异常表达的整合素在食管癌侵袭转移中的分子作用机制

Increasing evidences revealed that Integrins plays important roles in the progression of cancers by regulating cell adhesion and cell invasiveness. Our previous studies showed that Integrin α5 was over-expression in esophageal squamous cell carcinoma (ESCC) and its expression was related to poor overall survival of ESCC patients. Moreover, Integrin α5 could interact with Integrin β1 and Ezrin, and then participated in the progression of ESCC cells. More recently, by using RNA-Seq, Western blotting and bioinformation analysis methods, we found that 10 integrins such as Integrin β4 etc. were up-regulated and 2 integrins including Integrin β7 etc. were down-regulated in the ESCC tissues by comparing to the normal esophageal epithelium. Interestingly, expressions of other 8 integrins such as Integrin α10 etc. were stage-dependent. Moreover, certain Integrins-binding molecules were also alter-expression. These data indicated that Integrins might be widely involved in the progression of ESCC. However, the expression pattern and the biological roles of these dysregulated Integrins in the progression of esophageal cancer are still unknown. In addition, the biological roles and molecular mechanisms for the interaction between Integrins and their binding partners are needed to be discovered. In this proposal, by employing tissue microarray, immunohistochemical staining and real-time RT-PCR, we sought to identify the key Integrins which were significantly alter-expressed in the progression of esophageal cancer. After that, we will explore the roles and the underlying mechanisms of these Integrins and their binding partners in the invasiveness and metastasis of esophageal cancer cells by using in vitro and in vivo methods.

我们以往研究发现，整合素α5在食管癌中显著异常上调表达，与食管癌患者不良预后相关；同时和Ezrin、整合素β1等形成复合物，在食管癌侵袭中发挥重要作用。最近我们通过RNA-Seq和免疫印迹等实验手段进一步研究发现，与正常食管上皮相比，食管癌组织中β4等10种整合素显著上调表达，β7等2种显著下调表达，α10等8种呈现明显阶段特异性异常表达。此外，许多整合素的互作蛋白也出现显著表达异常，提示整合素广泛参与食管癌的恶性演进过程。然而，究竟哪些异常表达的整合素在食管癌发生发展，特别是侵袭转移中功能最重要，以及整合素与其互作蛋白间究竟怎样交互作用等重要科学问题均不清楚。鉴于此，我们提出本项目，拟通过联合运用免疫组化、定量PCR、定点突变及体内外系列功能实验，鉴定出在食管癌侵袭中发挥关键作用的整合素，继而在分子、细胞和整体动物模型三个层面，深入解析这些整合素在食管癌侵袭转移中的分子作用机制。

研究表明，整合素分子在肿瘤的发生发展中发挥重要作用。然而，食管癌中整合素分子的功能和分子作用机制均不清楚。在本项目中，我们通过RNA-Seq等实验手段研究发现，与正常食管上皮相比，食管癌组织中β4等10种整合素显著上调表达，β7等2种显著下调表达，提示整合素可能广泛参与食管癌的恶性演进过程。为此，我们优先选择了表达水平最高的四种整合素（α5、α6、β1和β4）进行了下一步研究，具体结果总结如下：.1）利用组织芯片、免疫组织化学等手段，研究发现整合素α5、α6、β1和β4蛋白的表达在食管癌中均较正常上皮显著上调，这些蛋白的表达水平可以作为食管癌患者预后预警的分子标志物。.2）综合使用RNAi、基因过表达以及系列细胞表型实验，研究发现，整合素α5、α6、β1或者β4的下调表达，均可以抑制食管癌细胞的恶性表型；反之，整合素α5、α6、β1或者β4的过表达，则可以促进食管癌的恶性表型。此外，磷酸化可以调控整合素α6的功能，磷酸化的整合素α6抑制食管癌细胞的生长与移动，而去磷酸化整合素α6则可促进食管癌细胞的上述行为。.3）为了进一步探讨这些整合素发挥作用的分子机制，我们研究发现，细胞粘附分子L1CAM是整合素β1的相互作用蛋白， L1CAM通过与整合素β1的相互作用，激活ERK/MAPK信号转导通路，进而上调骨架结合蛋白Ezrin的转录，最终促进食管癌细胞的恶性表型。而磷酸化整合素α6可以与抑癌基因Merlin相互作用，进而激活HIPPO通路，抑制食管癌细胞的生长和移动等行为。.4）我们还发现，一种新的大环内酯类药物F806，可以在体内外显著抑制食管癌细胞的生长，诱导细胞凋亡。而其发挥抑制肿瘤作用的其中一个重要机制是通过抑制整合素β1的活化。这些结果提示，整合素分子可能作为食管癌靶向治疗的靶点分子。.5）研究发现，整合素β4和α6是超级增强子相关基因，P63可以结合到超级增强子，调控这两个基因的转录。这为后续揭示整合素在食管癌等肿瘤中过表达的分子机制打下基础。.综上所述，整合素分子广泛参与食管癌发生发展的过程，是潜在的治疗靶点和预后预警分子标志物。