Prohibitin重组细胞骨架促进结直肠癌干细胞定向迁移的分子机制

The relapse and metastasis of colorectal cancer originate from cancer stem cells (CSC). In our previous experiments, we have found that prohibitin (PHB) was expressed strong positively in CSC with a high metastatic potential of human colorectal cancer, and the positive signals were mainly located at the leading-edge of the migrating cells accompanying with the aggregation of cytosketletonal proteins, such as actin filaments and microtubule. With immunofluorescent test of colorectal cancer tissues, PHB and ALDH1 (one of the biomarkers for CSC) was also co-immunuostained a few of migrating cells in the bulk of cancer cells out of cancerous gland profile. Therefore, we hypothesized that PHB might promote metastases through regulating the reconstruction of cytoskeleton with actin filaments and microtubule interaction to generate leading-edge of CSC involving in the enhancements of cellular motility. A series of evidences should be presented in this study in order to uncover the mechanism to initiate the migration of CSC. The experiments are associated with immune reaction of co-positioning assay, over-expression or RNA interference of PHB, signal pathways network interferences, establishing a model to evaluate migration of CSC by chemotaxis of extracelluar matrix or endothelial cells of blood vessel, CSC xenografted into nude mice and experimental metastases assay in vivo, and clinico-pathological analysis with prognostic survival. This study is aimed to elucidate the signal pathway of PHB triggering the reconstruction of cytoskeleton in accordance with the procedures of re-assemblying the cytoskeletonal components, then explore the chemotaxis of microenvironmental factors towords directional migration of colorectal CSC. A novel molecular mechanism would be proposed to interpret the metastasis of colorectal cancer underlying the directional migration of CSC.

肿瘤干细胞(CSC)是结直肠癌复发与转移的根源。我们前期发现结直肠癌高转移潜能CSC强表达Prohibitin(PHB)，其表达部位与细胞骨架蛋白F-actin、Microtubule表达的聚集相一致；免疫荧光检查癌组织发现癌性腺腔之外迁移的癌细胞共表达PHB与CSC标志物ALDH1，提示PHB诱导CSC细胞骨架肌动蛋白微丝-微管的重组，促进CSC的定向迁移。本项目拟通过免疫共定位、基因过表达、RNA干扰、信号网络综合干预、细胞外基质成分诱导CSC迁移模型、血管内皮细胞诱导CSC定向迁移模型、动物实验、临床病理和患者预后分析等系列研究，明确PHB触发结直肠癌CSC细胞骨架的重组过程，阐明PHB调节CSC细胞前导缘细胞骨架重组的信号通路，确定CSC定向迁移的微环境趋化作用，提出PHB诱导CSC定向迁移在结直肠癌转移中的分子机制。

肿瘤细胞从原发部位进入间质而发生转移，其具体转移的方向性不仍明确，即肿瘤细胞向何处去？细胞外因子作用于肿瘤细胞产生趋化作用，是通过哪些信号通路来实现的？肿瘤细胞是否会针对细胞外因子的趋化而产生方向改变？产生方向改变后，细胞内蛋白有没有发生相应改变。我们发现结直肠癌细胞在使用Prohibitin(PHB)免疫组化标记时，一些癌细胞的一端强表达PHB，另一些癌细胞不表达PHB；PHB表达于癌细胞的一侧，靠近腺腔表达PHB的癌细胞为向心性分布，靠近间质侧的癌细胞中强表达PHB呈浸润趋势，侵入间质与侵犯到血管内的癌细胞强表达PHB。离心性分布的临床结直肠癌患者是，预后差于向心性分布的患者。由此推测癌细胞内PHB发生重新聚集，强表达PHB的一端为癌细胞的前导缘，带动癌细胞向一定方向迁移，即定向迁移，促进了结直肠癌的转移。癌细胞定向迁移时，细胞内的PHB重新分布。因PHB位于线粒体内，意味着癌细胞定向迁移时，线粒体聚集于癌细胞的前导缘。Miro-1是线粒体上的跨膜蛋白，另一端结合Kinesin，Kinesin以微管为滑道。细胞外VEGF刺激癌细胞时，癌细胞前导管缘均朝向刺激侧，通过我们发明的细胞极性模型而观察到这一现象。癌细胞的定向迁移对VEGF呈时间与剂量依赖性，细胞外VEGF是促进癌细胞定向迁移的重要因子。VEGF仅与细胞膜上的受体NRP1结合，NRP1与PHB出现共定位。当PHB定位于癌细胞前导缘时，细胞内骨架重新结合，伪足延长，微管朝浸润侧而分布。因此，研究总体发现如下：细胞外因子VEGF与结直肠癌膜上的NRP1结合，NRP1诱发增强结合Kinesin，Kinesin随极向分布的微管而聚集于细胞前导缘，同时带动Miro-1螯合线粒体聚集于细胞前导缘，PHB得以释放至此处，诱发肌动蛋白微丝重塑，促进癌细胞迁移。癌细胞针对VEGF的刺激而发生方向改变，在肿瘤细胞发生EMT(肿瘤干性特征时)时更明显。本研究从癌细胞转移的基础细胞生物学改变、分子信号转导、细胞构象改变等方面均有重要发现，为抗转移治疗提供了理论基础。