Telomere dysfunction and telomerase reactivation in the etiology and progression of liver cancer

项目总结/摘要 端粒酶突变和端粒磨损是肝纤维化及其进展的主要危险因素 肝细胞癌（HCC）。然而，由于缺乏适当的模式和内在的困难， 研究人类端粒酶在生理相关的细胞，分子机制，负责肝脏 在由短端粒引起的DNA损伤的情况下的纤维化和癌症仍然是难以捉摸的。而端粒酶 基因敲除小鼠证实了端粒维持和DNA修复对肝功能的重要性， 在端粒受损的患者中，控制肝脏异常的分子机制仍然未知。 同样地，在端粒缩短后触发肝细胞衰竭的特定信号通路， DNA损伤的累积仍有待确定。此外，在启动子区的突变， 端粒酶逆转录酶组分（TERT）已被描述为初始的和最普遍的 HCC中的突变。虽然这些突变已被证明可以重新激活端粒酶，但端粒酶的功能相关性仍有待进一步研究。 肝细胞衰竭和转化过程中的这一过程还有待研究。 这项提案的重点是利用人类多能干细胞作为一个新的平台，以了解 突变的端粒酶、端粒缩短和DNA损伤的积累在不同的细胞中的有害作用， 肝细胞谱系。我们先前已经产生了具有几种疾病特异性的同源hPSC系。 端粒酶突变，并成功地衍生出端粒酶突变的人肝细胞和肝细胞。 星状细胞在体外，以下建立的协议，概括了这些谱系的体内发展。 在这里，提出了两个具体的目标，利用这个平台来了解分子的后果 端粒侵蚀，DNA损伤和端粒酶损伤对肝细胞功能的影响，并确定 在转型初期的作用。在目标1中，我们将确定端粒缩短的作用， 端粒酶受损的不同肝细胞系纤维化失败期间DNA损伤累积。我们 将决定DNA损伤的减轻、HNF 4 α的重新激活和p53的调节在多大程度上阻止了 端粒长度可变的端粒酶突变肝细胞中的纤维化触发。肝纤维化及其 进展为HCC是多细胞反应，我们将确定进行性端粒缩短的作用 在肝星状细胞的直接和旁分泌纤维化激活过程中。在目标2中，我们将研究 在HCC进展过程中，TERT启动子区突变的分子后果， 加剧了端粒受损造成的DNA损伤具体来说，我们将分析生物化学和功能 TERT启动子区突变对肝细胞功能和永生化的后果。 这些研究将确定肝纤维化的分子机制及其向HCC的进展， 突变端粒酶和DNA损伤的设置。我们独特的工具，结合我们在端粒酶方面的专业知识， DNA修复和干细胞生物学使我们处于一个理想的位置，在这一领域产生重大影响。

PROJECT SUMMARY/ABSTRACT Mutations in telomerase and telomere attrition are major risk factors for liver fibrosis and its progression to hepatocellular carcinoma (HCC). However, due to a lack of adequate models and intrinsic difficulties in studying human telomerase in physiologically relevant cells, the molecular mechanisms responsible for liver fibrosis and cancer in settings of DNA damage arising from short telomeres remain elusive. While telomerase knockout mice corroborate the importance of telomere maintenance and DNA repair for liver function, the molecular mechanisms that govern liver abnormalities in patients with damaged telomeres are still unknown. Likewise, the specific signaling pathways that trigger failure of hepatic cells following telomere shortening and accumulation of DNA damage remain to be determined. In addition, mutations in the promoter region of the telomerase reverse transcriptase component (TERT) have been described as the initial and most prevalent mutation in HCC. While these mutations have been shown to reactivate telomerase, the functional relevance of this process during failure and transformation of hepatic cells has yet to be interrogated. The focus of this proposal is to use human pluripotent stem cells as a novel platform to understand the detrimental effects of mutant telomerase, telomere shortening and accumulation of DNA damage in different hepatic cell lineages. We have previously generated isogenic hPSC lines harboring several disease-specific mutations in telomerase and have successfully derived telomerase-mutant human hepatocytes and hepatic stellate cells in vitro, following established protocols that recapitulate the in vivo development of these lineages. Here, two specific aims are proposed that utilize this platform to understand the molecular consequences of telomere erosion, DNA damage, and telomerase impairment for the function of hepatic cells, and to determine their role during early stages of transformation. In Aim 1 we will determine the role of telomere shortening and DNA damage accumulation during fibrotic failure of different hepatic cell lineages with impaired telomerase. We will determine the extent to which mitigation of DNA damage, reactivation of HNF4α, and modulation p53 prevent fibrotic triggering in telomerase-mutant hepatocytes with variable telomere lengths. As liver fibrosis and its progression to HCC are multicellular responses we will determine the role of progressive telomere shortening during the direct and the paracrine fibrotic activation of hepatic stellate cells. In Aim 2, we will investigate the molecular consequences of mutations in the TERT promoter region during progression of HCC, in settings of exacerbated DNA damage due to eroded telomeres. Specifically, we will analyze the biochemical and functional consequences of mutations in the TERT promoter region for hepatocyte function and immortalization. These studies will determine the molecular mechanisms of liver fibrosis and its progression to HCC in settings of mutant telomerase and DNA damage. Our unique tools, combined with our expertise in telomerase, DNA repair, and stem cell biology puts us in an ideal position to make a significant impact in this field.