Contribution of b-catenin controlled, intestinal stem cell-associated homeodomain transcription factors to cancer

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

Abstract

This study aims to identify novel molecular mechanisms by which TCF4 and beta-catenin genes control intestinal stem cells and how changes in the properties of these genes may lead to the development of bowel cancer.

The intestine represents an easily accessible developmental system which provides an attractive route to model biologically important intestinal processes such as; embryogenesis, tumorigenesis and stem cell biology. It also provides a unique opportunity for the direct identification and analysis of factors that contribute to development of both normal and tumour tissue.

The transcription factor, TCF4, has been shown to be critical for intestinal stem cell phenotype but how plasticity and self-renewal is regulated by TCF4 is poorly understood. Therefore, in vivo models of mouse gut-tissue-specific depletion of TCF4 on the APCMin, a mouse model of colon cancer background will be studied.

An in-depth understanding of the mechanisms that control differentiation of distinct cell lineages during normal development and cancer intestinal epithelial stem cells by Wnt/beta-catenin signalling, may lead to identification of novel therapeutic targets.

Given that beta-catenin signalling is deregulated in several different types of cancer, this may also lead to greater understanding of these tumours and of carcinogenesis in general.

Technical Summary

While the biological roles of canonical Wnt/b-catenin signalling in development and malignant evolution are well documented, understanding the molecular logic underlying these functionally distinct nuclear transcriptional programs remains a major challenge. Many proteins are associated with cytoplasmic b-catenin for regulation of Wnt/-catenin pathway activities, but the Lef/TCF family of transcription factors remain the sole focus as unambiguous DNA binding partners. However, in addition to Tcf/Lefs, the interaction of b-catenin with several other transcriptional co-regulators may be required for gene expression distinct patterns engage in development versus a malignant evolution.
Hypothesis
Developmentally important homeodomain transcription factors linked to b-catenin, which contribute to intestinal epithelial stem cell maintenance in the context of normal b-catenin status, are de-regulated in the transition to intestinal cancer stem cells.
Goals
1) To define how the vast and constantly varying spectrum of b-catenin protein-modifications is interpreted to produce alterations in cellular-phenotype.
2) To examine the functionally distinct actions of the TCF4 in response to b-catenin during the generation of diverse cell types from pluripotent precursors during development and carcinogenesis.
3) Characterisation any additional transcriptional strategies independent to and/or competitor to the TCF4:b-catenin to achieve the pleiotropic effects of the Wnt/b-catenin-signalling pathway.
Objectives and research plans
1) The yeast Ras recruitment system, already established by the applicant (2) will be carried out to isolate new-cofactors that bind to phosphorylated and/or un-phosphorylated b-catenin (Fig 1A). We speculate that this information could be shared with normal proliferating crypt cells including the IES cells and shared and distinct cofactors will be identified.
2) Stem cells are a pluripotent, self-renewing cell type that give rise to differentiated cells. To the stem cell fate determination in vivo, we intend to develop a novel in vivo model of mouse transgenic line in which the expression of H2B-GFP is controlled by a specific mouse intestinal stem cell marker, Musashi-1 promoter [Pmsi] (5,6) and with the gut-conditional villin-Cre mediated-recombination (Fig 1B).
3) The transcription factor, TCF4, a key feature of IES cells but how pluripotency and self-renewal is co-regulated by both TCF4 and b-Catenin is poorly understood. Therefore, in vivo models of conditional knockout of TCF4 (loss of function) and APCMin a gain of constitutive activation of b-catenin are proposed (Fig 1C).
4) Whether additional transcriptional strategies are required of the Wnt/b-catenin signalling pathway, to both stem cell and cancer cell function, TCF4DG, and APCMin crossed to the; Pmsi-H2B-GFP reporter mouse will be analysed.

Publications

10 25 50
 
Description The Centre for Cancer Research Nottingham
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact I've given lectures for summer schools on conjunctions with The Centre for Cancer Research Nottingham for years 2009 and 2010. My engagement was to inform young students who have actively sought information on stem cells, how the environments with particular alteration to intracellular signalling processes of intestinal stem cells biology, interacts with other cells, and how this may cause cancer and the fact that stem cells are not a dangerous entity but a natural part of our organism and essential for development maintenance.
 
Description Training & Teaching; MOL/MSc-Oncology course
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact I believed lecturing on the MSc oncology course and the cancer research summer school developed new subjects of training and teaching for postgraduate participants in mouse technology and stem cells biology.
 
