Epithelial ovarian cancer (EOC) is a common malignancy, usually diagnosed at an advanced stage, resulting in over 4,000 deaths each year in the UK. There is currently no National Screening Programme and no clinically approved diagnostic biomarker. Engrailed-2 (EN2) is a homeodomain-containing transcription factor, essential during embryological neural development, which is dysregulated in several cancer types. We have studied the biology and function of EN2, and have investigated the potential role as a biomarker in EOC tissue and bodily fluids. Elevated En2 mRNA expression levels and protein staining were observed in EOC tissue, but levels were much lower in benign tumours, and negligible in normal ovary. Therefore EN2 could be utilised as a diagnostic biomarker, particularly in high-grade serous tumours (HGSOC), however this would require an invasive biopsy specimen. Urine samples are more easily obtainable and EN2 protein was positive in 86% of newly diagnosed HGSOC, compared with female healthy controls, making it a potential non-invasive diagnostic biomarker and possible screening tool. In HGSOC patients who received neoadjuvant chemotherapy prior to surgery, elevated En2 mRNA levels and positive protein expression predicted a shorter progression-free survival and resistance to platinum chemotherapy. Such information could be used as a prognostic biomarker to guide post-operative treatment decisions. In normal adult Purkinje neurons EN2 is located in the nucleus at 33kDa, however EN2 was cytoplasmic and visualised at 43kDa or 50kDa in EOC, suggesting post-translational modification from the native state. En2 over-expression studies, including microarray analysis and cisplatin-challenge, suggested a role in the development of platinum-resistance. EN2 was also implicated in cell invasion and metastasis, via the process of epithelial-mesenchymal transition (EMT). EN2 appears to be actively involved in disease progression and treatment resistance in EOC, and further research determining its role in EMT, and the TGFβ receptor and Wnt signalling pathways is warranted. It also shows potential as a clinical biomarker, especially in urine where the non-invasive nature of the test and the potential for detection prior to any clinical signs or symptoms, make it a worthy candidate for ongoing research.

Acute myeloid leukaemia (AML) is a disorder characterised by the accumulation of blast cells or progenitors of one of several non-lymphoid haematopoietic cell lineages and is classified into two types: primary and secondary. HOX genes are over-expressed in both AML and other cancers. This over-expression is associated with an intermediate/unfavourable cytogenetic subset of AML. Although HOX over-expression is a common feature of AML, conventional knockout methods have failed to fully evaluate their functions due to their functional redundancy. We have applied an alternative approach by using a synthetic peptide called HXR9 to antagonise the interaction between HOX proteins and their cofactor PBX, which interacts with HOX proteins in groups 1-10. AML cell lines derived from different AML types express different subsets of HOX genes at different levels due to the heterogeneity of AML. It is showed for the first time that targeting the HOX-PBX interaction using HXR9 led to cell death of the tested AML cell lines. This cell death did not appear to be through apoptosis, as there were no signs of the caspase activation and nuclear fragmentation. Likewise, there was also no activation of key necrotic markers such as cypD and PARP1. Instead, cell death involved, at least in part, the expression of c-FOS and p21 in p53-independentmanner. In addition, HXR9 caused cell death in MEK/ERK and p38 independent pathways, but the JNK pathway exerted a resistant effect in K562 cells. It was found that inhibiting the Ca2+ downstream mediators CaM, PKC and HO-1 significantly sensitised tested AML cell lines to HXR9. Taken together, these findings indicate a novel cell death pathway in AML cells. In vivo modelling also showed that HXR9 could delay tumour growth in a mouse model of AML.

Within invasive breast cancers, lymphatic invasion is thought to be the first step tumour cells undertake when metastasizing through the lymphatic vasculature. The presence of lymphatic metastasis has been shown to stratify breast cancer phenotypes into distinct prognostic groups. The exact molecular mechanisms mediating tumour cell entry and dissemination within the lymphatic system remain unclear. We report the first identification of RORyt+-innate lymphoid tissue inducer (LTi) cells within the human breast cancer tumour microenvironment and the enrichment of lymphoid chemokines/chemokine receptor gene signature within an aggressive breast cancer subtype. The presence of these cells within the tumour microenvironment was shown to correlate with both an increased lymphatic vessel density (LVD) and tumour invasion into lymphatic vessels. We demonstrate the CCL21-dependent recruitment of LTi cells into breast tumours, the CXCL13-dependent interaction between the tumoural LTi and stromal cells and the downstream effect of the CXCL13 positive feedback loop in promoting lymphatic tumour cell motility via the RANK-RANKL axis. These data suggest a novel role for LTi cells in enhancing lymphatic invasion of tumour cells through modulation of the local lymphoid chemokine profile.

