SOX2, a predictor of survival in gastric cancer, inhibits cell proliferation and metastasis by regulating PTEN

Cancer Letters 358 (2015) 210–219

Contents lists available at ScienceDirect

Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

SOX2, a predictor of survival in gastric cancer, inhibits cell proliferation and metastasis by regulating PTEN Simeng Wang a,1, Jun Tie a,1,*, Rui Wang a,1, Fengrong Hu a, Liucun Gao b, Wenlan Wang c, Lifeng Wang d, Zengshan Li a, Sijun Hu a, Shanhong Tang a, Mengbin Li a, Xin Wang a, Yongzhan Nie a, Kaichun Wu a, Daiming Fan a,* a State key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, China b Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China c Department of Aerospace Hygiene and Health Service, School of Aerospace Medicine, Fourth Military Medical University, Xi’an, Shaanxi 710032, China d Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xi’an, Shaanxi 710032, China

A R T I C L E

I N F O

Article history: Received 3 October 2014 Received in revised form 8 December 2014 Accepted 19 December 2014 Keywords: SOX2 PTEN Gastric cancer Prognostic indicator Tumor metastasis

A B S T R A C T

Inconsistent results of SOX2 expression have been reported in gastric cancer (GC). Here, we demonstrated that SOX2 was progressively downregulated during GC development via immunochemistry in 755 human gastric specimens. Low SOX2 levels were associated with pathological stage and clinical outcome. Multivariate analysis indicated that SOX2 protein expression served as an independent prognostic marker for GC. Gain-and loss-of function studies showed the anti-proliferative, anti-metastatic, and proapoptotic effects of SOX2 in GC. PTEN was selected as SOX2 targets by cDNA microarray and ChIP-DSL, further identified by luciferase assays, EMSA and ChIP-PCR. PTEN upregulation in response to SOX2enforced expression suppressed GC malignancy via regulating Akt dephosphorylation. PTEN inhibition reversed SOX2-induced anticancer effects. Moreover, concordant positivity of SOX2 and PTEN proteins in nontumorous tissues but lost in matched GC specimens predicted a worse patient prognosis. Thus, SOX2 proved to be a new marker for evaluating GC outcome. © 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction Gastric cancer (GC) is the second most prevalent neoplastic disease in China; however, our understanding of the molecular circuitry that governs GC malignancy remains limited [1]. SOX2, a specific transcription factor during stomach development [2], has been identified to modulate a variety of biological processes ranging from the maintenance of gastric epithelial cell phenotypes [2] to preservation of stem cell pluripotency and embryonic development [3]. Recently, aberrant SOX2 expression has been reported to be involved in GC pathogenesis [4–6]. There are conflicting results regarding the extent to which SOX2 expression relates to gastric clinicopathological parameters. SOX2 is known as a stemness transcription factor, and some studies have reported that SOX2 is upregulated in GC and serves as an oncogene [7,8]. For instance, Matsuoka et al. [8] found that SOX2 was over-expressed in GC (SOX2-positive rate, 55%; 159/290);

* Corresponding authors. Tel.: +86 29 84771520; fax: +86 29 82539041. E-mail address: [email protected] (J. Tie); [email protected] (D. Fan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.canlet.2014.12.045 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved.

moreover, relative to SOX2-negative tumors, SOX2-positive tumors were associated with more extensive invasion, higher TNM stages, and worse prognoses. Paradoxically, other studies suggested that SOX2 was downregulated and exerted an anti-oncogene role in GC [4,5,9–12]. For example, Otsubo et al. [9] and Zhang et al. [13] found that SOX2 promoter methylation led to decreased SOX2 expression in GC and patients with SOX2-positive GCs demonstrated lower rates of lymph node metastasis, reduced tumor invasion extent, and longer survival than patients with SOX2-negative GCs. Motivated by this inconsistency, refined investigations are required for our understanding of SOX2 function in GC development and patient prognosis. Here, we discovered that SOX2 expression was progressively diminished during the initiation and progression of human GC through examining SOX2 expression in a set of 755 stomach tissues, including gastric normal mucosa, chronic superficial gastritis (CSG), intestinal metaplasia (IM), dysplasia (Dys), gastric cancer, and metastases. Notably, the present work illustrates that reduced SOX2 expression in GC compared with matched adjacent nontumorous tissues serves as a robust predictor of disease outcome. We first introduce a novel evaluation system that outperforms the existing ones for a stronger predictive power with regard to patient prognosis. The system not only stratified the correlations between

S. Wang et al./Cancer Letters 358 (2015) 210–219

gene-expression status and tumor pathological parameters but was also based on concordant analyses of tumor-matched nontumorous tissue and the tumor itself. Of significance, by enforcing SOX2 expression in GC cells, we demonstrated that SOX2 repressed proliferation, promoted apoptosis, and opposed metastasis. Specifically, we propose that SOX2 directly manipulates PTEN, resulting in p-Akt dephosphorylation, which is responsible for the antitumorigenic behavior of SOX2. Materials and methods Ethics statement All experimental procedures were approved by the Institutional Review Board of the Fourth Military Medical University. Written informed consent was obtained for all patient samples. Animal experiments were performed with the approval of the Institutional Committee for Animal Research and in conformity with national guidelines for the care and use of laboratory animals. Chromatin immunoprecipitation (ChIP)-DNA selection and ligation (DSL) promoter array A total of 5 × 107 MKN28-SOX2 cells in the logarithmic growth phase were crosslinked with 1% formaldehyde. Then, 500 μl of cell lysis buffer was added, and the cells were incubated on ice for 5 minutes. Chromatin was sonicated into 200–1000-bp fragments and centrifuged at 12,000 rpm at 4 °C for 15 minutes. The supernatant was collected, and 20-μl samples of fragmented DNA were designated as the experimental group, the control group, and the input. Subsequently, 5 μg of SOX2 rabbit mAb (5024s, Cell Signaling Technology) was added to the experimental group, and 5 μg of normal rabbit immunoglobulin G (IgG) (2727, Cell Signaling Technology) was added to the control group, followed by an overnight incubation at 4 °C. DNA–protein–antibody complexes were precipitated using protein A-coated beads (10001D, Invitrogen), eluted with elution buffer after washing, and digested overnight with proteinase K (P8102, NEB) at 65 °C. Finally, DNA fragments were purified and recovered (28006, Qiagen). Post-immunoprecipitation DNA and input were amplified. Total input DNA was labeled using Cy3, and the IP samples were labeled using Cy5. The amplified and labeled samples and ChIP-DSL H20K promoter array were hybridized overnight. After hybridization, the complex was washed at 42 °C with 2× saline–sodium citrate (SSC) solution containing 0.2% sodium dodecyl sulfate (SDS) and then at room temperature with 2× SSC solution for 5 minutes. After the slides dried, they were scanned with a LuxScan 10KA dual-channel laser scanner (CapitalBio). Microarray images were analyzed with LuxScan 3.0 image analysis

