Colon cancer occurs in over 150,000 men and women in the United States each year and is fatal about one third of the time. There are many well characterized genetic transformations which commonly occur during colon carcinogenesis, including mutations in the Adenometous Polyposis Coli (APC), Kirsten RAS (K-RAS) and p53 genes among many others. There are also many alterations in gene and protein expression which are not yet completely elucidated. We have identified kallikrein 6 (KLK6) as a gene whose expression is dependent on cancer associated proteins such as K-RAS, SRC and caveolin-1 (CAV-1). KLK6 is a member of the kallikrein protein family which consists of 15 secreted serine proteases. Like other types of proteases, kallikreins have been demonstrated to play a role in cancer progression and they hold promise as potential cancer biomarkers. The up-regulation of KLK6 was first identified by performing a microarray analysis on a mutant K-RAS transfected Caco2 cells. The activated K-RAS transfected cells expressed significantly more KLK6 than the mock transfected controls. Pathways downstream of K-RAS were found to induce KLK6 gene expression, including the p42/44 MAPK and the PI3-K/AKT pathways. Caveolae, plasma membrane associated structures, and their principle protein component, CAV-1, positively influence both KLK6 gene expression and KLK6 protein secretion in HCT116 colon cancer derived cells. Finally, it is shown that KLK6 increases the invasive potential of cells through laminin and Matrigel. Because KLK6 plays a role in cancer progression it may serves as a novel therapeutic target. Additionally, KLK6 holds potential for use as a serum biomarker for multiple types of cancer, including colon cancer for colon and other types of cancer.

No description available.

No description available.

Aberrant methylation of CpG island promoters is a frequent observation in cancer and is known to affect many genes, including tumour suppressor genes. Genes with methylated promoters are usually repressed and inactive, and there is good evidence that most genes that become methylated in cancer are already repressed in the normal tissues from which tumours arise. However, the methylation of some genes appears to arise at previously active loci, suggesting either a stochastic epigenetic event or that these genes are somehow predisposed to becoming methylated. The DNA mismatch repair gene MLH1 is expressed in normal colonic epithelial cells but methylated and down-regulated in some sporadic mismatch repair-deficient colon tumours. These tumours are almost invariably associated with the simultaneous methylation of multiple cancer-specific loci, termed the CpG island methylator phenotype (CIMP) and an activating mutation of BRAF (V600E), raising the possibility that a hypermethylator phenotype may arise in cancer in direct association with a specific genetic alteration. The possibility that MLH1-deficiency caused BRAF mutation was discounted as genetic deficiency of MLH1 is not associated with BRAFV600E. I explored the possibility that BRAFV600E might induce MLH1 methylation but found no evidence in support of this. I then focused on factors that might mediate CIMP gene methylation, of which MLH1 methylation is known to be a part. Bioinformatic analysis of the genes methylated in BRAFV600E colon tumours indicated a significant enrichment in binding sites for the transcription factor MAZ (MYC-associated zinc finger protein). I hypothesised that loss of MAZ might lead to MLH1 down-regulation and its subsequent methylation. In this thesis I provide evidence that both MAZ and MLH1 expression are deregulated during normal colonic epithelial differentiation. The down-regulation of MAZ by RNA interference led to a reduction in MLH1 expression and methylation of its promoter. I speculate that MLH1 methylation may be associated with BRAF mutation because transformation by BRAFV600E allows progenitor cells to undergo a degree of differentiation whilst maintaining their malignant proliferation. I speculate that it is during this process of differentiation that MLH1 becomes susceptible to methylation.

archives@tulane.edu / 1 / Margaret Brooks

In order to test the dietary effects of bioactive compounds present in whole grains, we decided to observe the effect of varying types of sorghum bran on colon cancer promotion. We used 40 rats consuming diets containing 6% fiber from either cellulose or bran from white (contains phenolic acids), brown (contains tannins), or black (contains anthocyanins) sorghum (n=10). Diets were fed for 10 wk, during which two azoxymethane (AOM) injections (15 mg/kg BW) were administered in wk 3 and 4. We observed that the total number of aberrant crypts (AC) and high multiplicity aberrant crypt foci (HMACF) were lower in rats consuming black (p < 0.04) and brown (p < 0.006) sorghum diets when compared to the cellulose diet, and that these decreases were an inverse function of diet antioxidant activity (ABTS). These observations led us to evaluate the effect of these diets on endogenous enzymatic activities (superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GPx), redox status as measured by reduced and oxidized glutathione, and cell cycle processes, proliferation and apoptosis, in the rat colon. Total SOD activity was higher (p < 0.04) in rats consuming black sorghum when compared to all other diets. A similar, but not significant, trend occurred in mitochondrial SOD. The white sorghum diet had enhanced (p < 0.02) CAT activity compared to the cellulose diet, but the black and brown sorghum diets were intermediate. Finally, all sorghum diets suppressed GPx activity relative to cellulose (p < 0.04). However, no changes were seen in levels of reduced and oxidized glutathione or the ratio of the two. The black sorghum fed rats had a lower proliferative index (p < 0.01) and zone (p < 0.04) compared to cellulose; brown and white sorghum rats were intermediate. Apoptotic index was highest in brown sorghum rats compared to cellulose (p < 0.03), while other sorghum diets were intermediate. These data suggest that the suppression of AC and HMACF formation in rats consuming sorghum bran may have resulted through the differential actions of the sorghum brans on endogenous antioxidant enzymes, which may affect colonocyte proliferation and apoptosis.

