The Regulatory Effect Of SOX2on Tumor Metastasis And Angiogenesis In Human Breast Cancer And Its Underlying Mechamism

Breast cancer is the first high incidence of malignant tumor in female patients, accounting for30%of new-onset malignant tumors for female. The statistics show that breast cancer is the most common malignant tumor for female and its incidence is increasing year by year in Beijing, Tianjin, Shanghai and other big cities. Prostate cancer is one of the common malignant tumors in Europe, the United States and other developed countries and regions. The mortality ranks in the second and the incidence is also increasing in the developing countries every year[2].Tumor metastasis is the key factor that compromises the prognosis of tumor patients and accounts for90%of tumor death. Metastasis is a multistep process by which a percentage of primary tumor cells acquire the ability to spread from their initial site to the surrounding normal tissues in the local areas or to secondary tissues/organs. Failure at any one of these steps can block the entire metastatic process. Since tumor metastasis is responsible for the majority of deaths for cancer patients, a better understanding of the molecular mechanism involved in tumor spreading process is important for preventing tumor metastasis and improving the prognosis of tumor patents.Angiogenesis plays an important role during the development of cancer. Traditional theory defines that tumor vasculatures are mainly developed through sprouting from pre-existing vessels by recruiting of endothelial progenitor cells (EPCs) from the bone marrow[9]. Recent study revealed that tumor may developed from a small population of tumor initiation cells that possesses "sternness" properties which shares the pluripotency, self-renewal as embryonic stern cells.SOX (sry-related high-mobility-group box-containing) gene family is a kind of gene of encoding transcription factor with HMG structure. It was firstly found by Gubbay et al in the mouse with Y chromosome deletion. The transcription factor SOX2is one of important members of SOX gene family. In recent years, many studies indicate that SOX2is inseparable with the tumorigenesis and the regulation of tumor stem cells. Some research prove that SOX2can promote tumor metastasis in many tumor tissues, but the underlying molecular mechanism remains unclear. To clarify SOX2’s effect in tumorigenesis, we used a series of experiments in vitro and in vivo to study the relationship between SOX2and tumorigenesis and the progression of tumor, and further investigated the underlying mechanism.Firstly, we tested the expression of SOX2in breast cancer tissues by immunohistochemical staining to elucidtate the effect of SOX2on tumorigenesis of human breast carcinoma. Secondly, we confirmed the effect of SOX2on tumor cell physiological functions and possible molecular mechanisms by in vitro experiments, including scratch assay and transwell chamber assay to test the migration ability of the cancer cells, ELISA, Western bloting and immunofluorescence methods to test the change of sectetion levels of metastasis associated protein and cytokines after overexpression or knockdown of SOX2. Then the transcriptional regulation ettect of SOX2on the target gene was proved by dual luciferase report system and CHIP methods. Finally, these results were verified in vivo.We established stable mouse breast cancer cell line4T1with Sox2down regulation (4T1-shSox2), immunofluorescence staining of CD31and LYVE of the xenografted tumor in situ showed that down-regulation of Sox2significantly reduces angiogenesis and lymphogenesis of tumor. By transplanting the bone marrow derived endothelial progenitor cells (MDEPCs) to BALB/C mice, we revealed that Sox2in the tumor cells promotes the recruitmrnt of this cells.By examing the expression of SOX2in50cases of normal breast or adjacent tissues and269cases of tumor tissues, we found that the expression level of SOX2is correlated with tumorigenesis of breast cancer, and the TNMstage and pathological grade.By examing the expression of SOX2in66cases of normal lymphoid tissues and49cases of lymph node metastasized with breast cancer cells, we found that SOX2is overexressed in the metastatic lymph nodes.By using scratch and tranwell test to detect the movements of breast cancer cell line MDA-MB-231and prostate cancer cell line DU145, we found that SOX2significantly promotes migration ability of tumor cells.By down-regulating or overexpressng SOX2in MCF-7and DU145cellsrespectively, we found that SOX2can promote the process of EMT in tumor cells by regulatingthe expression of EMT relative markers, such as E-cadherin and a-smooth actin.We detected the secretion level of TGF-βin supernatant of prostate cancer and breast cancer cells by ELISA and also detected the genes expression levels relative to TGF-P pathway in these two kinds of tumor cells by Western blotting. We found the regulation effect of SOX2on EMT is unrelated with TGF-β pathway.SOX2affects EMT through the classical WNT/β-catenin signal pathway. The main target gene is β-catenin. SOX2regulates the transcription by bining with the promoter sequence of β-catenin directly and also affects the expression of DKK3, DVL1and DVL3in WNT/β-catenin signal pathway.The promotion effect of SOX2on tumor cell metastasis was observed both in vitroand in the tumor bearing mice in vivo.Down-regulation of SOX2significantly reduced angiogenesis and lymphogenesis of breast cancer.SOX2promotes the expression of CD311, VEGF and LYVE in the xenografted tumor cells in situ. These results revealed that SOX2gene enhances tumor angioegenesis by regulateing tumor microenviroment via recruiting bone marrow derived endothelial progenitor cells as well as promoting tumor cells to form vessels.In summary, the conclusions are:The expression of SOX2is significantly correlated with clinical stage and pathological grade of breast cancer.SOX2may promote EMT process of tumor cells by regulation of WNT/β-catenin signal pathway.SOX2promotes the protein transportation to the nucleus to cause the transcription activation of downstream genes by binding with P-catenin promoter region directly to activiate its expression.SOX2regulates the expression of DKK3, DVL1and DVL3in WNT/β-catenin signal pathway.SOX2in tumor cells promotes angiogenesis by modulating the tumor microenvironment and promoting both tumor cells and MDEPCs to form vessels.ChIP assay further demonstrated the binding of SOX2protein to the region around the promoter of VEGF and LYVE, disclosing the underlying molecular mechanism for the regulatory effect of SOX2on angiogenesis.