Engineering and application of models for in vivo imaging of androgen receptor activity

Lead Research Organisation: Imperial College London
Department Name: Dept of Surgery and Cancer

Abstract

Androgens are the male sex hormones - the major androgen is testosterone. They are pivotal for male fertility and responsible for masculinisation of genitalia. However, they are also required for female fertility; and in both sexes they are important for bone development and act on the brain, heart, fat and hair follicles. Disorders associated with too much or too little androgen action are common. Severe defects can cause a genetic male to become undervirilised or even female in appearance. Less severe deficiencies, even the fall in testosterone associated with aging, can lead to male infertility, decreased muscle, weight gain and depression. Conversely, excess androgen action has been linked to Polycystic Ovarian Syndrome, female infertility, acne, baldness prostate cancer, and behavioural disorders. Patients are often treated with androgen replacement therapy or antiandrogens (which oppose androgen action). However, due to the number of possible target tissues these almost invariably produce side effects. These can include reduced sperm count, jaundice, changes in libido, hirsutism, acne, osteoporosis and increased risk of certain cancers. New therapies are constantly being developed with the aim of reducing these ?off-target? effects, while male contraceptives and HRT are being developed containing androgens. It is therefore vital we learn more about where and when these act in the body. Knowledge of their sites of action will enable tailoring of such therapies to reduce side-effects.

Androgens act by binding to androgen receptor protein, which In turn binds to specific DNA sequences (androgen response elements) in target genes, increasing the activity of these genes, which then causes growth or function of the relevant tissue. This project will exploit this by linking an androgen response element to a ?reporter? gene, activity of which can be easily detected by scanning. This activation will be detected, accurately located and measured by state-of-the-art 3-dimensional scanners uniquely available at Imperial College. We will use the system to determine where and when natural androgens are active in males and females ? this may highlight previously unknown sites of androgen action. We will also follow activation or inhibition of androgen signalling by known and novel therapeutic agents, including androgens, antiandrogens and novel therapies. As well as increasing knowledge of the role of androgens in development and aging of both sexes, this project will benefit patients with all of the conditions mentioned above since it will allow evaluation of new and more specific therapies.

Technical Summary

The many androgen target tissues include male internal and external genitalia, ovary, uterine endometrium and in both sexes bone, skeletal muscle, the CNS, adipose tissue and hair follicles. Disorders associated with excess of or deficiency in androgen action are common, including male and female infertility, pseudohermaphroditism, hypogonadism, benign and malignant prostate conditions, acne, baldness or hirsutism, sexual desire disorders and depression. Patients are often treated with androgens or antiandrogens as appropriate, but due to the number of targets these almost invariably produce side-effects. Androgens can exacerbate prostate conditions, suppress spermatogenesis and cause jaundice, hirsutism and acne. Antiandrogen therapy is associated with hepatic tumours, osteoporosis, loss of libido, impotence and gynaecomastia.

The major circulating androgen is testosterone, secreted by the testes, and in many target tissues this is converted to the more potent dihydrotestosterone. Both of these as well as weak adrenal androgens signal via the androgen receptor a ligand-activated transcription factor. This binds to specific response elements in the promoters of target genes and activates transcription of genes involved in growth and/or differentiation. We aim to exploit this by creating expression cassettes containing (i) the luciferase gene or (ii) the sodium iodide symporter gene under the control of an androgen response element, and create in vivo models expressing these reporters in all tissues. These will be used to visualize where and when the androgen receptor signalling pathway is activated in response to endogenous androgens during development and aging. Exogenous androgens, antiandrogens and selective AR modulators with therapeutic possibilities will also be tested under conditions mimicking those found in patients. Knowledge of their sites of action will predict possible side-effects hence suitability or limitations for therapy. Since imaging will be by non-invasive scanning using (i) luciferase imaging and (ii) more quantitative and accurate Positron Emission Tomography scanning for sodium iodide symporter activity, studies will be longitudinal and in real time. This study will increase knowledge of the role of androgens in differentiation, development and fertility of both sexes. Further, proposed crosses with models deficient in genes involved in androgen signalling will differentiate between the developmental actions of the major androgens testosterone and dihydrotestosterone, and elucidate crosstalk between androgen receptor and other steroid receptors. The model will also lend itself to many downstream applications such as the investigation of effects of hormonal environmental pollutants, androgen action in female fertility, and sexual dichotomy in obesity.

