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Carcinogenesis Advance Access originally published online on February 6, 2008
Carcinogenesis 2008 29(4):704-712; doi:10.1093/carcin/bgn031
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Macrophage inhibitory cytokine-1 activates AKT and ERK-1/2 via the transactivation of ErbB2 in human breast and gastric cancer cells

Kwang-Kyu Kim1,3, Jung Joon Lee1, Young Yang2, Kwan-Hee You3 and Jeong-Hyung Lee4,*

1 Molecular Cancer Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea
2 Department of Life Science, Sookmyung Women's University, Seoul, Republic of Korea
3 Department of Biology, Chungnam National University, Daejeon, Republic of Korea
4 Department of Biochemistry and Research Institute of Life Science, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea

* To whom correspondence should be addressed. Tel: +82 33 250 8519; Fax: +82 33 242 0459; Email: jhlee36{at}kangwon.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Macrophage inhibitory cytokine-1 (MIC-1) is a member of the transforming growth factor-β superfamily, which is overexpressed in a variety of human cancers, including breast and gastric cancer. The function of MIC-1 in cancer remains controversial and its signaling pathways remain poorly understood. In this study, we demonstrate that MIC-1 induces the transactivation of ErbB2 in SK-BR-3 breast and SNU-216 gastric cancer cells. MIC-1 induced a significant phosphorylation of Akt and ERK-1/2, and also effected an increase in the levels of tyrosine phosphorylation of ErbB1, ErbB2 and ErbB3 in SK-BR-3 and SNU-216 cells. The treatment of these cells with AG825 and AG1478, inhibitors specific for ErbB2 tyrosine kinase, resulted in the complete abolition of MIC-1-induced Akt and ERK-1/2 phosphorylation. Furthermore, the small-interfering RNA-mediated downregulation of ErbB2 significantly reduced not only the phosphorylation of Akt and ERK-1/2 but also the invasiveness of the cells induced by MIC-1. Our results show that ErbB2 activation performs a crucial function in MIC-1-induced signaling pathways. Further investigations revealed that MIC-1 induced the expression of the hypoxia inducible factor-1{alpha} protein and the expression of its target genes, including vascular endothelial growth factor, via the activation of the mammalian target of rapamycin (mTOR) signaling pathway. Stimulation of SK-BR-3 with MIC-1 profoundly induces the phosphorylation of mTOR and its downstream substrates, including p70S6K and 4E-BP1. Collectively, these results show that MIC-1 may participate in the malignant progression of certain human cancer cells that overexpress ErbB2 through the transactivation of ErbB2 tyrosine kinase.

Abbreviations: EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIF-1{alpha}, hypoxia inducible factor-1{alpha}; MIC-1, macrophage inhibitory cytokine-1; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol-3 kinase; rMIC-1, recombinant MIC-1; siRNA, small-interfering RNA; TGF-β, transforming growth factor-β; VEGF, vascular endothelial growth factor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Macrophage inhibitory cytokine-1 (MIC-1), which is identical to placental transforming growth factor-β, placental bone morphogenic protein, growth differentiation factor-15, prostate-derived factor or non-steroidal anti-inflammatory drug-activated gene-1, is a distant member of the transforming growth factor-β (TGF-β) superfamily (1,2). MIC-1 is synthesized as a 308-amino acid polypeptide, and the mature protein is secreted as a disulfide-linked homodimer comprising two 112-amino acid mature regions (3). MIC-1 is expressed quite widely, but under resting conditions, the placenta is the only tissue that expresses large quantities of MIC-1 (2). The epithelial cells express lower quantities of MIC-1 mRNA. MIC-1 expression is, however, dramatically increased in cases of inflammation, injury and malignancy (4). The principal function, the receptor and the signaling pathway of MIC-1 remain uncertain, although several of its biological activities have already been described (3,5–7). The role of MIC-1 in cancer also remains poorly understood. Increased MIC-1 expression is a common feature of many cancers, including cancers of the breast, colon, pancreas and prostate. Several studies have observed a major upregulation of MIC-1 mRNA and protein in cancer biopsies (8–10). High tumor expression is also associated with an increase in serum MIC-1 levels, which suggests that serum MIC-1 measurements might be utilized in the diagnosis and management of cancer (10–12). Serum MIC-1 levels are often markedly elevated in cases of metastatic cancer and appear to occur in parallel with the stage and extent of disease, particularly in cases of colorectal cancer (11). Paradoxically, a number of studies have described an antitumorigenic function for MIC-1, by which it induces apoptosis and inhibits the proliferation of several tumor cell lines (13–16). The overexpression of MIC-1 in HCT-116 colon and MDA-MB-468 breast carcinoma lines resulted in a reduction in cell viability, and nude mice xenograft models of the HCT-116 transfectants have been associated with a significant reduction in tumor size (13,16). These findings indicate that MIC-1 may negatively affect tumor growth. However, we have shown previously that MIC-1 can induce invasiveness of human gastric cancer cells via the activation of ERK-1/2 (17).

