Defining the role of FOSL2 in molecular adaptation to hypoxia in colorectal cancer

Lead Research Organisation: University of Nottingham
Department Name: School of Medicine

Abstract

Colorectal cancer is the third most common cancer in both men and women and leads to 500,000 deaths a year worldwide. As tumours grow they need blood vessels to transport oxygen and nutrients into the tumour. However tumours often outgrow their blood supply and contain regions that do not have enough oxygen or nutrients. These regions are frequently found in colorectal cancer. The colorectal tumour cells in these areas change at a molecular level so that they can survive in these "starving" conditions. The changes that occur make the cells resistant to chemotherapy and radiotherapy and make it more likely the tumour will spread. Patients with tumours that contain these regions of low oxygen are less likely to survive than patients without these regions. Thus it is important to find new alternative ways of killing cells in these regions with not enough oxygen or nutrients that can be used in combination with current approaches that are effective at killing the other regions. We think that we have found a new way of stopping the cells from adapting to the lack of oxygen and nutrients. In my previous work I tested the effect of interfering with 6000 genes and examined the effect of each of these on how cells survived and grew in an environment lacking oxygen. I also investigated whether patients with higher expression of any of these 6000 genes were less likely to survive. I found that 52 genes fit into both these categories inhibiting survival and growth in regions of low oxygen and also where high expression in a patients tumour meant the patient was less likely to survive. Interfering with one of these genes had the greatest effect on stopping tumour cells adapting to regions of low oxygen, preventing them from surviving and growing in these conditions in all of the tests. We think that by interfering with this gene we are inhibiting the changes that occur at a molecular level in response to the lack of oxygen and nutrients. We want to investigate the link between high expression of the protein this gene makes and the regions of low oxygen and colorectal cancer patient survival. This will let us see if high expression of this protein could be used to help understand which patients may need more aggressive treatments. We want to investigate how this gene helps tumour cells grow and survive in hypoxia. We will also examine the extent of the effect at the molecular level and at a cellular level to investigate the exact consequence of targeting this protein on colorectal cancer regions which lack of oxygen and nutrients. This will provide evidence for developing new inhibitors to target this therapy-resistant region of colorectal tumours.

Technical Summary

Colorectal cancer is the third most common cancer in both men and women and leads to 500,000 deaths a year worldwide. 1 in 3 colorectal tumours contains regions of low oxygen (hypoxia) that are found in most solid tumour types. Hypoxia is caused by high tumour metabolic and proliferative rates and aberrant vascularization. Clinically hypoxia is associated with worse patient survival and chemotherapy and radiotherapy resistance. Hypoxia also induces metastasis, a major cause of cancer death. Identifying novel ways to target hypoxic tumours is a key objective to improve therapeutic response for these patients. We carried out an unbiased induced-essentiality functional shRNA screen to identify novel regulators of hypoxic tumour cell survival in 2D and 3D cell-culture. FOSL2 was the top hit in these analyses where FOSL2 knockdown reduced cell viability in hypoxia. Using published colorectal cancer expression array data we identified that high FOSL2 expression is associated with worse patient survival. We have shown that FOSL2 expression is increased in hypoxia and that knockdown reduces the expression of the key hypoxia regulated gene CA9. FOSL2 binds with Jun proteins to form variants of the transcription factor complex activator protein-1 (AP-1). We hypothesise that FOSL2 promotes hypoxic cell growth and survival via transcriptional regulation of target genes and we will investigate this using 2D, 3D and in vivo xenograft models. We will identify the HIF1a and HIF2a -dependent and -independent gene expression roles of FOSL2. Furthermore a key FOSL2 regulated-transcript will be investigated for its mechanistic role in hypoxic tumour growth and survival. We will examine the associations between FOSL2 and patient survival, and hypoxia using colon cancer TMA. We will investigate FOSL2 as a key regulator of molecular adaptation to the hypoxic tumour microenvironment and as a target for development of inhibitors against the therapy resistant hypoxic tumour microenvironment.

