High-throughput Screening for Cushing Disease: Therapeutic Potential of Thiostrepton via Cell Cycle Regulation

• Published in: Endocrinology (Philadelphia) Vol. 165; no. 9
• Main Authors: Hakata, Takuro, Yamauchi, Ichiro, Kosugi, Daisuke, Sugawa, Taku, Fujita, Haruka, Okamoto, Kentaro, Ueda, Yohei, Fujii, Toshihito, Taura, Daisuke, Inagaki, Nobuya
• Format: Journal Article
• Language: English
• Published: US Oxford University Press 26.07.2024
• Subjects: Adrenocorticotropic hormone; Anticancer properties; Antitumor activity; Antitumor agents; Assaying; Bioactive compounds; Biosensors; Brain tumors; Cell culture; Cell cycle; Cushing syndrome; Cushing's disease; Cyclin-dependent kinase; Cyclin-dependent kinase 4; Cyclin-dependent kinases; Cyclins; Down-regulation; Flow cytometry; Fluorescence; High-throughput screening; Image acquisition; In vivo methods and tests; Kinases; Nervous system diseases; Neuroendocrine system; Neuroendocrine tumors; Pharmacology; Pituitary; Secretion; Thiostrepton; Transcriptomes; Xenotransplantation; thiostrepton; CDK inhibitor; Cushing disease; cell cycle; high-throughput screening
• ISSN: 1945-7170; 0013-7227; 1945-7170
• DOI: 10.1210/endocr/bqae089

Abstract Cushing disease is a life-threatening disorder caused by autonomous secretion of ACTH from pituitary neuroendocrine tumors (PitNETs). Few drugs are indicated for inoperative Cushing disease, in particular that due to aggressive PitNETs. To explore agents that regulate ACTH-secreting PitNETs, we conducted high-throughput screening (HTS) using AtT-20, a murine pituitary tumor cell line characterized by ACTH secretion. For the HTS, we constructed a live cell–based ACTH reporter assay for high-throughput evaluation of ACTH changes. This assay was based on HEK293T cells overexpressing components of the ACTH receptor and a fluorescent cAMP biosensor, with high-throughput acquisition of fluorescence images. We treated AtT-20 cells with compounds and assessed ACTH concentrations in the conditioned media using the reporter assay. Of 2480 screened bioactive compounds, over 50% inhibition of ACTH secreted from AtT-20 cells was seen with 84 compounds at 10 μM and 20 compounds at 1 μM. Among these hit compounds, we focused on thiostrepton (TS) and determined its antitumor effects in both in vitro and in vivo xenograft models of Cushing disease. Transcriptome and flow cytometry analyses revealed that TS administration induced AtT-20 cell cycle arrest at the G2/M phase, which was mediated by FOXM1-independent mechanisms including downregulation of cyclins. Simultaneous TS administration with a cyclin-dependent kinase 4/6 inhibitor that affected the cell cycle at the G0/1 phase showed cooperative antitumor effects. Thus, TS is a promising therapeutic agent for Cushing disease. Our list of hit compounds and new mechanistic insights into TS effects serve as a valuable foundation for future research.