Research On The Epigenetic Regulatory Functions And Mechanisms Of A Novel Tumor Suppressor Gene CXXC4in Gastric Cancer

Background:Gastric cancer (GC) is one of the most common malignancies in gastrointestinal system and the second leading cause of cancer-related death worldwide, which is a serious threat to human health. As an alternative to genetic changes, epigenetic regulatory disorder is attracting more and more attention in gastric carcinogenesis, which can contribute to the aberrant activation of oncogenic signalings. As the major player in the epigentic regulatory network, EZH2is frequently overexpressed in many human cancers. However, it remains unclear how EZH2contributes to the activation of some oncogenic signalings in gastric cancer.Objective:It has been confirmed that CXXC4is a negative regulator of the canonical Wnt/β-catenin signaling in renal cell carcinoma (RCC), however the detailed molecular mechanisms and biological functions remain poorly understood.We wonder whether CXXC4regulates Wnt signaling in gastic cancer. In the present study, we firstly proposed that CXXC4is the direct target of EZH2and a novel tumor suppressor gene in gastric cancer. EZH2promotes the activation of Wnt and MAPK signaling in gastric carcinogenesis through the downregulation of CXXC4expression. Next we attempt to demonstrate this hypothesis through a series of in vitro and in vivo experiments and clarify the biological functions of CXXC4gene and its tumor suppressive molecular mechanisms.Methods:Firstly, microarray analysis of gene expression before and after EZH2depletion was performed to screen the downstream targets of EZH2in human gastric cancer cells. After qPCR confirmation, we focused on CXXC4, the negative regulator of Wnt/β-catenin signaling for further investigation, whose expression was siginificantly upregulated after EZH2depletion. Next, we used Chromatin Immunoprecipitation (ChIP) assay to clarify the regulation of CXXC4by EZH2is direct or indirect. The correlation between the expression of EZH2and CXXC4in tissue levels was determined by immunohistochemistry (IHC) and mRNA in situ hybridyzation(ISH).We engineered CXXC4eukaryotic expression vector (pCMV-3Tag7-CXXC4) and CXXC4shRNA(pSUPERi-CXXC4) for cell transfection to observe the effect of exogenous CXXC4on cell phenotype. For example, cell proliferation ability was detected by MTS and soft agar assay. To further determine the long-term effect of CXXC4on gastric cancer cells, we engineered SGC7901cells to stably express CXXC4in a doxycycline-dependent manner and constructed stably CXXC4shRNA cell line.We established nude mice model to further verify the role of CXXC4in cancer cell growth in vitro.Furthermore,we constructed three essential CXXC4mutants using animo acid site-directed mutagenesis in the positions of CXXC4-Dvl-land CXXC4-Erkl/2interaction to throw light on the epigenetic regulatory mechanisms of CXXC4in tumor suppression via Co-Immunoprecipitaiton (Co-IP),Western Blot and ELISA assay. Results:In this study we firstly demonstrated that CXXC4is a novel target directly regulated by EZH2.EZH2inhibits CXXC4expression by directly binding to CXXC4promoter.CXXC4significantly inhibits cell growth both in vitro and in vivo through inactivating Wnt and MAPK signaling.We firstly proposed that CXXC4is a novel tumor suppressor gene. Furthermore, CXXC4depletion activates Wnt and MAPK signalings and promotes anchorage-independent cell growth. Mechanistically, CXXC4stabilizes the destruction complex of β-catenin to inhibit Wnt signaling and abogates the interaction of Erkl/2with MEK1/2to suppress MAPK activation. Finally, EZH2-promoted-activation of Wnt and MAPK signaling was impaired by CXXC4overexpression while CXXC4depletion depletion reversed EZH2siRNA-induced inhibition of Wnt and MAPK signaling. More importantly, CXXC4expression is downregulated in gastric cancer cells as well as primary carcinoma tissues and its downregulation is associated with shorter survival of patients with early stages of gastric cancer(p<0.05).Conclusions:CXXC4is a novel tumor suppressor gene directly regulated by EZH2and its expression is a significant prognosis factor for patients with early stages of gastric cancer. EZH2promotes the activation of Wnt and MAPK signalings in gastric carcinogenesis through downregulation of CXXC4expression.Innovative points:1. CXXC4is a novel tumor suppressor gene in human gastric cancer.2. CXXC4is a direct target of EZH2.3. CXXC4expression is a significant prognosis factor for patients with early stages of gastric cancer.4. CXXC4inhibits Wnt signaling by stabilizing the destruction complex of β-catenin.Two critical sites in its C-terminus are responsible for interaction with Dvl and degradation of P-catenin. 5. CXXC4inhibits MAPK signaling by directly binding to Erkl/2and abrogating the interaction of Erkl/2with MEK1/2.6. EZH2promotes the activation of Wnt and MAPK signalings in gastric carcinogenesis through downregulation of CXXC4expression.