Over-expression of parathyroid hormone-related protein (PTHrP) is commonly described in a number of different forms of cancer and it has been suggested that this over-expression leads to tissue-specific metastasis whereby primary tumours have a propensity to metastasise to one particular organ e.g. breast tumours metastasise to bone whereas gastrointestinal tumours favour the liver. The aim of my PhD was to examine the role PTHrP plays in cancer cell adhesion to the extracellular matrix (ECM), to explore a mechanism of action and to elucidate any tissue-specific differences to explain the apparent partiality during metastasis. In order to do this a small interfering RNA was used to silence PTHrP gene expression and expression vectors containing cDNA for PTHrP were used to create several stable PTHrP over-expressing cell lines. Analysis of cell adhesion revealed that regulating PTHrP expression caused changes in adhesion to the ECM proteins collagen type I, fibronectin and laminin in breast cancer cell lines. However although cell adhesion of gastrointestinal cancer cell lines to collagen type I and fibronectin was similarly affected, adhesion to laminin was unchanged by variations in PTHrP expression. The cell adhesion molecules integrins were subsequently investigated for their role in PTHrP mediated cell adhesion. Integrins are heterodimers composed of an α and β subunit and to date 24 different subunits have been identified. Each unique combination of subunits results in a different ligand specificity, which includes collagens, fibronectin and laminin. Analysis of integrin gene and protein expression in over-expressing cell lines and in cells where PTHrP had been silenced it was possible to demonstrate a link between expression of PTHrP and a number of different integrin subunits. One of the more significant findings was the observation that the integrin subunit α6 changed in parallel with PTHrP in breast but not gastrointestinal cancer cell lines. As laminin is a ligand for this subunit these results correlate with the results previously described regarding this ECM protein. The effects of PTHrP appeared to be mediated at the transcriptional rather than the translational level so integrin transcriptional activity was investigated using a reporter vector containing the coding region for firefly luciferase. The integrin subunit α5 had shown a link to PTHrP expression in both breast and gastrointestinal cell lines so the promoter region for this subunit was inserted into the reporter vector, which was then transiently transfected into PTHrP over-expressing cell lines. Subsequent examination of luciferase activity revealed a significant increase in promoter activity compared with control thus implicating PTHrP as a key factor in integrin gene transcription. The results described here suggest that PTHrP increases cell adhesion by inducing integrin gene transcription and is able to regulate expression of different subunits in different tissues thereby encouraging tissue-specific metastasis.

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Aims: The p53 regulatory network is crucial in directing the suppression of cancer formation and mediating the response to commonly used cancer therapies. Functional genetic variants in the genes comprising this network could help identify individuals at greater risk for cancer and patients with poorer responses to therapies, but few such variants have been identified as yet. Methods: We first develop and apply three different screens that utilize known characteristics of functional single nucleotide polymorphisms (SNPs) in the p53 network to search for variants that associate with allelic differences in (i) recent natural selection, (ii) chemosensitivity profiles, and (iii) the gender- and age- dependent incidence of soft-tissue sarcoma. Secondly, we study and explore the functional mechanisms associated with the identified variants. Results: We identify SNPs in the PPP2R5E, CD44, YWHAQ and ESR1 genes that associate with allelic differences in the age of tumour diagnosis (up to 32.5 years, p=0.031), cancer risk (up to 8.1 odds ratio, p=0.004) and overall survival (up to 2.85 relative risk, p=0.011) in sarcomas, ovarian and pancreatic cancers, and exhibit allelic differences in the cellular responses to cytotoxic chemotherapeutic agents (up to 5.4-fold, p=5.6x10^-47). Lastly, we identify candidate causal SNPs in those genes and describe the regulatory mechanisms by which they might affect human cancer. Conclusions: Together, our work suggests that the inherited genetics of the p53 pathway have a great potential to further define populations in their abilities to react to stress, suppress tumor formation and respond to therapies.

The paralogous multi-functional adaptor proteins YAP and TAZ are nuclear effectors of the Hippo pathway, a central regulator of developmental organ size control, tissue homeostasis and tumour suppression. YAP/TAZ target the TEAD transcription factor family to promote cell survival and inhibit apoptosis. TEAD proteins contain a DNAbinding domain and a YAP/TAZ interaction domain. PCR analysis of medaka fish TEAD cDNA revealed the presence of alternative TEAD splice-forms with variations at the C-terminus of the DNA-binding domain. Structural analysis indicated the YAPbinding domain of TEAD proteins is folded and globular. NMR spectroscopy showed that the TEAD binding domain of YAP does not contain secondary structure. YAP and TAZ both contain WW domains, which are small protein-protein interaction modules. Two YAP isoforms are known, YAP1 and YAP2 that contain one and two WW domains, respectively. To date, only a single WW isoform of TAZ has been described. PCR analysis of medaka TAZ cDNA identified both single WW and tandem WW isoforms of TAZ. NMR spectroscopy was used to characterise structural, conformational, and peptide binding features of the tandem WW domains from YAP and TAZ. The YAP WW2 solution structure confirms that the domain has the canonical anti-parallel β-sheet WW fold. WW1 of YAP and both WW domains of TAZ undergo conformational exchange. The region linking the two WW domains is flexible and allows interaction of both WW domains with peptides containing single and dual PPxY binding motifs. In addition to YAP and TAZ, tandem WW domains are also present in the core and upstream Hippo pathway proteins Salvador and Kibra. Both proteins contain one atypical WW domain; the tandem WW domains of these two proteins are unstable. Understanding structure and function of Hippo pathway components could contribute to drug development and will also contribute to knowledge of protein folding and interactions.

Acidic pH is a common characteristic of human tumours. It has a significant impact on tumour progression and response to therapies. In this thesis, we utilise mathematical modelling to examine the role of acidosis in the interaction between normal and tumour cell populations. In the first section we investigate the cell–microenvironmental interactions that mediate somatic evolution of cancer cells. The model predicts that selective forces in premalignant lesions act to favour cells whose metabolism is best suited to respond to local changes in oxygen, glucose and pH levels. In particular the emergent cellular phenotype, displaying increased acid production and resistance to acid-induced toxicity, has a significant proliferative advantage because it will consistently acidify the local environment in a way that is toxic to its competitors but harmless to itself. In the second section we analyse the role of acidity in tumour growth. Both vascular and avascular tumour dynamics are investigated, and a number of different behaviours are observed. Whilst an avascular tumour always proceeds to a benign steady state, a vascular tumour may display either benign or invasive dynamics, depending on the value of a critical parameter. Extensions of the model show that cellular quiescence, or non-proliferation, may provide an explanation for experimentally observed cycles of acidity within tumour tissue. Analysis of both models allows assessment of novel therapies directed towards changing the level of acidity within the tumour. Finally we undertake a comparison between experimental tumour pH images and the models of acid dynamics set out in previous chapters. This analysis will allow us to assess and verify the previous modelling work, giving the mathematics a firm biological foundation. Moreover, it provides a methodology of calculating important diagnostic parameters from pH images.