调控Rictor基因表达及其介导的肺癌细胞可逆性EMT和转移的分子机制

Metastasis is considered as one of the main causes of treatment failure and death in cancer patients. Epithelial-Mesenchymal Transition (EMT) has been shown to play a critical role in promoting metastasis in epithelium-derived carcinoma. Rictor, the unique component of mTORC2, has been reportedly involved in tumorgenesis, invasion and metastasis of many cancers. However, the regulatory mechanisms of Rictor expression and by which regulates Rictor-mediated EMT and metastasis in cancer cells remain unknown. Our previous results indicated that Rictor is overexpression in both human lung cancer tissues and cell lines tested and is regulated, at least in part, at transcriptional level. Additionally, knockdown of Rictor expression caused phenotype alterations through EMT, which is accompany with the impairment of migration and invasion ability in lung cancer cells. Moreover, we have cloned and identified, for the first time, the rictor core promoter and have been screening for the transcription factor(s) that regulates rictor gene transcription and expression. Thus, we hypothesize that the Rictor-regulating transcription factor(s) may involve in the reversible EMT procession and metastasis through regulating the formation of EMT-related Rictor complexes in lung cancer cells. This project would establish new relationships among the Rictor-regulating transcription factor(s), EMT and metastasis. The understanding of the regulator upstream of Rictor may afford an opportunity for the development of new inhibitors and the rational design of combination regimens based on different metastasis-related molecular targets, and finally prevents cancer metastasis.

上皮肿瘤中EMT的发生是引起侵袭和转移的重要途径。mTORC2特有组分Rictor可独立于mTORC2存在，参与多种肿瘤的发生、侵袭和转移，但其表达调控机制及上游调节子与EMT之间的联系尚未见报道。课题组基于Rictor在肺癌细胞和组织内高表达并受转录水平调控，沉默Rictor改变肺癌细胞的上皮和间质表型并抑制其体外迁移及侵袭能力的前期发现，首次克隆及确定了调控rictor转录的核心启动子。在此基础上，本项目将首先明确调控Rictor表达的转录因子，进一步推测该转录因子有可能通过调控Rictor表达及EMT相关的Rictor复合体形成而调节Rictor介导的肺癌细胞可逆性EMT及转移。通过本项目的研究，将首次阐明调控Rictor表达的转录因子及其与EMT、肿瘤转移之间的新联系，为开发针对肿瘤侵袭与转移新靶点的小分子抑制剂以及不同靶向的抑制剂联用治疗肿瘤转移提供重要的理论依据。

肺癌是严重威胁人类健康的重大疾病，发病率和死亡人数居恶性肿瘤的首位。侵袭与转移的发生是导致临床肺癌患者治疗失败和死亡的主要原因。上皮-间质转化（epithelial to mesenchymal transition，EMT）的发生是引起肿瘤侵袭和转移的重要途径。mTORC2特有组分Rictor可独立于mTORC2存在，参与多种肿瘤的发生、侵袭和转移，但其表达调控机制及上游调节子与EMT之间的联系尚未见报道。本项目发现Rictor在肺癌细胞和组织内高表达并受转录水平调控，沉默Rictor改变肺癌细胞的上皮和间质表型并抑制其体外迁移及侵袭能力。利用分子克隆、报告基因分析、ChIP、慢病毒感染介导的基因沉默及过表达等技术，本项目确定了Rictor的核心启动子及调控Rictor表达的转录因子KLF4。证实KLF4能直接结合Rictor核心启动子并调控其转录活性及Rictor基因表达。通过建立KLF4沉默的多株稳定肺癌细胞系、在肺癌细胞中过表达KLF4以及在稳定沉默KLF4的多株肺癌细胞株中回复表达Rictor，证实了基因沉默KLF4下调Rictor表达的同时，各稳定细胞株由间质表型向上皮表型转变，体外迁移和侵袭能力也相应地减弱；过表达KLF4增加Rictor启动子转录活性；而沉默KLF4后再回复表达Rictor则促使细胞向间质表型转变，体外迁移和侵袭能力也相应地增强。基因沉默KLF4抑制了EMT相关的Rictor/mTOR复合体形成。通过体内实验，证实了转录因子KLF4调控的Rictor表达对肺癌细胞转移的影响。本项目的研究首次阐明了调控Rictor表达的转录因子KLF4及其与EMT、肿瘤转移之间的新联系，为开发针对肿瘤侵袭与转移新靶点的小分子抑制剂以及不同靶向的抑制剂联用治疗肿瘤转移提供新思路。