beta-catenin Ser45 in development and disease

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

Mutations in the beta-catenin signalling pathway are found in many types of cancer. It is widely assumed that all these mutations activate this pathway. We are interested in the development of Wilms' tumours, kidney cancers found in young children that are the result of normal kidney development before birth going wrong. A subset of these tumours has mutations in beta-catenin. Interestingly, whereas there are four different mutations possible, almost all patients have exactly the same mutation, suggesting this mutation is specifically important for this type of cancer. In the work leading up to this proposal we have made mouse models that carry different mutant copies of beta-catenin, including the mutation found in Wilms' tumours. We found that this mutation resulted in kidney abnormalities that would fit with a role in Wilms' tumours but, surprisingly, this mutation did not seem to activate the protein. Unfortunately, these models were made in a way that differs from the way the gene is mutated in patients, and it is therefore not possible to conclude this mutation will have the same effect in tumours.

In this project we will therefore first make the mutation in exactly the same way as found in human tumours. We will repeat the analyses we have done before on the other model to see if the surprising findings we did are confirmed. We will study the effect of this mutation in mice, with an emphasis on the kidney and neuron / brain.

Technical Summary

It is assumed that oncogenic mutations found in beta-catenin result in activation of the pathway. We have a strong interest in the early steps in the development of Wilms' tumours, in particular the subset that is caused by loss of WT1 and activation of beta-catenin. Interestingly, almost all cases of Wilms' tumour with mutant beta-catenin carry a mutation affecting the Ser45 residue. To study the reason for this selection we have previously generated several mouse models that express different beta-catenin mutations from the Rosa26 locus. We found that the Ser45 mutant does not have an increased signalling capacity, despite being stabilized at the protein level as expected. Still, the Ser45 mutation has a biological effect on Wt1-deficient kidney mesenchyme cells which were driven a step closer towards Wilms' tumours. This finding suggests a complete new dimension to the role of beta-catenin mutations in cancer. However, the fact that our models express their mutant beta-catenin from Rosa26 means we cannot directly translate these finding to patients. In this project we will therefore generate a Cre-inducible Ser45 mutation in the endogenous mouse beta-catenin gene. We will test its capability to increase target gene transcription in ES cells as we did for the Rosa26 models. In vivo we will activate the expression of the mutant allele through a Cre deleter strain and analyse the phenotype in development and/or postnatal. To study the role of this mutation in Wilms' tumour development we will cross the mice with our kidney mesenchyme-specific Wt1 knockout. We will use genome-wide expression analysis to identify molecular changes that could explain the selection for this mutation in WT1-mutant Wilms' tumours. The mice will be followed for Wilms' tumour development and other phenotypes. Finally, we will specifically analyse any phenotypes in the neuronal system caused by the Ser45 mutation.

Planned Impact

The proposed project will have a potential impact on the following stake holders.

The academic community working on cancer genetics in general, and Wilms' tumour genetics in particular. The information gained in this project will increase our understanding of cancer genetics, Darwinian selection in tumourigenesis and the roles of specific cancer-related mutations in the origin of tumours.

The health sector. Better understanding of the earlier steps in cancer development will ultimately lead to improvement in cancer treatment options.

Cancer patients. Better understanding of the origins of cancer and identification of the processes that, when disturbed, lead to cancer development will allow the design of more efficient and more specific therapies. This will result in better treatments with fewer side-effects, leading to a significant increase in quality of life.

General public. As the public pays for MRC-funded research through their taxes there is an obvious right of information on what is done with the money.

The pharmaceutical industry. There is an important lack in sufficiently good animal models for cancer. The use of models that are available for design and testing of therapies have in many cases been found not to be predictive for the efficacy of a therapy in patients. As a result too many new drug candidates are failing to make it to the clinic. Models as proposed here that mimic the genetic events in cancer in patients as closely as possible will undoubtedly lead to better and more predictive models for these purposes.

