Colorectal cancer in the commonest internal malignancy in western society today. At least a third of the incidence is likely to be due entirely or in part to inherited genetic factors. Over the last 15 years several genes have been described in which germline mutation leading to increased colorectal cancer risk may occur. The commonest are Hereditary Non-Polyposis Colorectal Cancer (HNPCC), which accounts for around 1-4% of colorectal cancer diagnosis without polyposis and is caused by mutations in mismatch repair genes and Familial Adenomatous Polyposis caused by mutations in the AFC gene. In this thesis two related themes are addressed. Firstly I examine clinical, pathologic and molecular genetic information in kindreds recruited from family cancer clinics in order to investigate several relevant clinical problems relating to decisions regarding genetic testing and clustering of non-HNPCC families. Secondly, the group of individuals and families with multiple colorectal polyps without known genetic cause are investigated in several ways. A candidate gene analysis is undertaken looking for germline changes, an analysis of adenomas from such individuals for informative somatic changes is performed and I describe a new inherited syndrome of colorectal cancer and polyposis, MYH associated polyposis as well as the pathway of tumourigenesis in affected individuals.

Cetuximab is a monoclonal antibody targeting the epidermal growth factor receptor (EGFR) and is used for the treatment of metastatic colorectal cancer (mCRC) as monotherapy or in combination with chemotherapy. Recent clinical trials have shown little benefit in combining cetuximab with oxaliplatin in contrast to the positive interactions observed with irinotecan. This thesis aims to understand why a subset of colorectal cancers do not benefit from cetuximab and oxaliplatin combined treatment. In vitro drug combination assays showed that the addition of cetuximab to oxaliplatin resulted in antagonistic effects on cell proliferation as opposed to the synergism observed with 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan. Although both oxaliplatin and SN-38 increased the levels of reactive oxygen species (ROS), only oxaliplatin was able to induce ROS-mediated apoptosis via the activation of p38 Mitogen-Activated Protein Kinase (MAPK). RT-PCR oxidative stress array analysis revealed that oxaliplatin can produce ROS through the upregulation of the NADPH oxidase ROS generating enzyme Dual Oxidase 2 (DUOX2) and that cetuximab, by preventing DUOX2 induction, can inhibit ROS generation by oxaliplatin. Chromatin Immunoprecipitation (ChIP) of Signal Transducer and Activator of Transcription 1 (STAT1) following oxaliplatin treatment, indicated a role for this protein in the transcriptional upregulation of DUOX2 by direct promoter-binding. Cetuximab, by impairing STAT1 activation and DUOX2 upregulation, can antagonise ROS-mediated apoptosis by oxaliplatin through the activation of the p38 pathway. Microarray analysis was employed to identify deregulated genes in the p38 pathway following cetuximab and oxaliplatin treatment in order to better clarify the role that p38-regulated genes play in the negative interactions of these drugs. The results presented in this thesis highlight the importance of ROS generation for the cytotoxic effects of oxaliplatin and indicate that combination with ROS-reducing agents such as cetuximab can result in negative cellular effects. Understanding how these drug interactions lead to antagonistic effects will be useful for the optimisation of future therapeutic combinations in the clinical setting.

The mitogenic and anti-apoptotic effects of insulin-like growth factor-1 (IGF-1) are regulated by a family of insulin-like growth factor binding proteins (IGFBPs), particularly IGFBP-3. In vitro studies have demonstrated the importance of the IGF-1 axis in regulating the growth of breast cancer cells. Little is known, however, about the IGF-independent role of IGFBP-3 in breast cancer and the mechanisms regulating its production. We investigated the expression of IGFBP-3 in malignant and paired adjacent normal (n=53), and healthy normal (n=17) breast tissue samples using RT-PCR, immunohistochemistry and ELISA. We compared IGFBP-3 expression with other members of the IGF-I axis, other known tumorigenic genes and clinicopathological parameters. We also developed a novel tissue explant system using fresh normal and malignant breast tissue, with which we examined the in vitro effects of IGFBP-3 alone and in combination with known apoptotic agent, doxorubicin (n=6), on tissue viability and apoptosis. Results demonstrated universal high level of expression of IGFBP-3 mRNA in all types of breast tissue. There was no difference in level of expression between any of the three groups of breast tissue (approaching those seen in the liver where it is predominantly produced). 96% of samples also expressed IGFBP-3 protein. High levels of IGFBP-3 mRNA were associated with the presence of high grade ductal carcinoma-in-situ (p<0.0001), the pre-invasive stage in breast cancer. A significant correlation was also found between IGFBP-3 negative/weakly positive tumours and lymph node negativity (p<0.05), thereby supporting an association between IGFBP-3 and the development of invasive disease. There was, however, no significant correlation between IGFBP-3 expression and other clinicopathological parameters. The in vitro tissue explant system demonstrated that IGFBP-3 had little effect by itself on apoptosis. However, when used in combination with doxorubicin, a marked enhancement of apoptosis was seen in breast tumours. In contrast, less apoptosis was seen in normal breast tissue suggesting a protective effect. These divergent effects suggest a potential novel chemotherapeutic approach in the treatment of breast cancer. These findings suggest that IGFBP-3 may play a role in tumorigenesis. It may therefore be possible in future, that IGFBP-3 levels could be used in cancer risk assessment and prevention or infact, as markers of response to cancer treatments.

