Colorectal cancer, among other tumors, is characterized by elevated levels of prostaglandins due to the up-regulation of cyclooxygenase -2 (COX-2), a key enzyme in the eicosanoid biosynthesis pathway. Prostaglandin E2 (PGE2) is an important prostaglandin that exerts its biological function via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is the most important. The relevance of EP4 receptor to the carcinogenic process and the consequences of its interaction with PGE2 were explored in this dissertation.Despite the importance of the EP4 receptor in colon carcinogenesis, studies looking at the receptor expression during cancer progression have not been extensive. One study showed that the protein levels of EP4 receptor were elevated in colon cancer whereas another study indicated that mRNA levels were decreased in tumor compared to normal. We expanded these observations and now report that the elevated protein levels of EP4 receptor in cancer are due to increased translation of proteins.In addition, we identified S100P as a novel downstream target of the PGE2/EP4 receptor signaling pathway. S100P has been previously implicated in a number of gastro-intestinal cancers such as pancreatic, gastric and colon cancers. However, its regulation via the PGE2/EP4 receptor signaling pathway has never been investigated. Here, we show that PGE2 via the EP4 receptor signaling leads to the transcriptional activation of S100P and that this activation happens exclusively in the presence of CREB. In summary, this dissertation brings to light novel therapeutic targets which could be used as potential markers to stratify colon cancer patients as well as avenues for clinical intervention for the management of colon carcinogenesis.

Both Notch and the Wnt pathways are key regulators in maintaining the homeostasis in the intestine. Defects on the key tumor suppressor adenomatous polyposis coli, APC a gene in the Wnt pathway is most frequently mutated in colorectal cancer. Previous studies have indicated that there is a crosstalk between these two pathways. We investigate if there is correlation by first using bioinformatics to find Lef1/Tcf sites in several of the Notch pathway gene promoters. Bioinformatically we found that a lot of the genes contained theses sites controlled by the APC's destruction target β-catenin. By using semi quantitative PCR and western blot we found that Hes 1, Hes 7, JAG 2, MAML 1, Notch 2, NUMB, NUMBL, RFNG and LFNG was downregulated in HT29 colon cancer cells carrying a vector containing wild type APC. All but JAG 2 contains at least one Lef1/Tcf site in their promoter region. The results were verified in HT29 cells transfected with siRNA against β-catenin. We also investigated what would happen to the Lef1/Tcf target gene program of the Wnt pathway, if the Notch pathway was inhibited with the gamma-secretase inhibitor DAPT. Results showed no downregulution of β-catenin or its target gene Cyclin D1.Taken together, these results demonstrate that the Wnt pathway can be placed upstream of the Notch pathway and regulates the latter through β-catenin and the Lef1/Tcf target gene program. However, preliminary results indicate that there is no regulation of APC/β-catenin by the Notch pathway.

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Colorectal cancer poses a serious threat to the health and well-being of individuals, especially those at high risk or over the age of 50. Gone undetected, colorectal cancer is often fatal, however, preventive screening greatly reduces the number of people who may develop this disease. The Centers for Disease Control and Prevention developed the Colorectal Cancer Screening Demonstration Program in 2005 to assess the feasibility of a national colorectal cancer screening program serving low-income and un- or underinsured populations. Qualitative case study data from the Colorectal Cancer Screening Demonstration Program evaluation were analyzed in order to examine the start-up experiences of two of the programs involved. Results from this multiple case study document program models and describe facilitators, challenges, and participant perception of the expected impact on screening behavior. Further research on program implementation is needed to understand how program models perform and impact behavior once screening begins.

Timely access to end-of-life care is a growing problem. One under-referred group is adults who die shortly after cancer diagnosis. This group’s challenges include a lack of definition for short diagnosis-to-death (SDTD), and inability of health care providers to identify risks for SDTD. Research indicates socioeconomic factors may influence access to end-of-life care, though how is unclear. This study used recursive partitioning methods to define SDTD for decedent adults with colorectal cancer and identify socioeconomic predictors of SDTD. SDTD was defined as less than 18.5 days. Socioeconomic predictors included long-term care residence and community-level characteristics such as education, immigration, marital status, Aboriginal status, and income. Results showed existing SDTD timeframes may be too long to adequately understand the population’s needs, and indictors of risk may be unique for this population. Additional research could establish consistency for defining SDTD and clarify the utility of socioeconomic predictors for mitigating barriers to care.

