Epidemiological studies reveal vitamin D-insufficient individuals with low serum concentrations of 25-hydroxyvitamin D, the major circulating form of the vitamin, are at increased risk of developing colon cancer. Growth of colon cancer cells in vitro is inhibited by supraphysiologic doses of the active hormonal form of vitamin D, 1,25-dihydroxyvitamin D3. Unfortunately, doses of 1,25-dihydroxyvitamin D3 that might treat colon tumors in vivo also stimulate the vitamin's classic effects on calcium homeostasis, resulting in hypercalcemia. Our previously funded grant explored whether a glucuronide of 1,25-dihydroxyvitamin D3 could deliver the hormone directly to the colon to prevent tumor growth while reducing the risk of a hypercalcemic response. Conjugating a glucuronide to vitamin D molecules in a ¿-linkage renders it biologically inactive and resistant to mammalian digestive enzymes. Upon reaching the lower intestinal tract ¿-glucuronidase enzymes produced by resident bacteria cleave off the glucuronide and liberate the vitamin D molecule. Despite evidence that prolonged treatment with 1,25-dihydroxyvitamin D3 glucuronide was regulating gene expression almost exclusively in the lower intestine without causing hypercalcemia; our data reveal the treatment was unable to slow tumor growth in either of two murine colon cancer models tested. However, in CD1 mice treated with azoxymethane/dextran sodium sulfate, 25-hydroxyvitamin D3 glucuronide, included as a negative control, yielded a ca. 60% reduction in colon tumor load with no hypercalcemia. This treatment also increased expression of vitamin D-dependent genes in the colon. Our renewal proposal focuses on exploring the ability of 25-hydroxyvitamin D3 glucuronide to prevent the growth of, or treat existing, colorectal tumors. Aim 1 will mirror our earlier study using CD1 mice fed a Western-style diet, but we will now titrate and contrast different levels of 25-hydroxyvitamin D3 glucuronide compared to 25-dihydroxyvitamin D3 to determine doses required to inhibit colon tumor growth. Aim 2 will assess dietary 25-hydroxyvitamin D3 glucuronide or 25-hydroxyvitamin D3 in the same CD1 mouse model when treatment is delayed and the compounds are used on already developed colon tumors. Data from the prior grant period revealed the unexpected finding that 25- hydroxyvitamin D3 glucuronide is able to suppress colon tumor growth, alter colon gene expression and not induce hypercalcemia. The current proposal will provide much-needed basic information on the therapeutic potential of 25-hydroxyvitamin D3 glucuronide to slow the growth of, or treat, colon tumors. We are testing the possibility that studies linking low serum 25-hydroxyvitamin D with colon cancer are the result of a direct effect of 25-hydroxyvitamin D on colon cells, independent of being a precursor to 1,25-dihydroxyvitamin D.