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Proteolytic degradation of the extracellular matrix (ECM) by overexpressed endoproteases, notably the matrix metalloproteinase MMP-9 and legumain (human asparaginyl endopeptidase), contributes to the invasive and migratory phenotype of cancer cells and promotes metastases. These proteases represent valid biological targets in cancer for diagnostic and therapeutic purposes. In this research programme, novel MMP-substrate oligopeptide prodrugs of potent anticancer agents have been rationally designed for activation selectively in the tumour microenvironment. Prodrugs of experimental colchicine-based cytotoxic and vascular disrupting agents, experimental anthraquinone-based agents or clinically-active drugs, including epirubicin, have been synthesised by both solution- and solid-phase peptide methods, and characterised. Results of the preliminary cytotoxic and DNA-binding properties of examples of active agents are reported. Furthermore, the approach has been extended to legumain, which is overexpressed in colon, breast and ovarian cancers and is capable of extracellular proteolytic activity. A series of novel substrates of legumain has been designed and characterised by high resolution mass spectrometry. The new peptide substrates have been developed as fluorogenic molecular probes of legumain for diagnostic applications in the early detection of cancer. The novel substrates exploit the proteolytic activity of legumain to cleave uniquely at the C-terminus of asparagine residues. The new peptide substrates, exemplified by prototype fluorogenic probe TL11 (FAM-Pro-Ala-Asn-Leu-PEG-AQ) are efficient FRET substrates in which fluorescein-based (donor) fluorescence is fully quenched by an aminoanthraquinone residue (acceptor). Proof of principle has been demonstrated by use of recombinant human legumain which cleaves the substrate library efficiently as shown by fluorescence spectroscopic methods. The tetrapeptide sequence pro-X-asn↓-leu was shown to be sensitive when X= ala, ser or thr (wherein ↓indicates the legumain cleavage ‘hotspot'). The approach was also shown to be extendable to a therapeutic prodrug approach with the potential to selectively deliver potent agents to the tumour microenvironment.

Translocation of wild Asian elephants (Elephas maximus) is used extensively to mitigate human-elephant conflict (HEC) in Peninsular Malaysia since 1974. Very little is known about the fate of translocated elephants after relocation due to challenges in observing elephants in the dense rainforest. Advances in wildlife endocrinology suggest that faecal glucocorticoid metabolites (fGCM) can be used to study adrenal activity remotely, to assess the Hypothalamic-Pituitary-Adrenal (HPA) axis response towards stressors. The aim is to assess the impact of translocation on wild Asian elephants in Peninsular Malaysia using faecal endocrinology and GPS technology. The specific objectives are: (i) adapting hormone sampling methods for use under tropical field conditions, (ii) comparing fGCM concentrations between translocated and local resident elephants using enzyme immunoassay, and (iii) quantifying gastrointestinal parasite eggs and microflora ciliates in faecal samples to detect signs of immunosuppression. We found that Asian elephant’s fGCM (80 dungpiles, 685 subsamples) are stable up to eight hours in the field. From the monitoring of wild elephants at the release sites, between two months up to a year, translocated elephants (N=5) had lower fGCM concentrations in comparison to local resident elephants (N=4; Linear Mixed Models: t=-2.77, df=7.09, P=0.027). There were no differences in gastrointestinal parasite egg counts (P > 0.05) or microflora ciliate counts (P > 0.05) between translocated and local resident elephants. In conclusion, translocation does affect elephant physiology but this is in the opposite direction from that expected – a prolonged decrease rather than increase of adrenal activity. It is unknown if these conditions could cause immunosuppression, but it could adversely affect stress response and health of the elephant (e.g. adrenal insufficiency, chronic fatigue or Post-Traumatic Stress Disorder). When assessing HEC mitigation, conservation authorities and other stakeholders need to consider that translocation may not be the best solution for HEC, as it will have long-term consequences on elephants’ health.

