Research Article Oxidative Stress Induces ADAM9 Protein Expression in Human Prostate Cancer Cells Shian-Ying Sung,1,2 Hiroyuki Kubo,1,6 Katsumi Shigemura,1 Rebecca S. Arnold,4 Sanjay Logani,4 Ruoxiang Wang,1 Hiroyuki Konaka,1 Masayuki Nakagawa,6 Spiro Mousses,7 Mahul Amin,4 Cynthia Anderson,2 Peter Johnstone,2 John A. Petros,1,4 Fray F. Marshall,1 Haiyen E. Zhau,1 and Leland W.K. Chung1,3,5 1Molecular Urology and Therapeutics Program, Department of Urology, 2Department of Radiation Oncology, 3Winship Cancer Institute; Departments of 4Pathology, 5Biochemistry, and Hematology/Oncology, Emory University School of Medicine, Atlanta, Georgia; 6Department of Urology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan; and 7Translational Genomics Research Institute, Gaithersburg, Maryland Abstract and survival of cancer cells (1). Recent data suggest that chronic The ADAM (a disintegrin and metalloprotease) family is a inflammation of the prostate and production of reactive oxygen group of transmembrane proteins containing cell adhesive species (ROS) could contribute to DNA damage and genomic and proteolytic functional domains. Microarray analysis instability, which may facilitate subsequent progression of cancer cells (2). It also has become apparent that oxidative stress such as detected elevated ADAM9 during the transition of human LNCaPprostate cancer cells from an androgen-dependent to toxins, dietary fat consumption, or high level of androgen may be an androgen-independent and metastatic state. Using a important etiologic factors in the development and progression of prostate tissue array (N = 200), the levels of ADAM9 protein prostate cancer (3). Fisher et al (4) showed that oxidative and expression were also elevated in malignant as compared with osmotic stresses on tumor cells increase the shedding of pro- benign prostate tissues. ADAM9 protein expression was found heparin-binding epidermal growth factor (EGF), which is processed in 43% of benign glands with light staining and 87% of by proteins of the ADAM family, specifically ADAM9, ADAM10, and malignant glands with increasing intensity of staining. We ADAM17. There are concomitant inductions of ADAM9 (5) and found that ADAM9 mRNA and protein expressions were ROS (6) in prostate carcinogenesis. It seems that ROS can induce elevated on exposure of human prostate cancer cells to stress expression of the ADAMs via p38 mitogen-activated protein kinase activation (4). The induced ADAM proteins are responsible for conditions such as cell crowding, hypoxia, and hydrogen peroxide. We uncovered an ADAM9-like protein, which is release of processed heparin-binding EGF, which further promotes predominantly induced together with the ADAM9 protein by a cancer cell growth and survival through an EGFR-dependent brief exposure of prostate cancer cells to hydrogen peroxide. mechanism. Induction of ADAM9 protein in LNCaPor C4-2 cells can be Recent studies showed a relationship between the elevation of completely abrogated by the administration of an antioxidant, ADAMs and the progression and metastasis of cancer cells (7–11). ebselen, or genetic transfer of a hydrogen peroxide degrada- Among them, ADAM9 has been shown to possess potent biological tive enzyme, catalase, suggesting that reactive oxygen species activities. The metalloprotease domain of ADAM9 could cleave h a h (ROS) are a common mediator. The induction of ADAM9 by insulin -chain, tumor necrosis factor , gelatin, -casein, and stress can be inhibited by both actinomycin D and cyclohex- fibronectin (12, 13), and induce shedding of EGF, fibroblast growth imide through increased gene transcription and protein factor receptor 2IIIB (14), or heparin-binding EGF-like growth synthesis. In conclusion, intracellular ROS and/or hydrogen factor (4, 15). The disintegrin domain of ADAM9 contains an ECD peroxide, generated by cell stress, regulate ADAM9 expression. (Glu-Cys-Asp) motif, which may mediate cellular adhesion through a h (16) and a h integrins (17). The cytoplasmic tail of ADAM9 ADAM9 could be responsible for supporting prostate cancer 6 1 v 5 cell survival and progression. By decreasing ADAM9 expres- has potential SH3 binding motifs, which have been reported to interact with potentially important regulatory proteins, endophilin sion, we observed apoptotic cell death in prostate cancer cells. h (Cancer Res 2006; 66(19): 9519-26) 1 (SH3GL2), SH3PX1 (18), and mitotic arrest deficient 2 (19). The abilities of ADAM9 to degrade specific extracellular matrix substrates, release active growth factors, interact with key Introduction regulatory factors, and appear in the invasion front of several Prostate cancer progression, invasion, and metastasis are tumor metastases suggest that ADAM9 