The RB pathway in liver cancer

描述(申请人提供)：全球每年有超过60万人死于肝细胞癌。引起肝细胞癌的环境因素是众所周知的，包括乙型和丙型肝炎病毒(乙肝病毒和丙型肝炎病毒)的感染，接触黄曲霉毒素B1，以及过量饮酒。然而，目前还没有有效的治疗方法，而且肝癌患者的预后通常很差，总体中位生存期不到一年。Rb抑癌基因及其家族成员的p107和p130在几乎所有的人肝细胞癌中都处于功能失活状态。这种失活是由于CDK4抑制物p16沉默或CDK4伴侣Cyclin D1表达增加而导致的CDK4激酶活性的增加。此外，由乙肝病毒和丙型肝炎病毒产生的一些蛋白质可以使Rb家族成员失活，包括通过触发它们的降解。我们通过删除成年小鼠肝脏中的Rb家族基因来建立人类肝癌的小鼠模型，以模拟这种Rb家族蛋白的功能失活。Rb/p107/p130三重基因敲除(TKO)小鼠发生肝肿瘤，其组织学和基因表达谱类似于人肝癌。在TKO肝癌细胞中，通常被Rb家族抑制的E2F转录因子的活性很高。TKO肝细胞起源于前体细胞，而不是肝细胞，由于抑制其增殖的未知机制，肝细胞不能分裂。TKO肝癌模型提供了一个独特的体内系统来查询肝脏肿瘤发生的机制，并具体询问RB/E2F转录调控复合体如何控制肝癌的发展。我们的一般假设是，Rb通路的失活至少在一定程度上推动了癌症的发生 通过放松对E2F活动的管制，并在高度敏感的细胞群体中损害调控网络之间的平衡。具体地说，我们认为E2F活性的增加参与了促进突变细胞(包括肝脏前体细胞)扩张的途径，但也触发了负反馈循环，防止成熟肝细胞启动癌症并限制肝癌细胞的生长。我们将首先测试p21激活的特定假设 在TKO模型中通过E2F阻断肝细胞的增殖，从而阻止这些成熟细胞启动肝细胞癌。接下来，我们将测试Notch信号激活y E2F在肿瘤形成过程中限制肝癌细胞扩张的想法。最后，我们将研究E2F激活EZH2甲基转移酶促进肝脏前体细胞和肝癌细胞生长的可能性。为了验证这些假说，我们将在体内操纵Rb和E2F家族成员、p21和CDK2、Notch通路成员和EZH2在成人肝祖细胞、成熟肝细胞和肝癌细胞中的活性。对突变小鼠的这些实验将得到人体肝细胞体外分析的补充。我们的研究将确定诊断、检测和治疗肝细胞癌的新方法。此外，由于Rb途径的遗传、表观遗传和/或病毒失活在人类癌细胞中几乎是普遍的事件，这些研究通常与广泛的癌症患者相关。

DESCRIPTION (provided by applicant): More than 600,000 people die from Hepatocellular carcinoma (HCC) worldwide annually. The environmental factors that cause HCC are well known and include infection by hepatitis B and C viruses (HBV and HCV), exposure to Aflatoxin B1, and excessive alcohol intake. However, no effective treatments exist for HCC and the prognosis of HCC patients is usually poor, with an overall median survival of less than one year. The RB tumor suppressor and its family member's p107 and p130 are functionally inactivated in nearly all cases of human HCC. This inactivation is due to increased CDK4 kinase activity resulting from the silencing of the CDK4 inhibitor p16 or from increased expression of the CDK4 partner Cyclin D1. In addition, some proteins produced by HBV and HCV can inactivate RB family members, including by triggering their degradation. We generated a mouse model for human HCC by deleting RB family genes in the liver of adult mice to model this functional inactivation of RB family proteins. RB/p107/p130 triple knockout (TKO) mice develop liver tumors whose histology and gene expression profiles resemble human HCCs. The activity of E2F transcription factors, which are normally inhibited by the RB family, is high in TKO HCC cells. TKO HCCs initiate from progenitor cells and not hepatocytes, which do not divide due to unknown mechanisms that suppress their proliferation. The TKO HCC model provides a unique in vivo system to query the mechanisms of tumorigenesis in the liver and to specifically interrogate how RB/E2F transcriptional regulatory complexes control HCC development. Our general hypothesis is that inactivation of the RB pathway drives cancer initiation at least in part by deregulating E2F activity and compromising the balance between regulatory networks in acutely sensitive cell populations. Specifically, we propose that increased E2F activity engages pathways that promote the expansion of mutant cells, including liver progenitors, but also triggers negative feedback loops preventing cancer initiation from mature hepatocytes and limiting the growth of HCC cells. We will first test the specific hypothesis that activation of p21 by E2F in the TKO model blocks the proliferation of hepatocytes, thereby preventing HCC initiation from these mature cells. Next, we will test the idea that activation of Notch signaling y E2F limits the expansion of HCC cells during tumorigenesis. Finally, we will examine the possibility that activation of the EZH2 methyltransferase by E2F promotes the growth of both liver progenitors and HCC cells. To test these hypotheses, we will manipulate the activity of RB and E2F family members, p21 and CDK2, Notch pathway members, and EZH2 in adult liver progenitor cells, mature hepatocytes, and HCC cells in vivo. These experiments in mutant mice will be complemented by analyses of human liver cells ex vivo. Our studies will identify novel means to diagnose, detect, and treat HCC. In addition, because genetic, epigenetic, and/or viral inactivation of the RB pathway is a nearly universal event in human cancer cells, these studies are generally relevant to a broad cross-section of cancer patients.