background The extracellular matrix receptor integrin ανβ6 is known to potentiate breast cancer (BrCa) cell invasion, metastasis and tumour-trophic growth factor receptor crosstalk during tumourigenesis. Monoclonal antibody blockade of ανβ6 diminishes invasion in vitro and arrests BrCa tumour growth and metastasis in vivo. Aberrant integrin activation status has been implicated in progression to metastatic disease in BrCa; with differential internalisation and endocytic trafficking kinetics reported for active versus inactive integrin species in malignant disease. Despite its emerging potential for targeted therapy, little is known regarding regulation of integrin ανβ6-mediated activation and signalling during progression to an invasive, metastatic state. It is hypothesised that the aetiopathological significance of integrin ανβ6 during neoplastic transformation and malignant progression in BrCa is dependent specifically upon its activation status and associated conformation, since this active state will permit establishment of known integrin-mediated oncogenic signalling underpinning acquisition of a malignant phenotype, including activation of invasion and metastasis. results Canonical integrin activation studies using divalent cations and cognate ligand stimulation indicated antibodies 6.2E5 and 6.2G2 recognise activation-associated epitopes, which are also ligand-induced binding sites (LIBS) in live-labelled cells by FCM and IMF. However, their utility to discriminate the active fraction distinct from the total or inactive fractions of ανβ6 by IHC in primary BrCa samples could not be robustly established. Evaluation of the 6.2E5 and 6.2G2 epitopes in the MCF10 isogenic model revealed that relative surface abundance of these active epitopes determined by FCM was not significantly altered; but their subcellular redistribution upon neoplastic transformation and malignant progression was observed by IMF, implicating derailed internalisation and trafficking of active ανβ6 during breast tumourigenesis and metastatic disease progression. Proteomic interrogation and network analysis of the 2D-enriched adhesion assays identified 7 novel putative molecular regulators of a ligand-engaged, activated ανβ6-mediated adhesion environment (DMBT-1, MARCKS, MXRA5, SEPT6, SEPT9, MYH9, MYH10) in the BT-20 TNBC cell line. Functional validation of these candidate mediators of the "β6 adhesome" by siRNA strategies was not achieved due to inconsistent stable knockdown. Phosphoproteomic definition of LAP ligand-engaged, active ανβ6-mediated signalling ("β6 kinome") during receptor-ligand internalisation revealed EGFR-dependency for downstream ERK1/2 signal activation in BT-20 and SUM159, but not MDA-MB-468 TNBC cells. Kinase substrate enrichment analysis (KSEA) identified 5 novel putative mediators of downstream ανβ6 signalling (COT, MAPKAPK2, PDPK1, Nuak1, TBK1) and implicated Akt1 isoform-specific activation downstream of ανβ6-LAP internalisation. Following LAP-induced ανβ6 activation and internalisation, EGFR underwent phosphorylation at multiple known activation sites, including a residue (Thr693) critical for EGFR receptor internalisation; suggesting integrin ανβ6-EGFR reciprocity during respective receptor activation and internalisation. conclusion The active conformer of integrin ανβ6 may be studied using antibodies 6.2E5 and 6.2G2 in live-labelled cells by FCM and IMF. Subcellular redistribution of activation-associated epitopes during BrCa progression implicates derailed internalisation and intracellular trafficking kinetics of active ανβ6 during tumourigenesis, while protein expression studies identified 7 putative molecular regulators of ligand-engaged, active ανβ6-mediated adhesion. Integrin ανβ6-mediated signalling during internalisation revealed an ανβ6-EGFRAkt1 signalling axis during breast tumourigenesis and disease progression, while further understanding of integrin biology and growth factor receptor crosstalk may provide additional rationale for potential combination therapies in breast cancer.

The Hippo Pathway is a highly conserved regulator of tissue growth and size determination, limiting the activity of the transcriptional co-activator Yorkie (Yki), which promotes proliferation and inhibits apoptosis. Hippo signalling integrates and transduces cell polarity and cell-cell adhesion inputs thereby responding to the state of tissue architecture. The transmembrane apical polarity protein Crumbs (Crb) controls the activity of Yki by regulating Expanded (Ex), a protein that promotes Hippo signalling through kinase-dependent and -independent mechanisms to robustly inhibit Yki activity. Crb plays a dual role in the regulation of Ex by controlling its apical localisation, facilitating Yki inhibition, and by promoting Ex degradation, thus activating Yki. Crb regulates the stability of Ex by stimulating a phosphorylation-dependent ubiquitylation and proteasomal degradation. Characterisation of the precise mechanisms by which Crb regulates Ex has been the focus of this thesis. Based on candidates identified by mass spectrometry and from literature, the Casein Kinase 1 (CK1) family of kinases, and the deubiquitylating enzyme (DUB) Usp2 have both been identified as novel regulators of Ex stability. CK1s promote Ex phosphorylation and degradation, acting as Ex inhibitors, while Usp2 promotes Ex function by promoting its stabilisation. Furthermore, in a screen to identify DUBs that regulate Drosophila adult wing size, CG10889 has been established as a novel regulator of growth that interacts with members of the Hippo pathway.

