NIHRDataInsights/HRCSData huggingface dataset:
Title: Identification of genes required for the maintenance of AML cancer stem cells
Funder: Cancer Research UK

Abstract: Considerable evidence indicates that several human cancers are maintained and disseminated by a discrete sub-population of self-renewing tumor cells termed cancer stem cells (CSCs). In particular, evidence is compelling that acute myeloid leukemias (AMLs) display a hierarchical structure with leukemia initiating cells (LICs) constituting the self-renewing component, which gives rise to partially differentiated progeny without this potential. Elimination of the CSC component of a tumor is both necessary and sufficient for its eradication, and development of methods to selectively target CSCs constitutes and attractive strategy in cancer therapy. Based on comparison of the gene expression profiles of purified committed myeloid LIC populations from two murine AML models we have identified a core LIC signature of genes enriched in both MLL-AF9 overexpressing and Cebpa mutant LICs, the latter derived from a knockin mouse model developed by our group. We hypothesize that genes critical for LIC maintenance are contained within this signature, and propose to use it as a starting point for the development of small molecules that selectively target this cell population. To achieve this we have selected those genes within the common LIC signature that fulfill the following criteria: 1) they are not highly expressed in HSCs (increasing the likelyhood that they are not involved in HSC maintenance) and 2) they have molecular properties compatible with a small molecule or antibody targeting strategy (i.e. enzymatic activity, surface or extracellular location). We now aim to identify those genes within this group critical for LIC maintenance. To achieve this we will perform lentiviral shRNA knockdown experiments in Cebpa mutant LICs using hairpins directed against the “druggable” subset of LIC signature genes (using 4-5 shRNAs/gene). Using high-throughput sequencing of shRNAs maintained in LICs and subsequent bioinformatics analysis of the data we will identify those incompatible with LIC maintenance. Genes for which at least 2 hairpins are depleted will be considered hits. We will subsequently validate these hits in a second set of infection/shRNA retrieval experiments, at the same time testing whether the knockdown affects normal HSC maintenance. At the end of the project we expect to have identified a set of molecular targets that are specifically required for the malignant self-renewal, and which may then be used as the basis for development of small molecule or other drugs.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Dissecting the functions of the BRCT domains of the DNA integrity checkpoint protein Crb2
Funder: Cancer Research UK

Abstract: Failure to maintain genetic integrity can result in the inheritance of mutations, genetic disorders, cancer or cell death. To ensure genome stability, cells have a number of ways to deal with DNA damage. These include a range of different DNA repair mechanisms and DNA integrity checkpoints. It is becoming clear that checkpoint proteins not only regulate cell cycle progression but are also central to the repair of DNA damage and the facilitation of DNA replication. DNA integrity checkpoints thus have key roles in maintaining genome stability. As well as being components of gatekeeper pathways that prevent cacinogenesis, checkpoint proteins can also be exploited as targets for anti-cancer drugs. We have recently undertaken a structure/function analysis of the BRCT domains of the DNA integrity checkpoint protein Crb2 (related to human BRCA1 mutations in which result in a high proportion of breast and ovarian cancers). We have shown that at the molecular level, the BRCT domains have two distinct functions, namely dimerisation and phospho-protein binding. Independent disruption of these abilities results in different phenotypes, implying different cellular roles for the two functions. Although it is known that inability to dimerise results in loss of the checkpoint, the biochemical consequences of dimerisation of Crb2 are unknown. Additionally, the extent of interactions involving the phospho-protein binding motif is unkown. The objective of this project is to address these questions. Specifically, we will determine at the molecular level the biochemical consequences of Crb2 dimerisation and establish the identity and role of additional phosphoproteins that bind the Crb2 BRCT1 domain. The project will involve a comprehensive search for proteins that interact with Crb2, under conditions where Crb2 either dimerises or interacts with phospho-proteins. This will be undertaken using a combination of MudPIT analysis and a candidate approach (using bioinformatics and a screen of a phospho-peptide library using purifed BRCT domains). These interactions will be confirmed using immunoprecipitation and in vitro assays. From the proteins that we identify, we will select a few of the most interesting and identify the Crb2-interacting motifs/domains in each.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: A partnership between Rac, PAK and Rabs during junction disassembly and increased cell motility
Funder: Cancer Research UK

