Chemotherapy-Induced Activation of ADAM-17: a Novel Mechanism of Drug Resistance in Colorectal Cancer

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 measured 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 epidermal 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 following 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

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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 transfections tained from Sanofi-Synthelabo. A 1 mmol/L stock solu- ADAM-9, ADAM-10, ADAM-12, ADAM-15, ADAM-17, tion was prepared in injection water and stored at 4°C. and scramble control small interfering RNAs (siRNA) were 5-Fluorouracil (5-FU) was purchased from Sigma Che- obtained from Dharmacon. HCT116, HCT116-p53null, mical Co. A 10 mmol/L stock solution in 1× PBS was LoVo, RKO, and H630 CRC cells were seeded out in the

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appropriate media without penicillin-streptomycin. Twenty- TGF-α/VEGF ELISA four hours after seeding, siRNA transfections were done on An equal number of cells were plated into 24-well plates subconfluent cells incubated in unsupplemented Opti- and incubated for 24 hours. Cells were treated in each ex- MEM using the Oligofectamine reagent (both from Invi- periment for a particular period. Conditioned medium or trogen) according to the manufacturer's instructions. After serum was collected in vitro and in vivo, respectively, and 4 hours, 3× growth medium was added; cells were treated TGF-α or VEGF levels were analyzed according to the with oxaliplatin, 5-FU, or SN-38 1 hour following this. ELISA kit instructions (Calbiochem).

Generation of stable hemagglutinin-tagged ADAM-17 activity ADAM-17–overexpressing cells Excised tumors from HCT116 xenografts were homoge- The plasmid encoding the hemagglutinin (HA)-tagged nized in radioimmunoprecipitation assay buffer using an full-length mouse ADAM-17 (HA-ADAM-17) was ob- IKA Labortechnik homogenizer. After centrifugation of tis- tained as a kind gift from Dr. Atanasio Pandiella (Instituto sue homogenates, the supernatants were transferred to a de Microbiología Bioquímica and Centro de Investigación new tube and protein concentration was determined. del Cáncer) and has been previously described (23). The InnoZyme TACE Activity kit (Calbiochem) was used The pcDNA 3.1 empty vector (EV) was purchased from In- to measure ADAM-17 (TACE) activity in 500 μgofeach vitrogen. HCT116 cells were cotransfected with 10 μgof protein sample. Pure ADAM-17 was used as a positive con- HA-ADAM-17 construct or pcDNA 3.1 (EV) and 1 μgof trol (+TACE). a construct expressing a puromycin resistance gene (pIRE- Spuro3; Clontech) using GeneJuice transfection reagent Phospho-RTK array (Novagen). Stably transfected cells were selected and Activity of a panel of RTKs was detected using an anti- maintained in medium supplemented with 1 μg/mL puro- body-based array from R&D Systems. Antibodies against mycin (Life Technologies, Inc.). 42 different RTKs were prespotted in duplicate on nitrocel- lulose membranes, and cell lysates from EV control cells In vivo experiments and ADAM-17–overexpressing cells were incubated with Female BALB/c severe combined immunodeficient mice the membrane. Thereafter, a pan–anti-phosphotyrosine were maintained under sterile and controlled environmen- antibody was used to detect the phosphorylated tyrosine tal conditions (22°C, 50 ± 10% relative humidity, 12-h/ on activated RTKs. 12-h light/dark cycle, autoclaved bedding), with food and water ad libitum. Following 14 days of quarantine, Statistical analysis mice were included in our protocol. The experiment was Two-way ANOVA test was used to determine the signif- carried out in accordance with the Animals (Scientific Pro- icance of change in levels of apoptosis between different cedures) Act, 1986. To determine tumor volume, two axes treatment groups. All changes in levels of apoptosis that of the tumors were measured using digital Vernier calipers. are described as significant had P values that were <0.05 Tumor volumes were calculated using the following for- (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The nature mula: (longest tumor diameter) × (shortest tumor diame- of the interaction between GW280264X and chemothera- ter)2 × 0.5. HCT116 xenograft mouse models were py was determined by calculating the combination index established by s.c. inoculation of 2 × 106 HCT116 cells in- (CI) according to the method of Chou and Talalay (24). to the right and left flanks using Matrigel (BD Biosciences). CI values of <0.3, 0.3 < CI < 0.7, 0.7 < CI < 0.85, 0.85 < Tumors were allowed to grow until they reached ∼200 mm3 CI < 1, 1, and >1 indicate very strong synergism, strong (day 14), at which point the first group received placebo synergism, moderate synergism, slight synergism, an addi- (PBS) and the second group received 75 mg/kg 5-FU by tive interaction, and antagonism, respectively. CI values i.p. injection. Twenty-four hours after treatment, six mice were calculated from isobolograms generated using the were sacrificed in each of the two treatment groups. Whole CalcuSyn software program (Microsoft Windows). blood was collected from the axillary vessels, and tumors were harvested and snap frozen in liquid nitrogen. For Results analysis of the effect of ADAM-17 overexpression, HA- ADAM-17 or pcDNA 3.1 cells were s.c. implanted using Chemotherapy treatment results in an acute increase Matrigel. Tumors were allowed to grow until they in HER ligand shedding in CRC cells and xenografts reached size of approximately 50 to 100 mm3 (day 8), In view of our previous data showing that cytotoxic che- at which point the first group received placebo (PBS) motherapy activates an EGFR-mediated survival response and the second group received chemotherapy [5-FU (15 in CRC cells (5), we examined whether 5-FU treatment mg/kg, days 8–12 and 15–19) and oxaliplatin (2 mg/kg, induced the release of the prototypical EGFR ligand days 8 and 15)], and each treatment group contained TGF-α in culture medium of CRC cells. 5-FU–based regi- eight animals. The tumors were measured three times a mens [FOLFOX: 5-FU/leucovorin + oxaliplatin; FOLFIRI: week in two dimensions using a caliper. The statistical 5-FU/leucovorin + irinotecan (CPT-11)] represent the cor- significance was analyzed using the unpaired two-tailed nerstone of treatment of patients with advanced CRC. Student's t test. Twenty-four hours following treatment with 5-FU, we

