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Molecular Cancer Research 3:679-684 (2005)
© 2005 American Association for Cancer Research

Signaling and Regulation

Bone Morphogenetic Protein-2–Induced Transformation Involves the Activation of Mammalian Target of Rapamycin

Elaine M. Langenfeld1, Yingxin Kong2 and John Langenfeld2

1 Department of Surgery, Division of Thoracic Surgery, and 2 Department of Surgery, Division of Surgical Sciences, University of Medicine and Dentistry, New Jersey Robert Wood Johnson Medical School, New Brunswick, New Jersey

Requests for reprints: John Langenfeld, Department of Surgery, Division of Thoracic Surgery, University of Medicine and Dentistry, New Jersey Robert Wood Johnson Medical School, MEB 536, One Robert Wood Johnson Place, P.O. Box 19, New Brunswick, NJ 08903-0019. Phone: 732-235-7802; Fax: 732-235-8150. E-mail: langenje{at}umdnj.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Bone morphogenetic protein-2 (BMP-2) is an evolutionary conserved protein that is essential for embryonic development. BMP-2 is highly expressed in ~98% of human lung carcinomas with little expression in normal lung tissues. BMP-2 has been shown to enhance mobility, invasiveness, and metastasis of cancer cell lines. During development, BMP-2 induces the proto-oncogene phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway to regulate stem cell differentiation. We show that BMP-2 induces the phosphorylation of mTOR in A549 and H1299 lung cancer cell lines, which is attenuated by the PI3K antagonists LY-294002 and wortmannin. p70S6 kinase, which is a direct downstream target of mTOR, is also regulated by BMP-2 in lung cancer cell lines. We find that BMP-2 induces cyclin E in A549 and H1299 cells, which is mediated by the PI3K/mTOR signaling pathway. The regulation of cyclin E by BMP-2 occurs through a Smad 1/5–independent mechanism. Forced expression of BMP-2 in A549 cells (A549/BMP-2) induces transformation as shown by an increase in foci formation. The mTOR antagonist, rapamycin, prevented foci formation of the A549/BMP-2 cells. This study provides evidence that BMP-2-mediated transformation of lung cancer cells involves the activation of the PI3K/mTOR signaling pathway. (Mol Cancer Res 2005;3(12):679–84)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The bone morphogenetic proteins (BMP) were originally described for their ability to induce the entire cascade of endochondral bone formation when injected i.m. into mice (1). The BMPs are phytogenetically conserved proteins from insects to humans. BMP-2/4 mediates signaling by binding serine/threonine kinase type IA or IB together with a type II receptor (2). The receptor complex then phosphorylates Smad 1/5/8, which then activates the transcription of downstream targets. BMP-2 and BMP-4 are thought to mediate signaling through Smad 1/5/8; however, studies have shown that they can induce signaling independent of the Smads (3, 4). BMP-2 and the highly homologous BMP-4 are essential for embryonic development (5). During embryogenesis, BMP-2 and BMP-4 are required for the development of several organs, including the lungs (6-8). BMP-2 and BMP-4 promote cell survival, proliferation, migration, and self-renewal of embryonic stem cells (9-11).

BMP-2 is aberrantly expressed in ~98% of lung carcinomas (12). Studies have suggested that BMP-2 has an important role in regulating tumorigenesis. Coinjection of recombinant BMP-2 with A549 cells into nude mice enhanced tumor growth, whereas the BMP-2 antagonist, noggin, caused a decrease in growth (13). BMP-2 has also been shown to stimulate the development of a neovasculature in developing tumors (14). Both BMP-2 and BMP-6 have been reported to activate Smad 1/5 and promote tube formation of endothelial cells, suggesting that the BMPs directly activate endothelial cells (14, 15). BMP-2 was shown to increase the invasiveness of melanoma and lung cancer cell lines (13, 16). Forced expression of BMP-2 in A549 cells significantly increased lung metastasis in nude mice (17). Furthermore, a higher level of BMP-2 expression correlated with a worse survival in stage I non–small cell lung carcinomas.

