Retinoic Acid Mediates Regulation of Network Formation by COUP-TFII and VE-Cadherin Expression by TGFβ Receptor Kinase in Breast Cancer Cells

Tumor development, growth, and metastasis depend on the provision of an adequate vascular supply. This can be due to regulated angiogenesis, recruitment of circulating endothelial progenitors, and/or vascular transdifferentiation. Our previous studies showed that retinoic acid (RA) treatment converts a subset of breast cancer cells into cells with significant endothelial genotypic and phenotypic elements including marked induction of VE-cadherin, which was responsible for some but not all morphological changes. The present study demonstrates that of the endothelial-related genes induced by RA treatment, only a few were affected by knockdown of VE-cadherin, ruling it out as a regulator of the RA-induced endothelial genotypic switch. In contrast, knockdown of the RA-induced gene COUP-TFII prevented the formation of networks in Matrigel but had no effect on VE-cadherin induction or cell fusion. Two pan-kinase inhibitors markedly blocked RA-induced VE-cadherin expression and cell fusion. However, RA treatment resulted in a marked and broad reduction in tyrosine kinase activity. Several genes in the TGFβ signaling pathway were induced by RA, and specific inhibition of the TGFβ type I receptor blocked both RA-induced VE-cadherin expression and cell fusion. Together these data indicate a role for the TGFβ pathway and COUP-TFII in mediating the endothelial transdifferentiating properties of RA.     

Endothelial cadherins in cancer

Cadherins are cell adhesion receptors that play important roles in embryogenesis and tissue homoeostasis. Endothelial cells express various members of the cadherin superfamily, in particular vascular endothelial (VE-) cadherin, which is the main adhesion receptor of endothelial adherens junctions and neural (N-) cadherin, which is normally localized outside the junctions and may mediate adhesion between endothelial cells and non-endothelial cells. Dysregulation of cadherin expression has been implicated in tumor progression, in particular the loss of epithelial (E-) cadherin expression or function and the gain of N-cadherin. Moreover, more recently, aberrant expression of VE-cadherin was observed in certain cancer types. In breast carcinoma, VE-cadherin was shown to promote tumor cell proliferation and invasion through enhancing TGF-β signaling. Thus, in breast cancer, the cadherin switch involves another player, vascular endothelial cadherin, which is part of an intricate interplay of classical cadherins in breast cancer progression.     

Alteration of Interendothelial Adherens Junctions Following Tumor Cell–Endothelial Cell Interactionin Vitro

The integrity of the vascular endothelium is mainly dependent upon the organization of interendothelial adherens junctions (AJ). These junctions are formed by the homotypic interaction of a transmembrane protein, vascular endothelial cadherin (VE-cadherin), which is complexed to an intracellular protein network including α-, β-, and γ-catenin. Additional proteins such as vinculin and α-actinin have been suggested to link the VE-cadherin/catenin complex to the actin-based cytoskeleton. During the process of hematogenous metastasis, circulating tumor cells must disrupt these intercellular junctions in order to extravasate. In the present study, we have investigated the influence of tumor cell–endothelial cell interaction upon interendothelial AJ. We show that human breast adenocarcinoma cells (MCF-7), but not normal human mammary epithelial cells, induce a rapid endothelial cell (EC) dissociation which correlates with the loss of VE-cadherin expression at the site of tumor cell–EC contact and with profound changes in vinculin distribution and organization. This process could not be inhibited by metalloproteinase nor serine protease inhibitors. Immunoprecipitations and Western blot analysis demonstrate that the overall expression of VE-cadherin and vinculin as well as the composition of the VE-cadherin/catenins complex are not affected by tumor cells while the tyrosine phosphorylation status of proteins within the complex is significantly altered. Our data suggest that tumor cells modulate AJ protein distribution and phosphorylation in EC and may, thereby, facilitate EC dissociation.     