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found a dose-dependent increase in TGF-α ligand shed- Chemotherapy treatment results in an acute increase ding in HCT116 cells (Fig. 1A, top). In addition, shedding in ADAM-17 activity in CRC cells and xenografts of other HER ligands, such as the EGFR ligand AREG [sol- One mechanism by which EGFR can be activated is via uble AREG (sAREG)] and the HER3 ligand heregulin [sol- ADAM-mediated ligand shedding. Treatment with 5-FU uble heregulin (sHeregulin)], also increased significantly had no effect on TGF-α mRNA or AREG mRNA expression following treatment with 5-FU in HCT116 cell line (Fig. and resulted only in a moderate ∼2-fold increase in here- 1A, bottom). Importantly, these effects were also observed gulin mRNA expression level (Supplementary Fig. S2). in vivo, with statistically significant acute increases in hu- These results indicate that increased shedding of TGF-α, man TGF-α shedding into the circulation observed follow- AREG, and heregulin following 5-FU treatment is predom- ing treatment of HCT116 CRC xenograft-bearing mice inantly due to posttranslational mechanisms. With this in with 5-FU for 24 hours (Fig. 1B, top). In addition, the se- mind, we investigated whether a number of different AD- rum levels of AREG and heregulin were also increased in AM family members play a significant role in regulating the 5-FU treatment group compared with control (Fig. 1B, chemotherapy-induced EGFR activation and TGF-α shed- bottom). The relevance of HER ligand shedding for drug ding by using gene-specific siRNAs directed against resistance was shown by cotreating HCT116 cells with re- ADAM-9, ADAM-10, ADAM-12, ADAM-15, and ADAM- combinant TGF-α, AREG, EGF, or heregulin and 5-FU. 17 (Fig. 2A and B). Using quantitative real-time PCR, we Treatment with each HER ligand significantly protected found a decrease of ∼70% to 80% in ADAM-9, ADAM-10, cells from 5-FU–induced apoptosis and apoptosis induced ADAM-12, ADAM-15, and ADAM-17 mRNA expression by oxaliplatin and the active metabolite of irinotecan, following transfection with each siRNA in the LoVo and SN-38 (Fig. 1C, left and right; Supplementary Fig. S1). Tak- HCT116 cell lines (Supplementary Fig. S3). Specific down- en together, these data suggest that induction of TGF-α, regulation of ADAM-17 protein was also confirmed by AREG, and heregulin ligand shedding is an acute mecha- Western blotting (Fig. 2A). Interestingly, we found that nism of drug resistance in CRC cells. the increased TGF-α shedding and EGFR activity following