Background: Invasive fungal disease (IFD) is difficult to diagnose. For clinical trials the European Organisation for Research in Treatment of Cancer (EORTC) and the Mycology Study Group (MSG) criteria are useful but there are few data on their value in clinical practice. The aims of this study were to: (1) investigate the incidence and risk factors of IFD; (2) assess the utility of galactomannan (GM), β-D glucan (BDG), the UK consensus fungal PCR, and lateral flow device (LFD) assays together with the safety and feasibility of biopsy; (3) assess the role of cytokines in the diagnosis and prognosis of IFD; (4) establish the prevalence of baseline CT abnormalities, and assess diagnostic CT features and spectrum of radiological signs. Methods: Patients (N=203) were recruited prospectively and followed for a median (range) of 556 (12-730) days after chemotherapy or haematopoietic stem cell transplantation. Chest CT, Karnofsky score (KS), serum GM, and cytokine profiles were performed at baseline; during admission twice-weekly GM assays were performed on all patients. BDG, serum and whole blood consensus PCR, and LFD assays were performed on a selection of samples from different IFD categories. Neutropenic sepsis refractory to antimicrobials for ≥4 days triggered diagnostic CT and biopsy where feasible. All patients were on antifungal prophylaxis from admission. The revised EORTC/MSG criteria were used to diagnose IFD. Results: The cumulative incidence of proven/probable IFD at 6, 12, and 24 months was 16, 19, and 21%, respectively. Using GM or BDG alone (plus host and clinical evidence) the apparent incidence of proven/probable IFD was 11 and 16% respectively. The median time from index treatment to onset of IFD was 142, 17, and 41 days for proven mould, proven candida and probable IFD, respectively (P < 0.001). The 2-year overall survival (95% CI) were 45 (29-61)% for proven/probable IFD vs. 87 (77-97)% for no evidence of IFD (P < 0.001). Baseline CT abnormalities were found in 76 (38%) patients, 19 of which were EORTC signs. Risk factors for IFD on multivariate Cox regression were: EORTC CT signs (Hazard ration [HR] 4.3; 95% CI 1.9-9.8; P<0.001), IL-2R >834 pg/ml (HR 2.3; 95% CI 1.1-5.1; P=0.037), MCP-1 >841 pg/ml (HR 2.7; 95% CI 1.2-6.1; P=0.016), and KS <90 (HR 2.1; 95% CI 1.1-4.2; P=0.034) at baseline as well as monocytopenia >10 days (HR 2.6; 95% CI 1.3-5.4; P=0.009) and bacteraemia (HR 2.5; 95% CI 1.2-5.0; P=0.013). The sensitivity, specificity, PPV, and NPV for GM was 54, 71, 82, and 39%, respectively; coresponding values for BDG were 79, 55, 83, and 49%. The proportion of cases identified by both tests was 38%. The sensitivity of biopsy was 35% and PCR and LFD were less sensitive but more specific than GM or BDG. Conclusions: A multi-faceted approach is required to improve the diagnostic accuracy of IFD. No single assay was able to detect all the cases but the combination of BDG and GM seem to offer the best biomarker combination. Baseline chest CT, KS, and cytokine profile as well as monocytopenia and bacteraemia are important risk factors. (ClinicalTrials.gov number NCT00816088).

The transcription factor Nrf2 protects against cellular stress by inducing cytoprotective proteins. Activation of Nrf2 protects against inflammation and oxidative damage in disease models in vitro and in vivo. Nrf2 activation may be a good therapeutic strategy in these diseases. Some dietary components activate Nrf2, which may be partially responsible for their beneficial effects in preventing disease. In this study a novel organosulfur compound from garlic, diallyl pentasulfide (DAPS), was investigated. DAPS strongly activated the Nrf2 pathway. Furthermore, it was a much more powerful activator of heme oxygenase-1 than any diallyl sulfides reported to date. Nrf2 is regulated by Keap1, which targets it for degradation. Disruption of the Nrf2/Keap1 interaction results in Nrf2 activation. In this study, a novel cell-penetrating peptide, based on the Keap1-binding site of Nrf2, disrupted the Nrf2/Keap1 interaction, and activated the Nrf2 pathway. Furthermore, it demonstrated anti-inflammatory activity, significantly inhibiting LPS-induced TNF expression in THP-1 monocytes, suggesting that the interaction is a valid therapeutic target in inflammation. In cancer, Nrf2 plays a dual role. Activation of Nrf2 protects cells from carcinogens. However, once a tumour has developed, Nrf2 can be hijacked by cancer cells to induce chemoresistance. This study examined the role of Nrf2 in malignant melanoma cells. Nrf2 was found to be overexpressed in 11 human melanoma cell lines in comparison with melanocytes. Chemoresistance to dacarbazine, doxorubicin and cisplatin correlated with Nrf2 expression, and Nrf2 siRNA increased the susceptibility of M202 and SK-MEL-5 cells to cisplatin, suggesting that Nrf2 plays a role in chemoresistance in melanoma. In conclusion, this study has identified novel activators of Nrf2, including a dietary compound and a cell penetrating peptide which inhibits inflammation in vitro. In addition, Nrf2 inhibition sensitises melanoma cells to chemotherapy. These results suggest that targeting Nrf2 is a viable strategy in both inflammation and cancer.

