Pancreatic cancer is often referred to as the “silent killer“ due to the asymptomatic nature of the disease in the early stages and the extremely poor prognosis overall. The average one-year survival rate for PDAC patients is 24% (American Cancer Society, facts and figures, 2010), decreasing to 5%-6% over 5 years (WHO report, Pancreatic cancer, 2010). Only 20% of patients are suitable for surgical resection at the time of diagnosis and treatment options available to PDAC patients have not improved significantly over the past few decades. Thus novel therapeutic approaches are essential to treat this disease. Our experimental, clinical and pre-clinical data suggest integrin αvβ6 may be a suitable target. Bioinformatics studies using the Pancreatic Expression Database revealed that the β6 gene (ITGB6) was highly up regulated in pancreatic ductal carcinoma (PDAC) compared with normal pancreas. Further analysis carried out showed that there was a significant correlation between ITGB6 expression at the mRNA level and survival in a cohort of 292 PDAC patients. Immunohistochemistry analysis on two separate patient cohorts (n=118 and n=147) showed that normal pancreas lacked αvβ6 expression whereas 91% of PDAC tissues expressed αvβ6 at the protein level. There was no significant correlation between αvβ6 expression and survival at the protein level in both cohorts of patients tested. Flow cytometry and Western blotting analyses on a panel of PDAC cell lines confirmed expression of αvβ6 in PDAC cell lines. This study investigated the functional role of αvβ6 in PDAC cell lines. Antibody mediated function blockade of αvβ6 significantly inhibited proliferation in a dose dependent manner, specifically in αvβ6 positive PDAC cell lines. A significant reduction in migration and invasion was also observed in a panel of αvβ6 positive PDAC cell lines when treated with an αvβ6 function-blocking antibody. αvβ6 targeted antibody mediated therapy in combination with gemcitabine significantly inhibited tumour growth in a physiologically relevant pre-clinical subcutaneous xenograft model of PDAC. These data reaffirms that αvβ6 is a potential novel therapeutic target and an αvβ6 specific function-blocking antibody can be used as a novel agent to treat pancreatic adenocarcinoma patients.

Activating mutations of the kras proto-oncogene are found in more than 90% of human pancreatic ductal adenocarcinoma (PDAC) and can result in increased activity of the NF-κB pathway, leading to constitutive production of proinflammatory cytokines such as TNF-α. Pancreatic cancer progression occurs through a series of pre-invasive lesions, pancreatic intraepithelial neoplasias (PanIN lesions), which progress into invasive carcinoma. The aim of this thesis is to understand the autocrine role of TNF-α produced by premalignant epithelial cells in pancreatic tumour progression. This cytokine has already been shown to be involved in the progression of cancer. The major hypothesis therefore tested was that TNF-α secreted by pre-malignant epithelial cells promotes the early stages of pancreatic carcinogenesis by sustaining an inflamed microenvironment. In the spontaneous kras+/LSL-G12D; pdx1-cre mouse model of pancreatic cancer, concomitant genetic deletion of the TNF-α/IKK2 pathway substantially delayed pancreatic cancer progression and resulted in downregulation of the classical Notch target genes hes1 and hey1. Cell lines from the different PanIN bearing mice were established and used to dissect the cooperation between TNF-α/IKK2 and Notch signalling during PanIN progression. Optimal expression of Notch target genes was induced upon TNF-α stimulation of the canonical NF-κB signalling pathway, in cooperation with basal Notch signals. Mechanistically, TNF-α stimulation resulted in phosphorylation of histone H3 at the hes1 promoter and this signal was lost upon ikk2 genetic deletion. HES1 suppressed the expression of pparg, which encodes for the anti-inflammatory nuclear receptor PPAR-γ. Thus, crosstalk between TNF-α/IKK2 and Notch sustained an intrinsic inflammatory profile of the transformed cells. The treatment of PanIN bearing mice with rosiglitazone, a PPAR-γ agonist, also delayed PanIN progression. A malignant cell-autonomous, low-grade inflammatory process was shown to operate from the very early stages of kras-driven pancreatic carcinogenesis, which may cooperate with the Notch signalling pathway to promote pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by an intense desmoplastic stromal response that can comprise 60 to 80% of tumour volume and has been implicated to be a factor in promoting tumour invasiveness and the poor prognosis associated with this cancer type. It is now well established that pancreatic stellate cells, which are vitamin A storing cells found in the periacinar spaces of the stroma in the normal gland, are primarily responsible for this desmoplastic reaction. Studying the interaction between stellate cells and cancer cells could provide for a better understanding of the disease process. During the evolution of PDAC, the stromal proportion increases from 4% in the normal gland to up to 80%. We hypothesised that there is an optimal proportion of stellate cells and cancer cells that modulates tumour behaviour and we attempted to dissect out this probable ‘tipping point’ for stromal composition upon cancer cell behaviour using a well-established in vitro organotypic culture model of pancreatic cancer. The cancer cell-stromal cell interaction led to extra-cellular matrix contraction and stiffening; and an increase in cancer cell number. The stromal stellate cells conferred a pro-survival and pro-invasive effect on cancer cells which was most pronounced at a stellate cell proportion of 0.66-0.83. The expression of key molecules involved in EMT and metastasis such as E-Cadherin and β-catenin showed a reduction and this was found to be most significant again at a stellate cell proportion of 0.66-0.83. Stellate cells altered the genetic profile of cancer cells leading to differential expression of genes involved in key cellular pathways such as cell-cycle and proliferation, cell movement and death, cell-cell signalling, and inflammatory response. qRT-PCR confirmed the differential expression of the top differentially expressed genes and protein validation by immunofluorescence staining using PIGR as a candidate molecule confirmed the experimental findings in human PDAC specimens. This study demonstrates that the progressive accumulation of desmoplastic stromal cells has a tumour progressive (pro-survival, pro-invasive) effect on cancer cells in addition to stiffening (contraction) of the extracellular matrix (maximum effect when the stromal cell proportion is 60-80%). This is mediated through a number of signalling cascades and molecular targets. Dampening this tumour-promoting interaction between cancer and stromal cells by ‘multi-targeting’ agents may allow traditional chemo- and/or radiotherapy to be effective.