Methylation Of TIP30by TGF-β Promotes Metastasis And EMT In Esophageal Squamous Cell Cancer

Esophageal squamous cell cancer (ESCC) is a highly aggressive tumor with poorprognosis. China is one of the countries in which ESCC experienced a high incidenceand mortality which mainly due to tumor infiltration and invasion of surrounding tissue,adjacent tissue and remote organizations. In recent years, it has been found that theepithelial-mesenchymal transition (EMT) phenomenon, not only happened inmulticellular organisms during embryonic development, but also existed in a variety oftumorigenesis. Cancer cells spread and metasitasis through EMT.TIP30, also called CC3or HTIP2, is a putative tumor suppressor gene located onhuman chromosome11p15.1. It was originally identified by a differential displayanalysis of messenger RNA from the highly metastatic human variant small cell lungcancer (SCLC) versus less metastatic classic SCLC cell line.It has been previouslyreported that TIP30played important roles in liver cancer, prostate cancer, breast cancer,stomach cancer and other tumor diseases, such as inhibition of tumor growth, invasionand metastasis. The underlying molecular mechanism of TIP30inhibits tumormetastasis as well as the expression and function of TIP30in ESCC remains unknown.This study is focusing on the functions of TIP30gene and the underlyingmolecular mechanisms in ESCC.The subject can be divided into4parts,1. TIP30inhibit metastasis of ESCC in vivo and in vitroWe analyze the expression level of TIP30gene in11ESCC cell lines and onenormal immortalized esophageal epithelial cell line. To investigate the role of TIP30inregulation of tumorigenicity and metasatsis, lentiviruses encoding shRNA against TIP30was generated to inhibit TIP30expression and lentiviruses encoding TIP30wasgenerated to over expression TIP30expression.Tumorigenicity potentials of ESCC cellswere studied by soft agar colony formation assay, the tetrazolium salt modified experiment (MTS), colony formation assay and nude mice experiments. Wound-healingassays and transwell migration assays were used to determine migration and invationpotentials of ESCC cells.Results: Downregulation of TIP30gene expression in ESCC cell lines increasedcell proliferation, migration, invasion ability in vitro and promoted tumor growth,pulmonary metastases in vivo. Meanwhile, studies of upregulation of TIP30gene wereobserved vice versa.2. TGF-β downregulated TIP30expression by promoting methylation of TIP30We treated ESCC cell lines with TGF-β to induce cell undergo EMT, then weexamined expression of TIP30and EMT markers by RT-PCR and Westernblot, as wellas methylation status of TIP30by MSP. We analyze TIP30promoter region methylationstatus and expression level in11ESCC cell lines, one immortalized esophagealepithelial cell line by MSP and RT-PCR. We further determine the distribution andproportion of methylated sites by bisulfite squencing PCR (BSP).Results: TGF-β induced TIP30downregulation by methylation.TIP30promotermethylation was observed in5ESCC cell lines，18CpG islands was contained by MSPamplificiation and at least16CpG island were methelated by BSP analysis.3. TIP30inhibited EMT of ESCC by blocking β-catenin activityCells were infected with Lv-shTIP30or LvAcTIP and72huors later cells weretreated with TGF-β or not, then EMT markers were detected by RT-PCR and IFstaining. We used luciferase reporter assays to determine the transcriptional activity ofβ-catenin.Results: Downregulation of TIP30promted EMT in ESCC and upregulation ofTIP30may inhibit TGF-β incuced EMT; TIP30blocking β-catenin activity by inhibitingtranslocation of β-catenin to nucleus.4. The correlation between the TIP30expression/methylation and clinicalcharacters in ESCC tissuesThe expression of TIP30in100cases of ESCC were detected by ICH and themethylation status of TIP30in85cases were analyzed by MSP, then the correlation ofTIP30expresson/methylation and clinical characters were analyed by SPSS16.Results: The expression level of TIP30correlated with gender of patients, tumorsize and clinical stage.68/85(80%) ESCC cases showed methylation of TIP30promoter and methylation of TIP30was correlated with tumor metastasis and recurrence.Conclusion: In ESCC, TGF-β downregulates TIP30expression by promtesmethylation of its promoter; TIP30play an important role in inhibition tumor metastasisand EMT by blocking β-catenin activity; Downregulation of TIP30and methylation ofTIP30were correlated with clinical stage and metastasis in ESCC patients.