Fig. 1. Chemotherapy treatment results in increased ErbB ligand shedding in vitro and in vivo. A, HCT116 cells were treated with increasing doses of 5-FU for 24 h. Top, shedding of TGF-α into the medium was measured using a TGF-α ELISA kit; bottom, shedding of AREG and heregulin was assessed by Western blotting analysis. IC50 dose for 5-FU is shown. B, BALB/c nude mice were inoculated in both left and right flanks with HCT116 cells. When tumors reached 200 mm3, the mice were injected i.p. with PBS or treated with 75 mg/kg 5-FU for 24 h. TGF-α levels in serum were measured by ELISA. Shedding of AREG and heregulin was assessed by Western blotting analysis. C, left, HCT116 cells were cotreated with recombinant ErbB ligands and 2.5 μmol/L 5-FU for 48 h, and the cell cycle status of the cells was monitored by flow cytometry after PI staining.

Percentages of cells in sub-G1 phase are given. Right, Annexin V flow cytometry analysis of HCT116 cells treated with recombinant ErbB ligands and 5 μmol/L 5-FU for 48 h. Percentages of early apoptotic cells (Annexin V–positive and PI-negative) and late apoptotic cells (Annexin V– and PI-positive) are given. Representative results of at least three independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P <0.001.

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Fig. 2. Chemotherapy treatment results in increased ADAM-17 activity in vitro and in vivo. A, HCT116 and LoVo cells were transfected with 10 nmol/L ADAM-9 (A9), ADAM-10 (A10), ADAM-12 (A12), ADAM-15 (A15), or ADAM-17 (A17) or 10 nmol/L scramble control (SC) for 4 h and then treated with 5 μmol/L 5-FU (HCT116) or 2.5 μmol/L 5-FU (LoVo) for 24 h. Phospho-EGFR, EGFR, and ADAM-17 expression was detected by Western blotting. Equal loading was assessed by probing for β-tubulin. B, HCT116 and LoVo cells were transfected with 10 nmol/L ADAM-9, ADAM-10, ADAM-12, ADAM-15, or ADAM-17 or 10 nmol/L scramble control for 4 h and then treated with 5 μmol/L 5-FU (HCT116) or 2.5 μmol/L 5-FU (LoVo) for 24 h. TGF-α levels in serum were measured by ELISA. sTGF-α, soluble TGF-α; ns, not significant. C, HCT116 CRC cells were treated with 5 μmol/L 5-FU for 24 h. ADAM-17 activity was determined using a TACE/ADAM-17 fluorometric assay. D, BALB/c nude mice were inoculated in both left and right flanks with HCT116. When tumors reached 200 mm3, the mice were treated with placebo (PBS) or 75 mg/kg 5-FU for 24 h, with each treatment group containing six mice. ADAM-17 activity in tumor lysates was determined using an ADAM-17 fluorometric assay. ***, P < 0.001.

5-FU treatment was only abrogated by ADAM-17 gene si- RKO cell line, and the Kras wild-type/Braf wild-type and lencing, whereas silencing of the other ADAMs (ADAM-9, p53-mutant H630 cell line. We found that silencing of ADAM-10, ADAM-12, and ADAM-15) did not significantly ADAM-17 attenuated 5-FU–induced EGFR activation in affect 5-FU–induced TGF-α shedding and EGFR activation all three of these cell lines, indicating that these effects (Fig. 2A, top, and B). We next directly investigated the ef- are not p53, Kras, or Braf dependent (Fig. 3A). Impor- fect of chemotherapy treatment on ADAM-17 activity. We tantly, we found that shedding of TGF-α following 5-FU found that treatment with 5-FU significantly increased AD- treatment was ADAM-17 dependent in HCT116, LoVo, AM-17 activity in HCT116 cells (Fig. 2C). Importantly, this RKO, and H630 cell line using ADAM-17 siRNA was also observed in vivo, with a significant increase in AD- (Fig. 3B). In addition, 5-FU–induced AREG and heregulin AM-17 activity observed in HCT116 xenografts exposed to shedding was completely abrogated when ADAM-17 was 75 mg/kg 5-FU for 24 hours (Fig. 2D). These results corre- silenced in HCT116, LoVo, and H630 cell lines (Fig. 3C). lated strikingly with the effect of chemotherapy treatment We also determined the role of ADAM-17 in regulating on HER ligand shedding in vitro and in vivo (Fig. 1). an EGFR-mediated survival response following SN-38 and To rule out the possibility that the effects of ADAM-17 oxaliplatin (Supplementary Fig. S4). We found that silenc- were specific to the Kras mutant and p53 wild-type ing of ADAM-17 inhibited SN-38–induced TGF-α shed- HCT116 and LoVo models, we extended these studies into ding and EGFR activation in HCT116, LoVo, RKO, and three further CRC cell line models: a p53-null HCT116 H630 cell lines (Supplementary Fig. S4, top and bottom). daughter cell line, the Braf-mutant and p53 wild-type Similar data were obtained with oxaliplatin. Consistent