Vascular endothelial growth factor signaling in VE-cadherin expression and tube-like formation by rheumatoid arthritic synovial fibroblast-like cells

An increase in the vasculature is one of most representative changes in the synovial tissue of joints in rheumatoid arthritis (RA) and is closely associated with disease progression. Although the vasculatures are believed to be a result of VE-cadherin-dependent angiogenesis and a possible therapeutic target of the disease, synovial fibroblastic cells express VE-cadherin and form tube-like structures, suggesting that vasculatures in RA synovium may not simply result from angiogenesis. This paper analyzes a mechanism of VE-cadherin expression by rheumatoid arthritic synovial fibroblast-like cells (RSFLs) and their involvement in the tube-like formation. A representative angiogenic factor, vascular endothelial growth factor (VEGF), and its binding to a predominant receptor (VEGFR2) activated VE-cadherin expression and the signaling pathways of ERK/MAPK and PI3K/AKT/mTOR. Treatment of RSFLs with signaling pathway inhibitors, VEGFR2 siRNA and a VEGF-antagonizing mimicking peptide inhibited VE-cadherin expression dose-dependently. VEGF-stimulated tube-like formation by RSFLs on Matrigel was hindered by the mimicking peptide and inhibitor treatment. This data demonstrates that RSFLs activated by VEGF binding of VEGFR2 express VE-cadherin and formed tube-like structure under the control of ERK/MAPK and PI3K/AKT/mTOR pathways suggesting that the inhibition suppresses vascular development in RA synovium.     

Paracrine VEGF/VE-cadherin action on ovarian cancer permeability

Ascites formation associated with neoplasms is most likely due to increased vascular permeability, a process in which vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) plays a pivotal role. We hypothesized that tumor-derived VEGF/VPF modulates ascites formation through a paracrine effect on both tumor and peritoneal vessels. We investigated human vascular endothelial permeability using a newly developed dual-chamber permeability assay by co-culturing human umbilical vein cells with and without ovarian cancer cell lines (OVCAR-3, Hey-A8, and OCC-1) in the presence or absence of a human VEGF monoclonal antibody and VE-cadherin function-blocking antibody. This method permits determination of mechanisms by which substances released from neoplasms and other sources of vascular endothelial cell secretagogues modulate vascular permeability and likely other pathologic states.     

Retinoids arrest breast cancer cell proliferation: retinoic acid selectively reduces the duration of receptor tyrosine kinase signaling

Retinoic acid (RA) induces cell cycle arrest of hormone-dependent human breast cancer (HBC) cells. Previously, we demonstrated that RA-induced growth arrest of T-47D HBC cells required the activity of the RA-induced protein kinase, protein kinase Calpha (PKCalpha) [J. Cell Physiol. 172 (1997) 306]. Here, we demonstrate that RA treatment of T-47D cells interfered with growth factor signaling to downstream, cytoplasmic and nuclear targets. RA treatment did not inhibit epidermal growth factor (EGF) receptor activation but resulted in rapid inactivation. The lack of sustained EGFR activation was associated with transient rather than sustained association of the EGFR with the Shc adaptor proteins and activation of Erk 1/2 and with compromised induction of expression of immediate early response genes. Inhibiting the activity of PKCalpha, a retinoic acid-induced target gene, prevented the effects of RA on cell proliferation and EGF signaling. Constitutive expression of PKCalpha, in the absence of RA, decreased cell proliferation and decreased EGF signaling. RA treatment increased steady-state levels of the protein tyrosine phosphatase PTP-1C and all measured effects of RA on EGF receptor function were reversed by the tyrosine phosphate inhibitor orthovanadate. These results indicate that RA-induced target genes, particularly PKCalpha, prevent sustained growth factor signaling, uncoupling activated receptor tyrosine kinases and nuclear targets that are required for cell cycle progression.     

Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis.

