Angiogenin interacts with the plasminogen activation system at the cell surface of breast cancer cells to regulate plasmin formation and cell migration

Abstract

Angiogenin Breast cancerPlasminogen activation system Plasmin Cell migration A B S T R A C TAngiogenin (ANG), a 14-kDa pro-angiogenic secreted protein, has been shown to play a role in cell migration and tumor invasion, which involve proteolytic cleavage of plasminogen to generate plasmin. However, the mechanism by which ANG regulates plasmin formation and cell migration was not known. Our studies here detected elevated levels of secreted and cell surface-bound ANG in highly invasive metastatic breast cancer cells. ANG was also detected at very high levels in the tumor cells in infiltrating ductal carcinomas. By immunofluorescence and immunoprecipitation analysis, ANG was detected at the leading edges of the cell surfaces where it colocalized and interacted with members of the plasminogen activation system (PAS) such as annexin A2 (A2), calpactin (S100-A10) and urokinase plasminogen activator receptor (uPAR). Analysis of lipid raft (LR) and non-lipid raft (NLR) regions of the cell membranes showed the predominance of ANG, A2 and S100-A10 in the LR regions. In contrast, uPAR was detected predominantly in the NLR fractions, suggesting that ANG interacts with uPAR at the junctions of LR and NLR regions. ANG knockdown in T47D and MDA-MB-231 breast cancer cell lines did not affect the cellular expression of A2, S100-A10 and uPAR but decreased cell migration and plasmin formation. Neutralization of ANG with monoclonal antibodies similarly decreased the migration of MDA-MB-231 cells. In the presence of ANG, uPAR was observed to interact with uPA, which is necessary for plasmin formation. Conversely, in the absence of ANG, uPAR did not interact with uPA and FAK and Src kinases were observed to be dephosphorylated. Published by Elsevier B.V. All rights reserved.Abbreviations: ANG, angiogenin; A2, annexin A2; LR, lipid rafts; NLR, non-lipid rafts; PAS, plasminogen activation system; PAI, plasminogen activator inhibitor; uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor; S100-A10, calpactin S100-A10; VT, vitronectin.* Corresponding author. Tel.: þ1 847 578 8323. E-mail address: sujoy.dutta@rosalindfranklin.edu (S. Dutta).

Cantharidin Impairs Cell Migration and Invasion of Human Lung Cancer NCI-H460 Cells via UPA and MAPK Signaling Pathways

“…It is well known that MMP-2/-9 and UPA play important roles in cancer invasion and metastasis (13,14). Studies have shown that the transcription of MMP-2/-9 genes is regulated by upstream regulatory factors, including NF-ĸB p65, c-Jun and AP-1 (37-39).…”

“…Thus, MMP-2/-9 have been the focus on targeting of anticancer drug development due to their role that is associated with cancer cells' attachment, migration and invasion or metastasis (13). UPA has been shown to be involved in cancer cell migration (14) as cell migration is regulated by multiple factors, including signaling cascades, plasmin formation, which is generated by the proteolytic cleavage of plasminogen by UPA, plasminmediated proteolysis of the extracellular matrix and cell adhesion (15). Thus, the inhibition of migration and invasion of cancer cells, which is mediated by MMP-2/-9 or UPA, could be a preventive mechanism for cancer metastasis (13).…”

Three decades of research on angiogenin: a review and perspective

“…proteins [60,61] and cytoskeletal proteins [56,62,63]; (ii) cell proliferation, including follistatin [52,64], phospholipid scramblase 1 (PLSCR1) [57], histone H3 [65], RNH1 [50], four and a half LIM domains 3 (FHL3) [54,66], and ANG receptor that is a 170-kDa cellsurface protein expressed in human endothelial cells [67] or syndecan 4 in astrocyte cells [59]; and (iii) cell apoptosis, including p53 [51], MDM2 [51], heat shock factor 1 (HSF1) [58], and RNH1 [50]. A full identification of ANG-interacting proteins may help to draw the ANG interaction maps and to better understand its roles and mechanisms.…”

“…The interaction between ANG and cell-surface actin could also lead to the activation of several protease cascades, including the plasminogen activator/plasmin serine protease system and the matrix metalloproteinase system [92]. Recent data indicated that ANG interacts with uPAR, A2, and S100-A10 proteins at the junction of lipid raft and nonlipid raft regions of cell membranes, where ANG acts as a bridging molecule to facilitate indirect interactions between uPAR and the plasminogen receptor, A2, S100-A10 complex that is necessary for plasmin formation and cell migration [61,91,93]. Further studies identified a peptide (ANI-E) by phage-display technology that could specifically inhibit the interaction between ANG and actin and then ANG-induced angiogenesis [55,94].…”

p130Cas Scaffolds the Signalosome To Direct Adaptor-Effector Cross Talk during Kaposi's Sarcoma-Associated Herpesvirus Trafficking in Human Microvascular Dermal Endothelial Cells

“…The proximity ligation assay (PLA) was performed using the DuoLink in situ starter kit and PLA reagents (Sigma-Aldrich) to detect protein-protein interactions using fluorescence microscopy according to the manufacturer's protocol (54,55). Briefly, HMVEC-d cells were cultured in eight-well chamber slides either mock or KSHV infected (20 DNA copies/cell), fixed in 4% PFA for 15 min at room temperature, and blocked with DuoLink blocking buffer for 30 min at 37°C.…”