Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive “niches”. Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7), representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs). Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS) and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC) led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR) responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment.

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).

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.