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Title: Investigating roles for the deubiquitylating enzymes in the PtdIns3-K/PKB pathway in cancer
Author: Sacco, Joseph J. T.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2012
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Abstract:
Phosphatidylinositol 3-kinase (PtdIns3-K) signalling is a crucial survival pathway in multiple malignancies, and an exciting target for drug development. The pathway is subject to multiple regulatory mechanisms including ubiquitylation, a reversible process that can influence protein stability, localisation and activity. A family of approximately 80 deubiquitylating enzymes (DUBs) are responsible for the cleavage of ubiquitin and ubiquitin chains from protein substrates. The DUBs are attractive drug targets and have increasingly been implicated in cellular processes germane to malignancy, including the PtdIns3-K pathway. The aim of this study was to systematically identify DUBs involved in regulating the pathway, with a particular view to identifying potential novel drug targets. The project employed a DUB siRNA library to perform a series of RNAi screens using two complementary approaches. In the first approach, the effects of DUB depletion on the protein level of nine PtdIns3-K pathway components (p110α, p110β, p110γ, p85α, p170, PDK1, PTEN, Akt, and mTOR) were assayed by immunoblotting. DUBs whose depletion either increased or decreased the protein level of these components were subject to further validation. Of particular interest were five DUBs (PRPF8, TNFAIP3, USP32, USP34 and OTUD1) whose depletion decreased the level of PDK1. In addition, depletion of three closely related members of the Josephin family of DUBs (ATXN3, ATXN3L and JOSD1) resulted in an increase in the protein level of the tumour suppressor PTEN. In view of the potential clinical utility of upregulating PTEN, the latter DUBs were prioritised for further investigation. Initial investigation indicates that all three DUBs alter PTEN level at a transcriptional level. Moreover, the effects of ATXN3 appear to be independent of its known function in modulation of histone acetylation status. The second approach utilised a U2OS cell line stably transfected with EGFP tagged FOXO3, in which PtdIns3-K dependent FOXO3 translocation could be assessed by live-cell imaging. This enabled the design of a functional screen in which the effects of DUB depletion on downstream PtdIns3-K signalling were assessed. Among the DUBs identified in this screen was USP45, depletion of which enhanced nuclear translocation of FOXO3-EGFP in response to PtdIns3-K inhibition. Several DUBs regulating FOXO3-EGFP abundance were also identified in this screen, including USP1 and USPL1 whose depletion respectively decreased and increased FOXO3-EGFP levels. The screen additionally identified three DUBs (USP8, OTUD4 and DUB4) whose depletion was synthetically lethal with PI-103 treatment. In summary, several DUB modulators of the PtdIns3-K pathway were identified that, subject to further mechanistic and functional studies, may increase the options available for targeting this vital pathway in malignancy.
Supervisor: Clague, Michael J.; Coulson, Judy M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.579354  DOI: Not available
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