The mechanisms by which the BMPs regulate cancer cells are poorly understood. BMP-2/4 has been shown to regulate some of the same signaling pathways in both stem cells and human cancer cell lines. BMP-2 mediates self-renewal of embryonic stem cells (10) through its activation of Id-1. BMP-2 has also been shown to induce Id-1 expression in breast (18) and lung (17) cancer cell lines. Id-1 has been shown to stimulate proliferation of cancer cells (19) and to promote metastatic tumor growth of breast cancer cells (20). Forced expression of Id-1 was shown to immortalize primary human normal keratinocytes (21). BMP-2-induced differentiation of osteoblasts involves the activation of extracellular signal-regulated kinase 1/2 (22). BMP-2 also stimulates the phosphorylation of extracellular signal-regulated kinase 1/2 in lung cancer cell lines (17). Studies have shown that extracellular signal-regulated kinase 1/2 is a strong mitogen of cancer cells and promotes malignant transformation (23).

BMP-2 has also been shown to induce the proto-oncogene phosphoinositide 3-kinase (PI3K) in osteoblasts to regulate differentiation (24) and in cardiomyocyte to enhance contractility (25). Recently, the BMPs in combination with laminin-1 were shown to induce proliferation and colony formation of fetal pancreatic cells through PI3K and mitogen-activated protein kinase pathways (26). The activation of mammalian target of rapamycin (mTOR) is required for PI3K to mediate transformation (27). mTOR induces cell growth and survival, which occurs in part through the induction of G1 cyclins (28). mTOR signaling is mediated by inducing the translation of growth-promoting proteins (29). This study shows that BMP-2-induced activation of the PI3K/mTOR signaling pathway is conserved in lung cancer cells. We provide evidence that the activation of the PI3K/mTOR signaling pathway represents an important mechanism by which BMP-2 regulates transformation.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
BMP-2 Induces PI3K/mTOR Signaling Pathway
PI3K activates mTOR by inducing its phosphorylation. By Western blot analysis, we examined whether BMP-2 induced phosphorylation of mTOR in lung cancer cell lines. In both A549 (Fig. 1A) and H1299 (Fig. 1B) lung adenocarcinoma cell lines, BMP-2 caused an increase in phosphorylated mTOR within 10 minutes, which peaked by 20 minutes. To examine whether BMP-2-induced phosphorylation of mTOR is mediated by PI3K, cells were pretreated with the PI3K antagonist LY-294002. Cells treated with LY-294002 showed no increase in the level of phosphorylated mTOR following BMP-2 treatment (Fig. 1C).



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  		FIGURE 1. BMP-2 induces the activation of mTOR in A549 (A) and H1299 (B) lung cancer cell lines. Cells were treated with 10 ng/mL recombinant BMP-2 and phosphorylated mTOR (p-mTOR) was determined by Western blot analysis. C. A549 cells were pretreated for 1 hour with PI3K inhibitor, LY-294002 (20 µmol/L), before incubation with BMP-2. Experiments were done at least three times.

 
mTOR regulation of protein synthesis involves the phosphorylation of p70S6 kinase (p70S6K). p70S6K regulates cell growth and is a direct target of mTOR (30). Using an antibody that recognizes phosphorylated p70S6K, we assessed whether BMP-2 induces downstream mTOR mediators in the A549 (Fig. 2) and H1299 (data not shown) lung cancer cell lines. Similar to that seen with mTOR, BMP-2 induced phosphorylation of p70S6K within 10 minutes (Fig. 2A and B). The PI3K antagonists, wortmannin and LY-294002, blocked BMP-2-induced phosphorylation of p70S6K (Fig. 2A). To further verify that BMP-2-induced phosphorylation of p70S6K is mediated through the PI3K/mTOR signaling cascade, cells were pretreated with the mTOR antagonist rapamycin. Rapamycin completely attenuated BMP-2 phosphorylation of p70S6K in both A549 and H1299 lung cancer cell lines (Fig. 2B; data not shown).



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  		FIGURE 2. BMP-2 activates p70S6K through PI3K/mTOR pathway. A. A549 cells were treated with 10 ng/mL recombinant BMP-2 and phosphorylated p70S6K (p-p70S6K) was examined by Western blot analysis. PI3K was inhibited by pretreating cells for 1 hour with wortmannin (Wort; 100 nmol/L) or LY-294002 (Ly; 20 µmol/L) before being treated with 10 ng/mL BMP-2. B. A549 cells were treated with BMP-2 (10 ng/mL) and mTOR was inhibited with rapamycin (Rapa; 1 µg/mL). Experiments were done at least three times.

 
BMP-2 Induces Cyclin E Expression through PI3K/mTOR Signaling
mTOR is an evolutionary conserved signaling pathway that stimulates cell growth in yeast to humans (29). mTOR also shortens the G1-S phase of the cell cycle, which involves the regulation of G1 cyclins (28). We found that BMP-2 induces the expression of cyclin E in our lung cancer cell lines. The protein expression of cyclin E increased rapidly following BMP-2 treatment in the A549 cells, which lasted for at least 2 hours (Fig. 3A). Although not as robust, BMP-2 also increased the expression of cyclin E in the H1299 cells (Fig. 3C). BMP-2 did not induce the expression of cyclin D1 in either A549 or H1299 cells (Fig. 3E; data not shown). The cyclin inhibitor p21 was not found to be regulated by BMP-2 for at least 2 hours following treatment (data not shown). Consistent with previous reports, p27 was not expressed in the A549 cells. Both rapamycin and wortmannin inhibited BMP-2-induced cyclin E expression in both A549 and H1299 cells (Figs. 3F-H and 4A and B). These data show that BMP-2-induced expression of cyclin E occurs through the PI3K/mTOR signaling pathway.



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  		FIGURE 3. BMP-2 induces cyclin E through the PI3K/mTOR pathway. A549 (A) and H1299 (C) cells were treated with 10 ng/mL recombinant BMP-2 and cyclin E expression was examined by Western blot analysis. E. Immunoblot of cyclin D1 of A549 cells treated with 10 ng/mL BMP-2. Cyclin E immunoblot of A549 (F) and H1299 (G) cells treated with rapamycin (1 µg/mL) 2 hours before being treated with 10 ng/mL BMP-2. H. Cyclin E immunoblot of A549 cells pretreated with wortmannin (100 nmol/L) before BMP-2 (10 ng/mL) treatment. Experiments were done at least three times.

 


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  		FIGURE 4. Quantification of cyclin E expression in A549 cells treated with 10 ng/mL BMP-2 (A) and cells pretreated with 1 µg/mL rapamycin before being treated with BMP-2 (B). The level of cyclin E expression from each immunoblot was quantitated using NIH imaging. Columns, average of four experiments. *, P < 0.005, compared with control.

 
mTOR promotes the expression of proteins by enhancing translation (29). However, mTOR stimulates the translation of transcription factors, which could induce transcription. Using semiquantitative PCR, we examined whether BMP-2 induced the expression of cyclin E mRNA in A549 and H1299 cells. BMP-2 caused an ~2-fold increase in cyclin E mRNA in both A549 (Fig. 5A) and H1299 (data not shown) cells. Rapamycin did not inhibit BMP-2-induced expression of cyclin E mRNA (Fig. 5A). The p38 inhibitor, SB-203580, also did not inhibit BMP-2-induced expression of cyclin E mRNA (Fig. 5A). To further assess whether BMP-2 regulated translation of cyclin E, A549 cells were treated with cyclohexamide, which inhibits translation. BMP-2-induced cyclin E protein expression was attenuated in A549 cells treated with cyclohexamide (Fig. 5B). These data suggest that BMP-2 mediates cyclin E expression by enhancing its translation through mTOR signaling and promotes transcription of cyclin E, which occurs by another mechanism.