Guideline Title The Links between Transcription, beta-catenin/JNK Signaling, and Carcinogenesis
Description We have addressed the functional links reported between activated beta-catenin/JNK signaling pathways, their component genes, and their common targets, and discuss how alterations in the properties of these genes lead to the development of cancer.
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in clinical guidelines
Impact MDLinx Oncology Network connects healthcare professionals and patients to impact of medical oncology research knowledge and provides the pharmaceutical and preclinical and clinical societies with higher levels of education, research materials and interactive marketing
 
Description Exploring Wnt/b-catenin signalling pathways associated with thermal stimulation in taste cells.
Amount £9,994 (GBP)
Organisation University of Nottingham 
Department School of Biosciences
Sector Academic/University
Country United Kingdom
Start 01/2017 
End 08/2017
 
Description FLYWCH1-mediated control of let-7 microRNA expression.
Amount £250 (GBP)
Organisation European Molecular Biology Organisation 
Sector Learned Society
Country European Union (EU)
Start 09/2015 
End 09/2015
 
Description Genetic screens in intestinal/colon organoid (mini-gut) culture using the CRISPR-Cas9 system.
Amount £90,000 (GBP)
Funding ID NC/P001793/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Private
Country United Kingdom
Start 07/2017 
End 06/2020
 
Description International Corporation Program
Amount £75,000 (GBP)
Organisation King Abdulaziz University 
Sector Academic/University
Country Saudi Arabia
Start 10/2016 
End 09/2019
 
Description International Islamic Development Bank (IDB) Prize for Women?s Contribution to Development
Amount £80,000 (GBP)
Organisation Islamic Development Bank (IDB) 
Sector Private
Country Saudi Arabia
Start 07/2012 
End 07/2016
 
Description King Abdullah International Medical Research Centre king Saud bin Abdulaziz University
Amount £105,000 (GBP)
Organisation King Saud bin Abdulaziz University for Health Sciences (KSAU-HS) 
Sector Academic/University
Country Saudi Arabia
Start 01/2013 
End 12/2014
 
Description Pending application: Functional Association of microRNAs (miRNAs) Regulation of Metabolic Pathways: Intestinal/colon Cancer & Treatment
Amount £172,791 (GBP)
Funding ID Pending (Not yet decided) 
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2016 
End 05/2018
 
Description Studying stem cell dynamics in homeostasis and cancer using a combined biological and computational physics approaches in intestinal/colon organoids (mini-guts).
Amount $150,000 (USD)
Organisation Schlumberger Foundation 
Sector Charity/Non Profit
Country United States
Start 09/2017 
End 08/2020
 
Description University of Nottingham, Faculty of Medicine Research Accelerator Award
Amount £10,000 (GEL)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 04/2017 
End 11/2017
 
Description Vice Chancellor's Scholarship for Research Excellence (European Union)
Amount £60,000 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2015
 
Title A novel in vivo Lgr5 stem cell-reporter mouse model 
Description Initially, we intended to develop an in vivo model involving mouse transgenic line in which the expression of a reporter; the green fluorescent protein (GFP) fused to a basic nuclear histone protein, H2B is controlled by a previously identified mouse intestinal stem cell marker, Musashi-1 promoter and with conditional villin-Cre mediated recombination. Our data however after testing the Musashi promoter activity showed that Musashi activity is not restricted to intestinal stem cells. Then we applied similar strategy to label intestinal stem cells by bio-imaging techniques using Lgr5 (Leucine-rich repeat-containing G-protein coupled receptor 5) promoter fused to RFP for in vitro validation assays. Then, we cloned the 2.9kb Lgr5 proximal promoter by PCR using mouse genomic DNA and fused it to RFP for in vitro validation assays in cell culture. Our validation assays using colon cancer HCT116 and SW620 cells transfected with the Lgr5-promoter-RFP confirmed Lgr5 activity and positivity only in 1-2% of the cell population by fluorescent microscopy. Then, we constructed the transgenic vector in which the expression of the green fluorescent protein (GFP) fused to IRES is controlled by a specific mouse intestinal stem cell marker, Lgr5 promoter and with conditional Cre/LoxP mediated recombination strategy; e.g. villin-Cre. We have already generated few ES cell clones using Electroporation and subsequently genome screening assays based on PCR. These ES cells clones were subjected to Lentivial transduction that express Cre-recombinase and the expression of GFP confirmed by the fluorescent microscopy. Currently 2- ES clones have been used to generate chimera mice using the tetraploid (4N)-embryonic aggregation technique by the transgenic service facility of the University of Nottingham. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Stem cells are a pluripotent, self-renewing cell type that give rise to differentiated cells. To identify regulatory molecules that function in vivo to determine stem cell fate, we are developing a novel in vivo model involving mouse transgenic line in which the expression of GFP (reporter); is controlled by a specific adult mouse stem cell marker, Lgr5 promoter and with conditional villin-Cre mediated recombination. Once the transgenic mouse is equipped it will in principle allow the genetic tracking of normal and/or transformed Lgr5 cells and their offspring within established normal gut and/or tumour tissues. In longer term, we would cross the Lgr5 transgenic mouse with an APC single allele mutation (ApcMin/+) and a conditional homozygous deletion of Tcf4 (Tcf?G) generated in our lab to investigate the involvement of intestinal stem cells in tumourigenesis. Also, a conditional Lgr5- mouse model and its analysis would warrants publication in high impact journal. Furthermore, these mice could be used for carcinogenicity testing and drug screening. 
 
