Next, we explore whether USP8 inhibitor would have an effect on the clonogenic ability of AtT20 cells [Figure 3]a and [Figure 3]b.
To investigate whether USP8 inhibitor reduces cell viability by inducing apoptosis, flow cytometry analysis and apoptosis-related proteins analysis were performed.
AtT20 cells were incubated with USP8 inhibitor for 4 and 24 h to determine its effects on proopiomelanocortin (POMC) mRNA levels.
USP8, also designated as Ub-specific protease Y, is an ubiquitin isopeptidase that belongs to the USP family of cysteine proteases.
Two recent studies demonstrated that the USP8 gene is frequently mutated in corticotroph adenomas.[sup], ACTH adenomas harboring USP8 mutant had higher EGFR levels, expressed more POMC mRNAs, and had higher ACTH production than those with wild-type USP8.
Recently, it was also demonstrated that USP8 inhibitor impairs the growth of gefitinib-resistant and -sensitive nonsmall cell lung cancer cells by decreasing receptor tyrosine kinase (RTK) expression.[sup], Here, we explored the effect of the novel specific USP8 inhibitor, 9-ehtyloxyimino9H-indeno[1,2-b] pyrazine-2,3-dicarbonitrile, on murine AtT20 pituitary adenoma cells.
Moreover, we observed that the inhibitory effects of USP8 inhibitor on mouse ACTH-secreting pituitary adenoma cell viability are, at least in part, due to apoptosis induction, accompanied by increased cleaved caspase 3 and Bax and decreased Bcl-2 as previously reported in other experimental models.[sup], Arrest of tumor growth is often associated with induction of cell apoptosis, and USP8 inhibitor has been shown to induce apoptosis in tumor cells, in parallel with growth inhibition.
We finally investigated whether USP8 inhibitor had any effect on ACTH secretion in AtT20 cells.
In conclusion, USP8 inhibitor could inhibit proliferation, abolishes clonogenic ability, and induces apoptosis in pituitary corticotroph tumor cell-AtT20 cells.
Mutations in the deubiquitinase gene USP8 cause Cushing's disease.