br Materials and methods br

Materials and methods
Results
Discussion EZH2 is a subunit of the polycomb repressive complex, which trimethylates lysine 27 on histone 3, a repressive marker for gene expression. EZH2 is important for cancer cell proliferation, migration, and invasion, all of which are associated with cancer initiation, progression, and metastasis. More importantly, EZH2 is closely related to cancer stem cell properties and tumor-initiating cell function [26], [27]. In the present study, we identified that EZH2 and H3K27me3 were aberrantly overexpressed in ENKTL. Moreover, strong EZH2 Neostigmine Bromide was associated with increased tumor cell proliferation and showed significant prognostic effect in ENKTL. In agreement with previous reports, EZH2 overexpression is associated with cell proliferation, inhibition of apoptosis, and poor prognosis in a number of cancer types, including breast, ovarian, melanoma, and prostate, and hematopoietic malignancies [9], [12], [17], [28], [29]. H3K27me3 also was overexpressed in ENKTL cases and cell lines, but the clinicopathological significance is contrary to our expectations. H3K27me3 overexpression was correlated with lower proliferation rates and predicted a better prognosis in accordance with the fact that H3K27me3 expression is a superior prognostic indicator for clinical outcome in patients with breast, ovarian, and pancreatic cancers [30]. Previously, it was considered that the function of EZH2 is gene silencing through the methylation of H3K27. Several previous studies have shown a positive correlation between EZH2 and H3K27me3 activation, and both are inferior predictors in various cancers. Contrary to those previous data, our clinical samples show that expression of EZH2 and H3K27me3 is inversely correlated with each other and show a contrary effect on prognosis. These results highlight a noncanonical EZH2 function in ENKTL. Previous studies have also reported that EZH2 expression lacked an association with abundance of H3K27me3 in breast tumor subtypes [31], renal cell carcinoma [32], ovarian cancer, and pancreatic cancers [30]. In addition, it was reported that the expression of EZH2 was correlated with lower level of H3K27 methylation with enhanced PCNA expression, and high expression of H3K27me3 predicts better prognosis in non–small cell lung cancer [33]. In glioblastoma, AKT signaling activation leads to phosphorylation of EZH2 and inhibits its H3K27me3 enzymatic activity [34]. In glioblastoma multiforme and in a prostate cancer model, EZH2 has also been implicated in the methylation of nonhistone substrates and via binding and methylating STAT3, which promotes tumorigenesis of glioblastoma stem–like cells [34]. In ENKTL, EZH2 behaves unconventionally in that its promotion of growth is independent of its methyltransferase activity [12]. Phosphorylation of EZH2 by JAK3 mediates this switch from histone methyltransferase to transcriptional coactivator, leading to the up-regulation of a series of genes that are involved in DNA replication, cell cycle, biosynthesis, and invasiveness [23].Thus, pro-proliferative function of EZH2 in ENKTL is not completely dependent of its methyltransferase activity. STAT3 signaling pathway is hyperactive in various cancer types including ENKTL [21]. Gene expression profiling has revealed that members of the STAT3 pathway are differentially expressed in ENKTL tumor cells compared with normal NK cells [22], [35]. STAT3 activation often results from constitutive JAK3 phosphorylation at Tyr980. A recent study reported that EZH2 binds to and methylases STAT3, enhancing STAT3 activity in glioblastoma stem–like cells [34]. An analysis of the EZH2 promoter in the National Center for Biotechnology Information database identified that EZH2 contained 3 conserved STAT3-binding sites. Chromatin immunoprecipitation–polymerase chain reaction analysis also revealed that STAT3 signaling enhances EZH2 promoter activity in gastric cancer cells [17]. Furthermore, Yan et al [23] demonstrated that JAK3 phosphorylates EZH2, altering EZH2 activity and promoting the survival and proliferation of NK/T-cell lymphoma cells. All of the above studies are suggestive of potential interaction between EZH2 and STAT3. Tofacitinib, a JAK3 inhibitor, was reported to be able to induce G1 cell-cycle arrest and inhibited cell growth in Epstein-Barr virus–positive T- and NK-cell lines [36]. According to our data, tofacitinib not only decreased the expression of p-STAT3 but also decreased EZH2 expression and increased H3K27me3 expression. Thus, the JAK/STAT3 pathway may be an upstream signaling pathway controlling EZH2 and H3K27me3 expression in malignant NK/T cells [37].