Endometrial cancer (EC) is the most common gynaecological malignancy in the developed world and 4th most common women’s cancer in the UK. Although approximately 75% of women present with surgically resectable disease, 20% of these will relapse and 25% of initial diagnoses occur with metastatic disease. There are currently no validated biomarkers to assess treatment response or target molecular therapies, despite evidence for targetable aberrations in the literature. The PI3K pathway in particular is commonly mutated in EC and stathmin is a recently identified phosphoprotein associated with PI3K pathway activation and with prognostic significance in EC. I investigated biomarker development and novel pathways in EC in two ways: firstly, through a feasibility study of circulating tumour cell (CTC) enumeration and molecular profiling (MP) in patients with advanced endometrioid and non-endometrioid EC (EEC and NEEC); and secondly, through methylation and copy number variation (CNV) studies on formalin-fixed paraffin-embedded (FFPE) and fresh frozen (FF) EEC. CTCs have prognostic and predictive significance in a number of cancers, with increasing evidence on CTC MP. CTC enumeration and assessment of stathmin overexpression was performed on the validated Veridex CellSearch platform and shown to be feasible. In addition, CTC positivity was associated with worse survival and there was a subset of patients for whom a positive CTC count was predictive of outcome on chemotherapy. The second component focused on methylation and CNV analysis in the different phases of endometrial carcinogenesis from normal endometrium to atypical endometrial hyperplasia (AEH) and EEC. By extracting DNA from FFPE and FF EEC and using the Illumina Infinium HumanMethylation450 BeadChip, I was able to demonstrate these methods were feasible and that differential methylation and CNV was evident in the transition from normal endometrium through to AEH and EEC. This research provides a basis for further biomarker development and novel target selection in EC.

The receptor tyrosine kinase HER3, a pseudokinase in the epidermal growth factor receptor (EGFR) family, is involved in responses to ligands and in the acquired resistance against HER2-directed targeted therapy in breast cancer. In this thesis I aim to further investigate the structure/function relationships at the basis of HER2- HER3 heterodimerisation, particularly in response to inhibitor treatment. The data presented here shows that in HER2+ breast cancer cells treated with the HER2 inhibitor lapatinib, stimulation with the HER3 ligand NRG is able to not only rescue cytotoxicity, but even promote proliferation in a drug-growth factor cooperative manner. We show that in response to lapatinib treatment, inactive heterodimers of HER2-HER3 can form, and that these heterodimers adopt a different conformation to the canonical, asymmetric active heterodimer. Instead, lapatinib promotes a head-to-head, symmetrical heterodimer, in which the α-C helices of both receptors form the interface. For the formation of the inactive heterodimer, as well as for promoting ligandinduced signalling, we show that HER3 requires ATP-binding capability. Although there are implications that HER3 retains a measure of catalytic activity, I show that the ATP-binding requirement for dimerisation and signalling is independent of activity. To highlight this, I have identified a Src/Abl kinase inhibitor, bosutinib, which has high affinity to HER3. SKBR3 cells treated with bosutinib in the absence of exogenously added ligand show an increase in proliferation in 2D and 3D culture models. This indicates an importance for conformational stabilisation of the HER3 kinase domain in the formation of active signalling heterodimers. As a pseudokinase, the role of HER3 may be conceived as being of lesser importance than that of its heterodimerisation partners such as HER2. The structure/function studies presented here show that HER3 remains of great importance in providing an allosteric platform for HER2, together forming the HER2-HER3 oncogenic heterodimer.