Wnts are secreted morphogens that play pivotal roles in embryonic development, stem cell biology and a number of disease states including cancer. Most Wnts signal through a pathway that results in the stabilisation of an intracellular signalling molecule called β-catenin. In melanoma cells, Wnt/β-catenin signalling has been implicated as a key regulator of cellular invasion and metastasis. Using both transient and stable enhancement of Wnt/β-catenin signalling, I have found that mutation–based dysregulation of PI3K signalling dictates the invasive capacity of melanoma cell lines in response to Wnt3a stimulation. I demonstrate by confocal imaging that WNT3A facilitates perinuclear localisation of mitochondria with higher levels of mitochondrial networking and they show significant changes in the proteins of mitochondrial dynamics. Observed changes in mitochondrial fusion and fission proteins including MFN1, MFN2, OPA1 and DNM1L suggest that activation of Wnt/β-catenin signalling can increase mitochondrial fusion and decrease mitochondrial fission in melanoma cells. Cellular metabolic analysis using the Seahorse Bioscience XFe96 Analyzer suggests that Wnt/β-catenin mediated mitochondrial fusion may cause a global down-regulation of cellular energy metabolism in melanoma cells. This is supported by biochemical analysis of citrate synthase and lactate dehydrogenase activity. Knockout of -catenin removes the mitochondrial fusion effect in these cells and reverses any Wnt driven reduction in migration and metabolism suggesting that -catenin is able to control mitochondrial function and dynamics. We show that -catenin binds to the mitochondrial regulatory protein PARK2 in melanoma cells and subsequently blocks the autophagy dependency of melanoma cells. In summary, we demonstrate that activation of Wnt/β-catenin signalling in melanoma cells can lead to reduced cellular metabolism coupled with highly altered mitochondrial dynamics. This novel finding, controlled by -catenin, has potentially wide implications for understanding how certain context-dependent effects of Wnt/β-catenin signalling may be secondary to the regulation of mitochondrial dynamics and global cellular metabolism.

The aetiology of colorectal cancer, the third most prevalent cancer in the world, is linked to several risk factors, including age, genetic factors and diet. Seaweed consumption has been negatively correlated with colorectal cancer risk within Asian populations, and the anti-cancer properties of certain seaweed species have also been demonstrated within in vitro and animal models. The focus of this study was to examine the anti-cancer potential of selected Irish seaweed species, namely Ascophyl/um nodosum, Laminaria digitata, Palmaria palmata and U/va intestinalis using in vitro cell models of colon cancer. In order to account for the compositional changes occurring during gastrointestinal digestion, seaweed samples were subjected to in vitro simulation of gastric and pancreatic digestion. The chemopreventive properties of crude and digested seaweed treatments were tested using cell models, representing the key stages of colorectal cancer initiation, promotion and invasion. Of the tested seaweed species A. nodosum had the highest polyphenol-content and antioxidant power, both reduced by> 60% after in vitro gastrointestinal digestion. Yet, digested A. nodosum extracts had a strong anti-genotoxic activity, connected to up-regulation of a detoxifying enzyme GSTK-1. All crude and digested treatments had anti-proliferative effects in all cell types, inducing a necroptotic, mitochondrial cell death pathway with the up-regulation of AIF, JNK, Sax and PTEN. Digested extracts simultaneously induced the inflammatory or stress-related PI3K pathway, inhibiting the expression of Caspase-B and p53. The invasion and migration of metastatic HT115 cells was inhibited by P. palmata treatments both pre- and postdigestion, the up-regulation of an anti-invasive tissue inhibitor of metalloproteinases TIMP-2 was observed.

