| Colorectal cancer(CRC) is one of the most frequently diagnosed cancer s worldwide,which cause serious damage to human health. In recent years, the incidence and mortality of CRC have been increasing in our country and CRC have become a major public health problem. CRC has a high tendency to metastasis and more than 40% patients with CRC will eventually develop metastatic diseases. Metastatic CRC are usually resistant to existing therapies and contribute to the majority of CRC related mortality, being the largest challenge in CRC management. Thus a better understanding of the underlying mechanisms of metastasis in CRC will facilitate development of effective strategies for prevention and treatment of metastatic CRC. However, the molecular mechanism of CRC metastasis still remains largely unknown.Epithelial-mesenchymal transition(EMT) is generally considered as a crucial step in metastatic cascades of various types of malignancies including CRC, but the mechanism of EMT in CRC cells is obscure. Although there have been evidences showing that some EMT transcriptional factors(EMT-TFs) may directly executed the EMT process of CRC cells, how the expression of those EMT-TFs are regulated and how the EMT-inducing factors in tumor microenvironment activate those EMT-TFs are largely unknown. Researches concerning those above questions are likely to find promising preventive or therapeutic targets of metastatic CRC.Myocyte Enhancer Factor 2(MEF2) family of transcriptional factors have diversity of biological functions, and their critical roles in embryogenesis, muscular, cardiovascular and neurological systems have been long recognized. During recent years MEF2 members have been implicated in various cancer progression. Our lab has previously reported that MEF2 D, the fourth member of the MEF2 family, promotes hepatocellular carcinoma cell proliferation and leads to poor prognosis of patients. Thereafter, MEF2 D also has been implicated in lung cancer and osteosarcoma progression. These data imply that MEF2 D may serve as one potential “oncogene†in human cancers. However the roles of MEF2 D in CRC have not been investigated. In addition, these previous researches mainly focused on MEF2 D pro-proliferation effect, whether MEF2 D have other different functions that contribute to cancer progression remains largely unaddressed.We wonder whether MEF2 D has other novel functions to promote CRC, so we start this study and focus it on the roles and molecular mechanisms of MEF2 D in invasion and metastasis of human CRC.The main experiment designs and results of this study include:(1) In human CRC samples MEF2 D expression and its relationship with patients ’ clinicopathological characteristics are analyzed.Immunohistochemistry assays in 93 cases of clinical CRC specimens showed that MEF2 D was aberrantly overexpressed in cancerous tissues compared with adjacent noncancerous tissues and normal epithelia.Importantly, MEF2 D immunohistochemistry scores in primary tumors of the patients with lymph node, distant metastasis or cancer recurrence were significantly higher than scores of patients without metastasis or recurrence. Patients with MEF2 D positive tumors had poorer prognosis than patients with MEF2 D negative tumors. Moreover, MEF2 D immunohistochemistry scores negatively correlated with patients’ survival. These data implied a novel correlation of MEF2 D overexpression with cancer metastasis.(2) The roles of MEF2 D in invasion and metastasis of CRC cells were analyzed in vitro and in vivo.By comparing MEF2 D expression in different cancer cell lines, we found high MEF2 D expression correlates with mesenchymal phenotypes and invasive behavior of cancer cells.The direct role of MEF2 D in cancer cell invasion and migration were determined by MEF2 D knockdown or overexpression assays and results collectively suggested that MEF2 D plays a critcial role in cancer cell invasion and migration. Consitantly MEF2 D knockdown significantly reduced metastases of human CRC cells in experimetal animals.(3) The functions of MEF2 D in modulating cancer cell EMT were analyzed.Knockdown MEF2 D in mesenchymal and highly invasive CRC cell lines lead to loss of mesenchymal characters and increased epithelial markers’ expression. On the other hand, overexpression of MEF2 D in epithelial cancer cell lines not only decreased the expression of epithelial markers but also increased the expression of mesenchymal markes expression. Thus, MEF2 D can modulate cancer cell EMT phenotypes.(4) Target gene of MEF2 D in regulating EMT was detected and the molecular mechanism was investigated.We screened expression changes of EMT regulatory genes in multiple cancer cell lines after MEF2 D knockdown and found MEF2 D knockdown notably reduced ZEB1 expression. By overexpression assay, immunofluorescence assays and flowcytometry assay we determined that MEF2 D can regulate ZEB1 expression.Using chromatin immunoprecipitation(ChIP) and promoter reporter activities assay we found MEF2 D directly regulated ZEB1 transcription and facilitated acetylation of ZEB1 promoter histones by recruiting p300 to ZEB1 promoter.(5) Detecting the expression and function of MEF2 D under tumor microenvironment EMT inducing factors.Some well-known tumor microenvironment EMT-inducing factors,such as EGF and hypoxia were used to induce CRC EMT progress, and MEF2 D expressions in these conditions were analyzed. We found that MEF2 D expression is rapidly and significantly activated by those EMT signals. More importantly, in MEF2 D knockdown cells, these EMT processes were obviously impeded.(6) The role of MEF2 D in ZEB1 expression triggered by microenvironment factors was investigated.Under EMT-inducing factors treating, expression of MEF2 D and ZEB1 were analyzed dynamically, and results showed that MEF2 D expression increased more rapidly than that of ZEB1, so we speculated that MEF2 D may be essential in microenvironment factors triggered ZEB1 expression. To test this notion, MEF2 D knockdown cells and control cells were exposed to EMT-inducing factors. Results showed that MEF2 D knockdown significantly blocked microenvironment factors triggered ZEB1 expression. Consistently, ZEB1 expression couldn’t be detected in mouse liver metastatic tumor which derived from MEF2 D knockdown cells inoculation.The main conclusions of this study are:1. MEF2 D is aberrantly overexpressed in human CRC tissues and its expression correlates with cancer metastasis and poor prognosis.2. MEF2 D promotes CRC cell invasion, metastasis and EMT process.3. MEF2 D can regulate the critical EMT-TF ZEB1 expression transcriptionally.4. MEF2 D can rapidly response to multiple microenvironment signals and is essential in these signals triggered EMT process.5. Many microenvironment EMT inducing signals converge on MEF2 D and MEF2 D is a critical node to transduce microenvironment signals to some crucial EMT related genes. |
