Published August 8, 2016 Article An activated form of ADAM10 is tumor selective and regulates cancer stem-like cells and tumor growth Lakmali Atapattu,1* Nayanendu Saha,3* Chanly Chheang,1 Moritz F. Eissman,4 Kai Xu,3 Mary E. Vail,1 Linda Hii,1 Carmen Llerena,1 Zhanqi Liu,5 Katja Horvay,2 Helen E. Abud,2 Ulrike Kusebauch,6 Robert L. Moritz,6 Bi-Sen Ding,7 Zhongwei Cao,7 Shahin Rafii,7 Matthias Ernst,4 Andrew M. Scott,5 Dimitar B. Nikolov,3 Martin Lackmann,1 and Peter W. Janes1 1Cancer Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology and 2Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria 3800, Australia 3Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 4Cancer and Inflammation Laboratory and 5Tumor Targeting Laboratory, Olivia Newton-John Cancer Research Institute and School of Cancer Medicine, La Trobe University, Heidelberg, Victoria 3084, Australia 6Institute for Systems Biology, Seattle, WA 98109 7 Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10065 Downloaded from The transmembrane metalloprotease ADAM10 sheds a range of cell surface proteins, including ligands and receptors of the Notch, Eph, and erbB families, thereby activating signaling pathways critical for tumor initiation and maintenance. ADAM10 is thus a promising therapeutic target. Although widely expressed, its activity is normally tightly regulated. We now report prevalence of an active form of ADAM10 in tumors compared with normal tissues, in mouse models and humans, identified by our conformation-specific antibody mAb 8C7. Structure/function experiments indicate mAb 8C7 binds an active conformation jem.rupress.org dependent on disulfide isomerization and oxidative conditions, common in tumors. Moreover, this active ADAM10 form marks cancer stem-like cells with active Notch signaling, known to mediate chemoresistance. Importantly, specific targeting of active ADAM10 with 8C7 inhibits Notch activity and tumor growth in mouse models, particularly regrowth after chemotherapy. Our results indicate targeted inhibition of active ADAM10 as a potential therapy for ADAM10-dependent tumor development and drug resistance. on August 23, 2016 INT ROD UCT ION ADAM (a disintegrin and metalloprotease) transmembrane frequently overexpressed in cancers, correlating with aber- metalloproteases (MPs) catalyze the release of a range of cell rant signaling and poor patient prognosis, including cancers surface proteins, activating receptor tyrosine kinase (RTK), of the colon, lung, stomach, uterus, and ovary (Pruessmeyer Notch, cytokine, chemokine, and adhesion signaling path- and Ludwig, 2009). They are thus potent activators of key ways important in normal and oncogenic development. oncogenic pathways and recognized targets for multipathway Prominent oncogenic substrates include ligands and recep- inhibition (Murphy, 2008; Hartmann et al., 2013). The Journal of Experimental Medicine tors in the Notch, erbB, and Eph families, cytokines (TNF ADAM10 in particular acts as principal sheddase for and IL6), FAS ligand, Slit, L-selectin, and cadherins (Mur- Notch (Hartmann et al., 2002), Eph (Hattori et al., 2000; phy, 2008), which are all shed by one of two closely related Janes et al., 2005), and certain epidermal growth factor re- and widely expressed proteases, ADAM10 and ADAM17 (or ceptor (EGFR) ligands (Sahin et al., 2004), as well as E- and TACE [TNF converting enzyme]). These proteases are also N-cadherin (Reiss et al., 2005). The resemblance of ADAM10 and Notch-deficient mice, including embryonic defects in Dr. Lackmann died on 22 May 2014. somitogenesis, neurogenesis, and vasculogenesis (Hartmann *L. Atapattu and N. Saha contributed equally to this paper. et al., 2002; Saftig and Reiss, 2011), highlights a critical role Correspondence to Peter W. Janes: [email protected] for ADAM10 in canonical ligand-activated Notch signaling L. Atapattu’s present address is Children's Cancer Institute, Lowy Cancer Research in particular. Notch signaling is triggered by binding of cell Centre, University of New South Wales, Randwick, New South Wales 2031, Australia. surface–bound ligands, Delta-Like (1–4) or Jagged (1 and Abbreviations used: ADAM, a disintegrin and metalloprotease; CDR, complementarity 2), to Notch receptors (Notch1–4), which initiates ADAM- determining region; CRC, colorectal cancer; CSC, cancer stem cell; ECD, extracellular mediated shedding of both ligand (LaVoie and Selkoe, 2003) domain; EGFR, epidermal growth factor receptor; EMT, epithelial to mesenchymal and receptor extracellular domains (ECDs; Kopan and Ilagan, transition; GSI, γ-secretase inhibitor; HMW, high molecular