Background: A diagnosis of breast cancer and its subsequent treatment has a profound bio-psychosocial impact. There is a gap in the literature examining the impact on husbands of women with breast cancer, specifically in the Middle East. Aim: To achieve an understanding of the impact of having a wife with breast cancer undergoing chemotherapy on men's quality of life. Methods: A mixed-methods, repeated-measure design was used to collect data from Saudi husbands of women diagnosed with breast cancer at the beginning and at the end of chemotherapy. Instruments used were the Caregiving Appraisal Scale, Brief Cope Scale, and the WHOQOL-BREF Scale. A transitional stress and coping model helped to further explore the husbands perceived impact of their wives' cancer and chemotherapy upon their quality of life and how they appraised and coped with this impact. Results: The husbands of women with breast cancer perceived poor quality of life at the beginning and at the end of chemotherapy. Although the husbands appraised substantial negative aspects of their caregiving across both time points, they also appraised positive aspects. Husbands were struggling to cope with their wives' situations and tended to use more problem-focused coping strategies rather than emotion-focused and dysfunctional coping strategies. They used specific techniques such as social support and religion to cope with their wives' diagnosis and chemotherapy. Qualitative findings highlighted the complex role of culture in the caregiving appraisal and coping of husbands of women with breast cancer in Saudi Arabia. Conclusion: The findings provide new knowledge about the challenges, difficulties, and unmet needs of men in this situation within the cultural context of Saudi society. Nurses need to develop interventions to enhance the positive appraisal and emotion-focused coping strategies among the husbands to decrease the impact of their wife's illness upon their quality of life. Understanding and enhancing husbands' ability to cope may improve their quality of life. In addition, including the wives in future research would explore how the couple cope together and achieve optimal quality of life.

Background: Human epidermal growth factor receptor-2 (HER2) expression predicts response to anti-HER2 therapy in breast and gastric cancer. HER2 status is assessed by tumour biopsy but this may not be representative of the larger tumour mass or other metastatic sites; risking misclassification and selection of suboptimal therapy. The G3 designed ankyrin repeat protein (DARPin) binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. This research aims to assess the pre-clinical efficacy and safety of G3 DARPin HER2 PET and SPECT imaging. Methods: Hexahistidine (His6), histidine-glutamate (HE)3 and untagged-G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with 125I or site-specifically with 111In. BALB/c mice were injected with radiolabelled-G3 DARPins and biodistribution was evaluated. The lead construct, (HE)3-G3 was radiolabelled with 111In, 125I or 68Ga for assessment in mice bearing HER2-positive human breast tumour (BT474) xenografts. Mice received (HE)3-G3 at 50-100 times the human equivalent dose to assess acute toxicity. Results: (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged-G3 in non-tumour bearing mice when radiolabelled with 125I or 111In. In mice bearing HER2-positive tumour xenografts, 111In-(HE)3-G3 was better maintained in tumours and cleared faster from serum than 125I-(HE)3-G3, achieving superior tumour-to-blood ratios of 343.7±161.3 vs. 22.0±11.3 at 24 h, respectively. On microSPECT/CT 111In and 125I labelled (HE)3-G3, imaged HER2-positive tumours at 4 h post-administration but 111In-(HE)3-G3 had less non-specific uptake. 68Ga-(HE)3-G3 can image HER2-positive tumours at up to 2 h post-administration by PET scanning. (HE)3-G3 DARPin was well tolerated by mice treated at 50-100 times the human equivalent dose over 24 h. Conclusion: Radiolabelled (HE)3-G3 is a versatile radioligand with potential to acquire whole-body HER2 scans. (HE)3-G3 will be assessed in a regulatory standard pre-clinical toxicity study, prior to embarking on a first in human trial.

This thesis addresses heterotypic physical interactions between cancer cells and cancerassociated fibroblasts (CAFs) with different experimental approaches including timelapse movies, microarray and mass spectrometry analysis. Low-light and confocal imaging demonstrated the existence of direct physical interactions between cancer cells and fibroblasts. By combining light and SEM microscopy with siRNA methods it was shown that cancer cell and fibroblast contacts undergo a maturation process starting from filopodia to stable cadherin based interaction sites in which a stable cytoskeleton arrangement in fibroblasts plays an essential role. ROCK kinases stabilise cancer cell fibroblast interactions by stabilising the localisation of beta and p120-catenins at the interaction site. In addition, Src has a destabilising role on cancer cell-fibroblast interactions, independent of its ability to antagonise ROCK. Further it could be shown by analysing gene expression data of cancer cell-fibroblast co-cultures that atypical cadherins, protocadherin-7 (PCDH7) and protocadherin-9 (PCDH9), are expressed by both cell types. Cell migration assays using imprinted fibronectin chips and 3D spheroid assays of A431 and VCAF co-cultures showed that a stable expression of PCDH7 and PCDH9 seem to be important for cancer cells to move efficiently in certain environments in which they need the connection to fibroblasts. To understand the downstream signalling consequences of direct cancer cell fibroblast interactions additional experimental approaches as mass spectrometry and microarray were performed. Mass spectrometry analysis demonstrated that ERK kinase activity is up regulated in fibroblasts upon direct interaction with cancer cells. In addition to information including pathways activation and cell adhesion molecules and the better understanding of the nature of direct cancer cell-fibroblasts interactions, we obtained data by gene expression analysis. These data revealed a link of direct cancer cellfibroblast interactions to strong activation of interferon-regulated genes in both cell types, which gives an interesting link of cancer and inflammation.