Cancer Therapy: Preclinical Secretome Signature Identifies ADAM17 as Novel Target for Radiosensitization of Non–Small Cell Lung Cancer Ashish Sharma, Sabine Bender, Martina Zimmermann, Oliver Riesterer, AngelaBroggini-Tenzer, and Martin N. Pruschy Abstract Purpose: Ionizing radiation (IR) induces intracellular signaling processes as part of a treatment-induced stress response. Here we investigate IR-induced ADAM17 activation and the role of ADAM17-shed factors for radiation resistance in non–small cell lung cancer. Experimental Design: Large-scale secretome profiling was performed using antibody arrays. Secretion kinetics of ADAM17 substrates was determined using ELISA across multiple in vitro and in vivo models of non–small cell lung cancer. Clonogenic survival and tumor xenograft assays were performed to determine radio- sensitization by ADAM17 inhibition. Results: On the basis of a large-scale secretome screening, we investigated secretion of auto- or paracrine factors in non–small cell lung cancer in response to irradiation and discovered the ADAM17 network as a crucial mediator of resistance to IR. Irradiation induced a dose-dependent increase of furin-mediated cleavage of the ADAM17 proform to active ADAM17, which resulted in enhanced ADAM17 activity in vitro and in vivo. Genetic or pharmacologic targeting of ADAM17 suppressed IR-induced shedding of secreted factors, downregulated ErbB signaling in otherwise cetuximab-resistant target cells, and enhanced IR- induced cytotoxicity. The combined treatment modality of IR with the ADAM17 inhibitor TMI-005 resulted in a supra-additive antitumor response in vivo demonstrating the potential of ADAM17 targeting in combination with radiotherapy. Conclusions: Radiotherapy activates ADAM17 in non–small cell lung cancer, which results in shedding of multiple survival factors, growth factor pathway activation, and IR-induced treat- ment resistance. We provide a sound rationale for repositioning ADAM17 inhibitors as short-term adjuvants to improve the radiotherapy outcome of non–small cell lung cancer. Clin Cancer Res; 22(17); 4428–39. Ó2016 AACR. Introduction Radiotherapy along with chemotherapy or surgery is the primary treatment regimen for locally advanced non–small cell lung cancer (NSCLC). However, the response rates and clinical outcomes are still disappointing, with the 5-year survival rate being only approximately 15% (1–3). Ionizing radiation (IR) triggers multiple intracellular signaling processes as part of IR- induced stress responses that lead to the secretion of various para- and autocrine factors into the tumor microenvironment (4–6). These secreted components generate de novo resistance mechanisms during the course of the treatment and at the same time represent interesting targets to sensitize for IR-induced cell killing (7–9). Several studies have shown that targeting tumor- derived factors such as VEGF, PDGFA, TGFb, MMP-13, and SDF-1 counteract growth of carcinoma cells and sensitize them to irradiation (10–14). Growth and survival of NSCLC cells are often dependent on ectodomain shedding which includes the proteolytic cleavage of the extracellular part of membrane proteins primarily mediated by membrane-anchored metalloproteases, and results in release of various soluble growth factors and cytokines regulating cell proliferation and migration (15, 16). ADAMs (a disintegrin and metalloproteinase) are membrane-associated metalloproteinases with modular design and complex multidomain structure (17). Most of the members of the ADAM family (13 of 21 human ADAMs) have proteolytic activity, as well as domains with adhe- sive properties and a cytoplasmic domain involved in cell signal- ing (18). They are actively associated with the process of proteo- lytic "shedding" of membrane-bound proteins and hence the rapid modulation of key signals in the tumor microenvironment. ADAM-mediated shedding is both constitutive and inducible, depending on upstream kinase activation, intracellular Ca 2þ levels, membrane lipid composition, and other experimental and natural stimuli (19, 20). Multiple resistance mechanisms in NSCLC have been linked to the EGFR, which has therefore been regarded as a promising target for a combined treatment modality. However, a recent phase III trial for patients with stage IIIA or IIIB NSCLC (RTOG 0617) demonstrated that sole addition of the EGFR-directed mAb cetux- imab to concurrent chemoradiation and consolidation treatment Laboratory for Applied Radiobiology, Department of Radiation Oncology, University Hospital Zurich, University of Zurich Zurich, Switzerland. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). A. Sharma and S. Bender contributed equally to this article. Corresponding Author: Martin N. Pruschy, Department of Radiation Oncology, University Hospital Zurich, Ramistr. 100, Zurich ZH-8091, Switzerland. Phone: 414-4255-8549; Fax: 414-4255-4435; E-mail: martin.pruschy@usz.ch doi: 10.1158/1078-0432.CCR-15-2449 Ó2016 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 22(17) September 1, 2016 4428 Downloaded from http://aacrjournals.org/clincancerres/article-pdf/22/17/4428/2964287/4428.pdf by guest on 20 June 2022