Homeobox genes represent a class of transcription factors important in embryogenesis, organogenesis, cell growth and differentiation, and cell migration. However, there is little known about their role in regulating endothelial cell (EC) phenotype in response to proangiogenic factors secreted by breast cancer, although at least two homeobox genes (HOXD3 and HOXD10) have been implicated in inducing the angiogenic phenotype in ECs. We are therefore testing the hypothesis that the homeobox gene Gax regulates breast cancer-induced angiogenesis through its ability to regulate the expression of downstream target genes in ECs. Using in vitro tube formation assays, we have found that Gax expression inhibits in vitro angiogenesis. Moreover, by quantitative real time reverse transcriptase real time PCR, we have found that Gax expression is downregulated by proangiogenic factors, while cDNA micorarray analysis demonstrates that Gax downregulates pro-angiogenic adhesion molecules in ECs and upregulates the cyclin-dependent kinase inhibitor p19INK4D. More importantly, Gax expression downregulates NF-B activity in ECs. These observations will allow us to study the mechanism of Gax-mediated activation or repression of their expression to be studied and will form the basis for future studies that will examine in more detail the mechanism by which Gax activates downstream target genes and the detailed signaling pathways involved in this activation. Given the profound effect Gax has on endothelial cell activation, it is likely that these studies will identify new molecular targets for the antiangiogenic therapy of breast cancer. Ultimately, these same techniques will be applied to other homeobox genes implicated in regulating EC phenotype during breast cancer-induced angiogenesis.