Ubiquitin-mediated regulation of androgen receptor function: a potential therapeutic target in prostate cancer

Lead Research Organisation: Newcastle University
Department Name: Northern Institute for Cancer Research

Abstract

More than 10,000 men die annually in the UK from prostate cancer. The standard therapy for prostate cancer aims to block the androgen receptor signalling pathway by removing its activating hormone, androgen from the circulation and/or blocking androgen action. However, most cancers become resistant to treatment and become refractory to hormone manipulation. The outlook for these patients is poor.
A detailed functional analysis of the proteins that contribute towards signalling through the androgen receptor pathway should identify alternative, novel targets for treatment of this disease.
In this project we will focus on studying the enzymes that modify the function of proteins by directly attaching or removing a small protein called ubiquitin. Addition of ubiquitin, termed ubiquitination, or removal of ubiquitin from proteins, termed deubiquitination, are important events in regulating many of the functions of the androgen receptor.
Activation of the androgen receptor is critical to all the stages of prostate cancer, including later stages of the disease when standard treatments are ineffective. We aim to identify those proteins implicated in androgen receptor ubiquitination and deubiquitination and further determine the importance of these proteins in androgen receptor action. Our ultimate hope is that we can disrupt the enzymatic action of these proteins using small molecule chemical inhibitors. In this way we may be able to provide alternative treatments for those prostate cancer patients who no longer respond to standard treatments.
Consistent with our research Institute?s policy we will fully engage the wider public with our research findings through press communications, organised open days, school visits and attendance at charity events.

Technical Summary

Androgen ablation is widely used in prostate cancer (CaP) therapy. However, most tumours become resistant to treatment but still retain a functional androgen receptor (AR) signalling pathway.

Proteasomal turnover and transcriptional activity of the AR are subject to regulatory control by ubiquitination. Characterising the proteins that regulate ubiquitination of the AR may provide alternative targets for treatment of advanced CaP.

This study will determine the regulatory influence of ubiquitination on transcriptional activity and proteasomal degradation of the AR and further investigate the potential of using small molecule inhibitors to disrupt ubiquitin-mediated AR signalling in CaP. The aim is to identify novel protein within the AR protein transcriptional complex as alternative targets for therapeutic intervention in CaP.

This project will
(1) Investigate the degradation of wild type and mutant AR in CaP cell lines in response to ligand modulation and proteasomal inhibitors

(2) Apply small molecule inhibitors that modulate AR phosphorylation or interfere with AR chaperone binding to investigate AR degradation and function

(3) Determine the sites of ubiquitination of the AR both in vitro and in vivo

(4) Identify deubiquitinating enzymes linked to AR transcriptional activity

(5) Evaluate protein expression and genetic alterations of selected gene products that modulate AR ubiquitination in clinical material to determine their relevance to the initiation and progression of CaP
 
Description NCRI Biomarkers and Imaging Group
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description MICA
Amount £313,973 (GBP)
Funding ID BH161736 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2017 
End 01/2020
 
Description MRC Collaboration with Industry
Amount £500,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start  
 
Description Newcastle University + Cancer Research UK PhD Studentship
Amount £30,000 (GBP)
Organisation Newcastle University 
Sector Academic/University
Country United Kingdom
Start 10/2011 
End 09/2015
 
Description Newcastle University + Cancer Research UK PhD Studentship
Amount £30,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 09/2015
 
Description PCC Project grant
Amount £198,000 (GBP)
Organisation Prostate Cancer UK 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description Research Interaction
Amount £57,706 (GBP)
Funding ID BH164904 
Organisation CellCentric Ltd 
Sector Private
Country United Kingdom
Start 02/2017 
End 09/2018
 
Description Sir William Edmond Harker Foundation
Amount £120,000 (GBP)
Organisation Newcastle University 
Department Dr William Edmund Harker Foundation
Sector Charity/Non Profit
Country United Kingdom
Start 09/2011 
End 12/2015
 
Title AR REC MUT 
Description Purified recombinant forms of the AR mutated at specific amino acid sites 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact In vitro assays of ubiquitination and AR protein activity 
 
Title DUB KD LNCaP 
Description Integrated DUB siRNA expressing plasmid, for inducible knockdown in prostate cancer cells 
Type Of Material Cell line 
Provided To Others? No  
Impact Temporal control of DUB expression to evaluate importance in androgen receptor transcriptional control 
 
Title REC E3 LIGASE 
Description Purified forms of 4 E3 ligases 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact High throughput in vitro ubiquitination assays 
 
Title siRNA screening 
Description siRNA screen developed and applied including assay development to monitor expression of changes to androgen regulated genes 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact High throughput screening enabled with reproducible assay that is cost effective and simple 
 
