The growth and metastasis of tumors are heavily dependent on angiogenesis, but much of the transcriptional regulation of vascular endothelial cell gene expression responsible for angiogenesis remains to be elucidated. The homeobox gene Gax is expressed in vascular endothelial cells and inhibits proliferation and tube formation in vitro. We hypothesized that Gax is a negative transcriptional regulator of the endothelial cell angiogenic phenotype and studied its regulation and activity in vascular endothelial cells. Several proangiogenic factors caused a rapid down-regulation of Gax mRNA in human vascular endothelial cells, as did conditioned media from breast cancer cell lines. In addition, Gax expression using a replication-deficient adenoviral vector inhibited human umbilical vein endothelial cell migration toward proangiogenic factors in vitro and inhibited angiogenesis in vivo in Matrigel plugs. To identify putative downstream targets of Gax, we examined changes in global gene expression in endothelial cells due to Gax activity. Gax expression resulted in changes in global gene expression consistent with a quiescent, nonangiogenic phenotype, with increased expression of cyclin kinase inhibitors and decreased expression of genes implicated in endothelial cell activation and angiogenesis. Further analysis revealed that Gax downregulated numerous nuclear factor-k kB (NF-k kB) target genes and decreased the binding of NF-k kB to its target sequence in electrophoretic mobility shift assays. To our knowledge, Gax is the first homeobox gene described that inhibits NF-k kB activity in vascular endothelial cells. Because NF-k kB has been implicated in endothelial cell activation and angiogenesis, the down-regulation of NF-k kB-dependent genes by Gax suggests one potential mechanism by which Gax inhibits the angiogenic phenotype. (Cancer Res 2005; 65(4): 1414-24)

Tumors secrete pro-angiogenic factors to induce the ingrowth of blood vessels from the surrounding stroma, the end targets of which are vascular endothelial cells (ECs). The homeobox gene GAX inhibits angiogenesis and induces p21 WAF1/CIP1 expression in vascular ECs. To elucidate the mechanism through which GAX activates p21 WAF1/CIP1 expression, we constructed GAX cDNAs with deletions of the N-terminal domain, the homeodomain, or the C-terminal domain and then assessed these constructs for their ability to activate p21 WAF1/CIP1 . There was an absolute requirement for the homeodomain, whereas deleting the C-terminal domain decreased but did not abolish transactivation of the p21 WAF1/CIP1 promoter by GAX. Deleting the N-terminal domain did abolish transactivation. Next, we performed chromatin immunoprecipitation and found, ϳ15 kb upstream of the p21 WAF1/CIP1 ATG codon, an ATTA-containing GAX-binding site (designated A6) with a sequence similar to that of other homeodomain-binding sites. GAX was able to bind to A6 in a homeodomain-dependent manner and thereby activate the expression of a reporter gene coupled to this sequence, and this activation was abolished by mutating specific residues in this sequence. On the basis of the sequence of A6, we were then able to locate other ATTA-containing sequences that also bound GAX and activated transcription in reporter constructs. Finally, we found that the ability of these GAX deletions to induce G 0 /G 1 arrest correlates with their ability to transactivate the p21 WAF1/CIP1 promoter. We conclude that GAX activates p21 WAF1/CIP1 through multiple upstream AT-rich sequences. Given the multiple biological activities of GAX in regulating EC function, identification of a putative GAX-binding site will allow the study of how GAX activates or represses other downstream targets to inhibit angiogenesis.Angiogenesis is critical to the growth, invasion, and metastasis of human tumors because the diffusion of oxygen and nutri-

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