【作者】 李丽丽；

【导师】 陆军；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2015， 博士

【摘要】 上皮-间质细胞转化(Epithelial-Mesenchymal Transition,EMT)指的是一系列复杂的分子细胞程序,在这些进程中表皮细胞失去了它们的分化特征,包括细胞与细胞之间的黏合,平面极性,基顶极性以及运动性的缺乏,相反却获得了间质特性,包括运动性、侵袭性、以及被加强的对细胞凋亡的抗性。在不同的生物学时间和背景下发生的EMT作用和伴随现象也不相同,大致分为三种不同的类型即Type 1、Type 2和Type 3,分别涉及胚胎发育、器官纤维化和肿瘤进程。本论文的研究内容关注的是肿瘤进程相关的Type3型EMT。现有的研究已经证实,EMT在包括乳腺癌、前列腺癌和肺癌等多种癌症的浸润和转移过程中发挥着重要作用。在肿瘤发生发展过程中,具有极性的肿瘤上皮细胞由于EMT的作用,导致细胞之间的粘附能力下降,丧失了细胞与细胞之间的极性,最终通过细胞骨架重塑,变成了具有更强的移动能力的间质样细胞。由于细胞的迁移和侵袭能力提高,使间质样细胞很容易从基底膜上脱离,通过细胞外基质(Extracellular matrix,ECM)进入循环系统,并在远端器官形成转移灶,最终导致肿瘤细胞的转移。EMT的发生是多种转录因子和信号通路等因素相互作用的结果,其重要的分子标志是E-cadherin的表达下降和N-cadherin的表达上升。目前的研究已经证实有很多的转录因子通过调控E-cadherin和N-cadherin的表达从而参与了EMT程序的发生。例如Snail能够与SIP1以重叠的方式共同结合在E-cadherin的启动子上,导致E-cadherin的表达下降;同样的,锌指结合蛋白ZEB1和ZEB2也能直接与E-cadherin启动子上的元件相结合,抑制其表达;而Twist、ZEB1和MZF1等可以直接与N-cadhrin启动子结合,促使其表达上升。除此之外,多种细胞外信号也可以激活EMT程序,在这些下游的胞内信号通路和转录因子之间有非常重要的相互影响,比如转化生长因子(TGF-β)、细胞外信号调节激酶(ERK)、促分裂原活化蛋白激酶(MAPK)、Wnt等,这种串扰引导了这个复杂的程序,包括多种正负反馈循环。其中TGF-β是一种公认的EMT的重要诱导因子,通过经典的TGF-β/Smad信号通路和非Smad两种作用途径,在EMT进程中发挥重要作用。研究发现EMT的持续激活会导致细胞内逐渐的表观遗传改变,以维持细胞的间质状态,在表型上产生稳定的改变并产生细胞的谱系特性。研究还发现癌症上皮细胞能够通过启动EMT事件从而获得干细胞特征。SHON是奥克兰大学刘东旭课题组筛选出来的一种新的人类特异的分泌蛋白。刘东旭等人证实SHON在多种癌症细胞中呈现高表达,与乳腺癌症进程密切相关。但是SHON是通过什么方式促进乳腺癌进程,它又在其中发挥了怎样的作用,以及其具体的作用机制都不明确。我们的研究发现,在永生化的乳腺上皮细胞MCF10A和乳腺癌细胞MCF7中过表达SHON,能够导致细胞启动EMT进程,同时伴随着E-cadherin的表达上升和N-cadherin的表达下降,有效的提高细胞的迁移和侵袭能力,意味着SHON在乳腺癌进程中发挥了重要的作用。我们同样发现在乳腺上皮细胞中过表达SHON能够诱导CD44high/CD24low干细胞样群比例的增加,进一步的乳腺球形成实验也证明SHON能够促进乳腺上皮细胞进行非依赖性生长,这些都说明过表达SHON能够诱导乳腺上皮细胞获得干细胞特性。我们发现SHON能够上调很多EMT相关转录因子的表达,例如:Snail,TGF-β1,Twist等,进一步证明SHON可能在致癌性的EMT进程中发挥重要的作用。我们的研究还证实了SHON能够以旁分泌的方式激活TGF-β1信号通路,同样在TGF-β1诱导的EMT过程中也伴随着SHON表达量的增加。SHON和TGF-β1都属于分泌蛋白,能够被释放到胞外微环境中。我们证实了微环境中的SHON能够以旁分泌方式激活周边细胞的TGF-β1信号通路,而高表达的TGF-β1又能够进一步促进SHON的表达,从而形成一个正反馈调节过程,调控乳腺癌进程。我们的研究结果表明:SHON是一种新的EMT诱导因子,SHON能够通过激活TGF-β1信号通路调控乳腺癌细胞的EMT进程,促进乳腺癌细胞的迁移和侵袭,在乳腺癌进程中发挥重要作用。SHON有可能作为一种新的分子诊断标志成为分析乳腺癌预后的有效手段之一,并成为乳腺癌靶向治疗的新靶标。更多还原

