组蛋白去乙酰化酶6（HDAC6）在肿瘤发生和发展过程中的作用及机制研究

Histone deacetylase 6(HDAC6), a unique member of the mammalian HDAC family whose major substrates are non-histone proteins, has been involved in regulating several important cellular processes which are closely associated with tumor initiation and development, such as regulating cell skeleton, cellular response to various stress, transportation and degradation of ubiquitinated proteins, etc. This, together with the fact that overexpression of HDAC6 has been identified in numbers of human tumors, all suggest HDAC6 as a novel target for tumor therapy. Unfortunately, until now, there are only limited numbers of animal studies on HDAC6’s in vivo function in tumorigenesis. The aim of the current proposal is to dissect the roles and mechanisms of HDAC6 in tumor initiation and development, using a K-ras mutant driven mouse lung cancer model. First, we will investigate the effect of genetic ablation of HDAC6 on initiation and progress of mutant K-ras induced mouse lung cancer in vivo. We also have generated several HDAC6 knockin strains in which mutations have been introduced in different function domains of HDAC6 (e.g. both hdac catalytic domains and Znf-UBP domain ) . Those mice will be crossed to K-ras mutant strain to dissect the molecular mechanism by which HDAC6 is involved in tumorigenesis. In addition, we will also investigate the potential contribution of deacetylation of various known HDAC6’s substrates and their downstream pathways in tumorigenesis. Finally, we will extend our studies to preclinical models by testing the effect of HDAC6-specific small molecular inhibitors such as ACY-1215 in treating K-ras mutant induced mouse lung cancer.

组蛋白去乙酰化酶6（HDAC6）是HDAC家族的一个独特的成员，其主要作用底物是非组蛋白分子，参与很多重要细胞过程调控（如：调控细胞骨架结构，参与细胞对胁迫的应答，调控泛素化蛋白的降解等），这些都与肿瘤发生和发展密切相关。同时，HDAC6在多种肿瘤细胞中高表达，使得其可能成为抗肿瘤药物的新靶点。然而，迄今为此，运用动物模型系统性研究HDAC6在癌症中的功能机制却很少。本研究将以K-ras突变诱导的肺癌小鼠为模型，研究HDAC6在肿瘤发生和发展过程中的作用及机制。我们将首先研究小鼠中敲除HDAC6对肺癌的影响。我们也建立了不同品系的HDAC6点突变小鼠，用于研究HDAC6特定功能域影响肺癌的分子机制。我们还将进一步研究HDAC6是如何通过调控其去乙酰化底物和信号传导参与影响肺癌发生和发展的。在揭示HDAC6缺失抑制肿瘤的分子基础上，我们将探索利用特异靶向HDAC6的小分子抑制剂治疗肺癌的可能

组蛋白去乙酰化酶6(HDAC6)是HDAC家族的一个独特的成员,其主要底物是非组蛋白分子。其可能以正调控或负调控的方式参与多种肿瘤的发生和发展，但运用动物模型系统性研究HDAC6在癌症中的功能和机制却很少。本项目以K-ras-G12D突变条件性诱导小鼠肺癌为模型（简称KRAS肺癌），研究HDAC6在肿瘤发生和发展过程中的作用及分子和细胞机制，并为小分子抑制剂靶向HDAC6治疗KRAS肺癌提供了重要的临床前实验基础。. 通过把HDAC6基因敲除小鼠与不同肿瘤模型杂交，首次发现HDAC6敲除对KRAS肺癌与对照相比肿瘤数目明显减少，且病变程度低。利用CRISPR/Cas9建立了在HDAC6活性域(DD1或DD2)敲入突变使内源性HDAC6失活但其蛋白还正常表达的小鼠。结果发现，DD1和DD2分别突变导致了与HDAC6蛋白缺失类似的KRAS肺癌抑制，提示HDAC6的催化活性是其影响KRAS肺癌的主要原因。. 进一步研究HDAC6是如何通过调控其去乙酰化底物和信号传导参与影响肺癌发生和发展的，利用遗传学方法对其已知底物p53及微管蛋白进行了研究，也正在使用乙酰化蛋白质组和单细胞测序手段进行大规模组学分析。发现与p53缺失小鼠杂交后，与野生型一样，HDAC6敲除小鼠中KRAS肺癌肿瘤数目也显著增加。与微管蛋白乙酰化酶ATAT1敲除小鼠杂交的结果，提示HDAC6和ATAT1的共同底物可能参与了KRAS诱导肺癌发生。还构建了HDAC6条件性敲除小鼠，以研究HDAC6是直接在肿瘤细胞中还是在其他细胞中间接发挥抑制肿瘤的作用。. 发现Ricolinostat（HDAC6小分子抑制剂，ACY-1215）可以抑制K-ras-G12D驱动的肺癌，但在HDAC6敲除小鼠中没有影响，证明了该抑制剂的特异性。后续实验发现，该抑制剂对小鼠KRAS肺部肿瘤发生的影响作用于肿瘤发生早期而不是晚期。