Publications

10 25 50
 
Description 3Rs committee of Imperial College
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Participation in advisory committee
URL http://www3.imperial.ac.uk/research/animal-research
 
Description Imperial College Doctoral Training Account
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 09/2022
 
Description Project grant
Amount £212,000 (GBP)
Funding ID PG13-033 
Organisation Prostate Cancer UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2014 
End 08/2016
 
Description Research Grant
Amount £250,000 (GBP)
Organisation Urology Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 10/2018
 
Description Research Innovation Awards
Amount £255,817 (GBP)
Funding ID RIA15-ST2-014 
Organisation Prostate Cancer UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description Research Innovation Awards
Amount £588,628 (GBP)
Funding ID RIA17-ST2-017 
Organisation Prostate Cancer UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 03/2022
 
Title ARE-Luc mouse model 
Description Transgenic mice expressing androgen-responsive luciferase reporter gene in all tissues 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2016 
Provided To Others? Yes  
Impact Paper published (Dart et al, PLOS one, 2013) 
URL http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0071694
 
Title LNCaP/Luc 
Description A prostate cancer cell line with an integrated androgen-responsive luciferase reporter gene, allowing in vitro and in vivo (xenograft) imaging of androgen receptor activity 
Type Of Material Cell line 
Year Produced 2016 
Provided To Others? Yes  
Impact This allows the cell line to be used to detect androgen signalling in xenografts in living animals by in vivo imaging, throughout up to 36 days (in our experiments) this reducing the numbers of animals required. It could be used to monitor the effects of molecules or comounds aimed at the androgen signalling pathway 
 
Title LNCaP/Luc/PHB-cDNA 
Description Prostate cancer cells with integrated luciferase reporter plus doxycycline-inducible expression of prohibitin cDNA, suitable for use in cell culture in as xenografts 
Type Of Material Cell line 
Year Produced 2015 
Provided To Others? Yes  
Impact By allowing in vivo imaging of live mice throughout tumour progression reduces numbers of animals required. Demonstrated that increased prohibitin levels results in decreased growth of prostate tumours, hence proof of principle that increasing prohibitin levels could be a valid therapeutic strategy. 
 
Title LNCaP/Luc/PHB-siRNA 
Description Prostate cancer cells with integrated luciferase reporter plus doxycycline-inducible expression of prohibitin siRNA resulting in knockdown of PHB protein, suitable for use ionc ell culture in as xenografts 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact By allowing in vivo imaging of live mice throughout tumour progression reduces numbers of animals required. Demonstrated that decreased prohibitin levels results in increased growth of prostate tumours, hence proof of the hypothesis that decreasing amounts of corepressor proteins could lead to tumour progression. 
 
Title MCF7/TREx 
Description MCF breast cancer cell line in which it is possible to express a protein of interest under control of tetracycline 
Type Of Material Cell line 
Provided To Others? No  
Impact NOne as yet 
 
Description Campbell @ OHIO 
Organisation Ohio State University
Country United States 
Sector Academic/University 
PI Contribution Academic collaboration - submission of collaborative grant
Collaborator Contribution Academic collaboration - submission of collaborative grant. Also analysis of exisiting data
Impact Sunbmission of grant proposal (shortlisted for full application)
Start Year 2018
 
Description Carling group 
Organisation Imperial College London
Department MRC London Institute of Medical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration on use of PTEN-deficient prostate cancer mouse model, sharing of tissue resources (PCa patients), knowledge exchange, expertise
Collaborator Contribution Collaboration on use of PTEN-deficient prostate cancer mouse model, sharing of tissue resources (mouse), knowledge exchange, expertise
Impact Grant to Professor David Carling from Prostate Cancer UK Title Targeting the calcium/calmodulin-dependent protein kinase kinase 2 and AMP-activated protein kinase pathways in prostate cancer Reference RIA17-ST2-001 Organisation Imperial College London Total Awarded £235,582.00
Start Year 2014
 
Description Edinburgh Reproductive Sciences 
Organisation University of Edinburgh
Department MRC Centre for Reproductive Health
Country United Kingdom 
Sector Public 
PI Contribution Will provide ARE-luc mice for collaborative research
Collaborator Contribution Will provide expertise for reproductive research
Impact None yet
Start Year 2014
 
Description Gollnick ARE0luc, Roswell Park 
Organisation University at Buffalo
Department Roswell Park Cancer Institute
Country United States 
Sector Academic/University 
PI Contribution ARE-luc mice and expertise in use
Collaborator Contribution Expertise on AR signalling in immune system
Impact None yet
Start Year 2017
 
Description Guery lab 
Organisation National Institute of Health and Medical Research (INSERM)
Department Inserm/UPS UMR 1048 Institute of Metabolic and Cardiovascular Diseases (I2MC)
Country France 
Sector Public 
PI Contribution Provision of frozem mouse tissue
Collaborator Contribution Collaboration
Impact none yet
Start Year 2018
 
Description Hernandez lab 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Mouse tissues from out models
Collaborator Contribution Academic collaboration
Impact None yet
Start Year 2018
 
Description Prostate Cancer UK/Movember Centre of Excellence 
Organisation Institute of Cancer Research UK
Country United Kingdom 
Sector Academic/University 
PI Contribution We have a joint grant from Prostate Cancer UK. My team provides expertise and practical work on microRNAs as biomarkers in advanced prostate cancer, which will in future extend earlier to locally advanced and local.
Collaborator Contribution As above we have a joint Centre. The collaborators provide academic and practical input, including clinical expertise, access to approptiate clinical samples, and are involved int he study at all levels. J de Bono also co-supervises a PhD student in my group
Impact Centre grant
Start Year 2014
 