The family of ErbB receptor tyrosine kinases includes four members: epidermal growth factor receptor (EGFR)/ErbB1, ErbB2/Neu/HER2, ErbB3 and ErbB4. The binding of peptides of the EGF-related growth factor family to the extracellular domain of the ErbB receptors causes the formation of homo- and heterodimers. Ligand binding induces intrinsic receptor kinase activity, ultimately resulting in the stimulation of intracellular signaling cascades (18,19). An increasing body of evidence indicates that the primary function of ErbB2 is that of a co-receptor (20). Amplification or overexpression of the ErbB2 gene has been detected in many cancers, including human breast and gastric cancers, and is associated with increased tumor grade and shorter overall survival rates (21,22). ErbB2 activation is associated with an increase in the activity of receptor tyrosine kinase and tyrosine phosphorylation of the receptor (23,24). The transactivation of ErbB2 by prolactin, interleukin-6, CXCL-12 and lysophosphatidic acid has been identified as a ligand-independent mechanism in a variety of cancer cell types (25–28). Activated ErbB2 phosphorylates a host of downstream molecules, which in turn activate a variety of signaling cascades, including the phosphatidylinositol-3 kinase (PI3K)/Akt and RAS/mitogen-activated protein kinase cascade (19). For example, activated ErbB2 induces the hypoxia-inducible factor (HIF), a key transcription factor which activates the transcription of genes that regulate angiogenesis, cell survival, glucose metabolism and invasion (29) via the PI3K/Akt pathway (30) and vascular endothelial growth factor (VEGF) protein synthesis via signaling events involving ERK-1/2, PI3K/Akt, mammalian target of rapamycin (mTOR) and p70S6 kinase (31).

As described herein, we report that MIC-1 induced the transactivation of ErbB2 tyrosine kinase in human breast and gastric cancer cells, and this activation stimulated HIF-1{alpha} protein accumulation and the expression of its target gene via the PI3K/Akt/mTOR and ERK-1/2 signaling pathways. These novel observations provide additional support for the notion that MIC-1 may operate as a positive regulator of tumor progression in certain ErbB2-overexpressing tumors, including breast and gastric cancers.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cells and reagents
Human gastric cancer cell lines, AGS, MKN-28, MKN-45, SNU-1, SNU-5, SNU-216 and SNU-638, and breast cancer cell lines, MDA-MB-435, SK-BR-3 and MDA-MD-231 cells, were cultured as described previously (17,32). All cell lines were maintained in a humidified 5% CO2 atmosphere at 37°C. For hypoxic exposures, cells were placed in a hypoxia incubator (Thermo Electron Corp., Marietta, OH) maintained with 1% O2/5% CO2/balance N2 at 37°C. Specific antibodies to phospho-ERK-1/2 (Thr202/Tyr204), ERK-1/2, phospho-Akt (Ser473 and Thr308), Akt, phospho-mTOR (Ser2448), mTOR, phospho-4E-BP1 (Thr37/46), 4E-BP1, phospho-p70S6K (Thr389) and p70S6K were purchased from Cell Signaling Technology (Danvers, MA). EGFR (1005), ErbB2 (C-18), ErbB3 (C-17), phosphotyrosine (PY20) and topoisomerase-I (TOPO-I; C-15) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Human recombinant MIC-1 (rMIC-1) and antibody against HIF-1{alpha} were purchased from R&D Systems Inc. (Minneapolis, MN). AG825, AG1478, genistein, herbimycin A, wortmannin, rapamycin, PD98059, Gö6976, Akt inhibitor and bisindolylmaleimide-I were purchased from Calbiochem (San Diego, CA).

Western blot analysis
Cells were lysed with a buffer [50 mM Tris–HCl (pH 7.5), 1% Nonidet P-40, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 10 µg/ml aprotinin, 2 mM benzamidine, 50 mM NaF, 5 mM sodium orthovanadate and 150 mM NaCl]. To measure the expression level of HIF-1{alpha} protein, nuclear extracts were prepared using a commercial kit purchased from Pierce (Rockford, IL), according to the manufacturer's instructions. Proteins were separated onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a Hybond-P membrane (Amersham Biosciences, Buckinghamshire, UK). The membranes were blocked with 5% skim milk at room temperature for 2 h, and then incubated for 2 h with primary antibodies. After washing, the membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase. The signal was detected using the enhanced chemiluminescence system (Intron, Seongnam, Korea).

Immunoprecipitation
Cells that had been serum-starved for 12 h were stimulated in the presence or absence of 20 ng/ml of rMIC-1 for 5 min at 37°C. After stimulation, cells were extracted with a lysis buffer [50 mM Tris–HCl (pH 7.4), 1% Triton X-100, 5 mM EDTA, 300 mM NaCl, 0.02% sodium azide, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 5 mM sodium orthovanadate, 10 µg/ml pepstatin A, 10 µg/ml aprotinin and 2 mM benzamidine] on ice for 20 min. After centrifugation, supernatants were collected, and then were preabsorbed for 2 h with protein A/G agarose beads (Santa Cruz). After centrifugation at 2500 rpm for 5 min, the supernatants were incubated with anti-ErbB1, anti-ErbB2 or anti-ErbB3 antibody for 16 h at 4°C, followed by incubation with protein A/G agarose beads for 1 h at 4°C. The immunoprecipitates were washed three times with the lysis buffer, and then separated onto SDS-PAGE. The level of phosphorylated tyrosine was detected using phosphotyrosine antibody by western blotting.

Reverse transcription–polymerase chain reaction assays
Total RNA was extracted using the RNeasy Mini kit purchased from Qiagen (Valencia, CA), according to the manufacturer's instructions. Five micrograms of total RNA were transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) and aliquots of cDNA were used as template for polymerase chain reaction amplification of HIF-1{alpha}, VEGF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The following oligonucleotides were used as primers: (i) 5'-CTCAAAGTCGGACAGCCTCA-3' (sense) and 5'-CCCTGCAGTAGGTTTCTGCT-3' (antisense) (HIF-1{alpha}); (ii) 5'-GCTCTACCTCCACCATGCCAA-3' (sense) and 5'-TGGAAGATGTCCACCAGGGT C-3' (antisense) (VEGF); (iii) 5'-ACCACAGTCCATGCCATCAC-3' (sense) and (iv) 5'-TCCACCACCCTGTTGCTGTA-3' (antisense) (GAPDH). Thermocycling conditions for both primer sets were as follows: denaturation at 94°C for 5 min followed by 25 (HIF-1{alpha}), 27 (VEGF) and 21 (GAPDH) cycles, each consisting of 94°C for 30 s, 58°C for 30 s and 72°C for 1 min, and a final extension step at 72°C for 7 min. Polymerase chain reaction products were separated on 1.5% agarose gel and visualized by staining with ethidium bromide.

RNA interference experiments
The small-interfering RNAs (siRNAs) against human EGFR, ErbB2, ErbB3 and a non-specific siRNA control were purchased from Santa Cruz Biotechnology. SK-BR-3 cells were cultured to 60% confluency, and then transfected with the siRNA duplex using Lipofectamine plus reagent (Invitrogen), according to the manufacturer's recommended protocol. After 48 h of transfection, the cells were stimulated with rMIC-1 for 5 min and then were harvested to perform western blotting.

