mTOR在肿瘤中对染色质重塑的调控机制及其意义

Mechanistic target of rapamycin (mTOR) plays a central role in the regulation of eukaryotic cell growth and metabolism. Its deregulation drives uncontrolled tumor growth. mTOR pathway is a key anticancer drug target. Our previous studies showed for the first time that mTOR is localized in the nucleus and regulates chromatin remodeling. Using a novel chemical genomics method to screen for yeast rapamycin-sensitive genes, we identified several key chromatin remodeling molecules, providing a functional link between mTOR and chromatin remodeling. In response to nutrient and stress conditions, mTOR regulates chromatin modifications and functions such as transcription and genomic stability. Accordingly, we hypothesize that mTOR pathway deregulation in cancer leads to profound alteration in chromatin remodeling, promoting uncontrolled cancer growth while determining sensitivity to mTOR inhibitors. To test this hypothesis, we will carry out the following three specific aims: ① To systematically investigate how nutrient stimulation and inhibition by starvation or rapamycin through mTOR regulate chromatin remodeling; ② To understand the regulatory mechanisms of chromatin remodeling by mTOR; (3) Based on the molecular understanding of the aberrations in cancer chromatin remodeling, to identify surrogate biomarkers to guide mTOR-targeted therapy and develop rational combinational therapies. Successful implementation of these objectives should lead to new insights into the role of chromatin remodeling in cancer pathogenesis and help develop new strategies for cancer treatment.

雷帕霉素靶点蛋白mTOR是调控肿瘤细胞生长代谢的中心点，也是当今最重要的抗癌药物靶点之一。前期研究中，申请者首次发现mTOR定位于细胞核内以调节基因转录、染色质重塑；采用化学基因组学方法筛选雷帕霉素敏感基因，我们发现了几个与染色质重塑的关键分子； 在mTOR受外界刺激条件下调节染色质的多种修饰及调节染色质重塑复合物蛋白的磷酸化。据此我们提出科学假设，mTOR信号通路的激活导致染色质重塑从而促进肿瘤的生长。为了验证假设，我们拟开展以下三方面研究：①系统分析刺激及抑制等情况下，mTOR信号通路在肿瘤细胞中对染色质重塑的调节作用；②阐明mTOR通路调控染色质重塑的分子机制及其肿瘤生物学意义；③通过改变肿瘤染色质重塑关键分子的表达和功能，探索mTOR靶向治疗的敏感标志物与新方案。本项目的实施将有助于阐明肿瘤的发病机制，并为改善临床治疗奠定基础。

在本项目中，我们首先分析了TCGA数据库，发现染色质重塑复合物亚基ARID1A在肝癌所有的基因突变中位列第三，提示其在肝癌发生发展中起着重要作用。接着，在美国国立癌症研究所的CPTAC数据库中，我们发现肝癌中ARID1A 蛋白水平与 AKT–mTORC1 信号通路活性呈负相关；在TCGA数据库中，ARID1A的突变与AKT/mTORC1 信号通路中的关键基因的突变很少重叠，提示只要有其中一个基因的突变就能导致肝癌的发生发展。在实验中，mTOR抑制剂和激活剂分别上调和下调ARID1A蛋白表达；免疫共沉淀和PLA实验发现，mTORC1复合物中的Raptor与ARID1A主要在胞浆中相互结合，而且mTOR通过SCF泛素连接酶介导的泛素化-蛋白酶体途径来降解ARID1A蛋白，不影响ARID1A的mRNA表达；泛素-蛋白酶体途径诱导ARID1A蛋白降解；在水动力AKT激活肝癌模型上，我们证实了ARID1A过表达能够对抗/抑制AKT-mTOR激活导致的小鼠肝癌的形成及发展。通过染色质可及性测序（ATAC-seq）和RNA-seq的分析，我们发现受mTOR和ARID1A共同调控的染色质位点以Hippo通路居首位，而YAP是该通路的关键分子；当敲降ARID1A后，雷帕霉素调控YAP的效应被阻断，说明mTOR以ARID1A依赖的方式来调控染色质重塑及其对YAP的调控。在癌细胞系全库（CCLE）中，我们发现肝癌细胞对mTOR抑制剂坦罗莫司（Temsirolimus）的药敏性与染色质复合物的突变密切相关；实验证实，只有野生型ARID1A的肝癌细胞对雷帕霉素敏感，而突变型则不敏感，表明ARID1A是肝癌mTOR抑制剂的一个敏感性标志物。我们的研究阐明了mTOR调控ARID1A及其在肝癌中的作用及其分子机制，为临床肝癌mTOR抑制剂的应用提供了理论和实验依据。