Abstract: Conversion of adherent cells in solid tumours into de-differentiated, highly invasive tumour cells has major implications for the survival rate and the prognosis of cancer patients. A benign, solid tumour is self-contained, and in most cases can be clinically removed and successfully eradicated. The same prognosis is not true for malignant tumours. Identification of regulatory mechanisms involved in the transition between benign to malignant tumours may provide suitable strategies to minimize cell detachment and metastatic potential during tumorigenesis. In many different tumour types, there is a strong correlation between loss of cadherin-dependent cell-cell contacts (or reduced levels) and increased metastatic potential. Inappropriate activation of the small GTPase Rac is found in a broad range of tumours and is important for cell migration. We find that activation of Rac disrupts cell-cell adhesion by inducing internalization of cadherins. We have begun to elucidate the mechanisms via which active Rac perturbs cell-cell contacts. Interestingly, our recent results show that disruption of junctions by Rac can be reversed by inhibition of its target PAK or Rab11, a small GTPase that regulates early endocytic trafficking. This data is exciting in the context of tumour metastasis as PAK and endocytic trafficking are also key players in cell motility, by regulating the availability of growth factor and adhesion receptors at cell surface. Consistent with our data, Rac and PAK have been implicated in increased motility and invasiveness of different tumour cells. Thus, the pathway identified in our study may participate in the coordination of junction destabilization and increased motility observed during metastasis. The interplay between Rac, PAK and Rab11 is novel and significant to tumour progression. Here we will investigate the mechanisms required for junction disassembly and identify the: 1. Cellular and molecular mechanisms by which Rac/PAK/Rab11 interfere with junction stability 2. Functional consequences of PAK1 phosphorylation of junctional and trafficking proteins 3. Potential of PAK inhibition to ameliorate junction disassembly in different tumour cell lines This proposal will provide considerable insights into the intracellular mechanisms that lead to cadherin destabilization from junctions following Rac activation. By interfering with PAK function in human cancers, two major processes associated with malignancy will be impaired: de-differentiation (disassembly of cell-cell contacts) and metastasis (increased migration and invasion). Due to the wide-range of tumours in which Rac is up-regulated, our high-throughput screen of 700 tumour cell lines will validate the applicability of PAK inhibition as a therapeutic tool.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Characterisation of cytokinesis proteins, their regulation and cooperativity using biophysical and cell biological approaches
Funder: Cancer Research UK

Abstract: Cytokinesis is the last step of mitosis that separates a whole cell into two independent cells. For the stable maintenance of genome, cytokinesis must be tightly coupled with chromosome segregation both temporally and spatially. Microtubule bundle structures, such as the central spindle and midbody, play key roles in cytokinesis both by coordinating chromosome segregation with the cortical actin cytoskeletion and by working as a platform for dynamic vesicle trafficking events. Centralspindlin, a microtubule-bundling protein complex of a kinesin-like motor and a Rho-family GTPase-activating protein, plays critical roles both in the formation of microtubule bundles during cytokinesis and in the recruitment of various downstream factors to these structures. Failure of cytokinesis produces a tetraploid cell that can be an origin of aneuploidy, which is often associated with cancer. In addition, drugs that target microtubules or protein kinases that regulate centralspindlin have been used or are under development for anti-cancer chemotherapy. Although the list of proteins involved in cytokinesis is expanding, it is not yet complete. Moreover, our understanding of how multiple factors act cooperatively in the formation of the central spindle and midbody is very poor. Our research aims are to comprehensively detemine the proteins that interact with centralspindlin and to reveal how these proteins regulate or cooperate with centralspindlin in the formation and function of these microtubule bundle structures. These will provide novel insights into the early process of carcinogenesis and provide potential targets for anti-cancer chemotherapy. To achieve these aims, we will identify novel midbody proteins using a combination of proteomic and bioinformatic approaches coupled to high-throughput live cell observation. In parallel, we will further characterize a novel mechansims involving 14-3-3 protein and Aurora B kinase, which regulate centralspindlin. Specifically, we will identify protein kinases and phosphatases that cooperate with or counteract Aurora B using biochemical and cell biological approaches. To reveal how centralspindlin cooperates with another essential microtubule bundling protein PRC1/SPD-1, we will investigate the role of a direct interaction between them using cell biological and genetical approaches. To fully understand the actions of microtubule motors and bundling proteins, it is essential to observe how they interact with microtubules at the single molecule level. We will develop in vitro assays to visualize cooperative interaction of individual proteins with microtubules. We will also extend our in vivo observation of the movement of proteins along the central spindle microtubules, which we have succeeded in with centralspindlin, to other cytokinesis proteins.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS
Title: Epidemiological Study of BRCA1 and BRCA2 Carriers
Funder: Cancer Research UK