The tumor microenvironment consists of multiple cells types, including endothelial cells that line the tumor vasculature. Tumor vasculature is often abnormal and results in development of tissue ischemia, another contributing factor to the tumor microenvironment. Previous studies have demonstrated that ischemia influences epigenetic programming, but the mechanisms remained unclear and required further investigation. First, we profiled DNA methyltransferase (DNMT) expression and activity in human colorectal cancer cells (HCT116) under hypoxia or hypoglycaemia (mimicking ischemia). We found that DNMT1 and DNMT3b were significantly downregulated by hypoxia and hypoglycaemia, and DNMT3a was downregulated by hypoglycaemia. However, DNMT1 downregulation was p53-dependent. To examine if the changes in DNMT expression and activity translated to changes in DNA methylation patterns, we used bisulfite sequencing and examined the promoter region of p16. Hypoglycaemia significantly demethylated this region in both p53 wild-type and p53-null cells. Next, we used a genome-wide approach to discover what additional genes are hypomethylated by ischemia. Methylated DNA was immunoprecipitated and analysed with an Affymetrix promoter array, in parallel with an expression array. Ingenuity pathway analysis software revealed that a significant proportion of genes which were hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. We believe that hypoxia and hypoglycaemia may be driving changes in DNA methylation through dysregulation of DNMTs, resulting in cells acquiring a more mobile phenotype in ischemic regions. DNMT and histone deacetylase inhibitors are commonly used in research and some cancer therapies. Modifying epigenetic patterning with these inhibitors has been widely studied in cancer cells, but only briefly explored in the tumor’s vascular endothelium. We profiled the effect of these inhibitors on endothelial cell (EC) behaviour, and tested if combining them with a targeted anti-angiogenic therapy would augment the inhibition of angiogenesis. When the DNMT inhibitor 5-aza-2’-deoxycytidine was combined with sunitinib, inhibition of EC proliferation was enhanced compared to treatment with sunitinib alone. EC migration was also inhibited by the combination of these two inhibitors, but not in an additive manner. These studies have improved our understanding of how altering epigenetic patterning with ischemia and therapeutic inhibitors can influence colorectal cancer and endothelial cell behaviour. / Canadian Cancer Society Research Institute. The Cancer Research Society.

Dichloroacetate (DCA) it a metabolic reprogramming agent that is used to target the unique metabolism of cancer cells, but is not always effective in colorectal cancer cells. In HCT116 cells, DCA was unable to induce apoptosis, but did decrease proliferation when compared to untreated cells. A decrease in full length Mcl-1 protein expression 7 hours following DCA treatment did not correspond with changes in mRNA production or changes in expression of inhibitory binding partners, but may be due to altered proteasomal degradation. Similar reduction in levels of a lower molecular weight Mcl-1 band occurred, which did not result from alternative splicing or from caspase-mediated cleavage. Mcl-1 showed primarily nuclear localization within the cell, and expression changes in full-length Mcl-1 were seen in nuclear lysate but not cytoplasmic lysate after 7 hours of DCA treatment. Changes in nuclear Mcl-1 expression did not correspond with cell cycle arrest or progression. These results suggest that proteasomal degradation of Mcl-1 may be altered following treatment with DCA, and this change may be associated with decreased proliferation, independent of cell cycle arrest. This may indicate a novel role of nuclear Mcl-1 in response of colorectal cancer to DCA exposure. / Final thesis for Leanne Delaney in partial fulfillment of requirements for the degree of Master of Science in Biomedical Sciences / NSERC

Folic acid is a B vitamin involved in DNA CpG methylation. Mandated dietary fortification has led to a subsequent increase in blood folate concentration which has been correlated to a simultaneous spike in colorectal cancer incidence in Canada and the US. Several human colorectal cancer cell lines were cultivated under low (0 mg/L), standard (4 mg/L), and high (16 mg/L) folate conditions for seven days, then assessed for DNA methyltransferase1 protein expression, changes in DNA methylation, and ability to generate colonospheres in culture. Low folic acid levels generally led to reduced DNMT1 protein expression, CpG hypomethylation, and reduced colonosphere yield. High folic acid levels led to increased DNMT1 protein expression, CpG hypermethylation, and maintained colonosphere yield. This data demonstrates that varying levels of folic acid in vitro can influence the methylation status and cancer stem cell self-renewal ability of human colorectal cancer cells. / Canadian Cancer Society

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