ECE2017 Eposter Presentations: Pituitary and Neuroendocrinology Pituitary - Basic (13 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 , Carmelia M Monoranu 4 , Wolfgang Saeger 5 , Christian Hagel 5 , Sabine Herterich 6 , Cristina Ronchi 1 , Andreas Schlosser 2 , Martin Fassnacht 1, , Timo Deutschbein 1 & Silviu Sbiera 1
1Division of Endocrinology and Diabetes, University Hospital 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.
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 triggering 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 other putative USP8 targets that might explain the tumorigenesis and increased ACTH secretion of these cells. A literature search revealed several proteins with deregulated expression associated with corticotroph tumors that might be the result of increased USP8 deubiquitination (such as the transcription factors TR4 and CREB). Candidates were analyzed by IHC for their expression levels on FFPE tissue (corticotrophs (n=54), functionally inactive (n=19), somatotrophs (n=12) adenoma 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 analyzed proteins by IHC, 4 had an altered expression pattern between USP8 WT and mutated tumors, namely p27/kip1 (mean expression: 1±0.8 vs 0.4±0.6 (P=0.01), HSP90 (mean expression: 1.8±0.8 vs 0.7±0.8 (P=0.03), CRHR (mean expression: 0.4±0.5 vs 0.6±0.7 (P=0.2) and PRKACA (mean expression: 0.6±0.8 vs 1.1±0.6 (P=0.1). TUBE assays revealed an increased de-ubiquitination of Small Ubiquitin-Like Modifier 3 (SUMO3) in USP8mut 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.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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