Tumor-derived thrombin induces endothelial gap formation 1 Mutant B-Raf ( V 600 E ) 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 the current study, we investigated the mechanism by which the adhesion of melanoma cells to endothelium regulates adherens junction (AJ) integrity and modulates tumor transendothelial migration (TEM) by initiating thrombin generation. We found that the V600E B-Raf mutation in metastatic melanoma cells upregulated 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 V600E B-Raf and TF silencing attenuated the focal disassembly of AJ induced by tumor contact. Vascular endothelial-cadherin (VE-cadherin) disassembly was dependent on phosphorylation of p120-catenin on serine, S879, and VE-cadherin on tyrosine, Y658, Y685 and Y731, which can be prevented by treatment with the thrombin inhibitor, hirudin, or by silencing the thrombin receptor, protease-activated receptor-1(PAR-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 V600E B-Raf-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. Melanoma is the most invasive and metastatic form of skin cancer. Melanoma metastasis is a highly http://www.jbc.org/cgi/doi/10.1074/jbc.M115.696419 The latest version is at JBC Papers in Press. Published on October 26, 2015 as Manuscript M115.696419 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. at N ew Y rk M eical C olege on O cber 9, 2015 hp://w w w .jb.org/ D ow nladed from Tumor-derived thrombin induces endothelial gap formation 2 coordinated process, involving surviving blood’s mechanical shear force, tethering onto endothelium, developing shear-resistant adhesion, disrupting endothelial barrier function, and invading underlying tissues (1,2). Metastasis is accompanied by secretion of soluble factors including proteases and cytokines into the extracellular milieu, which perform autocrine or paracrine roles to further promote metastasis. Hematogenous dissemination of melanoma is clearly linked with thrombosis and activation of blood coagulation. Cancer patients often have abnormal blood coagulation with elevated levels of fibrinopeptide A (3). The initiation of blood coagulation is catalyzed by the tissue factor (TF)-factor VIIa pathway (4,5). The transmembrane protein TF is a single-chain, 263-amino acid membrane glycoprotein, which binds and allosterically activates factor VII. The TF-VIIa complex cleaves factor IX and X, leading to the generation of serine protease thrombin. TF is highly expressed on metastatic melanoma cell lines (6). In addition, tumor dissemination is associated with TF expression. Studies with the highly specific thrombin inhibitor, hirudin, have revealed that thrombin in the tumor microenvironment contributes to tumor metastasis, and that hirudin treatment markedly suppresses spontaneous tumor metastasis in vivo, prolonging mouse survival (7). B-Raf mutations have been identified in about 60%–80% of human melanomas, as well as in benign nevus specimens (8-11). A thymine to adenine transversion in exon 15, leading to a V600E substitution in the BRAF kinase domain occurs in ~ 60% of melanomas (9,10). BRAF is a component of the RAS–RAF–MEK–ERK signaling pathway that plays a critical role in cell proliferation, differentiation and survival (12). B-Raf may play critical roles in mediating constitutive activation of downstream kinases and gene transcription which facilitate melanoma adhesion, migration and proliferation (13,14). Targeted silencing of B-Raf downregulated the activity of mitogen-activated protein (MAP) kinase, intercellular adhesion molecule-1 (ICAM-1) and IL-8, while attenuating leukocyte-mediated melanoma extravasation and the development of lung metastases (15,16). Thrombin is a serine protease responsible for many homeostatic functions, including conversion of soluble fibrinogen into fibrin and activation of various intercellular signaling events in circulating blood cells via protease activated receptor-1 (PAR-1) (17). Under certain circumstances, thrombin exposure may result in cellular inflammatory responses, such as altered ICAM-1 expression on vascular endothelial cells and activation of polymorphonuclear neutrophils (PMNs), which help regulate hemostasis. By inducing VE-cadherin dimer dissociation, thrombin has been implicated in endothelial junction breakdown (17). As such, thrombin exposure induces VE-cadherin cytoplasmic phosphorylation which displaces p120-catenin and β-catenin from adherens junctions (AJ), thereby increasing endothelial permeability (18-21). VE-cadherin can be phosphorylated at a variety of tyrosine residues (20,21). Tyrosine sites 658, 685 and 731 of VE-cadherin may be phosphorylated in response to thrombin stimulation (19). AJ breakdown and endothelial barrier function loss play essential roles in regulating tumor metastasis. Tumor cell adhesion initiates a series of signaling cascades leading to the loss of endothelial junction integrity (22,23). However, it is unknown whether B-Raf overexpression in malignant melanoma cells can mediate endothelial junction breakdown by catalyzing thrombin production. In the current study, we sought to link B-Raf activity in melanoma cells with TF overexpression and thrombin generation. In addition, the effects of knockdown and ectopic expression of B-Raf on melanoma contact-induced endothelial junction disassembly, permeability increase and reduction of VE-cadherin-mediated cell-cell adhesion were evaluated in the presence of platelet-free plasma (PFP). The phosphorylation of VE-cadherin and p120 was assessed in response to melanoma adhesion in PFP. The roles of tumor-derived thrombin and VE-cadherin junction breakdown in regulating the routes of melanoma transendothelial migration (TEM) were investigated. EXPERIMENTAL PROCEDURES Reagents-Hirudin was from Sigma Aldrich (St. Louis, MO). Anti-VE-cadherin, anti-p120-catenin and anti-β-tubulin were purchased from Cell Signaling Technology (Massachusetts, MA). Alexa Fluor 488 F(ab’)2 goat anti-mouse IgG was from Invitrogen (Carlsbad, CA). TransIT 2020 was purchased from Mirus Bio LLC (Madison, WI). Super Signal West at N ew Y rk M eical C olege on O cber 9, 2015 hp://w w w .jb.org/ D ow nladed from Tumor-derived thrombin induces endothelial gap formation 3 pico chemiluminescence reagent and goat anti-mouse IgG horseradish peroxidase were obtained from Thermo Scientific (Rockford, IL). S879A-p120 cDNA was kindly provided by A. Reynolds (Vanderbilt University, Nashville, TN). It was fused to GFP to monitor transfection. Anti-p-Tyr-658 VE-cadherin and anti-p-Tyr-731 VE-cadherin antibodies were from Chemicon (Temecula, CA). Anti-p-Tyr-685 VE-cadherin, anti-Rab5, anti-B-Raf, anti-TF, and anti-phospho-serine antibodies were purchased from Abcam (Eugene, OR). Human TF antibody (polyclonal rabbit anti-human TF 4502) was obtained from American Diagnostica (Stamford, CT). Rabbit anti-K63-linked-polyubiquitin clone Apu3 and anti-ubiquitin FK2 clone antibodies were obtained from Millipore. Anti-B-Raf V600E (VE1) mouse monoclonal antibody was purchased from Ventana Medical Systems, (Tucson, AZ). A non-specific antibody (rabbit IgG fraction) to act as a control was from Dako (Denmark). Cell culture-HUVECs were obtained from the American Type Culture Collection (ATCC 1730-CRL) (Manassas, VA) and maintained in F12-K medium with 10% FBS, 30 μg/ml of endothelial cell growth supplement, 50 μg/ml heparin (Mallinckrodt Baker), and 100 U/ml penicillin-streptomycin (Biofluids) (Passages 5-8). A375M, UACC903 and Lu1205 melanoma cell lines (obtained from ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% FBS and 100 U/ml of penicillin-streptomycin. WM35 melanoma cells (provided by Dr. Meenhard Herlyn, Wistar Institute, Philadelphia, PA) were maintained in Roswell Memorial Park Institute (RPMI) media supplemented with 10% FBS and 100 U/ml penicillin-streptomycin. All cells were cultured in a humidified incubator at 37° C in 5% CO2. Amplification refractory mutation system (ARMS)-PCR-The ARMS-PCR primers were as follows: forward (Fo): 5’-CTCTTCATAATGCTTGCTCTGATAG-3’; reverse (Ro): 5’-GCCTCAATTCTTACCATCCAC-3’; forward wild-type identifying (Fiwt): 5’-GTGATTTTGGTCTAGCTACAGT-3’ and reverse mutation identifying (Rimut):5’-CCCACTCCATCGAGATTTCT-3’. PCR was performed in a 20 μl reaction volume containing 1 × buffer, 2 mM MgCl2, 1 unit of Hotstar Taq DNA polymerase (Qiagen Science, Valencia, CA), 200 μM each dNTP, 400 nM primer Fo, 200 nM primer Ro and Fiwt, 800 nM primer Rimut and 30 ng genomic DNA template. PCR amplification was carried out by denaturation at 95° C for 5 min, followed by 40 cycles at 95° C for 20 sec, 68° C for 20 sec, and 72° C for 20 sec with a final extension at 72° C for 5 min. PCR products were analyzed by 2% agarose gel electrophoresis. PFP preparation-Following the Pennsylvania State University Institutional Review Board (IRB)-approved protocols (no. 19311), fresh human blood was collected from healthy adults by venipuncture into a BD Vacutainer with sodium citrate as the anti-coagulant. To block t