Abstract: Background Germline mutations in the cancer predisposition genes BRCA1 and BRCA2 cause high lifetime risks of breast and ovarian cancer in female carriers. Predictive genetic testing for mutations in these genes in women with a strong family history of these cancers is now widespread. To provide adequate management of carriers, reliable estimates are required of the risks of cancers in mutation carriers, the extent of risk modification by lifestyle, genetic or other risk factors, and the protection offered by preventative strategies such as prophylactic surgery. As a basis for providing these estimates, a cohort of BRCA1 and BRCA2 mutation carriers has been established through clinical genetics centres in the U.K. and Eire. This study has accrued approximately 5,200 individuals to date. Relevance to Cancer The BRCA1 and BRCA2 genes are among the most important cancer susceptibility genes. Mutations in these genes cause high risks of breast and ovarian cancer, and significant risks of prostate, pancreatic and other cancers. Understanding the risks associated with these genes and how these risks are modified by other factors is central to management of women at high risk of cancer and their families. Aim The aim of this renewal is to extend the size of the study to 7,600 individuals; to follow-up the whole cohort with updated data on lifestyle risk factors, cancer incidence and mortality; and to obtain associated medical history and blood samples. This study also provides a resource for recruitment into other studies of BRCA1/2 carriers. Outline Information on lifestyle risk factors will be collected on both affected and unaffected carriers. Follow-up questionnaires will be completed at 2, 5 and 10 years; data on cancer incidence and mortality will also be obtained through national records. These data will allow prospective evaluation of lifestyle risk factors and cancer risk, both within EMBRACE and as part of the International BRCA1/2 Carrier Cohort Study (IBCCS). DNA samples will be collected on all recruits, and these will be used to identify genetic polymorphisms which modify cancer risk. In addition, serum samples will be collected as a resource for other studying hormone levels and other serum markers. Medical records collected on all affected individuals, to document tumour characteristics and treatment. Mammograms are being assembled to evaluate the effect of mammographic density on breast cancer risk in carriers.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Defining the 'bone metastasis niche'
Funder: Cancer Research UK

Abstract: Bone metastases are a devastating clinical consequence for patients with advanced breast and prostate cancer. The events leading to their development are poorly defined, and approaches to prevention and treatment currently limited. The majority of research in this area has focussed on established bone metastases. However, it is clear, that to prevent the development of new bone metastases, defining the events and pathways involved when tumour cells first arrive in bone will be critical. Through this programme we will test the general hypothesis that only a subset of the tumour cells that arrive in bone has the capacity to develop overt metastases. Of these, only those that locate to a specialised ‘bone metastasis niche’, created by osteoclasts and/or osteoblasts, which support their colonisation, survival and growth, will subsequently form a bone metastasis. Specifically we will determine: • Whether tumour cells that metastasise to bone localise to a specific ‘bone metastatic niche’. • Whether osteoblasts are critical components of the ‘bone metastasis niche’ and essential for tumour cell attachment, survival and growth in bone • Whether osteoclasts are a key component of the ‘bone metastasis niche’ and regulate the behaviour of tumour cells in the ‘niche’. Using in vivo models of breast and prostate cancer bone metastasis we will map the location of tumour cells in bone by multiphoton microscopy, determining the location of tumour cells in relation to osteoblasts and osteoclasts, and investigate whether accelerated or decreased bone turnover affects metastasis formation. The specific role of osteoblasts in this process will be determined by conditional knock-out of osteoblasts, whereas the role of osteoclasts will be identified by use of agents preventing osteoclast development or activity. Extensive analysis of bone and tumour cells will be carried out allowing us to characterise the cellular interactions that take place during bone metastasis development in the putative ‘bone metastasis niche’. Successful delivery of this programme will provide new knowledge of how cancer cells interact with their environment, allowing us to define the ‘bone metastasis niche’ in which they reside and to identify new approaches to modify the ‘niche’ to prevent tumour cell survival. This will have a major impact on approaches for prevention of bone metastasis as well as lead to improved therapeutic strategies to treat existing bone lesions.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: The contribution of heparan and chondroitin sulphates to ovarian cancer angiogenesis
Funder: Cancer Research UK