provokes cancer cell complex processes involving the interplay between cancer cells growth, invasion, and metastasis. This conclusion is further and microenvironment with tight regulations to ensure the growth supported by a recent report where ADAM9 knockdown mice, when crossed with transgenic mice bearing prostate carcinoma, exhibited reduced tumor growth and local invasion (14). Note: S-Y. Sung and H. Kubo contributed equally to this work. In this communication, we show increased ADAM9 expression in Requests for reprints: Shian-Ying Sung, Department of Urology, Emory University prostate cancer cells on androgen-independent progression and by School of Medicine, Suite 5100, Room B5101, 1365B Clifton Road, Atlanta, GA 30322. Phone: 404-778-3663; Fax: 404-778-3675; E-mail: [email protected] or Leland W.K. exposing prostate cancer cells to stress conditions. Our findings on Chung, Molecular Urology and Therapeutics Program, Department of Urology, Emory the roles of ROS in the induction of ADAM9 protein expression University School of Medicine, Atlanta, GA 30322. Phone: 404-778-3672; E-mail: provide a framework for the development of future antioxidant [email protected]. I2006 American Association for Cancer Research. therapies to arrest the growth of clinical human prostate cancer doi:10.1158/0008-5472.CAN-05-4375 and its distant metastasis. www.aacrjournals.org 9519 Cancer Res 2006; 66: (19). October 1, 2006 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cancer Research Materials and Methods confluence were treated with different concentrations of H2O2 for 0, 0.5, 1, 2, 4, and 8 hours. In some experiments, ebselen was added to the cells for Cell lines, cell culture, and chemical reagents. The androgen- 30 minutes before exposure to the H O . Cells were cultured and harvested dependent and androgen receptor–positive parental LNCaP human 2 2 4 hours later. To determine whether ebselen could affect ADAM9 expression prostate cancer cell line and its lineage-derived androgen-independent, induced by cell crowding, cells were first cultured under the crowding androgen receptor–positive, metastatic C4-2 and C4-2B sublines were used condition for 48 hours and then treated with ebselen (0.02-20 Amol/L) for for this study. Unless otherwise stated, these cell lines were cultured in 2 hours. T-medium (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine ADAM9 expression in clinical prostate cancer specimens. Clinical serum (FBS; Sigma, St. Louis, MO), 100 IU/mL penicillin G, and 100 Ag/mL prostate cancer tissue specimens were obtained following an institution- streptomycin at 37jC under 5% CO . Ebselen (2-phenyl-1, 2-benzisoselena- 2 approved protocol. Paraformaldehyde-fixed, paraffin-embedded tissues zol-3[2H]-one), actinomycin D, or cycloheximide (Sigma) was freshly were deparaffinized and rehydrated. Antigen retrieval was done in citrate reconstituted with DMSO before use. buffer (pH 6) using an electric pressure cooker at 120jC for 5 minutes. The Generation of human prostate cancer cell lines that stably express section was then exposed to 3% H O for 5 minutes to quench endogenous catalase. Construction and overexpression of catalase was done as 2 2 tissue peroxidase Iand then incubated for 30 minutes with the monoclonal previously described (20). In brief, human catalase cDNA was recovered anti-ADAM9 antibody (1:50 dilution). Immunohistochemical staining was from the pCI-neo vector and ligated into the HindIII site of the pZeoSV visualized with the Envision system (DAKO Corporation, Carpinteria, CA) vector. A clone containing the catalase in the sense orientation (pZeoSV/ and the results were independently evaluated by two pathologists (S.L and catalase) was verified by DNA sequencing. Transfection of the cDNA to M.A.). The anti-ADAM9 antibody from R&D Systems has been validated by prostate cancer cells was carried out with Fugene 6 (Roche Applied Science, several other groups to be specific for the detection of ADAM9 protein Indianapolis, IN) according to the instructions of the manufacturer. After expression in pancreatic cancer (8, 22), small-cell lung carcinoma (23), and 2 days, the cells were subcultured into 10-cm plates and treated with hepatic cancer (24). 0.4 mg/mL zeocin (Invitrogen) until individual colonies formed. Short hairpin RNA mediated gene silencing of ADAM9. To determine Quantitative reverse transcription-PCR. Quantitative reverse tran- the potential functions of ADAM9 protein in C4-2 cells, we transfected these scription-PCR (RT-PCR) was done with the iCycler thermal cycler system cells with a predesigned short hairpin RNA (shRNA; Invitrogen), which was (Bio-Rad, Hercules, CA). Total RNA (5 Ag) from cells at 80% to 95% shown to knock down specifically ADAM9