Published OnlineFirst October 24, 2014; DOI: 10.1158/1535-7163.MCT-13-1001 Molecular Cancer Cancer Biology and Signal Transduction Therapeutics The Effect of Disintegrin–Metalloproteinase ADAM9 in Gastric Cancer Progression Jeong Min Kim1,2, Hei-Cheul Jeung1,3, Sun Young Rha1,2,3, Eun Jeong Yu1,4, Tae Soo Kim1, You Keun Shin1, Xianglan Zhang1, Kyu Hyun Park1, Seung Woo Park2,3, Hyun Cheol Chung1,2,3, and Garth Powis5 Abstract Advanced gastric cancer is one of the most aggressive gastrointestinal malignancies, and ADAM (A disintegrin and metalloproteinase)-9 is a cell-surface membrane glycoprotein with oncogenic properties that is overexpressed in several cancers. Herein, we investigated the biologic mechanism of ADAM9 in the progression, proliferation, and invasion of gastric cancer. First, we detected ADAM’s expression, proces- sing, and protease activity in gastric cancer cells. Protease activity was moderately correlated with ADAM9 protein expression, but was better related to a processed smaller molecular weight (84 kDa) form of ADAM9. Knockdown of ADAM9 or specifically targeted monoclonal antibody (RAV-18) suppressed cancer cell proliferation and invasion in high ADAM9-expressing cells, not in low ADAM9-expressing cells. RAV- 18 showed in vivo antitumor activity in a gastric cancer xenograft model. Hypoxia (1% oxygen) induced ADAM9 expression and functional activity in low ADAM9-expressing gastric cancer cells that was inhibited by siRNA knockdown or RAV-18 antibody to levels in normoxic cells. Overall, our studies show that ADAM9 plays an important role in gastric cancer proliferation and invasion, and that while expressed in some gastric cancer cells at high levels that are responsive to functional inhibition and antitumor activity of a catalytic site–directed antibody, other gastric cancer cells have low levels of expression and only when exposed to hypoxia do ADAM9 levels increase and the cells become responsive to ADAM9 antibody inhibition. Therefore, our findings suggest that ADAM9 could be an effective therapeutic target for advanced gastric cancer. Mol Cancer Ther; 13(12); 3074–85. Ó2014 AACR. Introduction matrix (ECM), and "invasion" is the first step by which Gastric cancer is the second leading cause of cancer- cancer cells break away from the primary tumor and related mortality worldwide with a median survival of infiltrate surrounding stoma (1). Proteolysis has only around a year after diagnosis of advanced disease. emerged as a key posttranslational regulator of inva- This dismal prognosis mainlyresultsfromearlyinva- sion in gastric cancer (2, 3). However, the mechanism sion and high metastatic activity with an unknown and major players in invasion have not yet been fully molecular basis. Cancer metastasis is a complex, mul- elucidated. tistep process involving the interplay between cancer A disintegrin and metalloproteases (ADAM) are cells and surrounding components of extracellular modular type I transmembrane proteins that contain a metalloprotease and a disintegrin-like domain, and re- cent studies have demonstrated a relationship between 1Cancer Metastasis Research Center, Institute for Cancer Research, increased ADAMs and cancer progression (4). Twenty- Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, one members of ADAM family have been identified in 2 Republic of Korea. Brain Korea 21 Projects for Medical Science, Yonsei humans, 13 of which have functional proteases. ADAM9 University College of Medicine, Seoul, Republic of Korea. 3Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic is shown to possess potent biologic activities and is of Korea. 4Department of Biology, Baylor University, Waco, Texas. 5San- highly expressed in cancers, including prostate, pancre- ford-Burnham Research Institute Cancer Center, La Jolla, California. as, breast, and colon, associated with cancer progression Note: Supplementary data for this article are available at Molecular Cancer and poor clinical outcome (5–9). In prostate cancer, Therapeutics Online (http://mct.aacrjournals.org/). microenvironmental stress on tumor cells leads to shed- Corresponding Author: Hei-Cheul Jeung, Department of Internal Medi- ding of proheparin-binding epidermal growth factor cine, Gangnam Severance Hospital, Yonsei University College of Medicine, 211 Eonju-Ro (146-92 Dogok-Dong), Gangnam-Gu, Seoul 135-720, Korea. (EGF), which is processed by ADAM9. The metallopro- Phone: 82-2-2019-1242; Fax: 82-2-362-5592; E-mail: tease domain of ADAM9 also cleaves the insulin b-chain, [email protected] tumor necrosis factor (TNF)-a, transforming growth doi: 10.1158/1535-7163.MCT-13-1001 factor (TGF)-a, gelatin, b-casein, and so on, and induces Ó2014 American Association for Cancer Research. the shedding of EGF, fibroblast-growth factor receptor 3074 Mol Cancer Ther; 13(12) December 2014 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst October 24, 2014; DOI: 10.1158/1535-7163.MCT-13-1001 ADAM9 in Gastric Cancer 2IIIB, and heparin-binding EGF-like growth factor RNA extraction and RT-PCR (10, 11). Therefore, the abilities of ADAM9 to degrade Total RNA was extracted using TRizol reagent specific ECM substrates, release active growth factors, (Qiazen Science) according to the manufacturer’s pro- interact with key regulatory factors, and its expres- tocol. Briefly, 500 mL of TRizol was added to 1 Â 107 sion at the invasion edges of several cancer metastases cells, and after adding 200 mL chloroform, cell homo- suggest that ADAM9 regulates several cancer-related genates were centrifuged at 13,200 rpm for 25 minutes at processes—including cell growth and invasion. 