Quatitative proteomic analysis of castrate-resistant prostate cancer

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Prostate cancer is the most prevalent malignancy among adult men in the developed world and the second commonest cancer in the UK with an estimated 27,000 new cases diagnosed each year. Recent advances have improved early diagnosis and treatment, however many men relapse and develop aggressive forms of treatment resistant prostate cancer. Hormone treatment (androgen ablation) is commonly used for incurable cancer, but is often complicated by relapse with aggressive disease with tendency for metastases, resulting in a very poor prognosis. Research into prostate cancer focused on this area may in the future help us develop better treatments through a greater understanding of the underlying biology.

The mechanism by which prostate cancer becomes resistant to hormone treatments is not fully known. Recent advances in techniques looking at genes and proteins may shed light on the complex pathways by which cancer develops resistance to treatments. SILAC is a new way to accurately study thousands of proteins at one time. By combining the SILAC technique with available cutting-edge laboratory cell models of hormone resistant prostate cancer, I aim to explore important new proteins involved in prostate cancer relapses despite hormone treatment.

Professor Leung's group at the Beatson Institute for Cancer Research has developed exciting model systems of prostate cancer and by working with other research leaders at the Beatson including Dr Zanivan (expert on SILAC studies) and Dr Morrice (expert on protein analysis) I will analyse the protein changes that occur in hormone resistant prostate cancer. The ultimate goal is to identify proteins as drug targets to treat the aggressive, hormone resistant type of prostate cancer to improve quality of life and prognosis of the many men who suffer with this incurable disease.

Technical Summary

Aims: To study the proteome of castrate resistant prostate cancer (CRPC) and identify novel important proteins that can be exploited as potential therapeutic targets.
Objectives: To apply available in vitro and in vivo pre-clinical CRPC models to an unbiased proteomic analysis to identify novel proteins associated with CRPC.

Methods: Three Human PC cell lines and their CR isogenic derivatives will be selected and transfected with luciferase expressing constructs. Orthograft in vivo models will be used with castration to create a physiological environment for CRPC, compared to hormone naïve (HN) tumours. Tumours will be removed at the same stage as determined by luciferase signal comparing HN and CR tumours (all 3 pairs of cells). Experiments will be performed in triplicates to ensure accurate quantification and identification of proteins.

SILAC-based analysis of the tumours will be performed with peptides fractionated using filter-aided sample preparation and strong anion exchange chromatography. High-resolution mass spectrometry (MS) will be performed and data will be analysed for differential abundance of proteins common to the three HN/CR tumour pairs. Candidate proteins will be selected for validation.
Western blot and qRT-PCR, will test the abundance of candidate proteins in the CR cell lines. Knockdown of the candidate protein(s) in CR cell lines and orthografting will test reversibility of the CR phenotype. Immuno-staining of human tissue microarrays will attempt to confirm the protein(s) in clinical samples.
Contingency plan: If cells cannot be labelled by SILAC, a label-free approach may be used requiring quadruplicate orthograft models as quantification is less accurate than with SILAC.
Scientific Opportunities: Proteomic analysis of CRPC will generate large quantities of data, which may aid other research groups.
Medical Opportunities: Identification of commonly expressed important proteins in CRPC may provide ideal therapeutic drug targets.

Planned Impact

Prostate cancer is very common and despite increased awareness of this disease in the community, a significant proportion of presenting patients have incurable advanced cancer. For many of them, androgen ablation therapy is the current standard of care. Some patients receive radiotherapy as well. Even for those patients with disease deemed curable, as many as 30% of them will relapse over a period of 5-10 years.

The use of androgen ablation has been in practice for almost 60 years. The outcome has not changed despite significant efforts. The outlook for patients with CRPC is generally poor despite new hormonal agents and chemotherapy. Hence, the need to develop better treatment remains a very high priority. The fundamental issue remains the lack of comprehensive insight into the underlying reason for hormone escape. In this study, I will apply an unbiased method to study the proteome of prostate cancer during the transition from hormone responsive to hormone resistant status.
If successful, new tests can be developed to detect the formation of hormone resistant prostate cancer. Furthermore, proteins abnormally expressed in relapsed disease may be suitable targets for developing new therapies against.

