Genomic analysis of signaling by vitamin D3 and its analogs in head and neck SCC25

The hormonal form of vitamin D3, 1,25-dihydroxyvitamin D 3 (1,25(OH)2D3) is well known for its role in regulating calcium homeostasis. However, many studies have shown 1,25(OH) 2D3 and its analogue EB1089 have broad potential as anticancer agents due to their antiproliferative and pro-differentiating activities. Here, we examined the effects of 1,25(OH)2D3 and EB1089 on proliferation and target gene regulation of human head and neck squamous cell carcinoma (SCC) lines SCC4, SCC9, SCC15 and SCC25 and mouse AT84 SCC cells. A range of sensitivities to 1,25(OH)2D3 and EB1089 was observed, from complete G0/G1 arrest of SCC25 cells to only 50% inhibition of SCC9 cell growth. We have analyzed the molecular mechanisms underlying the antiproliferative effects of EB1089 on SCC25 cells by screening over 10,000 genes on cDNA and oligonucleotide arrays. These studies have identified ~200 novel target genes of 1,25(OH)2D3/EB1089, many of which are essential for normal DNA repair, are markers of cellular differentiation, or are key cell cycle regulators. One of these is the growth arrest and DNA damage (GADD45alpha) gene. Induction of the GADD45alpha gene and its encoded protein in EB1089-treated cells was confirmed by northern and western blotting, respectively. Moreover, while expression of proliferating cell nuclear antigen (PCNA) was reduced in EB1089-treated cells, coimmunoprecipitation studies revealed increased association between GADD45alpha and PCNA in treated cells, consistent with the capacity of GADD45alpha to stimulate DNA repair. Our data also show that amphiregulin, a member of the epidermal growth factor family, is a 1,25(OH)2D3 target gene, and suggest that its induction may contribute to the growth inhibitory effects of 1,25(OH) 2D3. In addition, we show that 1,25(OH)2D 3 analogues induce expression of the cyclin-dependent kinase inhibitor p27KIP1 in different cell types by inhibiting expression of subunits of the SCFSKP2 ubiquitin ligaThese studies (1) suggest that differences in action of the EB1089 and 1,25(OH)2D3 arise more from their relative sensitivities to metabolism and than from differing effects on VDR function, (2) substantially expand the number of known D3 target genes in cancer cells (3) enhance our understanding of the molecular mechanism controlling the antiproliferative effects of 1,25(OH)2D3 analogs in cancer (4) provide a number of marker genes that will be useful in assessing the sensitivity of tumor samples to the antiproliferative effects of 1,25(OH)2D 3