Endocrine Abstracts

Cushing’s Disease (CD) is a rare condition mostly caused by an ACTH-secreting pituitary tumor resulting in excess of cortisol release by the adrenal glands. Although pasireotide is the only pituitary-targeted drug approved to treat adult patients, many side effects are encountered during the clinical practice and a curative therapy for CD is still challenging. Recently, the discover of somatic mutations in the deubiquitinase USP8 gene in a subset of patients has shed new light on the crucial role played by the ubiquitin system in the modulation of corticotroph cells growth and hormone secretion. However, the anticancer potential of USP8 inhibition in the corticotrophs has been poor explored so far. Aim of this study was to characterize the in vitro responses induced by the USP8 inhibitor RA-9, in murine corticotroph AtT-20 cells and primary cultures from ACTHomas, and to compare them with those triggered by pasireotide. First, AtT-20 cells and ACTHomas were screened for USP8 mutations, all samples resulting wild type. Then, 48 h of incubation with RA-9 caused cell proliferation decrease (-24.3 ± 5.2%, P < 0.01 vs basal) and cell apoptosis increase (207.4 ± 75.3%, P < 0.05 vs basal) in AtT-20 cells, as observed with pasireotide. Interestingly, RA-9 significantly reduced ACTH secretion in AtT-20 cells (-34.1 ± 19.5%, P < 0.01 vs basal), as well as in 1 out of 2 primary cultures in vitro responsive to pasireotide (-40.3 ± 6% reduction at RA-9). Western blot experiments revealed that, similarly to pasireotide, RA-9 lowered phospho-ERK1/2 levels in AtT-20 cells (-52.3 ± 13.4%, P < 0.001 vs basal) and in all primary cultures regardless of their in vitro responsiveness to pasireotide (-32.9 ± 19.8% vs basal in the sensitive group, P < 0.001; -33.1 ± 11.8% vs basal in the resistant group, P < 0.05). On the contrary, upregulation of p27^kip1 was observed at 48 h of treatment with RA-9 only, both in AtT-20 cells (167.1 ± 36.7%, P < 0.05 vs basal) and one primary culture tested (168.4% vs basal), whilst phospho-CREB expression level was similarly halved in AtT-20 cells by both RA-9 and pasireotide. No synergic effect was observed in any experiments when RA-9 and pasireotide were co-administrated. Altogether, our data demonstrate that RA-9 is able to exert cytotoxic effects and inhibitory actions on cell proliferation and hormone secretion in tumor corticotrophs by modulating the expression of phospho-ERK1/2, phospho-CREB and p27^kip1. Moreover, although acting on different targets, RA-9 and pasireotide elicit a comparable spectrum of biological responses in vitro, therefore inhibition of USP8 might represent a novel mean to pharmacologically target ACTHomas in patients with CD.