To ensure engagement with this important research I will communicate with the research community through formal presentation of this work at national and international cancer research conferences. Locally I will participate in seminars and public events such as the annual science event at the Beatson Institute for Cancer Research. As a clinician I will also aim to engage the clinical community by seeking opportunities to present the work to a clinical audience both locally and at national Urology meetings.

Finally to ensure the maximum impact of the work for the future the research dataset will be submitted to the international Proteome Exchange consortium so as to be made available to multiple proteomics repositories, which can be accessed by researchers worldwide.

Publications

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Ahmad I (2016) Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer. in Proceedings of the National Academy of Sciences of the United States of America

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Salji M (2016) Non-clinical research - laboratory based in Journal of Clinical Urology

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Winchester C (2017) Gathering preliminary data in Journal of Clinical Urology

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Winchester C (2016) Writing a literature review in Journal of Clinical Urology

 
Title Peri-prostatic Fat Volume Measurement as a Predictive Tool for Castration Resistance in Advanced Prostate Cancer 
Description The surrounding region of the prostate comprising of periprostatic adipose tissue, including vessels draining the prostate can be quantified by the following method on T2 weighted MRI of prostate. Laterally the tissue is delineated by the first visible fascial boundary adjacent to the levator muscles, posteriorly by Denonvillier's facia (excluding the mesorectal fat) and anteriorly by the pubic symphysis, including the anterior venous plexus and retropubic fat, on each 3-mm slice from the bladder neck superiorly to prostate apex inferiorly. This delineates the region of surrounding adipose tissue around the prostate of similar signal and composition including local vasculature providing venous drainage of the prostate and seminal vesicles. Collectively, these represent the local visceral adipose-rich tumour macro-environment. This measurement may improve our current ability to predict patients who will respond less well to Androgen Deprivation Therapy prior to their commencing treatment. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2017 
Provided To Others? Yes  
Impact Publication of the paper describing the technique and its usefulness in comparison to current predictive clinical factors. 
URL http://www.sciencedirect.com/science/article/pii/S2405456917300287
 
Description BAUS June 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The British Association of Urological Surgeons Annual Congress June 2016 held in Liverpool BT Convention Centre. I presented a collaborative project entitled 'A Urinary Peptide Biomarker Panel to Identify Significant Prostate Cancer' winning the best academic paper award from BJU International. This was publicised on Twitter during the conference. I was subsequently invited as a speaker to present the work to the BAUS uro-oncology community at the BAUS section of oncology meeting (Nov 2016) as part of the biomarkers series of talks.
Year(s) Of Engagement Activity 2016
URL https://twitter.com/BURSTurology/status/747361597857927168
 
Description BAUS Oncology Nov 2016 urinary biomarkers talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited speaker to present prize winning talk on 'A Urinary Peptide Biomarker Panel to Identify Significant Prostate Cancer' to >100 professionals in uro-oncology. As part of the opening biomarker series of talks.
Year(s) Of Engagement Activity 2016
 
Description Genes and Cancer Conference (Cambridge) poster presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation of current work at international conference aimed at cancer researchers. Discussed my research with interested parties and Industry/Business representatives who also attended.
Year(s) Of Engagement Activity 2015
URL http://www.genesandcancer.org.uk/
 
Description Invited speaker at the CRUK Beatson Institute Public Open Evening March 2016 
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 Public/other audiences
Results and Impact I was an invited speaker to talk about Prostate Cancer and how my current research fits in to the wider picture of Prostate Cancer treatment entitled 'Dissecting Cancer: From Robotic Surgery to Molecular Biology' to a public audience of ~ 150 people comprising of local high school students, patient/relatives and interested public, at the CRUK Beatson Institute Public Open Evening March 2016.
Year(s) Of Engagement Activity 2016
 
Description Prostate Cancer Roadshow Open day invited speaker (CRUK Beatson Institute) 
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 Patients, carers and/or patient groups
Results and Impact 80 patients/families and careers and interested public attended for a Prostate Cancer Roadshow day sponsored by CRUK and MRC, there was discussion around current translational research including my own research and national clinical trials in prostate cancer in particular outcomes of the STAMPEDE trial and how this has influenced current standard of care in the treatment of advanced prostate cancer.
Year(s) Of Engagement Activity 2016