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with our earlier studies (5), oxaliplatin failed to increase models using ADAM-17 gene silencing. A significant supra- EGFR activity in HCT116 cell line, and no significant additive/synergistic increase in apoptosis was observed increase in TGF-α shedding following oxaliplatin was in ADAM-17–silenced CRC cells cotreated with 5-FU observed in this cell line. (Fig. 4A). Similar results were obtained when ADAM-17 Taken together, these results indicate that CRC cells siRNA was combined with SN-38 or oxaliplatin in this cell respond to chemotherapy by increasing ADAM-17 activity, line panel (Supplementary Fig. S5A). which further regulates HER ligand shedding and activa- We next assessed the effect of pharmacologic inhibition tion of EGFR. of ADAM-17 using a small-molecule dual ADAM-10/AD- AM-17 inhibitor, GW280264X, and compared this with a Inhibition of ADAM-17 activity, using siRNA or the specific ADAM-10 inhibitor, GI254023X. Physiologically pharmacologic inhibitor GW280264X, sensitizes CRC relevant doses that have been used in previous publica- cells to chemotherapy treatment tions were used (25). We found that GW280264X inhib- We next investigated the effect of ADAM-17 inhibition ited 5-FU–induced ADAM-17 activity, TGF-α ligand on chemotherapy-induced apoptosis in the CRC cell lines shedding, and EGFR activity in both HCT116 and LoVo

Fig. 3. ADAM-17 mediates ErbB ligand shedding and EGFR activity following 5-FU treatment in a panel of CRC cell lines. A, HCT116-p53null, RKO, and H630 cells were transfected with 10 nmol/L ADAM-17 (AD17) siRNA or 10 nmol/L scramble control for 4 h. CRC cells were thereafter treated with 5 μmol/L 5-FU (HCT116-p53null) or 2.5 μmol/L 5-FU (RKO and H630) for 24 h. Phospho-EGFR, total EGFR, and ADAM-17 expression levels were detected by Western blotting. Equal loading was assessed by probing for β-tubulin. B, CRC cells were transfected with 10 nmol/L ADAM-17 (AD17 siRNA) or 10 nmol/L scramble control siRNA for 4 h and then treated with 5 μmol/L 5-FU (HCT116 cells) and 2.5 μmol/L 5-FU (LoVo, H630, and RKO cells) for 24 h. Soluble TGF-α in the medium was measured using a TGF-α ELISA kit. **, P < 0.01. C. HCT116, LoVo, and H630 CRC cells were transfected with 10 nmol/L ADAM-17 (AD17 siRNA) or 10 nmol/L scramble control siRNA for 4 h and then treated with 5 μmol/L 5-FU (HCT116 cells) or 2.5 μmol/L 5-FU (LoVo and H630 cells) for 24 h. sAREG and sHeregulin in the media were detected by Western blotting analysis.

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Fig. 4. ADAM-17 mediates apoptosis following chemotherapy treatment in CRC cells. A, determination of apoptosis in CRC cells treated with combinations of ADAM-17 siRNA with chemotherapy. CRC cells were transfected with 10 nmol/L scramble control or 10 nmol/L ADAM-17 siRNA for 4 h and thereafter treated with 2.5 μmol/L 5-FU (HCT116 and HCT116-p53null), 1 μmol/L 5-FU (LoVo and RKO), and 0.5 μmol/L 5-FU (H630) cells for 72 h. The cell

cycle status of the cells was monitored by flow cytometry after PI staining. Percentages of cells in sub-G1 phase are given. Representative results of at least three independent experiments are shown. B, HCT116 and LoVo cells were incubated with increasing concentrations of GW280264X for 4 h and thereafter treated with 5 μmol/L 5-FU for 24 h. Top, ADAM-17 activity was measured using an ADAM-17 fluorometric assay; bottom, TGF-α ligand shedding in cell culture medium was assessed by ELISA. C, HCT116 and LoVo cells were incubated with increasing concentrations of GW280264X for 4 h and thereafter treated with 5 μmol/L 5-FU for 24 h. Phospho-EGFR and EGFR expression was detected by Western blotting. Equal loading was assessed by probing for β-tubulin. D, determination of apoptosis in CRC cells treated with combinations of GI254023X or GW280264X and chemotherapy. HCT116 and LoVo CRC cells were pretreated with 2.5 μmol/L GI254023X or 2.5 μmol/L GW280264X and combined with 2.5 μmol/L 5-FU for 72 h.

