Hee Doo Lee

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano‐sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage‐derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage‐derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome‐mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage‐derived exosomes were also shown to effectively suppress collagen‐induced activation of the mitogen‐activated protein kinase/extracellular signal‐regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking.

Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes

A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg‐Gly‐Asp (RGD) motif in its disintegrin‐like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15‐mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12‐myristate 13‐acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane‐associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvβ3 in an RGD‐dependent manner and suppress vitronectin‐ and fibronectin‐induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage‐derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome‐mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.—Lee, H. D., Koo, B.‐H., Kim, Y. H., Jeon, O.‐H., Kim, D.‐S. Exosome release of ADAM15 and the functional implications of human macrophage‐derived ADAM15 exosomes. FASEB J. 26, 3084–3095 (2012). www.fasebj.org

Integrin αvβ3-mediated transcriptional regulation of TIMP-1 in a human ovarian cancer cell line

We have previously reported that a disintegrin inhibits solid tumor growth and metastasis in mouse model [I.C. Kang, Y.D. Lee, D.S. Kim, A novel disintegrin salmosin inhibits tumor angiogenesis, Cancer Res. 59 (1999) 3754-3760; S.I. Kim, K.S. Kim, H.S. Kim, D.S. Kim, Y. Jang, K.H. Chung, Y.S. Park, Inhibitory effect of the salmosin gene transferred by cationic liposomes on the progression of B16BL6 tumors, Cancer Res. 63 (2003) 6458-462]. In this study, we have investigated the modulatory effect of a disintegrin, saxatilin, on the balance between MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in human ovarian cancer cell line MDAH 2774. Functional mechanism of the disintegrin-mediated transcriptional regulation of MMP-9 and TIMP-1 was examined in the ovarian cancer cell line. Saxatilin strongly induced TIMP-1 expression in dose- and time-dependent manners, while the disintegrin suppressed MMP-9 expression. Further analyses clearly indicated that interaction of the disintegrin and integrin alphavbeta3 results in the TIMP-1 promoter activation via c-fos to suppress TNF-alpha-induced cancer cell invasion. These results demonstrate that integrin alphavbeta3-mediated transcriptional regulation of MMP-9 and TIMP-1 is critical for suppressing the ovarian cancer cell invasion.

The ADAM15 ectodomain is shed from secretory exosomes

We demonstrated previously that a disintegrin and metalloproteinase 15 (ADAM15) is released into the extracellular space as an exosomal component, and that ADAM15-rich exosomes have tumor suppressive functions. However, the suppressive mechanism of ADAM15-rich exosomes remains unclear. In this study, we show that the ADAM15 ectodomain is cleaved from released exosomes. This shedding process of the ADAM15 ectodomain was dramatically enhanced in conditioned ovarian cancer cell medium. Proteolytic cleavage was completely blocked by phenylmethylsulfonyl fluoride, indicating that a serine protease is responsible for exosomal ADAM15 shedding. Experimental evidence indicates that the ADAM15 ectodomain itself has comparable functions with those of ADAM15-rich exosomes, which effectively inhibit vitronectininduced cancer cell migration and activation of the MEK/extracellular regulated kinase signaling pathway. We present a tumor suppressive mechanism for ADAM15 exosomes and provide insight into the functional significance of exosomes that generate tumor-inhibitory factors. [BMB Reports 2015; 48(5): 277-282]

Abstract A92: Tumor suppressive function of human macrophage-derived ADAM15 exosomes.

A disintegrin and metalloproteinase (ADAM) 15, the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed, human membrane protein. The present study demonstrates that macrophages derived from monocytes actively release ADAM15 into the extracellular space as an exosomal component. The exosomal ADAM15 release is further elevated by lipopolysaccharide stimulation, indicating that the exosome release is closely associated with immune cell differentiation and activation. ADAM15-rich exosomes display enhanced binding affinity for integrin alpha v beta 3, thereby interfering with the interaction between integrin alpha v beta 3 and vitronectin. ADAM15 exosomes significantly suppress vitronectin- and fibronectin-induced proliferation and migration of various tumor cells as well as tumor growth in vivo. Tumor suppressive function of exosomal ADAM15 is effectively blocked by monoclonal antibody specific for the extracellular domain of ADAM15. Experimental evidence shows that RGD-containing disintegrin-like domain, but not metalloproteinase activity, of ADAM15 is the responsible element for the tumor suppressive activity. Notably, proliferation and migration of macrophages are stimulated by the exosomes, indicating anti-tumor immunity. This work strongly suggests that a novel tumor suppressive mechanism is mediated by macrophage-derived ADAM15 exosomes. Citation Format: Hee Doo Lee, Yeon Hyang Kim, Doo-Sik Kim. Tumor suppressive function of human macrophage-derived ADAM15 exosomes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A92.

