Background
Gastric cancer (GC) is one of the highest incident cancers and the leading cause of cancer-related mortality worldwide [
1]. In China, GC has become the third highest cause of cancer death, and the relative 5-year survival rate for GC is less than 30% [
2,
3]. Adjuvant chemotherapy following a D2 resection is regarded as the standard curative treatments for localized GC, and the standard of care in the first line setting of metastatic disease remains a combination of fluoropyrimidine and platinum-containing chemotherapy [
4]. However, the long-term prognosis of GC remains unsatisfactory because of the high rate of recurrence and metastasis. Thus, it is important to investigate the molecular mechanisms of GC progression and to identify genes implicated and the driver mutations/genetic alterations.
The enhancer of zeste homolog 2 (Ezh2) belongs to the family of polycomb group genes (PcGs). It has emerged as a master regulator of cell division [
5], DNA repair [
6], autophagy [
7], signal transduction, and embryonic development [
8]. Thus, Ezh2 has multiple essential biological effects, and it is not surprising that its function (and often dysfunction) plays an important role in various diseases, especially in cancer [
9]. Increased Ezh2 expression has been reported in different types of cancers, including colorectal cancer [
10], hepatocellular carcinomas [
11], and lung cancer [
12,
13]. Recently Ezh2 protein has been disclosed overexpressed and is associated with several tumor proliferation and invasion-associated genes, as well as the prognosis of GC [
14‐
16]. A panel of genes including HOTAIR [
17], CCAT2 [
18], and TP53 [
19] that implicated in the EMT or pluripotent phenotype have been reported to be acting as the upstream moleculars of Ezh2 dysregulation in GC. These findings raised the question that what the downstream of functional Ezh2 is in cancer EMT phenotype and sphere-forming capacity, resulting in the carcinogenesis and progression of GC.
Herein, we demonstrate that Ezh2 expression is correlated with poor survival in GC. Besides promoting cell proliferation and invasion, Ezh2 has a pivotal role in CSC enrichment and EMT of GC. Most importantly, we show that PTEN is a direct target of Ezh2. Ezh2 binds to the PTEN locus and downregulates PTEN expression, which consequently activates the Akt pathway, stabilizes Vimentin, downregulates E-cadherin, and protects Sox2 and Oct4 from degradation, thus ultimately leads to the acquisition of EMT and pluripotent phenotype of GC cells. Taken together, our results provided an explanation for the aggressive nature of human tumors overexpressing Ezh2 through a mechanism that links Ezh2 to the key tumor suppressor PTEN. These findings of the role of Ezh2 in the progression of GC may imply Ezh2 as a prognostic factor and potential therapeutic target.
Methods
Human tissue specimens
The human tissue specimens in this study were collected from 156 patients with histologically confirmed GC, who had underwent prior surgical resection and received no pretreatment. These samples were obtained from the tissue bank of Fudan University Shanghai Cancer Center (FUSCC) between 2008 and 2010. Independently, a total of 105 GC patient samples were enrolled for IHC analysis; all patients’ formalin-fixed paraffin-embedded (FFPE) tissues were obtained from the department of pathology of FUSCC between 2005 and 2011. Follow-up was completed on November 30, 2016. The tumor grades were defined in accordance with the criteria outlined by the World Health Organization (WHO) Classification of Tumors of the Digestive System, 2010 edition [
20]. The study complied with the regulations of the Ministry of Health of China and the WHO Research Ethics Review Committee international guidelines for research involving human subjects and the Declaration of Helsinki on the ethical principles for medical research involving human subjects. The survival analysis results of 599 GC tissue samples from GEO cohort (GSE14210, GSE15459, GSE22377, GSE29272, and GSE51105;
n = 593) are available at the KMPlot database (
http://kmplot.com).
Chromatin immunoprecipitation assays
Chromatin immunoprecipitation (ChIP) assays were performed as previously described [
21]. Briefly, the cells were trypsinized and resuspended in lysis buffer, and nuclei were isolated and sonicated to shear the DNA to 500 bp–1 kb fragments (verified by agarose gel electrophoresis). Equal aliquots of chromatin supernatants were subjected to overnight IP with different antibodies as indicated or anti-flag as a negative control. DNA was extracted and the PTEN promoter, as well as the first exon, was amplified by PCR. Sequences of the PCR primers are listed in Additional file
1: Table S6.
Luciferase assays
Cells were transfected with the pGL3-based constructs containing the PTEN promoter plus the Renilla luciferase plasmid (pRL-TK). Then, the cells were harvested after 48 h for firefly/Renilla luciferase assays using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activities were normalized to the cotransfected pRL-TK plasmid (mean ± SD).
Other methods used in this study were described in previous publications and are listed in the supplementary information [
21‐
24].
Reproducibility
Each experiment was performed in triplicate, and the data are presented as the mean ± SD. The sphere formation assay, cell invasion assays, animal experiments, Western blot, and immunohistochemistry results are representative of three independent experiments.
Statistical analysis
All statistical analyses were performed using SPSS 22.0 (IBM, SPSS, Chicago, IL, USA) and GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). Statistical tests for data between group analysis included the χ2-test, Student’s two-tailed t test, and one-way ANOVA. DFS (disease-free survival) and OS (overall survival) curves were calculated with the Kaplan-Meier method and were analyzed with the log-rank test. The DFS rate was calculated from the date of surgery to the date of progression (local and/or distal tumor recurrence) or to the date of death. The OS rate was defined as the length of time between the diagnosis and death or last follow-up. Univariate and multivariate analysis were fit using a Cox proportional hazards regression model. A threshold of P < 0.05 was defined as statistically significant.
Discussion
In this study, we determined the pivotal role of Ezh2 in GC pathogenesis and its underlying mechanisms. We observed that elevated Ezh2 expression was directly correlated with GC tumorigenesis and progression. High expression of Ezh2 could be used to identify a greatly increased risk of recurrence or invasion in patients after surgery, which might serve as a valuable prognostic marker. We also found that in addition to facilitating the proliferation and invasion of GC cells, Ezh2 especially lead to the EMT and pluripotent phenotype of GC cells through mediating the Akt/PTEN signaling pathway. Collectively, our clinical and mechanistic evidence strongly suggested that dysregulated Ezh2 expression mediating abnormal Akt/PTEN signaling critically contributes to GC pathogenesis and aggressive behavior.
The present study demonstrated that Ezh2 was overexpressed in GC specimens. Similar results have been obtained by other investigations, although the number of tumors analyzed was different and those studies focused on only the protein level [
30,
31]. These findings provide support for a role of elevated Ezh2 protein expression in the tumorigenesis of GC. More importantly, we demonstrate for the first time the potential role of deregulated Ezh2 mRNA in promoting GC progression and unfavorable prognosis. We demonstrated that Ezh2 mRNA expression is significantly correlated with tumor stage and invasion. Further analysis demonstrated that Ezh2 mRNA, while not EZH2 protein expression, was an independent prognostic indicator for GC survival, strongly suggesting that GC patients with high Ezh2 expression should undergo follow-up at shorter intervals and be kept under close surveillance.
Our studies demonstrated that the toggle points protein of Akt signaling, PTEN, is transcriptionally regulated by Ezh2 in GC partly and contributes to the high-proliferation and invasiveness activity, EMT, and pluripotent phenotype. It was previously identified that PTEN/Akt signaling [
32] and Ezh2 [
33] are important regulators for the proliferation and invasion of gastric cancer cells. Here, we present a mechanism in consistent with these previous observations. Previous studies established the critical roles of PTEN/Akt signaling activity in tumor cell pluripotent phenotype [
34]. The present study is the first to demonstrate that PTEN/Akt signaling activity is associated with Ezh2 activation in human GC, suggesting a link between PTEN/Akt signaling inactivation and Ezh2 activation. By using well-established Ezh2 upregulation and downregulation systems, we were able to show that aberrant expression of Ezh2 significantly impacted PTEN expression and Akt phosphorylation in GC cells. We also found that Ezh2 expression is involved in PTEN promoter activity in GC cells, and the binding is functional, which we confirmed by using luciferase and ChIP assay and rescue experiments. However, Ezh2 is a histone methyltransferase and the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), but not a transcription factor that binds a specific DNA sequence/motif. Thus, there should be other factors that help Ezh2 to bind to PTEN promoter. It has been found that imatinib could cause drug resistance via recruitment of EZH2 to the promoter region of the PTEN and then downregulates PTEN’s transcripts in leukemia patients [
35]; moreover, cumulative evidences revealed that lncRNAs interacted with EZH2 and recruited it to genes’ promoter regions to repress genes’ expression [
36‐
38]. Furthermore, Chen NM and colleagues found that KRAS signaling was required for EZH2-mediated transcriptional activation of the inflammatory transcription factor nuclear factor of activated T cells 1 (NFATC1) in pancreatic ductal adenocarcinoma cells [
39]. All these suggest that drugs, lncRNA, and transcription factor, may contribute to the binding of EZH2 to PTEN promoter. Further studies may focus on the exactly molecular contribution to EZH2 recruitment to the promoter region of PTEN.
Besides its aforementioned role, the implication of a role for Ezh2 in GC tumorigenesis and development is supported by lines of evidence suggesting that Ezh2 is essential for cellular EMT in GC [
40‐
42]. In fact, Ezh2 regulates the expression of various EMT-related genes in various cancer types [
40‐
42], suggesting that Ezh2 is required for EMT during tumor progression. In addition to the present study is the first to provide evidence of a critical role for activated Ezh2 in gastric cancer pluripotent phenotype by regulating PTEN/Akt signaling; it also shows a tight correlation between Ezh2 expression and E-cadherin and Vimentin expression mediate by PTEN/Akt signaling, supporting a critical role for Ezh2-PTEN/Akt axis in cellular EMT. Given the identified pivotal roles of Ezh2 in these two critical aspects of cancer biology, promotion of GC progression by activation of Ezh2 is conceivable.
Aberrant Ezh2 expression occurs in many other solid tumors, but its effects and underlying mechanism in these cancers are currently unclear. Whether Ezh2 is involved in the oncogenesis and development of other human malignancies via the PTEN/Akt signaling is still worth exploring, and the results would help us to better understand the role of Ezh2 in cancer.
Conclusion
In summary, we demonstrated that Ezh2 is overexpressed in primary GC and its expression is correlated with the tumor burden and clinical outcome. In addition, besides promoting GC cell proliferation and invasion, we identified Ezh2 as a key regulator of GC cell EMT and pluripotent phenotype via activation of the PTEN/Akt pathway. Our data strongly suggested that Ezh2 can be used for prognostication for GC and it may be utilized as a therapeutic target. Further exploration is necessary for the mechanism underlying Ezh2 overexpression in GC, particularly the molecule(s) to which it binds when modulating tumor cell behavior. Therefore, Ezh2 provides new perspectives for research of cell regulation in gastric cancer and new targets for gastric cancer diagnosis and treatment.