Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel <i>USP8</i> Gene Variant
Cushing’s Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor, resulting in an excess of cortisol release by the adrenal glands. Somatic mutations in the deubiquitinases <i>USP8</i> and <i>USP48</i>, and in <i>...
| Main Authors: | , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-08-01
|
| Series: | Cancers |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2072-6694/13/16/4022 |
| _version_ | 1797524444551315456 |
|---|---|
| author | Donatella Treppiedi Anna Maria Barbieri Genesio Di Muro Giusy Marra Federica Mangili Rosa Catalano Emanuela Esposito Emanuele Ferrante Andreea Liliana Serban Marco Locatelli Andrea Gerardo Lania Anna Spada Maura Arosio Erika Peverelli Giovanna Mantovani |
| author_facet | Donatella Treppiedi Anna Maria Barbieri Genesio Di Muro Giusy Marra Federica Mangili Rosa Catalano Emanuela Esposito Emanuele Ferrante Andreea Liliana Serban Marco Locatelli Andrea Gerardo Lania Anna Spada Maura Arosio Erika Peverelli Giovanna Mantovani |
| author_sort | Donatella Treppiedi |
| collection | DOAJ |
| description | Cushing’s Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor, resulting in an excess of cortisol release by the adrenal glands. Somatic mutations in the deubiquitinases <i>USP8</i> and <i>USP48</i>, and in <i>BRAF</i> genes, have been reported in a subset of patients affected by CD. The aim of this study was to characterize the genetic profile of a cohort of 60 patients with ACTH-secreting tumors, searching for somatic mutations in <i>USP8</i>, <i>USP48</i>, and <i>BRAF</i> hotspot regions. Seven patients were found to carry <i>USP8</i> somatic mutations in the well-characterized 14-3-3 protein binding motif (<i>n</i> = 5 P720R, <i>n</i> = 1 P720Q, <i>n</i> = 1 S718del); 2 patients were mutated in <i>USP48</i> (M415I); no mutation was identified in <i>BRAF</i>. In addition, a novel <i>USP8</i> variant, G664R, located in exon 14, upstream of the 14-3-3 protein binding motif, was identified in 1 patient. Functional characterization of <i>USP8</i> G664R variant was performed in murine corticotroph tumor AtT-20 cells. Transient transfection with the <i>USP8</i> G664R variant resulted in a significant increase of ACTH release and cell proliferation (+114.5 ± 53.6% and +28.3 ± 2.6% vs. empty vector transfected cells, <i>p</i> < 0.05, respectively). Notably, USP8 proteolytic cleavage was enhanced in AtT-20 cells transfected with G664R USP8 (1.86 ± 0.58–fold increase of N-terminal USP8 fragment, vs. WT USP8, <i>p</i> < 0.05). Surprisingly, in situ Proximity Ligation Assay (PLA) experiments showed a significant reduction of PLA positive spots, indicating USP8/14-3-3 proteins colocalization, in G664R USP8 transfected cells with respect to WT USP8 transfected cells (−47.9 ± 6.6%, vs. WT USP8, <i>p</i> < 0.001). No significant difference in terms of ACTH secretion, cell proliferation and USP8 proteolytic cleavage, and 14-3-3 proteins interaction was observed between G664R USP8 and S718del USP8 transfected cells. Immunofluorescence experiments showed that, contrary to S718del USP8 but similarly to WT USP8 and other USP8 mutants, G664R USP8 displays an exclusive cytoplasmic localization. In conclusion, somatic mutations were found in <i>USP8</i> (13.3% vs. 36.5% incidence of all published mutations) and <i>USP48</i> (3.3% vs. 13.3% incidence) hotspot regions. A novel <i>USP8</i> variant was identified in a CD patient, and in vitro functional studies in AtT-20 cells suggested that this somatic variant might be clinically relevant in ACTH-secreting tumor pathogenesis, expanding the characterization of USP8 functional domains. |
| first_indexed | 2024-03-10T08:57:29Z |
| format | Article |
| id | doaj.art-65d0cf2452cf448b99eef64b164e8496 |
| institution | Directory Open Access Journal |
| issn | 2072-6694 |
| language | English |
| last_indexed | 2024-03-10T08:57:29Z |
| publishDate | 2021-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cancers |
| spelling | doaj.art-65d0cf2452cf448b99eef64b164e84962023-11-22T07:02:38ZengMDPI AGCancers2072-66942021-08-011316402210.3390/cancers13164022Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel <i>USP8</i> Gene VariantDonatella Treppiedi0Anna Maria Barbieri1Genesio Di Muro2Giusy Marra3Federica Mangili4Rosa Catalano5Emanuela Esposito6Emanuele Ferrante7Andreea Liliana Serban8Marco Locatelli9Andrea Gerardo Lania10Anna Spada11Maura Arosio12Erika Peverelli13Giovanna Mantovani14Department of Clinical Sciences and Community Health, University of Milan, 20122 Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, 20122 Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, 20122 Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, 20122 Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, 20122 Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, 20122 Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, 20122 Milan, ItalyEndocrinology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milan, ItalyEndocrinology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milan, ItalyDepartment of Pathophysiology and Transplantation, University of Milan, 20122 Milan, ItalyEndocrinology, Diabetology and Medical Andrology Unit, Humanitas Clinical and Research Center, IRCCS, 20089 