Abstract B42: Modulation of integrin‐mediated cell adhesion by ADAM15 disintegrin‐like domain

Integrin‐mediated cell adhesion on extracellular matrix works essentially on numerous physiological processes such as cell adhesion, migration and angiogenesis. As integrin ανβ3 and integrin α5β1 are the representative receptors for cell adhesion and due to their important function in cancer biology, the antagonists targeting the integrins has been sought for long. Lately, the interactions between ADAM (A Disintegrin And Metalloprotenase) 15, the only member in ADAM family with Arg‐Gly‐Asp (RGD) motif, and the ανβ3 and α5β1 intergins was observed. For that, we experimented on the cellular function of recombinant ADAM15‐derived disintegrin‐like domain containing Asp‐Typ‐Lys‐Arg‐Gly‐Asp (rNWKRGD) in integrin‐mediated cell adhesion. The binding affinity of HUVEC, COS‐1, MCF‐7 and MDAH 2274 cells to various extracellular matrix proteins were increased by the disintegrin‐like domain in a dose‐dependent manner, and the presence of inhibitory integrin α5β1 antibody thoroughly abrogated the rNWKRGD‐stimulated binding. By mutagenic analysis, it was founded that RGD motif is essential for the binding. In comparison, saxatilin, RGD disintegrin from snake venom, inhibited binding of cells to ECM proteins. Through flow cytometric analysis, we confirmed that β1 integrin on the cell surface was activated by rNWKGRD. All these results indicated that the activation of integrin α5β1 mediates the rNWKRGD‐stimulated cell adhesion. Therefore rNWKRGD could function as the potential activator of cell migration and proliferation. Although disintegrin is known as inhibitor of cell migration and proliferation, it is possible that ADAM15 disintegrin domain could have a regulatory role in integrin function such as angiogenesis and invasion of cancer cells. (This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (No. 2009‐0081759), KIST grant, and the Brain Korea 21 (BK21).) Citation Information: Cancer Prev Res 2010;3(1 Suppl):B42.

Abstract B56: Regulation of immune‐related genes by integrin signaling affects the cancer cell proliferation and invasion

Cytokines have been implicated in tumor proliferation and metastasis. Saxatillin, snake venom‐derived disintegrin, is known to suppress tumor progression in vivo and in vitro but its correlation with cytokine9s functions has not been known yet. We have investigated the role of immune‐related genes in cancer cell proliferation and metastasis in human ovarian cancer cell (MDAH 2774). We demonstrate that saxatilin, an integrin antagonist, mitigated MDAH 2774 proliferation and invasion by reducing the level of TNF‐ induced matrix metalloproteinase‐9 (MMP‐9) and IL‐8 expression in a dose‐dependent manner. And Immunoblot assay of nuclear extracts of the cancer cells implicated that signal transducer and activator of transcription (STAT) is related in integrin signaling pathway. We also observed the activity of human glioma cell‐invasion mediated by IL‐8, as examined by a Boyden chamber assay, involved the mechanism of enhancing the actin stress fiber formation. IL‐8 increased the phosphorylation of focal adhesion kinase (FAK), known to be the site of integrin clustering and nuclear translocation of STAT. We have shown here that interleukin‐8 promotes the cancer proliferation and that mRNA levels of immune‐related genes are regulated by disintegrin in human ovarian cancer cell line. These results demonstrate immune‐related genes as a mediator of metastasis and proliferation and disintegrin would contribute in slowing of cancer development by regulating integrin signaling. (This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea Government (No.2009‐0081759), KIST grant, and the Brain Korea 21 (BK21) program.) Citation Information: Cancer Prev Res 2010;3(1 Suppl):B56.