Malignant Mesothelioma (MM) is an aggressive tumour of the lung pleura with very poor patient prognosis. MM is unresponsive to all treatment regimes and over the next 30 years an estimated 100,000 fatalities will occur from this disease in Western Europe alone. MM is an extremely fibrous tumour containing abundant amounts of extracellular matrix, including collagen. Cell-matrix interactions are important for tumour progression and MM cells synthesise collagen as well as transforming growth factor-beta (TGF-beta), a key regulator of collagen production. Thiaproline, a proline analogue, was used to inhibit collagen production in MM cells in vitro and in vivo to test the hypothesis that collagen is important for MM growth. Murine MM cells (AC29) were incubated with increasing concentrations of thiaproline with and without TGF-beta1 (1ng/ml) and incorporation of tritiated thymidine and hydroxyproline levels measured as an index of cell proliferation and collagen production (nM hydroxyproline/106 cells) respectively. The effect of thiaproline on tumour growth (median weight, mg [range]) was determined in syngeneic mice subcutaneously injected in the flank with 106 MM cells. In vitro, 10mM thiaproline significantly reduced cell proliferation by over 65% (control 27400 3200; thiaproline 8950 1000 dpm/well, p 0.001) and basal and TGF-beta1-induced collagen production by over 50% (control 1.6 0.1; thiaproline 0.7 0.1, p 0.005) and 60% (TGF-beta1 3.8 0.2; TGF-beta1+thiaproline 1.3 0.1, p 0.005) respectively. At 10 days after injection of cells, 100mg/kg/day thiaproline reduced median tumour weight by over 80% (control 58 [30-105]; thiaproline 10.5 [5-12], p 0.01) but no significant difference was seen at 18 days. To summarise, thiaproline inhibited MM cell proliferation, collagen production and delayed tumour growth, suggesting an important role for collagen in MM growth. MM also secretes TGF-beta, which regulates cell growth and collagen production. MM cells produce 30 - 70 times more TGF-beta than untransformed normal mesothelial cells. To investigate the role of specific TGF-beta isoforms in MM cell proliferation and collagen production and to determine the relative importance in tumour growth, selective neutralising antibodies to TGF-beta1 and TGF-beta2 were used in conjunction with a pan-specific TGF-beta antibody which neutralised all TGF-beta isoforms. Through the use of the neutralizing antibodies it was determined that the predominant isoform produced by AC29 cells into conditioned medium was TGF-beta2. TGF-beta and control antibodies had no effect on AC29 cell proliferation or collagen production in vitro. TGF-beta and control antibodies (5mg/kg) were injected intraperitoneally into the murine flank model of MM tumour growth at three-day intervals until the end of the experiment, when the tumours were resected and weighed. TGF-beta2 antibody administered in vivo significantly decreased median tumour weight compared with PBS and control antibody (PBS control 67.5 [27-114], control antibody 57 [35-193], TGF-beta2 antibody 27 [5-51], p 0.001 for both controls). Pan-specific antibody reduced median tumour weight compared to PBS control (PBS control 46.5 [25-109], control antibody 36 [19-107], pan-specific TGF-beta antibody 30 [14-66], p 0.05 vs. PBS control). Antibodies against TGF-beta1 had no effect on tumour growth. Collagen analysis of the tumours revealed a significantly lower concentration of collagen in the pan-specific antibody treated tumours (PBS control 9.11 0.76, pan-specific TGF-beta antibody 4.03 0.68 nM hydroxyproline / mg tumour, p 0.0001). Collectively, this thesis has clearly demonstrated that TGF-beta-induced collagen production may be an important aspect of MM tumour growth, and inhibition of either collagen production or TGF-beta activity can delay MM tumour growth with a resultant decrease in tumour collagen content. A dual approach targeting both collagen and TGF-beta production may be of benefit in the treatment of this disease in which the current therapies are inadequate.

