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 highly 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.
Lung carcinoma is currently responsible for the highest cancer-related mortality worldwide, with overall 5-year survival approximating 15% (1, 2). Primary lung carcinoma can be largely classified as non–small cell lung carcinoma (NSCLC) and SCLC (1). Although early diagnosis of lung carcinoma remains challenging, the lack of effective approaches to prevent disease progression also persists. As a result of advances in cancer biology during the last few decades, a number of targeted agents have been developed, exemplified by erlotinib/gefitinib, which selectively inhibits the epidermal growth factor receptor (3). However, clinical application of these agents has provided only limited therapeutic benefits for patients with lung carcinoma (4–6), partially attributable to the compensatory effect of other cellular mechanisms exploited by the tumors for survival and progression (3). Therefore, identification and subsequent targeting of novel oncogenic pathways may provide an advantage to the current regimens used to treat lung carcinoma and consequently improve prognosis.
Artemin (ARTN) is a neurotrophic factor that belongs to the glial cell line–derived neurotrophic factor (GDNF) family of ligands (GFL). ARTN mediates survival, differentiation, and migration of various types of neurons (7, 8). ARTN signaling is reported to be transduced via cognate receptors GFRA3 and also GFRA1 (9), which stimulate the phosphorylation of the transmembrane receptor tyrosine kinase RET, to activate downstream mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways, among others (10). RET-independent signaling has also been observed for the GFL family via alternative partners, including integrins and neural cell adhesion molecule (10).
An increasing body of evidence has implicated ARTN in progression of carcinoma (8, 11–13). Elevated expression of ARTN predicted residual disease after chemotherapy, metastasis, and decreased overall survival in mammary carcinoma patients (8). ARTN expression is also positively correlated to high tumor grade and myometrial invasion in endometrial carcinoma (12). Forced expression of ARTN promoted survival, invasion, anchorage-independent growth, and xenograft tumor growth of both human mammary and endometrial carcinoma cells, whereas depletion or functional inhibition of ARTN inhibited these cellular activities (8, 12). ARTN and GDNF have also been reported to stimulate invasiveness but not proliferation of human pancreatic carcinoma cells (11, 14).
Herein, we show that ARTN promotes progression of NSCLC by enhancement of tumor growth and metastasis. We therefore propose that strategies targeting ARTN could potentially exert therapeutic benefit in human NSCLC.
Materials and Methods
pIRESneo3 empty vector, pIRESneo3-ARTN expression plasmid, negative control small interfering RNA (siRNA) construct pSilencer-CONTROL (previously designated as pSilencer-CK), and ARTN-specific siRNA construct pSilencer-ARTN were described previously (8).
Cell lines and cell transfection
The human NSCLC cell lines H1299, H2009, and A549 were obtained from and characterized by the American Type Culture Collection. Human NSCLC cell lines H1975 and H460 were generously provided by Professor William Wilson (University of Auckland). H1299 and H1975 were transfected using FuGENE HD Transfection Reagent (Roche Diagnostics) with pIRESneo3 or pIRESneo3-ARTN, respectively. Following 4-week selection in media containing 1200 μg/mL G418, pooled stable transfectants were designated as H1299-VEC, H1299-ARTN, H1975-VEC, and H1975-ARTN, respectively. H1299 or H1975 cells were also transfected with pSilencer-CONTROL or pSilencer-ARTN, respectively, generating stable cell lines H1299-CONTROL, H1299-siARTN, H1975-CONTROL, or H1975-siARTN.
Generation of chicken anti-ARTN antibody
Chicken anti-ARTN polyclonal antibody (ARTN-IgY) was generated as previously described (8). ARTN-IgY and preimmune chicken IgY (CON-IgY) were both used at 500 μg/mL for cell-based bioassays.
The BCL2 inhibitor YC137 (Calbiochem) was purchased from Merck KGaA. The specificity of YC137 for BCL2 has previously been shown elsewhere (15).
Western blot analysis
Western blot analysis was done as previously described (8, 16). Four milliliters of conditioned medium were generated from 106 cells incubated in serum-free medium for 48 hours and concentrated to 90 μL before denaturation in 2× protein loading buffer. Anti-ARTN antibody was purchased from R&D Systems, anti–β-actin antibody (A1978) was from Sigma-Aldrich, and anti-BCL2 antibody (51-6511GR) was from BD Biosciences. Chemiluminescence captured on films was imaged by GS-800 calibrated densitometer and processed by Quantity One software (Bio-Rad Laboratories).
In vitro assays
Total cell number, suspension culture, wound healing, and colony formation in soft agar assays were done as previously described with minor modifications as indicated (17, 18). Suspension culture and soft agar colony formation assays were done in 10% fetal bovine serum (FBS) medium. For total cell number assay, H1299 derivatives were 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 number and suspension culture assays, cells were collected after trypsinization for manual counting. For wound-healing assays, wounds were created in a 90% confluent cell monolayer using an inverted sterile 200 μL pipette tip in a continuous linear motion. The wounded cell monolayer was maintained in growth medium until the wounds in one of two compared groups were closed. The position of two frontlines of the cells migrating into the wounds was photographed at six to nine fixed locations on each day. For growth in three-dimensional Matrigel, 1 × 103 cells were plated in 10% FBS medium supplemented with 2% Matrigel in a 96-well plate. Matrigel-containing (2%) medium was renewed every 3 days until the experiment was terminated after 8 days. Cell number was quantified by Alamar blue as previously described (Invitrogen; ref. 19). Transwell migration and invasion assays were done as previously described with minor modifications (12). Twenty-four–well inserts (8-μm pore size; BD Biosciences) were coated with 2.5 μg/cm2 poly-d-lysine for both assays. For invasion assays, inserts were subsequently coated with Matrigel (BD Biosciences), diluted 1:40 with serum-free medium. Cells (2 × 104) were plated in serum-free medium on the upper side of each insert and allowed to migrate toward 10% FBS medium on the lower side. Migration assays were done for 9 hours for H1299 derivatives or 16 hours for H1975 derivatives. Invasion assays were done for 16 hours for H1299 derivatives or for 24 hours for H1975 derivatives. Cells on the lower side of inserts were fixed in ice-cold methanol, stained with 0.01% crystal violet, and counted. Phase-contrast micrographs were acquired with an Olympus DP70 digital camera attached to Olympus IX71 fluorescence microscope and analyzed with DPController v1.1 and DPManager v1.1 (Olympus America, Inc.).
Cell cycle analysis
Following serum depletion (0.2% FBS; 24 h for H1975 or 48 h for H1299), cells were harvested immediately or further cultured in 10% FBS medium for 24 or 48 hours before harvest. Samples were assessed using a FACSCalibur (BD Biosciences) as previously described (20). Data were analyzed using ModFit LT software (Verity Software House).
Apoptosis was induced by 0.5 μmol/L paclitaxel for H1975 cells or by 2.5 μmol/L paclitaxel for H1299 cells in serum-free media (0% FBS) for 24 hours. One half to 1 million cells were harvested and labeled using Annexin-V-FLUOS Staining kit (Roche Diagnostics) and subjected to flow cytometry as above. Data analysis was conducted using CellQuest Pro software (BD Biosciences). The Annexin V–propidium iodide (PI) double staining method allows differentiation between early and late apoptosis. High Annexin V positivity and low PI positivity indicate early apoptosis, whereas high Annexin V positivity and high PI positivity indicate late apoptosis. Total apoptosis is the addition of early and late apoptosis and represents the total apoptotic cell population.
Semiquantitative reverse transcription-PCR and quantitative PCR
Equal numbers of cells were plated in 10% FBS medium. On the next day, after removal of 10% FBS medium, cells were washed with PBS and incubated in serum-free medium for another 24 hours. RNA was then isolated from these cells for reverse transcription-PCR (RT-PCR) and subjected to quantitative PCR analyses (21). Semiquantitative RT-PCR was done as previously described (8) for which 0.5 μg total RNA (except 1 μg for ARTN) was used as template. Images of gel electrophoresis were acquired using UVP BioImaging Systems and LabWorks Image Acquisition and Analysis software version 4.0 (UVP, Inc.). Quantitative PCR was done as previously described and using the same primer sets (21). Changes in gene expression were expressed as fold change relative to the vector control.
Five millions cells were suspended in 150 μL Matrigel/PBS (1:1, v/v) and injected into the subscapular region of immunodeficient mice (Shanghai Slaccas Co.). Determination of tumor volume, S-phase entry (by bromodeoxyuridine incorporation), and apoptosis [by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) immunostaining] and H&E staining were done as previously described (21). Liver and lung of sacrificed mice were fixed in 4% paraformaldehyde in PBS for histologic examination.
Luciferase reporter assay
BCL2 P1 promoter reporter construct (2 μg/well; ref. 18) was transfected using FuGENE HD reagent into 5 × 104 cells in 12-well plates; 0.4 μg/well pSV–β-galactosidase expression plasmid was cotransfected controlling for transfection efficiency. The cells were harvested in reporter lysis buffer (Promega Co.) 36 hours after transfection. Luminescence and β-galactosidase activity were quantified as previously described (22).
All experiments were repeated at least thrice, and the results of one representative experiment are shown. All data are expressed as means ± SE of triplicate determinants. Data analysis was conducted using an unpaired two-tailed t test.
ARTN, GFRA3, and RET are expressed in human NSCLC
To determine the expression of components of the ARTN signaling pathway in NSCLC, the mRNA expression of ARTN, GFRA1-A3 (encoding GFRA1-A3), and RET was determined by semiquantitative RT-PCR in a panel of five human NSCLC cell lines: H1299, H1975, H460, H2009, and A549. As observed in Fig. 1A (left), ARTN, GFRA3, and RET were detected in all examined cell lines, although at dissimilar expression levels. The expression levels of the remaining GFRA members varied greatly between cell lines. GFRA1 was only detected in H1299, H460, and A549 cells, and GFRA2 was only detected in H1975 and A549 cells.
The Oncomine database (23) harbors microarray-generated data on gene expression in human cancers. In this database, we examined the expression of ARTN, GFRA1-A3, and RET in normal and neoplastic lung tissues (Supplementary Table S1). ARTN expression was significantly increased in carcinoid, NSCLC (adenocarcinoma and squamous cell carcinoma), and small cell carcinoma compared with normal lung (Supplementary Table S1a). Increased expression of ARTN was also correlated positively with dedifferentiation in squamous cell carcinoma, increased lung carcinoma grade, and progression to N-stage squamous cell carcinoma but inversely with M-stage lung carcinoma (Supplementary Table S1b). ARTN expression was correlated to tobacco use in patients with lung carcinoma and wild-type Ras genotype in NSCLC (Supplementary Table S1b). The highest expression of ARTN was repeatedly reported in squamous cell carcinoma compared with any other subtypes of lung carcinoma (Supplementary Table S1b). For instance, Bild et al. showed that ARTN expression was 96% greater in squamous cell carcinoma than adenocarcinoma.
Expression of GFRA1 varied between subtypes of lung carcinoma, and there was no clear correlation between expression of GFRA1 and progression to N-stage adenocarcinoma (Supplementary Table S1c). Expression of GFRA2 was reduced in most neoplastic (with carcinoid as the exception) compared with normal lung tissue (Supplementary Table S1a) and predicted differentiation and lower grade and stage of squamous cell carcinoma (Supplementary Table S1d). Expression of GFRA3 was elevated in carcinoid, adenocarcinoma, squamous cell carcinoma, and small cell carcinoma compared with normal lung (Supplementary Table S1a). Expression of RET was increased in adenocarcinoma compared with normal lung (Supplementary Table S1a). Adenocarcinoma exhibited higher expression of RET compared with other subtypes of lung carcinoma derived from squamous, large, and small cell types.
Forced expression of ARTN enhanced survival, anchorage-independent, and three-dimensional Matrigel growth of H1299 cells
To determine the effects of elevated ARTN expression in human NSCLC, we stably transfected the human NSCLC cell line H1299 with an expression plasmid encoding human ARTN (pIRESneo3-ARTN) or the empty vector plasmid pIRESneo3 to generate the stable cell lines H1299-ARTN or H1299-VEC, respectively. Transfected colonies were pooled to minimize any effects of potential clonal selection. Expression levels of ARTN mRNA and cellular ARTN protein were increased in H1299-ARTN cells compared with H1299-VEC cells (Fig. 1A, right). Concomitantly, the level of secreted ARTN protein was increased in the conditioned medium of H1299-ARTN cells in comparison with H1299-VEC cells (Fig. 1A, right).
We first determined the effects of forced expression of ARTN on total cell number and cell cycle progression in monolayer culture. No difference in total cell number was observed between H1299-VEC and H1299-ARTN cells in monolayer culture in 10% FBS media over 10 days (Fig. 1B, left). Concordantly, quantitative PCR analyses did not reveal any changes in genes regulating cell cycle progression (Supplementary Table S2). However, we observed an increase in total cell number of H1299-ARTN cells compared with H1299-VEC cells in serum-depleted (0.2% FBS) medium despite initial equal plating densities. By day 9, the number of H1299-ARTN cells compared with H1299-VEC cells was 48% greater (Fig. 1B, right). PI staining and flow cytometry analysis did not reveal any significant differences in cell cycle progression between H1299-VEC and H1299-ARTN cells following 48 hours of serum depletion (0.2% FBS) only or in combination with 24 or 48 hours of incubation in 10% FBS–containing medium, indicative that the differences observed in total cell number under serum-depleted condition may be due to different apoptotic rates (Fig. 1C; only data generated by 24-h incubation in 10% FBS–containing medium following 48-h serum depletion shown). To determine if forced expression of ARTN increased the survival of H1299 cells, we induced apoptosis with paclitaxel, which is used clinically to treat NSCLC (24). Flow cytometric analysis following Annexin V/PI counter-staining revealed that early, late, and total apoptosis of H1299-ARTN cells were, respectively, 48%, 57%, and 54% lower than H1299-VEC cells (Fig. 1C). Concordantly, quantitative PCR analyses revealed that the mRNA expression level of BCL2 in H1299-ARTN cells was significantly higher than that in H1299-VEC cells (Supplementary Table S2).
We next examined the effect of forced expression of ARTN on anchorage-independent growth, a characteristic hallmark of the transformed cell phenotype (25). H1299-ARTN cells formed 91% more colonies in soft agar than H1299-VEC cells (Fig. 2A). Growth of H1299-ARTN cells was 61% greater than H1299-VEC cells in three-dimensional Matrigel culture (Fig. 2B). Concordantly, H1299-ARTN cells exhibited an 82% increase in total cell number in suspension culture compared with H1299-VEC cells by day 9 (Fig. 2C). In contrast to the mostly organized spherical colonies formed in three-dimensional Matrigel by H1299-VEC cells, colonies formed by H1299-ARTN cells were significantly larger and acquired a stellate morphology. The enhanced growth and aggressive morphology exhibited by H1299-ARTN cells in three-dimensional Matrigel suggests that ARTN may stimulate invasion of NSCLC cells.
Forced expression of ARTN enhanced migration and invasion of H1299 cells
In migration assays, the number of H1299-ARTN cells that migrated in Transwell chambers was 131% more than H1299-VEC cells (Fig. 2D). In a wound-healing assay, H1299-ARTN cells closed scratch wounds significantly faster than H1299-VEC cells (Fig. 2D). In invasion assays, the number of H1299-ARTN cells invading in Transwell chambers was 128% more than H1299-VEC cells (Fig. 2D). Quantitative PCR analyses revealed that the expression level of TGFB1, a recognized invasion stimulator of NSCLC cells (26), was increased in H1299-ARTN cells compared with H1299-VEC cells (Supplementary Table S2).
Forced expression of ARTN increased tumorigenicity in a xenograft model
We next examined the effects of forced expression of ARTN in H1299 cells in a xenograft mouse model. Subcutaneous implantation of either H1299-VEC or H1299-ARTN cells into the subscapular region of immunocompromised mice resulted in formation of palpable and measurable tumors after a period of 1 week. Although xenograft tumors derived from either cell line did not exhibit significant increases in volume up to day 20, thereafter increases in the volume of H1299-ARTN–derived tumors were significantly greater, being 63% larger than H1299-VEC–derived tumors by day 30 (Fig. 3A). We also assessed S-phase entry by determination of bromodeoxyuridine incorporation or apoptosis by TUNEL staining, respectively, in tumor sections. H1299-ARTN–derived tumors exhibited a 45% greater S-phase entry and a 37% reduction in apoptotic nuclei compared with H1299-VEC–derived tumors (Fig. 3B and C). Histopathologic examination revealed that H1299-VEC–derived tumors were well-circumscribed masses formed by tumor cells of a grossly uniform size with clearly delineated margins to the surrounding tissues (Fig. 3D). In contrast, H1299-ARTN–derived tumors were loosely massed and/or contained areas of cavitation (Fig. 3D, top right). These areas contained large pleomorphic cells exhibiting irregular or multiple hyperchromatic nuclei and voluminous cytoplasm, with ill-defined margins to the surrounding tissues that often appeared mucinous (Fig. 3D). Concomitantly, H1299-ARTN–derived tumor cells were observed adjacent to neural fibers, suggesting perineural invasion (Fig. 3D, bottom left). We additionally observed metastatic nodules in lungs of mice with tumors derived from H1299-ARTN cells (two of six) but not with those from H1299-VEC cells (zero of six; Fig. 3D, bottom right). Thus, forced expression of ARTN increased NSCLC proliferation and survival, leading to increased growth and invasiveness of xenograft tumors, resulting in metastasis.
Forced expression of ARTN also enhanced oncogenicity of H1975 cells
We also stably transfected another human NSCLC cell line, H1975, with pIRESneo3-ARTN or the empty vector plasmid pIRESneo3 to generate the stable cell lines H1975-ARTN or H1975-VEC, respectively. Semiquantitative RT-PCR and Western blot analyses, respectively, showed that mRNA, cellular, and secreted protein expression levels of ARTN were significantly increased in H1975-ARTN cells relative to H1975-VEC cells (Fig. 4A).
Similar to our observations in H1299 cells, forced expression of ARTN did not significantly alter the number of H1975 cells in monolayer culture in 10% FBS medium by day 9 (Fig. 4A). However, under serum-depleted conditions, the number of H1975-ARTN cells was significantly higher than H1975-VEC cells from day 3 on; the number of H1975-ARTN cells was 58% higher in comparison with H1975-VEC cells on day 3 (Fig. 4A). This was despite cell number being decreased from the original plating number over time in serum-depleted media. Under serum-depleted conditions, early, late, and total apoptosis of H1975-ARTN cells were, respectively, 35%, 67%, and 43% lower than H1975-VEC cells (Fig. 4B). Forced expression of ARTN also improved survival of H1975 cells in response to treatment with paclitaxel (Fig. 4B).
H1975-ARTN cells formed 47% more colonies than H1975-VEC cells in soft agar (Fig. 4B). In migration assays, the number of H1975-ARTN cells that migrated into Transwell chambers was 102% more than H1975-VEC cells (Fig. 4B). In invasion assays, the number of H1975-ARTN cells observed in Transwell chambers was 98% more than H1975-VEC cells (Fig. 4B). H1975-ARTN cells also closed scratch wounds markedly faster than H1975-VEC cells in a wound-healing assay (Fig. 4C). Lastly, in three-dimensional Matrigel culture, growth of H1975-ARTN cells was 48% greater than H1975-VEC cells; H1975-ARTN cells formed larger and more disorganized colonies than H1975-VEC cells, which occasionally ceased growth and disappeared from the culture (Fig. 4C).
BCL2 mediates ARTN-stimulated oncogenicity in NSCLC cells
We examined the mechanism underlying the survival advantage conferred by forced expression of ARTN. In quantitative PCR analyses, we observed significant increases in mRNA level of the antiapoptotic gene BCL2 on forced expression of ARTN: a 165% increase in H1299-ARTN cells and a 356% increase in H1975-ARTN cells compared with the appropriate controls (Fig. 5A). Furthermore, use of a human BCL2 promoter reporter construct indicated that forced expression of ARTN stimulated BCL2 promoter activity: The level of BCL2 promoter activity in H1299-ARTN cells was 49% higher than that of H1299-VEC cells, whereas the level in H1975-ARTN cells was 383% higher than that of H1975-VEC cells (Fig. 5A). Concordantly, Western blot analyses revealed increases in BCL2 protein in H1299-ARTN and H1975-ARTN cells compared with the appropriate control cells (Fig. 5A).
We next quantitatively assessed colony formation of H1299-VEC and H1299-ARTN cells in soft agar and three-dimensional Matrigel following treatment with the BCL2 inhibitor YC137 (27). YC137 produced a dose-dependent reduction in soft agar colony formation of H1299-VEC cells (Fig. 5B). YC137 also largely abrogated the ARTN-stimulated enhancement in anchorage-independent growth of H1299-ARTN cells, indicative of BCL2 dependence of ARTN-stimulated soft agar colony formation. Furthermore, YC137 also largely abrogated the ARTN-stimulated enhancement of H1299-ARTN cell growth in three-dimensional Matrigel, although significant inhibition of H1299-VEC cell growth was also observed as expected (Fig. 5C; ref. 28).
Depletion of ARTN reduced anchorage-independent and three-dimensional Matrigel growth and motility of NSCLC cells
We additionally examined the functional outcomes of siRNA-mediated depletion of endogenous ARTN in the two NSCLC cell lines. We stably transfected H1299 cells with a siRNA construct targeting ARTN or a negative control siRNA construct to generate the stable cell lines H1299-siARTN or H1299-CONTROL, respectively. H1975 cells were similarly transfected to generate the stable cell lines H1975-siARTN or H1975-CONTROL. Semiquantitative RT-PCR and Western blot analyses showed that siRNA-mediated depletion of ARTN resulted in decreases in ARTN mRNA and protein compared with negative control siRNA in both H1299 and H1975 cells (Fig. 6A). Depletion of ARTN resulted in reductions in colony formation (32%), migration (54%), invasion (49%; Fig. 6A), and growth in three-dimensional Matrigel (31%; Fig. 6B) in H1299-siARTN compared with H1299-CONTROL cells. Similar results were observed between H1975-siARTN and H1975-CONTROL cells (Fig. 6A and B).
Functional inhibition of ARTN abrogated anchorage-independent and three-dimensional Matrigel growth and motility of NSCLC cells
We lastly assessed the effects of functional inhibition by antibodies to ARTN in NSCLC cell lines. Soft agar colony formation and three-dimensional Matrigel culture assays were done with H1299 or H1975 cells, which were treated with either a polyclonal chicken antibody against ARTN (ARTN-IgY) or preimmune chicken IgY (CON-IgY) as negative control. H1299 cells treated with antibody formed 23% less and significantly smaller colonies in soft agar than control-treated cells (Fig. 6C), and this effect was recapitulated in three-dimensional Matrigel culture (Fig. 6D). Additionally, H1299 cells treated with ARTN-IgY exhibited a reduction of 45% in migration and of 48% in invasion compared with control-treated cells (Fig. 6C). The reductions in colony formation in soft agar, growth in three-dimensional Matrigel, and motility were also observed in H1975 cells treated with ARTN-IgY compared with CON-IgY; a much larger proportion of ARTN-IgY–treated H1975 cells ceased growth and disappeared gradually in soft agar colony formation and three-dimensional Matrigel assays (Fig. 6C and D).
Increased expression of ARTN and one receptor, GFRA3, is observed in an unusual majority of lung malignancies, including carcinoid, adenocarcinoma and squamous cell carcinoma (two main subgroups of NSCLC), and SCLC. Additionally, expression of GFRA3 is detectable in the normal lung and bronchus of human and mouse (29, 30). RET expression has also been shown in normal human lung (30). These facts together suggest that an autocrine ARTN signaling pathway is functional in human lung and the excessive activity of this pathway may therefore promote disease progression in lung carcinoma. In contrast, amplification or expression of most other oncogenes, including epidermal growth factor receptor, has been reported only in some subtype(s) of lung carcinoma (31). We observed from Oncomine that expression of ARTN is strongly correlated with use of tobacco in patients with lung carcinoma. Concordantly, we observed the greatest differential expression of ARTN in squamous cell carcinoma. However, expression of ARTN is also elevated in other subtypes of NSCLC, including adenocarcinoma, which is the most common subtype observed in never smokers (32), carcinoid, and SCLC compared with normal lung tissues. We also observe that forced expression of ARTN in a xenograft model results in formation of cavitation associated with the presence of large multinucleated cells. These tumors microscopically resemble giant cell carcinoma observed in human lung, which rarely occurs but produces particularly rapid growth, and distant and extensive metastases, associated with more frequent recurrence after surgery and exhibit poorer response to current chemotherapeutic regimens and worse life expectancy compared with other subtypes of NSCLC (33, 34).
Although ARTN is not observed to stimulate mitogenesis of NSCLC cells in vitro, there is a clear enhancement of proliferation in vivo and a concomitant greater increase in volume of xenograft tumors derived from NSCLC cells exhibiting forced expression of ARTN. This finding is reminiscent of our previous observation with human mammary carcinoma cell lines and is presumably due to paracrine interaction of ARTN with the tumor microenvironment (8). Furthermore, enhanced invasion and metastasis of NSCLC cells with forced expression of ARTN is associated with increased expression of TGFB1. Elevated expression of TGFB1 is frequently associated with metastases compared with paired primary lung carcinoma, or primary tumors that have metastasized compared with those that have not, and predicts recurrence of disease and death (26, 35).
The relative level of ARTN expression in H1299 cells is not high compared with H1975 cells. However, depletion or inhibition of ARTN in H1299 cells decreases ARTN-stimulated cellular functions to similar extents to that observed in H1975 cells. The response of any particular cell to a ligand is not simply dependent on ligand concentration. For example, some ligands fully activate receptor signaling at only 5% occupancy of the receptor (36). It should also be noted that H1299 cells express both GFRA3 and GFRA1, whereas H1975 cells only express GFRA3. ARTN has been shown to stimulate the formation of a GFRA1-RET complex in addition to using GFRA3 (9). It may be that the presence of GFRA1 in addition to GFRA3 in the H1299 cells allows them to respond functionally at lower concentrations of ARTN than H1975 cells, which only express GFRA3. Furthermore, GFLs have been shown to bind to non-GFRA proteins (37, 38) and function independent of RET (39). Thus, the expression level of ARTN may not be predictive of the relative contribution of ARTN to oncogenic functions in different NSCLC cell lines.
ARTN-stimulated survival, clonogenic capacity, and three-dimensional Matrigel growth of NSCLC cells were shown to be mediated by increased expression of BCL2. Increased expression of BCL2 on forced expression of ARTN is also observed in human mammary and endometrial carcinoma cells (8, 12, 13). Nevertheless, this report has determined that BCL2 contributes to ARTN-stimulated oncogenicity in NSCLC. BCL2 exhibits increased expression and promotes cell survival in various human malignancies, including lung carcinoma (40, 41). It is therefore probable that ARTN-stimulated increases in BCL2 expression produce an in vivo tumorigenic advantage, as observed with xenografts of H1299-ARTN cells, which grow as larger tumors with increased carcinoma cell survival. To date, no correlation has been observed between BCL2 expression and response of NSCLC patients to chemotherapy (42–45). Nevertheless, BCL2 has been implicated in the development of chemoresistance in cell lines of human lung carcinoma (46–48). Furthermore, coapplication of ABT-737, a 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 ABT-737) have not only provided good preclinical activity as a single agent but also markedly enhanced apoptosis of human SCLC or NSCLC cell lines and produced regression of xenograft tumors derived from these cells in response to radiotherapies or chemotherapies (51). Currently, ABT-263 is in clinical trials in patients with SCLC (51). Given that ARTN produces a survival advantage in response to paclitaxel, it could be expected that inhibition of ARTN would also improve the response of NSCLC to chemotherapies. We also noticed elevated expression of ARTN, GFRA3, and RET in NSCLC cell lines under serum-depleted condition compared with 10% FBS condition (data not shown). We speculate that the ARTN signaling pathway may be induced to favor survival of NSCLC in response to cellular stressors, such as removal of growth stimuli, treatment with chemotherapeutic agents, or perturbation of tumorigenic signal transduction by targeted therapies. As such, inhibition of ARTN may be useful as an adjuvant therapeutic agent in human NSCLC.
Disclosure of Potential Conflicts of Interest
J. Kang, J.K. Perry, D-X. Liu, and P.E. Lobie have equity interests in Saratan Therapeutics Ltd. D-X. Liu and P.E. Lobie are inventors on 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 application 61/252513. T. Zhu and P.E. Lobie consult for Saratan Therapeutics Ltd.
Grant Support: Breast Cancer Research Trust and Foundation of Research, Science and Technology of New Zealand. X-J. Kong, Z-S. Wu, and T. Zhu were funded by the Hundred-Talent Scheme of Chinese Academy of Sciences, the National Natural Science Foundation of China (grant 30571030), and the National Basic Research Program of China (grant 2007CB914503). Z. Yin was funded by the National Key Scientific Program of China (grant 2007CB914801) and a National Outstanding Young Scientist Award of NSFC (30725015).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received December 3, 2009.
- Revision received April 2, 2010.
- Accepted April 10, 2010.
- ©2010 American Association for Cancer Research.