Vascular endothelial cadherin, VE-cadherin, mediates adhesion between endothelial cells and may affect vascular morphogenesis via intracellular signaling, but the nature of these signals remains unknown. Here, targeted inactivation (VEC-/-) or truncation of the beta-catenin-binding cytosolic domain (VECdeltaC/deltaC) of the VE-cadherin gene was found not to affect assembly of endothelial cells in vascular plexi, but to impair their subsequent remodeling and maturation, causing lethality at 9.5 days of gestation. Deficiency or truncation of VE-cadherin induced endothelial apoptosis and abolished transmission of the endothelial survival signal by VEGF-A to Akt kinase and Bcl2 via reduced complex formation with VEGF receptor-2, beta-catenin, and phosphoinositide 3 (PI3)-kinase. Thus, VE-cadherin/ beta-catenin signaling controls endothelial survival.     

Hydrostatic pressure influences morphology and expression of VE-cadherin of vascular endothelial cells

Bovine aortic endothelial cells (BAECs) were exposed to hydrostatic pressures of 50, 100, and 150 mmHg and changes in morphology and expression of vascular endothelial (VE)-cadherin were studied. After exposure to hydrostatic pressure, BAECs exhibited elongated and tortuous shape without predominant orientation, together with the development of centrally located, thick stress fibers. Pressured BAECs also exhibited a multilayered structure unlike those under control conditions and showed a significant increase in proliferation compared with control cells. Western blot analysis demonstrated that protein level of VE-cadherin were significantly lower under pressure conditions than under control conditions. Inhibition of VE-cadherin expression, using an antibody to VE-cadherin, induced the formation of numerous randomly distributed intercellular gaps, elongated and tortuous shapes, and multilayering. These responses were similar to those of pressured BAECs. The exposure of BAECs to hydrostatic pressure may therefore downregulate the expression of VE-cadherin, resulting in loss of contact inhibition followed by increased proliferation and formation of a multilayered structure.     

Stable RNA interference of ErbB-2 gene synergistic with epirubicin suppresses breast cancer growth in vitro and in vivo

Overexpression of human epidermal growth factor receptor-2 (Her2, ErbB-2) contributes to the progression and metastasis of breast cancer, implying that Her2 gene is a suitable target of RNA interference (RNAi) for breast cancer therapy. Here, we employed plasmid-mediated expression of 2 different Her2-shRNAs (pU6-Her2shRNAs) efficiently silenced the target gene expression on Her2 expressing SKBR-3 breast cancer cells in both mRNA and protein levels. Consequently, pU6-Her2shRNA increased apoptosis and reduced proliferation of SKBR-3 cells assayed by TUNEL and MTT, respectively. In vivo, intra-tumor injection of pU6-Her2shRNA inhibited the growth of SKBR-3 tumors inoculated subcutaneously in nude mice. Furthermore, pU6-Her2shRNA synergized the tumor suppression effect of epirubicin to SKBR-3 cells in vitro and implanted subcutaneously in nude mice. Therefore, we concluded that stable silencing of Her2 gene expression with plasmid expressing shRNA may hold great promise as a novel therapy for Her2 expressing breast cancers alone or in combination with anthracycline chemotherapy.     

Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-beta and retinoic acid-induced cancer cell death

Combination of retinoic acids (RAs) and interferons (IFNs) has synergistic apoptotic effects and is used in cancer treatment. However, the underlying mechanisms remain unknown. Here, we demonstrate that mitochondrial respiratory chain (MRC) plays an essential role in the IFN-beta/RA-induced cancer cell death. We found that IFN-beta/RA upregulates the expression of MRC complex subunits. Mitochondrial-nuclear translocation of these subunits was not observed, but overproduction of reactive oxygen species (ROS), which causes loss of mitochondrial function, was detected upon IFN-beta/RA treatment. Knockdown of GRIM-19 (gene associated with retinoid-interferon-induced mortality-19) and NDUFS3 (NADH dehydrogenase (ubiquinone) Fe-S protein 3), two subunits of MRC complex I, by siRNA in two cancer cell lines conferred resistance to IFN-beta/RA-induced apoptosis and reduced ROS production. In parallel, expression of late genes induced by IFN-beta/RA that are directly involved in growth inhibition and cell death was also repressed in the knockdown cells. Our data suggest that the MRC regulates IFN-beta/RA-induced cell death by modulating ROS production and late gene expression.     

Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling

To remodel endothelial cell-cell adhesion, inflammatory cytokine- and angiogenic growth factor-induced signals impinge on the vascular endothelial cadherin (VE-cadherin) complex, the central component of endothelial adherens junctions. This study demonstrates that junction remodeling takes place at a molecularly and phenotypically distinct subset of VE-cadherin adhesions, defined here as focal adherens junctions (FAJs). FAJs are attached to radial F-actin bundles and marked by the mechanosensory protein Vinculin. We show that endothelial hormones vascular endothelial growth factor, tumor necrosis factor α, and most prominently thrombin induced the transformation of stable junctions into FAJs. The actin cytoskeleton generated pulling forces specifically on FAJs, and inhibition of Rho-Rock-actomyosin contractility prevented the formation of FAJs and junction remodeling. FAJs formed normally in cells expressing a Vinculin binding-deficient mutant of α-catenin, showing that Vinculin recruitment is not required for adherens junction formation. Comparing Vinculin-devoid FAJs to wild-type FAJs revealed that Vinculin protects VE-cadherin junctions from opening during their force-dependent remodeling. These findings implicate Vinculin-dependent cadherin mechanosensing in endothelial processes such as leukocyte extravasation and angiogenesis.     

Mutant B-Raf(V600E) Promotes Melanoma Paracellular Transmigration by Inducing Thrombin-mediated Endothelial Junction Breakdown

Tumor invasiveness depends on the ability of tumor cells to breach endothelial barriers. In this study, we investigated the mechanism by which the adhesion of melanoma cells to endothelium regulates adherens junction integrity and modulates tumor transendothelial migration (TEM) by initiating thrombin generation. We found that the B-Raf(V600E) mutation in metastatic melanoma cells up-regulated tissue factor (TF) expression on cell membranes and promoted thrombin production. Co-culture of endothelial monolayers with metastatic melanoma cells mediated the opening of inter-endothelial spaces near melanoma cell contact sites in the presence of platelet-free plasma (PFP). By using small interfering RNA (siRNA), we demonstrated that B-Raf(V600E) and TF silencing attenuated the focal disassembly of adherens junction induced by tumor contact. Vascular endothelial-cadherin (VE-cadherin) disassembly was dependent on phosphorylation of p120-catenin on Ser-879 and VE-cadherin on Tyr-658, Tyr-685, and Tyr-731, which can be prevented by treatment with the thrombin inhibitor, hirudin, or by silencing the thrombin receptor, protease-activated receptor-1, in endothelial cells. We also provided strong evidence that tumor-derived thrombin enhanced melanoma TEM by inducing ubiquitination-coupled VE-cadherin internalization, focal adhesion formation, and actin assembly in endothelium. Confocal microscopic analysis of tumor TEM revealed that junctions transiently opened and resealed as tumor cells accomplished TEM. In addition, in the presence of PFP, tumor cells preferentially transmigrated via paracellular routes. PFP supported melanoma transmigration under shear conditions via a B-Raf(V600E)-thrombin-dependent mechanism. We concluded that the activation of thrombin generation by cancer cells in plasma is an important process regulating melanoma extravasation by disrupting endothelial junction integrity.     

Differential expression of VEGF-A mRNA by 17β-estradiol in breast tumor cells lacking classical ER-α may be mediated through a variant form of ER-α

