? ? Dietary sphingolipids such as ceramide and sphingosine promote apoptosis and protect against intestinal tumorigenesis. However, these sphingolipids can be converted within intestinal cells to sphingosine-1- phosphate (S1P), a potent mitogen and angiogenic factor. Thus, a delicate balance exists between these sphingolipid metabolites in gut epithelial cells, alterations of which may influence intestinal tumorigenesis. S1P is irreversibly degraded by the enzyme S1P lyase (SPL), which promotes apoptosis and is required for maximal apoptotic responses. SPL is highly expressed in gut epithelium, where it maintains low S1P levels and promotes normal cell turnover. However, we have found that SPL is downregulated in early Min mouse polyps and in human colon cancer. We hypothesize that SPL downregulation is an early event in intestinal tumorigenesis, representing a critical genetic change that leads to a biochemical switch favoring S1P accumulation in the intestinal mucosa, thereby activating mitogenic and angiogenic signals that contribute to tumor progression. To test this possibility, we have proposed four interrelated specific aims: 1. To characterize the changes in S1P metabolism that accompany intestinal tumorigenesis. We hypothesize that genetic and/or epigenetic changes leading to S1P accumulation are common and early events in intestinal tumorigenesis. Sphingolipid levels and the activity, expression and localization of enzymes involved in S1P metabolism will be compared in human intestinal polyps, CRC, Min mouse polyps of different stages and corresponding uninvolved tissues; 2. To establish whether SPL expression affects the growth characteristics of immortalized and malignant colonic epithelial cells in vitro. SPL expression will be modulated in malignant and nonmalignant colon epithelial cell lines using siRNA, adenoviral and stable expression systems. Effects on proliferation, apoptosis, migration and tumorigenicity and mechanism of SPL action will be determined. 3. To establish whether SPL downregulation increases intestinal tumorigenicity and alters the response to dietary sphingolipids. Available SPL knockout mouse models crossed into the Min mouse background will be used to explore the hypothesis that reduced SPL expression and increased S1P levels in mouse intestinal tissues will enhance the rate, incidence, growth, vascularity and invasive characteristics of intestinal polyposis and limit or reverse the chemopreventive effect of dietary sphingolipids; 4. To determine whether SPL downregulation is reversible early in intestinal tumorigenesis. We will investigate the hypothesis that SPL downregulation is the result of epigenetic changes involving DNA methylation at the SPL genomic locus. The proposed research plan should establish whether SPL downregulation is an early and reversible event that contributes to intestinal tumorigenesis. In accomplishing the proposed studies, we will move closer to our long term goal of employing chemopreventive measures to restore the delicate sphingolipid metabolic balance early in the process of tumorigenesis.Project Narrative Colon cancer is the second most common cancer of men and women and the third leading cause of cancer mortality. It is estimated that $8.4 billion per year is spent caring for patients with this disease in the US. Identifying the molecular and environmental determinants of disease incidence and progression is a critical goal that may help to reveal novel biomarkers for earlier detection and therapeutic intervention to help the 50% or more of patients suffering from advanced or refractory disease. If our central hypothesis is correct, the status of S1P metabolic genes may become informative biomarkers in predicting outcome and the response to therapeutic and dietary regimens. Our Research Plan should establish the validity of this hypothesis and provide information necessary for achieving our ultimate goal, which is to identify and validate novel therapeutic approaches that bypass or reverse the sphingolipid metabolic switch and restore normal S1P metabolism in premalignant and/or malignant cells. ? ? ?
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