Published OnlineFirst June 22, 2010; DOI: 10.1158/1078-0432.CCR-10-0014 Cancer Therapy: Preclinical Clinical Cancer See commentary p. 3319 Research Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer Joan N. Kyula, Sandra Van Schaeybroeck, Joanne Doherty, Catherine S. Fenning, Daniel B. Longley, and Patrick G. Johnston Abstract Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism. Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was mea- sured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting. Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-α, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epi- dermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor. Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC. Clin Cancer Res; 16(13); 3378–89. ©2010 AACR. Resistance to chemotherapy is a major barrier in the binding of the HER1-specific ligands [epidermal growth treatment of cancer. Recent studies including our own factor (EGF), transforming growth factor-α (TGF-α), and have shown that exposure to anticancer drugs or ionizing amphiregulin (AREG)] or ligands with dual specificity radiation can activate stress pathways, which trigger activa- [heparin-binding EGF (HB-EGF), β-cellulin, and epiregu- tion of multiple signaling pathways, such as those regulat- lin (EREG)] to the ectodomain of HER1 (9, 10). HER1 ed by the human epidermal receptor (HER) tyrosine and its ligand TGF-α constitute one of the best defined kinase family (1–5). autocrine loops in human tumors (6, 11), and their coex- The HER family of receptor tyrosine kinases (RTK) and pression correlates with aggressive disease and poor prog- their ligands are important regulators of tumor cell prolif- nosis in several types of tumors, including colorectal eration, angiogenesis, and metastasis (6–8). There are four receptors in the ErbB family: epidermal growth factor re- cancer (CRC). Recently, high AREG and EREG mRNA ex- ceptor (EGFR; HER1 or ErbB1), HER2 (neu or ErbB2), pression levels in Kras wild-type colorectal primaries have HER3 (ErbB3), and HER4 (ErB4). HER1 is activated by been correlated with response and survival benefit follow- ing treatment with cetuximab and irinotecan in advanced Authors' Affiliation: Drug Resistance Group, Centre for Cancer Research CRC (12). and Cell Biology, Queen's University Belfast, Belfast, Northern Ireland HER ligands are synthesized as transmembrane pre- Note: Supplementary data for this article are available at Clinical Cancer cursors that can be cleaved by cell surface proteases, Research Online (http://clincancerres.aacrjournals.org/). particularly members of the ADAM (a disintegrin and J.N. Kyula and S. Van Schaeybroeck contributed equally to this work. metalloproteinase) family. ADAM-mediated ligand shed- Corresponding Author: Patrick G. Johnston, Centre for Cancer Research ding results in enhanced juxtacrine and paracrine signal- and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland. Phone: 44-2890-972764; Fax: 44-2890- ing (13). ADAMs are synthesized as inactive precursors 972949; E-mail: [email protected]. containing a prodomain that blocks the activity of the doi: 10.1158/1078-0432.CCR-10-0014 catalytic domain. During transit through the secretory ©2010 American Association for Cancer Research. pathway, the prodomain of ADAMs is removed by 3378 Clin Cancer Res; 16(13) July 1, 2010 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst June 22, 2010; DOI: 10.1158/1078-0432.CCR-10-0014 ADAM-17 Activity and Chemotherapy Resistance preparedandstoredat4°C.SN-38wasobtainedfrom Translational Relevance Abatra, and a 2 mmol/L solution was prepared in DMSO and stored at 4°C. Resistance to chemotherapy is a major barrier in the treatment of colorectal cancer (CRC). In this study, we Cell culture show that cytotoxic chemotherapy treatment results in All tissue culture material was obtained from Invitro- an acute increase in ADAM-17 (a disintegrin and gen, unless otherwise stated. HCT116 and HCT116- in vitro in vivo metalloproteinase-17) activity and . p53null CRC cells were kindly provided by