Published OnlineFirst June 8, 2010; DOI: 10.1158/1535-7163.MCT-09-1077 Research Article Molecular Cancer Therapeutics Artemin-Stimulated Progression of Human Non–Small Cell Lung Carcinoma Is Mediated by BCL2 Jian-Zhong Tang1, Xiang-Jun Kong3, Jian Kang1, Graeme C. Fielder1, Michael Steiner1, Jo K. Perry1, Zheng-Sheng Wu4, Zhinan Yin5, Tao Zhu3, Dong-Xu Liu1, and Peter E. Lobie1,2 Abstract We herein show that Artemin (ARTN), one of the glial cell line–derived neurotrophic factor family of li- gands, promotes progression of human non–small cell lung carcinoma (NSCLC). Oncomine data indicate that expression of components of the ARTN signaling pathway (ARTN, GFRA3, and RET) is increased in neoplas- tic compared with normal lung tissues; increased expression of ARTN in NSCLC also predicted metastasis to lymph nodes and a higher grade in certain NSCLC subtypes. Forced expression of ARTN stimulated survival, anchorage-independent, and three-dimensional Matrigel growth of NSCLC cell lines. ARTN increased BCL2 expression by transcriptional upregulation, and inhibition of BCL2 abrogated the oncogenic properties of ARTN in NSCLC cells. Forced expression of ARTN also enhanced migration and invasion of NSCLC cells. Forced expression of ARTN in H1299 cells additionally resulted in larger xenograft tumors, which were high- ly proliferative, invasive, and metastatic. Concordantly, either small interfering RNA–mediated depletion or functional inhibition of endogenous ARTN with antibodies reduced oncogenicity and invasiveness of NSCLC cells. ARTN therefore mediates progression of NSCLC and may be a potential therapeutic target for NSCLC. Mol Cancer Ther; 9(6); 1697–708. ©2010 AACR. Introduction Therefore, identification and subsequent targeting of novel oncogenic pathways may provide an advantage to Lung carcinoma is currently responsible for the highest the current regimens used to treat lung carcinoma and cancer-related mortality worldwide, with overall 5-year consequently improve prognosis. survival approximating 15% (1, 2). Primary lung carcino- Artemin (ARTN) is a neurotrophic factor that be- ma can be largely classified as non–small cell lung carci- longs to the glial cell line–derived neurotrophic factor noma (NSCLC) and SCLC (1). Although early diagnosis (GDNF) family of ligands (GFL). ARTN mediates sur- of lung carcinoma remains challenging, the lack of effec- vival, differentiation, and migration of various types of tive approaches to prevent disease progression also per- neurons (7, 8). ARTN signaling is reported to be trans- sists. As a result of advances in cancer biology during the duced via cognate receptors GFRA3 and also GFRA1 last few decades, a number of targeted agents have been (9), which stimulate the phosphorylation of the trans- developed, exemplified by erlotinib/gefitinib, which se- membrane receptor tyrosine kinase RET, to activate lectively inhibits the epidermal growth factor receptor downstream mitogen-activated protein kinase and (3). However, clinical application of these agents has phosphatidylinositol 3-kinase pathways, among others provided only limited therapeutic benefits for patients (10). RET-independent signaling has also been observed with lung carcinoma (4–6), partially attributable to the for the GFL family via alternative partners, including compensatory effect of other cellular mechanisms integrins and neural cell adhesion molecule (10). exploited by the tumors for survival and progression (3). An increasing body of evidence has implicated ARTN in progression of carcinoma (8, 11–13). Elevated expres- sion of ARTN predicted residual disease after chemo- Authors' Affiliations: 1Liggins Institute and 2Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, therapy, metastasis, and decreased overall survival in University of Auckland, Auckland, New Zealand; 3Hefei National mammary carcinoma patients (8). ARTN expression is Laboratory for Physical Sciences at Microscale and School of Life also positively correlated to high tumor grade and myo- Sciences, University of Science and Technology of China; 4Department of Pathology, Anhui Medical University, Hefei, Anhui, P.R. China; and metrial invasion in endometrial carcinoma (12). Forced 5School of Life Sciences, Nankai University, Tianjin, P.R. China expression of ARTN promoted survival, invasion, an- Note: Supplementary material for this article is available at Molecular chorage-independent growth, and xenograft tumor Cancer Therapeutics Online (http://mct.aacrjournals.org/). growth of both human mammary and endometrial carci- Corresponding Author: Peter E. Lobie, Liggins Institute, University of noma cells, whereas depletion or functional inhibition of Auckland, Private Bag 92019, Auckland 