The mature bone morphogenetic protein-2 is aberrantly expressed in non-small cell lung carcinomas and stimulates tumor growth of A549 cells

Elaine M. Langenfeld2, Steve E. Calvano2, Fadi Abou-Nukta1, Stephen F. Lowry2, Peter Amenta1 and John Langenfeld1,3,4

1 Department of Surgery, Division of Thoracic Surgery
2 Department of Surgery, Division of Surgical Sciences
3 Department of Pathology, UMDNJ-Robert Wood Johnson Medical School, One Robert Wood Johnson Place, PO Box 19, New Brunswick, NJ 08903-0019, USA

4 To whom correspondence should be addressed Email: langenje{at}umdnj.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To help identify genes, which may regulate metastasis in lung cancer, we performed representational difference analysis between a patient-derived non-small cell lung carcinoma (NSCLC) and immortalized normal human bronchial epithelial cells. This analysis revealed that bone morphogenetic proteins-2/4 (BMP) mRNA was expressed in the lung carcinoma. BMP-2/4 are known to induce pluripotent cell differentiation, enhance cell migration and stimulate proliferation during embryonic development. Despite being powerful morphogens it is not known whether BMP-2/4 have significant biological activity in human carcinomas. Furthermore, it has not been established whether the mature active BMP-2/4 protein is aberrantly expressed in patient-derived tumors. The purpose of this study was to determine whether the expression of the mature BMP-2/4 protein is disregulated in human lung carcinomas and to establish whether it has adverse biological activity. This study reveals that the mature BMP-2 protein, but not BMP-4, is highly over-expressed in human NCSLC with little to no expression in normal lung tissue or benign lung tumors. The expression of BMP-2 localized specifically to the cancer cells. Recombinant BMP-2 stimulated in vitro, the migration and invasiveness of the A549 and H7249 human lung cancer cell lines. In vivo, recombinant BMP-2 enhanced the growth of tumors formed from A549 cells injected subcutaneously into nude mice. Furthermore, inhibition of BMP-2 activity with either recombinant noggin or anti-BMP-2 antibody resulted in a significant reduction in tumor growth. This study shows that expression of the mature BMP-2 protein is disregulated in the majority of NSCLC. BMP-2 enhancement of tumor cell migration and invasion, as well as stimulating tumor growth in vivo, suggests it has important biological activity in lung carcinomas.

Abbreviations: BMP, bone morphogenetic proteins-2/4; GFP, green fluorescent protein; NSCLC, non-small cell lung carcinoma; PBS, phosphate-buffered saline; RDA, representational difference analysis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lung cancer is the leading cause of cancer deaths in the US with over 150 000 people this year expected to die from this disease (1). Despite improvements in diagnosis and treatment, only 15% of lung cancer patients will survive 5 years (1) with the majority of patients succumbing due to metastatic disease. The mechanisms regulating lung cancer's ability to invade and metastasize are poorly understood. We hypothesized that genes, which are highly over-expressed in lung carcinomas in comparison with normal lung tissue, would identify genes, which enhanced metastasis of lung carcinomas. Using representational difference analysis (RDA) (2,3) we identified the expression of bone morphogenetic protein-2/4 (BMP) mRNA in a patient-derived non-small cell lung carcinoma (NSCLC). Since BMP-2/4 are powerful morphogens that have not been characterized in cancer, we further examined the expression and biological activity of BMP-2/4 in human lung carcinomas.

BMP were originally described as proteins that can induce endochondral osteogenesis following i.m. injection (4). The BMPs are members of the transforming growth factor (TGF) superfamily, which are a phylogenetically conserved group of proteins (5). There are 20 isotypes of the BMPs with BMP-2 and BMP-4 having 92% homology (6) and sharing similar biological activity. The BMPs are synthesized as inactive variable length precursor proteins (7,8). The precursor BMP-2 proteins are proteolytically cleaved, producing a mature C-terminal protein that is the active form (4,7). The mature BMP-2 protein is then secreted from the cell, which may then act in an autocrine and/or paracrine manner. BMP-2 protein signaling is mediated by transmembrane serine/threonine kinases called type IA, IB and type II receptors (912). In addition to inducing bone formation, the BMPs also stimulate proliferation, differentiation and migration of many different cell types (1316). BMP-2 stimulates ovarian granulosa cell differentiation and induces oestradiol production (17). BMP-2/4 also induces pluripotential mesenchymal differentiation, which is required for normal embryonic development of many organs including bone, lung, heart and kidney (18,19). Functional knockout of BMP-4 by homologous recombination in mice is lethal with animals containing no primordial germ cells (20). These prior studies demonstrate that BMP-2/4 are multipurpose cytokines that not only induce bone formation, but also have biological activity in many other tissues.

