title: Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration.
abstract: The exit of metastasizing tumor cells from the vasculature, extravasation, is regulated by their dynamic interactions with the endothelial cells that line the internal surface of vessels. To elucidate signals controlling tumor cell adhesion to the endothelium and subsequent transendothelial migration, we performed phosphoproteomic analysis to map cell-specific changes in protein phosphorylation that were triggered by contact between metastatic MDA-MB-231 breast cancer cells and endothelial cells. From the 2669 unique phosphorylation sites identified, 77 and 43 were differentially phosphorylated in the tumor cells and endothelial cells, respectively. The receptor tyrosine kinase ephrin type A receptor 2 (EPHA2) exhibited decreased Tyr(772) phosphorylation in the cancer cells upon endothelial contact. Knockdown of EPHA2 increased adhesion of the breast cancer cells to human umbilical vein endothelial cells (HUVECs) and their transendothelial migration in coculture cell assays, as well as early-stage lung colonization in vivo. EPHA2-mediated inhibition of transendothelial migration of breast cancer cells depended on interaction with the ligand ephrinA1 on HUVECs and phosphorylation of EPHA2-Tyr(772). When EPHA2 phosphorylation dynamics were compared between cell lines of different metastatic ability, EPHA2-Tyr(772) was rapidly dephosphorylated after ephrinA1 stimulation specifically in cells targeting the lung. Knockdown of the phosphatase LMW-PTP reduced adhesion and transendothelial migration of the breast cancer cells. Overall, cell-specific phosphoproteomic analysis provides a bidirectional map of contact-initiated signaling between tumor and endothelial cells that can be further investigated to identify mechanisms controlling the transendothelial cell migration of cancer cells.
authors: Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK. Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.
authors: Marie
authors: M
authors: Locard-Paulet
authors: Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
authors: Lindsay
authors: L
authors: Lim
authors: Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.
authors: Giulia
authors: G
authors: Veluscek
authors: Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.
authors: Kelly
authors: K
authors: McMahon
authors: Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
authors: John
authors: J
authors: Sinclair
authors: The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London SW3 6JB, UK.
authors: Antoinette
authors: A
authors: van Weverwijk
authors: Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK. Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.
authors: Jonathan D
authors: JD
authors: Worboys
authors: Centre for Evolution and Cancer and Centre for Molecular Pathology, Division of Molecular Pathology, The Institute of Cancer Research, 15 Cotswold Road, Sutton SM2 5NG, UK.
authors: Yinyin
authors: Y
authors: Yuan
authors: The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London SW3 6JB, UK.
authors: Clare M
authors: CM
authors: Isacke
authors: Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK. Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. claus.jorgensen@cruk.manchester.ac.uk.
authors: Claus
authors: C
authors: Jørgensen
mesh_terms: D002450:Cell Communication; D045744:Cell Line, Tumor; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism*; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism*; D009369:Neoplasms / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D040901:Proteomics; D036104:Receptor, EphA2 / Q000378:metabolism; D057705:Transendothelial and Transepithelial Migration
chemical_list: D009363:Neoplasm Proteins; D010750:Phosphoproteins; D036104:Receptor, EphA2