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  		FIGURE 5. A. BMP-2 induces cyclin E m-RNA that is not regulated through m-TOR. A549 cells were treated with vehicle control, 10 ng/mL of BMP-2, BMP-2 (10 ng/mL) and rapamycin (1 µg/mL), and BMP-2 (10 ng/mL) and SB-203580 (SB; 20 µmol/L) for 60 minutes. Semi-quantitative expression of cyclin E mRNA was determined using the light cycler. Data represents the average of at least 3 experiments and is depicted as the fold increase over control. *, P < 0.05, compared with vehicle control. B. BMP-2-induced cyclin E expression involves the regulation of translation. Immunoblot of A549 cells treated with BMP-2 (10 ng/mL) or BMP-2 (10 ng/mL) and cyclohexamide (CX; 20 µg). Experiments were done three times.

 
BMP-2 Induces Cyclin E Expression through a Smad-Independent Mechanism
BMP-2 can mediate signaling through Smad-dependent and Smad-independent pathways. We have shown previously that BMP-2 does not activate Smad 1/5 in A549 cells cultured in serum-free medium but does so in medium containing serum (17). We found that BMP-2 increases the expression of cyclin E in A549 cells cultured in serum-free medium (Fig. 6A). The same findings were found in the H1299 cells (data not shown). As expected BMP-2 did not induce the phosphorylation of Smad 1/5 in cells cultured in serum-free medium (Fig. 6A). BMP-2 also induced mTOR/p70S6K signaling pathway in cells cultured in serum-free medium (Fig. 6A). We have engineered A549 cells that do not activate Smad 1/5/8 signaling. The A549 cells were stably transfected with TobS3A (A549/Tob3SA). Tob3SA specifically binds Smad 1/5/8, which inhibits their activation by the BMP receptors (11, 31). BMP-2 does not induce the phosphorylation of Smad 1/5 or Id-1 in A549/Tob3SA cells even when cultured in medium containing serum (17). We found that BMP-2 induces the expression of cyclin E protein in the A549/Tob3SA cells (Fig. 6B). Consistent with BMP-2 regulating a Smad 1/5–independent pathway, BMP-2 also stimulated the phosphorylation of mTOR in the A549/Tob3SA cells (Fig. 6B). As expected, BMP-2 also induced cyclin E mRNA in the A549/Tob3SA cells (data not shown).



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  		FIGURE 6. BMP-2 regulates cyclin E through a Smad 1/5–independent mechanism. A. A549 cultured in serum-free medium were treated with 10 ng/mL BMP-2 and immunoblots for cyclin E, phosphorylated Smad 1/5 (p-Smad 1/5), and phosphorylated p70S6K were done. B. A549/Tob3SA cells cultured in DMEM/5% FCS were treated with 10 ng/mL BMP-2 and immunoblots for cyclin E and phosphorylated mTOR were done. Experiments were done at least twice.

 
BMP-2 Induces Foci Formation That Is Inhibited by Rapamycin
Forced expression of BMP-2 in A549 (A549/BMP-2) enhances metastatic growth in the lungs of nude mice in comparison with vector controls (17). The activation of the PI3K has been shown to induce transformation as shown by an increase in foci formation (27). We next examined whether BMP-2 enhanced foci formation. The A549/BMP-2 cells (Fig. 7B) formed significantly more foci than that of vector controls (Fig. 7A). To assess whether foci formation in the A549 and H1299 cell lines was mediated through mTOR, the cells were treated with rapamycin. Rapamycin completely inhibited foci formation in A549 cells (Fig. 7C; data not shown). In addition, rapamycin also prevented foci formation in the A549/BMP-2 cells (Fig. 7D). These data suggest that the activation of the PI3K/mTOR signaling pathway is involved in BMP-2-induced transformation.