BMP-2/4 mRNA has been shown by PCR to be expressed in a variety of human carcinoma cell lines (2123). However, it has never been established whether the mature BMP-2/4 proteins are aberrantly expressed in human-derived carcinomas in comparison with normal tissues. Furthermore, despite its significant morphogentic activity during embryogenesis a biological role of BMP-2/4 in human carcinomas has never been established. The purpose of this study was to determine whether the expression of the mature BMP-2/4 protein is disregulated in human lung carcinomas and to establish whether it has adverse biological activity in developing tumors. This study provides both in vivo and in vitro data suggesting that BMP-2 enhances lung tumorigenesis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines and acquisition of tissue specimens
The immortalized normal human bronchial epithelial cells (24) (BEAS-2B) and human lung cancer cell lines A549 and H7249 (25) were cultured in Dulbecco's Modified Eagles medium (DME) with 5% fetal bovine serum containing 1% penicillin/streptomycin, and 1% glutamine. Cells were kept in a humidified incubator with 5% CO2 at 37°C. Tissue from human lung carcinomas and a section of normal lung was obtained from patients who had their tumors surgically resected. The tissue was snap frozen in liquid nitrogen and stored in liquid nitrogen or at -70°C. Only samples with ischaemic time <30 min were examined. All sectioned samples were examined by a surgical pathologist and only samples without significant necrosis were used. The use of human tissues was approved by the Robert Wood Johnson Medical School Institutional Review Board.

Antibodies and recombinant proteins
Monoclonal anti-human BMP-2 antibodies MAB355 and MAB3551 (R&D Systems, Minneapolis, MN) were used to detect BMP-2 by western blot analysis. Goat anti-human BMP-2/4 (R&D Systems) was used for immunohistochemistry. Goat anti-human type IA, IB and II BMP receptor antibodies, mouse IgG antibody, and anti-human BMP-4 monoclonal antibody were obtained from R&D Systems. A goat anti-BMP-4 antibody was obtained from Santa Cruz (Santa Cruz, CA). Both anti-BMP-4 antibodies were used to assess BMP-4 expression by western blot analysis. Recombinant human BMP-2, recombinant human BMP-4 and recombinant noggin were purchased from R&D Systems. Recombinant proteins were reconstituted in phosphate buffered saline (PBS) with 0.1% gelatin. An anti-BMP-2 monoclonal antibody, which was a gift from the Genetics Institute (Cambridge, MA), was co-injected with the A549 cells into female NCJ nude mice.

Representational difference analysis
The RDA subtraction technique (2,3) was used to identify genes highly expressed in a Stage I adenocarcinoma of the lung obtained from a patient (tester), in comparison with immortalized normal human bronchial epithelial cells (24) (driver). In brief, total RNA was obtained by crushing frozen tissue samples with a mortar and immediately adding guanidine. RNA was fractionated in cesium chloride centrifuging in a SW 28 rotor (27 000 r.p.m.) for 16 h at 20°C. mRNA was purified using Oligo dT columns (Pharmacia, Peapack, NJ) and cDNA was obtained using the Pharmacia Time Saver cDNA synthesis kit (Pharmacia) as per the manufacturer's directions. cDNA was digested with Sau3AI endonuclease, R-linker ligated and amplified by PCR as described previously (2,3). The R-linkers were digested from the tester and removed using a microcon ym-30 column (Millipore, Bedford, MA). J-linkers were then ligated to the tester. The driver and tester cDNA were hybridized at 67°C for 20 h (driver in excess 100:1) and the subtracted tester cDNA amplified by PCR. A second round of subtraction was performed using N-linkers (driver in excess 800 000:1). The amplified PCR products were gel purified, cloned into blue script and sequenced at the core UMDNJ-RWJMS sequencing facility using an IBI Prism 377 DNA sequencer.

Migration assay
To examine whether BMP-2 enhanced the migration of lung cancer cells 50 000 A549 or H7249 cells were placed in the upper chamber of an 8 µ transwell migration chamber (Becton Dickinson, Bedford, MA). In the lower well was 300 µl of LHC serum free media (BioFluids, Rockville, MD) containing recombinant human BMP-2 (1, 10, 100 or 500 ng/ml) or an equal volume of PBS with 0.1% gelatin. After 24 h the filters were removed and the top of the filter was wiped with a cotton swab. Cells that had migrated through the filters were stained with Syto-16 intercalating dye (Molecular Probes, Eugene, OR). Five high power fields were counted using fluorescent microscopy. To inhibit BMP-2, 500 ng/ml of recombinant BMP-2 was incubated with 10 µg/ml of recombinant noggin in serum free media at 37°C for 1 h prior to placing the BMP-2 into the lower chamber.

Migration assay in monolayer culture
To assess whether BMP-2 stimulates the migration of tumor cells growing in monolayer culture, we examined whether A549 and H7249 cells growing on glass cover slips migrated toward Affi-Blue beads coated with recombinant human BMP-2. Recombinant human BMP-2 (R&D Systems) was coated with Affi-Blue agarose beads (Bio-Rad, Hercules, CA) as described previously (19,26). Briefly, 100 µl of the Affi-Blue agarose beads were incubated with 10 µl of BMP-2 (100 µg/ml) or PBS with 0.1% gelatin at 37°C for 2 h. The beads were then washed with PBS and reconstituted with 40 µl of PBS. Glass cover slips were coated with serum free media containing BSA, fibronectin and collagen (27) and 50 000 cells were plated per cover slip. After allowing the cells to attach for 12 h the cover slips were placed into a 6 well plate containing serum free media. Two microliters of the Affi-Blue agarose beads coated with recombinant BMP-2 or dilution buffer were placed in linear fashion next to the cover slips. Five days later the cells, which migrated off the cover slips were photographed.

Invasion assay
We examined whether BMP-2 enhanced the invasiveness of tumor cells by determining whether recombinant BMP-2 stimulated the migration of A549 and H7249 cells through the extracellular tumor matrix, Matrigel (Becton Dickinson). 100 µl of Matrigel was placed in the upper well of an 8 µ transwell migration chamber. 50 000 A549 or H7249 cells were placed in the upper chamber and 300 µl of LHC serum free media supplemented with recombinant human BMP-2 (1, 10, 100 or 500 ng/ml) or an equal volume of PBS with 0.1% gelatin was added to the lower well. After 48 h the Matrigel and the cells on the upper side of the filter were removed using a cotton swab. Cells that migrated through the filter were stained with Syto-16 nuclear dye and examined by fluorescent microscopy.

Growth assay
To determine the effects of BMP-2 on the monolayer growth of the A549 cells, 75 000 cells were plated onto 6 well tissue culture plates containing LHC serum free media. After allowing the cells to adhere for 12 h they were treated with recombinant BMP-2 or with vehicle alone (PBS with 1% gelatin). Two days later cells were detached and counted using a haemacytometer.

Western blot analysis
Tissue specimens in a modified RIPA (27) buffer were briefly sonicated on ice. Protein concentration was measured using the Bradford assay. Protein samples were prepared under reducing conditions. Total cellular protein was analyzed by a 15% SDS–PAGE, transferred to nitrocellulose (Schleicher and Schuell, Keene, NH) at 35 V for 16 h at 4°C. The blots were then incubated overnight at 4°C with the appropriate primary antibody in Tris-buffered saline with 1% Tween (TBST) and 5% non-fat dried milk. Specific proteins were detected using the enhanced chemiluminescence system (Amersham, Arlington Heights, IL). Relative BMP-2 expression was analyzed using NIH Imaging. Pixel density of the mature BMP-2 protein and actin were determined on each of the developed immunoblots. A relative actin value was determined in each sample by dividing its pixel density by the pixel density from the sample with the highest expression of actin. The sample with the highest actin level on the blot was given a value of 1 and the remaining samples as a fraction of that value. The relative BMP-2 expression was determined by dividing the BMP-2 pixel density by its relative actin value.

Northern blot analysis
The mRNA obtained from the driver and tester was size fractionated on a 1% agarose–formaldehyde gel in a MOPS (0.2 M 3-N-morpholino-propanesulfonic acid/0.05 M Na acetate/0.01 M EDTA) buffer. The mRNA was transferred to a nitrocellulose membrane by capillary transfer. The mRNA was cross-linked to the membrane using ultraviolet light. The subtracted BMP-2/4 cDNA was radiolabeled with 32P using the All-In-One Random Priming mix (Sigma, St Louis, MO). The probe was denatured by boiling and incubated with the blots in PerfectHyb Plus hybridization buffer (Sigma) for 12 h. Membranes were washed in high stringent conditions and exposed to Kodak XAR film with an intensifying screen.

RT–PCR
Total RNA was extracted from a patient-derived tissue sample using Trizol (Gibco, Rockville, MD). First strand cDNA was synthesized using the Advantage for PCR kit (Clontec, Palo Alto, CA) following the manufacturer's instructions. BMP-2 cDNA was amplified using primers (F) 5'-cct gag cga gtt cga gtt g-3', and (R) 5'-cac tcg ttt ctg gta gtt c-3' at 95°C for 1 min, 50°C for 1 min, 72°C for 2 min for 30 cycles. The expected size of the amplified BMP-2 was 230 bp. BMP-4 was amplified using primers (F) 5'-tac ctg aga cgg gaa gaa a-3', and (R) 5'-cca gac tga agc cgg taa ag-3' at 95°C for 1 min, 56°C for 1 min, 72°C for 2 min for 33 cycles. The expected size of the amplified BMP-4 was 211 bp. The amplified bands were gel purified and sequenced at the core UMDNJ-RWJMS sequencing facility using an IBI Prism 377 DNA sequencer.

Cloning and transfection of BMP-2
Full-length human BMP-2 cDNA (gift from Genetics Institute) was cloned into the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA) in both the sense (BMP-2-S) and anti-sense (BMP-2-AS) directions. A549 cells with forced expression of BMP-2-S were also co-transfected with a pCMV-GFP vector (BD Biosciences, Palo Alto, CA), which constitutively expresses green fluorescent protein (GFP). Transfection was performed by electroporation using 30 µg of BMP-2-S and 10 µg of GFP at 0.975 µF capacitance and 0.2 kV. Control cells were transfected with the pcDNA3.1 and the GFP expression vector (A549-GFP). Transfected cells were cultured in 5% FCS DME media containing 50 µg/ml of neomycin. After expanding the transfected A549 cells in selective medium, cells expressing GFP were bulk sorted using flow cytometry. To obtain A549 cells expressing BMP-2 in the anti-sense direction A549 cells were transfected with BMP-2-AS or pcDNA3.1 vector (A549-V) by electroporation. These cells were not co-transfected with the GFP expression vector and transfected cells were obtained by culturing in selective media. BMP-2-AS and A549-V subclones were obtained by limiting dilution in 96 well plates placing 1 cell for every 5 wells. Seven subclones of BMP-2-AS and A549-V were then selected.

Immunohistochemistry
Tissue samples were placed in O.C.T. (optimal cutting temperature) and snap frozen in liquid nitrogen. Four micron Cryostat sections were air dried before being fixed in cold acetone for 10 min. Sections were washed in cold 0.5 M PBS and intrinsic peroxidase was quenched with 0.03% periodic acid for 20 min at room temperature. Sections were then rinsed in cold PBS and 0.5% BSA/PBS was applied to the slides for 15 min in a humidified chamber. Biotinylated polyclonal BMP-2/4 antibody (R&D Systems) was applied at a concentration of 2 µg/ml in 1% BSA/PBS and incubated overnight at 4°C.

Negative controls were slides in which the samples were incubated overnight at 4°C with normal rabbit serum without primary antibody. In addition, we performed a competition assay by incubating the biotinylated BMP-2/4 antibody with recombinant human BMP-2 at a 1:10 M ratio for 2 h prior to the overnight incubation. Following the overnight incubations the slides were washed with cold PBS and incubated for 1 h in Streptavidin horseradish peroxidase (Dako, Carpinteria, CA) at a 1:500 dilution in 1% BSA/PBS. Slides were then counterstained in 0.7% Toluidine Blue. Two lung carcinomas and two normal lung carcinomas were examined for BMP-2 expression by immunohistochemistry.

Effect of BMP-2 on tumor growth in nude mice studies
To assess the effects of BMP-2 on tumor growth in vivo, A549 cells were co-injected subcutaneously into female NCJ athymic nude mice with recombinant BMP-2 or the BMP-2 antagonist noggin. Recombinant protein was delivered to the tumors using Affi-Blue agarose beads as described previously (19,28). In brief, 25 µg of Affi-Blue agarose beads were incubated with 20 µl of 100 µg/ml of BSA, recombinant human BMP-2, or noggin for 2 h, and then washed 3 times with PBS immediately prior to use. In separate experiments the beads were not washed prior to injection. The coated Affi-Blue agarose beads were co-injected with the 107 A549 cells subcutaneously into the flanks of NCJ nude mice. In a separate study 107 A549 cells were co-injected with 20 µg of an anti-BMP-2 monoclonal antibody reported to inhibit its activity (13). As a control A549 cells were co-injected with 20 µg of an isotype control antibody. Fourteen to nineteen days following injection the animals were killed and the tumors were removed and measured in three dimensions (length x width x depth). The mice studies were approved by the Robert Wood Johnson Medical School Institutional Animal Care and Use Committee.

Statistical analysis
Assessment of recombinant BMP-2 protein and recombinant noggin on tumor growth in athymic nude mice was analyzed from five independent experiments. In vivo studies using an anti-BMP-2 antibody were performed twice. All other experiments were performed at least 3 times. The size of tumors formed from A549 cells treated with recombinant BMP-2, recombinant noggin, or anti-BMP-2 antibody is reported as the mean ± SEM percentage of tumors formed from controls. Results were evaluated by one-way ANOVA using the Student–Newman–Keuls procedure for adjustment of multiple pairwise comparisons between treatment groups. Differences with P values <=0.05 were considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Representational difference analysis
The RDA subtraction technique was performed to identify mRNA that is uniquely or highly expressed in human lung carcinomas in comparison with normal bronchial epithelium. cDNA from immortalized human bronchial epithelial cells (BEAS-2B) was hybridized in excess (driver) to cDNA obtained from a patient-derived NSCLC (tester). The known genes identified by RDA were alpha-1-antitrypsin, cytokeratin 6 and BMP-2/4. We could not determine whether the sequenced cDNA was BMP-2 or BMP-4 because the amplified region (base pairs 766–863) is conserved in both. Alpha-1-antitrypsin has been reported previously to be over-expressed in 87% of human lung carcinomas (29). As BMP- 2/4 are powerful morphogens that have not been fully characterized in cancer, we further examined their expression and biological activity in human lung carcinomas.

BMP-2/4 mRNA expression in human lung carcinomas
The BMP-2/4 cDNA obtained from the RDA was labeled with 32P and hybridized to a northern blot containing the original mRNA from the BEAS-2B cells (driver) and NSCLC (tester). This blot revealed high expression of the BMP-2/4 mRNA in the tumor, with no expression found in the BEAS-2B cells (Figure 1a). RT–PCR confirmed that BMP-2 mRNA was not expressed in the BEAS-2B (Figure 1b, lane 2). We did identify BMP-2 expression in the A549 human lung cancer cell line (Figure 1c, lane 3). However, BMP-4 expression was detected by PCR in the BEAS-2B cells (data not shown). We next examined whether the BMP-2 and BMP-4 mRNA was expressed in other NSCLC. All four of the human lung tumors examined by RT–PCR demonstrated BMP-2 and BMP-4 expression (Figure 1c and d). Sequencing of the PCR product confirmed the primers were specific for BMP-2 or BMP-4.



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Fig. 1. RDA subtraction between NSCLC and IHBE cells demonstrated BMP-2/4 mRNA expression in NSCLC. (a) To confirm expression, the BMP-2/4 cDNA obtained from the RDA was labelled with 32P and hybridized to northern blot containing original mRNA from the NSCLC (lane 1) and IHBE (lane 2). Note the presence of a 1.7 kb transcript in NSCL but not in IHBE. (b) Expression of BMP-2 mRNA in the IHBE and A549 lung cancer cell line was assessed by RT–PCR. Lane 1, DNA 100 bp ladder; lane 2, IHBE cells; lane 3, A549 lung cancer cell line; lane 4, PCR without cDNA. Using RT–PCR expression of (c) BMP-2 and (d) BMP-4 mRNA was assessed in four human lung carcinomas, lanes 2–5.

 
The mature BMP-2 protein is highly over-expressed in human lung carcinomas
The BMP-2 and BMP-4 proteins are translated as precursor proteins and must be cleaved by a proprotein convertase to produce a mature active peptide (7). We examined whether the mature BMP-2 and/or BMP-4 proteins are expressed in patient-derived lung carcinomas. Immunoblots probed with a monoclonal BMP-2 antibody showed high expression of a 14 kDa mature BMP-2 protein in 11 of 12 NSCLC examined Figure 2a, lanes 2, 4, 6 and 8). In comparison, we found little to no expression of the mature BMP-2 protein in 10 normal lung tissue specimens (Figure 2a, lanes 3, 5 and 7). When the level of BMP-2 expression was assessed relative to the level of actin by quantitative scanning, BMP-2 expression was found to be at least 26 times higher in 11 of the 12 NSCLC when compared with that of normal lung tissue. Expression of BMP-2 was not increased in benign interstitial lung disease or a benign lung tumor (hamartoma) (data not shown). Recombinant human BMP-2 served as a positive control (Figure 2a, lane 1). BMP-2 was also found to be expressed in the A549 and H7249 human lung cancer cell lines (Figure 2c, lanes 2 and 3). The bone forming osteosarcomas have been reported previously to express BMP-2/4 by immunohistochemistry (Figure 2c, lane 1) (30). As expected, the SoAS osteosarcoma cell line also expressed a 14 kDa mature BMP-2 protein.



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Fig. 2. Representative immunoblot is demonstrating aberrant BMP-2 expression in NSCLC. (a) Immunoblots were probed with the MAB355 anti-BMP-2 monoclonal antibody. Recombinant BMP-2, lane 1; NSCLC, lanes 2, 4, 6 and 8; normal lung tissue, 3, 5 and 7. (b) Relative BMP-2 expression in normal lung tissue and NSCLC. (c) Mature BMP-2 is expressed in human lung cancer cell lines, SOAS osteosarcoma cell line, lane 1; A549 cells, lane 2; H7249 cells, lane 3. (d) Immunoblot probed with MAB3551 anti-BMP-2 specific monoclonal antibody. Recombinant BMP-2, lane 1; NSCLC, lanes 2, 4 and 6; normal lung tissue, lanes 3 and 5; hamartoma, lane 7. (e) Immunoblot probed with an anti-BMP-4 specific antibody. Hamartoma, lane 1; NSCLC, lanes 2, 4, 6 and 8; normal lung tissue, lanes 3, 5 and 7; recombinant BMP-4, lane 9. (f) Cell culture media was obtained from A549 cells transfected with full length BMP-2 cDNA (BMP-2-S) or vector alone (A549-GFP). Immunoblot of the media was probed with a monoclonal anti-BMP-2 antibody (MAB355). BMP-2-S, lane 1; A549-GFP, lane 2. (g) Immunoblot containing cell lysate from A549 subclones transfected with anti-sense BMP-2 cDNA (lanes 1–3) or vector alone (lanes 4–6) and probed with an anti-BMP-2 antibody (MAB355).

 
We found that the monoclonal anti-BMP-2 antibody (MAB355) used in the above experiments cross reacts with recombinant BMP-4 protein. We therefore probed the immunoblots with a second monoclonal anti-BMP-2 antibody (MAB3551), which is reported to be specific for BMP-2. Our studies confirmed that this antibody does not recognize recombinant BMP-4 protein (data not shown). The MAB3551 antibody also showed that the mature BMP-2 is over-expressed in NSCLC (Figure 2d, lanes 2, 4 and 6). The MAB3551 anti-BMP-2 antibody recognized predominately a 17 kDa mature BMP-2 protein, but also demonstrated expression of a 14 kDa mature protein in NSCLC (Figure 2d). The size difference may represent the level of glycosylation of the mature BMP-2 protein.

We next examined whether the mature active BMP-4 protein is expressed in human lung tumors. No detectable expression of the mature BMP-4 protein could be seen in either malignant or normal lung tissue when probing immunoblots with a BMP-4 specific monoclonal antibody (Figure 2b). Using a second antibody specific for BMP-4, we were still unable to detect expression of the mature BMP-4 protein (data not shown). To further verify the MAB355 anti-BMP-2 antibodies were recognizing the native mature BMP-2 protein, full-length human BMP-2 cDNA was transfected into the A549 cells (BMP-2-S). The cell culture media was obtained from the BMP-2-S and vector control A549 cells and western blot analysis performed. Immunoblots probed with the MAB355 anti-BMP-2 antibody showed higher expression of the 14 kDa BMP-2 protein in the BMP-2S cells (Figure 2f, lane 1) compared to controls (Figure 2f, lane 2). In addition, we transfected BMP-2 cDNA in the anti-sense direction into the A549 cells. All seven subclones containing the anti-sense BMP-2 cDNA had a lower expression of the 14 kDa protein on immunoblots probed in comparison with the seven subclones transfected with insertless vector (Figure 2g). Together the data demonstrate that the mature BMP-2, but not the mature BMP-4 protein, is highly over-expressed in human lung carcinomas.

To help determine which cells are expressing BMP-2 in the NSCLC, we examined BMP-2 expression in a NSCLC by immunohistochemistry. We found BMP-2 expression in the cytoplasm of the tumor cells (Figure 3a). BMP-2 was not found to be expressed in normal lung tissue or surrounding stromal cells. To confirm the antibody recognized BMP-2, the antibody was incubated with recombinant human BMP-2 prior to immunostaining. This led to a complete inhibition of the BMP-2 signal in the tumor specimen (Figure 3b).



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Fig. 3. (a) Immunohistochemistry localizing BMP-2 expression to the tumor cells. BMP-2 expression in a NSCLC demonstrating cytoplasmic staining of the tumor cells (arrowheads). The nuclei (N) of the tumor cells are non-reactive. (b) Pre-absorption of the BMP-2 antibody with recombinant human BMP-2 is non-reactive with the tumor cells (arrows). Original magnification x82.

 
Expression of BMP-2 receptors in human lung carcinomas
BMP-2 induces a physiological response through the activation of receptors specific for the BMPs. Intracellular activation begins with BMP-2 binding to either type IA or IB BMP receptors, which leads to the binding of this complex to the type II BMP receptor. The type II receptor phosphorylates the type I receptor, which then directly phosphorylates the Smad transcription factors (33). We found that the BMP type IA, IB and II receptors are expressed in both primary lung tumors and normal lung tissue. However, in contrast to the high level of expression of the BMP-2 ligand in primary lung cancers, we found the same level of expression of the type IA receptor between the lung carcinomas and normal lung tissue (Figure 4a). The expression of the type IB and type II receptors was lower in the lung tumor than that of normal lung tissue (Figure 4b and c). The IHBE, A549 and H7249 cells were also found to express type IA, IB and II receptors (Figure 4a–c, lanes 4–6). The data suggest that both cancer cells and normal lung tissue have the potential capability of being activated by the BMP-2 ligand.



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Fig. 4. BMP receptors are expressed in tumors and normal lung tissue. Immunoblots were incubated with antibodies specific for BMP receptor types (a) IA, (b) IB and (c) II; normal lung tissue, lanes 1–3; IHBE cells, lane 4; A549 cells, lane 5; H7249 cells, lane 6; NSCLC, lanes 7–9.

 
BMP-2 stimulates the migration and invasiveness of human lung cancer cells
BMP-2 and BMP-4 have been shown to stimulate the migration of non-cancerous human cells (13,14). Since migration is important for tumors to invade and metastasize, we examined whether recombinant BMP-2 stimulates the migration of the A549 and H7249 human lung cancer cell lines in vitro. When recombinant BMP-2 was placed in the lower well of a migration chamber, it caused a dose–responsive increase in the migration of both the A549 and H7249 cells (Figure 5a). The BMP-2 antagonist, noggin, completely inhibited the ability of BMP-2 to stimulate the migration of the A549 cells (Figure 5b). We next determined whether BMP-2 enhanced the migration of the lung cancer cell lines growing in monolayer cell culture. The A549 and H7249 cells were cultured on glass cover slips and placed in 6 well plates containing Affi-Blue agarose beads coated with PBS with 0.1% gelatin or recombinant BMP-2. After 5 days we assessed the number of cells, which had migrated off the cover slips and were growing in the 6 well plate. Representative photographs of the H7249 cells are shown in Figure 5 (c and d). We found only an occasional cluster of A549 or H7249 cells growing on the 6 well plates when cultured with control beads (Figure 5c). However, when A549 and H7249 were cultured with Affi-Blue agarose beads coated with recombinant BMP-2 we consistently found a large number of cells had migrated off the glass cover slips and were growing on the 6 well plate (Figure 5d).



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Fig. 5. BMP-2 stimulates the migration of A549 and H7249 human lung cancer cell lines. (a) 50 000 cells were added to the upper well and recombinant human BMP-2, 0, 1, 10, 100, 500 or 1000 ng/ml was added to the lower well of the transwell chamber. After 24 h cells which migrated through the filter were counted using fluorescent microscopy. (b) Noggin inhibits BMP-2 induced migration. Lower wells were treated with vehicle control, recombinant BMP-2 (500 ng/ml), or recombinant BMP-2 (500 ng/ml) incubated with recombinant noggin (10 µg/ml) prior to placing into lower well. (c and d) BMP-2 stimulates migration of tumor cells growing in monolayer culture. H7249 cells were cultured on glass cover slips and placed in six well plates containing Affi-Blue agarose beads coated with PBS with coated 0.1% gelatin or recombinant BMP-2. White arrow, edge of cover slip; black arrow, H7249 cells growing on the 6 well plate; star, agarose bead. (c) After 5 days only an occasional H7249 cell had migrated off the cover slip when cultured with control beads. (d) When cultured with Affi-Blue agarose beads coated with recombinant BMP-2 many H7249 cells migrated off the cover slips and were growing in the 6 well plate. Similar results were found using the A549 cells. (e) The upper well of a migration chamber was coated with Matrigel. 50 000 A549 or H7249 cells were placed in the upper well and recombinant BMP-2, 0, 1, 10, 100, 500 or 1000 ng/ml was added to the lower wells. After 48 h cells which migrated through the filter were counted using fluorescent microscopy. (f) A549 cells in serum free media were treated with 0, 1, 10 or 100 ng/ml of recombinant BMP-2 for 2 days. Cells were detached and counted using a haemacytometer.