Rozzano, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, 20122 Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, 20122 Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, 20122 Milan, ItalyDepartment of Clinical Sciences and Community Health, University of Milan, 20122 Milan, ItalyCushing’s Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor, resulting in an excess of cortisol release by the adrenal glands. Somatic mutations in the deubiquitinases <i>USP8</i> and <i>USP48</i>, and in <i>BRAF</i> genes, have been reported in a subset of patients affected by CD. The aim of this study was to characterize the genetic profile of a cohort of 60 patients with ACTH-secreting tumors, searching for somatic mutations in <i>USP8</i>, <i>USP48</i>, and <i>BRAF</i> hotspot regions. Seven patients were found to carry <i>USP8</i> somatic mutations in the well-characterized 14-3-3 protein binding motif (<i>n</i> = 5 P720R, <i>n</i> = 1 P720Q, <i>n</i> = 1 S718del); 2 patients were mutated in <i>USP48</i> (M415I); no mutation was identified in <i>BRAF</i>. In addition, a novel <i>USP8</i> variant, G664R, located in exon 14, upstream of the 14-3-3 protein binding motif, was identified in 1 patient. Functional characterization of <i>USP8</i> G664R variant was performed in murine corticotroph tumor AtT-20 cells. Transient transfection with the <i>USP8</i> G664R variant resulted in a significant increase of ACTH release and cell proliferation (+114.5 ± 53.6% and +28.3 ± 2.6% vs. empty vector transfected cells, <i>p</i> < 0.05, respectively). Notably, USP8 proteolytic cleavage was enhanced in AtT-20 cells transfected with G664R USP8 (1.86 ± 0.58–fold increase of N-terminal USP8 fragment, vs. WT USP8, <i>p</i> < 0.05). Surprisingly, in situ Proximity Ligation Assay (PLA) experiments showed a significant reduction of PLA positive spots, indicating USP8/14-3-3 proteins colocalization, in G664R USP8 transfected cells with respect to WT USP8 transfected cells (−47.9 ± 6.6%, vs. WT USP8, <i>p</i> < 0.001). No significant difference in terms of ACTH secretion, cell proliferation and USP8 proteolytic cleavage, and 14-3-3 proteins interaction was observed between G664R USP8 and S718del USP8 transfected cells. Immunofluorescence experiments showed that, contrary to S718del USP8 but similarly to WT USP8 and other USP8 mutants, G664R USP8 displays an exclusive cytoplasmic localization. In conclusion, somatic mutations were found in <i>USP8</i> (13.3% vs. 36.5% incidence of all published mutations) and <i>USP48</i> (3.3% vs. 13.3% incidence) hotspot regions. A novel <i>USP8</i> variant was identified in a CD patient, and in vitro functional studies in AtT-20 cells suggested that this somatic variant might be clinically relevant in ACTH-secreting tumor pathogenesis, expanding the characterization of USP8 functional domains.https://www.mdpi.com/2072-6694/13/16/4022Cushing’s diseaseACTH-secreting pituitary tumorUSP8USP48mutation |
| spellingShingle | Donatella Treppiedi Anna Maria Barbieri Genesio Di Muro Giusy Marra Federica Mangili Rosa Catalano Emanuela Esposito Emanuele Ferrante Andreea Liliana Serban Marco Locatelli Andrea Gerardo Lania Anna Spada Maura Arosio Erika Peverelli Giovanna Mantovani Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel <i>USP8</i> Gene Variant Cancers Cushing’s disease ACTH-secreting pituitary tumor USP8 USP48 mutation |
| title | Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel <i>USP8</i> Gene Variant |
| title_full | Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel <i>USP8</i> Gene Variant |
| title_fullStr | Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel <i>USP8</i> Gene Variant |
| title_full_unstemmed | Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel <i>USP8</i> Gene Variant |
| title_short | Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel <i>USP8</i> Gene Variant |
| title_sort | genetic profiling of a cohort of italian patients with acth secreting pituitary tumors and characterization of a novel i usp8 i gene variant |
| topic | Cushing’s disease ACTH-secreting pituitary tumor USP8 USP48 mutation |
| url | https://www.mdpi.com/2072-6694/13/16/4022 |
| work_keys_str_mv | AT donatellatreppiedi geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT annamariabarbieri geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT genesiodimuro geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT giusymarra geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT federicamangili geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT rosacatalano geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT emanuelaesposito geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT emanueleferrante geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT andreealilianaserban geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT marcolocatelli geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT andreagerardolania geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT annaspada geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT mauraarosio geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT erikapeverelli geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant AT giovannamantovani geneticprofilingofacohortofitalianpatientswithacthsecretingpituitarytumorsandcharacterizationofanoveliusp8igenevariant |