CAMPATH-1H is administered prior to haematopoietic stem cell transplantation (HSCT) to reduce risks of graft versus host disease (GvHD) by targeting CD52 antigens on T cells, resulting in their depletion. CAMPATH-1H is routinely used in HSCT using haematopoietic stem cells (HSC) from peripheral blood (PB) and bone marrow but not cord blood (CB). Data regarding in vivo and in vitro effects of CAMPATH-1H on immune cells is limited to PB T and B cells. Thus, we sought to determine whether a direct correlation between CD52 density and the depleting effects of CAMPATH-1H exists with fresh and frozen, resting and activated, PB and CB cells. CD52 expression was generally higher in resting CB than PB T cell subsets and B cells although CD52 levels were higher in PB natural killer cells. Furthermore, CAMPATH-1H depleted resting cells more effectively than activated cells with minimal or no necrosis. Higher percentages of apoptosis were noted in naïve CD4 and CD8 T cells with wild type/wild type genotype for caspase-8 (CASP8) gene promoter compared to donors with a single or double deletions, suggesting the potential contribution of CASP8 promoter polymorphism on sensitivity to the drug. CD52 was absent on HSC but upregulated during differentiation, implying that residual CAMPATH-1H could potentially impact on HSC differentiation by depleting CD52 expressing progenitors. This study provides evidence that low dose of CAMPATH-1H may be effective for cell depletion and prevent GvHD whilst allowing cell differentiation. Although the impacts of CAMPATH-1H on viability and differentiation of CB and PB cells were comparable, the use of CAMPATH-1H pre-CBT may not be ideal as it may further delay immune recovery and increase infection incidences. Therefore, this project provides a better understanding of CAMPATH-1H effects at the cellular and molecular level, with potential for clinical translation to achieve effective GvHD modulation while preserving GvL.

Tetanus neurotoxin (TeNT) is among the most poisonous substances on Earth and a major cause of neonatal death in non-vaccinated areas. There are approximately 300,000 cases reported worldwide each year, and the mortality rate is between 10-20%. In this work, I identified an extracellular matrix protein receptor for TeNT at the neuromuscular junction (NMJ) and developed a peptide inhibitor, which prevents tetanic paralysis in vivo in mice. TeNT binds to the NMJ with an extremely high affinity, yet the nature of its receptor complex was poorly understood. I showed that the presence of nidogens (also known as entactins) at the NMJ is the main determinant for TeNT binding. Nidogens are extracellular matrix (ECM) proteins, which are taken up into the endosomal carriers containing tetanus toxin binding fragment (HCT) in motor neurons. Inhibition of the HCT-nidogen interaction using a peptide originating from nidogen-1 abolishes HCT binding on these cells. TeNT causes slowly progressing local tetanus when it is injected intramuscularly into the triceps surae muscle in a low dose. When preincubated with the peptide originating from nidogen-1, TeNT injection does not alter the coordination of mice and the muscle force remains largely unchanged. Genetic ablation of nidogens prevented the binding of TeNT to neurons and the intact NMJ and protected mice from TeNT induced spastic paralysis. In my thesis I demonstrated for the first time, that an ECM protein accumulates and presents a neurotropic pathogen to the presynapse. This study follows recent studies showing that growth factors trigger downstream signalling more efficiently if they bind to certain ECM components – a new and rising concept in neuroscience.