Planned Impact

Direct beneficiaries will include, the academic community as proposed in the academic beneficiaries section, the scientists employed and funded by the research grant, the biotech and pharmaceutical industry, the University of Nottingham and long term colorectal cancer patients and patients with additional types of solid tumour which contain regions of hypoxia.
The scientists employed and funded by the grant to perform the research will directly benefit from training that they receive to do the work. The plan for the research is to hire 2 experienced researchers to fill the post-doc and bioinformatician-post-doc positions, however the breadth of research techniques and the variety of scientific challenges involved in the work necessitate that both individuals will require additional training and support. The skills developed in both positions are in demand in both industry and also academia and will provide a strong skill set base for their future careers.
The plan for the research is to identify and functionally validate 2 targets that are required for tumour cell growth and survival in hypoxia. Hypoxia is associated with poor patient outcome and chemotherapy and radiotherapy resistance in colorectal cancer and most other solid tumour types. The resistance of hypoxic regions of solid tumours to therapeutic regimes is a current stumbling block in cancer therapy. Therefore identifying novel ways to target hypoxic regions of tumours that induce apoptosis is key to overcoming therapy resistance and improving patient survival in combination with current treatment strategies. Further work will investigate the effect of combining such targeting with other chemotherapy and radiotherapy strategies in preclinical investigations. Furthermore this work will assess the utility of FOSL2 as a biomarker for poor patient outcome. These investigations will also be developed further examining FOSL2 not only as a prognostic indicator but also as a possible companion biomarker for any FOSL2 targeting small molecular inhibitor.
These are key long-term aim for the project. This will directly benefit the University of Nottingham, which will receive financial benefits from any patents filed upon the work based on its future commercialisation. Towards the mid-point/end of the project we will establish collaborations to identify novel inhibitors to target FOSL2 and FOSL2 regulated genes and pathways as a method of targeting the hypoxic tumour microenvironment (as described in the academic beneficiaries section). This would lead to investing in drug development projects with pharmaceutical companies or through establishing a spin-out company as discussed in the pathways to impact document. This will in the medium-term directly financially benefit the pharmaceuticals and biotech industry.
Long-term the work will have significant impact on the colorectal cancer patients and patients of other solid tumour types which contain regions of tumour hypoxia, by providing molecular targeted therapies to increase survival in these patients which currently have a worse outcome.
The main indirect Beneficiary is the UK economy. As recognised by organisations such as Campaign for Science and Engineering (CaSE), the UK has a strong record in science research despite low investment compared to other leading scientific nations. Maintaining high levels of high quality research is key in attracting inward financial investment into the UK. As stated by CaSE "Research and Innovation underpins a strong economy". It is therefore key for retaining the presence of the UK pharmaceuticals industry to have cutting edge high impact science in the UK with long-term clinical impact goals. Furthermore the training received by the 2 post doc scientists will also add to the UK economy as the skill sets developed during the term of the positions will increase the expertise available to academic and/or pharmaceutical institution

Publications

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Alfardus H (2017) MicroRNA Regulation of Glycolytic Metabolism in Glioblastoma. in BioMed research international

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Morotti M (2019) Hypoxia-induced switch in SNAT2/SLC38A2 regulation generates endocrine resistance in breast cancer. in Proceedings of the National Academy of Sciences of the United States of America

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Poser SW (2019) Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

 
Description BSc/MSci Cancer Sciences
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact I have as part of a small committee of 5 academics played a significant role in establishing a new research intensive BSc/MSci Cancer Sciences Undergraduate Course at the university of Nottingham. This involved course design and preparation, submission of the plans to the division, school and university, marketing etc. We are the majority of the way through this process (Business Case approved September 2017; Course Specifications in the final stages of acceptance -final corrections made and submitted December 2017) the course will begin in September 2019.This will have a significant economic impact initially through the establishment of the new course but also once the graduates of this new course make their way in the UK and international workforce. https://www.nottingham.ac.uk/ugstudy/courses/cancersciences/cancer-sciences-msci.aspx https://www.nottingham.ac.uk/ugstudy/courses/cancersciences/cancer-sciences-bsc.aspx
URL https://www.nottingham.ac.uk/ugstudy/courses/cancersciences/cancer-sciences-bsc.aspx
 