211

software (CapitalBio). The image signals were converted into digital signals, normalizing to median values. Genes with more than a twofold difference in fluorescence intensity ratio between the experimental group and the control group were regarded as candidate genes for SOX2 regulation. The ChIP-DSL H20K promoter array from human gastric cancer cells MKN28 with SOX2 overexpression (GSE45427) can be found at http://www.ncbi.nlm.nih.gov/geo/info/linking.html. Statistical analyses All data are presented as the mean ± SE and were analyzed using SPSS. The Fisher’s exact test was used for analysis of categorical data; the independent t test was used for continuous parametric data. Survival data were computed using the Kaplan– Meier method and compared between groups by the log-rank test. Tests were considered significant when the P values were less than 0.05. Detailed information is provided in Appendix S1.

Results SOX2 is downregulated during the progressive transformation of normal gastric cells into highly malignant derivatives To study the SOX2 expression profile during the developmental process of GC, we studied SOX2 expression in a set of 755 stomach tissues at typical stages throughout the gradual conversion of normal gastric cells into aggressive cells using tissue microarrays. We found that SOX2 expression gradually diminished as gastric cells evolved progressively from normal into invasive cancers through a series of premalignant states. Specifically, SOX2 levels in premalignant lesions, including intestinal metaplasia (IM) and dysplasia (Dys), were lower than those in either normal stomach mucosa or chronic superficial gastritis (CSG) but were higher than those in aggressive GC tissues (Fig. 1A). Of note, SOX2 was expressed at approximately equivalent levels in normal gastric mucosa and chronic superficial gastritis and were strongly positive in these tissues (Fig. 1B). To confirm our findings, we further examined SOX2 expression in 33 panels of adjacent nontumorous tissues, GC lesions, and metastases from 33 metastatic GC patients. Consistent with the above observations, 22 of 33 panels showed reduced SOX2 expression in GC

Fig. 1. SOX2 is downregulated during the progression from normal gastric cells to cancer cells. (A) Hematoxylin and eosin (H&E) stain of normal gastric tissues, tissues originating from CSG, CAG with IM, GC, and GC metastases. Immunohistochemical detection of SOX2 expression in consecutive tissues revealed positive staining in normal gastric tissues and CSG, slightly positive staining in CAG with IM, and negative staining in GC and distant metastases. Original magnification: 200×. (B) SOX2 expression is gradually diminished during the developmental process of human GC. 1P = 0.079 (CSG vs. normal gastric mucosa), 2P < 0.001 (CAG/IM/Dys vs. CSG), 3P < 0.001 (CAG/IM/Dys vs. normal gastric mucosa), 4P < 0.001 (GC vs. CAG/IM/Dys), and 5P = 0.046 (GC metastases vs. GC), Fisher’s exact test.

212

S. Wang et al./Cancer Letters 358 (2015) 210–219

Fig. 2. SOX2 downregulation in GC relative to matched nontumorous tissues correlates with poor prognoses. (A) H&E staining of patient-matched adjacent normal tissues, GC tissues, and metastases; 22 out of 33 metastatic GC patients exhibited reduced SOX2 in GC and metastases compared with matched nontumorous regions. (B) SOX2 expression in nontumorous tissues, primary gastric cancer sites, and metastases. SOX2 levels were downregulated from paired nontumorous tissues to GC lesions and further decreased or lost in corresponding metastases in 66.67% (22 of 33) of the cases (P < 0.001, one-way ANOVA); N: adjacent normal tissues; GC: gastric cancer; M: GC metastases (n = 33). (C) Kaplan–Meier survival curves for 92 GC patients (OD-CT-DgStm01-013 microarray) depict overall survival across group classification based on SOX2 expression inclination from tumor tissue to the paired nontumorous tissues. Decreased SOX2 expression in GC lesions relative to matched neighboring normal tissues was associated with poor prognoses (P = 0.002, log-rank test). (D) Western blots for SOX2 expression. Downregulated SOX2 was observed in 6 out of 8 surgical-resected human GC samples; T: tumor; N: normal neighboring tissues.

relative to matched nontumorous regions; SOX2 exhibited the most dramatic downregulation in paired GC metastases (Fig. 2A and B). On the basis of these results, we selected SOX2 for further exploration of its prognostic power in GC patients. Lower SOX2 expression in GC relative to matched nontumorous tissues correlates with poor patient prognosis Using immunohistochemical staining, we arrayed SOX2 in 203 pairs of stomach tumors and corresponding nontumorous tissues. The results showed that 87.68% of nontumorous tissues were SOX2 positive, while the ratio in GC regions was substantially lower at 37.93% (P < 0.001, Chisquare test). We analyzed the SOX2 profile obtained from GC tissues and found that a SOX2 profiling confined to GC alone, in general, had limited power to predict GC outcome (Fig. S1). To obtain an accurate estimate of how GC progression was associated with SOX2 expression, we co-analyzed SOX2 levels in

neighboring nontumorous tissues and identified the SOX2 signature according to the criteria of its expression inclination from paired nontumorous tissues to tumor parts, by which the patients with and without SOX2 downregulation were separately assigned to two subgroups. We found that, relative to nontumorous parts, SOX2 was downregulated in GC samples in 75.37% of the patients; in the remaining 24.63% of patients, SOX2 exhibited an either upregulated or unchanged manner. Furthermore, the Kaplan–Meier curves showed that patients harboring lower SOX2 in GC tissue compared with paired nontumorous regions were likely to exhibit shorter survival periods. The estimated median survival was 18 months, markedly shorter than its counterpart (18 months vs. 22 months; hazard ratio = 1.549; P = 0.002; Fig. 2C). Our data reflected that lower SOX2 expression in GC relative to matched nontumorous tissues was linked to larger tumor volumes, deeper tumor invasion, advanced tumor stages, and a higher incidence of lymph node metastasis (Table 1). Multivariate analysis indicated that SOX2 protein