Induction of apoptosis is the most studied cell death process and it is a tightly regulated physiological event that enables elimination of damaged and unwanted cells. Apoptosis can be induced via activation of either the intrinsic or the extrinsic signalling pathway. The intrinsic pathway involves activation of the mitochondria by stress stimuli, whereas the extrinsic pathway is triggered by ligand induced activation of death receptors such as Fas. Apoptosis induction via Fas activation plays an important role in the function of cytotoxic T lymphocytes and in the control of immune cell homeostasis. Several studies have shown that anticancer therapies require functional cell death signalling pathways. Irradiation based therapy has been successful in treatment of several malignancies but the usage of high doses has been associated with side effects. Therefore, low dose therapies, that either is optimized for specific delivery or administrated in combination with other treatments, are promising modalities. However, in order to achieve high-quality effects of such treatments, the death effector mechanisms involved in tumour eradication needs to be further explored. Importantly, tumour cells frequently acquire resistance to apoptosis, which consequently allows tumour cells to escape from elimination by the immune system and/or treatment. Interferons constitute a large family of pleotrophic cytokines that are important for the immune response against viruses and other microorganisms. The interferon signalling pathway mediates transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. Interferon has successfully been used in therapy against some tumours. However, several drawbacks have been reported, such as reduced sensitivity to interferon during treatment. The aim of this thesis was to elucidate mechanisms that mediate resistance to death receptor or interferon induced apoptosis in human tumour cell models, as well as investigate what molecular events that underlie cell death following radiation therapy of tumour cells. In order to elucidate mechanisms involved in acquired resistance to Fas- or interferon-induced apoptosis, a Fas- and interferon-sensitive human cell line, U937, was subjected to conditions where resistance to either Fas- or interferon induced apoptosis was acquired. Characterization of the Fas resistant cells showed that multiple resistant mechanisms had been acquired. Reduced Fas expression and increased cFLIP expression, which is an inhibitor of death receptor signalling, were two important changes found. To further examine the importance of these two alterations, clones from the Fas resistant population were established. The reduced Fas expression was determined to account for the resistant phenotype in approximately 70% of the clones. In the Fas resistant clones with normal Fas expression, the importance of an increased amount of the cFLIP protein was confirmed with shRNA interference. A cross-resistance to death receptor induced apoptosis was detected in the interferon resistant variant, which illustrates that a connection between death receptor and interferon induced apoptosis exists. Notably, interferon resistant cells also contained increased cFLIP expression, which were determined to mediate resistance to both interferon and death receptor mediated apoptosis. Finally, when cell death induced by irradiation treatment was investigated in HeLa Hep2 cells we could demonstrate that cell death was mediated by centrosome hyperamplification and mitotic aberrations, which forced the cells into mitotic catastrophes and delayed apoptosis. In conclusion, we have described model systems where selection for resistance to Fas or interferon induced apoptosis generated a heterogeneous population, where several signalling molecules were altered. Furthermore, we have shown that a complex cell death network was activated by irradiation based therapy.

(1) Human neuron specific enolase (NSE) was purified from human brain tissue and used as an immunogen in the production of murine monoclonal antibodies. It was also used as a standard preparation of NSE in the screening of monoclonal antibodies and in the investigations of an enzyme-linked immunosorbent assay (ELISA) for NSE. (2) Four monoclonal antibodies were raised to NSE. These did not cross react with the alpha isoenzyme of enolase (NNE). Approximately 76% of positive well supernatants reacted with both NSE and NNE. (3) All four monoclonal antibodies reacted in an ELISA but only two of the four reacted on an immunoblot. (4) The monoclonal antibodies were purified by precipitation by ammonium sulphate and by Protein A-affinity column chromatography. The latter resulted in immunoglobulin which was pure by SDS-PAGE. (5) The purified monoclonal antibodies were labelled with horseradish peroxidase by the periodate oxidation method, with ¹²⁵I using the Chloramine T and the Bolton-Hunter methods, and with biotin. (6) Labelling with biotin was the most effective method. (7) The labelled monoclonal antibodies were used in the investigation of a sandwich ELISA for NSE. These experiments showed that high binding occurred with no antigen present. It was surmised that monoclonal antibody-monoclonal antibody binding may be occurring. (8) The high no-antigen control was investigated in a number of ways including altering the assay pH, the detergent concentration, the type of solid support and by using Fab fragments in the assay instead of whole molecule.

