Angiogenin interacts with the plasminogen activation system at the cell surface of breast cancer cells to regulate plasmin formation and cell migration.
Angiogenin (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. Exogenous addition of recombinant ANG to ANG knocked down MDA-MB-231 cells restored FAK phosphorylation, uPAR interactions with uPA, plasmin formation as well as migration of these cells. Taken together, our results identified a novel role for ANG as a member of the uPAR interactome that facilitates the interaction of uPAR with uPA, leading to plasmin formation and cell migration necessary for tumor invasion and metastasis of breast cancer cells.
H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA. Electronic address: sujoy.dutta@rosalindfranklin.edu.
Sujoy
S
Dutta
H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA.
Chirosree
C
Bandyopadhyay
H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA.
Virginie
V
Bottero
H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA.
Mohanan V
MV
Veettil
H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA.
Lydia
L
Wilson
Department of Pathology, Advocate Lutheran General Hospital, Park Ridge, IL, USA.
Michael R
MR
Pins
H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA.
Karen E
KE
Johnson
H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA.
Case
C
Warshall
H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA.
Bala
B
Chandran
D001940:Breast / Q000378:metabolism / Q000473:pathology; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement; D005260:Female; D006801:Humans; D009361:Neoplasm Invasiveness / Q000473:pathology; D010958:Plasminogen / Q000378:metabolism*; D060066:Protein Interaction Maps; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D055293:Receptors, Urokinase Plasminogen Activator; D010958:Plasminogen; C046366:angiogenin; D012259:Ribonuclease, Pancreatic; D014568:Urokinase-Type Plasminogen Activator
Angiogenin; Breast cancer; Cell migration; Plasmin; Plasminogen activation system