ADAM15 is a multidomain multifunction transmembrane protein. It participates in protein ectodomain shedding via the metalloproteinase domain. ADAM15 also interacts with integrins via the disintegrin domain. ADAM15 mRNA is subject to complex processing, generating different isoforms as a result of alternative splicing. The splicing affects the intracellular domain (ICD) of the protein, generating distinct SH3 domain binding regions. The expression of specific splice forms correlates with breast cancer prognosis. We aimed to test if ADAM15 isoforms have distinct functions that may affect multiple aspects of cell behaviour in isoform-specific unique ways. We generated an isogenic panel expressing each ADAM15 isoform in MDA-MB-231 acells. Comparative characterisation of the panel demonstrated distinct differences between isoforms, such as catalytic function dependant or independent effects on proliferation rate, changes in cell size, isoform-specific reorganisation of the actin cytoskeleton. We discovered ADAM15 isoform-dependant upregulation of tight junction protein claudin1. Immunofluorescence analysis demonstrated co-localisation of ADAM15 with claudin1 and another tight junction protein ZO1 at cell-cell junctions. Further immunoprecipitation analysis showed potential complex formation with ADAM15 and ZO1. Pharmacological analysis showed claudin1 upregulation is mediated via PI3K/mTOR-pathway. Claudin1 upregulation depended on the metalloproteinase catalytic function of ADAM15, suggesting that ADAM15 isoforms have distinct substrates. Claudin1 was also found in the nucleus of MDA/ADAM15 A expressing cells where it could potentially function as a transcription factor. Focal adhesion (FA) turnover was influenced by ADAM15 isoform expression. In MDA/ADAM15 C expressing cells impairment of FA reassembling was observed, while ADAM15 D expression showed reduced FA disassembly. Comparison of spreading of ADAM15 isoform expressing cells on fibronectin (FN) and vitronectin (VN) revealed that most cell lines preferred FN over VN. Expression of ADAM15 E reduced this preference while ADAM15 A altered it in favour of VN over FN. Changes in subcellular localisation of β3, but not β1-integrin was observed.

The growth inhibitory actions of antihormones in the treatment of oestrogen receptor-positive (ER+) breast cancer are compromised by the development of resistance. There is emerging evidence that antihormones can rapidly induce expression of genes that enable cells to survive the initial impact of these agents and ultimately aid the acquisition of resistance. The aims of this thesis were to identify novel antihormone-induced pro-survival genes in a panel of ER+ breast cancer cell lines and to determine whether such genes contribute to the limited efficacy of antihormones during response and subsequently contribute to the emergence of resistant cell growth. Microarray analysis, together with a stringent filtering process, identified 14 pro-survival genes significantly induced by at least one antihormone treatment (10 day tamoxifen, fulvestrant or oestrogen deprivation) in ER+ MCF-7 breast cancer cells, with increased expression maintained into cell models of antihormone-resistance. Of these 15 genes, 5 (GABBR2, CLU, CTNND2, BCL3 and TSC22D3) were significantly induced by all antihormone treatments. PCR and/or Western blotting demonstrated antihormone-induced expression of these 5 genes in T47D (ER+/HER2-), BT474 and MDA-MB-361 (ER+/HER2+) cell lines. The role of BCL3 and CLU during antihormone response and resistance were next investigated. siRNA-mediated BCL3 knockdown had no effect on cell survival but reduced proliferation of tamoxifen-resistant (TAMR) and oestrogen deprivationresistant (XR) cells. Immunoprecipitation and immunofluorescence studies revealed nuclear localisation and direct association of BCL3 and p50 in TAMR and XR cells. However, during response, BCL3 was located in the nucleus and p50 in the cytoplasm. In contrast, siRNA-mediated CLU knockdown reduced proliferation of fulvestrant-treated MCF-7 cells but was without effect on the growth of resistant cells. To conclude,this thesis has identified one antihormone-induced gene (CLU), which appears to limit response, and a second (BCL3), which appears to promote the growth of antihormone-resistant cells, potentially via activation of NFκB-mediated gene transcription.

Intensity-modulated radiotherapy (IMRT) for gynaecological malignancies aims to reduce toxicity and improve tumour control. However, there are several barriers to its uptake in clinical practice. Amongst these are that of pelvic organ motion, whereby due to motion of the target organs on treatment there is a risk of geographical miss with IMRT. Secondly, although new IMRT techniques may improve bowel toxicity there is limited knowledge about dose-volume constraints for bowel, making it difficult to assess whether new techniques are likely to translate into clinical improvements. The purpose of this thesis is to address these problems. Methods Dose-volume constraints for late bowel toxicity are investigated initially through systematic review, followed by a dose-volume study based on toxicity data from pelvic radiotherapy patients. Pelvic organ motion is assessed in a systematic review examining organ motion patterns and potential strategies to account for this. Population-based and adaptive margin strategies are investigated in modelling studies for both definitive cervical cancer patients and post-hysterectomy patients. Results Initial systematic review of the literature, followed by the analysis of the toxicity and dosevolume data of 203 pelvic radiotherapy patients highlighted anal canal, bowel loops, bowel bag, sigmoid and large bowel as important organs at risk (OARs) for bowel toxicity. Dosevolume constraints were derived for these organs. Pelvic organ motion was found to be a significant problem for gynaecological IMRT. Adaptive margin strategies, such as plan-of-the-day, were demonstrated to achieve both CTV coverage whilst reducing dose to the OARs compared to standard margins and population-based margins. Conclusions Dose-volume constraints derived for late bowel toxicity, if validated with independent data, may be used to reduce bowel toxicity in future patients, and as a benchmark to assess the efficacy of new IMRT techniques. Adaptive strategies for gynaecological cancers appear a promising solution for organ motion management.

The Wnt, PI3-Kinase (PI3K) and MAP-Kinase (MAPK) cell signalling pathways play important roles in human prostate cancer (PCa). In this thesis, analysis of a human PCa tissue micro-array (TMA) constructed by the Welsh Cancer Bank demonstrated upregulation of markers associated with these pathways in PCa. There was also greater expression of these markers in high-risk tumours with some being predictive of biochemical recurrence following surgery. Furthermore, there is evidence to support cross talk between these pathways allowing clustering into low- and high-risk samples based on expression profiles. Targeted next generation sequencing (NGS) also demonstrated recurrent mutations of genes associated with these pathways in PCa. Conditional transgenic mouse models were employed to explore the complex communication between these pathways. The loss of Pten was incorporated as a means of activating the PI3K pathway, and mutated β-catenin and K-Ras as means of aberrant Wnt and MAPK signalling. This study provides the first evidence of crosstalk and cooperation between these pathways, resulting in a significant effect on prostate tumourigenesis. Mice with loss of Pten in addition to activated mutations of β-catenin and K-Ras (Triple mutants) have significant upregulation of all three pathways resulting in a shorter survival compared to single and double mutants. The feasibility of these models allows further specific gene profiles to be induced in the mouse, providing a platform for pre-clinical testing of novel therapeutic agents. The effect of deregulation of these pathways on the cancer stem cell (CSC) population was explored using fluorescence-activated cell sorting (FACS) and organoid culture. Compound mutant (doubles and triple) tumours have a greater number of CSC or enriched cells compared to single mutant or wildtype (WT) mice. Compound mutant tumours had greater organoid forming efficiency than single mutants however this was significantly inferior to WT cells. Overcoming the difficulty experienced in cultivating tumour organoids will help manufacture targeted drugs with the aim of forming a cryopreserved organoid library to facilitate precision medicine.

Both mouse double minute (MDM2) and prostate-specific membrane antigen (PSMA) are known to be associated with the progressive properties of cancer. Moreover, overexpression of both molecules has been implicated in an increase in the proliferation, migration and invasion of tumour cells. MDM2 is a negative regulator of tumour suppressor of p53 but also is known to play multiple p53-independent roles in many cancer types. PSMA was originally thought to be solely expressed in prostate tissues and overexpression prostatic cancers; however, recently its expression was reported in various other solid tumours, including those of the breast. Our work showed a possible link between these proteins following knockdown of each molecule in breast cancer cell lines, ZR-75.1 and MDA-MB-231, with targeted siRNA molecules. A decrease of MDM2 and PSMA led to a decrease in the proliferative, adhesive, migratory and invasive capacities of the cell lines. Additionally, knockdown of MDM2 and PSMA led to similar changes in secretion of matrix metalloproteinases (MMPs), with decreases in MMP2 and MMP8 being seen from both breast cell lines investigated. It was then seen that a link between the two protein could be mediated through the phosphorylation status of serine 473 on protein kinase B (AKT). PSMA knockdown in both breast cancer cell lines led to a decrease of AKT phosphorylation and thus a decrease in MDM2 serine 188. Additionally, it was found that MDM2 siRNA leads to an increase in c-JUN serine 63 phosphorylation, and that PSMA siRNA can lead to an increase at the same site, depending on the cell line. These results indicate that MDM2, AKT and PSMA may represent a new pathway which could be targeted for therapy for breast tumours and perhaps other types of cancer.

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