Abstract: Ovarian cancer is the fourth commonest cause of female cancer death and the commonest cause of gynaecological cancer death in the UK. Traditionally patients have been treated with surgery and platinum-based chemotherapy resulting, in advanced disease, in a median survival of approximately 3 years. Ovarian cancer is one of only a few diseases that respond to single agent anti-angiogenic therapy. Most angiogenic cytokines, such as FGFs and VEGF, require heparan sulphate (HS) for the activation of their receptors, while the role for chondroitin sulfate (CS) in angiogenic signalling is less defined. The degree and distribution of sulfation in HS determines the specificity and activity of the signalling complexes. Understanding how HS/CS contribute to ligand-induced angiogenic receptor activation is important, since it may represent new opportunities in designing anti-angiogenic drugs. Our proposal aims to investigate the distribution of HS/CS and proteoglycan/angiogenic receptor complexes in malignant and normal ovarian tissue in order to determine spatial localization of HS/CS and HS association with angiogenic complexes. Circulating endothelial cells (CECs) that are derived from tumour vessels reflect pathological angiogenesis. We will characterize human CECs and bone-marrow derived circulating endothelial progenitors (CEPs) and examine their HS composition and kinetics in ovarian cancer patients undergoing treatment with anti-angiogenic metronomic chemotherapy. Finally, we will investigate how reduced 6-O sulfation in endothelial cell HS affects angiogenic growth factor-induced cell functions, such as proliferation, adhesion, migration and tube formation. To further understand how HS sulfation patterns dictate endothelial cell responses to angiogenic cytokines we will investigate inhibitory activity of the unique series of structurally defined synthetic HS oligosaccharides that we have generated. HS/CS composition analysis and visualization of receptor/proteoglycan complex distribution in tissues will require immunofluorescence staining, biochemical analysis and FRET imaging. Flow cytometry will allow us to identify CECs/CEPs and their HS composition in blood samples from ovarian cancer patients. Modulation of 6-O sulfation in endothelial cells will be achieved by down-regulation of 6-O-sulfotranferases in these cells. Retroviral infection delivery of shRNAs or standard transfection methods will be used. Endothelial cell proliferation, migration, invasion and tube formation assays will be used to characterize endothelial cell functions or inhibitory activity of synthetic HS oligosaccharides. Altogether these experiments will highlight the role of HS/CS in ovarian cancer angiogenesis, thereby providing stronger rational for targeting HS/CS interactions in ovarian cancer in order to inhibit angiogenic process.


Research activity codes: 5.1 PHARMACEUTICALS;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Modelling the stromal regulation of tumour angiogenesis, progression and spread
Funder: Cancer Research UK

Abstract: Tumours consist not only of transformed cells but also contain stromal elements which have a major impact on tumour behaviour. Thus cancer growth and spread depends largely on angiogenesis, the development of new blood vessels, and this process, which involves the migration of endothelial and bone marrow derived (BMD) cells is regulated via changes in cell adhesion. Our previous work, has led to a greater understanding of the role adhesion molecules play in tumour angiogenesis. These studies have identified how integrins control pro-angiogenic growth factor receptors, such as VEGF-receptor 2, in this process. Signalling downstream of integrins and growth factor receptors converges on Focal Adhesion Kinase, (FAK), making it likely that this molecule is a central regulator of the effects we have observed. Until now the study of FAK in tumourigenesis has been restricted predominantly to tumour cells themselves. However, data indicate that, whilst FAK is up-regulated in the tumour cells of many cancers, pharmacological inhibition of FAK does not always affect tumour growth and its prognostic value is variable. We intend to elucidate the, hitherto uninvestigated, functional roles of stromal-FAK in tumourigenesis. We have compelling evidence that homozygous-deletion of FAK in the stromal compartment, in particular in endothelial cells, can inhibit primary tumour growth and angiogenesis, while paradoxically, enhancing metastasis. In contrast, partial-deletion of FAK in the tumour stroma actually can enhance tumour growth and angiogenesis. Together, our data indicate that stromal-FAK can regulate tumour growth and spread independent of its expression in tumour cells; an important demonstration of the pivitol role of stromal elements in modifying cancer behaviour. By exploiting novel genetic mouse models, that we have recently generated, where mutant forms of FAK will be expressed and endogenous-FAK deleted simultaneously in adult mouse endothelial, or BMD cells in vivo, we propose to uncover the specific functional roles of stromal-FAK in adult tumour growth, angiogenesis and spread. Additionally we will identify the effect of partial inhibition of FAK, using low doses of FAK-inhibitors, and ask if this could influence FAK-inhibitor efficacy. Lastly, we will establish the prognostic value of stromal-FAK in determining cancer outcome in human breast cancer. This is a fundamental science programme that will describe the precise roles of stromal-FAK in tumourigenesis and will provide rational basis for the clinical use of FAK in cancer diagnosis and treatment. Moreover it will have general significance in establishing how stromal elements can impact upon cancer behaviour.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS