Artemin-Stimulated Progression of Human Non–Small Cell Lung Carcinoma Is Mediated by BCL2

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 ligands, 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 neoplastic 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 expression 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).

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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 using a FACSCalibur (BD 48 hours and concentrated to 90 μL before denaturation in Biosciences) as previously described (20). Data were ana- 2× protein loading buffer. Anti-ARTN antibody was lyzed using ModFit LT software (Verity Software House). purchased from R&D Systems, anti–β-actin antibody (A1978) was from Sigma-Aldrich, and anti-BCL2 antibody Apoptosis (51-6511GR) was from BD Biosciences. Chemilumines- Apoptosis was induced by 0.5 μmol/L paclitaxel for cence captured on films was imaged by GS-800 calibrated H1975 cells or by 2.5 μmol/L paclitaxel for H1299 cells densitometer and processed by Quantity One software in serum-free media (0% FBS) for 24 hours. One half (Bio-Rad Laboratories). to 1 million cells were harvested and labeled using Annexin-V-FLUOS Staining kit (Roche Diagnostics) and In vitro assays subjected to flow cytometry as above. Data analysis was Total cell number, suspension culture, wound healing, conducted using CellQuest Pro software (BD Biosciences). and colony formation in soft agar assays were done as pre- The Annexin V–propidium iodide (PI) double staining viously described with minor modifications as indicated method allows differentiation between early and late

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apoptosis. High Annexin V positivity and low PI positiv- sion of ARTN, GFRA1-A3 (encoding GFRA1-A3), and ity indicate early apoptosis, whereas high Annexin V RET was determined by semiquantitative RT-PCR in a positivity and high PI positivity indicate late apoptosis. panel of five human NSCLC cell lines: H1299, H1975, Total apoptosis is the addition of early and late apoptosis H460, H2009, and A549. As observed in Fig. 1A (left), and represents the total apoptotic cell population. ARTN, GFRA3,andRET were detected in all examined cell lines, although at dissimilar expression levels. The ex- Semiquantitative reverse transcription-PCR and pression levels of the remaining GFRA members varied quantitative PCR greatly between cell lines. GFRA1 was only detected Equal numbers of cells were plated in 10% FBS medium. in H1299, H460, and A549 cells, and GFRA2 was only On the next day, after removal of 10% FBS medium, cells detected in H1975 and A549 cells. were washed with PBS and incubated in serum-free medi- The Oncomine database (23) harbors microarray- um for another 24 hours. RNAwas then isolated from these generated data on gene expression in human cancers. cells for reverse transcription-PCR (RT-PCR) and subjected In this database, we examined the expression of ARTN, to quantitative PCR analyses (21). Semiquantitative RT- GFRA1-A3,andRET in normal and neoplastic lung tis- PCR was done as previously described (8) for which 0.5 sues (Supplementary Table S1). ARTN expression was μg total RNA (except 1 μgforARTN) was used as template. significantly increased in carcinoid, NSCLC (adenocarci- Images of gel electrophoresis were acquired using UVP noma and squamous cell carcinoma), and small cell BioImaging Systems and LabWorks Image Acquisition carcinoma compared with normal lung (Supplementary and Analysis software version 4.0 (UVP,Inc.). Quantitative Table S1a). Increased expression of ARTN was also corre- PCR was done as previously described and using the lated positively with dedifferentiation in squamous cell same primer sets (21). Changes in gene expression were carcinoma, increased lung carcinoma grade, and progres- expressed as fold change relative to the vector control. sion to N-stage squamous cell carcinoma but inversely with M-stage lung carcinoma (Supplementary Table S1b). Xenograft studies ARTN expression was correlated to tobacco use in patients Five millions cells were suspended in 150 μL Matrigel/ with lung carcinoma and wild-type Ras genotype in PBS (1:1, v/v) and injected into the subscapular region of NSCLC (Supplementary Table S1b). The highest expres- immunodeficient mice (Shanghai Slaccas Co.). Determi- sion of ARTN was repeatedly reported in squamous cell nation of tumor volume, S-phase entry (by bromodeox- carcinoma compared with any other subtypes of lung yuridine incorporation), and apoptosis [by terminal carcinoma (Supplementary Table S1b). For instance, Bild deoxynucleotidyl transferase–mediated dUTP nick end et al. showed that ARTN expression was 96% greater in labeling (TUNEL) immunostaining] and H&E staining squamous cell carcinoma than adenocarcinoma. were done as previously described (21). Liver and lung Expression of GFRA1 varied between subtypes of of sacrificed mice were fixed in 4% paraformaldehyde lung carcinoma, and there was no clear correlation be- in PBS for histologic examination. tween expression of GFRA1 and progression to N-stage adenocarcinoma (Supplementary Table S1c). Expression Luciferase reporter assay of GFRA2 was reduced in most neoplastic (with carcinoid BCL2 P1 promoter reporter construct (2 μg/well; ref. 18) as the exception) compared with normal lung tissue was transfected using FuGENE HD reagent into 5 × 104 (Supplementary Table S1a) and predicted differentiation cells in 12-well plates; 0.4 μg/well pSV–β-galactosidase and lower grade and stage of squamous cell carcinoma expression plasmid was cotransfected controlling for (Supplementary Table S1d). Expression of GFRA3 was transfection efficiency. The cells were harvested in elevated in carcinoid, adenocarcinoma, squamous cell reporter lysis buffer (Promega Co.) 36 hours after transfec- carcinoma, and small cell carcinoma compared with tion. Luminescence and β-galactosidase activity were normal lung (Supplementary Table S1a). Expression quantified as previously described (22). of RET was increased in adenocarcinoma compared with normal lung (Supplementary Table S1a). Adenocarcino- Statistics ma exhibited higher expression of RET compared All experiments were repeated at least thrice, and the withothersubtypesoflungcarcinomaderivedfrom results of one representative experiment are shown. All squamous, large, and small cell types. data are expressed as means ± SE of triplicate determi- nants. Data analysis was conducted using an unpaired Forced expression of ARTN enhanced survival, two-tailed t test. anchorage-independent, and three-dimensional Matrigel growth of H1299 cells Results To determine the effects of elevated ARTN expression in human NSCLC, we stably transfected the human ARTN, GFRA3, and RET are expressed in NSCLC cell line H1299 with an expression plasmid en- human NSCLC coding human ARTN (pIRESneo3-ARTN) or the empty To determine the expression of components of the vector plasmid pIRESneo3 to generate the stable cell lines ARTN signaling pathway in NSCLC, the mRNA expres- H1299-ARTN or H1299-VEC, respectively. Transfected

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Figure 1. Effects of forced expression of ARTN in human NSCLC H1299 cells. A, left, mRNA levels of ARTN, GFRA1, GFRA2, GFRA3, and RET were determined by semiquantitative RT-PCR in five NSCLC cell lines. β-actin was used as loading control. Right, for comparison between stable cell lines H1299-VEC and H1299-ARTN, the level of ARTN mRNA was determined by semiquantitative RT-PCR; ARTN protein level was determined by Western blot analyses in cell lysates and conditioned media. β-actin was used as loading control where appropriate. Total cell number in 10% and 0.2% FBS medium (B), cell cycle progression in 10% FBS medium, and apoptosis induced by 2.5 μmol/L paclitaxel in 0% FBS medium for 24 h (C) were compared. **, P < 0.01; ***, P < 0.001.

colonies were pooled to minimize any effects of potential cells compared with H1299-VEC cells in serum-depleted clonal selection. Expression levels of ARTN mRNA and (0.2% FBS) medium despite initial equal plating densities. cellular ARTN protein were increased in H1299-ARTN By day 9, the number of H1299-ARTN cells compared cells compared with H1299-VEC cells (Fig. 1A, right). with H1299-VEC cells was 48% greater (Fig. 1B, right). Concomitantly, the level of secreted ARTN protein was PI staining and flow cytometry analysis did not reveal increased in the conditioned medium of H1299-ARTN any significant differences in cell cycle progression be- cells in comparison with H1299-VEC cells (Fig. 1A, right). tween H1299-VEC and H1299-ARTN cells following 48 We first determined the effects of forced expression of hours of serum depletion (0.2% FBS) only or in combina- ARTN on total cell number and cell cycle progression in tion with 24 or 48 hours of incubation in 10% FBS–con- monolayer culture. No difference in total cell number taining medium, indicative that the differences observed was observed between H1299-VEC and H1299-ARTN in total cell number under serum-depleted condition may cells in monolayer culture in 10% FBS media over 10 days be due to different apoptotic rates (Fig. 1C; only data (Fig. 1B, left). Concordantly, quantitative PCR analyses generated by 24-h incubation in 10% FBS–containing did not reveal any changes in genes regulating cell cycle medium following 48-h serum depletion shown). To progression (Supplementary Table S2). However, we ob- determine if forced expression of ARTN increased the served an increase in total cell number of H1299-ARTN survival of H1299 cells, we induced apoptosis with

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paclitaxel, which is used clinically to treat NSCLC (24). Forced expression of ARTN enhanced migration Flow cytometric analysis following Annexin V/PI and invasion of H1299 cells counter-staining revealed that early, late, and total apo- In migration assays, the number of H1299-ARTN cells ptosis of H1299-ARTN cells were, respectively, 48%, that migrated in Transwell chambers was 131% more than 57%, and 54% lower than H1299-VEC cells (Fig. 1C). H1299-VEC cells (Fig. 2D). In a wound-healing assay, Concordantly, quantitative PCR analyses revealed that H1299-ARTN cells closed scratch wounds significantly fas- the mRNA expression level of BCL2 in H1299-ARTN ter than H1299-VEC cells (Fig. 2D). In invasion assays, the cells was significantly higher than that in H1299-VEC number of H1299-ARTN cells invading in Transwell cham- cells (Supplementary Table S2). bers was 128% more than H1299-VEC cells (Fig. 2D). Quan- We next examined the effect of forced expression of titative PCR analyses revealed that the expression level of ARTN on anchorage-independent growth, a characteris- TGFB1, a recognized invasion stimulator of NSCLC cells tic hallmark of the transformed cell phenotype (25). (26), was increased in H1299-ARTN cells compared with H1299-ARTN cells formed 91% more colonies in soft H1299-VEC cells (Supplementary Table S2). agar than H1299-VEC cells (Fig. 2A). Growth of H1299-ARTN cells was 61% greater than H1299-VEC Forced expression of ARTN increased cells in three-dimensional Matrigel culture (Fig. 2B). tumorigenicity in a xenograft model Concordantly, H1299-ARTN cells exhibited an 82% in- We next examined the effects of forced expression crease in total cell number in suspension culture com- of ARTN in H1299 cells in a xenograft mouse model. pared with H1299-VEC cells by day 9 (Fig. 2C). In Subcutaneous implantation of either H1299-VEC or contrast to the mostly organized spherical colonies H1299-ARTN cells into the subscapular region of immu- formed in three-dimensional Matrigel by H1299-VEC nocompromised mice resulted in formation of palpable cells, colonies formed by H1299-ARTN cells were signif- and measurable tumors after a period of 1 week. Al- icantly larger and acquired a stellate morphology. The though xenograft tumors derived from either cell line enhanced growth and aggressive morphology exhibited did not exhibit significant increases in volume up to by H1299-ARTN cells in three-dimensional Matrigel day 20, thereafter increases in the volume of H1299- suggests that ARTN may stimulate invasion of NSCLC ARTN–derived tumors were significantly greater, being cells. 63% larger than H1299-VEC–derived tumors by day 30

Figure 2. Effects of forced expression of ARTN in H1299 cells. Colony formation in soft agar (A), growth in three-dimensional Matrigel culture (B), suspension culture (C), and migration and invasion and wound healing (D) were compared between H1299-VEC and H1299-ARTN cells. Images were taken at ×160 magnification. Scale bars, 100 μm. **, P < 0.01; ***, P < 0.001.

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(Fig. 3A). We also assessed S-phase entry by determination ing tissues that often appeared mucinous (Fig. 3D). of bromodeoxyuridine incorporation or apoptosis by TU- Concomitantly, H1299-ARTN–derived tumor cells were NEL staining, respectively, in tumor sections. H1299- observed adjacent to neural fibers, suggesting perineural ARTN–derived tumors exhibited a 45% greater S-phase invasion (Fig. 3D, bottom left). We additionally observed entry and a 37% reduction in apoptotic nuclei compared metastatic nodules in lungs of mice with tumors derived with H1299-VEC–derived tumors (Fig. 3B and C). from H1299-ARTN cells (two of six) but not with those Histopathologic examination revealed that H1299-VEC– from H1299-VEC cells (zero of six; Fig. 3D, bottom right). derived tumors were well-circumscribed masses formed Thus, forced expression of ARTN increased NSCLC prolif- by tumor cells of a grossly uniform size with clearly delin- eration and survival, leading to increased growth and in- eated margins to the surrounding tissues (Fig. 3D). In con- vasiveness of xenograft tumors, resulting in metastasis. trast, H1299-ARTN–derived tumors were loosely massed and/or contained areas of cavitation (Fig. 3D, top right). Forced expression of ARTN also enhanced These areas contained large pleomorphic cells exhibiting oncogenicity of H1975 cells irregular or multiple hyperchromatic nuclei and volumi- We also stably transfected another human NSCLC nous cytoplasm, with ill-defined margins to the surround- cell line, H1975, with pIRESneo3-ARTN or the empty

Figure 3. Forced expression of ARTN increased tumorigenicity in a xenograft model. H1299-VEC or H1299-ARTN cells were implanted s.c. into the subscapular region of immunodeficient mice. The mice were maintained for 30 d before euthanasia. A, tumor volume was measured every 3 d from 7 to 30 d after implantation and plotted. B and C, S-phase entry and apoptosis of tumors were determined. BrdUrd, bromodeoxyuridine. D, histologic features of tumors derived from H1299-VEC or H1299-ARTN cells and organs (lungs) with metastases were visualized following H&E staining. The circled area in the top right panel is a representative region enriched with pleomorphic tumor cells and shown as at greater magnification in the inset. Scale bars, 50 μm (inset in the top right) and 100 μm (remaining panels in D). **, P < 0.01.

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Figure 4. Forced expression of ARTN enhanced oncogenicity of H1975 cells. A, for comparison between H1975-VEC and H1975-ARTN cells, the level of ARTN mRNA was determined by semiquantitative RT-PCR; ARTN protein expression was determined by Western blot analysis in cell lysates and conditioned media. Total cell number in 10% FBS media and 0.2% FBS media (A), apoptosis (induced by 0.5 μmol/L paclitaxel in 0% FBS medium for 24 h), colony formation in soft agar, cell motility (B), and growth in three-dimensional Matrigel culture (C) were compared. *, P < 0.05; **, P < 0.01; ***, P < 0.001. PTX, paclitaxel. Scale bars, 100 μm. vector plasmid pIRESneo3 to generate the stable cell lines icantly higher than H1975-VEC cells from day 3 on; the H1975-ARTN or H1975-VEC, respectively. Semiquantita- number of H1975-ARTN cells was 58% higher in com- tive RT-PCR and Western blot analyses, respectively, parison with H1975-VEC cells on day 3 (Fig. 4A). This showed that mRNA, cellular, and secreted protein ex- was despite cell number being decreased from the orig- pression levels of ARTN were significantly increased in inal plating number over time in serum-depleted media. H1975-ARTN cells relative to H1975-VEC cells (Fig. 4A). Under serum-depleted conditions, early, late, and total Similar to our observations in H1299 cells, forced ex- apoptosis of H1975-ARTN cells were, respectively, pression of ARTN did not significantly alter the number 35%, 67%, and 43% lower than H1975-VEC cells (Fig. of H1975 cells in monolayer culture in 10% FBS medium 4B). Forced expression of ARTN also improved survival by day 9 (Fig. 4A). However, under serum-depleted of H1975 cells in response to treatment with paclitaxel conditions, the number of H1975-ARTN cells was signif- (Fig. 4B).

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H1975-ARTN cells formed 47% more colonies than than that of H1299-VEC cells, whereas the level in H1975- H1975-VEC cells in soft agar (Fig. 4B). In migration as- ARTN cells was 383% higher than that of H1975-VEC cells says, the number of H1975-ARTN cells that migrated into (Fig. 5A). Concordantly, Western blot analyses revealed Transwell chambers was 102% more than H1975-VEC increases in BCL2 protein in H1299-ARTN and H1975- cells (Fig. 4B). In invasion assays, the number of H1975- ARTN cells compared with the appropriate control cells ARTN cells observed in Transwell chambers was 98% (Fig. 5A). more than H1975-VEC cells (Fig. 4B). H1975-ARTN cells We next quantitatively assessed colony formation of also closed scratch wounds markedly faster than H1975- H1299-VEC and H1299-ARTN cells in soft agar and VEC cells in a wound-healing assay (Fig. 4C). Lastly, in three-dimensional Matrigel following treatment with three-dimensional Matrigel culture, growth of H1975- the BCL2 inhibitor YC137 (27). YC137 produced a dose- ARTN cells was 48% greater than H1975-VEC cells; dependent reduction in soft agar colony formation of H1975-ARTN cells formed larger and more disorganized H1299-VEC cells (Fig. 5B). YC137 also largely abrogated colonies than H1975-VEC cells, which occasionally ceased the ARTN-stimulated enhancement in anchorage- growth and disappeared from the culture (Fig. 4C). independent growth of H1299-ARTN cells, indicative of BCL2 dependence of ARTN-stimulated soft agar colony BCL2 mediates ARTN-stimulated oncogenicity in formation. Furthermore, YC137 also largely abrogated NSCLC cells the ARTN-stimulated enhancement of H1299-ARTN cell We examined the mechanism underlying the survival growth in three-dimensional Matrigel, although signifi- advantage conferred by forced expression of ARTN. In cant inhibition of H1299-VEC cell growth was also quantitative PCR analyses, we observed significant observed as expected (Fig. 5C; ref. 28). increases in mRNA level of the antiapoptotic gene BCL2 on forced expression of ARTN: a 165% increase in Depletion of ARTN reduced anchorage-independent H1299-ARTN cells and a 356% increase in H1975-ARTN and three-dimensional Matrigel growth and cells compared with the appropriate controls (Fig. 5A). motility of NSCLC cells Furthermore, use of a human BCL2 promoter reporter We additionally examined the functional outcomes of construct indicated that forced expression of ARTN siRNA-mediated depletion of endogenous ARTN in the stimulated BCL2 promoter activity: The level of BCL2 two NSCLC cell lines. We stably transfected H1299 cells promoter activity in H1299-ARTN cells was 49% higher with a siRNA construct targeting ARTN or a negative

Figure 5. BCL2 mediates ARTN-stimulated oncogenicity in NSCLC cells. A, mRNA, promoter activity, and protein expression of BCL2 was compared between H1299-VEC and H1299-ARTN or H1975-VEC and H1975-ARTN cells. B, colony formation of H1299-VEC and H1299-ARTN cells was compared in the presence of varied concentrations of the BCL2 inhibitor YC137. C, growth of H1299-VEC and H1299-ARTN cells in three-dimensional Matrigel in the presence of YC137 was compared. Scale bars, 100 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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control siRNA construct to generate the stable cell lines we observed the greatest differential expression of ARTN H1299-siARTN or H1299-CONTROL, respectively. H1975 in squamous cell carcinoma. However, expression of cells were similarly transfected to generate the stable cell ARTN is also elevated in other subtypes of NSCLC, lines H1975-siARTN or H1975-CONTROL. Semiquantita- including adenocarcinoma, which is the most common tive RT-PCR and Western blot analyses showed that subtype observed in never smokers (32), carcinoid, and siRNA-mediated depletion of ARTN resulted in decreases SCLC compared with normal lung tissues. We also ob- in ARTN mRNA and protein compared with negative serve that forced expression of ARTN in a xenograft control siRNA in both H1299 and H1975 cells (Fig. 6A). model results in formation of cavitation associated with Depletion of ARTN resulted in reductions in colony forma- the presence of large multinucleated cells. These tumors tion (32%), migration (54%), invasion (49%; Fig. 6A), and microscopically resemble giant cell carcinoma observed growth in three-dimensional Matrigel (31%; Fig. 6B) in in human lung, which rarely occurs but produces partic- H1299-siARTN compared with H1299-CONTROL cells. ularly rapid growth, and distant and extensive metas- Similar results were observed between H1975-siARTN tases, associated with more frequent recurrence after and H1975-CONTROL cells (Fig. 6A and B). surgery and exhibit poorer response to current chemotherapeutic regimens and worse life expectancy com- Functional inhibition of ARTN abrogated pared with other subtypes of NSCLC (33, 34). anchorage-independent and three-dimensional Although ARTN is not observed to stimulate mitogen- Matrigel growth and motility of NSCLC cells esis of NSCLC cells in vitro, there is a clear enhancement We lastly assessed the effects of functional inhibition by of proliferation in vivo and a concomitant greater increase antibodies to ARTN in NSCLC cell lines. Soft agar colony in volume of xenograft tumors derived from NSCLC cells formation and three-dimensional Matrigel culture assays exhibiting forced expression of ARTN. This finding is were done with H1299 or H1975 cells, which were treated reminiscent of our previous observation with human with either a polyclonal chicken antibody against ARTN mammary carcinoma cell lines and is presumably due (ARTN-IgY) or preimmune chicken IgY (CON-IgY) as to paracrine interaction of ARTN with the tumor micro- negative control. H1299 cells treated with antibody environment (8). Furthermore, enhanced invasion and formed 23% less and significantly smaller colonies in metastasis of NSCLC cells with forced expression of soft agar than control-treated cells (Fig. 6C), and this ARTN is associated with increased expression of TGFB1. effect was recapitulated in three-dimensional Matrigel Elevated expression of TGFB1 is frequently associated culture (Fig. 6D). Additionally, H1299 cells treated with with metastases compared with paired primary lung ARTN-IgY exhibited a reduction of 45% in migration carcinoma, or primary tumors that have metastasized and of 48% in invasion compared with control-treated compared with those that have not, and predicts recur- cells (Fig. 6C). The reductions in colony formation in rence of disease and death (26, 35). soft agar, growth in three-dimensional Matrigel, and The relative level of ARTN expression in H1299 cells is motility were also observed in H1975 cells treated with not high compared with H1975 cells. However, depletion ARTN-IgY compared with CON-IgY; a much larger pro- or inhibition of ARTN in H1299 cells decreases ARTN- portion of ARTN-IgY–treated H1975 cells ceased growth stimulated cellular functions to similar extents to that and disappeared gradually in soft agar colony formation observed in H1975 cells. The response of any particular and three-dimensional Matrigel assays (Fig. 6C and D). cell to a ligand is not simply dependent on ligand concen- tration. For example, some ligands fully activate receptor Discussion signaling at only 5% occupancy of the receptor (36). It should also be noted that H1299 cells express both Increased expression of ARTN and one receptor, GFRA3 and GFRA1, whereas H1975 cells only express GFRA3, is observed in an unusual majority of lung ma- GFRA3. ARTN has been shown to stimulate the forma- lignancies, including carcinoid, adenocarcinoma and tion of a GFRA1-RET complex in addition to using squamous cell carcinoma (two main subgroups of GFRA3 (9). It may be that the presence of GFRA1 in ad- NSCLC), and SCLC. Additionally, expression of GFRA3 dition to GFRA3 in the H1299 cells allows them to re- is detectable in the normal lung and bronchus of human spond functionally at lower concentrations of ARTN and mouse (29, 30). RET expression has also been shown than H1975 cells, which only express GFRA3. Further- in normal human lung (30). These facts together suggest more, GFLs have been shown to bind to non-GFRA pro- that an autocrine ARTN signaling pathway is functional teins (37, 38) and function independent of RET (39). Thus, in human lung and the excessive activity of this pathway the expression level of ARTN may not be predictive of the may therefore promote disease progression in lung carci- relative contribution of ARTN to oncogenic functions in noma. In contrast, amplification or expression of most different NSCLC cell lines. other oncogenes, including epidermal growth factor re- ARTN-stimulated survival, clonogenic capacity, and ceptor, has been reported only in some subtype(s) of lung three-dimensional Matrigel growth of NSCLC cells were carcinoma (31). We observed from Oncomine that shown to be mediated by increased expression of BCL2. In- expression of ARTN is strongly correlated with use of creased expression of BCL2 on forced expression of ARTN tobacco in patients with lung carcinoma. Concordantly, is also observed in human mammary and endometrial

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Tang et al.