No description available.

Barrett's oesophagus (BO) is the major precursor of oesophageal adenocarcinoma (OA) and we do not understand the dynamics of the evolution of BO in order to identify patients at high risk of cancer. Studies have proposed that BO is a monoclonal lesion, however recent work has shown that there are multiple independent clones present. Project 1: Determines the evolution of polyclonal dysplasia through sequencing and mapping clones onto tissue sections. I show that several cases are polyclonal but in each case only one clone progresses to cancer, suggesting oesophageal cancers are monoclonal outgrows from polyclonal Barrett's dysplasia. Project 2: Aims to understand the clonal relationship between cells in glands displaying basal crypt dysplasia-like atypia (BCDA), as it is unclear whether those cells in the upper part of the gland arise from the same stem cell that generates the gland bases. Glands displaying BCDA show a common mutation between the dysplastic base and non-dysplastic surface suggesting a common cell of origin. Project 3: 50% of patients who undergo oesophagectomy for OA develop post-oesophagectomy Barrett's (neo- BO) within 3-5 years possibly due to a field effect, wherein pre-neoplastic cells remain post-resection in histologically normal areas of epithelium predisposing the patient to cancer recurrence. Here I show that no genetic link between the neo-BO and the cancer is present. Immunohistochemical analysis shows that neo- Barrett's glands are gastric in nature. Project 4: The stem cell dynamics and clonal expansion rates of BO are unknown. Here I employed diversity analysis of methylation patterns of CpG islands in the promoter regions of non-expressed genes as a molecular clock. My data suggests that 3-4 stem cells are found in each Barrett's gland. Methylation patterns within a gland were less diverse compared to adjacent and distant glands, suggesting BO is characterized by long periods of stasis followed by bursts of clonal expansions.

The Receptor Tyrosine Kinase (RTK) Met, overexpressed or mutated in cancer, plays a major role in cancer progression and represents an attractive target for cancer therapy. This study aimed to investigate whether PI3K plays a role in Met oncogenicity. Three cell models were used: (i) NIH3T3 cells expressing WT Met or the constitutively active mutant M1268T Met; (ii) U87MG glioblastoma cells, with endogenous WT Met constitutively activated due to an autocrine loop; (iii) A549 lung cancer cells expressing endogenous WT Met, activated upon binding exogenous HGF. Met dependent Rac1 translocation to the plasma membrane, actin cytoskeleton organisation, cell migration, anchorage independent growth in soft agar and tumour growth were studied in the presence of inhibitors of pan-PI3K / mTOR, various PI3K Class I isoforms, mTOR or Akt, or following siRNA knock-down of PI3K isoforms. We report that PI3K class I (but not class III) regulates Met dependent cell migration. The PI3K class I isoforms required varies among the cell models. Interestingly, the combined inhibition of all p110 Class I isoforms lead to the strongest reduction of Met dependent cell migration. Met dependent phosphorylation of Akt, an effector of PI3K class I, is reduced upon endocytosis inhibition, suggesting that Met signals to PI3K Class I on endosomes. Our results indicate that mTOR is responsible for Met dependent anchorage independent growth and tumour growth in vivo. Surprisingly, PI3K class I (and class III) are not required. Moreover, Rac1 is required for Met dependent mTOR activation, (phosphorylation of mTORC1's effector, p70 S6K) subcellular translocation of mTOR and anchorage independent growth. Finally, our results suggest that this Met-Rac1- mTOR pathway occurs on endosomes. Thus while PI3K class I regulates Met dependent cell migration, mTOR regulates Met driven anchorage independent growth and in vivo tumorigenesis. Thus PI3K Class I / mTOR may be targeted in Met driven cancers.

