Inhibition of GSK 3β Activity Is Associated with Excessive EZH2 Expression and Enhanced Tumour Invasion in Nasopharyngeal Carcinoma

Abstract

BackgroundEnhancer of zeste homolog 2 (EZH2) has been shown to contribute to tumour development and/or progression. However, the signalling pathway underlying the regulation of EZH2 in nasopharyngeal carcinoma (NPC) remains unclear. Since EZH2 contains the putative Glycogen synthase kinase 3 beta (GSK3β) phosphorylation motif ADHWDSKNVSCKNC (591) and may act as a possible substrate of GSK-3β, it is possible that inactivation of GSK3β may lead to excessive EZH2 expression in NPC.MethodWe first examined the expression of EZH2 and phosphorylated GSK3β (p-GSK3β) by immunohistochemical staining in NPC samples. Then, we evaluated the interaction of GSK3β and EZH2 using immunoprecipitation and immune blot. Moreover, we determined the effect of inhibition of GSK3β activity on EZH2 expression and tumor invasiveness in NPC cell lines in vitro. Finally, we evaluated the invasive properties of NPC cells after knocking down EZH2 expression with EZH2 siRNA.ResultsWe found that expression of EZH2 correlated with phosphorylated GSK3β (p-GSK3β) at Ser 9 (an inactivated form of GSK3β) in human nasopharyngeal carcinoma (NPC) samples. We also provided evidence that GSK3β is able to interact with EZH2 using immunoprecipitation and immune blot. Furthermore, we found that inhibition of GSK3β activity can lead to upregulation of EZH2 in NPC cell lines in vitro, with enhanced local invasiveness. By knocking down EZH2 expression with EZH2 siRNA, we found that these invasive properties were EZH2 dependent.ConclusionOur findings indicate that GSK3β inactivation may account for EZH2 overexpression and subsequent tumour progression, and this mechanism might be a potential target for NPC therapy.

Osteopontin Promotes EZH2 Expression and Tumor Progression in Nasopharyngeal Carcinoma

“…Overexpression of EZH2 is a marker of advanced and metastatic disease in many solid tumors, including prostate cancer and NPC [18]. Previously, we reported that EZH2 expression was significantly enhanced in NPC tissues, which was regulated by GSK-3β [16]. In this study, we found that EZH2 expression was significantly associated with the TNM stage of NPC, suggesting that EZH2 may play a critical role in the progression of NPC.…”

“…Immunohistochemical staining was performed as previously reported [16]. Briefly, human tissue sections were stained for the expression of OPN (1:200) and EZH2 (1:200; Cell Signalling, Danvers, Mass., USA) and detected by streptavidin-biotin-horseradish peroxidase complex formation (Zhongshanjinqiao, Beijing, China).…”

“…qRT-PCR was performed as we previously reported [16]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as inner control for PCR amplification of transcripts.…”

“…Cell migration and invasion were detected by transwell invasion assay, which was performed as described elsewhere [16]. Briefly, CNE-1 cells were grown in serum-free medium until 90-100% confluency was reached.…”

Nasopharyngeal Cancer

“…These patterns are widespread and have great biological significance [96]. Excessive EZH2 expression by the inhibition of GSK-3β activity enhances NPC tumor invasion [100]. The overexpression of BRD7 inhibits NPC cell growth and the cell cycle by transcriptional regulation, which is achieved by BRD7 binding to lysine-acetylated peptides derived from histone H3 with K9 or K14 acetylated and from histone H4 with K8, K12, or K16 acetylated.…”