Beta-estradiol (17beta-E2) augments VEGF-A expression in various estrogen targeted organs and cells including breast tumor derived cell lines, via an ER-alpha mediated pathway. Ironically, 17beta-E2 is able to regulate some genes via ER-alpha independent pathways. In the present study, we sought to determine whether 17beta-E2 can modulate VEGF-A expression in absence of ER-alpha, and therefore, three different cell lines including ER-alpha+ MCF-7, and ER-alpha SKBR-3 and HMEC were used for this study. The present study demonstrates that 17beta-E2 also induces VEGF-A mRNA expression in ER-negative SKBR-3 breast tumor cells in a manner similar to that observed in ER-positive MCF-7 cells. Blocking the induced-expression by antiestrogen ICI 182,780 indicates the induction pathway is ER dependent. While ER-alpha mRNA is absent in both HMEC and SKBR-3 cells, the impact of estrogen was found only in SKBR-3 cells, suggesting the existence of an analogue to ER-alpha or overlapping signal in these cells. Consistent with this suggestion, the present studies demonstrate the existence of an ER-alpha(var2) protein in MCF-7 and in SKBR-3 cells. This variant is predominantly localized in the nuclei of SKBR-3 cells. Importantly, specific binding of 17beta-E2 by these cells suggest the ER-alpha(var2) may act as active receptor in SKBR-3 cells.     

Tyrosine phosphorylation of VE-cadherin and claudin-5 is associated with TGF-β1-induced permeability of centrally derived vascular endothelium

Breakdown of the inner blood-retinal barrier and the blood-brain barrier is associated with changes in tight and adherens junction-associated proteins that link vascular endothelial cells. This study aimed to test the hypothesis that transforming growth factor (TGF)-β1 increases the paracellular permeability of vascular endothelial monolayers through tyrosine phosphorylation of VE-cadherin and claudin-5. Bovine retinal and human brain capillary endothelial cells were grown as monolayers on coated polycarbonate membranes. Paracellular permeability was studied by measuring the equilibration of (14)C-inulin or fluorescence-labelled dextran. Changes in VE-cadherin and claudin-5 expression were studied by immunocytochemistry (ICC) and quantified by cell-based enzyme linked immunosorbent assays (ELISA). Tyrosine phosphorylation of VE-cadherin and claudin-5 was studied by ICC, immunoprecipitation and Western blotting. We found that exposure of endothelial cells to TGF-β1 caused a dose-dependent increase in paracellular permeability as reflected by increases in the equilibration of (14)C-inulin. This effect was enhanced by the tyrosine phosphatase inhibitor orthovanadate and attenuated by the tyrosine kinase inhibitor lavendustin A. ICC and cell-based ELISA revealed that TGF-β1 induced both dose- and time-dependent decreases in VE-cadherin and claudin-5 expression. Assessment of cell viability indicated that changes in these junction-associated proteins were not due to endothelial death or injury. ICC revealed that tyrosine phosphorylation of endothelial monolayers was greatly enhanced by TGF-β1 treatment, and immunoprecipitation of cell lysates showed increased tyrosine phosphorylation of VE-cadherin and claudin-5. Our results suggest that tyrosine phosphorylation of VE-cadherin and claudin-5 is involved in the increased paracellular permeability of central nervous system-derived vascular endothelium induced by TGF-β1.     

Endothelial induced EMT in breast epithelial cells with stem cell properties

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.     

Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells

Tissue transglutaminase (TGase) exhibits both a GTP binding/hydrolytic capability and an enzymatic transamidation activity. Increases in TGase expression and activation often occur in response to stimuli that promote cellular differentiation and apoptosis, yet the signaling mechanisms used by these stimuli to regulate TGase expression and activation and the role of TGase in these cellular processes are not well understood. Retinoic acid (RA) consistently induces TGase expression and activation, and it was shown recently that RA-induced TGase expression was inhibited in NIH3T3 mouse fibroblasts co-stimulated with epidermal growth factor (EGF). Here we investigate whether EGF also antagonized RA-induced TGase expression in breast cancer cells. We found that EGF stimulation affected TGase expression and activation very differently in these cancer cells. Not only did EGF fail to block RA-induced TGase expression, but also EGF alone was sufficient to potently up-regulate TGase expression and activation in SKBR3 cells, as well as MDAMB468 and BT-20 cells. Inhibiting phosphoinositide 3-kinase activity severely diminished the ability of EGF and RA to increase TGase protein levels, whereas a constitutively active form of phosphoinositide 3-kinase potentiated the induction of TGase expression by EGF in SKBR3 cells. Because EGF is an established antiapoptotic factor, we examined whether the protection afforded by EGF was dependent on its ability to up-regulate TGase activity in SKBR3 and BT-20 cells. Exposure of cells to a TGase inhibitor or expression of a dominant-negative form of TGase potently inhibited EGF-mediated protection from doxorubicin-induced apoptosis. Moreover, expression of exogenous TGase in SKBR3 cells mimicked the survival advantage of EGF, suggesting that TGase activation is necessary and sufficient for the antiapoptotic properties of EGF. These findings indicate for the first time that EGF can induce TGase expression and activation in human breast cancer cells and that this contributes to their oncogenic potential by promoting chemoresistance.     

