate E2F1 and increase the expression of its target gene p73 but not E2F2.27 These findings were supported by our data that periostin restoration markedly upregulated the expression of p73 and SENP8 in gastric cancer cells. In this study, we found that periostin restoration in gastric cancer cells significantly up-regulated the expression of another E2F1 target gene p14ARF. Accumulated evidences have demonstrated that p14ARF can promote Mdm2 degradation and physically block interaction of Mdm2 with p53, leading to p53 stabilization.23–26 Taken together, periostin induces cell apoptosis by both p53-dependent (Rb/E2F1/p14ARF/Mdm2/p53) and -indenpdent (Rb/E2F1/p73) pathways. Additionally, periostin stabilizes E-cadherin protein through accelerating Mdm2 degradation via the Rb/E2F1/p14ARF signaling, leading to inhibition of tumor cell invasiveness.

In summary, we have demonstrated that epithelial cell-derived periostin is frequently down-regulated in primary gastric cancer tissues and functions as a potential tumor suppressor. Our data are consistent with a model (Fig. 6G) in which epithelial cell-derived periostin contributes to inhibition of cell growth and invasiveness through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14ARF/Mdm2 signaling.

Materials and Methods

Clinical samples

A total of 25 primary gastric cancer tissues, as well as matched normal gastric mucosa (n = 8) and metastatic lymph nodes (n = 10) were randomly obtained from the First Affiliated Hospital of Xi’an Jiaotong University School of Medicine. None of these patients received any therapeutic intervention before surgery. All tissues were histologically examined by 2 senior pathologists at Department of Pathology of the Hospital. Informed consent was obtained from each patient before the surgery, and the study protocol was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University School of Medicine.

Immunohistochemistry (IHC)

Paraffin-embedded sections (5 μm) were deparaffinized and rehydrated in a graded series of ethanol, and washed in distilled water. After antigen retrieval and blocking, the sections were incubated with anti-periostin (Abcam) and anti-phospho-Rb (p-Rb) (Epitomics, Inc..) antibodies overnight at 4°C. Immunodetection was performed with the Streptavidin-Peroxidase system (ZSGB-bio, Beijing China) according the manufacture's protocol. 3,3′-diaminobenzidine was used as chromogen. Slides were then counterstained with hematoxylin. To insure the comparability of immunohistochemical staining, a common reference standard was included to serve as an internal or intra-assay control in each batch. The level of antigen was scored in a double-blinded manner (i.e., without knowing the tumor stage of the case), and −, ±, +, ++ reprints negative, weak, moderate, and strong staining, respectively.

Cell culture, plasmid construction and transfection

Human gastric cancer cell lines BGC823 and SGC7901, as well as human embryonic kidney epithelial cell line 293T were cultured in RPMI 1640 and DMEM media supplemented with 10% fetal bovine serum, respectively. Hypoxia culture was simulated by treating cells with 150 μM cobalt dichloride as described previously.40

To construct a periostin expression plasmid, the full open reading frame of human periostin variant 2 cDNA (NM_001135934.1) was cloned into pcDNA3.1 (−) mammalian expression vector with a Myc-His tag. BGC823 and SGC7901 cells were transfected with periostin expression plasmid (pcDNA3.1-POSTN) or empty vector (pcDNA3.1) using X-tremeGENE HP DNA Transfection Reagent (Roche Applied Science, Germany) according to the instructions of the manufacture. After 48 h of transfection, the transfectants were selected in a medium containing 0.2 mg/ml of G418 for 14 d Stable cell clones were isolated and confirmed by Western blotting analysis of periostin protein. The lentiviral vector encoding hairpin RNA sequences were used to specific knock-down periostin. The hairpin sequences used in this study were presented in Table S1. The lentiviral particles and stably transfected cell lines were generated as previously described.41 The siRNA targeting Rb sequence (siRb) was 5′-GCU CAA AGA ACC AUA UAA ATT-3′, and control siRNA sequence (siNC) was 5′-UUC UCC GAA CGU GUC ACG UTT-3′.

Cell viability and colony formation assays

Cell viability was determined by the MTT assay. Briefly, cell clones stably transfected with pcDNA3.1-POSTN or empty vector (200/well) were seeded and cultured in 96-well plates for 1, 3, 5, 7, and 9 d At the indicated times, 20 μl of 0.5 mg/ml MTT (Sigma, Saint Louis, MO) was added into the medium and incubated for 4 h, followed by adding 150 μl of DMSO for additional 15 min. The plates were then read on a microplate reader using a test wavelength of 570 nm and a reference wavelength of 670 nm. Three triplicates were done to determine each data point.

For colony formation assay to examine cell growth in monolayer culture, cells (200/well) transfected with plasmid were seeded in the medium containing 0.1 mg/ml G418 in 6-well plates. After 14 d of culture, surviving colonies (≥50 cells per colony) were fixed with methanol and stained with 0.5% crystal violet, and the colonies were then counted. The experiments were performed in triplicate.

Cell cycle and apoptosis assays

For cell cycle assay, cells transiently transfected with different plasmids were harvested at 48 h or 72 h when the confluence reached ∼90%, and washed twice with PBS. Cells were then fixed in ice-cold 70% ethanol for at least 30 min, and stained with propidium iodide solution (50 μg/mL propidium iodide, 50 μg/mL RNase A, 0.1% Triton-X, 0.1 mM EDTA). Cell cycle distributions were assessed based on DNA contents by FACS using a Flow Cytometer (BD Biosciences, NJ). For apoptosis analysis, the indicated cells were harvested, washed with PBS, suspended in binding buffer, and sequentially stained with Annexin V-FITC Detection Kit (Roche Applied Science, Penzberg, Germany) by flow cytometer according to the manufacturer's protocol. Each experiment was performed in triplicate.

