Ubiquitin specific peptidase 8 (USP8) in human corticotroph pituitary tumors- possible targets and mode of action

Isabel Weigand; Lisanne Knobloch; Jens T Vanselow; Jorg Flitsch; Camelia Maria Monoranu; Wolfgang Saeger; Christian Hagel; Sabine Herterich; Cristina L Ronchi; Andreas Schosser; Martin Fassnacht; Timo Deutschbein; Silviu Sbiera

doi:10.1530/endoabs.56.GP198

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Endocrine Abstracts (2018) 56 GP198 | DOI: 10.1530/endoabs.56.GP198

ECE2018 Guided Posters Pituitary Basic (8 abstracts)

Ubiquitin specific peptidase 8 (USP8) in human corticotroph pituitary tumors- possible targets and mode of action

Isabel Weigand ¹ , Lisanne Knobloch ¹ , Jens T Vanselow ² , Jörg Flitsch ³ , Camelia Maria Monoranu ⁴ , Wolfgang Saeger ⁵ , Christian Hagel ⁵ , Sabine Herterich ⁶ , Cristina L Ronchi ^1, , Andreas Schosser ² , Martin Fassnacht ¹ , Timo Deutschbein ¹ & Silviu Sbiera ¹

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Author affiliations

¹Division of Endocrinology and Diabetes, University Hospital, University of Wuerzburg, Wuerzburg, Germany; ²Rudolf Virchow Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany; ³Neurosurgery, University Hospital of Hamburg-Eppendorf, Hamburg, Germany; ⁴Institute of Pathology, University of Wuerzburg, Wuerzburg, Germany; ⁵Institute of Neuropathology, University Hospital Hamburg-Eppendorf, Hamburg, Germany; ⁶Central Laboratory, University Hospital, University of Wuerzburg, Wuerzburg, Germany; ⁷Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, UK.

Recently, somatic, heterozygous mutations in the gene encoding the deubiquitinase USP8 have been identified in 30–60% of corticotroph tumors. These mutations were found to hinder binding of 14-3-3 proteins, increasing its deubiquitinating activity. One substrate is Epidermal Growth Factor Receptor (EGFR), USP8 triggers EGFR recycling and increased EGFR signaling. However, tumors harboring mutations in USP8 are smaller than WT tumors, raising the debate if EGFR, as a potent growth factor, is the only substrate of USP8 in these tumors. We aimed to identify putative USP8 targets that might explain the tumorigenesis and increased ACTH secretion of these cells. We performed a literature search to identify putative USP8 targets, revealing several proteins with deregulated expression associated with corticotroph tumors that might be the result of increased USP8 deubiquitination (e.g. the transcription factors TR4 and CREB). Candidates were analyzed by IHC for their expression levels on FFPE tissue (pituitary tumors: corticotrophs (n=85), functionally inactive (n=19), somatotrophs (n=12) and normal pituitary glands (n=5)). We further metabolically labeled, transiently transfected the murine corticotroph cell line AtT-20 with USP8 WT or mutant plasmids and performed Tandem-Ubiquitin-Binding-Entity (TUBE)-assays, followed by nanoLC-MS/MS analysis to identify changes in poly-ubiquitinated protein abundance. Of the 10 proteins analyzed by IHC, 3 had an altered expression pattern between USP8 WT and mutated tumors, namely p27/^kip1 (mean expression: 1±0.7 vs 0.4±0.6 (P=0.003)), AVPR1b (mean expression: 0.8±0.6 vs 0.3±0.4 (P=0.0045)), phospho-CREB (mean expression: 0.7±0.7 vs 1.3±0.9 (P=0.0165)). TUBE assays revealed an increased de-ubiquitination of Small Ubiquitin-Like Modifier 3 (SUMO3) in USP8^mut transfected cells, suggesting co-occurrence of another post-translational protein modification. In conclusion, these results suggest a much more complicated mechanism of action of the identified mutations in USP8, with sumoylation adding another dimension to the regulation of a USP8 mediated equilibrium between degradation and recycling.