1023, New Zealand. Phone: ARTN inhibited these cellular activities (8, 12). ARTN 64-9-9232125; Fax: 64-9-3737497. E-mail: [email protected] and GDNF have also been reported to stimulate invasive- doi: 10.1158/1535-7163.MCT-09-1077 ness but not proliferation of human pancreatic carcinoma ©2010 American Association for Cancer Research. cells (11, 14). www.aacrjournals.org 1697 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst June 8, 2010; DOI: 10.1158/1535-7163.MCT-09-1077 Tang et al. Herein, we show that ARTN promotes progression of (17, 18). Suspension culture and soft agar colony formation NSCLC by enhancement of tumor growth and metastasis. assays were done in 10% fetal bovine serum (FBS) medi- We therefore propose that strategies targeting ARTN could um. For total cell number assay, H1299 derivatives were potentially exert therapeutic benefit in human NSCLC. seeded at 3 × 103 cells per well in 10% FBS medium and 2.5 × 104 cells per well in 0.2% FBS medium. For total cell Materials and Methods number and suspension culture assays, cells were collect- ed after trypsinization for manual counting. For wound- Plasmid constructs healing assays, wounds were created in a 90% confluent pIRESneo3 empty vector, pIRESneo3-ARTN expres- cell monolayer using an inverted sterile 200 μL pipette sion plasmid, negative control small interfering RNA tip in a continuous linear motion. The wounded cell mono- (siRNA) construct pSilencer-CONTROL (previously layer was maintained in growth medium until the wounds designated as pSilencer-CK), and ARTN-specific siRNA in one of two compared groups were closed. The position construct pSilencer-ARTN were described previously (8). of two frontlines of the cells migrating into the wounds was photographed at six to nine fixed locations on each Cell lines and cell transfection day.Forgrowthinthree-dimensionalMatrigel,1×103 The human NSCLC cell lines H1299, H2009, and A549 cells were plated in 10% FBS medium supplemented with were obtained from and characterized by the American 2% Matrigel in a 96-well plate. Matrigel-containing (2%) Type Culture Collection. Human NSCLC cell lines H1975 medium was renewed every 3 days until the experiment and H460 were generously provided by Professor William was terminated after 8 days. Cell number was quantified Wilson (University of Auckland). H1299 and H1975 were by Alamar blue as previously described (Invitrogen; ref. transfected using FuGENE HD Transfection Reagent 19). Transwell migration and invasion assays were done (Roche Diagnostics) with pIRESneo3 or pIRESneo3-ARTN, as previously described with minor modifications (12). respectively. Following 4-week selection in media contain- Twenty-four–well inserts (8-μm pore size; BD Biosciences) 2 ing 1200 μg/mL G418, pooled stable transfectants were des- were coated with 2.5 μg/cm poly-D-lysine for both as- ignated as H1299-VEC, H1299-ARTN, H1975-VEC, and says. For invasion assays, inserts were subsequently coat- H1975-ARTN, respectively. H1299 or H1975 cells were also ed with Matrigel (BD Biosciences), diluted 1:40 with transfected with pSilencer-CONTROL or pSilencer-ARTN, serum-free medium. Cells (2 × 104) were plated in se- respectively, generating stable cell lines H1299-CONTROL, rum-free medium on the upper side of each insert and al- H1299-siARTN, H1975-CONTROL, or H1975-siARTN. lowed to migrate toward 10% FBS medium on the lower side. Migration assays were done for 9 hours for H1299 de- Generation of chicken anti-ARTN antibody rivatives or 16 hours for H1975 derivatives. Invasion as- Chicken anti-ARTN polyclonal antibody (ARTN-IgY) says were done for 16 hours for H1299 derivatives or for was generated as previously described (8). ARTN-IgY 24 hours for H1975 derivatives. Cells on the lower side of and preimmune chicken IgY (CON-IgY) were both used inserts were fixed in ice-cold methanol, stained with 0.01% at 500 μg/mL for cell-based bioassays. crystal violet, and counted. Phase-contrast micrographs were acquired with an Olympus DP70 digital camera BCL2 inhibitor attached to Olympus IX71 fluorescence microscope and The BCL2 inhibitor YC137 (Calbiochem) was purchased analyzed with DPController v1.1 and DPManager v1.1 from Merck KGaA. The specificity of YC137 for BCL2 has (Olympus America, Inc.). previously been shown elsewhere (15). Cell cycle analysis Western blot analysis Following serum depletion (0.2% FBS; 24 h for H1975 or Western blot analysis was done as previously described 48 h for H1299), cells were harvested immediately or fur- (8, 16). Four milliliters of conditioned medium were gen- ther cultured in 10% FBS medium for 24 or 48 hours before erated from 106 cells incubated in serum-free medium for harvest. Samples were assessed