Supplementary Material for: Ubiquitin-Specific Protease 8 Mutant Corticotrope Adenomas Present Unique Secretory and Molecular Features and Shed Light on the Role of Ubiquitylation on ACTH Processing

Background: Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have recently been shown to occur in ACTH-secreting pituitary adenomas, thus calling attention to the ubiquitin system in corticotrope adenomas. Objectives: Assess the consequences of USP8 mutations and establish the role of ubiquitin on ACTH turnover in human ACTH-secreting pituitary adenomas. Methods: USP8 mutation status was established in 126 ACTH-secreting adenomas. Differences in ACTH secretion and POMC expression from adenoma primary cultures and in microarray gene expression profiles from archival specimens were sought according to USP8 sequence. Ubiquitin/ACTH coimmunoprecipitation and incubation with MG132, a proteasome inhibitor, were performed in order to establish whether ubiquitin plays a role in POMC/ACTH degradation in corticotrope adenomas. Results: USP8 mutations were identified in 29 adenomas (23%). Adenomas presenting USP8 mutations secreted greater amounts of ACTH and expressed POMC at higher levels compared to USP wild-type specimens. USP8 mutant adenomas were also more sensitive to modulation by CRH and dexamethasone in vitro. At microarray analysis, genes associated with endosomal protein degradation and membrane components were downregulated in USP8 mutant adenomas as were AVPR1B, IL11RA, and PITX2. Inhibition of the ubiquitin-proteasome pathway increased ACTH secretion and POMC itself proved a target of ubiquitylation, independently of USP8 sequence status. Conclusions: Our study has shown that USP8 mutant ACTH-secreting adenomas present a more “typical” corticotrope phenotype and reduced expression of several genes associated with protein degradation. Further, ubiquitylation is directly involved in intracellular ACTH turnover, suggesting that the ubiquitin-proteasome system may represent a target for treatment of human ACTH-secreting adenomas.

研究背景：近期研究证实，泛素特异性蛋白酶8（ubiquitin-specific protease 8, USP8）基因体细胞突变可发生于促肾上腺皮质激素（adrenocorticotropic hormone, ACTH）分泌型垂体腺瘤中，由此引发学界对促肾上腺皮质激素腺瘤中泛素系统的关注。
研究目的：评估USP8突变的生物学效应，并明确泛素在人ACTH分泌型垂体腺瘤中对ACTH周转代谢的调控作用。
研究方法：本研究对126例ACTH分泌型垂体腺瘤样本进行USP8突变状态检测。根据USP8基因序列分型，对比分析腺瘤原代培养样本中ACTH分泌水平与阿黑皮素原（pro-opiomelanocortin, POMC）表达差异，以及存档标本的基因芯片表达谱。通过泛素/ACTH免疫共沉淀实验，以及使用蛋白酶体抑制剂MG132进行孵育，验证泛素是否参与促肾上腺皮质激素腺瘤中POMC/ACTH的降解过程。
研究结果：本研究在29例腺瘤中检测到USP8突变，检出率为23%。与USP8野生型腺瘤相比，携带USP8突变的腺瘤分泌更多ACTH，且POMC表达水平更高。体外实验显示，USP8突变腺瘤对促肾上腺皮质激素释放激素（corticotropin-releasing hormone, CRH）与地塞米松的调控敏感性更强。基因芯片分析结果表明，USP8突变腺瘤中与内体蛋白降解、膜组分相关的基因表达下调，同时精氨酸加压素受体1B（arginine vasopressin receptor 1B, AVPR1B）、白细胞介素11受体亚基α（interleukin 11 receptor subunit alpha, IL11RA）以及成对样同源盒转录因子2（paired-like homeodomain transcription factor 2, PITX2）的表达也出现降低。抑制泛素-蛋白酶体通路可提升ACTH分泌水平，且POMC本身可作为泛素化修饰的靶标，该现象与USP8序列状态无关。
研究结论：本研究证实，携带USP8突变的ACTH分泌型垂体腺瘤呈现更典型的促肾上腺皮质激素表型，且多种与蛋白质降解相关的基因表达水平降低。此外，泛素化修饰直接参与细胞内ACTH的周转代谢过程，提示泛素-蛋白酶体系统可作为人ACTH分泌型垂体腺瘤的潜在治疗靶点。