AKT协同NOTCH信号诱导小鼠肝内胆管癌发病机制研究

Hepatocelluar carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the two major types of malignancy occurring in the liver. In the past decades, the incidence of ICC has been increasing in China. However, the molecular mechanism of ICC carcinogenesis is still unknown. Multiple studies have shown that that Notch signaling plays a role in cell fate determination, stem cell renew and tumor formation, studies also shown that NOTCH can induce cell apoptosis. AKT is a major mediator of cell survival through direct inhibition of pro-apoptotic signals. In our recent study, using a mouse model of hepatocyte fate tracing in combination of electron microscopy, we showed that activated NOTCH and AKT signaling cooperate to convert normal hepatocytes into biliary cells that act as precursors of rapidly progressing, lethal ICCs. Activation of NOTCH signaling is able to induce ICC development over long latency, the tumorigenesis process was significantly accelerated when co-activated with AKT signaling. In addition, we consistently noticed that NICD injected mice developed very few tumor lesions. In striking contrast, NICD/AKT mice developed hundreds of lesions throughout the liver. We hypothesized that hepatocyte-BEC metaplasia initiated by NOTCH cascade could be toxic to the hepatocytes, and NICD induced hepatocyte apoptosis through p53/p21 signaling, which finally led to the elimination of the majorities of these cells. The apoptosis induced by NICD might be protected by AKT/mTOR signaling via Foxo1 and Mcl-1, which contributes to the accelerated ICC development in NICD/AKT mouse model. In the present application, we propose to systematically study the molecular mechanism of NICD/AKT induced ICC formation that may be targetable for anti-ICC therapy.

原发性肝癌按病理学分型可分为肝细胞型肝癌（HCC）、肝内胆管细胞癌（ICC）及混合型肝癌。在我国ICC的发病率逐年上升，但其发病机制尚不清楚。有研究表明NOTCH信号一方面能促进肿瘤发生和干细胞分化，另一方面又能促进细胞凋亡。AKT常通过其抗凋亡作用促进肿瘤进展。我们前期研究表明，NOTCH信号的单独活化能缓慢诱导ICC发生，NOTCH和AKT信号的共活化能快速诱导ICC进展，同时我们还发现AKT信号对ICC进展的促进作用可能与其抗凋亡能力相关。由此我们提出ICC发病机制假设：在诱导ICC发生同时，NOTCH能通过p53/p21信号诱导部分癌变肝细胞发生凋亡，减缓自身诱发的ICC进展。而AKT能通过Foxo1和Mcl-1信号阻断NOTCH的凋亡诱导作用，从而促进ICC进展。本项目拟在小鼠模型中分析AKT协同NOTCH信号促进ICC发病的具体机制，为后续ICC靶向治疗研究提供依据。

研究分为三个部分。研究的第一部分，我们通过高动力尾静脉注射癌基因组合（活化NOTCH(NICD), shP53和IDH1R132）的方式使FVB小鼠肝脏内形成肝内胆管细胞癌（ICC）。尾静脉注射12周后，小鼠肝脏内开始出现肿瘤。18周后，小鼠肝脏表面出现多发囊性肿瘤样病变。该ICC肿瘤模型具有ICC患者标本肿瘤的组织学特征，如不规则的腺体增生，同时所有NICD/shP53/IDH1R132C肿瘤细胞均表达胆系标志物CK19。本研究表明，IDH1R132突变体可以与其他癌基因或抑癌基因共同作用，促进ICC发生进展。该成果为研究IDH1R132突变在ICC发生中的具体作用提供了新型小鼠模型。研究的第二部分，我们通过高动力尾静脉注射方式癌基因组合（myr-AKT和YapS127A），建立了另一个ICC小鼠模型。在该模型中，我们对比了mTOR抑制剂MLN0128与ICC标准化疗方案（吉西他滨联合奥沙利铂）的治疗效果。研究结果发现，吉西他滨联合奥沙利铂针对晚期AKT/YapS127A ICC疗效有限。与之对比，MLN0128能使晚期AKT/YapS127A ICC部分消退，同时MLN0128的早期给药能使AKT/YapS127A ICC肿瘤稳定。因此本部分研究表明，MLN0128具有抗ICC的潜力，它可能优于吉西他滨联合奥沙利铂对ICC的治疗，尤其在Akt/mTOR信号活化的ICC中的潜力更大。第三部分，我们进行了一部分胃癌相关研究，评估了TRAIL信号在胃癌治疗中的可行性。首先观察了rhTRAIL对胃癌细胞的杀伤效果。结果显示rhTRAIL对9株胃癌细胞有不同程度的生长抑制和凋亡诱导作用，这种作用呈现时间和剂量依赖性。胃癌细胞对rhTRAIL治疗的敏感性与细胞中线粒体凋亡通路的激活、TRAIL受体的上调以及抗凋亡蛋白c-IAP1、c-IAP2、Livin和Mcl-1的下调相关。在细胞和小鼠皮下移植瘤模型中，紫杉醇（PTX）能通过抑制MAPK信号显著增强rhTRAIL的细胞治疗敏感性，该部分研究为PTX用于治疗TRAIL非敏感胃癌提供了理论依据。