RNA localisation is essential for the establishment of body axes in the developing Drosophila embryo. Transcripts of certain maternal genes are transported along microtubules and translated in restricted regions of the oocyte to define these axes. RNA from one such gene, gurken (grk) is first localised to the oocyte posterior, where translated Grk signals to the soma to specify the follicle cells in this region to adopt a posterior fate. Subsequently these posterior follicle cells signal back to the oocyte triggering cytoskeletal reorganisation. The nucleus and grk RNA are then transported to the dorsal-anterior region of the oocyte, where Grk signals to specify the surrounding cells to become dorsal. This two-stage grk localisation thereby determines both the anterior-posterior and dorsal-ventral axes. This thesis describes a random mutagenesis screen designed to identify novel genes on chromosome arm 3R that are involved in grk RNA localisation in the oocyte. We observed the distribution of endogenous grk RNA in vivo using a fluorescent construct. From 4943 mutagenised lines scored, I recovered 38 with reproducible grk mislocalisation and 78 lines with other defects in oogenesis. After phenotypic analyses and complementation testing, these mutants were grouped and the affected genes identified using whole genome sequencing and recombinational mapping. I focus on three groups of mutants from the screen that showed a grk mislocalisation phenotype. Mutations in karyopherin-beta3, a nuclear import factor gene, and in CG9925, encoding a TUDOR-domain containing protein, cause reduced Grk translation and axis misspecification. In two new mutant alleles of CG5508/minotaur, which encodes a protein involved in phospholipid biosynthesis, Grk translation is reduced and transposon expression levels are elevated, a typical phenotype of mutants for piRNA pathway genes. Mutants of these three genes, despite diverse predicted gene functions, share many phenotypes. I describe detailed characterisation of these novel mutants and suggest how they may all be affecting grk mRNA localisation due to disruptions in the mechanisms of piRNA biogenesis and the protection of genome integrity.

Isocitrate dehydrogenase (IDH) genes 1 and 2 are frequently mutated in acute myeloid leukemia (AML), lower-grade glioma (LGG), and cholangiocarcinoma (CC). In these three malignancies, mutant IDH status is associated with increased 2-hydroxyglutarate (2-HG) production and a DNA hypermethylation phenotype, implicating altered epigenome dynamics in the aetiology of these cancers. Here I show that the IDH variants in chondrosarcoma (CS) are also associated with a hypermethylation phenotype, supporting the role of mutant IDH-produced 2-HG as an inhibitor of TET-mediated DNA demethylation. The associated gene expression profile is also investigated, highlighting the need for a better understanding of DNA methylation-mediated transcriptional regulation. The generated methylation data is additionally harnessed to reveal novel copy number variants in CS. Meta-analysis of the AML, LGG, CC and CS methylation data identifies cancer-specific effectors within the retinoic acid receptor activation pathway among the hypermethylated targets. By analysing sequence motifs surrounding hypermethylated sites across the four cancer types, and using chromatin immunoprecipitation and western blotting, I identify the transcription factor EBF1 as an interaction partner for TET2, in the first description of a targeted demethylation pathway. In an effort to assess whether patient-derived tumour xenografts (PDXs) are suitable models for epigenetic research in rare and common cancers, such as osteosarcoma (OS) and colon cancer, respectively, I compare PDXs to their matched patient tumour and reveal that an average of only 2.7% of the assayed methylome undergoes major methylation changes with xenografting. In addition, no further changes are identified in subsequent PDX generations, making these models highly suitable for expansion of rare tumours and preclinical drug screening. Finally I propose a model to inform future study design and statistically dilute those methylation shifts identified in PDXs.

Vulval lichen sclerosus (LS) and high-grade intraepithelial neoplasia (VIN 3) are two common and distressing diseases. Significant morbidity is caused by symptoms of persistent pruritus and surgical treatment of skin areas suspicious of malignancy. The risk of developing cancer in a background of LS and VIN 3 is poorly defined. The methods currently available for clinical assessment of the vulva are limited. There is abundant research on the application of the LASER Doppler technique - laser Doppler Flowmetry (LDF) - showing changes in perfusion within the small blood vessels of the skin as a useful parameter for more accurate disease classification. There is also research on immunohistochemical microvessel density (MVD) studies showing increases in blood supply in tissues prone to develop cancer or as a prognostic marker of cancer outcome. The Laser Doppler perfusion imager (LDPI) provides a rapid, real time, non-invasive and non-contact method to measure skin blood flow in an area as opposed to a single point by the LDF, making the LDPI more suitable for application to the vulva. This thesis reports for the first time, the application of the LDPI to the vulva. Initially the LDPI was applied to the clinically normal vulva to study perfusion variance related to menstrual cycle, age and local skin temperature provocation. The application was then extended to vulval disease, LS and VIN 3, and validated against morphological differences in MVD. The LDPI and MVD studies suggest that in VIN 3 there is an actual increase in skin perfusion. In LS the situation is more complex and suggests that the LDPI measured perfusion at a greater depth than the MVD. Studies on base line perfusion variance of vulval LS to topical therapy show that there is no overall difference in baseline perfusion in spite of symptom improvement. Temperature provocation studies suggest differences in skin blood flow response in diseased compared to the normal vulva.