Sonic Hedgehog pathway may be target in Cushing’s disease
USP8 mutations may activate signaling tied to ACTH production
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Mutations in the USP8 gene appear to switch on a signaling pathway, called Sonic Hedgehog (SHH), in pituitary tumors that cause Cushing’s disease, according to a study involving patient tumor data and laboratory cell experiments. These results suggest that targeting this pathway could help guide future treatment research for patients whose tumors carry such mutations.
The study, “The Sonic Hedgehog Pathway is Activated in Cushing’s Disease with USP8 Mutations,” was published in Biochemical Genetics by researchers in China.
Cushing’s disease is caused by tumors in the pituitary gland at the base of the brain. These tumors produce excess adrenocorticotropic hormone (ACTH), which signals the adrenal glands to release cortisol. In excess, cortisol can lead to a range of symptoms. Some ACTH-producing pituitary tumors in Cushing’s disease carry mutations in the USP8 gene, but their effects are not fully understood.
Study examines Sonic Hedgehog signaling
This study tested whether mutations in the USP8 gene can activate the SHH signaling pathway, which regulates cell growth and development. Gene expression — which genes are switched on or off — was compared using a dataset from 12 people with Cushing’s disease, including eight without and four with USP8 mutations.
A total of 730 genes were differentially expressed between the two groups: 436 were more active (upregulated), and 294 were less active (downregulated) in tumors with USP8 mutations compared with those without such mutations. Many of the upregulated genes were linked to SHH signaling, suggesting that the pathway may be abnormally activated in USP8-mutated tumors.
The researchers then focused on Smoothened (SMO), a protein that controls the SHH signaling pathway. In the lab, both normal (wild-type) and mutant USP8 proteins could bind to SMO, but mutant USP8 interacted more strongly. USP8 is a deubiquitinating enzyme, meaning it removes ubiquitin, a molecular “tag” that normally marks proteins for degradation. Mutant USP8 removed more ubiquitin from SMO, which was associated with activation of the SHH signaling pathway.
As a result, several genes linked to SHH signaling were upregulated in lab cells expressing mutant USP8. The researchers also observed increased GLI2 activity, a transcription factor that switches genes on and off after SHH is activated. In cells expressing mutant USP8, GLI2 levels increased in the nucleus, the cell’s control center, allowing it to activate target genes more effectively.
Human tumors support lab findings
To confirm these findings in human tumors, the researchers examined nine tumor samples with USP8 mutations and nine without. Tumors with USP8 mutations contained significantly more SMO-positive cells than wild-type tumors. The average proportion of SMO-positive cells was more than twice as high as in tumors without mutations (0.316 vs. 0.142).
To test whether blocking SMO could reduce ACTH production, the researchers treated tumor cells from four patients with cyclopamine, an SMO inhibitor. Genetic testing showed that two tumors carried mutations in USP8 while two did not. In tumors with USP8 mutations, cyclopamine appeared to reduce levels of both ACTH and POMC, a precursor from which ACTH is produced, but the sample size was too small for statistical analysis.
Further tests showed that GLI2 bound directly to the promoter region of the POMC gene. A promoter is a DNA sequence that controls whether a gene is switched on. Among three predicted binding sites, GLI2 attached to one specific location with particularly strong binding, supporting a direct link between SHH signaling and POMC gene expression.
Overall, the study showed that mutations in the USP8 gene activate SHH signaling in part by preventing SMO from being degraded. This activates GLI2, which, in turn, upregulates POMC and increases ACTH production. “The findings from our study thus support the development of new treatment strategies for Cushing’s disease,” the researchers wrote.