We have previously shown that dietary fish oil and the fiber pectin protect against colon cancer in rats by increasing apoptosis induced by reactive oxygen species (ROS) at the initiation stage of tumorigenesis. We hypothesized that fish oil would incorporate into the cardiolipin of colonic mitochondrial membranes, creating an environment in which butyrate, a fermentation product of pectin, would also increase ROS and lead to apoptosis, as evidenced by decreased mitochondrial membrane potential (MMP), enhanced caspase-3 activity and cytochrome c translocation from the mitochondria, thus protecting against colon cancer by removing DNA damaged cells at the promotion stage of carcinogenesis. Sixty rats were provided a diet containing 15% corn or fish oil for 11 wk and injected with azoxymethane (AOM) or saline at wk 3 and 4. At wk 11, colonocytes were exposed to +/- butyrate ex vivo for 30 or 60 min. ROS and MMP were measured using fluorescence microscopy, and cytochrome c concentration and caspase-3 activity were measured using ELISA assays. Cardiolipin fatty acid enrichment was measured via TLC and GC. Butyrate increased ROS (p<0.0001) regardless of diet or treatment group. In colonic crypts from fish oilconsuming rats, butyrate reduced MMP (p=0.05). However, butyrate had no effect on MMP if the rats were consuming corn oil. In colonocytes from rats consuming fish oil, butyrate decreased mitochondrial cytochrome c (11%; p=0.02) concomitant with an increase in caspase-3 activity (17%; p=0.04) in the distal colon. In fish oil-fed animals, the n-3 fatty acids DHA and EPA were incorporated into cardiolipin at the expense of n-6 fatty acids. Regression analysis revealed a positive relationship between DHA (R=0.49, p=0.03) and EPA (R=0.59, p=0.02) and cytosolic cytochrome c content. As the percentage of DHA and EPA in the cardiolipin increased, the level of cytochrome c in the cytosol increased. These relationships were not seen in rats consuming corn oil and suggest that these results, induced only by the combination of butyrate with fish oil, may lead to increased apoptosis at the promotion stage of colon carcinogenesis via a mitochondria-mediated mechanism.

Colorectal cancer is the second leading cause of death from cancer (men and women combined) in the U.S. and Canada. The mainstay of treatment remains surgical resection and although new agents are constantly emerging to treat colorectal cancer, to date none of the agents have been successful at curing patients with advanced disease. In recent years there has been an increasing interest in the notion that cancers are organized as a hierarchy with the cancer-initiating cell (C-IC or cancer stem cell) existing at the apex. The C-ICs only represent a subset of the total tumour cells; however, research indicates that they are responsible for both the initiation and maintenance of tumour growth. In the studies presented here, we determined that human colon cancers are organized in a hierarchical manner. Furthermore, we prospectively isolated a subset of colon cancer-initiating cells (CC-ICs) based on the expression of the cell surface marker, CD133. The identification of CC-ICs has led to a number of questions concerning the molecular mechanisms driving these cells. Functionally all C-ICs are characterized by their ability to: i) generate a xenograft that histologically resembles the parent tumour from which it was derived, (ii) be serially transplanted in a xenograft assay thereby demonstrating the ability to self-renew and, (iii) generate daughter cells that possess some proliferative capacity but are unable to maintain the cancer because they lack intrinsic regenerative potential. It is becoming evident that cancer cells evolve as a result of their ability to hijack normal self-renewal pathways, a process that can drive malignant transformation. Studying self-renewal in the context of cancer and C-IC maintenance will lead to a better understanding of the mechanisms driving tumour growth. In this work we demonstrate that the inhibitors of differentiation genes (Id1 and Id3) play a central role in driving self-renewal in the CC-IC subset. Furthermore, we demonstrate that this effect is partially mediated through the cdk-inhibitor, p21cip1/waf1.

Colorectal cancer is the second leading cause of death from cancer (men and women combined) in the U.S. and Canada. The mainstay of treatment remains surgical resection and although new agents are constantly emerging to treat colorectal cancer, to date none of the agents have been successful at curing patients with advanced disease. In recent years there has been an increasing interest in the notion that cancers are organized as a hierarchy with the cancer-initiating cell (C-IC or cancer stem cell) existing at the apex. The C-ICs only represent a subset of the total tumour cells; however, research indicates that they are responsible for both the initiation and maintenance of tumour growth. In the studies presented here, we determined that human colon cancers are organized in a hierarchical manner. Furthermore, we prospectively isolated a subset of colon cancer-initiating cells (CC-ICs) based on the expression of the cell surface marker, CD133. The identification of CC-ICs has led to a number of questions concerning the molecular mechanisms driving these cells. Functionally all C-ICs are characterized by their ability to: i) generate a xenograft that histologically resembles the parent tumour from which it was derived, (ii) be serially transplanted in a xenograft assay thereby demonstrating the ability to self-renew and, (iii) generate daughter cells that possess some proliferative capacity but are unable to maintain the cancer because they lack intrinsic regenerative potential. It is becoming evident that cancer cells evolve as a result of their ability to hijack normal self-renewal pathways, a process that can drive malignant transformation. Studying self-renewal in the context of cancer and C-IC maintenance will lead to a better understanding of the mechanisms driving tumour growth. In this work we demonstrate that the inhibitors of differentiation genes (Id1 and Id3) play a central role in driving self-renewal in the CC-IC subset. Furthermore, we demonstrate that this effect is partially mediated through the cdk-inhibitor, p21cip1/waf1.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21446号 / 医博第4413号 / 新制||医||1032(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 萩原 正敏, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM