Vascular endothelial growth factor (VEGF) signaling
When we refer to VE-cadherin it is necessary to describe VEGF receptors (VEGFRs) because they represent the most important pathway to regulate VE-cadherin function and turn-over. VEGFR-2 (flk-1/KDR) and VEGFR-1 (FLT-1) are the most important receptors in this pathway although at least five different VEGF isoforms are also known in human. These receptors are stimulated by vascular endothelial growth factor (VEGF-A) and represent a crucial regulatory system of endothelial growth in a normal cell physiology context; in contrast VEGF is also an endothelial-specific mitogen and potent activator of vascular permeability secreted by tumor cells [
24]. VEGFR-2 is highly expressed in vascular ECs thus forming the primitive tubular vessels called vasculogenesis. In contrast, VEGFR-1 is also highly expressed in tumor cells with capacity to form VM; this is the case of malignant melanoma [
25]. As mentioned above, VEGF-A promotes vascular permeability by weakening adherent junctions and tight junctions, resulting in transient opening of the endothelial cell-cell contacts [
26]. In fact, VEGF-A promotes tyrosine phosphorylation of VE-cadherin and its binding to the partner β-catenin, plakoglobin and p120, via Src-dependent mechanism [
27]. VE-cadherin is inhibited following its phosphorylation in mice deficient in Src [
27]. VE-cadherin may also be associated with and inhibit VEGFR-2 phosphorylation and subsequent internalization [
28]. This association promotes the phosphorylation of components of the adherent junctions by Src, thus impairing the integrity of the endothelial barrier and promoting tumor cell extravasation and diffusion in pathological models [
29]. In addition, VEGF-A mediates phosphorylation of VE-cadherin internalization through sequential activation of Src, the nucleotide exchange factor of Vav2, Rho GTPase Rac, leading to a downstream effect on the serine/threonine kinase P21 protein (Cdc42/Rac)-activated kinase 1 (PAK). Finally, phosphorylated PAK phosphorylates VE-cadherin, triggering their internalization [
26]. Moreover, VEGF signaling reduces the association between VE-cadherin and p120-catenin promoting clathrin-dependent VE-cadherin endocytosis [
30]; binding of p120 to VE-cadherin prevents its internalization, while p120 silencing leads to degradation of VE-cadherin, and loss of cell-cell contacts [
30].
VEGF binding to VEGFR-1 can enhance other variety of signaling pathways. Activation of tyrosine kinase Src and extracellular regulatory kinases 1 and 2 (ERK1/2) leads to cancer cell invasion and migration. In melanoma, VEGFR-1 seems to promote VM via PI3K/PKC pathway. VEGFR-1 also mediates angiogenesis through activation of PI3K/Akt pathway [
7,
8]. Apart from this, VEGFR-1 is necessary for the expression of VE-cadherin [
25]. As for VEGFR-3, it has been proved in melanoma that endothelin-1 (ET-1) can enhance its expression and also the expression of its ligands VEGF-C and -D. The signaling pathway involves ET-1 binding to its receptor ETBR, which activates hypoxia-inducible factors. The binding also leads to phosphorylation of VEGFR-3 (probably mediated by Src and β-arrestin-1), which becomes able to trigger the activation of MAPK signaling cascade. All of these events lead finally to the promotion of cell migration and VM [
31].
Different evidences support the fact that VEGF expression is determined by EphA2 in breast cancer and pancreatic islet carcinoma cells. Cyclooxygenase-2 (COX-2) also stimulates the expression of VEGF in other tumor cell lines [
7‐
9].
Cyclooxygenase-2 catalyzes the reaction that converts arachidonic acid into primarily prostaglandin E2 (PGE2). This molecule binds to prostanoid receptors (EP1-4) that activate protein kinase C (PKC) and epidermal growth factor receptor (EGFR) signaling pathways. Both of them lead to decreased apoptosis and increased tumor proliferation, invasion and angiogenesis. In particular, PKC signaling up-regulates VEGF expression [
7‐
9]. In breast cancer, elevated COX-2 expression results in the increased ability to form VM networks, while its knockdown leads to a reduced VM. Importantly, VM capability of breast cancer cells with low COX-2 can be restored if PGE2 is added to the culture [
32]. It has been reported that EP3, but not EP4, modulates VM in breast cancer [
33].
On the contrary, VEGF/VEGFR-1 signaling can be inhibited by the pigment epithelium-derived factor (PEDF), a glycoprotein that belongs to the family of serine protease inhibitors. Furthermore, PEDF induces tumor cell differentiation and apoptosis, yet probably prevents VM development, since it is usually down-regulated in aggressive tumor cells. Even more, PEDF silencing favors VM in melanoma [
7,
8].
Tissue factor (TF) and TF pathway inhibitors (TFPI-1/2) are involved in VM as well. Specifically, knockdown of TFPI2 inhibited MMP-2 activity, suggesting a role for TFPI2 in extracellular matrix remodeling associated with VM channel formation [
7,
8].
Finally, an opposite role of VEGF signaling in VM has been proposed too. All the evidence presented so far indicates that VEGF promotes VM, but it has been suggested that, on the contrary, VM might increase in the absence of this signaling pathway: VEGF would promote angiogenesis, while VEGF blockade could enhance some other strategies for tumor cell survival, including VM [
34].
VE-cadherin and EphA2 in VM
EphA2 has also been shown to be related to VM. EphA2 is a protein tyrosine kinase whose phosphorylation and activity depend on the binding of ephrin-A1, although it has been reported that EphA2 can also be constitutively active in some tumor cells [
35]. As VE-cadherin, EphA2 was found to be expressed only in highly aggressive tumors, where it was tyrosine-phosphorylated. When cells were cultured on a three-dimensional matrix and labeled with anti-phosphotyrosine antibodies, the staining showed that tyrosine-phosphorylation was present mainly in the areas of tubular network formation. General inhibitors of protein tyrosine kinases as well as specific silencing of EphA2 hindered the development of vascular networks, suggesting a potential role for phosphorylated EphA2 in this process [
35]. In VM, VE-cadherin and EphA2 co-localize at the plasma membrane, specifically in cell-to-cell contact regions. Knockdown of VE-cadherin resulted in a reorganization of EphA2 location, which seemed to move into the cytoplasm. Moreover, there was a decrease in EphA2 phosphorylation. On account of these results, it seems that VE-cadherin may help EphA2 translocate to the plasma membrane, [
36,
37].
PI3K up-regulates both the activity and expression of matrix metalloproteinase-14 (MMP-14) in highly aggressive cells. MMP-14 in turn activates MMP-2, and finally cleaves laminin 5γ2 chain to produce the γ2’ and γ2x fragments, which are secreted to the extracellular matrix to promote migration in various tumor cell types, like breast, colon carcinomas and hepatoma [
38]. More precisely, they activate the secretion of γ2’ and γ2x pro-migratory fragments leading to VM in melanoma, gallbladder and ovarian carcinomas [
39‐
41]. Furthermore, poorly aggressive melanoma cells (which cannot normally engage in VM) could form vasculogenic-like networks when seeded on collagen gels that had been pre-conditioned by highly aggressive melanoma cells. Aggressive cells were removed before apparent formation of tubular networks, but the examination of the a-cellular matrices showed the presence of laminin-positive patterned networks. If these matrices where treated with antibodies anti-laminin-5γ2 chain before seeding the poorly aggressive melanoma cells, they could no longer develop tubular networks. Altogether, these results confer great importance to this signaling cascade and laminin-5γ2 in particular.
As for FAK, over-expression of EphA2 caused an increase of MMP-2 in a way dependent on FAK [
42]. FAK itself is highly phosphorylated at positions Y397 and Y576 in highly aggressive melanoma, but not in poorly aggressive melanoma cells; this post-translational changes are indicative of a fully active FAK. FAK-related non-kinase (FRNK) can interact with focal adhesion proteins but it lacks kinase activity, so it is considered a dominant-negative FAK protein. Expression of FRNK in aggressive melanoma decreased invasiveness, migration and ability to form tubular networks on collagen gels. It also reduced the levels of phosphorylated ERK1/2, whose inhibition decreased the levels of urokinase as well as the activity of MMP-14 and MMP-2. These results hint at a signaling cascade where EphA2 promotes VM via FAK and ERK, meeting the PI3K pathway at MMP-14 and leading henceforth to the cleavage of laminin-5γ2 [
43].
Notch/Nodal
Nodal/Notch belongs to the superfamily of the transforming growth factor β (TGF-β). It is essential during embryonic development, since it maintains undifferentiated status of embryonic stem cells in order to ensure growth and development. Notch is a trans-membrane receptor with four different isoforms (Notch1-4), all of which undergo cleavage by γ-secretase after binding of one out of five possible ligands: Delta-like-1/3/4 and Jagged1/2. Notch intracellular domain (NICD) is released into the cytoplasm and it is imported to the nucleus, where it regulates gene expression, including expression of Nodal. Notch signaling pathway is involved in differentiation, although cell fate depends on cellular context. Also Notch is associated with the development of vascular networks in embryonic stages, so it may have a relationship with VM in cancer. Nodal binds several membrane receptors (Cripto-1, activing-like kinase receptors (ALK4/7), ActRIIB, and/or TGF-β receptor I and II), causing phosphorylation of SMAD2/3, enabling its association with SMAD4 and their translocation to the nucleus to activate the expression of Nodal target genes, including LEFTY, which is responsible of negative feedback in this signaling pathway, as it directly inhibits Nodal.
It has been reported that disruption of the Notch signaling cascade with γ-secretase inhibitors stabilizes VM networks in melanoma, that is, Notch might have an attenuating effect in VM [
44]. Nevertheless, there are evidences supporting a positive relationship between Notch and VM: Notch-4 was shown to be highly expressed in several aggressive melanoma cell lines capable of VM, where it seemed to play a crucial role in the up-regulation of Nodal expression as well. Furthermore, antibody-mediated blocking of Notch signaling resulted in an impairment of VM ability, which could be restored with the addition of Nodal [
45]. Nodal expression normally stops after cell differentiation during human development, but it can be rescued in tumor cells, for instance, melanoma and breast cancer cells. This pathway is related to tumor progression, aggressiveness, VM and VE-cadherin expression in aggressive melanoma cells [
15,
16].
Hypoxia
Oxygen depletion is a common situation in growing tumors; therefore tumor cell survival and, consequently, malignancy are often dependent on their adaptation to hypoxia. Hypoxia-inducible factors (HIFs) and hypoxia responsive elements (HRE) play a crucial role in this context; HIFs are transcription factors stable in hypoxic conditions, while HRE are the genomic regions where HIF bind to mediate gene expression. These transcription factors are stabilized during conditions of hypoxia and they are critical to induce genes for tumor cell adaption to a hostile and changing microenvironment.
Notably, some potential hypoxia target genes containing HRE are involved in VM, such as VEGF-A, VEGFR-1, EphA2, Twist, Nodal, COX-2 and VE-cadherin [
8,
46]. The later has been found to contain up to six HRE upstream of the promoter [
6]. As a result, hypoxia has been reported to promote VM in a wide variety of tumor cell lines [
47‐
49]. In murine models of melanoma, VM development in conditions of ischemia was significantly increased. Moreover, there is a positive relationship between expression of HIF-1α and VEGF in ischemic tumor cells [
50]. Instead of VEGF, in human fibrosarcoma, HIF-1α promoted VM by up-regulating Neuropilin-1 (NRP-1), a VEGF receptor and co-receptor of VEGFR-2. Unexpectedly, silencing of NRP-1 completely disrupted tumor formation in fibrosarcoma [
51].
In human melanoma, hypoxia induced over-expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2), which in turn increased VE-cadherin expression [
17]. VE-cadherin was also up-regulated by HIF-1α in esophageal carcinoma [
18]. To summarize, hypoxia is an essential trigger for vascular signaling pathways in the process of vasculogenic mimicry. Hypoxia may also influence VM through BNIP3, a protein that belongs to the family of Bcl-2. Expression of BNIP3 is remarkably up-regulated under hypoxia, allowing its contribution to cell migration and VM development in melanoma. BNIP3 enhanced these processes by modulating the organization of the actin cytoskeleton while BNIP3 knockdown completely inhibits VM, changed cell size and shape, driving to formation of actin stress fibers, and reduced tight and adherents junctions [
52].
In glioblastoma, HIF-1α expression is mediated by mammalian target of rapamycin (mTOR). Specific inhibition or silencing of mTOR disrupted VM formation, especially under hypoxia but also in normoxic conditions, since some VM signaling molecules (HIF-1α, MMP-2 and MMP-14) were down-regulated [
47]. In 2007, A. Le Bras and col [
53] showed that the expression of VE-cadherin is controlled and regulated by a basal, non-endothelial specific promoter, containing six putative hypoxia responsive elements (HRE) which are binding sites for HIF-1α and HIF-2α. Consistent with this, HIF-2α (but not HIF-1α) regulated the expression of VE-cadherin in hypoxia as well as in normoxia. HIF-1α also induced the expression of VE-cadherin and modulated VM in esophageal carcinoma cells [
18]. Following knockdown of HIF-1αVM in inhibited and the expression of VM-related genes reduced, for example EphA2, VE-cadherin or laminin-5γ2 but not MMP-2 in vitro and also in vivo [
18]. Finally, it should be noted that mediating gene expression is not the only way by which hypoxia can influence VM: HIF-1α is known to stabilize NICD [
54], whose role was explained above.
Galectin-3
Galectins are carbohydrate-binding proteins whose functions include cell adhesion and migration [
9]. Galectin-3 (Gal-3) was proven to have oncogenic and angiogenic properties, being up-regulated, for example, in metastatic melanoma and breast cancer cells. Gal-3 silencing reduced invasiveness and inhibited VM formation in melanoma through down-regulation of the expression of some endothelial markers, such as VE-cadherin, together with the release of interleukin-8 (IL-8) was found to decrease. IL-8 is pro-angiogenic interleukin which modulates the expression of MMP-2, whose important role in VM was mentioned above [
55]. Gal-3 mediates gene expression by inhibiting early growth response protein 1 (EGR-1), which loses its ability to bind (and thereby to repress) VE-cadherin and IL-8 promoter regions [
55]. Therefore, the presence of Gal-3 allows the transcription of EGR-1 target genes. Consistent with this, gene expression microarrays analysis after silencing Gal-3 showed that Gal-3 regulates the expression of multiple genes, and has a negative influence on endothelial markers aberrantly expressed in highly aggressive melanoma cells such as VE-cadherin, IL-8, fibronectin-1, endothelial differentiation sphingolipid G-protein receptor-1 and MMP-2 [
56]. It has been shown that shRNA of Gal-3 decreased VE-cadherin, and IL-8 promoter activity due to enhanced transcription of factor early growth response-1 (EGR-1) [
57].
Wnt family
The Wingless (Wnt) family proteins is related to a large range of physiological processes, including embryonic patterning, cell proliferation, migration, cell differentiation. It plays an important role in endothelial cell differentiation, vascular development and angiogenesis [
58], especially Wnt5a, a member of the non-canonical Wnt signaling. However, the role of Wnt5a in cancer is still being debated: in certain settings has been described as a tumor suppressor, but it is generally involved as a pro-metastatic factor [
59].
Wnt5a is known to promote cell migration by modulating several proteins of the cytoskeleton, providing tumor cells with abilities in relocation. Moreover, Wnt5a releases intracellular calcium, activating calcium-dependent proteins such as protein kinase C (PKC), which is essential for invasion in cancer cells. All these properties, among others, suggest that Wnt5a is a cancer-promoting molecule [
60]. PKCα has been shown to be involved with Wnt5a in EMT and VM in ovarian cancer cells, where the expression of Wnt5a and PKCα were correlated and PI3K levels were enhanced upon up-regulation of Wnt5a [
61]. Over-expression of Wnt5a mediates VM formation in ovarian cancer and lung cancer [
62,
63]. In 2015, Lisha Qi et al. [
62] reported that Wnt3a expression in HT29 colon cancer cells promoted the capacity to form tube-like structures and increased the levels of proteins involved in VM such as VEGFR-2 and VE-cadherin. In addition, the antagonist Dickkopf-1(Dkk-1) reverted the capacity to form VM and decreased the expression of VEGFR2 and VE-cadherin in Wnt3a-overexpressing cells.
On the other hand, Wnt5a may perform as a tumor suppressor in certain cancers by inhibiting β-catenin-mediated transcription via several different molecules, preventing the expression of potential oncogenes. For this reason, the opposing effects of Wnt proteins remain controversial [
60].
Epithelial Mesenchymal Transition (EMT) in VM
EMT is a dynamic biological process where polarized epithelial cells lose their epithelial properties and gain typical characteristics of mesenchymal cells [
64]. The best known regulator of EMT is Transforming Growth Factor-beta (TGF-β), whose role in EMT is well-established [
64,
65]. In 2008, Myriam Labelle and col [
66] showed that VE-cadherin is induced in EMT in mammary tumor cells and it is also aberrantly expressed in invasive human breast carcinomas. In addition, VE-cadherin influenced the levels of SMAD2 phosphorylation and the expression of TGF-target genes. Thus, VE-cadherin might promote tumor progression by contributing to tumor angiogenesis as well as by enhancing tumor cell proliferation via TGF-β signaling. Recently, it has been reported that Zinc finger E-box binding homeobox 2 (ZEB2) fosters VM by TGF-β induction of EMT in HCC where ZEB2 over-expression significantly enhanced cell mobility and VM formation. Up-regulation of ZEB2 increased VE-cadherin, VEFGR-2 and VEGFR-1 expression as well as MMP-2 and MMP-9 [
67]. ZEB1 down-regulation decreased the expression of VE-cadherin and VEGFR-2 in colorectal carcinoma, which are characteristic of ECs. In conclusion, ZEB1 promote VM formation by inducing EMT in colorectal carcinoma through the inhibition of VE-cadherin.
Other protein that has been identified to have a prominent role in EMT is Twist1. This protein binds DNA using similar E-box sequence motifs repressing E-cadherin and up-regulating mesenchymal markers expression; over-expression of Twist1 significantly enhanced cell mobility, invasiveness and promoted VM formation in HepG2 cells while chromatin immunoprecipitation showed that Twist1 binds to the VE-cadherin promoter and enhances its activity [
68].
Cyclic adenosine monophosphate (cAMP)
cAMP is an essential second messenger involved in a number of cellular processes, such as cell growth and differentiation. It has also been linked to VM in cancer [
69,
70]. Firstly, a rise in cAMP levels reduced VM in cutaneous and uveal melanoma. This inhibition appeared to be dependent mostly on the exchange protein directly activated by cAMP (Epac), but not on protein kinase A. Secondly, VM impairment was associated with an inhibition of ERK and PI3K/Akt signaling [
70,
71], both of which were previously mentioned as important participants in the vascular pathways.
Apart from vascular signaling, cAMP may be involved in Notch signaling during endothelium development, so there might be an association with VM, too. The exact relationship between cAMP and Notch in endothelial cell differentiation remains unknown, but cAMP has been shown to modulate presenilin-1 (a component of γ-secretase) in neurons [
72].
Besides the different signaling described above other key elements regulating VM are presented in Table
2.
Table 2
VM inducer and suppressor molecules
VM inducers | References |
HIF1α | |
Twist1 | |
Nodal | |
Wnt5a | |
VE-cadherin | |
EphA2 | |
Laminin 5γ2 | |
CD133 | |
VEGFR-1/2/3 | |
|
|
PECAM1 | |
Desmoglein 2 | |
VM suppressors |
FAK-related nonkinase | |
PEDF | |
cAMP | |
TIMP-2 | |
AP-2α | |
miR-26b (EphA2) | |
miR-200a (EphA2) | |
miR-1236 (PI3K) | |
miR-27a-3p (VE-cadherin) | |
miR186 (Twist1) | |
hsa-mir-299–5p | |
miR-409-3p | |
miR-124 | |
|
miR-Let-7f | |