mTOR信号通路新型直接抑制因子DEPTOR在肺癌发生中的作用研究

Lung cancer is the most common cancer and the leading cause of cancer death. Thus, it is significant to investigate the molecular pathogenesis of lung carcinogenesis for the prevention and treatment of lung cancer. The PI3K/AKT/mTOR pathway is constitutively active in many lung cancer patients. Therefore, targeting the PI3K/AKT/mTOR pathway is considered as an attractive approach for effective lung cancer treatment. DEPTOR was recently identified as an mTOR direct inhibitor which plays critical roles in the regulation of cell proliferation and survival. In cell culture settings, DEPTOR acts as a tumor suppressor and an oncogene. However, the physiological functions of DEPTOR and its roles in tumorigenesis in vivo are totally unknown. To this end, we have generated both Deptor knock-out (KO) and knock-in (KI) mouse models. For the KI mouse model, it expresses DeptorS275A (equivalent to human S287A), a DEPTOR mutant resistant to SCFβTrCP-mediated degradation, which should enhance the inhibition to mTOR pathway. In this proposed study, we are going to elucidate the physiological role of DEPTOR in lung carcinogenesis and determine whether it is a tumor suppressor gene or an oncogene via 1) lung cancer cell lines using CRISPR-Cas9 system to KO/KI DEPTOR, and 2) mouse lung cancer models (Deptor KO/KI), acting in combination with Kras activation (KrasG12D) and p53 loss (p53fl/fl), and 3) tumor tissue samples from patients with lung cancer.

肺癌是发病率和死亡率最高的恶性肿瘤，分子水平阐明肺癌的发病机制，对肺癌防治具有重大意义。众多肺癌患者中PI3K/AKT/mTOR信号通路都异常激活，因此靶向此通路被认为是治疗肺癌的一条有效途径。DEPTOR是新发现的mTOR直接抑制因子，在细胞增殖和存活中发挥重要的调节作用。体外细胞水平研究显示DEPTOR表现出抑癌和原癌基因双重性，然而，它在体内肿瘤发生中的功能完全不清楚。为此我们制备了其失活敲除鼠和增强型突变体敲入鼠。预实验结果显示：Deptor敲除促进Kras突变诱导的肺肿瘤发生；DEPTOR水平与肺癌患者预后正相关。本项目拟：1）在细胞水平敲除或敲入DEPTOR，研究其对肺癌细胞增殖和存活的影响；2）在小鼠水平敲除或敲入Deptor，研究其对Kras突变和p53失活诱导肺癌发生的影响；3）在临床水平研究其与肺癌患者预后的关系。从而确定DEPTOR在肺癌发生中的功能及其抑癌/原癌性。

DEPTOR是mTOR信号通路的直接抑制因子，通过与mTOR直接结合，抑制mTORC1和mTORC2的活性，原则上DEPTOR可以抑制整个mTOR信号通路。因此，DEPTOR基本上可以认为是一个肿瘤抑制基因。但是，由于mTORC1可以通过S6K1-IRS1轴线负反馈调控mTORC2/AKT活性，这样就赋予了DEPTOR原癌基因特性。大量体外细胞学研究显示，DEPTOR在细胞增殖和存活中发挥重要的调节作用，表现出抑癌和原癌基因双重性，然而，它在体内肿瘤发生中的功能完全不清楚。本项目发现：1）DEPTOR失活（siRNA敲低或CRISPR-Cas9敲除）通过激活mTORC1和mTORC2明显促进肺癌细胞的增殖和存活；2）DEPTOR直接与EGFR结合抑制其激酶活性，从而抑制EGFR-mTOR信号通路，同时敲低mTORC1下游S6K1和mTORC2的下游AKT、或EGFR均可以挽救DEPTOR敲除导致的增殖和存活增强的表型；3）DEPTOR失活通过激活mTORC2-AKT、诱导转录因子ZEB1和SLUG，促进EMT转换，从而增强肺癌细胞的迁移和侵袭能力，同时敲低ZEB1和SLUG可以挽救DEPTOR敲除导致的迁移和侵袭能力增强的表型；4）Deptor敲除通过激活EGFR-mTOR信号通路明显加速LSL-K-RasG12D;p53fl/fl;Sftpc-CreERT2小鼠原发型肺癌的发生和发展，并缩短小鼠的生存期；5）DEPTOR水平与肺癌患者的临床预后呈明显正相关。总之，本项目确定了DEPTOR在肺癌发生中充当抑癌蛋白作用，失活DEPTOR通过激活EGFR-mTOR信号通路明显促进肺癌的发生和发展。