Retinoic acid induces expression of SLP-76: expression with c-FMS enhances ERK activation and retinoic acid-induced differentiation/G0 arrest of HL-60 cells

Retinoic acid (RA) is known to cause MAPK signaling which propels G0 arrest and myeloid differentiation of HL-60 human myeloblastic leukemia cells. The present studies show that RA up-regulated expression of SLP-76 (Src-homology 2 domain-containing leukocyte-specific phospho-protein of 76 kDa), which became a prominent tyrosine-phosphorylated protein in RA-treated cells. SLP-76 is a known adaptor molecule associated with T-cell receptor and MAPK signaling. To characterize functional effects of SLP-76 expression in RA-induced differentiation and G0 arrest, HL-60 cells were stably transfected with SLP-76. Expression of SLP-76 had no discernable effect on RA-induced ERK activation, subsequent functional differentiation, or the rate of RA-induced G0 arrest. To determine the effects of SLP-76 in the presence of a RA-regulated receptor, SLP-76 was stably transfected into HL-60 cells already overexpressing the colony stimulating factor-1 (CSF-1) receptor, c-FMS, from a previous stable transfection. SLP-76 now enhanced RA-induced ERK activation, compared to parental c-FMS transfectants. It also enhanced RA-induced differentiation, evidenced by enhanced paxillin expression, inducible oxidative metabolism and superoxide production. RA-induced RB tumor suppressor protein hypophosphorylation was also enhanced, as was RA-induced G0 cell cycle arrest. A triple Y to F mutant SLP-76 known to be a dominant negative in T-cell receptor signaling failed to enhance RA-induced paxillin expression, but enhanced RA-induced ERK activation, differentiation and G0 arrest essentially as well as wild-type SLP-76. Thus, SLP-76 overexpression in the presence of c-FMS, a RA-induced receptor, had the effect of enhancing RA-induced cell differentiation. This is the first indication to our knowledge that RA induces the expression of an adapter molecule to facilitate induced differentiation via co-operation between c-FMS and SLP-76.     

CD44 variant,but not standard CD44 isoforms,mediate disassembly of endothelial VE-cadherin junction on metastatic melanoma cells

Loss of endothelial adherens junctions is involved in tumor metastasis. Here, we demonstrate that, in the metastatic Lu1205 melanoma cells, expression of the CD44 variant CD44v8-v10 induced junction disassembly and vascular endothelial (VE)-cadherin phosphorylation at Y658 and Y731. Short interfering RNA (siRNA)-mediated CD44 knockdown or sialic acid cleavage reversed these effects. Moreover, microspheres coated with recombinant CD44v8-v10 promoted endothelial junction disruption. Overexpression of CD44v8-v10 but not of standard CD44 (CD44s) promoted gap formation in the non-metastatic WM35 melanoma cells, whereas CD44 knockdown or neuraminidase treatment dramatically diminished melanoma transendothelial migration. Endothelial cells transfected with the phosphomimetic VE-cadherin mutant Y658E supported transmigration of CD44-silenced Lu1205 cells. Our findings imply that CD44 variant isoform (CD44v) but not CD44s regulates endothelial junction loss, promoting melanoma extravasation.