The staff on the project. Biomedical sciences increasingly require a multitude of techniques and approaches to answer a scientific question. The experimental models used become more sophisticated as technical possibilities improve. For both named staff this project will allow the extension of their expertise. For Dr. Ozdemir this project will help to further establish her reputation in the Wilms' tumour and mouse model field. She will be able to increase her expertise in the generation of genetically modified mouse models using the latest technologies, and introduce her to next-generation sequencing approaches. Mrs Sheraz has so far been running the mouse colony of the Hohenstein lab and been involved in developing and optimizing in vitro culture methods for mesenchymal kidney progenitor cells. This project will allow her to become experienced in all steps in the generation and analysis of transgenic mouse models.

Publications

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Munro DAD (2017) Refuting the hypothesis that semaphorin-3f/neuropilin-2 exclude blood vessels from the cap mesenchyme in the developing kidney. in Developmental dynamics : an official publication of the American Association of Anatomists

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Hohenstein P (2015) The yin and yang of kidney development and Wilms' tumors. in Genes & development

 
Description PhD student funded from MSc biomedical sciences
Amount £18,000 (GBP)
Organisation University of Edinburgh 
Sector Academic/University
Country United Kingdom
Start 11/2014 
End 10/2017
 
Title V5-beta catenin mouse ES cells 
Description These cells carry a V5 epitope tag on the N-terminus of the endogenous beta-catenin gene. They were an intermediate step in the generation of the delSer45 mouse model in this proposal. 
Type Of Material Cell line 
Provided To Others? No  
Impact These cells can be used for a variety of in vitro proteomic approaches and could be turned into mice for in vivo proteomics (not part of this proposal) 
 
Title mouse embryonic stem cells with Ser33Tyr mutation in beta-catenin 
Description This cell line was made as a control for the delSer45 mutation which was an intermediate step in the generation of the delSer45 mice from this project. 
Type Of Material Cell line 
Provided To Others? No  
Impact These ES cells can be used to generate Ser33Tyr mutatn beta-catenin m ice (not part of this proposal) and can be used as an in vitro system for further analysis in their own right. 
 
Title mouse embryonic stem cells with constitutive delSer45 mutation in beta-catenin 
Description We generated mouse ES cells heterozygous and homozygous for the delSer45 mutation in the endogenous beta-catenin gene that we currently are analysing 
Type Of Material Cell line 
Provided To Others? No  
Impact This ES line is an intermediate step in the generation of delSer45 knock-in mice we proposed in the project, and which can be used as an experimental in vitro system for further analysis 
 
Title WT1 expression in breast cancer disrupts the epithelial/mesenchymal balance of tumour cells and correlates with the metabolic response to docetaxel 
Description RNA profiles of breast cancer cells (MDA-MB-157) were generated by deep sequencing on the Illumina HiSeq 2000 platform. Untreated MDA-MB-157 cells, MDA-MB-157 cells transduced with a lacZ control vector, and MDA-MB-157 cells transduced with a lentiviral vector carrying a Wt1 shRNA were sequenced (titled untreated, lacZ and Wt1 respectively). 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
 
Description 7th course Developmental Biology, Institute Curie, Paris 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I gave a talk at the 7th international course for developmental biology, organised by the Institue Curiein Paris
Year(s) Of Engagement Activity 2016
 
Description Blog post NC3Rs 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Wrote blogpost about how transgenic technologies can help the 3Rs.
Year(s) Of Engagement Activity 2017
URL https://www.nc3rs.org.uk/news/when-3rs-and-transgenic-technologies-meet
 
Description Coming of Age: The Legacy of Dolly at 20 public lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact At a series of public lectures to celebrate the 20th birthday of Dolly the Sheep I discussed kidney research with the public
Year(s) Of Engagement Activity 2016
 
Description Consensus Meeting on Renal Progenitors and Kidney Regeneration 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I gave a talk at this meeting organised to discuss the many different views in the field about renal progenitor cells.
Year(s) Of Engagement Activity 2016
 
Description ILAR Roundtable 'gene editing to modify naimal genomes for reserarch - scientific and ethical considerations' 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Round Table organized by the National Academy of Sciences in the USA to discuss the impact of genome editing on animal use in science.
Year(s) Of Engagement Activity 2015
URL http://nas-sites.org/ilar-roundtable/roundtable-activities/gene-editing-to-modify-animal-genomes-for...
 