Cell migration and invasion assays

Cell migration and invasion assays were assessed by transwell chambers (8.0 μm pore size; Millipore, MA) pre-coated with rat tail tendon collagen type 1 (0.5 mg/mL) on the lower surface. For cell invasion assay, chambers were coated with Matrigel (4 × dilution; 15 μl/well; BD Bioscience, NJ). Cell clones stably transfected with pcDNA3.1-POSTN or empty vector were starved overnight and then seeded in the upper chamber at a density of 1×106 cells/ml in 200 μl of medium containing 0.5% FBS. Medium with 10% FBS (1 ml) was added to the lower chamber. After a 24- or 48-h incubation (depending on the cell type), non-migrating or non-invading cells in the upper chamber were removed using a cotton swab, and migrating or invading cells were then fixed in 100% methanol and stained with crystal violet solution (0.5% crystal violet in 2% ethanol). Photographs were taken randomly for 5 fields of each membrane. The number of migrating/invading cells was expressed as the average number of cells per microscopic field over 5 fields.

Scratch wound-healing assay was also performed to assess the migratory ability. Briefly, cells were cultured in standard medium until the confluence reached ∼90% on the day of transfection. After 48 h of transfection, cells were starved with medium containing 0.5% serum overnight, and the wounds were then scratched. Cells were subsequently cultured in medium containing 1% serum and the widths of wound were measured and photographed. Each experiment was performed in triplicate wells.

Western blot analysis

Cells were lysed on ice in prechilled RIPA lysis buffer containing protease inhibitors (Roche Diagnostics, Mannheim, Germany). Cells for ubiquitin assays were lysed in ubiquitin-preserving buffer (20 × stock buffer contains 2.5 ml 2-mercaptoethanol, 10.43 ml water, 0.5 ml 50% glycerol, 10 ml 10% SDS, 1.56 ml 1M Tris pH 6.8 and 0.0025 g bromophenol blue) as previously described.42 Fifty micrograms of protein from each sample were separated on SDS-PAGE gels and transferred onto PVDF membranes. The membranes were then incubated with primary antibodies. Anti-periostin and anti-Smac were purchased from Abcam. Anti-phospho-AktSer473 (p-Akt) were purchased from Bioworld Technology, co, Ltd. Anti-p53 and anti-Mdm2 were purchased from Santa Cruz Biotechnology, Inc.. Anti-E-cadherin, anti-phospho-RbSer811 (p-Rb) and anti-Rb were purchased from Epitomics, Inc.. Anti-GAPDH were purchased from Abgent, Inc.. Anti-p21 were purchased from Cell Signaling Technology, Inc.. This was followed by incubation with species-specific HRP-conjugated secondary antibodies from ZSGB-BIO, and immunoblotting signals were visualized using the Western Bright ECL detection system (Advansta, CA).

Enzyme linked immunosorbent assay (ELISA)

The secreted recombinant periostin in the supernatant was detected by ELISA. Briefly, equal BGC823/V, /C10 and /E7 cell clones were cultured and the supernatant was collected after 48 h. A 96-well plate was coated with rabbit anti-human periostin antibody (2 μg/ml). The collected supernatant was then added and incubated at 4°C overnight. Subsequently, the plate was incubated with mouse anti-His antibody at 37°C for 1 h, followed by HRP-conjugated goat anti-mouse antibody. TAB was used as chromogen, and the plate was read on a microplate reader using a test wavelength of 570 nm. Three triplicates were done to determine each sample.

RNA extraction and quantitative RT-PCR (qRT-PCR)

Total RNA was isolated from cell lines using RNAiso Plus (Takara Inc.., Dalian, P.R. China) according to the instructions of the manufacturer. Total RNA was converted to cDNA using PrimeScript RT reagent Kit (Takara Inc.., Dalian, P.R. China). Quantitative RT-PCR (qRT-PCR) was performed on a CFX96 Thermal Cycler DiceTM real-time PCR system (Bio-Rad Laboratories, Inc.., CA) using SYBR Premix Ex TaqTM (Takara Inc.., Dalian, P.R. China) according to the instructions of manufacturer. The mRNA expression of each gene was normalized to 18S rRNA cDNA. Each sample was run in triplicate. The primer sequences were presented in Table S2.

Statistical analysis

Data were compared using Student's t-test (SPSS statistical package 16.0, Chicago, IL). A P < 0.05 was considered to be statistically significant. All values were expressed as the mean ± SE. Unless indicated, data shown in the figures are representatives.

Funding Statement

This work was supported by the National Natural Science Foundation of China (No. 81171969, 81272933 and 81372217), the Fundamental Research Funds for the Central Universities, and the Program for New Century Excellent Talents in University (No. NCET-10–0674).

Author's Contributions

P.H. conceived, guided and supervised the research project. P.H. and H.L. designed the experiments. H.L., R.L., J.F., Q.Y., J.S. and P.C. performed the experiments. M.J., B.S. and P.H. provided tumor samples and research materials. H.L., R.L. and P.H. analyzed the data. H.L. and P.H. wrote the manuscript. All authors contributed to and approved the final version of the manuscript.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplemental Materials

Supplemental data for this article can be accessed on the publisher's website.

Supplementary_Materials

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Supplementary Materials

Supplementary_Materials
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