S. Wang et al./Cancer Letters 358 (2015) 210–219

213

Table 1 Clinicopathological correlations of SOX2 downregulation in 203 gastric cancer patients. Clinicopathological features

No. of cases

%

SOX2 Down-regulation

Age (years) ≥60 <60 Gender Male Female Tumor size ≥5 cm <5 cm Invasion depth T1, 2 T3, 4 Stage I, II III, IV Lymph node metastasis Positive Negative Lauren classification Diffuse Other types

P value No down-regulation

79 124

38.92% 61.08%

60 95

19 29

1.000

132 71

65.02% 34.98%

102 53

30 18

0.730

100 103

49.26% 50.74%

83 72

17 31

0.032

42 161

20.69% 79.31%

26 129

16 32

0.023

65 138

20.02% 67.98%

42 113

23 25

0.008

153 50

75.37% 24.63%

123 32

30 18

0.022

97 106

47.78% 52.22%

77 76

20 30

0.254

expression might serve as an independent prognostic marker for GC (P = 0.01, HR = 2.07, 95% CI 1.21–3.89; Table S1). To extend our findings, we detected SOX2 expression in a panel of 8 paired surgical-resected human GC/nontumorous samples together with 8 human gastric cell lines using western blotting. Consistently, SOX2 was expressed at significantly lower levels in the majority (6/8) of GC resections compared with the paired nontumorous tissues (Fig. 2D). Among the 8 gastric cell lines, SOX2 was only detectable in KATOIII GC cells and immortal gastric cells GES-1 (Fig. S2A). Taken together, SOX2 expression is largely downregulated in GC cells and patients. Downregulated SOX2 within GC lesions compared with paired nontumorous tissues was strongly predictive of a poor prognosis. SOX2 inhibits proliferation, promotes apoptosis, and impedes metastasis in vitro and in vivo Given these inverse correlations between SOX2 levels and GC malignancy, we identified the potential anticancer roles of SOX2. We utilized a SOX2 lentiviral vector to enforce SOX2 expression in MKN28 cells. Western blotting and q-PCR assays indicated that infection with SOX2 lentiviruses resulted in enforced expression of SOX2 compared with the negative control (Fig. S2B and C). Enforced SOX2 expression inhibited proliferation in vitro and in vivo (Fig. 3A). Flow cytometric analysis for apoptotic cell number revealed an increased number of apoptotic MKN28 cells infected with SOX2 lentiviruses compared with the empty-vector group (Fig. 3B). Furthermore, tumorigenic tissues of nude mice were subjected to H&E and immunohistochemical staining for SOX2 and Ki-67. We observed fainter Ki-67 staining in tumors derived from MKN28SOX2 cells compared with the control group (11% vs. 43%; P < 0.001, Chi-square test; Fig. 3C). SOX2-induced apoptosis was also demonstrated by TUNEL staining in tumorigenic tissues of nude mice. TUNEL positivity was stronger in tumors generated from MKN28SOX2 cells (Fig. 3C). Conversely, ectopic overexpression of SOX2 did not affect the cell cycle (data not shown). Instead, SOX2 reduced invasion and motility, as demonstrated by transwell assays and tail vein injection experiments in nude mice (Fig. 3D). The antiproliferative, anti-metastatic, and proapoptotic effects of SOX2 were further validated in another gastric cancer cell, SGC7901

(Fig. S3). Hence, SOX2-imposed GC suppression is associated with disruption of GC cell growth and metastatic dissemination and induction of GC cell death. Our additional work revealed that SOX2 knockdown in KATOIII cells enhanced proliferative, tumorigenic, and metastatic potential but dampened apoptosis in vitro and in vivo (Fig. S4). The data further confirmed the tumor suppressive role of SOX2 in GC. SOX2 directly regulates the PTEN/AKT pathway in human GC The rationale for the functional contributions of SOX2 is based on its ability to target multiple effectors involved in these biological processes. To uncover the underlying mechanisms, first we sought the direct targets of SOX2 using ChIP-DSL [14,15] and cDNA microarrays. Then, 718 protein-coding gene promoters were obtained by ChIP-DSL, and 320 differentially expressed genes following SOX2 enforced expression were harvested via cDNA microarrays. Of the 11 overlapping genes, PTEN was selected as the most putative direct-target of SOX2 (Fig. 4A). Guided by bioinformatics, we predicted two SOX2 binding sites in the PTEN promoter: site 1, GTGCTAAGAC (−576 to −567), and site 2, CGATTGTGATCCGAC (−491 to −477; Fig. S5A). As an initial test for verification, we cloned the intact PTEN promoter into luciferase constructs. When introduced into MKN28-SOX2 cells, the constructs with intact PTEN promoter increased luciferase activity, suggesting a straight transcriptional modulation of SOX2 over the PTEN promoter. Additionally, reporter assays revealed that mutations of predicted binding site 1 or in combination with site 2 abrogated responsiveness to SOX2 in MKN28SOX2 cells, while no responsive repression was discerned in the case of a site 2 mutation alone, demonstrating the specificity of site 1 as the solitary SOX2 binding site in the PTEN promoter (Fig. 4B). Furthermore, EMSA results demonstrated the validity of site 1 (Fig. 4C). Finally, we applied ChIP-sequencing to confirm SOX2 binding to the PTEN promoter (Fig. 4D, Fig. S5B). We then correlated SOX2 expression with PTEN protein expression in our paired human GC/nontumorous tissue samples. PTEN, similar to SOX2, mostly exhibited a repressed expression in GC but was overwhelmingly elevated in nontumorous tissues. We found that PTEN downregulation was associated significantly with downregulated SOX2 protein (Fig. 5A). We further evaluated the PTEN expression profile in GC relative to the nontumorous portions with