Title CRC ex-plant cultures 
Description Predicting clinical response to anticancer drugs remains a major challenge in cancer treatment. Emerging reports indicate that the tumour microenvironment and heterogeneity can limit the predictive power of current biomarker-guided strategies for chemotherapy. We use tumour explants maintained in a conditional medium which are presenting the functional response model of tumour ecosystem. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2018 
Provided To Others? No  
Impact CRC tumour explant culture can emerge as a powerful platform for enabling personalized medicine. 
 
Title Conditional TCF4 knockout mouse line 
Description Here, by employing in vivo tissue specific-Cre/LoxP-recombination techniques for Tcf4 (Tcf7l2) we aimed to ablate its function; to demonstrate that Tcf4 regulates intestinal homeostasis and cancer by crossing homozygous floxed-Tcf4(fl/fl) to gut-specific Villin-Cre (Villin-Cre+:Tcf4fl/fl or a gut-specific Tcf4 deletion) mice. TCF4 is a key regulator of proliferation for intestinal progenitor stem cells, as loss of TCF4 leads to death of mice shortly after birth. We had previously generated the conditional TCF4 knockout DNA plasmid, by which the exons encoding the HMG-DNA binding domain are flanked by loxP sequences, using recently developed BAC/PAC recombinant technology. We have then transfected the linearised DNA into mouse ES cells by electroporation. We have also confirmed of insertion of a specific targeted single copy by Southern blot and PCR analysis. However, ES cells harbouring conditional TCF4 knockout could be useful for the functional analysis of TCF4 in ES cell self-renewal and pluripotency properties studies using Lentiviral expressing vectors with Cre-GFP and GFP expression that we have already developed in the lab. The ES clone has been injected into the tetraploid (4N) embryos (blastocysts) of recipient mice by the Transgenic Facility in the Biomedical Services Unit, University of Nottingham. We have now obtained hetrozygous Tcf4 single allele floxed-Fred mice. Heterozygous mice are intercrossed to yield homozygous mutant mice. Hetrozygous Tcf4 (fl/+) recently been crossed to Flp-recombinase mice to remove the NeoR allele and subsequently will be crossed to specific gut Cre (villin-Cre). 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact The use of the Cre-LoxP system to obtain conditional, mosaic, inducible and ubiquitous deletion of a floxed gene. In order to obtain mice carrying a cell type specific deletion of the gene of interest, mice carrying the floxed gene of interest are bred with mice expressing Cre recombinase under a cell type specific promoter. The offspring show a deletion of the floxed gene only in cells expressing the Cre recombinase. TCF4 floxed mice (TCF4f/f) has been crossed to Flp-recombinase mice to remove the NeoR allele and subsequently crossed to specific gut Cre (villin-Cre). Heterozygous mice will be intercrossed to yield homozygous mutant mice. Therefore, the Tcf4fl/fl mouse line facilitate to study the role of Tcf4 in different tissue. Also, a conditional intestinal TCF4 K/O mouse generation and its analysis within our MRC funding scope would warrants publication in high impact journal. 
 
Title Technology to develop more efficient and more precise Chromatin Immunoprecipitation (ChIP) assays 
Description We modified the conventional ChIP assay to detect protein complex binding to known promoter DNA in cells. In this procedure, Dynabeads(R) M-280 Streptavidin was used to capture biotinylated minimal-promoter-DNA-protein complex. Biotinylated-promoter are transfected in cells with all controls and DNA-binding proteins are fixed as previously described (Nateri et al., 2005). Specifically binding of specific protein in larger scale could be detected using 2D Gel Electrophoresis, Western Blotting and Mass Spectrometry. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact 1) Easier & faster analysis and experimental times 2) better controlling process 3) higher specificity 
URL http://www.ncbi.nlm.nih.gov/pubmed/23661244
 