weight; IF, immunofluores- cence; IHC, immunohistochemistry; IP, immunoprecipitation; LMW, low molecular weight; MMP, matrix MP; MP, metalloprotease; MPB, maleimide-PEG2-biotin; NICD, © 2016 Atapattu et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www .rupress .org notch intracellular domain; PDI, protein disulfide isomerase; ROS, reactive oxygen /terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial– species; RTK, receptor tyrosine kinase; WB, Western blot. Share Alike 3.0 Unported license, as described at http ://creativecommons .org /licenses /by -nc -sa /3 .0 /). The Rockefeller University Press $30.00 Supplemental Material can be found at: J. Exp. Med. 2016 Vol. 213 No. 9 1741–1757 http://jem.rupress.org/content/suppl/2016/07/28/jem.20151095.DC1.html 1741 www.jem.org/cgi/doi/10.1084/jem.20151095 Published August 8, 2016 2009). Shedding of the notch ECD provides the signal for which, mAb 8C7, specifically recognized the substrate-bind- γ-secretases to cleave and release the Notch intracellular do- ing C domain and inhibited ADAM10-mediated cleavage of main (NICD), acting as transcriptional activator for an exten- Eph receptor ligands (ephrins) and ephrin/Eph-dependent sive set of genes, regulating cell proliferation, differentiation, signaling and cell segregation in vitro (Atapattu et al., 2012). epithelial to mesenchymal transition (EMT), and cell sur- We thus sought to test the mechanism of action of 8C7 and its vival (Kopan and Ilagan, 2009). Deregulated Notch signaling efficacy for tumor growth inhibition. We find 8C7 specifically promotes the progression of solid cancers (Ranganathan et binds a CxxC-dependent, active form of ADAM10, which al., 2011) by driving angiogenesis (Roca and Adams, 2007) we detect preferentially in tumors compared with normal and maintaining undifferentiated, cancer stem cells (CSCs), tissues. Moreover, the 8C7-recognized, active ADAM10 par- thought to initiate and sustain tumor growth and pro- ticularly marks CSC-like cells with high Notch activity, and mote metastasis and chemoresistance (Espinoza et al., 2013; 8C7 treatment inhibits Notch signaling and tumor growth Giancotti, 2013). However, pan-specificγ -secretase inhibi- in mouse models, particularly regrowth after chemotherapy. tors (GSIs) blocking NICD release (Groth and Fortini, 2012) cause severe intestinal toxicity, likely reflecting the diversity RES ULTS of γ-secretase targets (Dikic and Schmidt, 2010). Similarly, Preferential targeting of ADAM10 in tumors small-molecule inhibitors blocking the ADAM protease ac- We tested our anti-ADAM10 mAb 8C7 in a LIM1215 col- tive site failed clinical development, initially because of, at orectal cancer (CRC) xenograft model, which displays high Downloaded from least in part, off-target effects, reflecting the close structural levels of ADAM10, Notch, Eph, and erbB receptors. We first similarity of this site in all matrix MPs (MMPs; DasGupta et analyzed targeting to tumors and other tissues of tumor-bear- al., 2009; Saftig and Reiss, 2011). In support, more specific ing mice by injection of Alexa-labeled 8C7 and subsequent ADAM inhibitors, with limited MMP targets, show no ad- analysis of tumor and organ tissues by immunofluorescence verse effects associated with MMP inhibition, such as fibro- (IF) microscopy. Remarkably, despite 8C7 recognizing both plasias (Fridman et al., 2007). mouse and human ADAM10 (Atapattu et al., 2012), it se- The ADAM ECD contains an N-terminal pro-se- lectively bound to tumors, particularly to cells near vessels jem.rupress.org quence followed by MP (M), disintegrin (D), cysteine-rich (marked with rhodamine-lectin) and near the tumor rim, (C), transmembrane, and cytoplasmic domains (Hartmann with only slight or undetectable binding to other organs et al., 2013). Proteolytic specificity is not simply caused by (Fig. 1, A and B). In comparison, a commercial ADAM10 a typical substrate cleavage signature, but relies on noncata- antibody (MAB946) recognizing both human and mouse lytic interactions of the substrate with the ADAM C domain ADAM10 strongly stained multiple tissues (Fig. 1 B, last col- to position the substrate for effective cleavage (Smith et al., umn), consistent with the ubiquitous expression of ADAM10. on August 23, 2016 2002; Janes et al., 2005, 2009). In addition, emerging evidence We also recovered 8C7-bound ADAM10 from tumors and suggests that ADAM17 is regulated by adopting latent and organs of mice injected with 8C7, or PBS as control, by in- active ECD conformations, dependent on redox state, be- cubating protein extracts with protein A Sepharose. ADAM10 cause mild reducing or