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<p>Cushing’s disease (CD) is a neuroendocrine disorder characterized by excessive adrenocorticotropic hormone (ACTH) secretion from pituitary adenomas, closely associated with ubiquitin-specific protease 8 (USP8) mutations. This study aimed to investigate whether USP8 mutations activate the Sonic Hedgehog (SHH) pathway by deubiquitinating its key component Smoothened (SMO), thereby promoting ACTH production in CD. mRNA expression profiles of USP8-mutated and wild-type CD patients (GEO dataset GSE132982) were analyzed for differentially expressed genes, followed by GO and KEGG pathway enrichment. USP8 mutant/wild-type plasmids were transfected into HeLa cells. Co-immunoprecipitation and Western blot evaluated USP8-SMO interaction and SMO ubiquitination. RT-qPCR, luciferase reporter assays, and nuclear translocation analysis assessed SHH pathway gene expression and GLI2 transcriptional activity. Immunohistochemistry detected SMO/SHH proteins in 9 USP8-mutated and 9 wild-type pituitary adenomas. Primary tumor cells were treated with the SMO inhibitor cyclopamine, and POMC mRNA/ACTH secretion were measured by RT-qPCR/ELISA. Chromatin immunoprecipitation (ChIP) verified GLI2 binding to the POMC promoter. SHH pathway genes were significantly dysregulated in USP8-mutated CD. Mutant USP8 enhanced SMO interaction, reduced SMO ubiquitination, and upregulated SHH target genes, promoting GLI2 nuclear translocation/activity. SMO-positive cells were more frequent in mutated tumors, with unchanged SHH expression. Cyclopamine suppressed POMC/ACTH specifically in mutated cells, and ChIP confirmed GLI2-POMC promoter binding. USP8 mutations activate the SHH pathway via SMO deubiquitination, driving GLI2-mediated POMC transcription and excessive ACTH secretion in CD. Targeting the SHH pathway may offer a novel therapeutic strategy for USP8-mutated Cushing’s disease.</p>

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The Sonic Hedgehog Pathway is Activated in Cushing’s Disease with USP8 Mutations

  • 期刊论文

  • 发表日期：

    2026年5月8日

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  • Shan Huang,
  • Yihua Xu,
  • Xiang Zhou,
  • Jiahuan Lu,
  • Xiaoyun Cao

摘要

Cushing’s disease (CD) is a neuroendocrine disorder characterized by excessive adrenocorticotropic hormone (ACTH) secretion from pituitary adenomas, closely associated with ubiquitin-specific protease 8 (USP8) mutations. This study aimed to investigate whether USP8 mutations activate the Sonic Hedgehog (SHH) pathway by deubiquitinating its key component Smoothened (SMO), thereby promoting ACTH production in CD. mRNA expression profiles of USP8-mutated and wild-type CD patients (GEO dataset GSE132982) were analyzed for differentially expressed genes, followed by GO and KEGG pathway enrichment. USP8 mutant/wild-type plasmids were transfected into HeLa cells. Co-immunoprecipitation and Western blot evaluated USP8-SMO interaction and SMO ubiquitination. RT-qPCR, luciferase reporter assays, and nuclear translocation analysis assessed SHH pathway gene expression and GLI2 transcriptional activity. Immunohistochemistry detected SMO/SHH proteins in 9 USP8-mutated and 9 wild-type pituitary adenomas. Primary tumor cells were treated with the SMO inhibitor cyclopamine, and POMC mRNA/ACTH secretion were measured by RT-qPCR/ELISA. Chromatin immunoprecipitation (ChIP) verified GLI2 binding to the POMC promoter. SHH pathway genes were significantly dysregulated in USP8-mutated CD. Mutant USP8 enhanced SMO interaction, reduced SMO ubiquitination, and upregulated SHH target genes, promoting GLI2 nuclear translocation/activity. SMO-positive cells were more frequent in mutated tumors, with unchanged SHH expression. Cyclopamine suppressed POMC/ACTH specifically in mutated cells, and ChIP confirmed GLI2-POMC promoter binding. USP8 mutations activate the SHH pathway via SMO deubiquitination, driving GLI2-mediated POMC transcription and excessive ACTH secretion in CD. Targeting the SHH pathway may offer a novel therapeutic strategy for USP8-mutated Cushing’s disease.