NIHRDataInsights/HRCSData huggingface dataset:
Title: Elucidating the mechanism by which the SHROOM2 locus on Xp22.2 contributes to colorectal cancer aetiology.
Funder: Medical Research Council

Abstract: Colorectal cancer (CRC) is a common cancer but incidence could be reduced by early detection or prevention with drugs and/or dietary constituents/additives. Understanding CRC causation would enable development of preventative agents and risk biomarkers. Through GWAS, we discovered a locus on Xp22.2, harbouring SHROOM2, the focus of this proposal. We plan to take this GWAS "hit" from sequence interrogation, through to understanding molecular mechanism and function in relevant tissues, into in vivo human and model organisms Pilot data provide compelling rationale for study of SHROOM2 in CRC initiation and biology. The association between Xp22.2 and CRC risk is robust (p=7.46x10-10). Although effect size is modest (OR 1.07), we identified an eQTL with striking effects on expression, explaining most of the variance in SHROOM2 expression in human normal large bowel epithelium. By re-sequencing, we found a putative causative variant exhibiting enhanced association with CRC risk and even stronger eQTL effects. We show siRNA knockdown of SHROOM2 inhibits CRC cell growth in vitro. We found Xp22.2 is associated in males with retinal pigment changes, previously linked with CRC risk. These exciting data represent the first discovery of an eQTL at a cancer risk locus with such a dramatic effect on gene expression. We plan a coherent body of work to define the role of SHROOM2 in CRC carcinogenesis and aim to identify drug targets for prevention and new risk variants for use in risk profiling strategies:- resequencing SHROOM2 region; characterising risk and expression correlates of newly identified variants; study molecular mechanisms of SHROOM2 regulation by various techniques - RNAseq, ChIP, methylation; deplete/over-express SHROOM2 in CRC lines and cultured human colonic epithelial organoids to study cancer-relevant biological phenotypes; generate SHROOM2 deleted model organisms to study phenotypic effects, effect in chemical carcinogenesis and other genetic (APCmin) models.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Regulation of CD38 by IRF4: Implications for prognostication and therapeutic exploitation in CLL.
Funder: Medical Research Council

Abstract: Chronic lymphocytic leukaemia (CLL) follows a heterogeneous disease course and this presents a challenge in clinical management. A number of prognostic factors that can help predict disease progression and guide treatment have been identified. Of these, CD38 expression conveys a poorer prognosis with CD38+ve patients experiencing a shorter time to first treatment and reduced median survival. Understanding how CD38 drives disease pathogenesis and what regulates CD38 expression at a genetic and functional level could lead to a better understanding of its application as a prognostic marker, and to the identification of novel therapeutic targets. A genome wide association study has identified a risk allele for CLL in the 3'UTR region of IRF4, a master regulator of B cell differentiation. IRF4 dysfunction has already been implicated in the pathogenesis of CLL, and we have demonstrated in a study of over 800 patients that this risk allele is an independent predictor of poor prognosis (HR 1.6, p<0.006). Vitally, we also demonstrated an association between the IRF4 risk allele and CD38 expression, with carriers of the risk allele more likely to have CD38+ve disease (p=0.004). The CD38 promoter region contains a putative binding region for IRF4, suggesting that IRF4 regulates CD38 expression. We predict that the IRF4 risk allele codes for a variant that affects IRF4 protein levels, which ultimately affects promoter binding to downstream transcriptional targets including CD38. Using ChIP and EMSA techniques, we will demonstrate and quantify this binding, and its effects on CD38 expression. We will use RNA interference techniques to investigate the effect of IRF4 knockdown on CD38 expression, and to examine the effect of IRF4 expression levels on cytotoxic sensitivity. Experiments will be conducted in an immortalised B cell line and on primary ex vivo CLL lymphocytes, co-cultured on a fibroblast system designed to mimic the in vivo tumour microenvironment.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: MICA: Well-characterised monoclonal antibody-photosensitiser conjugates for the treatment of head and neck cancer
Funder: Medical Research Council

