The role of this study was to further understand the mechanism of RA mediated endothelial transdifferentiation in SKBR-3 breast cancer cells. We have previously shown that RA induces the expression of genes associated with the endothelial lineage and allows for interaction of RA treated SKBR-3 cells with HUVEC cells in Matrigel. We have shown that ER81 and SOX9 are necessary but not sufficient for inducing VE-cadherin expression. In this study, we have shown that a physiologic dose of RA regulates many genes. Some of the affected pathways are the cancer growth regulation pathway, cardiovascular development, and the hematological and coagulation pathways. Genes associated with proliferation and cancer are decreased, as is expected with RA treatment 
. As we have previously shown, genes associated with the endothelial lineage are induced 
. These results may help explain the mixed results of RA treatment in clinical trials. In individuals with specific molecular profiles, RA treatment, while decreasing cell growth, may also push cells along deleterious differentiation pathways, such as the endothelial pathway. This differentiation may promote vasculogenic mimicry, and thus, an alternative mechanism of tumor vascularization. We utilized several markers of endothelial transdifferentiation – VE-cadherin, COUP-TFII, NRP1, EfnB2, TFPI2, and COX1 - and analyzed the roles of VE-cadherin, COUP-TFII, and NRP1 as master regulators of vasculogenic mimicry. Knockdown of VE-cadherin expression using siRNA was unable to inhibit the expression of endothelial genes when analyzed using both qPCR and microarray analysis. Surprisingly, the loss of VE-cadherin seemed to promote the expression of some pro-angiogenic genes in RA treated SKBR-3 cells. However, down-regulation of VE-cadherin expression is one of the first steps of angiogenesis; thus, this may, in fact, promote further branching morphogenesis. VE-cadherin siRNA also potentiated the RA mediated increase in coagulation/hematological factors, which is in line with the current understanding of the role of VE-cadherin in the coagulation cascade 
. The loss of VE-cadherin did not have an effect on the growth regulatory genes that were affected by RA treatment. We also used siRNA technology to knockdown COUP-TFII and NRP1 and study the expression of endothelial-related genes on the qPCR level. Neither of these genes appears to play a role in regulating the expression of endothelial specific genes.
Given the importance of kinases in various steps of vasculogenesis and angiogenesis, we wanted to know whether kinases played a role in regulating the expression of endothelial-related genes. For these studies, we used VE-cadherin as a marker for endothelial transdifferentiation. We find that treatment of SKBR-3 cells with genistein results in a loss of VE-cadherin expression, which is similar to that observed in HUVECs treated with genistein 
. Another pan-kinase inhibitor, SD705701, had similar effects on VE-cadherin expression at much lower concentrations. To determine whether tyrosine kinases are involved in this process, we utilized a receptor tyrosine kinase array to determine the activity of kinases following RA treatment. We found that RA treatment leads to a loss of tyrosine kinase activity. These results indicate that while kinase activity is necessary for VE-cadherin expression, it is independent of receptor tyrosine kinases.
Following closer analysis of our microarray data, we identified several members of the TGF-β family of cytokines that are induced as a result of RA treatment. SD705701 is known to have activity against serine/threonine kinases, and genistein, while inducing the expression of TGF-β has also been shown to inhibit TGFβR1 mediated phosphorylation of p38 MAPK 
. TGF-β treatment, alone, is unable to induce VE-cadherin expression; however, inhibition of TGFβR1 activity using the specific kinase inhibitor, SB431542, inhibits RA-induced VE-cadherin expression. These results imply that activity of TGFβR1 is necessary but not sufficient for VE-cadherin expression, and presumably complete endothelial transdifferentiation. Additionally, the activation of ALK1 type I receptors induces the phosphorylation of SMAD1, SMAD5, and transcription of Id1, while activation of ALK5 receptors induces the phosphorylation of SMAD2 and the transcription of PAI-1 
. ALK1 has been shown to stimulate endothelial proliferation and migration, while ALK5 activation inhibits these processes. Our microarray analysis indicates that Id1 expression is induced as a result of RA treatment. Thus, the ALK1 type I receptor may also play a role in RA induced endothelial transdifferentiation.
We have also shown that network formation and cell fusion are distinct processes during vasculogenic mimicry. VE-cadherin expression, as expected, plays a role in mediating the formation of cell-cell adhesions and cell fusion. Loss of VE-cadherin expression either directly by VE-cadherin siRNA or indirectly via inhibition of TGFβR1 kinase is able to inhibit cell fusion in Matrigel, but does not appear to affect the formation of primitive network-like structures. On the other hand, loss of COUP-TFII expression using siRNA is unable to inhibit cell fusion following RA treatment, but profoundly inhibits network formation. Proper vascular formation by endothelial cells requires both processes. Network formation without cell fusion may not allow for the proper conductance of nutrients to a tumor. In the same way, cell fusion without network formation will not allow the lumen-like spaces formed by RA treated SKBR-3 cells to properly connect with the host vasculature. Thus, both processes may have to occur to allow for the deleterious effects in cancer patients.
The presence of vasculogenic mimicry is a negative prognosticator of patient outcomes in human cancers. We have previously identified a dietary agent, vitamin A, in regulating this process in breast cancer. We now propose that VE-cadherin expression induced by RA is regulated by ER81, SOX9, and TGFβR1 activity, while network formation is regulated by COUP-TFII ().
Schematic representation of factors regulating RA-mediated endothelial transdifferentiation.