214

S. Wang et al./Cancer Letters 358 (2015) 210–219

Fig. 3. SOX2 inhibits proliferation, promotes apoptosis, and impedes metastasis in vitro and in vivo. (A) SOX2 obstructs MKN28 cell proliferation in vitro, as determined using WST-1 assays (P = 0.009, two-way ANOVA, n = 3) (Left). MKN28-SOX2 and control cells were subcutaneously injected into nude mice. The median weight of the MKN28SOX2-derived tumors was notably less than that of the control group (P = 0.009, Student’s t test; n = 7) (Middle). Tumor images clearly reflect the anti-tumorigenic effects of SOX2 in vivo (Right). (B) Apoptosis was examined by flow cytometry via Annexin and PI staining. The bar chart displays a substantial rise in the apoptotic rate of MKN28 cells with SOX2 overexpression compared with the control group (P = 0.031, Student’s t test, n = 3). (C) SOX2 dampens proliferation and apoptosis in vivo. The panel sets show that the number of TUNEL-positive cells in the SOX2-expressing group was higher than that in the control group. Ki-67 staining in tumors generated from MKN28SOX2 cells was weaker than in the control group. (D) Re-expression of SOX2 inhibited cell migration and impeded metastasis in MKN28 cells. The bars reveal sharp contrasts in the number of invasive cells between MKN28-SOX2 and control cells (P < 0.001, Student’s t test, n = 3). The incidence of metastasis in mice that received intravenous tail injections with MKN28-SOX2 cells was notably lower than those in control groups.

GC outcome in 203 cases. These PTEN expression patterns conferred poor prognoses (Fig. S6A). Additionally, in pursuit of clinical advantages for GC prognostication, we categorized GC patients into 4 groups according to the combined expression level of SOX2 and

PTEN. Kaplan–Meier analyses showed significantly distinct survival patterns among the four subgroups, among which the patients with coordinately downregulated SOX2 and PTEN showed the lowest median survival (Fig. S6B). Thus, these results highlighted the value

S. Wang et al./Cancer Letters 358 (2015) 210–219

215

Fig. 4. PTEN is the direct target of SOX2 in human GC. (A) A Venn diagram of overlapping genes from cDNA microarrays and ChIP-DSL for the tentative targets of SOX2. Eight genes were found to be upregulated (red), and 3 were downregulated (green). (B) Verification of PTEN as a SOX2 target via luciferase reporter assays. Constructs with an intact PTEN promoter or SOX2-binding site 1 resulted in enhanced luciferase activities in SOX2-expressing MKN28 cells, while those carrying either mutant site 1 or were mutant in two binding sites resulted in strongly repressed luciferase activities. In contrast with MKN28-SOX2 cells, MKN28 cells failed to show any luciferase activity after transfection with any of the above constructs (*P = 0.005, **P = 0.007, ***P = 0.007, ****P = 0.009, n = 3). (C) EMSA for identification of SOX2-binding sites in the PTEN promoter. Nuclear protein extracts are prepared from MKN28-SOX2 cells and were incubated with a biotin-labeled DNA probe, followed by chemiluminescent EMSA. The unlabeled probe served as a cold competitor, and its mutant served as a negative control. The specificity of the SOX2 binding motif was confirmed by the ability of a SOX2specific antibody to block the DNA complex shift. An arrow indicates the SOX2 DNA-binding complex. Probe 1 was designed exclusively for the SOX2-binding site 1 and probe 2 for the SOX2-binding site 2. (D) ChIP-PCR confirmed that SOX2 binds to the PTEN promoter in vivo. Amplification of the PTEN promoter sequence from ChIP DNA validated the binding of SOX2 to the PTEN promoter. Sonicated input DNA and H2O lanes served as positive and negative controls, respectively. The product sequences were terminally verified to represent PTEN promoter following gene-sequencing analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of an appropriate prognosis profile in GC, which is represented by a combined signature of SOX2 with PTEN expression. Moreover, PTEN is known to mediate Akt activation via dephosphorylation. We further confirmed the relationship between SOX2 and PTEN/AKT in human GC cell lines. SOX2-overexpressing MKN28 cells exhibited remarkable PTEN upregulation and p-Akt dephosphorylation, yet without a clear attenuation of total Akt protein expression. Although siRNA-mediated PTEN downregulation substantially enhanced p-Akt, neither SOX2 nor total Akt proteins showed altered expression in GC cells (Fig. 5B and Fig. S2C and D). Similar results were observed in SGC7901 cells (data not shown). These observations supported the hypothesis that PTEN is transcriptionally modulated by SOX2. Ultimately, to verify whether SOX2 mediated GC cell growth and metastasis inhibition through PTEN, we deployed a PTEN loss-offunction strategy in SOX2-expressing MKN28 cells and demonstrated that PTEN inhibition increased proliferative, metastatic, and antiapoptotic abilities in vitro and in vitro (Figs. 5C, D and 6). Collectively, our findings indicated that PTEN upregulation and subsequent p-Akt dephosphorylation mediated SOX2 anticancer effects in GC.

