Quatitative proteomic analysis of castrate-resistant prostate cancer

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.

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.

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.