Abstract A087: Angiogenin (ANG) localized at the leading edges of breast cancer cells regulates plasmin formation by plasminogen activation complex (PAC) and cell migration

Abstract

Angiogenin (ANG), a 14-kDa pro-angiogenic protein, had been shown to play a role in cell migration and tumor invasion processes, 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 detected elevated levels of secreted ANG and cell surface-bound ANG in highly invasive metastatic breast cancer cells. By immunofluorescence and immunoprecipitation analysis, ANG was detected at leading edges of the cell surfaces where it colocalized and interacted with members of the plasminogen activation complex (PAC) such as Annexin A2 (A2), Calpactin (S100A10) and Urokinase plasminogen receptor (uPAR). Analysis of lipid raft (LR) and non-lipid raft (NLR) regions of the cell membranes showed the predominance of ANG, A2 and S100A10 in the LR regions. In contrast, uPAR was detected only in the NLR fractions, suggesting that ANG interacts with uPAR at 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, S100A10 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 on cell surfaces, uPAR was observed to interact with UPA, which is known to be necessary for proteolytic cleavage of plasminogen by UPA to form plasmin. However, in the absence of ANG, uPAR interacted exclusively with PAI, which is known to inhibit plasmin formation by blocking UPA mediated proteolytic cleavage of plasminogen. Assessment of phosphorylation states of cellular FAK and Src kinases under these conditions further showed that both FAK and Src are dephosphorylated in the absence of ANG. Taken together, our results identified a novel regulatory role of ANG as a bridging protein that facilitates the interaction of uPAR with UPA, which leads to plasmin formation and cell migration necessary for tumor invasion and metastasis of breast cancer cells. These studies also suggest that ANG could serve as a bio-marker for identifying the leading edges of metastatic breast cancer cells and targeting ANG could be a novel method to block breast cancer invasiveness. Citation Format: Sujoy Dutta, Chirosree Bandyopadhyay, Lydia Wilson, Case Warshall, Virginie Bottero, Karen E. Johnson, Bala Chandran. Angiogenin (ANG) localized at the leading edges of breast cancer cells regulates plasmin formation by plasminogen activation complex (PAC) and cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A087.