Figure 6. Effects of depletion or inhibition of ARTN in NSCLC cell lines. A, the level of ARTN mRNA was determined by semiquantitative RT-PCR; cellular ARTN protein expression was determined by Western blot analyses. Colony formation, cell migration, and invasion (A) and growth in three-dimensional Matrigel (B) were compared between H1299-CONTROL and H1299-siARTN cells or between H1975-CONTROL and H1975-siARTN cells. In addition, colony formation (C), migration and invasion (C), and growth in three-dimensional Matrigel (D) were compared between H1299 or H1975 cells treated by ARTN antibody (ARTN-IgY) or preimmune IgY (CON-IgY) at 500 μg/mL. Images were taken at ×160 magnification. Scale bars, 100 μm. *, P < 0.05; **, P < 0.01.

1706 Mol Cancer Ther; 9(6) June 2010 Molecular Cancer Therapeutics

Artemin Stimulates Oncogenicity of NSCLC via BCL2

carcinoma cells (8, 12, 13). Nevertheless, this report has de- We speculate that the ARTN signaling pathway may be termined that BCL2 contributes to ARTN-stimulated onco- induced to favor survival of NSCLC in response to cellular genicity in NSCLC. BCL2 exhibits increased expression stressors, such as removal of growth stimuli, treatment and promotes cell survival in various human malignancies, with chemotherapeutic agents, or perturbation of tumori- including lung carcinoma (40, 41). It is therefore probable genic signal transduction by targeted therapies. As such, in- that ARTN-stimulated increases in BCL2 expression pro- hibition of ARTN may be useful as an adjuvant therapeutic duce an in vivo tumorigenic advantage, as observed with agent in human NSCLC. xenografts of H1299-ARTN cells, which grow as larger tumors with increased carcinoma cell survival. To date, no Disclosure of Potential Conflicts of Interest correlation has been observed between BCL2 expression – and response of NSCLC patients to chemotherapy (42 J.Kang,J.K.Perry,D-X.Liu,andP.E.Lobiehaveequityinterests 45). Nevertheless, BCL2 has been implicated in the devel- in Saratan Therapeutics Ltd. D-X. Liu and P.E. Lobie are inventors on opment of chemoresistance in cell lines of human lung car- PCT application PCT/NZ2008/000152 and U.S. provisional application – 61/234,902. P.E. Lobie is also the inventor on the U.S. provisional applica- cinoma (46 48). Furthermore, coapplication of ABT-737, a tion 61/252513. T. Zhu and P.E. Lobie consult for Saratan Therapeutics Ltd. pharmacologic inhibitor of the BCL2 family, enhances the cytotoxicity of paclitaxel and gefitinib in NSCLC cell lines (49, 50). Both ABT-737 and ABT-263 (an oral version of Grant Support ABT-737) have not only provided good preclinical activity Breast Cancer Research Trust and Foundation of Research, Science and as a single agent but also markedly enhanced apoptosis of Technology of New Zealand. X-J. Kong, Z-S. Wu, and T. Zhu were funded human SCLC or NSCLC cell lines and produced regression by the Hundred-Talent Scheme of Chinese Academy of Sciences, the of xenograft tumors derived from these cells in response to National Natural Science Foundation of China (grant 30571030), and the National Basic Research Program of China (grant 2007CB914503). radiotherapies or chemotherapies (51). Currently, ABT-263 Z. Yin was funded by the National Key Scientific Program of China (grant is in clinical trials in patients with SCLC (51). Given that 2007CB914801) and a National Outstanding Young Scientist Award of NSFC (30725015). ARTN produces a survival advantage in response to pacli- The costs of publication of this article were defrayed in part by the taxel, it could be expected that inhibition of ARTN would payment of page charges. This article must therefore be hereby marked also improve the response of NSCLC to chemotherapies. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We also noticed elevated expression of ARTN, GFRA3, RET and in NSCLC cell lines under serum-depleted condi- Received 12/03/2009; revised 04/02/2010; accepted 04/10/2010; tion compared with 10% FBS condition (data not shown). published OnlineFirst 06/08/2010.

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1708 Mol Cancer Ther; 9(6) June 2010 Molecular Cancer Therapeutics

Artemin-Stimulated Progression of Human Non−Small Cell Lung Carcinoma Is Mediated by BCL2

Jian-Zhong Tang, Xiang-Jun Kong, Jian Kang, et al.

Mol Cancer Ther 2010;9:1697-1708. Published OnlineFirst June 8, 2010.

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