USP8 inhibitor RA-9 reduces ACTH release and cell growth in tumor corticotrophs

in Endocrine-Related Cancer
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  • 1 D Treppiedi, Department of Clinical Sciences and Community Health, University of Milan, Milano, Italy
  • 2 G Di Muro, Department of Clinical Sciences and Community Health, University of Milan, Milano, Italy
  • 3 G Marra, Department of Clinical Sciences and Community Health, University of Milan, Milano, Italy
  • 4 A Barbieri, Department of Clinical Sciences and Community Health, University of Milan, Milano, Italy
  • 5 F Mangili, Department of Clinical Sciences and Community Health, University of Milan, Milano, Italy
  • 6 R Catalano, Department of Clinical Sciences and Community Health, University of Milan, Milano, Italy
  • 7 A Serban, Endocrinology Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
  • 8 E Ferrante, Endocrinology Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
  • 9 M Locatelli, Department of Pathophysiology and Transplantation, University of Milan, Milano, Italy
  • 10 A Lania, Endocrinology, Diabetology and Medical Andrology Unit, Humanitas Clinical and Research Center, IRCCS, Milan, Italy
  • 11 M Arosio, Department of Clinical Sciences and Community Health, University of Milan, Milano, Italy
  • 12 A Spada, Department of Clinical Sciences and Community Health, University of Milan, Milano, Italy
  • 13 E Peverelli, Department of Clinical Sciences and Community Health, University of Milan, Milano, Italy
  • 14 G Mantovani, Department of Clinical Sciences and Community Health, University of Milan, Milano, Italy

Correspondence: Erika Peverelli, Email: erika.peverelli@unimi.it
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Cushing’s Disease (CD) is a rare endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor. Pasireotide is the only pituitary-targeted drug approved for adult patients. Nevertheless, many side effects are encountered and a curative therapy is still challenging. Ubiquitin Specific Peptidase 8 (USP8) plays a crucial role in the modulation of corticotroph cells growth and ACTH secretion. Here, we explored the anticancer potential of the USP8 inhibitor RA-9 in USP8-wild type human tumor corticotroph cells and murine AtT-20 cells. Our results showed that RA-9 causes cell proliferation decrease (-24.3±5.2%, P<0.01) and cell apoptosis increase (207.4±75.3%, P<0.05) in AtT-20 cells, as observed with pasireotide. Moreover, RA-9 reduced ACTH secretion in AtT-20 cells (-34.1±19.5%,P<0.01), as well as in AtT-20 cells transfected with USP8 mutants, and in 1 out of 2 primary cultures in vitro responsive to pasireotide (-40.3±6%). A RA-9 mediated decrease of pERK1/2 levels was observed in AtT-20 cells (-52.3±13.4%, P<0.001), comparable to pasireotide, and in primary cultures, regardless of their in vitro responsiveness to pasireotide. Upregulation of p27 was detected upon RA-9 treatment only, both in AtT-20 cells (167.1±36.7%, P<0.05) and 1 primary culture tested (168.4%), whilst pCREB level was similarly halved in AtT-20 cells by both RA-9 and pasireotide. Altogether, our data demonstrate that RA-9 is efficient in exerting cytotoxic effects and inhibitory actions on cell proliferation and hormone secretion by modulating the expression of pERK1/2, pCREB and p27. Inhibition of USP8 might represent a novel strategy to target both USP8-wild type and USP8-mutated tumors in CD patients.