The cell cycle status of the cells was monitored by flow cytometry after PI staining. Percentages of cells in sub-G1 phase are given. Representative results of at least three independent experiments are shown. ***, P < 0.001.

cells (Fig. 4B and C). In contrast, the ADAM-10–specific majority of drug concentrations, with most CI values be- inhibitor GI254023X had only a marginal effect on tween 0.5 and 1.0 (Supplementary Fig. S7A and B). These 5-FU–induced TGF-α shedding and EGFR activation (Sup- results further highlight the importance of ADAM-17 as a plementary Fig. S6). In addition, when GW280264X was key mediator of resistance to chemotherapy and strongly combined with 5-FU, SN-38, or oxaliplatin in HCT116 suggest that ADAM-17–targeted agents may be novel and LoVo cells, significant supra-additive/synergistic drugs for use in conjunction with existing chemotherapy increases in apoptosis were observed (Fig. 4D; Supplemen- regimens in patients with CRC. tary Fig. S5B and C). In contrast, GI254023X had no effect on chemotherapy-induced apoptosis (Fig. 4D; Supple- Effect of ADAM-17 overexpression on growth/survival mentary Fig. S5B and C). We next determined the level of CRC cells and xenografts of synergy between GW280264X and chemotherapy using To complement our gene silencing and inhibitor stud- the method of Chou and Talalay to calculate CI values. For ies, we developed HCT116 cell line models that stably the combination of GW280264X with 5-FU and SN-38, CI overexpress HA-tagged ADAM-17. Two stable clones with values <0.7 were observed for majority of concentrations, different levels of ADAM-17 overexpression were identi- indicative for strong synergism in HCT116 and LoVo cells fied: HA-ADAM-17 3 (AD3) and HA-ADAM-17 4 (AD4; (Supplementary Fig. S7A and B). The combination of Fig. 5A). ADAM-17 activity in clones AD3 and AD4 were GW280264X with oxaliplatin was also synergistic for the approximately 2.5- and 35-fold higher compared with EV

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(Fig. 5A, right, top). Importantly, TGF-α, AREG, and here- and evaluated their response to combined treatment with gulin shedding (Fig. 5A, left and right, bottom) and EGFR 5-FU and oxaliplatin (Fig. 5C). The growth of ADAM-17– and HER3 activation (Fig. 5A) in the ADAM-17–overex- overexpressing CRC xenografts was more rapid than pressing clones were significantly increased compared with control xenografts. Importantly, overexpression of active the EV cell line, with the more highly ADAM-17–overex- ADAM-17 in vivo attenuated the antitumor activity of 5- pressing clone (AD4) exhibiting greater TGF-α shedding FU/oxaliplatin combination treatment (Fig. 5C). Western and EGFR activation. In vitro, ADAM-17 overexpression blot analysis confirmed that the ADAM-17–overexpressing protected cells from 5-FU–induced apoptosis (Fig. 5B; xenografts expressed HA-tagged ADAM-17 (Fig. 5D, top). Supplementary Fig. S8B) and also apoptosis induced by Furthermore, ADAM-17 activity levels were significantly oxaliplatin and SN-38 (Supplementary Fig. S8A and B). higher (∼4-fold) in ADAM-17–overexpressing tumors, We next determined the effect of increased expression of and this was associated with increased EGFR phosphoryla- ADAM-17 on the growth of human HCT116 xenografts tion in these tumors compared with the EV xenografts