Title Yeast based two-hybrid Ras Recruitment System (RRS) to identify proteins that interact in a phosphorylation dependent manner 
Description We have modified and developed a genetic screening system called; Ras Recruitment system (RRS) to isolate protein(s) that are interacting to another (bait protein) in a phosphorylation-dependent-manner. Use of conventional yeast two hybrid systems in defining protein-protein interactions is highly limited because the interaction must take place in the nucleus for the subsequent activation of the reporter gene. The RRS uses the growth defective yeast strain CDC25-2, deficient in Ras activity. Ra 
Type Of Material Biological samples 
Year Produced 2013 
Provided To Others? Yes  
Impact As previously described and in brief, nuclear ß-catenin is a multi-functional protein that acts in the context of canonical Wnt signalling pathway to modulate transcription of many downstream target genes. We hypothesised that in addition to TCF/LEFs, the interaction of ß-catenin with several other transcriptional co-activators/repressors is required for gene regulation. With MRC funding support, we have identified a number of proteins that interact with ß-catenin in the yeast in phosphorylation 
 
Title Mini-Gut or Intestinal Organoids 
Description The development of appropriate in vitro systems and the accompanying tools for gene manipulation is an important step to reduce animal use in our research fields. In 2009, Sato et al. developed a novel primary culture system that uses proliferative intestinal fragments (crypts) from mouse to establish long-term in vitro culture of intestinal epithelium containing adult stem cells and all differentiated cell types. This system is known as 'Mini-Gut' or 'Intestinal Organoids' and we and a number of groups already established in the lab to address gene functions; a good example is our study that identify the regulatory function of beta-catenin interaction partners such as CIP-1 and CIP-2 in the Wnt signalling pathway. 
Type Of Material Data analysis technique 
Year Produced 2013 
Provided To Others? Yes  
Impact As outlined above, the system is initially developed in Hans Clevers laboratory and we have also established it in our Nottingham lab... The potential impact of this establishment in 3Rs is on Replacement and Reduction. 
 
Title Spheroid-formation (Colonosphere) Assay for in vitro Assessment and Expansion of Stem Cells in Colon Cancer 
Description The colonosphere formation assay is an in vitro technique to test for clonogenic growth potential of colorectal cancer (CRC) cells and their stem cell-like characteristics. 
Type Of Material Data analysis technique 
Year Produced 2014 
Provided To Others? Yes  
Impact The colonosphere formation assay was previously described by several other laboratories, and we have optimized the culture condition to facilitate colorectal cancer research through immunofluorescence staining of colonospheres. Furthermore, this protocol may also allow screening of libraries of compounds to identify bona fide CRC-CSC differentiation inducers. The protocol is under peer review for publication by JoVE. 
 
Description Analysis of WNT/b-catenin signalling gene expression and potential role in Acute Myeloid Leukaemia (AML) cells 
Organisation Jeddah University
PI Contribution We determine the potential implications of FLYWCH1 versus some of Wnt/ß-catenin target gene expression and potential impact on AML cell growth, etc.
Collaborator Contribution Through an international collaborative research programme, they intend to provide some funding for a PG student and our visit to their institute.
Impact When we award a research grant from Jaddeh University for a collaborative project we expect there to be a formal collaboration agreement in place before the project starts.
Start Year 2018
 
Description Exploring Wnt/b-catenin signalling pathways associated with thermal stimulation in taste cells. 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution This proposal outlines a new collaboration within UoN, taking current expertise in sensory perception (Dr Rebecca Ford), and cancer genetics/stem cell research (Nateri), to investigate cell signalling pathways associated with sensory reception in the oral cavity.Practical aspects of this project is taken place in my laboratory (QMC) where there is scope for Dr Ford to develop practical skills in cell culturing techniques for personal development and scientific understanding. The main body of practical work (culturing of taste cells and gene expression profiling) conducted by a researcher under my supervision.
Collaborator Contribution Dr Rebbeca Ford (Bio-sciences/Food Science/Sensory Science Group) will write the resulting paper. Dr Ford will also use pilot data to support a grant application to BBSRC or MRC or her early career academic fellowships. Dr Ford is an early career researcher and has published work from her PhD in multimodal flavour perception and individual variation in sensory perception utilizing flavour chemistry, psychophysical sensory methods and functional magnetic resonance brain imaging techniques.
Impact School of Biosciences (RAA) Research Accelerator Award: £9,944
Start Year 2017
 
Description FLYWCH1 and F-box in tumour xenograft 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Lentiviral gRNAs and overexpressing constraints and vectors
Collaborator Contribution validated the function of proposed genes using xenografts
Impact No outcome yet, the collaboration began recently.
Start Year 2018
 
