可溶性CEA诱导结直肠癌中内皮祖细胞归巢促进肿瘤血管新生的机制探讨

It has been evidenced that endothelial progenitor cells (EPCs) are involved in tumor development, metastasis or recurrence and angiogenesis. However, the molecular mechanisms of tumor angiogenesis process are poorly understood. The applicant in the study of the Youth Fund of National Natural Science Foundation firstly reported that vascular endothelial growth factor (VEGF) phosphorylated the HDAC7 protein through activation of PKD1 signaling pathway in EPCs, which leaded to the HDAC7 protein nucleocytoplasmic transport, thus affecting the ability of angiogenesis mediated by EPCs. Further preliminary research about this project application suggests that the cell surface of EPCs separated from peripheral blood of patients with colorectal cancer expresses CEA receptor, and the exogenous CEA promotes the migration of EPCs to subcutaneous xenografts of colon cancer in mice. Therefore, we assume that soluble CEA in colorectal cancer is an important part of the tumor microenvironment, and blood CEA can be combined with CEAR on the surface of EPCs, prompting bone marrow-derived EPCs homing to the tumor site. Soluble CEA exerts regulatory effects probably by influencing the intracellular PKD1 and integrin αvβ3 signaling pathways, which subsequently changes the biological function of the EPCs, and takes part in the induction of tumor angiogenesis. This project will further elucidate the mechanisms of EPCs involved in tumor angiogenesis in order to provide a new strategy for blocking tumor angiogenesis radically by using the EPCs homing as the target.

已知内皮祖细胞（EPCs）参与肿瘤发展、转移或复发及血管新生，但是其参与肿瘤血管新生的机制未明。申请人在国家自然科学基金青年基金的研究中首次发现：VEGF可以激活EPCs中PKD1信号，通过HDAC7蛋白磷酸化及其核浆转运，影响EPCs介导的血管形成能力。围绕本项目申请开展的预实验进一步提示：结直肠癌患者外周血中分离的EPCs细胞膜表面存在CEA受体，动物实验中外源性CEA促进EPCs向小鼠皮下肠癌移植瘤周围迁移。本项目据此推测：在结直肠癌患者血液中CEA可与EPCs表面的CEAR结合，促使骨髓源性EPCs归巢至肿瘤部位，通过影响细胞内PKD1及整合素αvβ3信号通路改变EPCs的生物学功能，从而参与诱导肿瘤的血管新生。本项目将有望阐明EPCs 参与肿瘤血管新生的机制，为以EPCs归巢为靶点阻断肿瘤血管生成治疗提供新的策略。

已知内皮祖细胞（EPCs）参与肿瘤发展、转移或复发及血管新生，但是其参与肿瘤血管新生的机制未明. .该项目的主要研究内容如下，我们探讨了mCRC患者初始治疗前CEA水平与西妥西单抗疗效相关性及其机制研究；我们也探讨了结直肠癌中EGF调控CEA表达促进肿瘤增殖及其机制，并探讨结直肠癌中外源性CEA对EGFR通路介导的细胞增殖的影响。.结果证实了mCRC患者治疗前CEA升高组较CEA正常组在接受西妥西单抗治疗后获得较好的DCR。 EGF能促进LOVO细胞CEA表达，并伴有CEAR下调；EGF通过EGFR诱导LOVO细胞AKT、ERK磷酸化和促CEA高表达。EGF刺激CRC细胞AKT、ERK磷酸化激活与RAS和BRAF突变状态无关，而与EGFR表达相关；EGF促进CEA高表达细胞系Lovo增殖，同时上调CEA表达，西妥昔单抗对其抑制效应不明显；西妥昔单抗抑制CEA低表达细胞系CACO2细胞增殖，EGF对其促增殖效应不明显；结直肠癌细胞小鼠皮下移植瘤模型中发现，EGF 促进肿瘤生长并上调肿瘤组织中CEA的表达，西妥昔单抗抑制肿瘤生长并下调CEA表达；EGF上调Lovo细胞CEA的表达是通过Ras/Raf/MEK/ERK通路，而不是PI3K/Akt/mTOR通路；下调Lovo细胞CEA的表达可阻断EGF促细胞增殖的效应；上调CACO2细胞CEA表达可以阻断西妥昔单抗抑制细胞增殖的效应。外源性CEA对结直肠癌细胞CACO2细胞增殖无明显影响，但CEA可以影响西妥昔单抗抑制细胞增殖效应；细胞免疫荧光提示外源性CEA能与CACO2细胞粘附；同时过表达CEA和EGFR的293T细胞中存在CEA和EGFR结合。我们的研究项目发现EGF通过上调结直肠癌细胞中CEA的表达促肿瘤增殖。结直肠癌中外源性CEA可能通过与EGFR结合影响西妥昔单抗抑制肿瘤增殖效应。.本项目通过结直肠癌临床样本检测、体内外实验进一步验证CEA诱导EPCs归巢促进结直肠癌肿瘤血管新生作用，然后通过功能研究以及EPCs 的调控机制研究，明确细胞内的 EGF与CEA的表达信号通路调控作用，作为丰富EPCs在肿瘤发生发展中的作用奠定理论基础，为寻找诊断标志物或治疗靶点，以及判断预后和转归提供依据。