 
To assess whether BMP-2 may enhance the invasiveness of cancer cells, we examined if BMP-2 stimulated the migration of the A549 and H7249 lung cancer cells through the tumor matrix Matrigel. Recombinant BMP-2 again produced a dose–responsive increase in the migration of both the A549 and H7249 cells through the Matrigel coated chambers (Figure 5e).

In vitro growth effects of BMP-2 on the A549 cells
We next assessed the effects of recombinant BMP-2 on the growth of the A549 cells in vitro. We found that recombinant BMP-2 caused only minimal growth suppression of the A549 cells. In serum free conditions, BMP-2 at 100 ng/ml caused a 15% decrease in growth of the A549 cells after 2 days (Figure 5f). Lower concentrations of BMP-2 had no effect on the growth of the A549 cells. These results are consistent with a previous report showing BMP-2 inhibited the growth of the A549 cells (25).

BMP-2 enhances in vivo tumor growth
As BMP-2 is a secreted protein, we hypothesized that it may affect the growth of the A549 cells differently in vivo than it does in vitro. To answer this question, we assessed tumor growth of the A549 cells in athymic nude mice treated with recombinant BMP-2 or by inhibiting BMP-2 activity with noggin or an anti-BMP-2 antibody. Noggin coated Affi-Blue agarose beads were co-injected with the A549 cells subcutaneously into nude mice. Noggin has a high binding affinity for BMP-2 and BMP-4 preventing their activation of the BMP receptors (34). This method of delivering noggin in vivo has been shown in several other model systems as an effective means to inhibit BMP-2/4 activity (3537). Noggin-treated 549 cells (n = 14) (Figure 6a, 3) consistently formed tumors, which were less than half the size of A549 cells treated with BSA (n = 15) (Figure 6a, 2). Co-injection of A549 cells with an anti-BMP-2 monoclonal antibody reported previously to inhibit BMP-2-induced migration of smooth muscle cells (13), (n = 4) (Figure 6b, 2) also produced an ~60% reduction in tumor growth when compared with mice co-injected with a control antibody (n = 4) (Figure 6b, 1).



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Fig. 6. BMP-2 enhances tumor growth in vivo. (a) 107 A549 cells were co-injected subcutaneously into nude mice with 25 µg Affi-Blue agarose beads coated with 100 µg/ml of recombinant human BMP-2 (1), BSA (2) or noggin (3). Tumors were measured 14–19 days following injection. (b) Anti-BMP-2 antibody inhibits tumor growth. The A549 cells were co-injected into nude mice with 20 µg of an isotype control antibody (1) or 20 µg of an anti-BMP-2 antibody (2).

 
A549 cells co-injected into nude mice with Affi-Blue agarose beads coated with recombinant human BMP-2 (n = 15) (Figure 6a, 1, Table I) formed tumors which were ~50% larger than that of A549 cells treated with BSA (n = 15). Tumors were stained with hematoxylin and eosin and examined by a surgical pathologist for the presence of bone and/or cartilage. There was no evidence of bone and/or cartilage in any of the tumor formed from A549 cells treated with recombinant BMP-2. Together the data suggest that BMP-2 produced from the A549 cells enhances tumor growth in vivo, which is not associated with the formation of bone.


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Table I. BMP-2 enhances tumor growth in vivo

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Using RDA we identified the expression of BMP-2 mRNA in a patient-derived NSCLC. We subsequently showed high levels of expression of the mature active BMP-2 protein in all of the NSCLC examined, with little to no expression in normal lung tissue. Although prior studies have shown BMP-2 expression in human carcinomas the presence of the active mature protein has not been reported. It is also not clear whether the mature BMP-2 protein expression is aberrantly expressed in comparison to normal tissue. Prior studies were performed predominately on cell lines and BMP expression was assessed mainly by PCR. BMP-2 mRNA has been shown to be expressed in carcinoma cell lines from a variety of different cell types including gastric, ovarian, prostate, pancreatic and breast (2123). The highly homologous BMP-4 mRNA has also been shown to be expressed in human carcinoma cell lines (23,29). BMP-2 protein expression in patient-derived carcinoma has been performed in only a few studies and has been assessed using immunohistochemistry. These studies have suggested higher expression of BMP-2 in pancreatic (22) and head and neck tumor (40) samples in comparison with normal tissues. However, the antibody used in these studies recognized both BMP-2 and BMP-4 and is not specific for the mature protein. This present study shows the mature BMP-2 protein is highly over-expressed in perhaps all patient-derived lung carcinomas, and that mature BMP-4 protein is not significantly expressed in human lung carcinomas.