Description Immunohistochemical expression of Catenin interacting proteins (CIPs) in human colorectal adenoma: using tissue microarrays (TMA) 
Organisation University of Nottingham
Department School of Clinical Sciences Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution In our lab, we perform the immunostaining for CIPs and analyzing both quantitative and semi-quantitative methods to score TMA sections and studying their correlation with Wnt/b-catenin.
Collaborator Contribution Our collaborator's lab recently constructed four TMA blocks from over 400 specimens. They provide us the slides/ sectioned from these 4- blocks.
Impact Tissue microarray (TMA) holds promise as a high-throughput method for the analysis of biomarkers in tissue specimens. In this study, we want to assess CIPs (currently CIP1 and CIP2) as possible biomarkers in colorectal adenomas using TMA. Currently we aimed to use these data for ongoing publications and pilot data for research grant applications..
Start Year 2010
 
Description In situ hybridisation analysis of novel Catenin Interacting Proteins (CIPs) expression pattern in embryonic and adult mouse tissues 
Organisation Cancer Research UK
Department Cancer Research UK London Research Institute (LRI)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution For collaboration no.1; we prepare probes for in situ hybridization and provide mice tissues (embryonic, gut adult normal and tumour tissues) which were fixed in 4% PFA and blocked and then sending off to the experimental histopathology lab for examination. Data will be sent to us and all images will be captured in our lab and examined for gene expression analysis.
Collaborator Contribution (Collaboration no.1) Dr Bradley Spencer-Dene (BSD) conducted for in-situ hybridization assays using their established non-radioactive labelling techniques for hybridization of mRNA expression for novel b-catenin interacting proteins within the adult tissue sections and whole mounts. cDNA probe and other required materials are provided from my lab.
Impact The work from Dr Bradley Spencer-Dene collaboration (no.1), will be included in a manuscript which is under submission for CIP-1/b-catenin data; he has already provided some essential in situ hybridization data as the CIP-1 expression pattern has not been addressed since identified in mammals
Start Year 2009
 
Description Investigation the diverse biological activity of ß-catenin through characterisation of novel ß-catenin interacting protein-2 (CIP-2) 
Organisation University of Nottingham
Department School of Molecular Medical Sciences Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution For collaboration no.2, our joined student is entirely working in my lab and he is uploading experimental data in the Molecular Medicine School Postgraduate Research Progression System and we (MI & I) review the student progression/ performance in a regular monthly meeting
Collaborator Contribution (Collaboration no.2) Professor Mohammed Ilyas (MI) act as co-supervisor for our joined international student. MI has a long track record of studying colorectal cancer and the role of Wnt signalling in both human cancers and in mouse models. His current interests lie in downstream targets of Wnt signalling which complement the proposed study. He is also a fully trained Histopathologist and providing human colorectal carcinoma and adenoma samples with technical expertise on mutational analysis of CIP1 gene.
Impact We have recently published the following articles together; Albasri A et al., 2009; J Pathol. 218(1):57-65. Ibrahem S et al., 2014; Int J Exp Pathol. 95(6):392-400.
Start Year 2008
 
Description Modelling Colorectal Cancer using CRISPR-Cas9-mediated Engineering in Cultured Human Intestinal/colon Organoids 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution We generate mutant organoids for cancer associated genes, then screen and validation of small molecule compounds selectively targeting mutant organoid for differentiation and/or apoptosis induction.
Collaborator Contribution Dr Koo's lab assist with CRISPR/Cas9-mediated mutagenesis method for organoid cultures
Impact We are using the CRISPR-Cas9 lentivirus for efficient chromosomal integration of CRISPR components in organoids. So far, we have obtained pLV vector which features the Cas9 nickase fused to the 4-OHT inducible receptor domain ERT2 (mutated hormone binding domain of the human estrogenic receptor), Puromycin and GFP, for selection/ monitoring stable cell populations that are present in organoids. Currently aimed for a pilot study and to apply research grant(s).
Start Year 2015
 
Description The Intestinal Organoid Culture System 
Organisation Royal Netherlands Academy of Arts and Sciences
Department Hubrecht Institute
Country Netherlands 
Sector Academic/University 
PI Contribution The organoid culture forms from single intestinal stem cells and grow by branching. These technical approach would allow us to not only test the effect of activated beta-catenin signalling and it new interaction protein FLYWCH1 that suppress number of microRNAs, on adult intestinal/colonic stem cells mediated Villi growth but also determine whether distinct miRNA genes are targeted by beta-catenin/FLYWCH1 in the presence and absence of activated beta-catenin and FLYWCH1 during normal and carcinogenesis.
Collaborator Contribution Dr Rob Vries, in his laboratory at Hubrecht Institute, Netherland, contributed in training of one of my group member; how to culture and maintain the murine organoid culture from single intestinal stem cells and crypts in which we have already established the technology in our Nottingham laboratory.
Impact We are now extending the technology to apply different conditional genetically modified intestinal crypts from transgenic mice and outcome the work will be published in a manuscript that is in completion.
Start Year 2011
 