4C,andtheaqueousphaseswerecollected.TotalRNA A previous report has shown that the transcription was precipitated with the same volume of isopropanol of ADAM9 is upregulated in gastric cancer (12); how- for 60 minutes at À20C and centrifuged for 30 minutes. ever, its biologic function and potential as a thera- The pellets were washed once with 70% ethanol and peutic target are largely unknown. In this study, we suspended in RNase-free water. The quantity of total investigated the biologic role of ADAM9 in gastric RNA was measured using a NanoDrop spectrophoto- cancer invasion, and explored a therapeutic approach meter (Thermo Scientific). of antimetastasis treatment for high ADAM9-ex- To synthesize the cDNA, 4 mg of total RNA from pressing cancers using an ADAM9-specific inhibitory each cell line was mixed with the oligo-dT primer and antibody. incubated at 65C for 10 minutes. After adding the Super- Script II, 5Â first strand buffer, 100 mmol/L DTT, and 10 mmol/L dNTP mix to the RNA/oligo-dT mixture, a Materials and Methods reverse transcription process was performed at 42C Reagents and antibodies for 90 minutes. The remaining RNA was hydrolyzed Matrigel was supplied by BD Biosciences. Recombinant by incubation at 65C for 15 minutes in 0.1 N NaOH. The ADAM9 protein was from R&D Systems Inc. ADAM9 reaction was then neutralized by the addition of an equal antibodies were from Cell Signaling Technology for volume of 0.1 N HCl. immunoblot and GeneTex for immunohistochemistry. Amplification reactions were performed in an Eppen- Antibodies of hypoxia-inducible factor-1a (HIF1a) and dorf Mastercycler Grandient (Eppendorf). PCR was per- total/phospho forms of EGFR and ERK were obtained formed using 1 mg of cDNA, 2.5 mmol/L dNTPs, 1.5 Â from Santa Cruz Biotechnology. Antibodies against mmol/L 10 PCR buffer with MgCl2, and 5 U Taq poly- GAPDH and a-tubulin were purchased from Abcam and merase (Invitrogen) to total 50 mL of reaction volume. The Sigma-Aldrich. Total and phospho-Akt antibodies were primer sequences used were as follows: ADAM9 (for- purchased from Cell Signaling Technology. RAV-18, an ward: 50-AGT GGC GGG AAA AGT TTC TT-30; reverse: anti-ADAM9-specific blocking antibody, was kindly pro- 50-CCA GCG TCC ACC AAC TTA TT-30). The conditions vided by Macrogenics Inc. for PCR amplification were as follows: 94C for 2 minutes followed by 94C for 40 seconds, 60C for 50 seconds and Cell culture 72C for 1 minutes, then 72C for 2 minutes, and repeated The human gastric cancer cell lines AGS, NCI-N87, for 30 cycles. PCR products were separated on ethidium Hs746T, and KATO-III and the cervical cancer cell line bromide gels containing 1.2% agarose. HeLa, positive control of ADAM9, were purchased from the American Type Culture Collection (ATCC) in 2012. Exposure to hypoxic conditions SNU-1, -5, and -16 in 2006, SNU-216, -638, and -668 in When cells reached 80% confluence in a 25 cm2 flask, 2007, SNU-484 in 2008, MKN-1, -28, -45, and -74 were the flask was placed in a hypoxic chamber (Personal supplied from the Korean Cell Line Bank (KCLB). O2/CO2 Incubator; Aspect). Cells were exposed to 1% Above cell lines were authenticated by standard short O2,5%CO2,and37 C for 24 hours. After that, cells were tandem repeat (STR) DNA typing methodology before collected and transferred to a Matrigel-coated 24-well being purchased from the ATCC and KCLB. YCC-1, -2, plate for invasion assay. Invaded cells were stained and -3 were established in 1989, YCC-6 and -7 were using 0.5% crystal violet and counted after 24 hours. established in 1994, and YCC-9, -10, -11, and -16 were Protease activity assay, immunobloting, and prolife- established in 2000 by the Cancer Metastasis Research ration assay were conducted using cells exposed to Center at Yonsei University College of Medicine (Seoul, hypoxic conditions in the same way. Korea) from the ascites or peripheral blood (YCC-16) of patients with advanced gastric cancer. YCC cell lines Immunoblotting were authenticated by STR in July 20, 2012, that had Subconfluent cells were harvested and suspended in a been outsourced to the KCLB. All cells were maintained whole cell lysis buffer [50 mmol/L Tris (pH 8.0), 10% in RPMI-1640 (Nissui) and supplemented with 10% glycerol, 2 mmol/L EDTA, 5 mmol/L NaF, 1% NP-40, FBS (Omega Scientific, Inc.), 100 U/mL ampicillin, 150 mmol/L NaCl, 1 mmol/L sodium orthovanadate, 100 mg/mL streptomycin, and 2 mmol/L glutamine 1 mmol/L NaF, 10 mg/mL aprotinin, 10 mg/mL leupeptin, (Gibco) in 5% CO2 humidified atmosphere at 37 C.