【Abstract】 Epithelial-mesenchymal transition(EMT) is a biological phenomenon occurring in specific physiological or pathological conditions, in which cells of plasticity transform into the mesenchymal cells capable of migration. EMTs are classified into three different biological subtypes, i.e., Type 1, Type 2 and Type 3, based on the biological context in which they occur in the processes of embryonic development, organ fibrosis or tumorigenesis. The purpose of this study focuses on the tumor-associatied Type3 EMT. The existing studies have shown that EMT plays an important role in the process of invasion and metastasis in many kinds of cancers, including breast, prostate and lung cancer. The term EMT refers to a complex molecular and cellular program by which epithelial cells shed their differentiated characteristics, including cell–cell adhesion and lack of motility, and acquire the mesenchymal features, including motility and invasiveness. EMT can promote the cells to detach from the basement membrane and disperse through the extracellular matrix into the circulatory system, resulting in tumor metastasis.EMT is a process in which a variety of factors are involved, including many transcription factors and signaling pathways. A hallmark of EMT is a decline in the expression of E-cadherin and an increase of N-cadherin. The present study has confirmed that there are a lot of transcription factors involved in the occurrence of EMT program by regulating the expression of E-cadherin and N-cadherin. For example, Snail could combined to E-cadherin together with SIP1 in the form of overlap in E-cadherin promoter, resulting in a decline in the expression of the E-cadherin; Likewise, the zinc finger protein ZEB1 and ZEB2 also affect directly with E-cadherin promoter element, and inhibit the expression of E-cadherin; Similarly, there are some factors that can improve the expression of N-cadherin through acting directly on its promoter, including Twist, ZEB1 and MZF1.In addition, a variety of extracellular signals can also activate the EMT program, there is a very important "crosstalk" between transcription factors and the intracellular signaling pathways, such as the TGF-β, ERK, MAPK and Wnt. The crosstalk may induce a variety of positive and negative feedback loops. TGF-β is the main and the best characterized inducer of EMT. It functions through two pathways of Smad-dependent and Smad-independent. Some studies found that the activation of EMT could induce epigenetic changes gradually to maintain the state of mesenchymal cells, and generate stable change in the phenotype. The study also found that the epithelial cells could get stem cell characteristics through EMT events.SHON is identified as a novel secreted hominoid oncogene and expressed in most human cancer cell lines, and is oncogenic for human mammary carcinoma cells. But the mechanism through which SHON promotes the process of breast cancer is not clear.We report in this thesis that over-expression of SHON in immortalized human mammary epithelial cell MCF10 A and human mammary cancer cell MCF7 was competent for the obtainment of the enhanced ability of cell migration and invasion, and induced the occurrence of EMT, along with the change of the EMT markers E-cadherin and N-cadherin. We also found that ectopic over-expression of SHON in human mammary epithelial cells may induce an EMT program in epithelial cells and potentiate the stem-like properties, characterized by increased number of CD44high/CD24 low population and mammosphere forming ability. Furthermore, SHON has also been shown to upregulate many important regulators of EMT, such as Snail, TGF-β1 and Twist. Based on these previous data, we speculate that SHON may play a role in breast cancer progression through induction of an oncogenic EMT program.Our study also showed that SHON activated the TGF-β signaling, and SHON itself was induced by TGF-β signaling in mammary epithelial cells. Presumably, SHON may interact with TGF-β, resulting in self-autocrine feedback. These date suggest that SHON regulates the EMT via a SHON-TGFβ-SHON feedback loop in breast cancer progression. At the same time, we synthesized the functional monoclonal antibodies of SHON in inhibitory type, and we found that it can effectively inhibit the activation of TGF-β1 signaling pathways caused by SHON, and further affect the SHON-TGFβ-SHON feedback loop.In this report, we provide evidence that SHON is a novel inducer of EMT in breast cancer. SHON induces an EMT program in epithelial cells via TGF-β1 signaling pathways, and promotes cell migration and invasion, and plays an improtant role in breast cancer progression. These results indicate that the latent value of SHON as a new molecular diagnostic marker for clinical diagnosis in human breast cancer, also provide theoretical basis for the development of targeted drugs for breast cancer.更多还原