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  		FIGURE 7. BMP-2-enhanced foci formation is inhibited with rapamycin. Limiting dilution cloning assay was done in A549 cells stably transfected with vector alone (A549/Vector; A) and A549 cells stably transfected with BMP-2 (A549/BMP-2; B). Average of three experiments done in duplicate. Limiting dilution cloning assay of A549/Vector (C) and A549/BMP-2 (D) cells treated with (+) and without (-) rapamycin (1 µg/mL). *, P < 0.05, compared with vector control.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
This study reveals that BMP-2 induces the PI3K/mTOR signaling cascade in lung cancer cell lines. The avian sarcoma virus 16 is a retrovirus that causes hemangiosarcomas in chickens and transforms chicken embryo fibroblasts (30). Avian sarcoma virus 16 encodes v-3pk that is derived from the catalytic subunit of PI3K (30). Activation of the PI3K/mTOR pathway has been shown to promote cell growth, proliferation, cell survival, and motility of human cancer cell lines (29, 32). mTOR activation is essential for PI3K transformation but not for other oncogenes (27). mTOR functions predominantly by regulating components of translation, particularly those requiring binding the ribosome to the mRNA (29). Translational initiating factors guide the mRNA to the ribosome. The 4E-binding proteins form a complex with translational initiating factors that inactivate their activity. Phosphorylation of 4E-binding proteins by mTOR releases it from the complex allowing translation to occur (29). Forced expression of eIF4E or eIF4G is transforming in immortalized rodent cells (33). Ectopic expression of eIF4E transforms immortalized human epithelial cells as shown by an increase in foci formation (34). We also found that BMP-2 phosphorylates p70S6K, which is a direct target of mTOR. Phosphorylation by mTOR activates p70S6K, which then phosphorylates the ribosomal protein S6 (29). p70S6K also regulates translation and is important for cell growth.

mTOR in mammalian cells is regulated by nutrients, energy metabolism, and cytokines (29). Growth factor–induced regulation of mTOR is mediated by PI3K. Phosphatase and tensin homologue is a phosphatidylinositol-3,4-bibisphosphate phosphatase that counteracts PI3K activity by dephosphorylating phosphatidylinositol-3,4-bibisphosphate 2 and 3 (29). Inactivation of phosphatase and tensin homologue enhances PI3K activity. Dysregulation of PI3K/mTOR signaling occurs in ~74% of lung carcinomas (35). Mutations, which constitutively activate PI3K, occur in ~30% of colon cancer but occur only rarely in lung carcinomas (36). Inactivation of phosphatase and tensin homologue is also an infrequent mechanism that activates PI3K in lung cancer (37). The present study suggests that BMP-2 may also be an important regulator of PI3K/mTOR signaling in lung carcinomas.

Forced expression of BMP-2 in A549 cells (A549/BMP-2) significantly enhanced metastatic tumor growth following i.v. injection into nude mice (17). In this study, we showed that A549/BMP-2 cells formed more foci than that of vector controls, consistent with BMP-2-inducing transformation. Rapamycin completely inhibited foci formation of A549/BMP-2, suggesting that mTOR signaling is essential for BMP-2-induced transformation. We show that BMP-2-induced mTOR phosphorylation can occur independent of Smad 1/5 activation. Smad-dependent and Smad-independent signaling may be required for BMP-2-mediated transformation. BMP-2 induction of Id-1 expression in lung cancer cell lines occurs through a Smad 1/5 mechanism (17). The activation of Smad 1/5 signaling seems to be essential for BMP-2 to promote tumor growth in the lungs of nude mice following tail vein injections (17). These studies suggest that BMP-2 mediates transformation through both Smad 1/5–dependent and Smad 1/5–independent mechanisms.

We found in lung cancer cell lines that BMP-2 induces the expression of cyclin E that is mediated through mTOR signaling. Prior reports in other cancer cell lines have shown that PI3K/mTOR signaling increases the translation of cyclin D1 (38). In our cells, BMP-2 did not induce cyclin D1 protein or mRNA. Other studies have also shown that mTOR can preferentially regulate cyclin E. mTOR has been shown to induce cyclin E expression in B-cell chronic lymphocytic leukemia cells (39) and NIH cells (40). Mice livers containing a conditional deletion of the ribosomal protein S6 failed to proliferate or induce cyclin E following partial hepatectomy (41). The livers formed active cyclin D-CDK4 complexes, suggesting that mTOR signaling was specific for cyclin E. We found that rapamycin blocked BMP-2-induced protein expression of cyclin E but not the mRNA. This is consistent with mTOR regulating cyclin E through a translational mechanism. BMP-2 also caused an increase in cyclin E mRNA. This suggests that BMP-2 regulates cyclin E through both PI3K/mTOR signaling pathway and another signaling pathway that increases cyclin E mRNA.

