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

Lead Research Organisation: University of Birmingham
Department Name: Cancer Sciences

Abstract

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.

Technical Summary

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%.

Planned Impact

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.
 
Description Eliz Challenor Scholarship
Amount £16,000 (GBP)
Organisation University of Birmingham 
Sector Academic/University
Country United Kingdom
Start 09/2016 
End 08/2017
 
Description Investigating the role of PRMT5 in cancer
Amount £1,495,856 (GBP)
Funding ID 25500 
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2018 
End 04/2024
 
Description Modelling breast cancer in mice via CRISPR-mediated in vivo gene editing
Amount £12,251 (GBP)
Funding ID Wellcome Trust ISSF 
Organisation University of Birmingham 
Sector Academic/University
Country United Kingdom
Start 08/2018 
End 08/2019
 
Description UoB RDF fund - Does arginine methylation of RUVBL1 regulate the drug sensitivity of triple negative breast cancers
Amount £4,793 (GBP)
Organisation University of Birmingham 
Sector Academic/University
Country United Kingdom
Start 12/2017 
End 08/2018
 
Title Intra-nipple injection of Adenoviral Cre into genetically enginerred mice 
Description We are establishing a protocol to induce Cre-mediated gene deletion in the mouse mammary gland of genetically engineered mice. We have found that the MMTV-Cre transgenic strain that is often used to induce Cre-mediated gene recombination in the mammary gland is expressed early during development throughout the developing embryo. This can be problematic if genetic deletion of the gene being studied (as in our case with PRMT1) results in embryonic lethality. To circumvent this, and to enable the study of PRMT1 in adult tumour baring mice, we are refining a protocol that enable deletion of PRMT1 specifically in the ducts of the mammary gland through the intra-nipple injection of Cre-expressing viruses. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact This methodology, whilst being developed by the Krause lab based at Harvard Medical School (http://www.jove.com/video/50692/intraductal-injection-for-localized-drug-delivery-to-mouse-mammary), but has not been widely implemented in the UK. By establishing this protocol, we will be able to disseminate out expertise to other researchers. In line with this, I am a member of SEARCHbreast, a resource to facilitate sharing of archived material and methodologies from in vivo breast cancer models. In addition, by using Cre-expressing virus that targets specific cell type of the mammary gland (luminal cells verses basal myoepithelial cells), we will be able to start asking questions regarding the cell of origin of cancer development. This model also has the advantage of being able to conduct intra-animal controls. Here, control virus is injected one side of the animal, and Cre-expressing virus on the other. Furthermore, by exogenously expressing Cre via a virus, one less animal strain (i.e. the Cre transgenic strain) is required, thus reducing the complexity of breeding strategies and number of unwanted animals due to incorrect genotypes. Together, these factors mean a refinement of experimental design and a reduction in animal numbers. 
URL http://www.jove.com/video/50692/intraductal-injection-for-localized-drug-delivery-to-mouse-mammary
 
Description Astra Zeneca PRMT5 projects 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution This was a studentship awarded directly to Astra Zeneca Oncology department, of which only one was available, of which myself and Dr. James Lynch at AZ competitively obtained. It will be critical in helping to build up industrial links, particularly concerning PRMT5 and the DNA damage response. The student will be hosted for 7/8th of her time in my lab, and I am her main supervisor.
Collaborator Contribution Astra Zeneca have financially contributed to bench fees and student stipend. They will provide tool compounds to investigate DNA damage pathways, and the student will be visiting AZ to conduct a CRISPR screen. We have monthly meeting with her co-supervisor at AZ to facilitate a good working relationship and development of the project.
Impact None yet.
Start Year 2018
 
