缺氧微环境诱导的EMT在涎腺腺样囊性癌细胞干性获得及血管生成拟态中的作用

Vasculogenic mimicry(VM) - some tumor cells take on certain characteristics of vascular endothelial cells and line the tumor's blood vessels. This ability of tumors is required for tumor cells to invade surrounding tissue and metastasize to distant sites -the deadly hallmarks of cancer. Numerous studies have demonstrated that the degree of tumor VM has prognostic impact in different malignancies. Recently, evidence for the direct involvement of cancer stem-like cells in tumor VM formation has been provided in human tumors. In addition, tumor hypoxic microenvironment can induce epithelial-to-mesenchymal transition (EMT) and is closely associated with the stemness of cancer stem-like cells. Based on these evidences and our previous data, we hypothesize that tumor hypoxic microenvironment induces VM through EMT induction and stenmness achievement. To test this hypothesis , the molecular mechanism of EMT induced by tumor hypoxic microenvironment regulating the stemness and VM potential of cancer stem-like cells in adenoid cystic carcinoma (ACC) of salivary gland will be investigated in vivo and in vitro using zinc finger nuclease (ZFN), gene transfer, PCR microarry, microRNAs microarry and protein Chip, MeDIP and ChIP technology. Meanwhile, the special stemness markers of cancer stem-like cells involved in VM formation in ACC will be defined.The results could provide a new strategy and target for inhibiting the growth, invasion,and metastasis of ACC in the future.

肿瘤缺氧微环境促进血管生成拟态（VM），VM在恶性肿瘤生长和侵袭转移中发挥重要作用。最新研究显示，肿瘤干细胞样细胞参与VM；而肿瘤缺氧微环境既能诱导EMT发生，又与干细胞干性密切相关。本课题在原有研究基础上，结合最新进展提出假设：肿瘤缺氧微环境通过诱导EMT发生赋予肿瘤细胞干性，从而促进VM形成。拟采用ZFN、基因导入、微阵列分析、蛋白芯片分析、MeDIP和ChIP等技术，通过体外细胞培养、3D血管形成模型、斑马鱼和NOD/SCID小鼠模型和临床标本，探讨缺氧微环境诱导的EMT在涎腺腺样囊性癌肿瘤细胞干性获得中，哪些分子表达和表观遗传改变发挥关键作用？它们通过哪些关键信号通路促进VM形成？缺氧微环境诱导的EMT赋予涎腺腺样囊性癌干性获得介导VM的特异标记物是什么？结果将从EMT赋予肿瘤细胞干性的新视野，诠释缺氧微环境促进涎腺腺样囊性癌VM形成的分子机制，为防治涎腺腺样囊性癌转移提供新策略。

缺氧微环境诱导的EMT发生能够赋予肿瘤干细胞特性，在肿瘤侵袭和转移中扮演重要的角色。而肿瘤干细胞又能够分化为血管内皮细胞，介导血管生成拟态（vasculogenic mimicry,VM）。但是，目前，肿瘤缺氧微环境诱导的EMT发生赋予肿瘤细胞干性特征，从而在血管生成拟态中发挥作用的分子机制不清楚。本项目按照原计划，在体外细胞株、动物模型以及涎腺腺样囊性癌（salivary adenoid cystic carcinoma, SACC）临床标本上，分别研究了缺氧微环境促进涎腺腺样囊癌EMT发、缺氧微环境诱导的EMT在涎腺腺样囊性癌细胞干性获得以及缺氧微环境诱导的EMT在涎腺腺样囊性癌干细胞样细胞血管生成拟态中的分子机制。结果显示，在涎腺腺样囊性癌标本中，VM表达与患者TNM分期、复发和转移密切相关，与患者性别、年龄、部位、病理分型无相关性。在成功建立涎腺腺样囊性癌血管生成拟态体外3D培养体系上，发现缺氧微环境促进涎腺腺样囊性癌EMT发生、干细胞特性获得和血管生成拟态形成；缺氧条件下，VEGF刺激促进涎腺腺样囊性癌细胞VE-cadherin表达，血管生成拟态形成增加，同时细胞干性标记物CD44和ALDH1的表达增加；抑制VEGF表达，涎腺腺样囊性癌细胞VE-cadherin表达降低，CD44和ALDH1表达降低，血管生成拟态形成减少，EMT发生和细胞干性获得可能是缺氧条件下VEGF调控血管生成拟态形成的重要机制。进一步发现，CD133表达与涎腺腺样囊性癌患者VM表达以及局部复发和远处转移具有正相关性，CD133+ SACC干细胞样细胞通过促进VM的形成诱导了肿瘤EMT形成，从而增加肿瘤的侵袭和迁移能力。研究结果将丰富了肿瘤EMT发生赋予干性促进转移的理论，为涎腺腺样囊性癌寻找新的抗血管治疗和抑制侵袭、转移提供新靶点，为开发新型靶向药物提供新的思路和策略。