Bert Vogel- Blocking ADAM-17 activity, using small interfering stein (Johns Hopkins University, Baltimore, MD) and RNA or a small-molecule inhibitor, significantly in- maintained in McCoy's 5A medium. LoVo CRC cells, creased apoptosis following chemotherapy treatment. supplied by AstraZeneca, were grown in DMEM. The We further show that overexpression of ADAM-17 in- RKO and H630 CRC cells were provided by the National creases activity of the human epidermal receptors and Cancer Institute (Bethesda, MD) and maintained in other prosurvival receptors, such as insulin-like growth DMEM. All medium was supplemented with 10% dia- factor-I receptor and vascular endothelial growth factor lyzed FCS, 50 μg/mL penicillin-streptomycin, 2 mmol/L receptor, and that this results in resistance to chemo- L-glutamine, and 1 mmol/L sodium pyruvate (Invitro- therapy treatment in CRC tumors. Thus, targeting gen). All cells were grown in a humidified atmosphere ADAM-17 in conjunction with existing chemotherapy with 5% CO2 at 37°C. treatments may enhance response rates in patients with advanced CRC by blocking the activity of multi- Flow cytometric analysis and cell death measurement ple prosurvival receptors. Flow cytometry was done as previously described (5). Annexin V analysis and apoptosis measurement μ furin-like proprotein convertases (14). Several studies have Cell pellets were resuspended in 100 L of 1× binding buffer. Annexin V stain (5 μL) was added to each sample shown that members of the ADAM family, such as ADAM-9, along with 5 μL of propidium iodide (PI) stain (50 μg/mL; ADAM-10, ADAM-12, ADAM-15, and ADAM-17, may be 1:20 dilution in PBS of stock), and samples were incubat- involved in regulating HER1 activation via proteolytic pro- ed in the dark at room temperature for 15 minutes. After cessing of HER1 ligand precursors (1, 3, 15, 16) and that incubation, 320 μL of 1× binding buffer was added to each these metalloproteinase-dependent mechanisms can be sample before analysis on the EPICS XL flow cytometer. activated by cellular stress (1). Of these, ADAM-17 has been suggested to be the major HER ligand “sheddase.” Studies by Western blotting various groups have shown that ADAM-17 deficiency abro- Western blot analysis was carried out as previously gates the shedding of TGF-α, HB-EGF, EREG, AREG, and described (5). Immunodetections were done using anti- heregulin (17–22). EGFR (clone 13; Pharmingen, BD Biosciences), anti- The aims of this study were to investigate whether ex- heregulin (R&D Systems), anti-AREG (R&D Systems), posure to chemotherapy treatment results in increased and anti–insulin-like growth factor (IGF)-I (Santa Cruz Bio- HER ligand shedding and whether this survival response technology) mouse monoclonal antibodies in conjunction was associated with resistance to chemotherapy treat- with a horseradish peroxidase–conjugated anti-mouse sec- ment. We have also investigated the mechanism by ondary antibody (Amersham). Anti–phospho-EGFR which chemotherapy triggers HER ligand shedding, par- (Tyr1068;Calbiochem),anti–ADAM-17 (Pharmingen, BD ticularly the role of ADAM proteases in regulating this Biosciences), anti–phospho-IGF-IR (Calbiochem), anti– survival response. phospho-vascular endothelial growth factor receptor 1 (VEGFR1; Calbiochem), anti–phospho-VEGFR2/3 (Cal- biochem), and anti–platelet-derived growth factor receptor Materials and Methods β (PDGFRβ; Calbiochem) rabbit polyclonal antibodies were used in conjunction with a horseradish peroxidase– Materials conjugated anti-rabbit secondary antibody (Amersham). All chemicals and reagents of Analar grade were ob- Equal loading was assessed using β-tubulin (Sigma) tained from BDH Laboratory Supplies unless otherwise mouse monoclonal primary antibodies. The SuperSignal stated. GI254023X and GW280264X were provided by chemiluminescent system (Pierce) or ECL Plus (Amer- GlaxoSmithKline. A 10 mmol/L working solution of sham) was used for detection. GI254023X and GW280264X in DMSO was prepared, aliquoted, and stored at −70°C. Oxaliplatin was ob- Small interfering RNA