Description Establishment of intra ductal injections 
Organisation Netherlands Cancer Institute (NKI)
Country Netherlands 
Sector Academic/University 
PI Contribution We are injecting lenti-cre into PRMT1 Floxed animals crossed to the PYMT oncogenic transgene to monitor tumour growth after PRMT5 deletion in luminal cells. We are monitoring recombination by using the mTmG system that means luminal cells turn from red to green after recombination.
Collaborator Contribution This is a collaboration with Prof. Jos Jonkers, Group Leader at the Netherlands Cancer Institute. He has established a protocol to promote Cre-mediated recombination of luminal cells of the mammary duct via intra ductal injection of lenti-Cre. My postdoc went to the NKI to learn this challenging technique from his animal technicians, and has spent a number of months perfecting it in our laboratory. We are only one of two laboratories in the UK that are routinely performing intra ductal injections. Jos Jonker is also providing us with purified lenti-Cre for these experiments.
Impact No output yet.
Start Year 2015
 
Description Ingrid Dreveny USP11 
Organisation University of Nottingham
Department School of Chemistry Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We are characterising PRMT1-mediated methylation of USP11, making methylation sites and establishing biological significance.
Collaborator Contribution Dr. Dreveny has provided expert advice on the study of USP11 and has provided us with recombinant protein enabling in vitro methylation assays analysis of wild type and methyl-deficient USP11. This has been important as USP11 is a large protein and very difficult to purify. Her expertise in protein purification, and thus providing this regent has been invaluable for the continuation of the project. She has also conducted in silico analysis of how methyl-USP11 can influence protein structure, and has advise on setting up USP11 DUB assays within my lab.
Impact No output yet.
Start Year 2016
 
Description James Dowden, PRMT1 inhibitors 
Organisation University of Nottingham
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution We have trained James Dowden's PhD student in PRMT1 vitro methylation assays which has enabled small scale analysis of putative PRMT1 inhibitor compounds.
Collaborator Contribution James Dowden has synthesised a series of PRMT1 inhibitors for analysis.
Impact No Outputs yet
Start Year 2015
 
Description SILAC PRMT1 project 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We devised the intellectual questions and generated the cells lines required (including molecular biology cloning steps). We frequently communicate regarding seeking advice on assay design. We visit Dr Skehel at least once a year.
Collaborator Contribution Dr Mark Skehel has advised us on technical aspects of setting up SILAC labelling in our lab. He has analysed samples for incorporation and advised us when necessary. He has provided us with mass spectrometry data as a no-fee collaborations, thus substantially reducing costs for this part of the project.
Impact This is an onging project and outcomes have yet to be made.
Start Year 2015
 
Description CRUK fundraising event for Business in Birmingham 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Supporters
Results and Impact Invited to attend a charitable dinner to raise money for Cancer Research at University of Birmingham through the influence of CRUK and Businesses for Birmingham. I sat on a table with cancer survivors/patients, and influential fundraisers, and publicised the research we are doing, and how CRUK are supporting it. It has raised awareness of research in Birmingham and will hopefully help to fund-raising efforts.
Year(s) Of Engagement Activity 2018
 
Description CRUK legacy visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Supporters
Results and Impact 100 supporter of CRUK who were thinking of leaving sizeable donations to the charity in their Will were invited to the Institute. As part of this, my laboratory conducted a lab tour to the group to explain the type of research we were doing and to highlight the importance of funding for this to occur. The organiser of the event reported that many of the visitors appreciated the science we were conducted and understood the need for financial support.
Year(s) Of Engagement Activity 2016
 
Description College assembly - Pecha Kulcha style presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact I represented our Institute at the College Assembly and presented our recent findings published in Cell Reports in a lay format and in the Pecha Kulcha style. Here, slides are automatically rolling and change every 20 seconds. The premise was to increase awareness of research in the Institute of Cancer and Genomic Sciences to other academics and non-academic working at the University of Birmingham
Year(s) Of Engagement Activity 2018
 
Description Lab tour for Woman in Business - CRUK organised 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Supporters
Results and Impact 13 local business woman visited our Insititute to hear about the cancer research work that is being funded by CRUK. We provided a lab tour and hands-on experience of working in lab. We raised awareness of the type of research my lab is doing and highlighted how expensive it is to do research. One participant asked about great writing and was amazed that the researchers wrote them! The participants throughly enjoyed their lab tour.
Year(s) Of Engagement Activity 2018