P720R USP8 Mutation Is Associated with a Better Responsiveness to Pasireotide in ACTH-Secreting PitNETs

SIMPLE SUMMARY: Somatic mutations in USP8 gene have been identified in 11–60% of patients with ACTH-secreting pituitary tumors. This study investigated the impact of USP8 mutations on corticotroph tumor cells responsiveness to the treatment with the somatostatin analog pasireotide. Although we found that the somatostatin receptor targeted by pasireotide, SSTR5, was upregulated in cells transfected with USP8 mutants, pasireotide failed to reduce ACTH secretion in cells expressing mutated USP8, except for the mutation P720R. Overall these data demonstrate that different USP8 mutations exert opposite effects on the responsiveness of ACTH-secreting pituitary tumors to pasireotide. ABSTRACT: Somatic mutations in the ubiquitin specific peptidase 8 (USP8) gene have been associated with higher levels of somatostatin (SS) receptor subtype 5 (SSTR5) in adrenocorticotroph hormone (ACTH)-secreting pituitary neuroendocrine tumors (PitNETs). However, a correlation between the USP8 mutational status and favourable responses to pasireotide, the somatostatin multi-receptor ligand acting especially on SSTR5, has not been investigated yet. Here, we studied the impact of USP8 mutations on pasireotide responsiveness in human and murine corticotroph tumor cells. SSTR5 upregulation was observed in USP8 wild-type primary tumor cells transfected with S718del USP8 mutant. However, cell transfection with S718del USP8 and C40-USP8 mutants in in vitro sensitive cultures from USP8 wild-type tumors abolished their ability to respond to pasireotide and did not confer pasireotide responsiveness to the in vitro resistant culture. Pasireotide failed to reduce ACTH secretion in primary cells from one S718P USP8-mutated tumor but exerted a strong antisecretory effect in primary cells from one P720R USP8-mutated tumor. In agreement, AtT-20 cells transfection with USP8 mutants led to SSTR5 expression increase but pasireotide could reduce ACTH production and cyclin E expression in P720R USP8 overexpressing cells, only. In situ Proximity Ligation Assay and immunoflurescence experiments revealed that P720R USP8 mutant is still able to bind 14-3-3 proteins in AtT-20 cells, without affecting SSTR5 localization. In conclusion, P720R USP8 mutation might be considered as a molecular predictor of favourable response to pasireotide in corticotroph tumor cells.