This study suggests that tumors express both a glycosylated (17 kDa) and non-glycosylated (14 kDa) mature BMP-2 with the non-glycosylated form being the most highly over-expressed in lung carcinomas. The MAB355 anti-BMP-2 antibody was produced against an Escherichia coli-derived mature human BMP-2 protein. Although this antibody recognized predominately a 14 kDa protein, it did recognize a 17 kDa protein on some of the immunoblots (Figure 2c). The MAB3551 monoclonal antibody was raised against a glycosylated mature human BMP-2 protein (NSO-derived recombinant human BMP-2). The predicted size of BMP-2 under reducing conditions is 14 kDa; however, following glycosylation BMP-2 is reported to be 17 kDa (4). Prior studies have shown that glycosylated and non-glycosylated mature BMP-2 proteins have equivalent biological activity, in vivo (31,32).

Our studies suggest that BMP-2 has important biological activity in human lung carcinomas. The natural BMP-2 inhibitor, noggin, caused a significant reduction of tumor growth of the A549 cells in nude mice. Although noggin could potentially inhibit other BMP proteins affecting tumor growth, our study supports the conclusion that the inhibition was specific for BMP-2. Noggin has a high affinity to BMP-2 and BMP-4 with a much lower affinity to BMP-7 (34). Noggin has also been shown to inhibit growth differentiation factor-5 (GDF-5) and GDF-6. However, our studies did not reveal expression of the mature BMP-4 protein in patient-derived lung carcinomas. Our preliminary studies thus far (not shown) have not detected significant expression of BMP-7 or GDF-5 protein in human lung tumors. Furthermore, the findings that an anti-BMP-2 antibody also inhibited tumor growth and that recombinant BMP-2 enhanced tumor growth in vivo support the hypothesis that BMP-2 has a significant role in lung tumorogenesis.

The addition of recombinant BMP-2 to the A549 cells injected into nude mice did not induce bone or cartilage formation. The reason for this apparent discrepancy of BMP-2 in vivo activity is not known. However, with our increasing knowledge of BMP-2, it is becoming clear that not all biological activity associated with BMP-2/4 involves the formation of bone or cartilage. BMP-2 has recently been shown to be involved in the stimulation of ovulation (17). BMP-2/4 also induces the migration and differentiation of precursor cells that are not involved in bone or cartilage formation. The biological response of BMP-2 may depend not only on the particular cell types present, but may vary depending on the presence of other cytokines. BMP-2 has been shown to have a trophic effect on locus coeruleus neurons only in the presence of other neuronal growth factors (41). Future studies will most likely define even a broader role of the BMPs in postnatal tissues.

We demonstrated that BMP-2 stimulates the migration of lung cancer cell lines in vitro. Therefore, BMP-2 may enhance the invasiveness of a tumor by an autocrine mechanism. At high concentrations BMP-2 suppressed the growth of the A549 cells, suggesting that it may inhibit the growth of cancer cells. As BMP-2 enhances tumor growth in vivo this would suggest that BMP-2 also acts in a paracrine manner. Although several other studies have shown that BMP-2 inhibits growth in vitro (25,42,43), this effect may depend on the presence of other cytokines. Ide et al. (44), showed that BMP-2 inhibited the growth of the LNCap prostate carcinoma cell in the presence of androgen, while in the absence of androgen BMP-2 stimulated growth (44). BMP-2 has been shown to enhance proliferation during early wound healing (45). BMP-2 also stimulates the proliferation of growth plate chondrocytes (46). Therefore, the effects of BMP-2 on cell growth may vary depending on the cell culture conditions. Further studies are needed to better define the autocrine effects of BMP-2 on human tumorigenesis.

There are several potential mechanisms through which BMP-2 could enhance carcinogenesis in a paracrine manner. BMP-2 is known to induce stem cell differentiation, enhance migration of vascular smooth muscle cells and monocytes, and stimulate the production of an extracellular matrix (1316,47). More recently, BMP-2 has been shown to stimulate VEGF production from a murine preosteoblast like cell line which promotes angiogenesis in fetal mouse bone explants (48). Any of these paracrine mechanisms could have an adverse effect on the progression of cancer.

The data show that the mature BMP-2 protein is aberrantly expressed in the majority of NSCLC. We provide both in vitro and in vivo data demonstrating that BMP-2 may have important biological activity in human lung carcinomas, which enhances lung tumorigenesis. Further studies are needed to define the specific mechanisms activated by BMP-2 in human carcinomas.


    Acknowledgments
 
We would like to thank Patrick Martin (Institute Curie, Centre Universitaire) for his assistance with the RDA. We also thank Ramsey Foty (UMDNJ-RWJMS) for his careful review of this manuscript. This study has been funded in part by the NIH K22 grant # CA91919-01A1, The New Jersey State Commission of Cancer Research and UMDNJ Foundation to J.L.


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 Introduction
 Materials and methods
 Results
 Discussion
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Received December 19, 2002; revised April 24, 2003; accepted June 6, 2003.