Pancreatic cancer (PC) is known to be highly resistant to chemotherapy. Transporters, which regulate the influx and efflux of substrates across the plasma membrane, may play a role in PC drug resistance. ABC transporters are a large family of transmembrane proteins with diverse physiological functions, several of which play major roles in cancer drug resistance. Given that 90% of PC express a mutant K-ras oncogene and that PC are highly hypoxic, I postulated that constitutive K-ras activation and/or hypoxia may correlate with ABC transporter expression, which in turn may promote drug resistance in PC. Using normal and PC cell lines either overexpressing mutant K-ras or subjected to hypoxic treatment, mRNA expression was profiled for 48 ABC transporters. My findings indicate that expression of mutant K-ras and hypoxic treatment, as well as long-term exposure to chemotherapy, may contribute to the development of drug resistance in PC cells in part by inducing the expression of ABC transporters. Similar to ABC transporters, I investigated whether amino acid transporters would mediate drug resistance in PC. The Xc⁻ amino acid transporter (Xc⁻) mediates cellular uptake of cystine for the biosynthesis of glutathione, a major detoxifying agent. Because the Xc⁻ has been regulates the growth of various cancer cell types, and Xc⁻ is expressed in the pancreas, I postulated that the Xc⁻ may be involved in growth and drug resistance in PC. The Xc⁻ transporter is differentially expressed in normal pancreatic tissues and is overexpressed in PC in vivo. Using PC cell lines, I found that cystine uptake via the Xc⁻ was required for growth and survival in response to oxidative stress, and that expression of the Xc⁻ correlated with gemcitabine resistance. Accordingly, inhibition of Xc⁻ expression via siRNA reduced PC cell proliferation and restored sensitivity to gemcitabine. I also identified the anti-inflammatory drug sulfasalazine as a mixed inhibitor of the Xc⁻, which acts to inhibit cell proliferation via reducing Xc⁻ activity and not by reducing NFKB activity. My findings thus indicate that the Xc⁻ plays a role in PC growth in partby contributing to glutathione synthesis to promote PC cell proliferation, survival, and drug resistance. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

In this study, we searched for significant hypo and hyper methylation CpG (5'-C-phosphate-G-3') probes from The Cancer Genome Atlas (TCGA) datasets. First, the relationship between hypo and hypermethylation pattern in significantly expressed genes associated in pancreatic ductal adenocarcinoma (PDAC) was analyzed using computational methodologies in R package. This was done by combining DNA methylation (DM) and gene expression (GE) information, and their corresponding metadata (i.e., clinical data and molecular subtypes) and saved as R files. Next, examination of differentially methylated CpG sites (DMCs) between two groups (normal vs tumor) was identified gene sets. From this analysis, we found nine (09) overexpressed hypomethylated and six (06) under expressed hypermethylated genes near significant CpG probes. Results from this work will shed light on the relationship between CpG methylation and gene expression associated with PDAC.

Pancreatic cancer (PDAC) affects 4400 Canadians each year and with 5year survival rates <8%, there is clearly an unmet need for new therapeutic approaches for treating this deadly disease. Herein I report the development of a surgical model of PDAC that recapitulates many of the hallmarks of human disease and has an immune infiltrate consisting of T cells and suppressive regulatory T cells and myeloid derived suppressor cells. This model allows the exploration of new therapeutics that can be used in combination with surgical resection of primary tumours. Furthermore, I propose that the use of neoadjuvant administration of a prime-boost oncolytic vaccine targeting a pancreatic tumour associated antigen (TAA) - mesothelin - could potentiate pancreatic tumour specific immune responses to improve patient prognosis. We demonstrate that immune tolerance to this self antigen can be broken by the complete depletion of circulating Tregs at the time of vaccination, which leads to the activation of a population of CD8+ T cells responsive to mesothelin. We demonstrate that these T cells respond to mesothelin expressing tumour cells ex vivo, and that CD8+ T cells are recruited to the site of tumour challenge. However, despite the generated CD8+ T cell response, oncolytic vaccine strategies targeting mesothelin provide no protection against Pan02 tumours, or against other mesothelin expressing murine tumour lines. I demonstrate that this is not through common tumour escape mechanisms, nor through the upregulation of suppressive immune populations. Any efficacy observed was found to be provided solely by depletion of Tregs, as the depletion of CD8+ T cells did not reduce protection from tumour outgrowth in vaccinated mice. While mesothelin represents a promising target, it is not an ideal target for oncolytic vaccine platforms, potentially due to its nature as a self antigen.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The aggressiveness and therapeutic recalcitrance of this malignancy has been attributed to multiple factors including the influence of an active desmoplastic stroma. How the stromal microenvironment of PDAC contributes to the fatal nature of this disease is not well defined. In the analysis of clinical specimens, we observed diverse expression of the hypoxic marker carbonic anhydrase IX and the lactate transporter MCT4 in the stromal compartment. These stromal features were associated with the epithelial to mesenchymal phenotype in PDAC tumor cells, and with shorter patient survival. Cultured cancer- associated fibroblasts (CAFs) derived from primary PDAC exhibited a high basal level of hypoxia inducible factor 1a (HIF1 alpha) that was both required and sufficient to modulate the expression of MCT4. This event was associated with increased transcription and protein synthesis of HIF1a in CAFs relative to PDAC cell lines, while surprisingly the protein turnover rate was equivalent. CAFs utilized glucose predominantly for glycolytic intermediates, whereas glutamine was the preferred metabolite for the TCA cycle. Unlike PDAC cell lines, CAFs were resistant to glucose withdrawal but sensitive to glutamine depletion. Consistent with the lack of reliance on glucose, CAFs could survive the acute depletion of MCT4. In co-culture and xenograft studies CAFs stimulated the invasive potential and metastatic spread of PDAC cell lines through a mechanism dependent on HIF1a and MCT4. Together, these data indicate that stromal metabolic features influence PDAC tumor cells to promote invasiveness and metastatic potential and associate with poor outcome in patients with PDAC.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the five leading causes of cancer-related deaths in the West, and this, largely, is due to metastatic disease. In order to better understand PDAC metastatic spread and identify novel therapeutic targets, we analysed the proteome of primary tumours and matched lymph node (LN) metastases. As frozen specimens of metastatic lesions are scarce, we examined formalin-fixed paraffin-embedded (FFPE) tissues. Whilst such tissue is in routine diagnostic use, the cross-linkages induced by fixation have, in the past, precluded proteomic investigation for research purposes. Recent technological advances have, however, overcome this technical limitation. Using laser capture microdissection (P.A.L.M system), we isolated malignant epithelia from seven FFPE primary PDAC tumours and matched LN metastases. Following dissection, samples were analysed in duplicate using Multidimensional Protein Identification Technology (MudPIT); this resulted in the identification of 1504 proteins, 854 of which were common to all samples analysed. Comparison of the obtained proteins with data from previous proteomics studies on pancreatic tissue, pancreatic juice, serum and urine resulted in a less than 30 % overlap, indicating that our study has expanded the current database of proteins expressed in this malignancy substantially. Statistical analysis further showed that 115/854 proteins (13.5%) were significantly differentially expressed (g-value ≥ 3.8). Two proteins, S100P and 14-3-3 sigma, with highly significant g-values were confirmed to be significantly differentially expressed (S100P: p = 0.05 and 14-3-3 sigma: p < 0.001) 4 in a larger series of 55 cases of matched primary PDAC and LN metastases using immunohistochemistry. We chose to investigate further the roles of S100P in lymphatic invasion in vitro and in vivo. By co-culturing a Panc1 S100P-overexpressing clone (S5L), or a vector control clone (V3L), with human dermal lymphatic endothelial cells (HDLEC), we were able to show that different receptors mediate S5L adhesion to resting and activated HDLEC as opposed to V3L; and that the presence of S5L cells in these co-cultures significantly increased permeability at one (p = 0.02), four (p = 0.002) and eight (p = 0.007) hours post-seeding, and significantly increased translymphatic endothelial migration at 72 hours (p = 0.006). Using the V3L and S5L cell lines, which were transduced to express luciferase, we also created an orthotopic mouse model of PDAC, as well as experimental metastatic mouse models, in CD1 nude mice. These models were used to evaluate the effects of S100P on primary tumour growth, metastasis and site-specific growth. S100P was only found to significantly increase primary tumour growth in this model (n = 10 animals/group), both by bioluminescence (p = 0.002) and tumour weight (p = 0.01). No metastases (spontaneous and/or experimental) were seen however. Thus, this model can be used to evaluate the anti-tumour efficacy of novel therapies to S100P in the future.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, and the 5th most common cause of cancer death in the UK. One of the peculiarities of this malignancy is its ability to invade nerves, a process called perineural invasion (PNI). PNI is found in almost 100% of PDAC, and is associated with poor prognosis, tumour recurrence and generation of pain. However, the molecular bases of PNI remain largely unknown. We investigated the molecular alterations underlying the neuro-epithelial interactions in PNI using one and two dimensional liquid chromatography – mass spectrometry (1D and 2D LC-MS) of laser microdissected PNI and non-PNI cancer from formalin fixed, paraffin embedded PDAC tissues. We also performed 1D LC-MS analysis of invaded and non-invaded nerves from the same cases. In addition, we developed an in vitro model of PNI using a co-culture system comprising PC12 cells, a rat pheochromocytoma cell line, as the neuronal element and PDAC cell lines. The overall proteomic profiles of PNI and non-PNI cancer appeared largely similar; of very few deregulated proteins, we have validated the up-regulation of antiapoptotic protein Olfactomedin 4 in PNI cancer using immunohistochemistry. In contrast, nerve samples demonstrated widespread molecular alterations characteristic of neuronal plasticity upon invasion by cancer cells. Immunohistochemistry confirmed the up-regulation of VGF in nerves from PDAC and chronic pancreatitis (CP) specimens compared to normal pancreas, as well as in invaded compared to non-invaded nerves in PDAC tissues. Furthermore, VGF expression strongly correlated with pain in CP; similar analysis in PDAC cases is still pending. Using the in vitro co-culture model, several PDAC cell lines were able to induce PC12 cells neuronal plasticity including survival, neurite extension as well as VGF expression, recapitulating thus the changes observed in human tissues. PDAC-induced PC12 plasticity was not mediated via NGF, a neurotrophin acting upstream of VGF and thought to be involved in the neuro-epithelial interactions. The induction of VGF expression was shown not to be necessary for PC12 cell survival, however, it contributed to the neurite extension induced by PDAC cell lines. In summary, based on proteomics analysis and in vitro modelling, we show the complex and intricate involvement and crosstalk of both tumoral and neural elements that are activated during perineural invasion in pancreatic cancer.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer death in Western countries. The PDAC tumour microenvironment (TME) is characterized by a dense stromal reaction, consisting of many cell types including fibroblasts and immune cells. The chemokine receptor, CX3CR1 forms a high-affinity axis with its unique ligand CX3CL1 and is expressed on monocytes, macrophages and T cells. CX3CR1 is also present on pancreatic malignant cells, where it has been associated with metastasis formation. The aim of my project is to investigate the role of CX3CR1 in the progression and development of pancreatic cancer in a genetically engineered mouse model of PDAC, the CX3CR1GFP/GFPLSL-KRASG12D/+LSL-Trp53R172H/+Pdx1-Cre (CKPC) mouse. In these mice, the CX3CR1 protein is not functional but they express GFP. I have found that the absence of CX3CR1 in KPC mice has no effect in their lifespan and response to chemotherapy. Comparison of the immune infiltrate of the tumours revealed that the lack of CX3CR1 causes a significant decrease in T cells and a possible increase in myeloid cells in CKPC mice compared to KPC mice. Expression analysis of several inflammatory cytokines in the TME showed a significant difference in IL-10 between KPC and CKPC mice. There was also a significant increase in levels of, CX3CL1, both locally and in the plasma. Finally, we performed RNA-seq on KPC and CKPC tumours. My analysis revealed 607 differentially-expressed genes, some of which encoded other chemokines or protein regulating the immune system. In particular, I observed the upregulation of Cxcl10 and Cxcl12, and the downregulation of Gata3 and S100a4, which could explain the decrease in T cells in the TME of CKPC mice. In conclusion, although the lack of CX3CR1 modifies the TME in this genetic model of PDAC, these changes do not affect the lifespan or the response to chemotherapy.

INTRODUCTION: Pancreatic cancer remains the fourth leading cause of cancer-related deaths with approximately 5% five-year survival and 3 months of median survival. The survival rate of pancreatic cancer has not improved substantially over the past 40 years. Therefore, a novel potential treatment for pancreatic cancer is urgently needed. Recently, a cell surface receptor, Neuropilin-2 (NRP-2), was found to competitively bind either stimulatory angiogenic ligands such as vascular endothelial growth factor-A (VEGFA) or inhibitory class 3 Semaphorin-3F (SEMA3F) ligands. Knowing that angiogenesis is necessary for pancreatic tumor growth, elucidating the role of NRP2 in angiogenesis may lead to curative treatment for pancreatic cancer. OBJECTIVES: Previously, NRP-2 has been shown to be expressed by human cells of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal forms of pancreatic cancer. Additionally, knockdown of NRP-2 in vivo inhibited PDAC tumorigenesis. In our current study, we aimed to investigate the role of endothelial cell derived-Nrp-2 in PDAC-associated tumor angiogenesis. Furthermore, we studied the efficacy of SEMA3F as a potential inhibitory factor for pancreatic tumor growth. METHODS: To investigate the role of Nrp-2 in tumor-derived angiogenesis, we injected Panc0H7 cells, a C57BL/6 syngeneic mouse PDAC cell line, orthotopically into the pancreas of Nrp-2+/+, Nrp-2+/-, and Nrp-2 -/- mice and compared tumor growth and angiogenesis. We next injected control adenovirus (Ad-control) or SEMA3F adenovirus (Ad-3F), which actively encodes SEMA3F in vivo, followed by orthotopic injection of Panc0H7 cells into C57BL/6 mice three days later. We studied the efficacy of SEMA3F as a potential treatment for pancreatic cancer by comparing the tumor growth and tumor-associated angiogenesis of the two groups of adenovirus-treated mice. RESULTS: Our results showed that Panc0H7 tumors were significantly smaller in Nrp-2-deficient mice as compared to that of Nrp-2-intact mice. Furthermore, tumor microvessel density was significantly lower in Nrp-2-knockout mice compared to wild-type mice, while there was no difference in tumor weight or angiogenesis between wild-type and Nrp2 heterozygous mice. Our results also demonstrated that pancreatic tumors harvested from SEMA3F-treated mice were significantly smaller than the tumors from the control-treated mice. Furthermore, micrometastases were detected in the livers of mice treated with Ad-control but not in the Ad-3F group. CONCLUSIONS: Taken together, our results suggested that NRP2 might facilitate in vivo angiogenesis and tumor growth. Furthermore, SEMA3F could be a potential treatment to inhibit the growth and metastases of pancreatic tumors.

Obesity is a known risk factor for many types of cancer including pancreatic. Calorie restriction (CR), an anti-obesity diet regimen, has potent anticancer effects that may be mediated through its ability to reduce serum metabolic hormones and protumorigenic cytokines such as insulin-like growth factor (IGF)-1. IGF-1 is a metabolic hormone responsive to nutrient status that activates the inflammatory, cancer-related pathway, nuclear factor (NF)-[kappa]B. For this report, we tested the hypothesis that CR, via regulation of IGF-1, inhibits pancreatic tumor cell growth through modulation of NF-kB activation and protumorigenic gene expression. Male athymic nude mice were randomized to either a control diet consumed ad libitum (n=15) or a 30% CR diet (n=15) for 17 weeks, at which time, mice were injected with human pancreatic cancer cells (MiaPaca) and tumor growth was monitored for 6 weeks. Translocation of p65, a regulatory element of NF-[kappa]B, and expression of its downstream gene targets were analyzed in excised tumors. CR mice weighed less, (p<0.05), and had smaller tumors (p=0.022) relative to controls. Tumors from CR mice, relative to controls, demonstrated significant decreases in NF-[kappa]B downstream genes CCND1, RELA, Survivin, VEGF, and XIAP. These findings parallel our previous studies in pancreatic tumors from mouse origin, and suggest that the inhibitory effects of CR on MiaPaca pancreatic tumor growth are associated with decreased NF-kB activation. / text

One of the current goals in cancer research is to discover and validate novel molecular targets that may be useful for diagnostic and therapeutic purposes in fighting this disease. The PRL phosphatases (PRL-1, PRL-2, and PRL-3) are low molecular weight protein tyrosine phosphatases with unknown biological function(s) that have gained attention from cancer researchers in the past couple of years, mainly due to reports that these phosphatases may play important roles in tumor progression and metastasis. Motivated by the particular urgent need for molecular targets in pancreatic cancer this work was undertaken to determine what role PRL proteins played in pancreatic cancer biology and to determine if targeting PRLs would be effective in treating this disease. In this dissertation, it was found that both PRL-1 and PRL-2, but not PRL-3 are upregulated in pancreatic adenocarcinomas, suggesting that some cancer cells are dependent upon their activity for continued proliferation and survival. To validate this hypothesis, siRNAs were used in cell-based assays to evaluate the biological consequences of PRL-1 and/or PRL-2 inhibition. It was found that perturbations in PRL phosphatase signaling result in reduced proliferation, migration and especially the ability to grow in soft agar. Oligonucleotide microarray analysis revealed that many Erk and/or Akt dependent stress and growth factor inducible genes were differentially regulated between pancreatic cancer cells treated with PRL-targeting siRNA and their non-targeting siRNA treated counterparts. Subsequently, PRL knockdown was found to alter serum induced as well as amino acid deprivation induced Akt and Erk phosphorylation in multiple pancreatic cancer cell lines, suggesting that PRLs function upstream of these key pathways. Interestingly, we show that PRL proteins in cell free assays exhibit higher activity on doubly phosphorylated phosphatidylinositol substrates than tyrosine-phosphorylated peptides, suggesting that the biological substrate(s) might include non-protein molecules. These data support the hypothesis that PRL-1 and PRL-2 might play important biological roles in pancreatic cancer cells and further studies should be undertaken to determine the usefulness of these phosphatases as potential molecular biomarkers and targets.