Description Defining the role of STAT3 in astrocyte adaptation to hypoxia as therapeutic target in glioblastoma (CoI)
Amount £119,734 (GBP)
Funding ID GN-000537 
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 09/2018 
End 08/2020
 
Description Investigating the role of FOSL2 in transcriptional heterogeneity and its regulation by kinases in hypoxic colorectal cancer
Amount £40,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2020 
End 03/2022
 
Description Collaboration with Kazan Federal University, Kazan, Russia Albert A. Rizvanov, Svetlana F. Khaiboullina. 
Organisation Kazan Federal University
Country Russian Federation 
Sector Academic/University 
PI Contribution Expertise and intellectual input. We are developing a research project together and writing a research review on microRNA post transcriptional regulation of NLRP3 inflammasome in the immunopathologies.
Collaborator Contribution Expertise, access to equipment and facilities intellectual input. We are developing a research project together and writing a research review on microRNA post transcriptional regulation of NLRP3 inflammasome in the immunopathologies.
Impact We have published a review article: MicroRNA Post-transcriptional Regulation of the NLRP3 Inflammasome in Immunopathologies Frontiers in Pharmacology 2019-05-01 | journal-article DOI: 10.3389/fphar.2019.00451Part of ISSN: 1663-9812
Start Year 2018
 
Description Collaboration with University of Bath, Centre for Therapeutic Innovation, Bath, UK, Albert A. Banafshe larijani 
Organisation University of Bath
Country United Kingdom 
Sector Academic/University 
PI Contribution We are hosting a researcher from the lab of Banafshe larijani to learn techniques and expertise in the development in a novel approach for investigating the role of HIF proteins in hypoxia in patient tumour samples.
Collaborator Contribution The collaborators are developing new tools for FRET/FLIM investigation of the role of HIF proteins in hypoxia in patient tumour samples.
Impact We have generated preliminary data and developed a grant that we will submit to the MRC as a program grant in the May grant round.
Start Year 2019
 
Description Pedro Fuentes-Visiting PhD student from Institut de Recerca Hospital Vall d´Hebron (VHIR) 
Organisation Vall d' Hebron Research Institute
Country Spain 
Sector Academic/University 
PI Contribution Pedro Fuentes-Visiting PhD student from Institut de Recerca Hospital Vall d´Hebron (VHIR). Pedro joined us to learn 2 of the scientific techniques that we have optimised and to develop his research in line with this and investigate the role of hypoxia on the molecular mechanism he was investigating.
Collaborator Contribution Pedro generated additional research data that will contribute to his PhD the understanding of metastasis in cancer and a research publication.
Impact None as yet
Start Year 2017
 
Description miR193BHG-Ivan Lab 
Organisation Indiana University
Country United States 
Sector Academic/University 
PI Contribution We used siRNA and qRT-PCR to identify that the long intergenic noncoding RNA (lincRNA) transcribed from the miR193BHG locus on chr16p13.12 termed NORS (Noncoding Oxygen-Sensitive Regulator of Sterol/Steroid Biogenesis) was regulated at a transcriptional level by FOSL2 in conditions of low oxygen (hypoxia).
Collaborator Contribution We contributed this work to a larger study being led at the University of Indiana in the Ivan lab investigating noncoding transcripts which modulate sterol and steroid biosynthesis in low oxygen conditions
Impact We have submitted a manuscript to Nature Communications which is under review, NCOMMS-18-25313A: Title: NORS: A LncRNA Regulator of the Cellular Sterol Biosynthesis Program
Start Year 2017
 
