Tumor growth depends on a variety of secreted growth and angiogenic factors. Cellular responses to many of these factors rely on the presence of cell surface heparan sulfate proteoglycans, which serve as co-receptors for several signaling pathways. Mounting evidence suggests that inhibition of heparan sulfate biosynthesis could bock the ability of tumor cells to respond growth and angiogenic stimuli. This hypothesis will be tested by altering heparan sulfate synthesis in tumor cells and model organisms. The novel molecular target in this proposal is the biosynthetic pathway for heparan sulfate. Blocking or reducing the expression of specific genes in the pathway by genetic strategies is needed to validate specific enzymatic targets for eventual pharmacologic intervention. Screening technologies are in place to identify compounds derived from natural products and synthetic libraries. To achieve the long-term goal of developing novel chemotherapeutic agents, we have the following specific aims: 1. Validate the heparan sulfate biosynthetic pathway as a target for cancer drug therapy. Previous studies of Chinese hamster ovary cells suggest that ablation of heparan sulfate formation prevents tumor formation in athymic mice. To extend these studies to more common tumor lines, heparan sulfate biosynthesis will be altered genetically using classic mutagenesis, anti-sense methods, new RNA interference procedures, and chimeraplasty (RNA/DNA hybrids). These studies will focus on EXT1 and NDST1 since these enzymes are responsible for the polymerization of the polysaccharide chain and initiation of all downstream modification reactions. 2. Identify new targets in the heparan sulfate biosynthetic pathway. Although many of the genes that encode the biosynthetic enzymes for heparan sulfate assembly have been identified, several critical components have not. To characterize the role of these other components and to study their role in tumor formation, new mutants of CHO cells will be isolated in the C5 epimerase and EXT2, a subunit of the co- polymerase complex. RNAi methods recently developed for Drosophila tissue culture cells will be employed to determine the function of D-Ext2, D-epimerase, and developed for Drosophila tissue culture cells will be employed to determine the function of D-Ext2, D-epimerase, and D-Ext1 in heparan sulfate biosynthesis. 3. Identify and characterize inhibitors of heparan sulfate biosynthesis. A high throughput screening method was been developed to identify compounds that inhibit heparan sulfate biosynthesis in cultured cells. To date, over 60,000 samples have been screened from the Natural Products Branch of the Developmental Therapeutics Program of the NCI and active extracts have been identified. Parallel screening of synthetic libraries is planned. Assays for screening drug candidates using sensitized genetic backgroups in Drosophila will be established and inhibition of PTEN-mediated overgrowth will be measured. The molecular target of active compounds will be examined by characterizing intermediates that accumulate in cells or tissues after drug treatment. Identification of active components coupled with large scale synthesis would allow us to test if the compounds have anti-tumor activity in mice.
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