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.
From the Key Laboratory of Luminescence and Real Time Analytical Chemistry, Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China, the Department of Bioengineering, Pennsylvania State University, University Park, Pennsylvania 16801, and pxz122@swu.edu.cn.
Pu
P
Zhang
From the Key Laboratory of Luminescence and Real Time Analytical Chemistry, Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.
Shan
S
Feng
the Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, Shanghai 200433, China.
Gentao
G
Liu
the Shanghai Pulmonary Hospital, Tongji University School of Medicine, 507 Zhengmin Road, Shanghai 200433, China.
Heyong
H
Wang
From the Key Laboratory of Luminescence and Real Time Analytical Chemistry, Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.
Huifeng
H
Zhu
From the Key Laboratory of Luminescence and Real Time Analytical Chemistry, Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.
Qiao
Q
Ren
From the Key Laboratory of Luminescence and Real Time Analytical Chemistry, Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.
Huiyuan
H
Bai
the Department of Bioengineering, Pennsylvania State University, University Park, Pennsylvania 16801, and.
Changliang
C
Fu
the Department of Bioengineering, Pennsylvania State University, University Park, Pennsylvania 16801, and.
Cheng
C
Dong
D015703:Antigens, CD / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000378:metabolism*; D020868:Gene Silencing; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008545:Melanoma / Q000378:metabolism*; D009154:Mutation; D009362:Neoplasm Metastasis; D010539:Permeability; D010766:Phosphorylation; D010949:Plasma / Q000378:metabolism; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D012878:Skin Neoplasms / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism*; D054875:Ubiquitination; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D048493:Proto-Oncogene Proteins B-raf; D034741:RNA, Small Interfering; D013917:Thrombin; D013925:Thromboplastin; D000099152:Cadherin 5; C482119:BRAF protein, human
V600EB-raf; VE-cadherin; adherens junction; focal adhesion; thrombin; tissue factor; tumor metastasis