Hsp90调控USP8通路在ACTH型垂体瘤中的作用机制研究

Cushing Disease (CD) is ACTH-produced pituitary adenomas that occur predominantly in young adults and puberty with high immortality. However, its pathological mechanism is still not clear. Our preliminary data demonstrate that, 1, mutation of USP8 (deubiqutinase) is closely associated with CD. 62.5% CD patients have the USP8 mutation. Mutation of USP8 can induce EGFR protein stabilization and signal transduction leading upregulation of the ACTH expression; 2, Hsp90 and Hsp70 are upregulated in the CD tumor tissues compared to the parental control; 3, the results of Gst-pull down assay indicate that USP8 can interact with Hsp90 in the ACTH-type cell line. We therefore, hypothesize accordingly that mutation USP8 could be activated by Hsf1-meidated heat shock response, which lead to CD tumor growth. In this grant application, we will focus on: 1, dissection the regulatory mechanism of Hsp90 on mutation USP8; 2, the upregulation of the expression of ACTH and EGFR by Hsp90 pathway in ACTH tumor cell lines. Our aim is to dissect the key regulatory association between Hsf90 and USP8mutation on ACTH tumorigenesis and being targets for potential therapy of ACTH pituitary adenomas.

ACTH型垂体瘤(库欣病)是一种严重威胁青壮年人生命的神经内分泌疾病，其发病机制仍不太清楚。我们前期研究发现：①USP8(去泛素化酶8)基因突变与ACTH型垂体瘤发生密切相关，62.5%的病人携带USP8(突变位点)；②Hsp90、Hsp70和Hsf1在人ACTH垂体瘤组织中表达升高；③USP8蛋白与Hsp90蛋白能体外结合。由此，我们推测Hsp90参与USP8突变介导的ACTH型垂体瘤生长调控。为此，本研究将重点研究：①Hsp90对USP8基因突变的调控机制；②Hsp90上调垂体EGFR信号通路和ACTH产生的机制。本研究将揭示ACTH型垂体瘤中Hsp90调控USP8基因通路的关键机制，为临床靶向治疗提供理论依据。

本课题前期工作揭示USP8基因突变与库欣病发生有明显的相关关系，并且发现USP8参与调控EGFR蛋白质稳定和信号通路传导调控。已报道Hsp90参与多种肿瘤细胞内EGFR蛋白稳定和生物活性，我们前期结果显示Hsp90 在ACTH垂体瘤组织内表达增高，并且Hsp90与USP8蛋白间存在相互作用。因此推测Hsp90可能通过与USP8蛋白结合，动态调控ACTH垂体瘤细胞中EGFR的活性，继而促进垂体瘤的生长。因此本课题聚焦于明确Hsp90在ACTH垂体瘤组织中的表达量以及与USP8突变的相关性，应用鼠AtT20垂体瘤细胞系，检测Hsp90对垂体瘤细胞分泌及USP8通路的调控机制。1）明确Hsp90在ACTH垂体瘤组织中的表达量以及与USP8突变的相关性:利用华山医院神经外科大样本、标准化的垂体瘤标本库收集的ACTH垂体瘤标本，按USP8突变及野生进行分类，分别用免疫印迹、定量RT-PCR和免疫组化测定Hsp90的表达。2）应用鼠AtT20垂体瘤细胞系，观察Hsp90对垂体瘤细胞分泌的调控。①抑制Hsp90蛋白表达对垂体瘤细胞体外生长的抑制作用：应用shRNA 或热休克蛋白抑制剂（17-AAG）抑制鼠AtT20垂体瘤细胞系中的Hsp90活性，检测Hsp90对垂体瘤细胞生长，细胞迁徙的影响，细胞凋亡的调控作用，并对下游EGFR，POMC及ACTH的表达量的改变。根据上述体外功能验证实验结果，初步探索Hsp90对库欣病USP8通路调控的分子机制及Hsp90受抑制后对ACTH的调控机制。