TO DOWNLOAD A PDF OF THE REPORT, PLEASE CLICK HERE
Report date - 20/01/20
Bradleigh Whitton undertook this four year, laboratory based, doctoral research project with funding provided jointly through The Urology Foundation and Wessex Medical Research. Further funding was obtained from The Gerald Kerkut Charitable Trust (www.kerkut-trust.org.uk). Bradleigh commenced work in October 2015, within Dr Simon Crabb’s laboratory in the Cancer Sciences Unit at the University of Southampton. Co-supervisors were Professor Graham Packham, also within the Cancer Sciences Unit, and Dr Haruko Okamoto, who moved during the project from the Centre for Biological Sciences at Southampton to a new role within the University of Surrey.
This project investigated a novel aspect of prostate cancer biology with potential relevance for therapeutic exploitation. Prostate cancer remains critically dependant on ‘signalling’ through the androgen receptor at all stages. Hormonal therapy addresses this in patients with advanced disease to inhibit disease growth and clinical progression. Evolving alterations within a prostate cancer, linked to androgen receptor function, is central to many forms of hormonal therapy resistance in later stages of the disease.
Within our supervisory team, Dr Okamoto has expertise in the biology of a group of proteins that form ‘cellular pumps’ called vacuolar ATPases. These are complex multi-protein structures that are important in maintaining cellular homeostasis and in particular in maintaining a healthy acid/base balance within cells. This is critical for normal cellular function and if altered can contribute to various disease processes. There is emerging data to support that this can be a factor in the development of some cancers.
This project proposal was based on some initial evidence suggesting that V-ATPases might be important in the development of some prostate cancers. In this project, Bradleigh has investigated a central hypothesis that V-ATPases would activate and drive activity of the androgen receptor in prostate cancer. If confirmed, then they might be relevant potentially to response to current hormonal therapies and V-ATPases might represent a therapeutic target in their own right.
We proposed a pre-clinical cell and molecular biology project undertaken across the Crabb, Okamoto and Packham laboratories within the University of Southampton. The project developed through three parallel work streams which are outlined in the Key Findings section below.
There were no major problems or complications and essentially the project ran to plan.
There were no major problems or complications and essentially the project ran to plan. One challenge that we identified was in the availability of suitable commercial antibodies for testing components of the V-ATPase multi-protein complex. These are used routinely in projects like this to measure the levels of proteins in cells. Bradleigh optimised experimental conditions for some of these to allow them to be used. For others he developed alternative experiments to allow us to test the level of expression of some of these proteins through other methods. Addressing technical challenges is an important aspect of developing as a scientist in this field (and so the challenges were good training opportunities!)
The practical experimental work undertaken within Haruko Okamoto’s lab was mostly completed prior to her departure to Surrey and so did not affect the smooth running of the project. A few final experiments were undertaken after, with Haruko returning to assist with supervision. She remained a fully active member of the supervisory team through video conferencing.
A central component of this project was to use prostate cancer cell line models to determine whether there is a direct interaction between the androgen receptor, which is the critical biological driver of prostate cancer, and V-ATPases. We have established that experimental depletion of V-ATPase function, with specific chemical inhibitors, impacts both on the activity of the androgen receptor and also its expression in prostate cancer cell lines. This was seen at both the protein and RNA message level of target genes for the androgen receptor. (This mirrors the approach to measuring prostate cancer activity in the clinic through levels of a protein called ‘prostate specific antigen’ (PSA)). Furthermore, we found that there was an increase in V-ATPase expression (the amount in the cells) as a result of androgen receptor stimulation. This was of significant central importance to this project as it has established a novel mechanism of androgen receptor functional interaction and confirmed the central basis for the project. It suggested that there is a bi-directional impact between the androgen receptor and V-ATPases in this disease.
The detail of this molecular interaction continued to form a central component of this project. We performed experiments to artificially deplete various components of the V-ATPase protein complex and thus dissect the mechanics of its interaction with the androgen receptor. This included ‘CRISPR’ experiments to edit the genome of prostate cancer cell lines as a model system to explore these concepts further. We also tested chemical inhibition of V-ATPase activity in prostate cancer cell lines. We found this to be effective in blocking the growth of these cells and to be complementary to conventional hormonal treatments such as enzalutamide.