Fig. 5. ADAM-17 overexpression protects CRC tumors from apoptosis via increasing ADAM-17 activity, ErbB ligand shedding, and HER1/3 phosphorylation. A, left, Western blot analysis of HA, phospho-EGFR, EGFR, phospho-HER3, sAREG, and sHeregulin expression levels in pcDNA 3.1 (EV control), HA-AD3, and HA-AD4 clones. Right, top, ADAM-17 activity assay was carried out in pcDNA 3.1 (EV), HA-AD3, and HA-AD4 stable clones; bottom, serum TGF-α levels were measured by ELISA for HA-AD3 and HA-AD4 and compared with levels in the pcDNA 3.1 (EV) control. B, flow cytometric analysis of apoptosis in pcDNA 3.1 (EV), HA-AD3, and HA-AD4 cells following either no treatment or treatment with 2.5 μmol/L 5-FU for 72 h. The cell cycle status of the cells was monitored by flow cytometry after PI staining. Percentages of cells in sub-G1 phase are given. Representative results of at least three independent experiments are shown. C, growth rate of pcDNA 3.1 and HA-AD4 xenografts in BALB/c severe combined immunodeficient mice. Mice were either untreated (PBS) or treated with a chemotherapy regimen [5-FU (15 mg/kg, days 8–12 and 15–19) and oxaliplatin (2 mg/kg, days 8 and 15)]. Differences in growth were determined using Student's t test and by calculating subsequent P values. *, P < 0.05; **, P < 0.01. Asterisks, significance of the differences between pcDNA 3.1 and HA-AD4 chemotherapy-treated xenografts. D, top, Western blot showing EGFR activation, cleaved caspase-3, and HA expression in EV xenografts compared with AD4 xenografts following PBS or 5-FU/oxaliplatin treatment; bottom, ADAM-17/TACE activity assay was carried out in EV xenografts and compared with AD4 xenografts following PBS or 5-FU/oxaliplatin treatment.

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Fig. 6. ADAM-17 regulates activity of several survival receptors such as VEGFR1, VEGFR2/3, and IGF-IR. A, human phospho-RTK array in HCT116 cells. HCT116 cells were transiently transfected with EV or HA-ADAM-17 expression construct for 24 h followed by protein extraction. The cell extracts were incubated with membranes containing antibodies to 42 different RTKs. The membranes were washed and incubated with a cocktail of biotinylated detection antibodies to measure the levels of active kinases. The RTKs that phosphorylation changed following 24 h of transfection with ADAM-17 construct are illustrated. B, Western blot analysis of HA, phospho-VEGFR2/3, phospho-VEGFR1, phospho-IGF- IR, and phospho-PDGFRβ expression levels in pcDNA 3.1 (EV control), HA-AD3, and HA-AD4 clones. Equal loading was assessed by probing for β-tubulin. C, expression levels of soluble IGF-I (sIGF-I) and soluble VEGF (sVEGF) in EV control, HA-AD3, and HA-AD4 clones were assessed by Western blotting and ELISA, respectively. D, HCT116 cells were incubated with 2.5 μmol/L GW280264X (GW) for 4 h and thereafter treated with 5 μmol/L 5-FU for 24 h. Soluble IGF-I and soluble VEGF ligand shedding in cell culture medium was assessed by Western blotting (left) and ELISA (right). ***, P < 0.001.

(Fig. 5D). Consistent with our previous findings, we found assessed the phosphorylation status of 42 RTKs in that ADAM-17 activity levels increased significantly in EV HCT116 cells, 24 hours following transient transfection controls and ADAM-17–overexpressing HCT116 xeno- with ADAM-17, using a human phospho-RTK array kit grafts following treatment with 5-FU and oxaliplatin (Fig. 6A). In addition to EGFR, we found increased phos- (Fig. 5D, bottom). Moreover, chemotherapy-induced AD- phorylation levels of VEGFR2/3, IGF-IR, PDGFR, the AM-17 activity levels were associated with increased EGFR Ephrin receptors and developmental tyrosine kinase in phosphorylation (Fig. 5D, top). In addition, chemothera- ADAM-17–overexpressing HCT116 cells. In contrast, py-induced caspase-3 activation was abrogated in the phosphorylation levels of hepatocyte growth factor recep- ADAM-17–overexpressing CRC xenografts compared with tor were reduced following transient transfection with EV xenografts. Collectively, these data indicate that AD- ADAM-17 (Fig. 6A; Supplementary Fig. S9). The VEGFRs, AM-17 activity regulates the sensitivity of CRC tumors to PDGFR, and IGF-IR are key regulators of colorectal tumor standard chemotherapy treatment, further indicating that angiogenesis, lymphangiogenesis, tumor growth, and pro- combining ADAM-17 inhibitors with chemotherapy could liferation and have emerged as important targets in CRC be a potential novel strategy for the treatment of CRC. (26–28). So, we next validated our array results for these receptors by Western blotting analysis using phospho- Active ADAM-17 activates several growth factor RTKs, specific VEGFR1, VEGFR2/3, IGF-IR, and PDGFR anti- such as IGF-IR and VEGFR bodies that reflect the activation state of the receptors. We To further investigate the mechanisms of ADAM-17– found that VEGFR2/3, VEGFR1, and IGF-IR activity was sig- mediated resistance to chemotherapy treatment, we nificantly increased in the stable ADAM-17–overexpressing

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cell lines AD3 and AD4 compared with the EV cell line lated 5-FU–induced, SN-38–induced, and oxaliplatin- (Fig. 6B). Activity of PDGFRβ was only slightly increased induced HER ligand shedding and EGFR activation in AD clones compared with the EV cell line. Moreover, in a broad panel of CRC cell lines, irrespective of p53, we found that shedding of the VEGF and IGF-I ligands Kras, or Braf mutational status. Furthermore, a synergis- was significantly increased in cells stably overexpressing tic activation of apoptosis was observed in vitro when ADAM-17 (Fig. 6C). We subsequently assessed whether ADAM-17 siRNA or the small-molecule ADAM-10/ VEGF and IGF-I shedding occurred following chemo- ADAM-17 inhibitor GW280264X was combined with therapy treatment and if this was also regulated by ADAM- chemotherapy treatment in this panel of CRC cells. In 17. We found that 5-FU treatment resulted in increased contrast, the specific ADAM-10 inhibitor GI254023X VEGF and IGF-I ligand shedding and that this was abrog- had no effect on chemotherapy-induced apoptosis. Taken ated in the presence of the ADAM-10/ADAM-17 inhibitor together, our findings suggest that chemotherapy treat- GW280264X (Fig. 6D). Taken together, these results ment results in acute upregulation in ADAM-17 activity, indicate that through its ability to shed multiple growth which promotes EGFR ligand shedding and an EGFR- factors, ADAM-17 is an important regulator of several mediated prosurvival response following chemotherapy key survival pathways following treatment of CRC cells treatment. Thus, targeting ADAM-17 in combination with with chemotherapy. chemotherapy could represent an important treatment strategy for patients with metastatic CRC. Discussion To complement our gene silencing and small-molecule inhibitor studies, we further examined the importance of We previously reported that CRC cells respond acutely ADAM-17 activity as a mediator of resistance to chemo- to chemotherapy by activating a HER-mediated survival therapy treatment using ADAM-17–overexpressing response and are thereby sensitized to HER inhibitors HCT116 cell lines. ADAM-17–overexpressing clones (5). In light of this, we investigated the mechanisms by showed increased ADAM-17 activity; TGF-α,AREG,and which HERs are activated in response to chemotherapy heregulin ligand shedding; and EGFR/HER3 activation. in CRC cells. Initially, we assessed shedding of TGF-α Moreover, we showed for the first time that ADAM-17 and other HER ligands following exposure to 5-FU treat- can also regulate shedding of other growth factors, such ment both in vitro and in vivo. We found that 5-FU treat- as IGF-I and VEGF, and subsequently regulates activity of ment resulted in statistically significant increases in their respective receptors, IGF-IR and VEGFR. Of note, the human TGF-α, AREG, and heregulin shedding both in cul- enhanced tumor growth of AD4 PBS xenografted mice ture medium of HCT116 cells and in serum of mice bear- compared with EV PBS xenografted mice may be the result ing human HCT116 xenografts. Furthermore, addition of of ADAM-17 regulating growth factor shedding and activ- exogenous EGFR ligands to the culture medium resulted in ity of multiple survival receptors that promote xenografts decreased 5-FU–induced cell death, showing the function- growth. It may be that ADAM-17 should be added to a al relevance of HER ligand shedding following chemother- growing list of nononcogenes that could be exploited as apy treatment. Hagan et al. (2) showed that radiation an anticancer drug target (29). Importantly, the clones therapy can increase shedding of TGF-α in serum of pa- with increased ADAM-17 activity levels had a decreased re- tients treated for hormone-refractory prostate cancer. Our sponse to chemotherapy treatment compared with the EV study is the first to show increased HER ligand shedding in clones. Moreover, overexpression of ADAM-17 protected the context of chemotherapy treatment in CRC. HCT116 xenografts from the growth-inhibitory effects of Several reports have indicated that different ADAMs, chemotherapy and abrogated chemotherapy-induced apo- such as ADAM-9, ADAM-10, ADAM-12, ADAM-15, and ptosis in vivo. Collectively, these results further indicate ADAM-17, can induce EGFR activation by cleaving the ec- that ADAM-17 is an important regulator of chemotherapy todomain of six ligands of EGFR (13), resulting in their resistance, and suggest that targeting this ADAM in con- shedding and ability to activate the receptor in an auto- junction with chemotherapy may have therapeutic poten- crine and paracrine manner. Hence, we determined the tial for the treatment of CRC tumors. effect of ADAM-9, ADAM-10, ADAM-12, ADAM-15,and Although a number of studies have shown additive in- ADAM-17 gene silencing on TGF-α shedding and EGFR teractions when chemotherapy was combined with the activity following 5-FU treatment. In both HCT116 and ADAM-10/ADAM-17 inhibitor INC3619 in lung cancer, LoVo cell lines, complete inhibition of 5-FU–induced breast cancer, and head and neck xenograft models TGF-α shedding and EGFR activation was only observed (6, 30), no underlying mechanism behind this interaction following ADAM-17 silencing. More importantly, we was provided. Our data show for the first time that chemo- found that ADAM-17 activity was potently upregulated therapy treatment can result in potent increases in ADAM- following 5-FU treatment in CRC cells and that chemo- 17 activity in CRC cells and that this protease thereby therapy (the clinically relevant 5-FU and oxaliplatin regulates resistance to chemotherapy treatment. Blocking combination) significantly increased ADAM-17 activity this survival response, using small-molecule ADAM-10/ in human HCT116 xenograft models. These results corre- ADAM-17 inhibitors, resulted in synergistic increase in ap- lated with the results of the TGF-α ELISA both in vitro optosis, and this was irrespective of p53, Kras, or Braf mu- and in vivo. Moreover, we found that ADAM-17 regu- tational status. One other study has investigated the role of