Description The role of Epithelial-Myofibroblast interactions in regulating Murine Intestinal Homeostasis and Cancer 
Organisation Eberhard Karls University of Tubingen
Department Interfaculty Institute of Biochemistry
Country Germany 
Sector Academic/University 
PI Contribution More recently we have generated conditional Tcf4 knockout mouse line in which could be useful to study the function of TCF4 in different tissue types. We cross the floxed- TCF4 with MS-Cre line which provided by our collaborator. Then we validate/ and analyze the depletion of TCF4 in myofibroblasts and its consequences in intestinal tissue by IHC, biochemical and cell and molecular analysis.
Collaborator Contribution Our collaborator supply the SM-CreERT2 (ki) Transgenic mouse line to study the role of epithelial-myofibroblast interactions regulated by TCF4 in intestinal normal and cancer development.
Impact Currently aimed for a pilot study and to apply a research grant(s)
Start Year 2015
 
Description Validation of Wnt-small molecule inhibitors 
Organisation Nexigen GmbH
Country Germany 
Sector Private 
PI Contribution the methodology and other applications of the University's in vivo and in vitro screening system; the biological/pathological consequences of small molecules delivery; and the mechanism(s) and molecules to control the signalling pathway(s) required for early neoplasia and gastrointestinal repair
Collaborator Contribution NEXIGEN possesses certain proprietary business, scientific, technical information, trade secrets and know how relating to its technologies and screening platforms, research and development programs and products
Impact structurally confidential
Start Year 2012
 
Title CIP-1 (FLYWCH1): a novel microRNA inhibitor 
Description Nothing is known about CIP-1 in mammalian cells, and our data suggest CIP-1 functions as a negative regulator of several cancer associated micro-RNAs and it is expressed in the stem cell compartment. However we are currently consulting the technology transfer office and the patent office at the University of Nottingham, in order to assess the protection of this discoveries. 
IP Reference  
Protection Protection not required
Year Protection Granted
Licensed No
Impact Till now we run a basic science project mainly working on mouse tissue, thus translational impact on human health is likely to take several years to develop. If the project is realised in human then this could lead to the use of miRNA expression analysis in biomarker design and the identification of future medication targets.
 
Title Conditional Knockout and Transgenic mouse lines 
Description The heterozygous floxed Tcf-4 (Tcf4fl/fl) mouse took us almost 2-years to obtain a germline transmitted F1 offspring that carry the Tcf-4 targeted gene. We have now fully established the hetrozygous floxed Tcf-4 line crossed to the gut-specific recombinase Cre- line (Villin-CRE) at the Biomedical Services Unit (BMSU) in the University of Nottingham. These in vivo models of conditional knockout of intestinal Tcf4 (loss of function) and constitutive activation of ?-catenin (gain of function on the ApcMin/+ mouse background) are being studied and under way to be completed. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Initial development
Year Development Stage Completed 2010
Development Status Actively seeking support
Impact We used the mouse as our experimental system, since mice are advantageous due to their well studied anatomy, physiology, behaviour and molecular genetics, as well as their ready availability, and therefore the physiological roles of the gene of interest is applicable to human disease. 
 
Title Novel beta-Catenin-Interacting Protein no.1 (CIP1): Expression, Function and Regulation in Cancer cells & Cancer therapy 
Description There are no reports on the in vivo function of CIP1, while we are currently striving for obtaining substantial knowledge of the regulatory circuitry of adult intestinal epithelial stem cells. Recent studies have revealed that intestinal/colon tumour contain CIP-1-cancer stem cells as counterparts of CIP1-CBCs in normal intestine and CIP1 exerts its influence on intestinal homeostasis through regulation of RNA-splicing and miRNAs biosynthesis. Currently we secured an international studentship for running this project and actively seeking funding from other resources. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Refinement. Non-clinical
Year Development Stage Completed 2013
Development Status Actively seeking support
Impact The proposed project will provide a better general understanding of how stem cells are regulated in the intestine and other tissues as well as how tissue homeostasis and tumorigenesis is maintained. This will benefit the scientific community.the most direct pathway to impact for this type of research is publication of the scientific findings in the lead academic journals in the field. This includes publications in Cancer Cell, J Exp Med, and Nature BioTechnology and in the pubmed database. 
 
