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

项目摘要/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 至肝细胞癌(HCC)。然而，由于缺乏适当的模型和内部困难， 在生理相关细胞中研究人类端粒酶，研究与肝脏有关的分子机制 短端粒引起的DNA损伤导致的纤维化和癌症仍然难以捉摸。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， 端粒受损患者肝脏异常的分子机制仍不清楚。 同样，端粒缩短和端粒缩短后触发肝细胞衰竭的特定信号通路也是如此。 DNA损伤的累积仍有待确定。此外，该基因启动子区域的突变 端粒酶逆转录酶组件(TERT)已被描述为最早也是最普遍的 肝细胞癌中的突变。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 肝细胞衰竭和转化过程中的这一过程还有待研究。 这一提议的重点是利用人类多能干细胞作为一个新的平台来了解 突变的端粒酶、端粒缩短和DNA损伤在不同疾病中的不利影响 肝细胞系。我们之前已经产生了含有几种疾病特异性的等基因hPSC系 端粒酶的突变和成功获得端粒酶突变的人肝细胞和肝细胞 在体外培养星状细胞，遵循概括这些血统在体内发展的既定方案。 在这里，提出了两个具体的目标，利用这个平台来理解分子后果 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 它们在转型的早期阶段所起的作用。在目标1中，我们将确定端粒缩短和 端粒酶受损的不同肝细胞系纤维化过程中DNA损伤的积累。我们 将决定在多大程度上减轻dna损伤、重新激活hnf4α和调节p53 端粒长度可变的端粒酶突变肝细胞中的纤维化触发。AS肝纤维化及其临床意义 进展到肝癌是多细胞反应，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激活过程中。在目标2中，我们将调查 肝细胞癌进展过程中TERT启动子区域突变的分子后果 加剧了端粒侵蚀造成的DNA损伤。具体地说，我们将分析生化和功能 TERT启动子区域突变对肝细胞功能和永生化的影响。 这些研究将确定肝纤维化及其进展为肝细胞癌的分子机制 突变端粒酶的设置与DNA损伤。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，可以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.