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Lung cancer is the most lethal malignancy worldwide and late diagnosis is a significant factor contributing this. The Liverpool Lung Project (LLP) aims to reduce lung cancer mortality through the development of a molecular-epidemiological risk assessment model which will facilitate early detection of lung cancer and thus early intervention. LLP encompasses retrospective and prospective sub-studies. DNA methvlation is an epigenetic modification with key role in gene transcriptional control, embryonic development, imprinting and cancer. A large number of studies in lung cancer have revealed abnormal DNA methylation patterns involving a variety of genes. The aim of this study was to construct and evaluate a panel of DNA-methylation biomarkers which can be applied to bronchial washings (BWs) material and assist in diagnosis of lung cancer. The discovery and validation process followed the guidelines set by the NCI- Early Detection Research Network (EDRN) and the CR-UK diagnostic biomarker roadmap. Specific objectives included (a) the discovery of promoter targets with high frequency of hypermethylation in primary lung cancer, (b) the development of a highly sensitive and specific DNA methylation assay fitting to clinical standards and (c) the validation of these targets in BWs utilising a longitudinal retrospective case-control design. Targets from previous high throughput approaches were validated in an independent set of 48 non-small cell lung cancer samples and paired normal tissues using Pyrosequencing methylation analysis (PMA). PMA confirmed significant hypermethylation in the primary NSCLC tissue for the following promoters: RASSF1, p16, WT1, CYGB, RARB, CDH13, DAPK, p73, TMEFF2, and TERT. In addition, immunohistochemical staining for p16 and WT1 was performed in a 20 non-small cell lung cancer samples. Quantitative methylation-specific PCR (qMSP) assays were subsequently developed and tested for reliability and robustness for these ten candidates. These assays were used to screen 655 BWs from the Liverpool Lung Project (LLP) subjects divided into a training (194 cases and 214 Controls) and validation (139 cases and 109 controls) sets. Multifactor Dimensionality Reduction (MDR) was used to select the best subset of the markers with good discrimination. Analysis in the training BWs set demonstrated significant differences in the detected hypermethylation frequency in cases over controls for RASSF1 p16, WT1, CYGB, RARB and TERT. The diagnostic efficiency of this panel was evaluated in the independent validation set. A logit method was used to obtain the sensitivity and specificity of the six markers. LogicF analysis demonstrated that the top five predictors were WT1, cytology, RASSF1, TERT and p16. The overall performance the latter panel demonstrated no diagnostic bias in different groups of gender, age or smoking status. While cytology alone provides a 49.5% sensitivity, 99.5% specificity, the addition of the four methylation markers provided 76.2% sensitivity, 92.3% specificity (AUC=0.89). Although the diagnostic efficiency of this panel must be improved by incorporating additional promoters, our findings clearly demonstrate the impact of DNA methylation- based assays in the diagnosis of cytologically occult lung neoplasms.

Lung cancer is the most common cause of cancer deaths worldwide. Major advances in the understanding of the molecular pathogenesis of lung cancer may lead to new strategies for early detection, diagnosis, staging and therapy that hold promise for improving lung cancer outcomes. MicroRNAs (miRNAs) are 19 to 25 nucleotide-long non-coding RNAs that regulate gene expression by binding complementary sequences of target mRNAs. Many studies have reported that alterations in miRNA expression are involved in the development of several human tumours. MicroRNAs could also constitute a new class of blood-based biomarkers useful for cancer detection and prognosis definition. Moreover, miRNAs released in the bloodstream can also have a role in targeting tumour cells promoting growth and invasiveness. The intent of this project is to characterize tissue and plasmatic miRNA expression of early lung cancer patients, and to assess their functional role in lung carcinogenesis. Low Dose Computed Tomography (LDCT) detected lung tumours and paired normal lung tissues were first profiled for miRNAs expression. In particular we found miRNAs associated with clinic-pathological characteristics in both tumour and normal lung tissue. Plasma samples from the same patients were compared with those of disease-free individuals. Specific diagnostic and prognostic miRNA signatures, composed by 24 miRNAs, were defined in plasma samples of lung cancer patients collected before and at the time of disease detection. To understand if circulating miRNAs have a functional role in tumour development, their activity was assessed in lung tumour and normal bronchial epithelial cells. Preliminary results suggested that miR-486~5p and miR-660 act as tumour suppressor miRNAs, while miR-197 and miR-28-3p behave as oncogenic miRNAs. Overall, miRNA profiling and related functional studies would provide greater knowledge of the regulatory events underlying the development and progression of lung cancer and lead to the identification of new therapeutic targets for this malignancy.

The evasion of apoptosis is a common feature of various malignancies and often depends on the overexpression of Bcl-2 family antiapoptotic proteins. Resistance to chemotherapy and radiotherapy is promoted because these tumour cells have a higher threshold for apoptosis-promoting signals than their normal cell counterparts. This has lead to the development of BH3 mimetic compounds that target the survival proteins of the Bcl-2 family.

This study aims to identify and explore the experiences of people living with advanced lung cancer and their palliative care professionals across one Health and Social Care Trust in Northern Ireland. The objective was to obtain a greater understanding of decision-making in palliative and end of life care by professionals and patients. The study sample consisted of two purposively selected groups: professionals (n;10) working in one community specialist palliative care team; and patients (n;12) diagnosed with primary lung cancer, being cared for by the community palliative care team. Qualitative data were collected during two focus group meetings with professionals (each lasting 60 minutes); and two individual interviews with each of the 12 patients (mean duration 48 minutes). Interview schedules were informed by the Llewellyn Thomas (1995) framework. They explored practical and ethical challenges regarding decision-making; socio demographic and socio political factors influencing decision making; and how to promote high quality palliative and end of life care. Patient data were analysed using Interpretative Phenomenological Analysis (IPA) (Smith et aI., 2009). Analysis of patient interviews revealed three cross-cutting themes: the preciousness of time, the importance of communication and the maintenance of hope. Emergent themes from the patient findings are presented in the framework of narrative chronology, i.e. beginnings, middles and endings/futures, so as to mirror the patient's journey through illness. Frank's (1995) narrative typology of restitution, chaos .and quest were used to explore how patients told their stories. The focus group data were also analysed using IPA (Smith et aI., 2009). Super-ordinate themes identified were: time, communication and decision-making. Findings from the study provide insights into the patients' experiences of lung cancer, what influenced decision-making throughout the illness trajectory. It is hoped that messages from this research may help to inform end of life care policy initiatives and professional practice within palliative care.

To improve and provide novel insights for the treatment of Non-Small Cell Lung Cancer (NSCLC) and rectal adenocarcinoma, an innovative approach using gene expression profiling has been used to identify key differences in the transcriptome between clinically responding and non-responding patients. Oligonucleotide microarrays were used to profile the expression of 22,283 transcripts in the NSCLC study (Aberdeen Microarray in Lung Cancer Study-1, AMLUCS1) and 54,675 transcripts in the rectal adenocarcinoma study (Aberdeen Microarray in Rectal Cancer Study-1, AMRECS1), and both studies represent the first of their type performed. In NSCLC, with bioinformatics analysis a set of 17 genes was identified whose expression was highly correlated with clinical response to platinum based chemotherapy (PtBC) and the ability of this ‘predictive gene set’ to correctly classify an independent set of patients for response was demonstrated.  Expression of the cross class lysosomal protease inhibitor Serpin B3 showed a highly significant  correlation with extent of clinical response (R= -0.978, p<0.0001) and was identified as an outlier (50 fold increase in non-responding versus responding NSCLCs).  Previous cell line studies had identified serpin B3 as inhibitor of cell death and a subsequent analysis of 1007 genes involved in the regulation of cell death identified other lysosomal protease inhibitors cystatin C and cystatin SN and provided evidence to support the common inhibition of classical apoptosis in NSCLC and hence an ‘apoptosis resistant phenotype’.  In an independent set of NSCLC patients it was demonstrated that protein expression of serpin B3 was significantly correlated with response and when combined with protein expression of cystatin C and its target protease cathespsin B was able to independently predict response to PtBC (OR 17.8, 95% C.I. 2.0-162.4, p=0.010).  These results identify the lysosomal protease inhibitors and the lysosomal cell death pathway as important determinants of clinical response in NSCLC.  These molecules and this pathway has not been hitherto suspected or investigated as a therapeutic target and in further investigations we demonstrated the therapy independent prognostic impact of serpin B3 protein expression in NSCLC, thereby implicating serpin B3 in disease pathogenesis. Contrasting prognostic impacts were found in lung adenocarcinomas (HR for death at 5 years = 0.43 (0.18-0.93, p=0.04)) and squamous cell carcinomas (HR for death at 5 years = 2.09 (1.03-4.72-0.93, p=0.03)).  Further investigations suggested that this may relate to an inhibitory role for serpin B3 in invasion and metastasis in squamous cell carcinoma but not adenocarcinoma.

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Lung cancer is the commonest cancer killer worldwide. This is mainly due to the rapid development of drug resistance and early metastatic dissemination of the disease. SRC family kinases (SFKs) are frequently over-expressed in various cancers and have been implicated in tumorigenesis through their ability to promote cancer cell proliferation, survival and invasiveness. Therefore, we sought to evaluate the involvement of SFKs in lung cancer biology and assess the possible therapeutic benefits of their inhibition, either alone or in combination with additional treatments. We demonstrate that SRC family kinases are over-expressed and activated in-vitro in a panel of lung cancer cell lines as compared to immortalised normal lung epithelial cells. SRC, FYN and LYN are expressed in a high proportion of lung cancer but not normal lung tissue sections. Furthermore, enhanced expression of LYN correlates with poor patients’ survival. Dasatinib is a novel SRC/ABL inhibitor which effectively blocks SFKs activity at nanomolar concentrations. Dasatinib reduces cell numbers in 10 out of 11 NSCLC cell lines tested, which correlates with a strong inhibition of DNA synthesis and cell proliferation, while apoptosis is moderately enhanced only in a few cell lines. Interestingly, dasatinib potently induces autophagy in all NSCLC cell lines tested. This appears to be a pro-survival mechanism as autophagy inhibition using chemical compounds or siRNA-mediated depletion of ATG5 sensitises NSCLC cells to dasatinib through enhanced apoptosis. Lastly, our results indicate that SFKs have both overlapping and isoform-specific functions in NSCLC cell biology, as demonstrated by the effects of siRNA-mediated knockdown of SRC, YES, FYN or LYN. Our results suggest that inhibition of SRC family kinases alone or in combination with autophagy inhibitors may be a beneficial therapeutic strategy in the management of lung cancer patients.