Discussion Early reports varied with regard to the relationship between SOX2 expression and GC patient outcome. The reasons for these differences may lie in the different approaches, tissue sources, and small sample sizes used. In most cases, the progressive transformation of GC starts from normal gastric mucosa, and basically experiences sequential pathological changes of pre-cancerous stage to early stage GC and advanced GC, finally ending up with diffused GC metastases. In the current study, we used the largest sample size of more than 700 cases and multi-center specimens with various pathological stages involved in GC development, which is the most comprehensive and systematic study to date for SOX2 expression in GC. The results showed an essential expression pattern of SOX2 in the GC evolutionary process. This is fresh evidence that incipient premalignant gastric tissues often experience a gradual loss of SOX2 before the acquisition of tumorigenic characteristics, which is supported by previous studies [4,5,9–12] and the data from GENEVESTIGATOR (https://www.genevestigator.com/gv/). However, some scholars who observed SOX2 over-expression in GC cells [7,8]

216

S. Wang et al./Cancer Letters 358 (2015) 210–219

Fig. 5. PTEN inhibition alters GC cell proliferation and apoptosis via the PTEN/AKT pathway. (A) Expression signature of SOX2 and PTEN. SOX2 expression in line with PTEN was globally downregulated in GC tissue but upregulated in related nontumorous tissues. PTEN downregulation was closely associated with downregulated SOX2 protein in human GC (P < 0.001, Chi-square test). Down regulation: Relative to nontumorous parts, the target gene was downregulated in GC samples; No down regulation: Relative to nontumorous parts, the target gene was upregulated or unchanged in GC samples. (B) Immunoblots for SOX2, PTEN, p-Akt, and total Akt protein in MKN28-SOX2 and MKN28-SOX2-si-PTEN cells. Tubulin was used as a loading control. SOX2 overexpression in MKN28 cells led to PTEN expression and Akt dephosphorylation. PTEN knockdown affected only Akt dephosphorylation, leaving the total Akt protein stable. (C) Blocking PTEN reversed the anti-proliferation effects of SOX2 in SOX2-expressing MKN28 cells in vitro via WST-1 assays (P = 0.015, two-way ANOVA, n = 3; Left). Tumors derived from PTEN-knockdown MKN28-SOX2 cells showed increased weights (P = 0.017, Student’s t test, n = 7; Middle). Tumor images obtained from nude mice injected subcutaneously with MKN28-SOX2 cells with or without PTEN suppression showed that PTEN deletion initially augments subcutaneous tumor growth (Right). (D) Apoptotic cells in response to PTEN suppression were detected via flow cytometry using Annexin and PI staining. PTEN inhibition reversed SOX2-dependent apoptosis in vitro (P = 0.031, Student’s t test, n = 3).

or GC stem cells [16] believed that SOX2, as a stem cell marker, should have a tumor-promoting effect. In fact, as a pluripotency transcription factor, it is possible to play an anti-tumor effect through regulating tumor suppressor genes in specific tissues and

conditions. This phenomenon is also observed in another stem transcription factor, KLF4, which is decreased in many cancers, such as diffuse GC [17], breast cancer [18–20], colon cancer [21–23], lung cancer [24], ovarian cancer [25], prostate cancer [26,27],

S. Wang et al./Cancer Letters 358 (2015) 210–219

217

Fig. 6. PTEN attenuates proliferation, apoptosis, and metastasis in vivo. (A) PTEN expression was observed in tumor tissues originating from the control cells, while reduced p-Akt expression was found in the tissues. Intensely positive staining of p-Akt was confined to PTEN-negative tumors. The intensity of Ki-67 expression was specifically enhanced in tumorigenic tissues from the PTEN-defect tumor group, in which the TUNEL staining conversely became weak (n = 7). The original magnification: 200×. (B) PTEN repression promoted migration and invasion in vitro (P < 0.001, Student’s t test, n = 3). (C) Loss of PTEN stimulated GC metastasis. Arrows indicate the metastatic foci. The original magnification: 200×. Nude mice transplanted with PTEN-knockdown MKN28-SOX2 cells had a higher incidence of distant metastasis.

esophageal cancer [28], and bladder cancer [29], and plays negative roles. In addition, SOX2 serves as an oncogene in lung cancer [30] and prostate cancer [31] but as an anti-oncogene in GC, which may be because different organizational origin tumors have different molecular mechanisms of tumorigenesis and development. This phenomenon is common in tumor development. For example, P21 is a tumor suppressor in GC and colorectal cancer but a tumor promoter in prostate and ovarian cancer [32]. In further studies, we found that SOX2 was only detectable in KATOIII GC cells and the immortal gastric cells GES-1. Tsukamoto et al. [6] found similar results. However, SOX2 expression has been detected in AGS and MKN45 cells by Otsubo et al. [9] and Hutz et al. [7], whereas no SOX2 expression was found in these two cell lines

in our study. This difference may be due to the cell source, culture conditions, passage number, and even the SOX2 antibodies and experimental conditions used. For example, SOX2 was found to be expressed in MKN7 cells in research by Hutz et al. [7] but not by Otsubo et al. [9]. Therefore, we believe that the heterogeneity of results is reasonable and acceptable. It is worth mentioning that almost all studies have reported a lack of SOX2 expression in MKN28 cells, which is the reason we used MKN28 cells as the model in this study. To avoid having the experimental results influenced by cell heterogeneity, we also performed functional studies using SGC7901 cells, and the results agreed with those of MKN28 cells. Previous studies found that SOX2 was expressed in the side population of cells derived from MKN45 [33], as well as SGC7901 [16]