Title Patients tumour ex vivo platform 
Description The ability to predict patient tumour response to cytotoxic or target defined therapeutic agents remains a challenge and that the tumour microenvironment and heterogeneity can limit the predictive power of current biomarker-guided strategies for chemotherapy. In our model, tumour explants derived from patients can be maintained in a conditional medium over 7-8 days. We have recently used this system to validate the toxicity of defined drug and the work is under submission. The idea and medium/ resources resulted from work supported by NC3Rs and MRC but mainly developed by a sponsored international PhD student. 
Type Support Tool - For Medical Intervention
Year Development Stage Completed 2018
Development Status Actively seeking support
Impact Further development will initially lead to significant collaborations and researcher/clinician/biotech exchange programme and in the long term can emerge as a powerful platform for enabling personalized medicine. 
 
Title Technology to develop more efficient and more precise Chromatin Immunoprecipitation (ChIP) assays 
Description We modified the conventional ChIP assay to detect protein complex binding to known promoter DNA in cells. In this procedure, Dynabeads® M-280 Streptavidin was used to capture biotinylated minimal-promoter-DNA-protein complex. Biotinylated-promoter are transfected in cells with all controls and DNA-binding proteins are fixed as previously described (Nateri et al., 2005). Specifically binding of specific protein in larger scale could be detected using 2D Gel Electrophoresis, Western Blotting and Mass Spectrometry. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Small-scale adoption
Year Development Stage Completed 2011
Development Status Under active development/distribution
Impact 1) Easier & faster analysis and experimental times 2) better controlling process 3) higher specificity 
 
Description 3D CELL CULTURE TECHNOLOGICAL INNOVATION AND CLINICAL SUCCESS 
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 Meeting & hearing from industry and research leaders on the latest in 3D cell culture, Insights into high throughput screening, and the use of CRISPR in 3D cultures, organoid technology for personalized models of drug discovery
Year(s) Of Engagement Activity 2017
 
Description Anticancer Drug Action and Drug Resistance: from Cancer Biology to the Clinic Conference in Florence, Italy. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact This conference had presentations ranging across the identification of novel drug targets; non-oncogene addiction and synthetic lethality; discovery of drug resistance mechanisms; the development of combinatorial therapies; and related translational topics, across a wide spectrum of cancer types. It was a great opportunity to attend; especially for my PhD students very much benefitted by the varied sessions during the conference, oral sessions and poster sessions working in different types of cancer, basics and translational research activities. One of my student presented a poster which was related to MRC funding project, entitled; "Roles and Mechanisms of NANOG-mediated Drug Resistance in Human Colorectal Cancer Cells".
Year(s) Of Engagement Activity 2015
 
Description Cancer Research Nottingham 2017 Annual Symposium 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The one-day CRN event included presentations from local Cancer scientists to the latest development/achievements of Cancer & Stem cell research in Nottingham, including a few key cancer scientists as external speakers. I have also invited Dr Robert Vries, from Utrecht, The Netherlands as a keynote speaker on Organoids for drug development and diagnostics.
Year(s) Of Engagement Activity 2017
URL https://www.nottingham.ac.uk/pharmacy/events/2016-17/cancer-research-nottingham-2017-annual-symposiu...
 
Description EPITHELIA 2014/ Germany, Heidelberg 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact a lot of important topics in Epithelial biology were discussed and developed new collaborations, etc..,

generated lot of collaborations in exchanging of resources, materials, and technologies
Year(s) Of Engagement Activity 2014
 
Description Goodbye Flat Biology: Models, Mechanisms and Microenvironment conference, Harnack House, Berlin, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact The meeting was interested to all those who are using cancer cell lines, patient-derived tissue samples or primary cultures for the study of tumour biology, bioengineering and biochemistry, drug target validation, compound and antibody screening, toxicology, and patient-tumour profiling. I was particularly participated through a range of interactive opportunities, including a a 'Meet the Expert' session, and a Round Table Discussion.
Year(s) Of Engagement Activity 2016
 
Description Home - Cell Symposia - CRISPR: From Biology to Technology and ... 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The symposium was a great opportunity to meet leading CRISPR researchers, learn about exciting unpublished work, and to gain practical insights into applying CRISPR technology to our own research.
Year(s) Of Engagement Activity 2017
URL http://www.cell-symposia.com/crispr-2017/
 
Description International Conference on Biological and Environmental Sciences, Mansoura, EGYPT 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I gave a 45minute presentation on "Activated beta-catenin-JUN/NANOG defines CRC initiating cells. Over 300 people from different academic fields were attended in my talk and also there were many general and scientific questions after my speech and during our visits.

