VE-PTP participates in vasculogenic mimicry by preventing autophagic degradation of VE-cadherin

Aberrant extra-vascular expression of VE-cadherin has been observed in metastasis associated with Vasculogenic Mimicry (VM); we have recently shown that in VM prone (VM+) tumor cells VE-cadherin is mainly in the form of pVE-cadherin in Y658 allowing an increased plasticity that potentiates VM development. As excessive VE-cadherin phosphorylation is regulated by the phosphatase VEPTP in endothelial cells in the current study we analysed its role in this aberrant phenotype in malignant tumor cells. We show that human malignant melanoma cells VM+, also express VE-PTP although at lower levels than endothelial cells. The complex VE-PTP/VE-Cadherin/p120-catenin act as a safeguard to prevent VE-cadherin degradation by autophagy. Indeed, silencing of VE-PTP results in complete degradation of VE-cadherin with the features of autophagy and increases the global p120 tyrosine phosphorylation status. In summary, we show that VE-PTP is involved in VM formation and disruption of VE-PTP/VE-Cadherin/p120 complex results in enhanced autophagy in aggressive VM+ cells.


Introduction
The term vasculogenic mimicry (VM) describes the formation of perfusion pathways in tumors by highly invasive, genetically deregulated tumor cells: vasculogenic because they distribute plasma and may contain red blood cells and mimicry because the pathways are not blood vessels and merely mimic vascular function. While VM formation is clearly a marker of highly invasive tumor phenotype, mechanisms by which these structures may contribute to adverse outcome are not well understood. It has been proposed that VM formation may facilitate tumor perfusion and the physical connection between VM and blood vessels may also facilitate hematogeneous dissemination of tumor cells. There is a strong association between the histological detection of VM patterns in primary uveal and cutaneous melanomas and subsequent death from metastasis (McLean et al., 1997;Warso et al., 2001), consistent with the in vitro observations that these patterns are generated exclusively by highly invasive tumor cells (Maniotis et al., 1999). ECs express various members of the cadherin superfamily, in particular vascular endothelial (VE-) cadherin (VEC), which is the main adhesion receptor of endothelial adherent junctions. Aberrant extra-vascular expression of VE-cadherin has been observed in certain cancer types associated with VM (Hendrix et al., 2001). VE-PTP (vascular endothelial protein tyrosine phosphatase) is an endothelial receptor-type phosphatase whose name was coined for its prevalence to the bind to VE-cadherin (Nawroth et al., 2002). VE-PTP poise endothelial barrier through helping homotypic VE-cadherin to keep at minimum basal endothelial permeability (Wessel et al., 2014). Knockdown of VE-PTP increases endothelial permeability and leukocyte extravasation. VE-PTP also counterbalances the effects of permeability-increasing mediators such as VEGF, which increase endothelial permeability and leukocyte trafficking, by dephosphorylating VEcadherin at Tyr658 and Tyr685, leading to stabilization of VE-cadherin junctions (Orsenigo et al., 2012;Wallez et al., 2007). We also have shown that FAK inhibition enabled Kaiso to suppress the expression of its target genes and enhanced Kaiso recruitment to KBS-containing promoters (CCND1 and WNT 11). We observed that silencing of VE-PTP induced a significant reduction of CCND1 and WNT 11 (Kaiso-dependent genes) ( Figure 1C) and disrupted VM formation ( Fig 1D) suggesting that VE-PTP was also involved in the intracellular dynamic of VE-cadherin resulting in the regulation of kaisodependent genes. To evaluate the correlation between the levels of VE-PTP and VE-cadherin we performed a western after siVE-PTP in MUM 2B ( Fig 1E) and found an almost complete disappearance of VE-cadherin and phoshoVE-cadherin suggesting that VE-PTP was involved in VE-cadherin stability and needed for phosphoVE-Cadherin to reach the nucleus, as indirectly suggested the results obtained in Fig 1C and D. Interestingly, a completely different situation was found in primary endothelial cells HUVEC where siVE-PTP lead to an accumulation of phosphoVEC in both cytosolic and nuclear compartments ( Figure 1F). These results suggested that VE-PTP in malignant melanoma cells was protecting phosphoVEC from degradation.

VE-Cadherin and VE-PTP form a complex with p120 catenin in melanoma cells
The VE-cadherin-catenin complex provides the backbone of adherens junction in the endothelium. Nonetheless, in non-endothelial cells the proteins interacting with VE-cadherin have not been identified. Using a coIP approach to analyze the VEcadherin and VE-PTP interacting proteins we shown that VE-Cadherin form a weak complex with VE-PTP in MUM2B ( Figure 2A) compared with HUVECs ( Fig 2B).
Surprisingly, the presence of p120-catenin in complex with VE-PTP was much higher in MUM2B cells as compared to HUVEC cells (Figures 2B and 2C) suggesting that VE-PTP might be involved in the control of p120-catenin phosphorylation status in melanoma cells. To analyze the possible impact of p120 on VE-cadherin stability in MUM2B, we performed a cytosol-nucleus subfractionation assay after silencing p120 and we found that p120 protect the stability of VE-cadherin ( Fig 2D). Finally, we performed a coIP of p120 after siVE-PTP in MUM 2B cells, and we observed that binding of VE-cadherin to VE-PTP was lost and resulted in increased global tyrosine phosphorylation of p120, suggesting that VE-PTP safeguard of VE-Cadherin/p120 binding in VM + cells ( Fig   2F) and p120-catenin is likely to be a substrate for VE-PTP. These results are compatible with the increased phosphop120 (as result of VE-PTP inactivation) being responsible of complex dissociation to initiate ve-cadherin proteolysis.

VE-Cad/VE-PTP complex dissociation enhanced autophagy
In view of elevated expression of VE-PTP in aggressive melanoma cells and his association with VE-Cadherin/p120 association, we investigated the possible implication of autophagy in these models. To dissect the mechanism leading to VE-Cadherin degradation after the inhibition of VE-PTP, we treat MUM 2B with proteasome inhibitor lactacystin and did not prevent the degradation of VE-Cadherin after VE-PTP disabling ( Figure 3A). Macroautophagy (referred to simple as "autophagy") is a homeostatic "self-eating" pathway that has been conserved among eukaryotic cells. Is a lysosomal-associated process which intracellular components, small portions of cytosol or receives chaperone-associated cargoes are engulfed in double-membrane vesicles, called autophagosomes, to be degraded with lysosomal hydrolyses (Dikic and Elazar, 2018). Recently, different studies report that the formation of VM was promoted by bevacizumab-induced autophagy in GSCs, which was associated with tumor resistance to antiangiogenic therapy through high expression of VEGFR-2 (9) . In our setting, inhibition of the fusion of autophagosomes and lysosomes with chloroquine suppressed the degradation of VE-cadherin after siVE-PTP ( Fig 3A). Even more, the levels of the mTOR substrate p-p70 (as a readout od mTOR activity and autophagy status) decreased in the MUM 2B K.O cells ( Figure 3B), suggesting that VE-cadherin/VE-PTP complex might be restraining autophagy. We then performed an electron microscopy experiment in MUM 2B and C8161 in siVE-PTP or MUM 2B knockout for VE-cadherin; we shown an enhanced autophagic morphology after the VE-PTP silencing or VE-cadherin knockout cells, suggesting that the presence of either protein out of the complex triggered autophagy. To confirm the implication of autophagy on VE-cadherin degradation, we perfomed an experiment to quantify autophagosomes formation under the same conditions described above after transfection LC3-GFP, and we observed that autophagosomes (LC3-GFP puncta) increased following silencing of VE-PTP in both MUM 2B and C8161 VM + cells ( Fig   3D). By analysing the cBioPortal database, a platform of 48333 tumour samples, we found that high mRNA levels of PTPRB were inversely associated with the expression of two key genes involved in autophagy, LAMP1 and ATG7 in uveal melanoma (figure S1B), suggesting that in patients this interaction might be relevant to determine autophagy features of the tumor. and C) and the loss of this complex (after siVE-PTP silencing) leads to p120catenin increased phosphorylation and VE-cadherin degradation. Globally these results shed light to a new mechanism to control VM through the balance between VE-PTP/VE-cadherin .

Reagents and antibodies
The following reagents were used: Chloroquine 20

In vitro angiogenesis assay
The effect of siVE-PTP on the formation of tube-like structures in Matrigel (BD Lentiviral particles for the best two sgRNAs in terms of allelic disruption (GGCAGGCGCCCGATGTGGCG and GATGATGCTCCTCGCCACATC).
Consequently, the next day IP lysates were incubated for 2 hours at 4ºC with 50 µl of of Dynabeads™ Protein G for Immunoprecipitation (ThermoFischer). Dynabeads were washed 3 times with low salt 120 mM lysis buffer and 2 times with high salt 300 mM lysis buffer. All lysates were separated by dodecyl sulfate-polyacrylamide (7,5%, Biorad) gel electrophoresis and transferred to PVDF membrane (Pall laboratory) by semi-wet blotting.

Electron microscopy
The   immunoprecipitation of p120 revealed after inhibition of VE-PTP produce a high increase onto p120 phosphorylation and decrease in the union of VE-cadherin.   what is the union of p120 with VE-cadherin as well as VE-PTP. Surprisingly, p120 have a high union with VE-PTP and VE-cadherin, d) cytosol-nucleus subfracionation with scb and sip120 shown that the stability of VE-cadherin as well the phosphorylation of Y658 is p120-dependent, e) finally, immunoprecipitation of p120 revealed after inhibition of VE-PTP produce a high increase onto p120