218

S. Wang et al./Cancer Letters 358 (2015) 210–219

cells, suggesting that it may be a marker of GC stem cells. This result does not contradict our results because GC stem cells are a minor fraction of the population. Even if SOX2 was expressed in the few cancer stem cells, it was not detected in the overall GC population. Then, in MKN45 cells, as well as in SGC7901 cells, it was not easy to detect. As mentioned before, Tsukamoto et al. [6] and we did not observe SOX2 expression in MKN45 cells. The predictive effects of SOX2 on patient survival were well documented in the present study. Our compelling paradigms illustrate that SOX2 levels exclusively confined to GC tissue failed to accurately predict GC outcome. However, given that we collected specimens from GC and paired nontumorous tissues together for evaluation, decreased SOX2 expression in GC lesions was an independent predictor of patient survival. Therefore, additional analyses of SOX2 levels in nontumorous tissues to achieve a general understanding of SOX2 status restricted to GC specimens were determined to improve the credibility and accuracy of the predictive value of SOX2. Because no similar results have been reported for GC or other types of cancer, our data underscore that SOX2 expression is highly informative for either the likelihood of an early GC event or disease progression. This unique method could be readily extrapolated to studies of other tumor biomarkers. In this study, SOX2 might aptly be characterized as a tumor suppressor. A rich body of evidence has shown that deletion or loss of SOX2 activity stimulates GC tumorigenesis and progression. First, we have described a succession of gradually downregulated SOX2 expression during the transformation of normal gastric cells into malignant cells. Second, a diverse class of GC cells shows little SOX2 expression, apart from KATOIII, in which the mechanisms of positive SOX2 expression are unclear. Third, additional experiments showed that SOX2 counteracted proliferation and metastasis, which can be altered via disruptions of SOX2 expression. Finally, recent results have reflected a specific dedication of Helicobacter pylori (HP) infection to SOX2 downregulation [10,12,34]. It is acknowledged that HP infection is one of the leading causes of GC, and thus, our observations imply that HP may potently foster tumorigenic activity by blocking SOX2 expression, exemplifying the tumor suppressive role of SOX2 in turn. Although previous studies have reported the oncogenic effects of SOX2 in lung [35], breast [36], and prostate cancers [37], these inconsistencies could be explained by the complexity of the SOX2 regulatory circuits based on diverse cancer origins. Conversely, although SOX2 has also been discovered to have growth-inhibitory effects [9], the precise mechanisms are poorly understood. These reports and our observations raise important questions regarding the distinct signaling downstream of SOX2, with an emphasis on direct targets of the transcription factor, which may have a profound effect on its biological functions. Here, two highthroughput genome-wide analyses and bioinformatics were deployed to screen SOX2 targets. The results showed that PTEN might be directly transactivated by SOX2. As expected, PTEN mRNA and protein levels increased in response to SOX2 overexpression. Considering that a central node of PTEN signaling downstream is Akt/PKB, a serine/threonine kinase whose levels are limiting for the activation of the Akt-mediated survival cascades, even tiny changes in cellular PTEN are sufficient to affect this critical signaling core [38]. Supporting this view, in the present work, SOX2 enforced expression in MKN28 cells resulted in reduced Akt phosphorylation in accordance with induced PTEN expression. The p-Akt dephosphorylation affects diverse cellular roles, which include cell survival, growth, proliferation, and migration [39]. In addition, Akt/PKB signaling has been reported to preside over a number of apoptotic programs [40]. It has been shown that loss of PTEN function in SOX2overexpressing MKN28 cells causes constitutive activation of Akt and leads to their pro-proliferative, anti-apoptotic and pro-metastatic properties. Implicit in our discussion is a novel notion that

SOX2-dependent PTEN upregulation may directly orchestrate downstream p-Akt dephosphorylation. Normally, genomic instability, such as mutations [41] and PTEN promoter methylation, may confer the loss of PTEN [42]. However, recent findings indicate that PTEN mutations and methylation are rare events in human GC, so rare that the exact PTEN mutation is not known except for one reported incidence, which is extremely lower than the odds of negative PTEN expression [43,44]. Thus, the hypothesis that SOX2 might regulate PTEN transcription is attractive because both the absence of SOX2 and the inhibition of PTEN have been proven to allow the anti-tumor activities and other similar effects on GC cells. It is reasonable to believe that PTEN could be regulated at least in part by SOX2. In agreement with our hypothesis, PTEN expression was observed in accordance with SOX2 expression in both GC cells and clinical samples. Furthermore, SOX2 overexpression elicited an increase in PTEN levels, suggesting SOX2 involvement in PTEN expression. Together with the results from luciferase report assays, EMSA and Chip-DSL, our data demonstrated that SOX2 regulated PTEN transcription. However, our data do not eliminate the possibility that other transcription factors might also contribute to the regulation of PTEN expression. The first lines of evidence claim that PTEN was also transcriptionally regulated by TGFβ in human breast cancer, prostate cancer, and glioblastomas [45], followed by the discovery of numerous factors, including PPARγ [46], EGR-1 [47], p53 [40], and MYC [48]. However, only two out of these transcription factors, TGFβ [49] and MYC [50], were reported to have aberrantly elevated expression levels in human GC, and they all function to promote GC carcinogenesis. Because their expression and function are opposite to that of PTEN in GC, it is less likely that they directly regulate PTEN. Therefore, SOX2 might serve as a major regulator of PTEN transcription in human GC. The 10 other candidate genes might function in a manner similar to the anti-cancer effects of SOX2. All of these genes should be further investigated in the future. To date, the consistency of our results from multiple reliable models for profiling study and the convergence of our associated studies in human GC samples and murine specimens together argue for a major influence of SOX2 on PTEN downregulation. Our discovery opens up an emerging tenet for transcriptionally regulatory circuits over PTEN in human GC. Overall, our work indicates exciting breakthroughs not only elucidating the mechanism of SOX2 downstream signaling but also covering the molecular details of PTEN transcriptional regulation in GC. As the first report that SOX2 mediated p-Akt dephosphorylation through PTEN, its discovery yields a succession of the SOX2/ PTEN/Akt pathway, defects which impinge on cellular behaviors against growth and metastasis in GC. Nevertheless, despite the advances in our understanding of SOX2 regulatory circuits, further experiments are required to substantiate whether PTEN upregulation is the solitary target of SOX2 function. Moreover, the tenants of why the cell cycle was free of abnormalities have yet to be addressed in full. It will also be important to further define the role of crosstalk between the SOX2/PTEN/Akt signaling with other distinct PTEN substrates in the ultimate tumor suppression of SOX2 upregulation. Accordingly, we anticipate that with holistic clarity of proof-toconcept mechanistic support, SOX2 will become both a rational indicator of GC prognosis and a promising target for GC gene therapy.

Acknowledgments We are indebted to Dr. Li Xu for critical advice on manuscript revision. This work was supported by the National Natural Science Foundation of China (No. 81071763 and No. 81272649) and the National Key and Basic Research Development Program of China (No. 2010CB529302).

S. Wang et al./Cancer Letters 358 (2015) 210–219

Conflict of interest The authors declare no competing financial interests.

Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2014.12.045.