Abstract: The proposal involves a translational study of novel photosensitiser-monoclonal antibody conjugates for targeted treatment of cancer using photodynamic therapy (PDT). The targeted conjugates will be tested in a clinically relevant animal model of head and neck cancer which is a key clinical application for PDT. A clinically tested human anti-angiogenic antibody available will be used, SIP(L19), which binds to the extra domain B (EDB) of fibronectin. This peptide is undetectable in plasma and normal tissues, but over-expressed on neovasculature of many solid tumours. Therefore SIP(L19) photosensitiser conjugates will exert their effect primarily by destruction of tumour vasculature. Vasculature targeting is likely to be considerably more effective compared to tumour cell targeting, due to the accessibility of the antigen to blood borne antibody conjugates, and because destruction of one blood vessel kills many thousands of tumour cells. The key objectives are to optimise a new generation of well characterised photosensitser-antibody conjugates incorporating regioselective chemistry, and to demonstrate selective tumour destruction. Previous work on photosensitiser-antibody conjugates has employed coupling via lysine residues giving an average number of photosensitisers distributed randomly on the antibody surface, including the binding region. In contrast, we have recently developed methodology to selectively attach precisely two photosensitisers to SIP(L19) in a region remote from the binding site, and shown that immunoreactivity and photodynamic activity are retained in cell models. Using modified linkers it should be possible to attach up to six photosensitisers per antibody which should improve therapeutic efficacy. In proof-of-principle in vivo studies, we have shown that a similar photosensitiser conjugated non-regioselectively to lysine residues of SIP(L19) can eradicate sub-cutaneous murine carcinomas.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS
Title: Modelling ADP-ribosyltransferases as therapeutic targets in cancer therapy
Funder: Medical Research Council

Abstract: This application describes complementary experimental approaches in Dictyostelium, mouse and human cells to investigate three outstanding questions with regards to the mechanisms by which ADP-ribosyltransferases (ARTs) maintain genome stability following exposure to DNA damaging agents: a) How do alternate DNA repair pathways compensate for defective single strand break repair induced by disruption of ARTs? b) What is the long-term impact of disrupting ARTs, alone or in combination with DNA damage induced by IR, on genome stability? c) How do HR-defective cells become refractory to treatment with ARTi. Redundancy between different ARTs will be assessed in MEFs disrupted for multiple ARTs, or by depletion of ARTs in human cells using siRNA. The role of specific repair pathways in this redundancy (e.g. BER, NHEJ, HR etc.) will be tested by depleting selected repair pathway components in the appropriate genetic background by siRNA, or chemical inhibition. The ability to tolerate and resolve DNA damage will be assessed using standard assays including sensitivity of cells to DNA damage, assessing DNA repair kinetics by comet assays, and recruitment of repair factors to DNA lesions by cell imaging and chromatin fractionation technologies. The impact of disrupting ARTs on genome stability will be assessed in Dictyostelium using cell-based assays to determine mutation frequencies at specific loci, or whole genome sequencing to assess the types and frequency of global mutations. Genetic screens to identify genes that when de-regulated render HR-defective cells resistant to ARTi will be performed by generating mutation libraries in HR-defective backgrounds and selecting for cells that tolerate ARTi.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: The role of miRNAs in regulation and signalling induced by a new oncogenic kinase, LMTK3
Funder: Medical Research Council

Abstract: Aims: To establish the role of miRNAs in the regulation and signaling induced by the new oncogenic kinase LMTK3, and to understand their role in estrogenic signaling and tumorigenesis. Objectives: 1. To identify LMTK3/FOXO3a-regulated miRNAs using the UCSC genome browser, miRNA expression profiling arrays, chromatin immunoprecipitation and luciferase reporter assays. We also wish to establish whether these miRNAs regulate LMTK3 expression post-transcriptionally aiming to reveal an active feedback loop. 2. To study the effects of miRNA-induced regulation on the direct/indirect actions of LMTK3, such as estrogen receptor-alpha (ERa) regulation, cell proliferation/viability and endocrine resistance. 3. To assess the clinical relevance of FOXO3a-regulated and/or LMTK3-targeting miRNAs, by correlating levels of pre- and mature miRNAs with LMTK3 and ERa expression, in well characterised patient cohorts (n=1210) with comprehensive survival data. Methodology: This project uses a range of cell and molecular biology techniques, bioinformatics and tissue handling techniques, which are well established in the host laboratory. Scientific and Medical Opportunities This proposal derives from the interplay of two recent publications from the host laboratory and aims to elucidate a novel mechanism of LMTK3 regulation. Estrogen driven pathways are crucial to breast tumorigenesis and LMTK3 plays an important role here. We wish to study how the interaction between the ERa and LMTK3 is regulated and controlled by microRNAs, which may help us better understand resistance to hormonal therapies. This work which will not occur in isolation (an LMTK3 based drug discovery strategy has also commenced) has broad and far-reaching basic science strengths, and thus its ultimate impact to patients may not be immediately evident. However, the elucidation of a new regulatory system has the potential to produce broad, fundamental insights into new basic science processes.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Impact of targeting myeloid PI3Kg on the pancreatic tumour stroma compartment and the response to chemotherapy.
Funder: Medical Research Council

