Angiogenesis is critical to tumor progression. The homeobox gene GAX inhibits angiogenesis in vascular endothelial cells (ECs). We have identified a microRNA (miR-130a) that regulates GAX expression and hypothesized that it plays a major role in modulating GAX activity in ECs. A 280-bp fragment from the GAX 3-untranslated region (3-UTR) containing 2 miR-130a targeting sites was observed to be required for the rapid downregulation of GAX expression by serum and proangiogenic factors, whereas the activity of the GAX promoter did not vary with exposure to serum or proangiogenic factors. This same 280-bp sequence in the GAX 3-UTR cloned into the psi-CHECK2-Luciferase vector mediated serum-induced down-regulation of the reporter gene when placed 3 of it. Finally, forced expression of miR-130a inhibits GAX expression through this specific GAX 3-UTR sequence. A genome-wide search for other possible miR-130a binding sites revealed an miR-130a targeting site in the 3-UTR of the antiangiogenic homeobox gene HOXA5, the expression and antiangiogenic activity of which are also inhibited by miR-130a. From these data, we conclude that miR-130a is a regulator of the angiogenic phenotype of vascular ECs largely through its ability to modulate the expression of GAX and HOXA5. IntroductionAngiogenesis is critical to the growth, invasion, and metastasis of human tumors. Key to this process is the vascular endothelial cell (EC), 1 which in health responds to a balance between proangiogenic and antiangiogenic factors secreted by various cells to maintain blood vessel homeostasis. This balance determines whether ECs become angiogenic in response to normal physiologic signals in processes as diverse as wound repair, the menstrual cycle, embryogenesis, and organogenesis. 2,3 During carcinogenesis, tumors hijack angiogenesis by secreting proangiogenic factors to supply themselves with the oxygen and nutrients necessary for their continued growth, a transition known as the "angiogenic switch." 2,3 Because targeting angiogenesis has emerged as a promising avenue of treatment for malignancies, 4 understanding the transcriptional regulation of the angiogenic phenotype in ECs has become increasingly important.Not surprisingly, given their diverse roles in development, homeobox genes are involved in the regulation of this transition between the resting and "activated," or "angiogenic," phenotype in ECs. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Proangiogenic homeobox genes [5][6][7][8]14,20 and antiangiogenic homeobox genes have been described, 12,13,[15][16][17][18] as have differences in homeobox gene expression and function in ECs from different vascular beds. 17,19,21,22 The diverged homeodomain gene GAX (also known as MEOX2) is also expressed in ECs, 12 and previous work in our laboratory has implicated GAX in inhibiting nuclear factor-B (NF-B) signaling as well as angiogenesis in ECs, both in vitro and in vivo. 10,13,17 Most recently, we reported that GAX induces G 0 cellcycle arrest by activating the expression of p21 WAF...

Angiogenesis, the formation of new capillaries from pre-existing vessels, is essential for tumour progression. Angiostatin, a proteolytic fragment of plasminogen that was first isolated from the serum and urine of tumour-bearing mice, inhibits angiogenesis and thereby growth of primary and metastatic tumours. Radiotherapy is important in the treatment of many human cancers, but is often unsuccessful because of tumour cell radiation resistance. Here we combine radiation with angiostatin to target tumour vasculature that is genetically stable and therefore less likely to develop resistance. The results show an antitumour interaction between ionizing radiation and angiostatin for four distinct tumour types, at doses of radiation that are used in radiotherapy. The combination produced no increase in toxicity towards normal tissue. In vitro studies show that radiation and angiostatin have combined cytotoxic effects on endothelial cells, but not tumour cells. In vivo studies show that these agents, in combination, target the tumour vasculature. Our results provide support for combining ionizing radiation with angiostatin to improve tumour eradication without increasing deleterious effects.

gax, a diverged homeobox gene expressed in vascular smooth muscle cells (VSMCs), is down-regulated in vitro by mitogen stimulation and in vivo in response to vascular injury that leads to cellular proliferation. Recombinant Gax protein microinjected into VSMCs and fibroblasts inhibited the mitogen-induced entry into S-phase when introduced either during quiescence or early stages of G1. Overexpression of gax with a replication-defective adenovirus vector resulted in Go/G 1 cell cycle arrest of VSMCs and fibroblasts. The gax-induced growth inhibition correlated with a p53-independent up-regulation of the cyclin-dependent kinase inhibitor p21. Gax overexpression also led to an association of p21 with cdk2 complexes and a decrease in cdk2 activity. Fibroblasts deficient in p21 were not susceptible to a reduction in cdk2 activity or growth inhibition by gax overexpression. Localized delivery of the virus to denuded rat carotid arteries significantly reduced neointima formation and luminal narrowing. These data indicate that gax overexpression can inhibit cell proliferation in a p21-dependent manner and can modulate injury-induced changes in vessel wall morphology that result from excessive cellular proliferation.

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Adult vascular smooth muscle cells dedifferentiate and reenter the cell cycle in response to growth factor stimulation. Here we describe the molecular cloning from vascular smooth muscle, the structure, and the chromosomal location of a diverged homeobox gene, Gax, whose expression is largely confined to the cardiovascular tissues of the adult. In quiescent adult rat vascular smooth muscle cells, Gax mRNA levels are down-regulated as much as 15-fold within 2 h when these cells are induced to proliferate with platelet-derived growth factor (PDGF) or serum growth factors. This reduction in Gax mRNA is transient, with levels beginning to rise between 8 and 24 h after mitogen stimulation and returning to near normal by 24 to 48 h. The Gax down-regulation is dose dependent and can be correlated with the mitogen's ability to stimulate DNA synthesis. PDGF-AA, a weak mitogen for rat vascular smooth muscle cells, did not affect Gax transcript levels, while PDGF-AB and -BB, potent mitogens for these cells, were nearly as effective as fetal bovine serum. The removal of serum from growing cells induced Gax expression fivefold within 24 h. These data suggest that Gax is likely to have a regulatory function in the G0-to-G1 transition of the cell cycle in vascular smooth muscle cells.

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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)

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