References [1] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, 2014, CA Cancer J. Clin. 64 (2014) 9–29. [2] Y. Tani, Y. Akiyama, H. Fukamachi, K. Yanagihara, Y. Yuasa, Transcription factor SOX2 up-regulates stomach-specific pepsinogen A gene expression, J. Cancer Res. Clin. Oncol. 133 (2007) 263–269. [3] Y.W. Fong, C. Inouye, T. Yamaguchi, C. Cattoglio, I. Grubisic, R. Tjian, A DNA repair complex functions as an Oct4/Sox2 coactivator in embryonic stem cells, Cell 147 (2011) 120–131. [4] X.L. Li, Y. Eishi, Y.Q. Bai, H. Sakai, Y. Akiyama, M. Tani, et al., Expression of the SRY-related HMG box protein SOX2 in human gastric carcinoma, Int. J. Oncol. 24 (2004) 257–263. [5] T. Tsukamoto, K. Inada, H. Tanaka, T. Mizoshita, M. Mihara, T. Ushijima, et al., Down-regulation of a gastric transcription factor, Sox2, and ectopic expression of intestinal homeobox genes, Cdx1 and Cdx2: inverse correlation during progression from gastric/intestinal-mixed to complete intestinal metaplasia, J. Cancer Res. Clin. Oncol. 130 (2004) 135–145. [6] T. Tsukamoto, T. Mizoshita, M. Mihara, H. Tanaka, Y. Takenaka, Y. Yamamura, et al., Sox2 expression in human stomach adenocarcinomas with gastric and gastric-and-intestinal-mixed phenotypes, Histopathology 46 (2005) 649–658. [7] K. Hutz, R. Mejias-Luque, K. Farsakova, M. Ogris, S. Krebs, M. Anton, et al., The stem cell factor SOX2 regulates the tumorigenic potential in human gastric cancer cells, Carcinogenesis 35 (2014) 942–950. [8] J. Matsuoka, M. Yashiro, K. Sakurai, N. Kubo, H. Tanaka, K. Muguruma, et al., Role of the stemness factors sox2, oct3/4, and nanog in gastric carcinoma, J. Surg. Res. 174 (2012) 130–135. [9] T. Otsubo, Y. Akiyama, K. Yanagihara, Y. Yuasa, SOX2 is frequently downregulated in gastric cancers and inhibits cell growth through cell-cycle arrest and apoptosis, Br. J. Cancer 98 (2008) 824–831. [10] S. Asonuma, A. Imatani, N. Asano, T. Oikawa, H. Konishi, K. Iijima, et al., Helicobacter pylori induces gastric mucosal intestinal metaplasia through the inhibition of interleukin-4-mediated HMG box protein Sox2 expression, Am. J. Physiol. Gastrointest. Liver Physiol. 297 (2009) G312–G322. [11] T. Otsubo, Y. Akiyama, Y. Hashimoto, S. Shimada, K. Goto, Y. Yuasa, MicroRNA-126 inhibits SOX2 expression and contributes to gastric carcinogenesis, PLoS ONE 6 (2011) e16617. [12] V. Camilo, R. Barros, S. Sousa, A.M. Magalhaes, T. Lopes, A. Mario Santos, et al., Helicobacter pylori and the BMP pathway regulate CDX2 and SOX2 expression in gastric cells, Carcinogenesis 33 (2012) 1985–1992. [13] X. Zhang, H. Yu, Y. Yang, R. Zhu, J. Bai, Z. Peng, et al., SOX2 in gastric carcinoma, but not Hath1, is related to patients’ clinicopathological features and prognosis, J. Gastrointest. Surg. 14 (2010) 1220–1226. [14] J. Keilwagen, J. Grau, I.A. Paponov, S. Posch, M. Strickert, I. Grosse, De-novo discovery of differentially abundant transcription factor binding sites including their positional preference, PLoS Comput. Biol. 7 (2011) e1001070. [15] Y.S. Kwon, I. Garcia-Bassets, K.R. Hutt, C.S. Cheng, M. Jin, D. Liu, et al., Sensitive ChIP-DSL technology reveals an extensive estrogen receptor alpha-binding program on human gene promoters, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 4852–4857. [16] T. Tian, Y. Zhang, S. Wang, J. Zhou, S. Xu, Sox2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer, J. Biomed. Res. 26 (2012) 336–345. [17] M. Krstic, S. Stojnev, L. Jovanovic, G. Marjanovic, KLF4 expression and apoptosisrelated markers in gastric cancer, J. BUON 18 (2013) 695–702. [18] H. Xiao, H. Li, G. Yu, W. Xiao, J. Hu, K. Tang, et al., MicroRNA-10b promotes migration and invasion through KLF4 and HOXD10 in human bladder cancer, Oncol. Rep. 31 (2014) 1832–1838. [19] K. Akaogi, Y. Nakajima, I. Ito, S. Kawasaki, S.H. Oie, A. Murayama, et al., KLF4 suppresses estrogen-dependent breast cancer growth by inhibiting the transcriptional activity of ERalpha, Oncogene 28 (2009) 2894–2902. [20] T. Nagata, Y. Shimada, S. Sekine, R. Hori, K. Matsui, T. Okumura, et al., Prognostic significance of NANOG and KLF4 for breast cancer, Breast Cancer 21 (2014) 96–101. [21] W. Tang, Y. Zhu, J. Gao, J. Fu, C. Liu, Y. Liu, et al., MicroRNA-29a promotes colorectal cancer metastasis by regulating matrix metalloproteinase 2 and E-cadherin via KLF4, Br. J. Cancer 110 (2014) 450–458.