Description Characterisation of a Novel Series of Small Molecule Androgen 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution This is a joint funded project between AZ and the MRC (to commence in Jan 2009 for 3 years). this will further strengthen interactions between my research group and AZ.
Collaborator Contribution This is a joint funded project between AZ and the MRC (to commence in Jan 2009 for 3 years). this will further strengthen interactions between my research group and AZ.
Impact This is a joint funded project between AZ and the MRC (to commence in Jan 2009 for 3 years). this will further strengthen interactions between my research group and AZ.
Start Year 2007
 
Description Development of a Ubiquitin Proteome Consortium 
Organisation Netherlands Cancer Institute (NKI)
Country Netherlands 
Sector Academic/University 
PI Contribution Reagent distribution, expertise
Collaborator Contribution Specialist expertise in deubiquitinase assays
Impact Ongoing discussion for financial support from Cancer Research UK. Identification of a novel deubiquitinase enzyme in prostate cancer cells.
Start Year 2010
 
Description Haendler Collaboration 
Organisation Bayer
Country Germany 
Sector Private 
PI Contribution Exchange of unique reagents
Collaborator Contribution Expert advice, provision of unique reagents
Impact Speeded up reseach programme
Start Year 2008
 
Description High throughput siRNA library screening 
Organisation Cellectricon
Country Sweden 
Sector Private 
PI Contribution Technology input and experience in prostate biology, including small-medium scale siRNA library screens.
Collaborator Contribution Methodology gained from G0601123 will be applied in a high throughput screening format developed by Cellectricon
Impact Methodology gained from G0601123 will be applied in a high throughput screening format developed by Cellectricon
Start Year 2007
 
Description NEPAF Collaboration 
Organisation One North East, Newcastle
Country United Kingdom 
Sector Public 
PI Contribution Provision of unique reagents and expertise
Collaborator Contribution Interegation of post-translational modification of the androgen receptor
Impact NEPAF - North East Proteome Analysis Facility - has conducted some proteomics analysis for our group on this project for which they received payment. However, we are currently in negotiation with NEPAF to seek a commitment from them to provide financial support to develop a particular area of related research. At this point no funding has been secured.
Start Year 2007
 
Description Tokai - anti-androgen therapy 
Organisation Tokai Pharmaceuticals Inc
Country United States 
Sector Private 
PI Contribution Identified a deubiquitinase enzyme that is targetted by small molecule anti-androgen produced by Tokai
Collaborator Contribution Provide small molecules for study in collaboration
Impact Funded research in our laboratories to explore Tokai small molecules and to develop more potent small molecules that are beneficial in prostate cancer therapy
Start Year 2015
 
Description Ubiquitin Probes 
Organisation Netherlands Cancer Institute (NKI)
Department Division of Cell Biology
Country Netherlands 
Sector Hospitals 
PI Contribution Biological assay development and application
Collaborator Contribution Synthesis of selected probes and antibody generation
Impact publication Chemistry Molecular and Cell Biology
Start Year 2010
 
Description Cancer fund raising event 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Participants in your research and patient groups
Results and Impact Short talk on research highlights to date

Positive interaction with patient groups and stimulated their further interest to lobby government , MPs and specific prostate charities to devote more monies to this research area
Year(s) Of Engagement Activity 2008,2009,2010
 
Description Charity Fun Run 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Short presentation of research to large audience (primarily female) of between 3000-6000 audience. Including interviews with local press and local radio.

Informal discussions with smaller groups of cancer sufferers/ survivors and patient representative group
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description Institute Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 3-4 hour planned session, 2-3 times yearly, on a Saturday morning. Visits of approx 100 members of public. Short presentations and demonstrations of equipment by 4-6 research groups. Closing with 1 to 1 discussions over coffee/tea.

Contact maintained with several individual members of public
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description Movember Fundraising - Prostate Cancer Awareness 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Approx 100 workers from Proctor and Gamble PLC made aware of prostate cancer (briefly mentioned other cancers) risk factors and nature of research programme to combat disease

2012 gave two talks to different groups and this stimulated a series of talks for 2013 and additional events related to fundraising and public awareness of prostate cancer
Year(s) Of Engagement Activity 2012,2013
URL http://www.uk.pg.com http://uk.movember.com/
 
Description School visit - Gaughan 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Gaughan as RC UK Fellow had meetings with local teachers to discuss workshops for school students. Presentations from Gaughan to teachers. Outreach being extended to presentations to school children.

Continuing programme to encourage students from underachieving schools to engage with science
Year(s) Of Engagement Activity 2008,2009,2010