There is increasing evidence that BMP-2 has a role in tumorigenesis. We now show that BMP-2 regulates PI3K/mTOR pathway, which is known to have an important role regulating growth of human carcinomas. This study further shows that BMP-2 uses some of same signaling pathways in cancer as it does in promoting growth during development.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cell Culture
The A549 and H1299 lung cancer cell lines were cultured in DMEM (Sigma Aldrich, St. Louis, MO) with 5% fetal bovine serum containing 1% penicillin/streptomycin and 1% glutamine. A549 cells were transfected with pcDNA3 vector alone or Tob3SA (17). Transfected cells were selected with neomycin.

Antibodies, Proteins, and Reagents
Primary antibodies against phosphorylated mTOR, phosphorylated p70S6K (Upstate Biotechnology, Lake Placid, NY), p21 (Santa Cruz Biotechnology, Santa Cruz, CA), cyclin E (Santa Cruz Biotechnology), cyclin D1 (Santa Cruz Biotechnology), phosphorylated extracellular signal-regulated kinase 1/2 (Promega, Madison, WI), and actin (Sigma, St. Louis, MO) were used for Western blot analysis. Recombinant human BMP-2 was purchased from R&D Systems (Minneapolis, MN). To inhibit PI3K, LY-294002 (Calbiochem, La Jolla, CA) and wortmannin (Sigma, St. Louis, MO) were used. Rapamycin (Calbiochem) was used to inhibit mTOR and PD-98059 (Upstate Biotechnology) to inhibit MEK. SB-203580 (Calbiochem) was used to inhibit p38 signaling.

Western Blot Analysis
Total cellular protein was obtained using RIPA buffer, analyzed by SDS-PAGE, and transferred to nitrocellulose (Schleicher & Schuell, Keene, NH) at 35 V for 16 hours at 4°C. The blots were then incubated overnight at 4°C with the appropriate primary antibody. Specific proteins were detected using the enhanced chemiluminescence system (Amersham, Arlington Heights, IL).

Short Interfering RNA
Synthetic short interfering RNA and transfection kit (siPORT) were purchased from Ambion (Austin, TX). Cells (35,000) were transfected with 50 µmol/L short interfering RNA using siPORT according to the manufacturer's instructions. Scrambled short interfering RNA was used as a control. RNA was collected 48 hours after transfection.

RNA Extraction and Semiquantitative PCR
RNA was extracted using RNeasy kit as per manufacturer's instructions (Qiagen, Valencia, CA). cDNA was made using Advantage RT for PCR kit (BD Biosciences Clontech, Palo Alto, CA) and then treated with DNase. Real-time PCR was done using the LightCycler (Roche). SYBR Green was used to detect double-stranded DNA. The HotStart Taq Polymerase kit (Qiagen) was used for the PCR mix with 20 µmol/L primers. Samples were denatured at 95°C for 15 seconds, annealing for 20 seconds, and extension at 72°C for 20 seconds for 35 cycles. DNA contamination was tested by PCR of reverse transcription samples. Negative control includes reagents, except cDNA. Expression was normalized to actin using the formula: 2{Delta}CT. Primers for cyclin E are 5'-CCTTATGCGAAACAACTGGAA-3' (forward) and 5'-CCTGGCTCTCTGAAGACCTTT-3' (reverse).

Foci Formation
Five hundred cells were plated into six-well plates in DMEM/5% FCS for 2 weeks. The cells were then stained with Diff-Quick (Imeb, Inc., San Marcos, CA). The total number of colonies formed were counted. The experiments were done in duplicate at least three times.

Statistical Analysis
To evaluate multiple groups, a one-way ANOVA followed by Fisher least significant difference post hoc test was used to compare individual means. To compare two groups, a Student's t test was used. Differences with Ps < 0.05 were considered statistically significant.


   Notes
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 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Grant support: NIH K22 grant CA91919-01A1 (J. Langenfeld) and Thoracic Surgery Foundation.

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 8/ 4/05; revised 10/29/05; accepted 11/21/05.


   References
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 Introduction
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 Discussion
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 References
 

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