Invasion assays
The ability of cells to invade through Matrigel-coated filters was determined using a modified 24-well Boyden chamber (Corning Costar, Cambridge, MA; 8 µm pore size) as described previously (17,32). Briefly, SK-BR-3 cells transfected with ErbB2 siRNA or control siRNA were seeded at a density of 5 x 104 cells in 100 µl RPMI 1640 containing 10% fetal bovine serum in the upper compartment of transwell, and then exposed to 20 ng/ml of rMIC-1 for 24 h. To determine the effect of wortmannin, PD98059 and Akt inhibitor, SK-BR-3 cells seeded in the upper compartment were treated with the inhibitors for 30 min prior to the stimulation with rMIC-1. The cells that had not penetrated the filter were completely wiped out with a cotton swabs, and the cells that had migrated to the lower surface of the filter were fixed with methanol. Then the cells were stained and counted in five randomly selected microscopic fields (x100) per filter.

Statistical analysis
Statistical significance was determined using Student t-tests and analysis of variance. Results were considered to be significant at P < 0.05. Correlation of ErbB2 expression with MIC-1-induced activation of ERK-1/2 and Akt was analyzed with the non-parametric Spearman rank order correlation (Spearman R).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
MIC-1 activates the Akt and ERK-1/2 in some human gastric and breast cancer cells
We have previously demonstrated that MIC-1 induces the phosphorylation of ERK-1/2 and MEK1/2, an upstream kinase of ERK-1/2, in certain human gastric cancer cell lines, including SNU-216 cells (17). In order to further investigate the MIC-1 signaling pathway, we initially assessed the effects of MIC-1 on ERK-1/2 and Akt phosphorylation in a variety of human gastric cancer cell lines, AGS, MKN-28, MKN-45, SNU-1, SNU-5, SNU-216 and SNU-638, and breast cancer cell lines, MDA-MB-435, SK-BR-3 and MDA-MB-231. The cells were treated with 20 ng/ml of rMIC-1 for the indicated times, after which the activation of Akt and ERK-1/2 was determined via western blot analysis. The representative results are provided in Figure 1. Of the tested gastric cancer cell lines, SNU-216 cells evidenced the most profound response to MIC-1 with regard to Akt and ERK-1/2 activation (Figures 1A and 3B). The gastric cancer cells other than the SNU-1 cells, for example MKN-45 cells, evidenced weak Akt and ERK-1/2 phosphorylation in response to rMIC-1 (Figure 1A). SNU-1 cells exhibited a barely detectable level of MIC-1-induced Akt and ERK-1/2 phosphorylation. Of the tested breast cancer cells, the treatment of SK-BR-3 cells with rMIC-1 resulted in the profound activation of both ERK-1/2 and Akt (Figures 1B and 3B), and this activation occurred in a dose-dependent manner (Figure 1C). On the other hand, MDA-MB-231 and MDA-MB-435 human breast cancer cells barely responded to MIC-1 treatment (Figure 1B).


Figure 1
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  		Fig. 1. MIC-1-induced activation of Akt and ERK-1/2 in human gastric and breast cancer cell lines. Western blot of cell lysates from human gastric cancer MKN-45 and SNU-216 cells (A), and human breast cancer MDA-MB-231 and SK-BR-3 cells (B). The cells were stimulated with 20 ng/ml of rMIC-1 for indicated periods of time in serum-free medium. Phosphorylation of ERK-1/2 (left panel) and Akt (right panel) was determined by western blot. Representative autoradiographic exposures are shown. The bottom of each panel represents the densitometric quantification of the MIC-1-induced phosphorylation of ERK-1/2 and Akt from three different experiments. (C) Western blot of cell lysates from SK-BR-3 cells stimulated with various concentrations of rMIC-1. The cells were stimulated with the indicated concentrations of rMIC-1 for 5 min in serum-free medium. Phosphorylation of ERK-1/2 (left panel) and Akt (right panel) was determined by western blot. Representative autoradiographic exposures are shown. The bottom of each panel represents the densitometric quantification of the MIC-1-induced phosphorylation of ERK-1/2 and Akt from three different experiments.