Abstract: Pancreatic ductal adenocarcinoma (PDA) has one of the highest mortality rates of any malignancy and effective treatment is major unmet need. A marked infiltration of myeloid cells into the stromal compartment and the generation of a desmoplastic stromal reaction is a particular characteristic of PDA and is thought to play a key role in disease progression and the poor response to therapy.Consequently, there is an urgent need to develop effective therapies targeting the stromal compartment in PDA. Aim: The proposed study aims to identify and target myeloid signalling pathways that promote pro-tumourigenic myeloid cell functions and desmoplasia in pancreatic cancer. Objectives and planned methodologies: This study will: 1. Use pre-clinical pancreatic tumour models in combination with genetic tools to inhibit gene expression (e.g. PI3Kg) in tumour associated macrophages. 2. Generate and use primary pancreatic stellate cells and primary macrophages in co-culture assays to characterise the interaction between these two stromal cell types. 3. Use a proteomic approach to identify PI3Kg downstream effectors regulating macrophage conversion, tumour immunity, PSC activation and desmoplasia. 4. Evaluate identified factors in vitro and in vivofor their impact on tumour immunity and desmoplasia formation. 5. Test in pre-clinical mouse PDA models whether blockade of PI3Kg and/or the newly identified downstream effectors in combination with standard chemotherapy (gemcitabine or 5FU) improves therapeutic outcome. Results: The preclinical testing of potential therapies will identify treatment options that can be translated into clinical trials. In using both genetic and pharmacological approaches, this programme of work maximises the likelihood of successfully identifying specific novel targets that are necessary for the tumour promoting functions of macrophages and provide the rationale for developing new anti-cancer therapeutic approaches.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Estrogen receptor beta is an important modulator of hormone-related carcinogenesis in the human prostate.
Funder: Medical Research Council

Abstract: I aim to increase understanding of the role of ERb in the development and progression of PCa; to identify whether ERb is a potential therapeutic target and to identify a biomarker of ERb activity. I will explore the mechanisms of ERb activity and the role of FoxA1. 1. Mechanistic analysis of ERb activity in cell lines and prostate tumour tissue. Using PCa cell lines, ERb will be stimulated with known agonists and ERb, AR and FoxA1 mapped by ChIP-seq. In addition, stable inducible short hairpin (sh)ERb expressing cells will be generated to silence ERb expression. I will assess the impact of ERb loss on AR and FoxA1 binding. Under all conditions cell growth will be assessed with or without AR agonists and antagonists. Ex vivo prostate tissue culture will be performed in ERb and AR agonist and antagonist conditions, and transcription factor mapping by ChIP-seq conducted in the tissue to assess ERb mechanistic activity within the context of stromal-epithelial interactions. 2. Identify a protein biomarker demonstrating a functional ERb complex. Using the cell lines (Aim 1), I will manipulate ERb using specific agonists or inducible shRNA. Total RNA will be collected at multiple time points and RNA seq conducted. Genes differentially regulated under activation and inhibition conditions will be integrated with ERb ChIP-seq data to identify direct ERb target genes. Candidates will be analysed using IHC on a series of PCa TMAs, with the aim of discovering a biomarker representing a functional ERb complex. 3. Determination of the role of FoxA1 and FoxA1 mutations with respect to ERb in PCa. ERb and FoxA1 interaction assays will be conducted using RIME. I will assess dependence on FoxA1 by silencing it then mapping ERb binding by ChIP-seq. Using validated zinc finger nucleases (ZFN) to target the endogenous FoxA1 genomic locus, stable PCa cell lines with mutant FoxA1 will be generated. The effect of mutant FoxA1 on growth, ERb and AR binding will be determined.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS
Title: Multiscale Molecular Imaging of Immune Cell Variety in the Malignant Microenvironment in Premetastatic Tissue
Funder: Medical Research Council

Abstract: Aims and objectives To assess the hypothesis of premetastatic tissue priming and the role of immune cells and associated chemokines during tumour cell migration and seeding. Methodology A syngeneic mouse model for breast cancer metastasis will be established using the cell line 4T1, known to preferentially metastasise to the lungs. As control, non- and low-metastatic 4T1 clones will be used, all equipped with a fusion protein allowing for both, fluorescence and radionuclide mediated imaging. A labelled antibody will serve for parallel visualisation of monocyte activity indicating S100A9 expression. For visualisation of cell interactions and the influence of S100A9 on premetastatic niche (PN) formation, intravital and ex-vivo microscopy (incl. FRET, FLIM) will be performed. This will inter alia allow for the first in-vivo visualisation of the aggregation of S100A8 and A9 to form the active S100A8/A9 heterodimer and evaluation of the influence of mon- and dimers on the malignant microenvironment. PN tissue will be identified by signs of inflammation (S100A9) without tumour cell associated signals in whole-body imaging and further examined ("optical biopsy"). PN-associated macrophages will be characterised and transferred to transcriptome profiling, screening for potential target genes for intervention. Scientific and medical opportunities This study will result in a model system for further study of the metastatic process in general. As a paradigm, the impact and regulation of S100A8/A9, discussed as a major promoter of the PN, will be evaluated. This and the identification of PN-associated macrophages with regards to their specific makeup and role within this process might reveal novel opportunities to impair tumour metastasis.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: A mouse model of Chromosomal instability in Cancer
Funder: Medical Research Council

