【作者】 李雪霏；

【导师】 向荣； 李娜；

【作者基本信息】 南开大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 乳腺癌是在女性患者中发病率排名第一的恶性肿瘤,占女性新发恶性肿瘤的30%。据统计结果显示,在北京、天津、上海等大城市里,乳腺癌是最常见的女性恶性肿瘤,同时其发病率是逐年上升的。前列腺癌是一种在欧美等发达国家和地区常见的恶性肿瘤,死亡率列在第二位,在发展中国家的发病率也呈逐年递增的趋势[2]。肿瘤转移是评估肿瘤患者预后的关键因素,同时由转移引发的肿瘤患者的死亡占所有死亡病例的90%。肿瘤转移是由多个步骤依次发生而引发的综合性结果,其中包括一些肿瘤细胞获得从原发灶散布到初始转移位置的能力、浸润到周围正常的组织以及转移到远端的转移组织或者器官等。其中,任何一个过程的失败都会影响整个转移的过程。因为肿瘤的转移是肿瘤致死的主要病因,所以如果能更清楚的了解肿瘤转移发生过程中的潜在的分子机制将有助于找到有效治疗肿瘤的靶点,为临床有效防治肿瘤的转移以及提高患者的预后提供可能。血管生成在肿瘤的发生中起到了重要的作用,也是伴随肿瘤转移的一个必要的过程。传统的观点认为,肿瘤的新生血管系统是肿瘤细胞通过招募骨髓来源的内皮祖细胞(endothelial progenitor cells (EPCs) from the bone marrow)而形成的[9]。近年的研究表明肿瘤可能是起源于一小部分肿瘤起始细胞,这样的小部分细胞表现出干细胞特性,同时具有多向分化、自我更新等胚胎干细胞的特征。SOX (sry-related high-mobility-group box-containing)基因家族是一类具有HMG特征性结构的编码转录因子的基因,它最早是由Gubbay等在Y染色体缺失小鼠中发现的[12]。其中转录因子SOX2是SOX基因家族的一个重要成员。近年许多研究表明SOX2与肿瘤的发生以及肿瘤干细胞的调控密不可分[13]。一些研究表明,在许多肿瘤组织中证实SOX2具有促进肿瘤转移的作用[14],然而其潜在的分子机制尚有待深入探讨。为了明确SOX2在肿瘤病因中所起到的作用,我们利用体内体外一系列实验方法,研究SOX2与肿瘤发生及发展的关系,进而研究了SOX2参与肿瘤转移及新生血管生成调控的分子机制,为探讨更好的临床治疗肿瘤新靶点提供了理论基础。首先我们利用免疫组织化学染色的方法对乳腺癌组织中的SOX2进行检测,明确SOX2在乳腺癌发生及发展中的作用；其次利用体外实验进一步证实SOX2对肿瘤细胞转移特性的影响并进一步探讨了潜在的分子机制,其中包括运用划痕实验和Transwell实验检测细胞的迁移能力、利用ELISA和蛋白印迹以及免疫荧光等实验方法检测了SOX2过表达或低表达肿瘤细胞中转移相关蛋白及细胞因子的分泌水平的变化；然后运用双荧光报告系统和CHIP方法证明SOX2对目的基因的转录调控方式；最后利用小鼠原位接种及转移模型对其调控作用进行了体内实验的验证。另外,我们在BAL/C小鼠的乳腺癌细胞株4T1中建立沉默SOX2的稳定细胞系,并在体内成瘤,建立荷瘤小鼠模型,取小鼠肿瘤组织进行切片制作,通过免疫荧光方法染色检测沉默SOX2之后肿瘤组织中的CD31和LYVE的表达变化。我们利用小鼠骨髓移植的方法,将带有红色荧光的小鼠的骨髓细胞移植给经过放射线照射并被破坏造血系统的正常C57小鼠体内,并使其造血系统重建。再利用荷瘤小鼠模型,给小鼠接种带有绿色荧光的E0771细胞,这样收取肿瘤组织做荧光染色分析可以发现肿瘤的新生血管系统的来源,即来源于宿主骨髓来源的内皮祖细胞,还是来源于肿瘤细胞的自分化。我们通过对50例正常乳腺或癌旁组织和269例肿瘤组织中SOX2的表达检测,我们发现,SOX2对乳腺癌的发生有促进作用,乳腺癌组织中SOX2的表达水平与其临床分期和病理分级呈正相关。通过对66例正常淋巴组织和49例乳腺癌转移淋巴结的SOX2表达的免疫组化染色检测及统计学分析发现,SOX2在有乳腺癌细胞转移的淋巴结中明显高表达。通过利用划痕实验和tranwell细胞侵袭实验对乳腺癌细胞系MDA-MB-231和前列腺癌细胞系DU145的运动能力检测,我们发现SOX2对细胞的迁移和侵袭能力有明显的促进作用。利用沉默SOX2的MCF-7细胞和分别过表达SOX2的MCF-7和DU145细胞,通过对EMT相关标志物E-钙粘蛋白和α-肌动蛋白在肿瘤细胞蛋白表达水平的检测,发现SOX2促进肿瘤细胞发生EMT的过程。通过ELISA检测前列腺癌和乳腺癌细胞上清中TGF-β1和TGF-β2的分泌水平,以及在这两种肿瘤细胞中检测其它TGF-β通路相关基因的表达发现SOX2对EMT的调控不是通过TGF-β通路进行的。SOX2是通过与WNT/β-catenin信号通路的经典途径对EMT产生作用,主要目的基因是β-catenin。并且是通过直接结合到β-catenin启动子序列的方式对其转录进行调控。并且能影响WNT/β-catenin信号通路相关基因DKK3、 DVL1和DVL3的表达。SOX2对肿瘤细胞的促转移作用不仅仅体现在体外实验中,同样也发生在小鼠乳腺癌原位荷瘤模型中。SOX2可以通过促进肿瘤细胞对肿瘤微环境中骨骼来源内皮祖细胞的招募作用促进肿瘤的血管新成过程。SOX2促进肿瘤细胞中CD31、LYVE和VEGF的高表达,说明肿瘤组织SOX2基因不仅能够修饰肿瘤微环境,而且同时促进肿瘤细胞和骨髓来源的内皮祖细胞共同参与肿瘤新生血管的生成。通过以上实验,我们得到主要结论如下：SOX2与乳腺癌的发生、临床指标特别是转移显著相关。SOX2能通过调控WNT/β-catenin信号通路对肿瘤细胞的EMT过程起到促进作用。SOX2能通过直接结合于β-catenin启动子序列的方法激活其表达,同时促进其入核。SOX2调节WNT/β-catenin信号通路中DKK3,DVL1和DVL3的表达水平。SOX2促进肿瘤细胞和骨髓来源的内皮祖细胞共同参与肿瘤新生血管的生成。SOX2可以直接结合于VEGFB和LYVE启动子附近区域,进而对这两个基因的转录进行调控。更多还原

【Abstract】 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.更多还原