Pancreatic cancer is an aggressive disease with poor prognosis and a high fatality rate. Gemcitabine, the standard first-line chemotherapy for advanced disease, has negligible effects, necessitating the development of new therapies. We previously demonstrated that deletion of the anti-apoptotic gene E1B19K (AdΔ19K) in a replication-selective adenoviral mutant, caused synergistically-enhanced cell-killing when combined with low-dose DNA-damaging drugs in pancreatic cancer xenograft models. To delineate the cellular pathways targeted by the combination treatment we employed AdΔ19K and gemcitabine or irinotecan, with the goal of identifying cellular factors that are essential for the synergistic cell-killing. We hypothesised that AdΔ19K and DNA-damaging drugs act synergistically to deregulate cell-cycle mechanisms. Pancreatic cancer cell death induced by AdΔ19K and DNA-damaging drugs is apoptotic and time-dependent. AdΔ19K could not block DNA-damage responses (DDR) elicited by the drugs, despite virus-mediated degradation of the DDR factor Mre11. Mre11 siRNA-mediated knockdown augmented the synergistic cell death. Mitotic-index analysis in synchronised cells and immunofluorescence microscopy suggested that AdΔ19K promotes mitotic entry of gemcitabine-treated DNA-damaged cells. Moreover, AdΔ19K inhibited drug-induced accumulation of Claspin, a DDR protein whose degradation is required for checkpoint recovery. Treatment with AdΔ19K and gemcitabine accelerated Claspin degradation, and siRNA-mediated Claspin knockdown enhanced the synergistic cell death. Time-lapse microscopy in histoneH2B mCherry-expressing cells showed that AdΔ19K enhanced gemcitabine-induced mitotic catastrophe, characterised by prolonged mitosis, chromosome missegregation errors, cytokinesis failure and formation of multinucleated cells. Moreover, live-cell imaging revealed that the majority of cells treated with AdΔ19K and gemcitabine die before mitotic entry. 5 These findings suggest that E1B19K-deleted adenoviruses cannot prevent cell-cycle checkpoint responses elicited by DNA-damaging drugs, but enhance drug-induced cell death by downregulating DDR factors, such as Mre11 and Claspin. Additionally, the virus enhances mitotic catastrophe of DNA-damaged cells escaping cell-cycle checkpoints, eventually leading to increased apoptosis. Through these studies cellular pathways and factors involved in the synergistic cell killing were identified, that could be explored in the future to develop improved targeted therapies for pancreatic cancer.

Gastrointestinal malignancies such as liver and pancreatic cancers are the deadliest due to late detection and drug resistance. Nucleoside analogues, like Gemcitabine, are the conventional therapy despite their little impact on survival and off-target toxicity. A novel class of nucleoside analogues able to evade drug resistance mechanisms has been developed by the Guindon group and biologically screened in our lab. Some of these proprietary molecules were further equipped with a lipoate moiety designed to target cancer cell metabolism. LCB2151 and LCB2179 have emerged as the lead molecules in this class, with an IC50 of 10-15 µM in the Gemcitabine-resistant human pancreatic (Capan-2 & Panc-1) cancer cell lines. The focus of this project is deciphering the cellular mechanisms activated by LCB2151 in these pancreatic cancer lines. A series of biased molecular approaches, like gene expression profiling, and unbiased large throughput proteomic and metabolomics analyses were applied to identify potential targets and affected pathways. Results collectively show that LCB2151 evades drug resistance mechanisms, increases pro-apoptotic markers and impairs mitochondrial respiration as early as 6 hours posttreatment. Furthermore, MS/MS analyses reveal that LCB2151 alters the levels of several metabolites in the central carbon metabolism pathway and identifies the citric acid cycle enzyme α-ketoglutarate dehydrogenase as a potential molecular target of LCB2151. Understanding the exact mechanism of action of our lead molecule along with extensive testing on murine cancer models, will surely pave its way to clinical testing and evaluation.

Background: Current targeted therapies in pancreatic cancer have been ineffective. The tumor stroma, including intra- and peri-tumoral inflammation and fibrosis, is increasingly implicated in pancreatic cancer. Pancreatic cancer is characterized by a highly fibrotic tumor environment resulting in stromal resistance to chemotherapy. Peroxisome proliferator-activated receptor-alpha (PPARα), a ligand-activated nuclear receptor/transcription factor, is a negative regulator of inflammation. In PPARα deficient mice, stromal processes inhibit tumor growth, resulting in dormant tumors. The presence of PPARα in the tumor cells as well as in the host is necessary for unabated tumor growth. Objective: We hypothesized that blocking the PPARα pathway with a small molecule PPARα antagonist (NXT) may prevent pancreatic cancer progression by targeting tumor cells as well as non-neoplastic cells in the tumor microenvironment. Methods: Growth inhibitory activity of the PPARα antagonist was assessed in murine as well as human pancreatic tumor cell lines (Panc0H7 and BxPC3) and in a murine macrophage cell line (RAW 264.7). Cell viability was determined by trypan blue exclusion assay. AKT, P-AKT, PCNA, BAX, and p27 levels were analyzed by western blot analysis. Cell cycle changes were detected by flow cytometry. Cellular senescence was determined by senescence-associated β-gal (SA-β-gal) staining. Results: The PPARα antagonist inhibited cell growth in macrophages and in pancreatic tumor cells as confirmed by reduced protein level expression of PCNA and activated AKT. Treatment of the PPARα antagonist was non-cytotoxic to tumor cells. Inhibition of PPARα induced cell cycle arrest at G0/G1 in tumor cells and macrophages. The induction of cellular senescence was observed in pancreatic cancer cells. Interestingly, we observed a reduction in protein level expression of BAX, a marker for apoptosis, and p27, an inhibitor of the cell cycle. Conclusion: We now demonstrate that a PPARα antagonist exerts its anti-growth activity by inducing G0/G1 cell cycle arrest, thereby inducing cellular senescence without cell death. These findings provide a mechanism for the anti-tumorigenic activity of PPARα inhibition, and the rationale to use PPARα antagonists as a novel therapeutic approach to pancreatic cancer. / 2016-11-03T00:00:00Z