Abstract: Chromosomal instability (CIN), characterised by large-scale structural and numerical mutations, is a feature of many cancers. However, the specific causes of CIN and selective consequences of specific CIN mutations (such as single-copy deletions or duplications) are largely unknown. I propose to develop a mouse model of CIN using transposons to deliver loxP sites pseudo-randomly throughout the genome. Recombination between distant sites has been demonstrated previously, and will result in a variety of deletions, duplications and translocations. These may drive tumorigenesis ab initio, but I shall also cross the CIN mice to Apc-mutant mice to investigate the effects of CIN subsequent to tumour initiation. I may also cross the mice to Cenpe mutants to cause numerical chromosome abnormalities in addition to the structural abnormalities caused by my mouse model. I shall assess the effects of CIN on tumorigenesis in the mouse intestines in terms of numbers and types of tumour, including the promotion of invasion and metastasis. Specific, recurrent chromosomal-scale changes that promote tumour growth will be identified and compared with the equivalent changes in human colorectal tumours. These data will provide evidence for chromosomal regions that affect tumour growth through copy number change, or specific alterations such as the production of fusion genes or gene-gene translocations.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Exploring c-Met and HER3 crosstalk by nano-proximity imaging for understanding its clinical significance in lung adenocarcinoma.
Funder: Medical Research Council

Abstract: The study aims to explore the interaction between two receptors, c-Met and HER3 in lung cancer, in order to understand their role in the development of EGFR-tyrosine kinase inhibitor resistant adenocarcinoma. This will be achieved by using a lung cancer cell model developed in the sponsoring laboratory. This model represents common clinical phenotypes of EGFR (TKI Resistant/sensitive and wildtype). We will image the interaction between c-Met and HER3 in vitro, using this cell model, under basal conditions or with c-Met inhibition. The sponsoring laboratory has proof-of-principle data demonstrating quantification of hetero-dimerisation/oligomerisation of HER family receptors using Förster (Fluorescence) resonance energy transfer (FRET). In addition we will use these cells to develop xenograft tumours in mice, to compare c-Met inhibitor exposed or naive animals. These groups will be assessed for tumour cell proliferation and other tumoral properties, and very importantly, to explore how the c-Met and HER3 interaction is modulated in vivo. The study will finally use archival NSCLC adenocarcinoma sub-type patient specimens (resected primary tumours and regional lymph nodes) to explore the putative interaction between c-Met and HER3 in metastasis and its potential role as a clinical tool in the context of a prognostic model that could predict responsiveness to c-Met inhibitors.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Molecular Mediators of Mammographic Density
Funder: Medical Research Council

Abstract: Aim: To identify pathways modified by tamoxifen to better understand the biology of mammographic density (MD), provide markers to predict a protective response to tamoxifen and ultimately identify new, potentially targetable, molecules that could be used to reduce breast cancer risk. Objectives: (i) To use in-vitro models of primary human breast fibroblasts to dissect the effect of tamoxifen on stromal cell function, using a candidate gene and genome-wide approach. Primary fibroblasts will be isolated from human breast tissue of high MD and treated with tamoxifen (+/- oestradiol), or vehicle control. Fibroblast function will be analysed by: measuring proliferation; analysing key candidate molecules including LOX enzymes, collagen I, fibronectin and SMA by RT-PCR and western blotting (WB); TGF-beta expression and signalling by ELISA and WB; evidence of adipocyte differentiation (expression of leptin and CD36), and the expression and role of ER receptors. A genome-wide approach will also be taken using RNA-Seq (Illumina HiSeq facility) with a minimum of 5 matched treated: control samples. (ii) Establish the impact of the biological pathways identified in (i) on fibroblast-epithelial cross-talk using co-culture systems 2D and 3D systems will be used to assess the functional significance of pathways identified on normal and pre-malignant epithelial cell proliferation and invasion using knock-down and antibody inhibition approaches. (iii) Assess the power of the biological readout generated in (i) and (ii) to predict response to tamoxifen in terms of 1) change in MD and 2) risk of breast cancer. We will access tissue from the IBIS-1 Preventive and MD Trial and the Manchester Prevention Study, which provide data on MD response and breast cancer risk. The project will provide broad laboratory experience with exposure to bioinformatics analysis and the results generated may identify new markers of tamoxifen response and novel targets for prevention


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS