Twist1直接调控细胞骨架维持上皮-间充质转变促进肿瘤转移的机制研究

Epithelial-mesenchymal transition(EMT) regulated by Twist1 is one of the important mechanisms of cell shaping such as tumor metastasis and vasculogenic mimicry formation. The preliminary study found that Twist1 could combine with the promoter of cytoskeletal proteins Tubulin and Actin and regulate the expression of them. In addition, we observed that specific hypoxic condition can induce cells with high expression of Twist1 to undergo stress-induced suspension without apoptosis. Therefore,the molecular mechanisms and cellular effects may be related to tumor hematogenous metastasis.Based on these observations presented, we further identify the binding sites of Twist1 in the promoter region of the cytoskeletal protein, role of transcriptional activation of Twist1 and its biological effects. Truncated promoter reporter gene analysis, quantitative ChIP, sequence observation of living cells, the cytoskeleton depolymerization - polymerization experiments, and the tumor tissue quantitative ChIP of tumor-bearing animal will be performed to prove that Twist1 can regulate the expression of conserved cytoskeletal protein in tumor cells,induce and inhibit cytoskeleton polymerization and remodeling persistently.Liver metastases and their matching primary tumor tissue will be selected to perform quantitative ChIP and clinical data analysisto verify the molecular mechanism.This study will help to clarify a new molecular mechanism and clinical significance of regulation of cytoskeleton by Twist1.

Twist1介导的EMT是肿瘤转移和血管生成拟态形成等细胞塑形的重要机制之一。前期工作发现Twist1可与细胞骨架蛋白Tubulin和Actin的启动子结合并调节二者的表达，也观察到特定的缺氧可诱导高表达Twist1的细胞发生应激性悬浮且不凋亡，因此考虑该分子机制和细胞效应可能与肿瘤血道转移有关。本研究在此基础上进一步证明Twist1对细胞骨架蛋白启动子区的结合位点、转录激活作用和生物学效应，分别利用截短体启动子报告基因分析、定量ChIP、活细胞序列观察、细胞骨架解聚-聚合实验和荷瘤动物肿瘤组织定量ChIP等方法来证明Twist1可以调节肿瘤细胞内保守细胞骨架蛋白的表达，诱导并持续维持细胞骨架解聚与重构，介导肿瘤细胞呈悬浮状态存活而促使其血道转移；配对选取临床原发/转移肝癌组织行定量ChIP并分析临床资料以验证该分子机制。该项目有助于阐明Twist1调控细胞骨架的新分子机制及其临床意义。

肿瘤细胞骨架重塑性是肿瘤恶性行为产生的原因之一，其调控机制并不明了。目前的研究发现细胞骨架的改变可与细胞转分化机制，与肿瘤血道转移密切相关，也是重要的药物研究靶标。本研究观察到Twist1可以直接调控细胞骨架相关蛋白Tubulin和Actin，进一步研究发现该调控为转录抑制作用；通过一系列的分子水平和细胞水平的检测发现，血清中的凝血酶可以通过PAR1通路激活Twist1对细胞骨架的作用，并可通过钙流效应激活细胞内下游信号转导，促进肿瘤细胞骨架解聚而发生细胞悬浮作用促进血道转移，对该通路的抑制可以使肿瘤细胞转向失巢凋亡，在分子、细胞和动物模型三个层面对上述研究进行了阐释。这些发现进一步完善了细胞骨架调节蛋白的重要上游信号，为肿瘤细胞骨架重塑作用提供了重要的线索。基于这些重要的分子机制发现，课题组进一步开发了基于细胞骨架调控通路相关的抗肿瘤药物，筛选了一批有较好抗肿瘤活性的先导化合物和天然分子，其中一项已申报国家I类新药临床试验批文。研究工作发表论文2篇，达到申请书额定指标，申请结题。