Description Prostate Cancer UK/Movember Centre of Excellence 
Organisation University College London Hospital
Department University College London Hospitals Charity (UCLH)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We have a joint grant from Prostate Cancer UK. My team provides expertise and practical work on microRNAs as biomarkers in advanced prostate cancer, which will in future extend earlier to locally advanced and local.
Collaborator Contribution As above we have a joint Centre. The collaborators provide academic and practical input, including clinical expertise, access to approptiate clinical samples, and are involved int he study at all levels. J de Bono also co-supervises a PhD student in my group
Impact Centre grant
Start Year 2014
 
Description Rennie collaboration 
Organisation Vancouver Prostate Centre
Country Canada 
Sector Hospitals 
PI Contribution ARE-luc mouse and imaging expertise
Collaborator Contribution New AR inhibitors and signalling experitse
Impact None yet
Start Year 2014
 
Description Trento - SBMA model 
Organisation University of Trento
Department Centre for Integrative Biology
Country Italy 
Sector Academic/University 
PI Contribution Provide ARE-luc mice and expertise in in vivo imaging
Collaborator Contribution Expertise and reagents of spinal bulbar muscular atrophy, Grant to study same
Impact Maria Pennuto awarded grant from Muscular Dystrophy Association
Start Year 2017
 
Description Address at March for men 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact At Prostate cancer UK's March for Men, held in London, C Bevan addressed to attendees, A Shibakawa and C Bevan participated in a "science station", and C Bevan was interviewed for social media channels
Year(s) Of Engagement Activity 2017
URL https://prostatecanceruk.org/get-involved/march-for-men
 
Description Annual Meeting of ARTP, the French Association for Prostate Cancer Research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Questions and much discussion

Several approaches from people interested in collabroating
Year(s) Of Engagement Activity 2013
URL http://www.iartp.org/
 
Description Conference - Androgens 2014: Precision Medicines Targeting Androgen Signalling 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact 2 days of talks and discussion, Speakers and attendees from UK, Europe, USA, Australia, Canada. Work funded by relevant grants was presented both orally and as poster

Collaborations initiated regarding ARE-luc mouse. Ditto for Prohibitin. Industrial involvement in repressors project
Year(s) Of Engagement Activity 2014
URL http://www.precisionmedicines.com/
 
Description Donor visits 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Between 3 and 12 lay people who donate to charities funding cancer research attended the dept for a lab tour and talks describing the kind of research we undertake and its implications.

Donors pledged funding either in general or for other projects ongoing
Year(s) Of Engagement Activity 2010,2011
 
Description Dublin Steroid Cancer Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Charlotte Bevan an invited speaker at Dublin Steroid Cancer Conference
Year(s) Of Engagement Activity 2018
 
Description Endocrinology conference (Edinburgh) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Postdoctoral research associate rpesented poster at the British Endocrine Society 2015
Year(s) Of Engagement Activity 2015
URL http://www.endocrine-abstracts.org/ea/0038/ea0038p161.htm
 
Description Lab tour and related online video by Movember/Norhampton Saints 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact Lab visit by funders (Movember) and supporter (Rugby personalities), plus charity Prostate cancer UK. Filming, online video, press releases
Year(s) Of Engagement Activity 2016
 
Description Lab tours 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Supporters
Results and Impact Lab tours, with demonstrations including hand-on activities, plus a talk on our research to lay audience and quesiton session
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
 
Description PCUK Lab tour 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact Lab tour organised by Prostate Cancer UK, attended by their supporters. Resulted in an ongoing relationship with new donors for the charity
Year(s) Of Engagement Activity 2016
 
Description Patient session at RCP 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact Patients, carers and other stakeholders attended a public session at the royal College of Physicians at which I , along with 2 clinicians and a patent advocate, gave talks and participated in a discussion panel. Discussion continued until well after the stated close time, and there was a lot of engagement
Year(s) Of Engagement Activity 2016
 
Description Prostate Cancer National Research Conference for stakeholders 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Over 300 patients/carers/health professionals attended a research day at which members of the group had a "stall" for informal discussion of their research

Excellent feedback was received which the Prostate Cancer Charity is currently evaluating. This may become a regular event
Year(s) Of Engagement Activity 2011
 
Description Selected talk at Androgens 016 by AD 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk at international conference, followed by discussion with interested attendees
Year(s) Of Engagement Activity 2016
 
Description Selected talk at Androgens 2016 by AB 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk at international conference, followed by discussion with audience and ongoing discussion with interested attendees
Year(s) Of Engagement Activity 2016
 
Description Seminar at CARDIFF UNI 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Talk, followed by afternoon of discussion with interested attendees
Year(s) Of Engagement Activity 2017
 
Description Talk at Aberdeen Uni 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact talk sparked questions and discussion afterwards.

Asked to be external examiner for student
Year(s) Of Engagement Activity 2013
 
Description Talk at Queen's University Belfast 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact talk sparked questions and discussion afterwards.

New contacts made with QUB researchers
Year(s) Of Engagement Activity 2013
 
Description Talk to Cancer Support Group, Maggie's 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Patients, carers and/or patient groups
Results and Impact Talk to a meeting of a cancer support group at Maggie's Cancer Centre. Also a consultation on ethics of biopsy
Year(s) Of Engagement Activity 2018