Description BBC Radio Nottingham interview and article in local newspaper 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact There was an article in the local newspaper (Nottingham post) and I was interviewed on BBC Radio Nottingham during drive time at 17:15 about our work in particular one of the projects funded by Breast Cancer Now investigating the role of pH regulation by bicarbonate transporters in metastasis in breast cancer. In the interview we discussed breast cancer generally the progress of the research and the future clinical impact this may have. The intended purpose was to help the general public understand the research, why it is important and the future benefits which may arise.
Year(s) Of Engagement Activity 2017
URL http://www.nottinghampost.com/news/nottingham-news/university-professor-secures-90k-funding-461004
 
Description Invited Speaker- 3. Fudan University Cancer Hospital, Shanghai 15th November 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker to give a seminar Fudan University Cancer Hospital, Shanghai 15th November 2018. The intended purpose was to identify and establish new collaborations to present our research to the field to receive feedback about our work and update other researchers in the immediate and related fields on our progress, publicising our progress and ideas.
Year(s) Of Engagement Activity 2018
 
Description Invited speaker- West China Hospital, Sichuan University, Tumour Centre, Chengdu, China-13th November 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker to give a seminar at West China Hospital, Sichuan University Tumour Centre, Chengdu, China-13th November 2018. The intended purpose was to identify and establish new collaborations to present our research to the field to receive feedback about our work and update other researchers in the immediate and related fields on our progress, publicising our progress and ideas.
Year(s) Of Engagement Activity 2018
 
Description Lund University-invited speaker, seminar February 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker to give a seminar at Lund University February 2018. The intended purpose was to identify and establish new collaborations to present our research to the field to receive feedback about our work and update other researchers in the immediate and related fields on our progress, publicising our progress and ideas. A new collaboration was established with Dr Yvonne Ceder, Lund University Medicon Village, Sweden, this is at an early stage.
Year(s) Of Engagement Activity 2018
 
Description Open day University of Nottingham Wonder 2017 
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 Public/other audiences
Results and Impact We had an interactive stand at the open day explaining the research that we did to understand regions of low oxygen in tumours. The stand enabled the general public to look at regions of low oxygen tumour sections under the microscope and had a number of exhibits explaining the work we do in the lab. 4 members of my group (including the MRC funded post doc) and myself participated discussing and answering questions about our research and more generally about cancer. 100+ people visited us. In addition to the impact this had on the audience it helped the researchers gain a different perspective on their work and inspired them in their research going forward. The lay advert was as follows:
Tackling tumours
Come and see how cancer researchers are trying to understand
hard to treat regions of tumours. Look at these down a microscope
and see if you can guess where they might be. Suitable for 5 years up.
Year(s) Of Engagement Activity 2017
URL http://www.nottingham.ac.uk/wonder/
 
Description Open day University of Nottingham Wonder 2019 
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 Public/other audiences
Results and Impact We had an interactive stand at the open day explaining the research that we did to understand breast cancer. The stand enabled the general public to look at histological section of breast tumours under the microscope and had a number of exhibits explaining the work we do in the lab. 2 members of my group including myself participated discussing and answering questions about our research and more generally about cancer. 100+ people visited us. In addition to the impact this had on the audience it helped the researchers gain a different perspective on their work and inspired them in their research going forward.
Year(s) Of Engagement Activity 2019
URL https://www.nottingham.ac.uk/wonder/
 