We also undertook work on experimental models of hormonal therapy resistance that mirror the changes seen when established prostate cancer therapies become ineffective clinically (termed castration resistant prostate cancer (CRPC)). By undertaking these experiments we tested the potential relevance of V-ATPases in this important clinical setting for which we require better therapeutic interventions in the clinic. We found that if we inhibited V-ATPase activity then the activity of mutated androgen receptor forms, that can contribute to CRPC, had reduced activity.
Secondly, we have completed work on structural abnormalities of subunits of the V-ATPase protein complex that may be relevant in cancer. Bradleigh generated and tested ‘clones’ of V-ATPase subunits. This work was undertaken in yeast experimental models. These were constructed with the insertion of ‘mutations’ of the basic DNA code. By doing so we were able to test the impact of abnormal forms of V-ATPases seen in some cancers including prostate cancer. We demonstrated that some of these abnormal V-ATPase forms impact its functional state to make it abnormally active. Greater understanding of the underlying alterations in V-ATPase structure in cancers is a potential route to working out how this might be targeted therapeutically in the future. It has potential relevance in other cancers also.
Thirdly we developed data on the genomics of V-ATPase subunit derangement in publically available genomic databases of prostate cancer and compared these to other cancers. This was done in collaboration with Dr Matthew Rose-Zerilli a cancer geneticist within the Faculty of Medicine in Southampton to allow us to identify the specific genetic abnormalities that underpin the role of V-ATPase function in prostate cancer. We found that in a small proportion of cases we were able to identify genetic alterations in the genes that code for the protein subunits that make up the V-ATPase protein complex. This implies that potentially there is an underlying abnormality in V-ATPase itself that contributes to development of prostate cancers in some cases.
Bradleigh Whitton was appointed to undertake this project for a nationally advertised PhD studentship. He had recently completed a Bachelor of Science degree, with first class honours, from the University of Surrey.
Bradleigh Whitton was appointed to undertake this project for a nationally advertised PhD studentship. He had recently completed a Bachelor of Science degree, with first class honours, from the University of Surrey.Bradleigh worked at a high level, making very satisfactory progress towards completion of his doctoral studies on time. He mastered multiple diverse techniques and became increasingly able to function independently and to trouble shoot his experiments. His thesis was examined in November 2019 by Dr Mark Coldwell from the University of Southampton and Professor Hing Leung from the University of Glasgow. He passed, requiring only minor corrections to the thesis, and has been awarded his PhD. Bradleigh has since taken up a post as a Scientific Officer in Target Validation and Immuno Oncology Drug Discovery within the Division of Cancer Therapeutics at the Institute for Cancer Research.
Bradleigh is currently drafting two manuscripts for the data produced from this project that will be submitted to peer reviewed journals in the coming months. We have also co-authored a review on the role of V-ATPases in cancer (Vacuolar ATPase as a potential therapeutic target and mediator of treatment resistance in cancer. Whitton B, Okamoto H, Packham G, Crabb SJ. Cancer Med. 2018 Aug;7(8):3800-3811).
Prior to this project, there was limited data to support a role for V-ATPase in prostate cancer biology. This project has shown that there is bi-directional interaction between it and the androgen receptor. Furthermore this is therapeutically targetable in experimental models that include examples that model CRPC which is a lethal disease state. Furthermore, we have undertaken genomic studies to indicate that some prostate cancers have genomic alterations in V-ATPase and that such alterations confer functional differences in V-ATPase activity. These findings have led us to a conclusion that V-ATPase is a potential therapeutic target in this disease that warrants further investigation.
As described above, our findings indicate that the molecular interaction between V-ATPase and the androgen receptor could be developed as a novel therapeutic target for prostate cancer. We have shown that this includes pre-clinical models of CRPC for which outcomes remain poor and that a therapeutic approach of this nature might combine appropriately with conventional hormonal therapies such as enzalutamide.
The next step in the development of this concept would be to undertake a project focussed specifically on pre-clinical aspects of V-ATPase as a therapeutic target. The chemical approaches we have used were with compounds that are likely to be unsuitable as drugs. We would therefore wish to develop our understanding of chemistry relating to agents that can target the specific subunits of the V-ATPase complex that we have identified as relevant to prostate cancer. This would lead to in-vivo model testing including combination therapeutics studies. Further work would also be valuable in understanding how one might target such an approach to susceptible prostate cancers to try to understand, before clinical evaluation, how one might develop a ‘predictive biomarker’ for benefit.