Tumour angiogenesis is a hallmark of cancer. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in endothelial cells (ECs) survival, proliferation and migration. FAK has several tyrosine phosphorylation sites thought to be involved in FAK function but the requirement of phosphorylation of these residues in vivo is unknown. We have generated mice where endogenous FAK is deleted simultaneously with the expression of nonphosphorylatable FAK-Y397F or FAK-Y861F mutated or wild type forms of FAK in adult endothelium in order to test this. My data show that EC-FAK-Y397FKI mice present with decreased tumour angiogenesis (in sygeneic B16F0, CMT19T and LLC) but impaired B16F0 and CMT19T tumour growth only, with increased tumour hypoxia. FAK-Y397F tumour endothelium is not perfusion, leakage or vascular maturation defective. This mutation affects VEGF-, PlGF- and bFGF-driven angiogenesis in vivo and VEGF+Ang2 administration is able to partially rescue this phenotype ex vivo. In contrast, endothelial FAK-Y861F mutation leads to an initial delay in B16F0 tumour angiogenesis, that subsequently resolves, and does not affect B16F0 tumour growth. LLC and CMT19T tumour growth and angiogenesis are not affected by the endothelial FAK-Y861F mutation; neither are tumour blood vessel perfusion, leakage, vascular maturation or tumour hypoxia. VEGF-, PlGFand bFGF-driven angiogenesis in vivo and ex vivo was not affected by the endothelial FAK-Y861F mutation, whereas increased in vivo angiogenesis was triggered by Ang2 administration. Lastly, to understand whether cytokine profiles that might affect angiocrine signalling are affected differentially in FAK-Y397F vs FAK-Y861F endothelial cells, I show that CCL1 and CCL2 are increased in FAK-Y397F but IL- 13, IL-1F3, CCL4, IL-1F1, CCL2 and others are increased in FAK-Y861F endothelial cells. Overall my data indicates that endothelial-specific FAK mutations on two phosphorylation sites has different effects on tumour angiogenesis, tumour growth, growth factor stimulated angiogenesis in vivo and ex-vivo and cytokine production.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that localises to focal adhesions. FAK is crucial for many cellular processes that are disturbed in malignancy, including proliferation, cell cycle, cell survival, adhesion, and migration. Mouse models have shown that FAK is involved in tumour formation and progression. Other studies demonstrated a functional correlation between FAK expression, tumour progression and malignancy in human cancer, making FAK a potentially important therapeutic target. Several FAK inhibitors have been developed most of which target the FAK kinase function. However, FAK may predominantly act as a scaffolding molecule rather than as a kinase, therefore, disruption of FAK’s interaction with protein binding partners could be a good strategy to inhibit some cancer processes. The identification and characterisation of novel FAK interactions may help to uncover important molecular mechanisms that, in turn, regulate key cellular processes involved in tumour formation and/or progression. Disruption of their function, or inhibition of their binding to FAK, will define their roles and identify whether they are good anti-cancer targets. In this thesis work, I set out to identify novel binding partners of FAK, and study the role of a sub-set of these in tumour biology by impairing them in squamous cell carcinoma cancer cells in vitro. To do this I employed protein microarray and phage display methodologies using FAKΔ375 and FAK-FERM recombinant proteins as bait, respectively. I identified a number of novel proteins that interact directly with FAK. Then I set out to characterise some of these proteins. The first of these, Axl, is a protein receptor tyrosine kinase that has previously been linked with tumour progression and metastasis in number of human cancers. I confirmed the interaction between FAK and Axl in SCC cells and showed that the FAK-Axl interaction is predominantly a scaffolding function of FAK, which seems to be unregulated, at least by any of the major phosphorylation events characterised for FAK. I also found that Axl controls cell spreading, cell polarisation and invasive migration in this cancer cell lines. The second protein I characterise is the autophagy protein Ambra1. I found that Ambra1 is required for selective targeting of active Src to the autophagy pathway – a process that SCC cancer cells use when they are under adhesion stress, such as when FAK is deleted. Thus, Axl and Ambra1 are potentially important proteins in SCC biology. They bind to FAK and function at cell adhesions to promote cancer-associated cellular processes. Analysis of FAK binding proteins may be a useful strategy to discover proteins that function in various aspects of cancer cell behaviour.

Understanding the perfusion of blood and drugs in tumours is fundamental to foreseeing the efficacy of treatment regimes and predicting tumour growth. In particular, the dependence of these processes on the tumour vascular structure is poorly established. The objective of this thesis is to derive effective equations describing blood, and drug perfusion in vascular tumours, and specifically to determine the dependence of these on the tumour vascular structure. This dependence occurs through the interaction between two different length scales - that which characterizes the structure of the vascular network, and that which characterizes the tumour as a whole. Our method throughout is to use homogenization as a tool to evaluate this interaction. In Chapter 1 we introduce the problem. In Chapter 2 we develop a theoretical model to describe fluid flow in solid tumours through both the vasculature and the interstitium, at a number of length scales. Ultimately we homogenize over a network of capillaries to form a coupled porous medium model in terms of a vascular density. Whereas in Chapter 2 it is necessary to specify the vascular structure to derive the effective equations, in Chapter 3 we employ asymptotic homogenization through multiple scales to derive the coupled equations for an arbitrary periodic vascular network. In Chapter 4, we extend this analysis to account for advective and diffusive transport of anticancer drugs delivered intravenously; we consider a range of reaction properties in the interstitium and boundary conditions on the vascular wall. The models derived in Chapters 2–4 could be applied to any drug type and treatment regime; to demonstrate their potential, we simulate the delivery of vinblastine in dorsal skinfold chambers in Chapter 5 and make quantitative predictions regarding the optimal treatment regime. In the final Chapter we summarize the main results and indicate directions for further work.