The role of PRMT1-mediated arginine methylation during breast cancer development.

Despite recent advancements in sophisticated imaging systems and screening programs, breast cancer is still the third most common cause of cancer death in the UK. A major reason for this is that treatment of breast cancers often leads to acquired drug resistance. Consequently there is still a real need to identify and develop novel therapeutic strategies.
Protein function is dynamically modulated by the addition of chemical groups through a process called post-translational modifications. PRMTs are enzymes responsible for a type of protein modification called arginine methylation which is known to regulate many diverse processes within the cell. Recently, a growing body of evidence has implicated a role for PRMTs in cancer. This is particularly true within the breast; breast cancers overexpress PRMTs, and key proteins involved in the development of breast cancer including estrogen receptor are targeted for methylation. Because of this, we and others have shown that by removing PRMTs from breast cancer cells we can slow their growth. This is really exciting as specific PRMT inhibitors are currently in development; we therefore hope that in the long term drug targeting of PRMTs could be a new way in which to treat breast cancer.
Despite this potential we still know very little about how PRMTs change a normal breast cell to a cancer cell. To help investigate this, we have recently made a number of discoveries and identified new proteins targeted for modification by PRMTs. We now want to take this further and address the following questions:

1. Can we identify breast cancer specific proteins that are targeted for modification by PRMT1 and are these modifications important for cancer cell growth?
2. What role does methylation play in changing the behaviour of proteins that we have identified as new PRMT1 substrates? What significance does this have in breast cancer biology?
3. Can we show that PRMT1 is a good target in which new drugs might be made against?

Altogether, this research will substantially increase our understanding of how arginine methylation impacts on breast cancer biology. This is important as we hope that our findings will pave the way for the development of new anti-breast cancer drugs and help identify new cancer specific methylated proteins that could be used as biomarkers for disease.

We hypothesise that PRMT1-mediated methylation of specific proteins contributes to breast cancer initiation and/or progression. Furthermore, we hypothesise that genetic disruption of the prmt1 loci will reduce oncogenic Her2 penetrance and tumour burden. The first objective is to quantitatively identify and characterise breast cancer specific PRMT1 substrates through SILAC coupled to an affinity purification process that we have already validated. We will then build on preliminary data to further understand the role of PRMT1-mediated methylation of USP7 and USP11 through a combination of methyl-site mapping, mutational analysis and the generation of cell lines ectopically expressing proteins proficient or deficient in methylation. These tools will then be used to study the effects of USP7/11 methylation on DUB activity and the ability of USP7/11 to engage the DNA damage response. We will also establish if USP7/11 mediated stabilisation of PRMT1 is a mechanism of methyltransferase regulation through the use of in vitro methylation assays.

In order to explore the transforming potential of PRMT1 we will utilise the 3D acinar formation in matrigel assay that we have already established. To understand the in vivo significance of PRMT1 in the transformed mammary gland, I will use my expertise in utilising genetically modified mice in the study of tumourigenesis. We will cross the conditional prmt1 floxed (prmt1f/f) mouse with a well-established Her2 mouse model of human breast cancer (MMTV-Her2-IRES-Cre) and monitor tumour initiation and burden by generating the MMTV-Her2-IRES-Cre;PRMT1 F/F strain. The breeding and experimental scheme we have devised clearly shows that we are committed to ensuring reduction and refinement of all our animal procedures through design of appropriate experiments. A statistical significance of 5% will be used for data obtained and a power of 80%.

This proposal focuses on basic biosciences and will improve our understanding about the mechanism of breast cancer and will therefore ultimately contribute to the diagnosis and treatment of breast cancer. Main beneficiaries will be:

The academic community.
Within the arginine methylation community, there are a small number of groups focusing on breast cancer (eg. Dr Jocelyn Cote in Ottawa, Canada and Muriel Le Romancer, Lyon, France). Our approach of biochemical characterisation coupled with in vivo analysis using mouse models will greatly benefit their own research by providing experimental tools and new knowledge in the field. I have also established collaborations with Dr. William Muller (Montreal, Canada) who researches EGFR signalling in the transformed breast. Results from this project could potentially lead to further collaboration in the future.

Clinicians
By identifying novel breast cancer methyl-specific proteins and through the subsequent development of modification-specific antibodies, we will be providing Clinical Research Fellows research tools in which to potentially establish new cancer biomarkers. This will greatly facilitate research into this area and could be of clinical benefit to cancer patients (see below).

Pharmaceutical Industry/Biotech companies/medicinal chemists.
By genetically demonstrate a requirement for PRMT1 in breast cancer we will be validating PRMT1 as a target for drug design. This will therefore be highly beneficial to the biotechnology sector and our chemistry collaborators (Dr. James Dowden, University of Nottingham). The development of clinically relevant PRMT1 inhibitors could in the long term stimulate economic growth. Furthermore, given the implication of deregulated arginine methylation in several disease processes (cancer, CVS, viral pathogenesis) the scope for clinical use of PRMT1 inhibitors is far reaching.

Cancer Patients
Breast cancer is still a disease associated with morbidity and mortality with a real need to identify novel targets for rational drug design. The long-term implications for our research for the cancer patient is two fold: Firstly, chemical inhibition of PRMT1, or small molecule manipulation of the effects of arginine methylation could help identify new drugs for cancer treatment. Secondly, research tools that we will develop (methyl-specific antibodies) might be useful in the diagnosis or staging of breast cancer, and would also identify patients that may respond to anti-PRMT1 compounds. Untimely this could lead to a more personalised approach to cancer therapy and reduce unwanted side effects, improve clinical efficacy and increase the quality of life and well being of patients.

The NHS and Cancer Charities
These are major stakeholders in the search for better diagnosis strategies and more personalised cancer diagnosis. Cancer treatment and palliative care is a major financial burden on the NHS, a better understanding of disease processes will ultimately lead to the development of tailor-made therapeutic strategies thus limiting disease relapse and after-treatment care. Our research will also be used to educate charitable organisations and fundraisers on the science behind breast cancer research and keep them informed of advancements in the field.

Transferable skills gained by staff working on the project.
The post-doctoral scientist employed on this project will develop extensive project management skills required for in vivo research and acquire a skill sets highly desirable for future employment prospects. With help from myself, the post-doc will also gain additional general project management skills that are obtained from running a research project, which are transferable to many other disciplines in addition to the scientific employment sector. The post-doc will gain communication and supervision/teaching skills that will be transferable to any management discipline.