In this project we study the role of RAGE in the melanoma and pancreatic cancer progression. Based on published studies, we hypothesized that RAGE localization in melanoma varies with different cellular architectures. To test this hypothesis, we utilized an in vitro spheroid model and a lung colonization mice model to compare the RAGE localization in 3D architecture vs 2D monolayer culture. RAGE was found at the cell surface in WM115 and B16F10 spheroids, whereas RAGE is mostly distributed intracellularly in WM266. We also observed that RAGE is present at the surface of B16F10 melanoma cells within tumor nodules in the lungs of mice colonized with B16F10 cells. Previously, our group has demonstrated that RAGE promotes pancreatic tumor cell survival under normoxic conditions, upon gemcitabine administration. Hypoxia is also associated with increased tumor aggressiveness. Based on published reports, we hypothesized that RAGE upregulation under hypoxic conditions contributes to autophagy and migration in pancreatic cancer cells. We observed that autophagy decreases after RAGE inhibition by FPSZM1. Moreover, we observed decreased cell migration after RAGE blockage, indicating that RAGE also mediates migration under hypoxia. We also investigated Advanced Glycation Endproducts (AGEs) on proliferation and migration of pancreatic cancer cells. Based on published reports, we hypothesized that RAGE activation by AGEs contributes to the proliferation and migration in pancreatic cancer cells. We employed ribose modified BSA to activate RAGE in the murine KPC 5517 pancreatic cancer cell line. We observed that AGE-treated samples showed significant increase in migration but no change in proliferation. As RAGE is involved in the progression of melanoma and pancreatic cancer, our results will help researchers to better understand the biology of RAGE. Our research can help to design RAGE-specific antibodies and inhibitors that could target RAGE more effectively. Moreover, our findings on AGE-RAGE interactions, and on the role of RAGE in pancreatic cancer progression under hypoxia, may contribute to reduce the progression of pancreatic cancer. Our results showing that a RAGE inhibitor can reduce autophagy and migration of pancreatic tumor cells, suggest that FPS-ZM1 could be utilized as a potential therapeutic aid for the treatment of pancreatic cancer.

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.

No description available.

Pancreatic ductal adenocarcinoma is a highly desmoplastic tumour, and non-malignant stromal cells contribute to progression and treatment resistance. Inflammatory cells in particular are known drivers of carcinogenesis, and macrophages are one of the most abundant inflammatory leucocytes. Therefore, exploring how macrophages drive tumour progression in pancreatic cancer would not only aid in understanding disease biology but could also offer insight to novel treatment strategies. Results presented in this thesis show macrophages secrete factors that drive epithelial-to-mesenchymal transition, promote invasion and lead to expression of checkpoint inhibitors. To determine what factors were driving this phenotype, the serine protease inhibitor SerpinB3 was initially explored, as it was highly upregulated in cancer cells cultured with conditioned media from macrophages. However, SerpinB3 gene overexpression and knockdown did not confirm a direct role for this gene in mediating migration and invasion. Further investigation revealed macrophages were secreting the cytokine oncostatin M, which was driving a metastatic phenotype through activation of the STAT3 pathway. Expression of oncostatin M receptor was upregulated in cancer cells following culture with macrophage conditioned media and conferred a worse prognosis in patient samples. STAT3 pathway activation by oncostatin M led to increased invasion in vitro, particularly of the highly tumourigenic cancer stem cell population, and increased metastasis in vivo. Moreover, oncostatin M mediated expression of the immune 'checkpoint' inhibitors on the surface of pancreatic cancer cells. Using antibody and small molecule inhibitors, reversion of these signalling pathway effects were seen and preliminary data from in vivo assays showed decreased metastasis formation with cytokine receptor antibody inhibition. Overall, the findings in this thesis contribute to emerging knowledge of how tumour associated macrophages drive tumour progression in pancreatic adenocarcinoma. Not only do they promote invasion and metastatic potential through oncostatin M secretion, but also potentiate inherent biological properties of cancer stem cells and assist in immune tolerance. In addition, results provide preliminary data to support a rationale for clinical targeting of macrophage-derived oncostatin M in pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer death in the United States, with a five-year survival rate under 5%. Given the disease’s deadliness, increasing our understanding of the molecular nature of the pancreatic cancer is key to developing more effective preventive measures and treatments. Dietary energy restriction (DER) has been shown to have potent anticancer effects in pancreatic cancer, but the mechanism of action has yet to be completely elucidated. Here we investigate the potential of altered microRNA expression as a mechanism by which DER exerts its anticancer effect. Using the Exiqon microRNA Array, we identified several microRNAs of interest for further study. This includes microRNA (mir) 669c, a known regulator of glutathione-S transferases (linked to carcinogen metabolism and oxidative stress) that increases with age. To our knowledge, this is the first exploration of the effects of DER (which is known to suppress oxidative stress and other processes associated with aging and cancer) on microRNA expression. These findings may provide the initial steps towards identifying novel targets for pancreatic cancer prevention or treatment. / text

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.

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.