Description Invited seminar Cincinnati Children's Hospital Medical Center 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited visit to CCHMC to give seminar and discuss future collaborations.
Year(s) Of Engagement Activity 2017
 
Description Invited seminar LUMC Leiden, the Netherlands 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited seminar at the Leiden University Medical Center, Leiden, the Netherlands
Year(s) Of Engagement Activity 2017
 
Description Invited talk at BSDB/Nordic meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk at joined meeting of the British Society for Developmental Biology and Scandinavian developmental biologists
Year(s) Of Engagement Activity 2017
 
Description Lobbying reception Scottish Parliament 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Kidney research UK organized a lobbyng reception in the Scottish parliament to discuss the importance of kidney research with MSPs, patients and other researchers.
Year(s) Of Engagement Activity 2016
 
Description Organiser and speaker at 3rd international workshop on the biology of WT1 
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 Organized the 3 international workshop in the biology of WT1 and spoke on the meeting
Year(s) Of Engagement Activity 2015
 
Description Roslin Institute Open Day 
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 I was involved in discussion about the use of animals in research as well as presenting the imaging work from my own laboratory.
Year(s) Of Engagement Activity 2017
 
Description Talk at MRC Toxicology Unit, Leicester 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited seminar
Year(s) Of Engagement Activity 2016
 
Description Talk at Nephrotools meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Ta\lk at a meeting of the EU funded Nephrotools consortium
Year(s) Of Engagement Activity 2015
 
Description Teaching at 10th Advanced Paediatric Oncology Course (APOC), Edinburgh, United Kingdom 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Teaching at this course for paediatric oncology nurses
Year(s) Of Engagement Activity 2017
 
Description Visit Kidney research UK delegation to Edinburgh 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact A delegation from Kidney research UK visited Edinburgh to discuss renal research with all kidney researchers
Year(s) Of Engagement Activity 2016
 
Description Visiting undergrad students Wyoming 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact I discussed the effects of Brexit on science funding and networking with visiting undergrad students from Wyoming
Year(s) Of Engagement Activity 2018
 
Description Welcoming committee 
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 At the open doors day Roslin Institute I was welcoming visitors to the building, while my lab was demonstrating the culture of embryonic mouse kidneys on our time-lapse imaging system
Year(s) Of Engagement Activity 2016
 
Description hosting seminar Andy McMahon 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact hosted an external seminar in the institute
Year(s) Of Engagement Activity 2015
 
Description hosting seminar Robert Kelsh 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact hosted external seminar from Robert Kelsh
Year(s) Of Engagement Activity 2015
 
Description in conversation with session on Roslin Open Doors day 
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 General public had 'in conversation with' sessions with scientists from The Roslin Institute to discuss their science. I have focused my sessions on the use of animals in research, and in kidney development and disease in particular.
Year(s) Of Engagement Activity 2014
 
Description invited seminar at Crick Institute Mill Hill 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact invited seminar
Year(s) Of Engagement Activity 2016
 
Description regenerative Medicine Therapy network development meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact A meeting to discuss the development of a national network on Renal Regenerative Medicine with the support of Kidney Research UK
Year(s) Of Engagement Activity 2016
 
Description student talk at high school 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact talk at local high school discussing animal use in science in the context of kidney development, kidney stem cells, kidney disease and imaging
Year(s) Of Engagement Activity 2015
 
Description visit Biomed students Wyoming 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact A group of undergrad students from Wyoming visited the institute and I gave a talk about 'being a scientist' and our kidney research in particular
Year(s) Of Engagement Activity 2017