TERT Promoter Mutations and Telomerase Reactivation in Cancer Cells

摘要 端粒酶是一种RNA-蛋白质复合体，它复制线形染色体的末端(端粒) 人类和其他真核生物。端粒的维持是细胞持续增殖所必需的，而且在 大多数人类癌症是通过重新激活端粒酶催化亚单位的基因来实现的 (端粒酶逆转录酶或TERT)。重新激活的触发因素一直是个谜，直到2013年， 两个研究小组报告了TERT基因启动子的突变存在于多种肿瘤类型中，但 而不是在邻近的健康组织中。最近我们报道了这些TERT启动子突变确实是 与较高的TERT蛋白产量、较高的端粒酶活性和较长的端粒有关 尿路上皮癌(UC)，患者存活率低。然而，人们对此知之甚少。 启动子突变导致TERT激活的分子机制阻碍了进展 在基础科学和临床应用方面都是如此。现在，我们打算解开TERT的机制 UC和肝细胞癌(HCC)细胞系的激活。这些细胞系中的许多都是杂合子 TERT启动子突变--即细胞中的两个TERT基因中只有一个有突变。因此，我们将 比较这两个等位基因以测试RNA聚合酶II的占有率，表观遗传沉默的标志， 和mRNA的产生，允许关于TERT基因沉默和重新激活的特定假设 测试过。为了超越关联并测试因果关系，我们将使用CRISPR-Cas9基因组编辑来插入 或者清除TERT启动子突变，并评估这是否足以改变TERT的产生。如果 启动子突变还不够，我们会灭活组蛋白甲基转移酶PRC2等因子 (它增加了抑制性染色质标记)通过击倒或通过添加有效的小分子 抑制剂和复活测试。此外，生物信息学(基因表达数据)揭示了 其他参与基因再激活的成分将被测试。这个项目的长期目标是 了解被抑制和激活的TERT基因的状态以及参与端粒酶的步骤 在癌症中重新激活。这项基础科学研究将提供有关癌症生物学的信息，并可能 提供与临床相关的信息。

Summary Telomerase is an RNA-protein complex that replicates the very ends (telomeres) of the linear chromosomes of humans and other eukaryotes. Telomere maintenance is required for continuous proliferation of cells, and in most human cancers this is achieved by reactivating the gene for the catalytic subunit of telomerase (telomerase reverse transcriptase or TERT). The trigger for reactivation remained mysterious until 2013, when two research groups reported mutations in the promoter of the TERT gene present in multiple tumor types but not in adjacent healthy tissue. Recently we reported that these TERT promoter mutations were indeed associated with higher production of TERT protein, higher telomerase enzyme activity, and longer telomeres in urothelial cancer (UC), and associated with poor patient survival. However, very little is known about the molecular mechanisms by which the promoter mutations lead to TERT activation, which has impeded progress both in basic science and in clinical applications. Now we propose to unravel the mechanisms of TERT activation in UC and hepatocellular carcinoma (HCC) cell lines. Many of these cell lines are heterozygous for the TERT promoter mutation – i.e., only one of the two TERT genes in the cell has the mutation. Thus, we will compare the two alleles to test for differences in RNA polymerase II occupancy, marks of epigenetic silencing, and production of mRNA, allowing a specific hypothesis about TERT gene silencing and reactivation to be tested. To move beyond association and test for causation, we will use CRISPR-Cas9 genome editing to insert or erase TERT promoter mutations and assess whether this is sufficient to switch TERT production. If the promoter mutation is not sufficient, we will inactivate factors such as the histone methyltransferase PRC2 (which adds repressive chromatin marks) either by knockdown or by adding a validated small-molecule inhibitor and test for reactivation. Furthermore, bioinformatics (gene expression data) reveals candidates for other components involved in gene reactivation that will be tested. The long-term goal of this project is to understand the state of the repressed and activated TERT genes and the steps involved in telomerase reactivation in cancer. This basic science study will provide information about cancer biology and is likely to give information that is clinically relevant.