Research ArticleCancer Biology

Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration

See allHide authors and affiliations

Science Signaling  09 Feb 2016:
Vol. 9, Issue 414, pp. ra15
DOI: 10.1126/scisignal.aac5820

You are currently viewing the abstract.

View Full Text

Log in to view the full text

Log in through your institution

Log in through your institution

Phosphoproteomics of metastasis

During the process of metastasis, cancer cells migrate into and then out of the bloodstream to colonize the target tissue. Locard-Paulet et al. investigated signaling events that occurred when breast cancer cells were cocultured with endothelial cells to mimic the early events associated with exit from the bloodstream. Detailed analysis of the phosphorylation status of a specific site on the receptor EPHA2, which can mediate cell repulsion or attraction, showed decreased phosphorylation of that site in cancer cells upon endothelial contact. Phosphorylation of this site was found to be critical for EPHA2-mediated inhibition of transendothelial migration. Furthermore, a highly metastatic breast cancer cell line that targets the lung exhibited altered phosphorylation dynamics at this site and had enhanced adhesion to and migration through endothelial cell monolayers in culture. Thus, this study provides a rich source of candidates for exploration of cancer cell metastasis and identifies altered phosphorylation dynamics of EPHA2 as a mechanism that may enhance breast cancer cell exit from the bloodstream.


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 Tyr772 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-Tyr772. When EPHA2 phosphorylation dynamics were compared between cell lines of different metastatic ability, EPHA2-Tyr772 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.

View Full Text

Stay Connected to Science Signaling