Isabel Weigand1, Lisanne Knobloch1, Jörg Flitsch2, Wolfgang Saeger3, Camelia M Monoranu4, Kerstin Höfner1, Sabine Herterich5, Roman Rotermund2, Cristina L Ronchi1,6, Michael Buchfelder7, Markus Glatzel3, Christian Hagel3, Martin Fassnacht1,5,8, Timo Deutschbein1, Silviu Sbiera1. 1. Division of Endocrinology and Diabetes, Department of Internal Medicine I, University Hospital, University of Wuerzburg, Wuerzburg, Germany. 2. Department of Neurosurgery, University Hospital of Hamburg-Eppendorf, Hamburg, Germany. 3. Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 4. Department of Neuropathology, Institute of Pathology, University of Wuerzburg, Wuerzburg, Germany. 5. Central Laboratory, University Hospital Wuerzburg, Wuerzburg, Germany. 6. Institute of Metabolism and System Research, University of Birmingham, Birmingham, United Kingdom. 7. Department of Neurosurgery, University of Erlangen-Nuernberg, Erlangen, Germany. 8. Comprehensive Cancer Center Mainfranken, University of Wuerzburg, Wuerzburg, Germany.
Abstract
CONTEXT: Cushing disease (CD) is a rare disorder with severe sequels and incompletely understood pathogenesis. The underlying corticotroph adenomas harbor frequently somatic mutations in the ubiquitin-specific peptidase 8 (USP8) gene. These mutations render USP8 hyperactive and prevent client proteins from degradation. OBJECTIVE: To investigate the impact of USP8 mutations on proteins deregulated in CD. DESIGN: One hundred eight pituitary adenomas (75 corticotroph [58 USP8 wild type (WT) and 17 USP8 mutated], 14 somatotroph, and 19 nonfunctioning) were investigated by immunohistochemistry. All evaluated proteins [USP8, arginine vasopressin receptor 1b and 2, corticotropin-releasing hormone receptor, cAMP response element-binding protein (CREB), p27/kip1, cyclin E, heat shock protein 90 (HSP90), orphan nuclear receptor 4, epidermal growth factor receptor, histone deacetylase 2, glucocorticoid receptor, cyclin-dependent kinase 5 and Abelson murine leukemia viral oncogene homolog 1 enzyme substrate 1] were known to be deregulated in CD. Furthermore, AtT20 cells were transfected with USP8 to investigate the expression of possible downstream proteins by immunoblot. RESULTS: Whereas most of the investigated proteins were not differentially expressed, the cell-cycle inhibitor p27 was significantly reduced in USP8 mutated corticotroph adenoma (H-score 2.0 ± 1.0 vs 1.1 ± 1.1 in WT adenomas; P = 0.004). In contrast, the chaperone HSP90 was expressed higher (0.5 ± 0.4 vs 0.2 ± 0.4; P = 0.29), and the phosphorylation of the transcription factor CREB was increased in USP8 mutated adenomas (1.30.5 ± 0.40.9 vs 0.70.5 ± 0.40.7; P = 0.014). Accordingly, AtT20 cells transfected with the USP8 P720R mutant had higher phosphorylated CREB (pCREB) levels than WT transfected cells (1.3 ± 0.14 vs 1 ± 0.23; P = 0.13). CONCLUSIONS: We could demonstrate that USP8 mutations are associated with deregulation of p27/kip1, HSP90, and pCREB. These findings suggest that these proteins are direct or indirect clients of USP8 and could therefore be potential targets for therapeutic approaches in patients with CD.
CONTEXT: Cushing disease (CD) is a rare disorder with severe sequels and incompletely understood pathogenesis. The underlying corticotroph adenomas harbor frequently somatic mutations in the ubiquitin-specific peptidase 8 (USP8) gene. These mutations render USP8hyperactive and prevent client proteins from degradation. OBJECTIVE: To investigate the impact of USP8 mutations on proteins deregulated in CD. DESIGN: One hundred eight pituitary adenomas (75 corticotroph [58 USP8 wild type (WT) and 17 USP8 mutated], 14 somatotroph, and 19 nonfunctioning) were investigated by immunohistochemistry. All evaluated proteins [USP8, arginine vasopressin receptor 1b and 2, corticotropin-releasing hormone receptor, cAMP response element-binding protein (CREB), p27/kip1, cyclin E, heat shock protein 90 (HSP90), orphan nuclear receptor 4, epidermal growth factor receptor, histone deacetylase 2, glucocorticoid receptor, cyclin-dependent kinase 5 and Abelson murineleukemia viral oncogene homolog 1 enzyme substrate 1] were known to be deregulated in CD. Furthermore, AtT20 cells were transfected with USP8 to investigate the expression of possible downstream proteins by immunoblot. RESULTS: Whereas most of the investigated proteins were not differentially expressed, the cell-cycle inhibitor p27 was significantly reduced in USP8 mutated corticotroph adenoma (H-score 2.0 ± 1.0 vs 1.1 ± 1.1 in WT adenomas; P = 0.004). In contrast, the chaperone HSP90 was expressed higher (0.5 ± 0.4 vs 0.2 ± 0.4; P = 0.29), and the phosphorylation of the transcription factor CREB was increased in USP8 mutated adenomas (1.30.5 ± 0.40.9 vs 0.70.5 ± 0.40.7; P = 0.014). Accordingly, AtT20 cells transfected with the USP8P720R mutant had higher phosphorylated CREB (pCREB) levels than WT transfected cells (1.3 ± 0.14 vs 1 ± 0.23; P = 0.13). CONCLUSIONS: We could demonstrate that USP8 mutations are associated with deregulation of p27/kip1, HSP90, and pCREB. These findings suggest that these proteins are direct or indirect clients of USP8 and could therefore be potential targets for therapeutic approaches in patients with CD.
Authors: Ananth P Abraham; Rekha Pai; Daniel L Beno; Geeta Chacko; Hesarghatta Shyamasunder Asha; Simon Rajaratnam; Nitin Kapoor; Nihal Thomas; Ari G Chacko Journal: Endocrine Date: 2021-10-18 Impact factor: 3.633
Authors: Pietro Locantore; Rosa Maria Paragliola; Gianluca Cera; Roberto Novizio; Ettore Maggio; Vittoria Ramunno; Andrea Corsello; Salvatore Maria Corsello Journal: Int J Mol Sci Date: 2022-06-19 Impact factor: 6.208
Authors: Mateusz Bujko; Paulina Kober; Joanna Boresowicz; Natalia Rusetska; Natalia Zeber-Lubecka; Agnieszka Paziewska; Monika Pekul; Grzegorz Zielinski; Andrzej Styk; Jacek Kunicki; Jerzy Ostrowski; Janusz A Siedlecki; Maria Maksymowicz Journal: J Clin Med Date: 2021-01-20 Impact factor: 4.241