Description Organiser-British Association Of Cancer Research (BACR) Tumour Microenvironment Meeting 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was an organiser of the 2019 BACR Tumour Microenvironment meeting, July 1st-3rd 2019, Nottingham Conference Centre, Nottingham, UK The meeting opened with a keynote session from Gabriele Bergers who presented an outstanding story regarding the immunomodulatory role of endothelial cells, in particular high endothelial venules. The Vascular-tumour interactions session was filled by Michele De Palma, Richard Kolesnick, Martin Brown and Andrew Reynolds. The second day started with the Immunology with David Lyden, Munitta Munthana, Jacqui Shields and John Stagg enchanting us in the Immune-tumour Interactions session. Day 3 was opened with our second keynote lecture by Adrian Harris who entertained us with his current work regarding metformin, metabolism and glutamine dependency in cancer. These theme was continued by Alan McIntyre, Ester Hammond and Tony Ng. Kurt Ballmer-Hofer welcomed us into the world of VEGF-Receptors before we went Into the Clinic with Stuart Farrow and Lindy Durrant. The conference welcomed nearly 190 guests, speakers and sponsors from across the globe, including each of the major cancer centres in the UK. We were delighted to see the number of postgraduate students, and early career researchers at the meeting and our invited speakers were joined to present their work as selected speakers from the submitted abstracts. Our lively poster sessions were indeed a highlight, and poster prizes (with generous sponsorship from EACR) to Ute Jungwirth, Miriea Sueca Comes and Brian Lee. Excellent feedback was received from attendees and speakers with the local organising team (Andrew Benest, David Bates, Stewart Martin, Hester Franks, Alan McIntyre and Sarah Storr) .
Year(s) Of Engagement Activity 2019
URL https://www.bacr.org.uk/news/tumour-microenvironment
 
Description Public open day Launch of Nottingham Breast Cancer Research Centre- Sunday 25 March, 10am - 3pm 2018 
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 Public/other audiences
Results and Impact We had an interactive lab tours at the open day explaining the research that we did to understand regions of low oxygen in tumours. The lab tours enabled the general public to look at regions of low oxygen tumour sections under the microscope and had a number of exhibits explaining the work we do in the lab. 6 members of my group (including the MRC funded post doc) and other departmental researchers of the dept i manage and myself participated discussing and answering questions about our research and more generally about cancer. 70+ people visited us in the lab. In addition to the impact this had on the audience it helped the researchers gain a different perspective on their work and inspired them in their research going forward.

In addition i also presented a lay overview of the research we are doing to an audience of 250+.
Year(s) Of Engagement Activity 2018
URL https://www.nottinghampost.com/news/health/nottingham-lead-fight-against-breast-1381253
 
Description Public open day- Lab tours as part of the Nottingham Breast Cancer Research Centre Open day- Sunday 28th October 
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 We had an interactive lab tours at the open day explaining the research that we did to understand regions of low oxygen in tumours. The lab tours enabled the general public to look at regions of low oxygen tumour sections under the microscope and had a number of exhibits explaining the work we do in the lab. 6 members of my group (including the MRC funded post doc) and other departmental researchers of the dept i manage and myself participated discussing and answering questions about our research and more generally about cancer. 70+ people visited us in the lab
Year(s) Of Engagement Activity 2018
URL https://www.nottingham.ac.uk/research/groups/nottinghambreastcancerresearchcentre/documents/nbcrc-se...
 
Description Public outreach podcasts of the research from my research group and the Centre for Cancer Sciences, University of Nottingham 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We have established public outreach podcasts (explaining the key information at a level for the general public to understand) for the research from my research group including that funded by this award and the Centre for Cancer Sciences, University of Nottingham. This is to increase public understanding of the research that we carry out. The primary audience is regional however the material is open access.
Year(s) Of Engagement Activity 2019
URL https://www.nottingham.ac.uk/ccs/podcasts.aspx
 
Description The Breast Cancer Now Toby Robins Breast Cancer Research Centre-Institute of Cancer Research- UK- Thursday 21st September 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Invited Speaker at The Breast Cancer Now Toby Robins Breast Cancer Research Centre-Institute of Cancer Research- UK- Thursday 21st September 2017. The intended purpose was to present our research to the field to receive feedback about our work and update other researchers in the immediate and related fields on our progress, publicising our progress and ideas.
Year(s) Of Engagement Activity 2017