Table 1.
Top 25 Endothelial Related Genes Regulated by RA.
Table 2.
Endothelial-Related Genes Regulated by RA and VE-cadherin.
Figure 1.
VE-cadherin, COUP-TFII, and NRP1 are not master regulators of retinoic acid mediated vasculogenic mimicry.
Following siRNA transfection with luciferase control or siRNA targeting VE-cadherin, COUP-TFII, or NRP1, cells were treated with control ethanol or RA for 48 hours. qPCR analysis was performed to assess knockdown of VE-cadherin (a), COUP-TFII (b), NRP1 (c), COX1 (d), TFPI2 (e), and EFNB2 (f). Samples have been labeled as follows: 1 - Control, Luciferase siRNA; 2 - RA, Luciferase siRNA; 3 - Control, VE-cadherin siRNA; 4 - RA, VE-cadherin siRNA; 5 - Control, COUP-TFII siRNA; 6 - RA, COUP-TFII siRNA; 7 - Control, NRP1 siRNA; 8 - RA, NRP1 siRNA.
Figure 2.
Inhibition of VE-cadherin expression occurs with treatment of pan-kinase inhibitors but is independent of tyrosine kinase activity.
Pre-treatment of SKBR-3 cells for 1 hour with 10 µM, 50 µM or 100 µM of Genistein prior to a 48 hour RA treatment results in a loss of VE-cadherin expression and tyrosine phosphorylation (a). Pre-treatment with the novel pan-kinase inhibitor, SD705701, also results in a loss of VE-cadherin expression in a dose-dependent manner (b). A Human Phospho-Receptor Tyrosine Kinase array demonstrates that RA treatment results in the loss of tyrosine kinase phosphorylation (c). A key for this array is as follows: Control - A1, A2, A23, A24, F1, F2, F23, F24; EGFR - B1, B2; EphB4 - E9, E10; ErbB2 - B3, B4; ERBB3 - B5, B6; EGFR - B11, B12, B13, B14; Dtk - B23, B24; Mer - C1, C2; Tie-2 - D1, D2; TrkA - D3, D4; VEGFR2 - D11, D12. Inhibition of ERBB2 activity with AG825 is unable to inhibit VE-cadherin expression (d). Samples are labeled as follows: 1 - Control; 2 - RA; 3 - Control, Ethanol; 4 - RA, Ethanol; 5 - Control, 100 µm Genistein; 6 - RA, 100 µM Genistein; 7 - Control, DMSO; 8 - RA, DMSO; 9 - Control, 50 µM AG825; RA - 50 µM AG825. Inhibition of DTK expression with siRNA (e) is unable to inhibit VE-cadherin expression (f).
Figure 3.
TGFβR1 kinase activity is necessary for VE-cadherin expression.
A one hour pre-treatment with SB431542, a TGFβR1 kinase specific inhibitor, prior to a 48 hour RA treatment results in a loss of VE-cadherin expression in a dose-dependent manner (a). Treatment with SB431542 decreases PAI-1 activity, confirming TGFβR1 blockade (b).The samples are as follows: 1 - Control; 2 - RA; 3 - Control, 10 µM SB431542; 4 - RA, 10 µM SB431542; 5 - Control - 50 µM SB431542; 6 - RA, 50 µM SB431542. Treatment with 10 ng/ml of TGFβ, alone, does not induce VE-cadherin expression in the absence of RA treatment (c).
Table 3.
TGFβ Family Members Regulated by RA Treatment.
Figure 4.
COUP-TFII is necessary for network formation while TGFβR1 activity is necessary for cell fusion.
Low power images (20×) of control siRNA transfected untreated SKBR-3 cells show that they grow as clusters of individual cells in Matrigel (a) without any cell fusion (inset, 40×). Upon RA treatment, the cells begin to form networks (b) with cell fusion (inset). Loss of VE-cadherin expression using VE-cadherin siRNA does not affect untreated SKBR-3 cells (c, inset). However, a loss of VE-cadherin expression with concomitant RA treatment results in a preservation of network formation (d), but a loss of cell fusion (inset). Like the VE-cadherin siRNA treatment, COUP-TFII siRNA treatment does not affect the growth patterns of untreated SKBR-3 cells (e, inset). However, treatment with COUP-TFII siRNA does not affect the fusion of RA treated SKBR-3 cells (f, inset), but it does inhibit network formation (f). Untreated SKBR-3 cells also grow as clusters of unfused individual cells in the absence of RA treatment (g, inset), and RA treatment results in network formation and cell fusion (h, inset). Pre-treatment with 50 µM SB431542 does not affect the growth patterns of untreated SKBR-3 cells (i, inset). Pre-treatment with SB431542 prior to RA treatment does not alter network formation (j), but inhibits cell fusion (j, inset).
Figure 5.
Schematic representation of factors regulating RA-mediated endothelial transdifferentiation.