Throughout this visit, I've been offered to supervise a PhD student with their financial support.
Year(s) Of Engagement Activity 2010
 
Description Molecular Analysis for Personalised Therapy, Symposium, Paris, France 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact MAP 2018 was a joint initiative of Cancer Research UK, UNICANCER and ESMO and aims to provide oncologists with expert guidance on interpreting genomic alterations to design tailored treatment programmes for their patients.
Year(s) Of Engagement Activity 2018
 
Description NCRI cancer conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The NCRI cancer conference interested to all cancer biologists, Postgraduates, Researchers, Biotechs & industry representatives. The NCRI is the UK's largest cancer symposium showcasing the latest advances in cancer research.
Year(s) Of Engagement Activity 2017
URL http://abstracts.ncri.org.uk/abstract/flywch1-a-novel-transcription-factor-with-tumor-suppressor-act...
 
Description Selective targeting of human colon cancer stem-like cells - a 3D screening system for differentiation therapy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The Cardiff annual symposium brings together national leading researchers in cancer stem cells and cancer. In this event keynote speaker are presenting focused cancer research as well as selected abstracts from early career researchers. Miss Mehreen Ahmed who is a PhD student in the lab presented her drug screening data and this led to the new collaborations.
Year(s) Of Engagement Activity 2017
URL https://www.eventbrite.co.uk/e/european-cancer-stem-cell-research-institute-research-symposium-2017-...
 
Description Selective targeting of human colon cancer stem-like cells -a 3D screening system for differentiation therapy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The NCRI cancer conference interested to all cancer biologists, Postgraduates, Researchers, Biotechs & industry representatives. The NCRI is the UK's largest cancer symposium showcasing the latest advances in cancer research.
Year(s) Of Engagement Activity 2017
URL https://conference.ncri.org.uk/2017-programme/
 
Description Spandidos meeting, Greece, Athens 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact facilitated scientific communication and may talk raised lot of questions and discussions afetrwards

Generated new collaborations, etc
Year(s) Of Engagement Activity 2014
 
Description Stem Cells and Organoids Symposium London 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact This symposium provided a forum for researchers in this field, allowing them to exchange knowledge, and reinforce the network in stem cell research.
Year(s) Of Engagement Activity 2016
 
Description Summer School on New Developments in Translational Cancer Research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact 40-50 participants from different schools and companies attended in my talk; a general overview of animal models used for diseases and in particular in cancer studies.

The Cancer Research Summer School follow the mixing teaching (overview) presentations on topics in the field with practical demonstrations of technology and equipment.
Year(s) Of Engagement Activity 2009
 
Description The EMBO Meeting 2015, Birmingham, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The conference covered new topics for the entire range of the life sciences; from studies of molecules and the cell all the way up to investigation of larger, complex biological systems. One of my PhD student has presented a poster which was also awarded the EMBO travel prize, entitled; 'FLYWCH1-mediated control of let-7 microRNA expression'. This is a new project resulted from MRC grant.
Year(s) Of Engagement Activity 2015
 
Description The Spanish National Cancer Research Centre (CNIO) visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact I gave a 45minute presentation on "links between signalling, transcription and colorectal cancer". About 80-100 researchers and scientists were attended form different research groups in CNIO. Yes, there were many questions through audiences after my presentation and ultimately my visit was extended to discuss individually with few group leaders.

Yes, there was some indication sand encouraging impacts on research collaboration.
Year(s) Of Engagement Activity 2009
 
Description The annual Cancer Research Nottingham symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact The 2018- September Cancer Research Nottingham (CRN) Symposium program featured industrial, medical and scientific sessions of interest to all members of the Cancer Research Priority Area at University of Nottingham. Once again, this year CRN symposium program proved to be so successful and undoubtedly, provided a friendly meeting where our delegates and invited speakers have a chance to interact on both a scientific and social basis.
Year(s) Of Engagement Activity 2018
 
Description the 2nd Annual Stem Cell Congress and the Cell & Gene Therapy Congress 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Development of gene and advanced therapies, Application of novel genome-editing tools inc. CRISPR & TALEN, Innovative technologies for development and administration inc. gene therapy delivery, Cell & gene therapy commercialisation strategies and business models, etc.
Year(s) Of Engagement Activity 2015
 
Description the Seventh Affiliated Hospital of Sun Vat-Sen University, Shenzhen, China 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact For the purpose of attending the Seventh Affiliated Hospital of Sun Vat-Sen University opening ceremony and speaking at the International Academic Leader Forum.
Year(s) Of Engagement Activity 2018