The RB pathway in liver cancer

描述（由申请人提供）： 全世界每年有超过60万人死于肝细胞癌（HCC）。引起HCC的环境因素是众所周知的，包括B和丙型肝炎病毒（HBV和HCV）感染，暴露于黄曲霉毒素B1和过量饮酒。然而，对于HCC没有有效的治疗方法，并且HCC患者的预后通常很差，总体中位生存期不到一年。 RB肿瘤抑制因子及其家族成员的p107和p130在几乎所有人HCC病例中功能失活。这种失活是由于CDK 4抑制剂p16的沉默或CDK 4伴侣细胞周期蛋白D1的表达增加导致的CDK 4激酶活性增加。此外，由HBV和HCV产生的一些蛋白质可以破坏RB家族成员，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家族基因来模拟RB家族蛋白的功能性失活，从而生成了人肝癌的小鼠模型。RB/p107/p130三重敲除（TKO）小鼠发生肝肿瘤，其组织学和基因表达谱类似于人HCC。通常被RB家族抑制的E2 F转录因子的活性在TKO HCC细胞中很高。TKO HCC起源于祖细胞而不是肝细胞，由于抑制其增殖的未知机制，肝细胞不分裂。TKO HCC模型提供了一个独特的体内系统来查询肝脏中肿瘤发生的机制，并具体询问RB/E2 F转录调控复合物如何控制HCC的发展。 我们的一般假设是RB通路的失活至少部分地驱动了癌症的发生 通过解除E2 F活性的调节并损害急性敏感细胞群体中调节网络之间的平衡。具体来说，我们提出E2 F活性的增加参与了促进突变细胞（包括肝祖细胞）扩增的途径，但也触发了负反馈回路，阻止癌症从成熟肝细胞开始并限制HCC细胞的生长。我们将首先检验p21的激活 TKO模型中的E2 F阻断了肝细胞的增殖，从而阻止了HCC从这些成熟细胞开始。接下来，我们将测试Notch信号转导y E2 F的激活限制肿瘤发生期间HCC细胞的扩增的想法。最后，我们将研究E2 F激活EZH 2甲基转移酶促进肝祖细胞和HCC细胞生长的可能性。为了验证这些假设，我们将在体内操纵RB和E2 F家族成员、p21和CDK 2、Notch通路成员和EZH 2在成人肝祖细胞、成熟肝细胞和HCC细胞中的活性。突变小鼠中的这些实验将通过离体人肝细胞分析来补充。 我们的研究将确定诊断、检测和治疗HCC的新方法。此外，由于RB途径的遗传、表观遗传和/或病毒失活几乎是人类癌细胞中的普遍事件，因此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.