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ADAM-17 in CRC and only examined the interaction be- enhance response rates in patients with advanced CRC tween an ADAM-17 inhibitor and EGFR-targeted agents and thereby improve survival rates compared with those (31); the current study is the first to examine the interac- obtained with combined EGFR monoclonal antibody tion between ADAM-17 and cytotoxic chemotherapy in inhibition (cetuximab)/chemotherapy treatment (33). CRC. We have previously shown that the death receptor – ligand tumor necrosis factor related apoptosis-inducing li- Disclosure of Potential Conflicts of Interest gand can increase TGF-α shedding and HER1/HER2 activity and that this may be regulated by ADAM-17 in CRC, P.G. Johnston: shareholdings, Fusion Antibodies and GlaxoSmithKline; indicating that activation of ADAM-17 may be a common consultancy, AstraZeneca, Pfizer, Roche Pharmaceuticals, Merck, Amgen, Bristol-Myers Squibb, and Ortho Biotech; contracted research, AstraZeneca prosurvival response following treatment with a range of and Amgen. The other authors disclosed no potential conflicts of interest. cytotoxic and apoptosis-inducing agents (32). Merchant et al. showed that ADAM-17 is overexpressed in primary and metastatic CRC compared with normal colonic epi- Acknowledgments thelium, further highlighting the importance of ADAM- We thank GlaxoSmithKline for supplying us with GW280264X and 17 as a potential target in CRC (31). GI254023X and Dr. Atanasio Pandiella for providing us with the ADAM- In conclusion, our findings provide strong evidence that 17 construct. CRC tumors respond to chemotherapy by activating AD- AM-17, which results in increased growth factor shedding Grant Support and activation of growth factor receptor–mediated prosurvival response. Furthermore, we provide strong evidence Cancer Research UK, Medical Research Council, and Ulster Cancer that enhanced ADAM-17 activity and HER ligand shed- Foundation. The costs of publication of this article were defrayed in part by the ding result in resistance to chemotherapy treatment in payment of page charges. This article must therefore be hereby marked CRC tumors. Moreover, therapies targeting ADAM-17 advertisement in accordance with 18 U.S.C. Section 1734 solely to (and thereby the activity of multiple RTKs, such as EGFR, indicate this fact. HER3, IGF-IR, and VEGFR) in conjunction with existing Received 01/04/2010; revised 04/01/2010; accepted 04/15/2010; chemotherapy treatments (FOLFOX and FOLFIRI) may published OnlineFirst 06/22/2010.

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Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

Joan N. Kyula, Sandra Van Schaeybroeck, Joanne Doherty, et al.

Clin Cancer Res 2010;16:3378-3389. Published OnlineFirst June 22, 2010.

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