219

[22] H.Y. Chen, Y.M. Lin, H.C. Chung, Y.D. Lang, C.J. Lin, J. Huang, et al., miR-103/107 promote metastasis of colorectal cancer by targeting the metastasis suppressors DAPK and KLF4, Cancer Res. 72 (2012) 3631–3641. [23] D. Li, Z. Peng, H. Tang, P. Wei, X. Kong, D. Yan, et al., KLF4-mediated negative regulation of IFITM3 expression plays a critical role in colon cancer pathogenesis, Clin. Cancer Res. 17 (2011) 3558–3568. [24] Y. Zhou, W.L. Hofstetter, Y. He, W. Hu, A. Pataer, L. Wang, et al., KLF4 inhibition of lung cancer cell invasion by suppression of SPARC expression, Cancer Biol. Ther. 9 (2010) 507–513. [25] O. Yoon, J. Roh, Downregulation of KLF4 and the Bcl-2/Bax ratio in advanced epithelial ovarian cancer, Oncol. Lett. 4 (2012) 1033–1036. [26] Y.N. Liu, W. Abou-Kheir, J.J. Yin, L. Fang, P. Hynes, O. Casey, et al., Critical and reciprocal regulation of KLF4 and SLUG in transforming growth factor betainitiated prostate cancer epithelial-mesenchymal transition, Mol. Cell. Biol. 32 (2012) 941–953. [27] J. Wang, R.F. Place, V. Huang, X. Wang, E.J. Noonan, C.E. Magyar, et al., Prognostic value and function of KLF4 in prostate cancer: RNAa and vector-mediated overexpression identify KLF4 as an inhibitor of tumor cell growth and migration, Cancer Res. 70 (2010) 10182–10191. [28] Y. Tian, A. Luo, Y. Cai, Q. Su, F. Ding, H. Chen, et al., MicroRNA-10b promotes migration and invasion through KLF4 in human esophageal cancer cell lines, J. Biol. Chem. 285 (2010) 7986–7994. [29] S. Ohnishi, S. Ohnami, F. Laub, K. Aoki, K. Suzuki, Y. Kanai, et al., Downregulation and growth inhibitory effect of epithelial-type Kruppel-like transcription factor KLF4, but not KLF5, in bladder cancer, Biochem. Biophys. Res. Commun. 308 (2003) 251–256. [30] B. Laflamme, PRKCI and SOX2 drive lung cancer, Nat. Genet. 46 (2014) 325. [31] A.P. Rybak, D. Tang, SOX2 plays a critical role in EGFR-mediated self-renewal of human prostate cancer stem-like cells, Cell. Signal. 25 (2013) 2734–2742. [32] T. Abbas, A. Dutta, p21 in cancer: intricate networks and multiple activities, Nat. Rev. Cancer 9 (2009) 400–414. [33] J. Liu, L. Ma, J. Xu, C. Liu, J. Zhang, J. Liu, et al., Spheroid body-forming cells in the human gastric cancer cell line MKN-45 possess cancer stem cell properties, Int. J. Oncol. 42 (2013) 453–459. [34] K. Matsuda, K. Yamauchi, T. Matsumoto, K. Sano, Y. Yamaoka, H. Ota, Quantitative analysis of the effect of Helicobacter pylori on the expressions of SOX2, CDX2, MUC2, MUC5AC, MUC6, TFF1, TFF2, and TFF3 mRNAs in human gastric carcinoma cells, Scand. J. Gastroenterol. 43 (2008) 25–33. [35] C.M. Rudin, S. Durinck, E.W. Stawiski, J.T. Poirier, Z. Modrusan, D.S. Shames, et al., Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer, Nat. Genet. 44 (2012) 1111–1116. [36] S. Stolzenburg, M.G. Rots, A.S. Beltran, A.G. Rivenbark, X. Yuan, H. Qian, et al., Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer, Nucleic Acids Res. 40 (2012) 6725–6740. [37] X. Jia, X. Li, Y. Xu, S. Zhang, W. Mou, Y. Liu, et al., SOX2 promotes tumorigenesis and increases the anti-apoptotic property of human prostate cancer cell, J. Mol. Cell Biol. 3 (2011) 230–238. [38] T. Maehama, J.E. Dixon, PTEN: a tumour suppressor that functions as a phospholipid phosphatase, Trends Cell Biol. 9 (1999) 125–128. [39] B.D. Manning, L.C. Cantley, AKT/PKB signaling: navigating downstream, Cell 129 (2007) 1261–1274. [40] V. Stambolic, D. MacPherson, D. Sas, Y. Lin, B. Snow, Y. Jang, et al., Regulation of PTEN transcription by p53, Mol. Cell 8 (2001) 317–325. [41] S. Wang, J. Gao, Q. Lei, N. Rozengurt, C. Pritchard, J. Jiao, et al., Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer, Cancer Cell 4 (2003) 209–221. [42] J. Li, C. Yen, D. Liaw, K. Podsypanina, S. Bose, S.I. Wang, et al., PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer, Science 275 (1997) 1943–1947. [43] G. Fei, M.P. Ebert, C. Mawrin, A. Leodolter, N. Schmidt, K. Dietzmann, et al., Reduced PTEN expression in gastric cancer and in the gastric mucosa of gastric cancer relatives, Eur. J. Gastroenterol. Hepatol. 14 (2002) 297–303. [44] K. Sato, G. Tamura, T. Tsuchiya, Y. Endoh, K. Sakata, T. Motoyama, et al., Analysis of genetic and epigenetic alterations of the PTEN gene in gastric cancer, Virchows Arch. 440 (2002) 160–165. [45] D.M. Li, H. Sun, TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta, Cancer Res. 57 (1997) 2124–2129. [46] L. Patel, I. Pass, P. Coxon, C.P. Downes, S.A. Smith, C.H. Macphee, Tumor suppressor and anti-inflammatory actions of PPARgamma agonists are mediated via upregulation of PTEN, Curr. Biol. 11 (2001) 764–768. [47] T. Virolle, E.D. Adamson, V. Baron, D. Birle, D. Mercola, T. Mustelin, et al., The Egr-1 transcription factor directly activates PTEN during irradiation-induced signalling, Nat. Cell Biol. 3 (2001) 1124–1128. [48] T. Palomero, M.L. Sulis, M. Cortina, P.J. Real, K. Barnes, M. Ciofani, et al., Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia, Nat. Med. 13 (2007) 1203–1210. [49] B.R. Achyut, L. Yang, Transforming growth factor-beta in the gastrointestinal and hepatic tumor microenvironment, Gastroenterology 141 (2011) 1167–1178. [50] D.Q. Calcagno, V.M. Freitas, M.F. Leal, C.R. de Souza, S. Demachki, R. Montenegro, et al., MYC, FBXW7 and TP53 copy number variation and expression in gastric cancer, BMC Gastroenterol. 13 (2013) 141.