 
EGFR family tyrosine kinase and PI3K inhibitors block MIC-1-induced ERK-1/2 and Akt activation
In order to study in greater detail the signaling pathways induced by MIC-1, we assessed the effects of a variety of pharmacological inhibitors on the MIC-1-induced phosphorylation of Akt and ERK-1/2 in SNU-216 and SK-BR-3 cells (Figure 2). The SNU-216 cells were pretreated with a variety of inhibitors for 30 min prior to MIC-1 treatment. These include tyrosine kinase inhibitors (genistein and herbimycin A), protein kinase C inhibitors (Gö6976 and bisindolylmaleimide-I), a specific PI3K inhibitor (wortmannin) and a specific MEK1/2 inhibitor (PD98059). MIC-1-induced ERK-1/2 activation was significantly abolished by the PI3K inhibitor, wortmannin and the tyrosine kinase inhibitor, genistein. However, herbimycin A, Gö6976 and bisindolylmaleimide-I did not affect MIC-1-induced ERK-1/2 activation (Figure 2A). Similarly, MIC-1-induced Akt phosphorylation was inhibited significantly by wortmannin and genistein (Figure 2B). Genistein and wortmannin also significantly suppressed MIC-1 induced Akt and ERK-1/2 activation in SK-BR-3 cells (Figure 2C).


Figure 2
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  		Fig. 2. Effect of various kinase inhibitors on the activation of ERK-1/2 and Akt induced by MIC-1. (A) SNU-216 cells were pretreated with PD98059 (PD, 10 µM), bisindolylmaleimide-I (BIM, 1 µM), Gö6976 (1 µM), genistein (10 µM), herbimycin A (1 µM), wortmannin (500 nM) or vehicle (dimethylsulfoxide) for 1 h, and then stimulated with 20 ng/ml of rMIC-1 for indicated periods of time. The bottom of each panel represents the densitometric quantification of the MIC-1-induced phosphorylation of ERK-1/2 from three different experiments. (B) SNU-216 cells were pretreated with genistein (10 µM), herbimycin A (1 µM), wortmannin (500 nM) or vehicle (dimethylsulfoxide) for 1 h, and then stimulated with 20 ng/ml of rMIC-1 for indicated periods of time. The bottom of each panel represents the densitometric quantification of the MIC-1-induced phosphorylation of Akt from three different experiments. (C) SK-BR-3 cells were pretreated with genistein (10 µM), wortmannin (500 nM) or vehicle (dimethylsulfoxide) for 1 h, and then stimulated with 20 ng/ml of rMIC-1 for 5 min. (D) SK-BR-3 cells were pretreated with the indicated concentrations of AG1478 and AG825 for 1 h, and then stimulated with 20 ng/ml of rMIC-1 for 5 min. In these experiments, whole-cell lysates were used to determine the levels of ERK-1/2, phosphorylated ERK-1/2, Akt and phosphorylated Akt by the western blot analysis.

 
In order to examine whether the EGER family tyrosine kinases involve MIC-1-induced signaling pathways, we employed EGFR family tyrosine kinase inhibitors, AG1478 and AG825 (Figure 2D). It has been determined that AG1478 inhibits the EGFR and ErbB2 kinases (33), and AG825 is an ErbB2-selective tyrosine kinase inhibitor (34). AG1478 and AG825 completely abolished MIC-1-induced Akt and ERK-1/2 activation. Collectively, these results show that ErbB2 tyrosine kinase may be involved in MIC-1-induced Akt and ERK-1/2 activation in these cancer cells.

MIC-1 induces the phosphorylation of EGFR family tyrosine kinase
As the MIC-1-induced activation of Akt and ERK-1/2 was inhibited by ErbB2 tyrosine kinase inhibitors, we attempted to determine whether ErbB2 is involved in MIC-1-induced Akt and ERK-1/2 activation. We initially determined the expression levels of EGFR, ErbB2 and ErbB3, which have been known to play important roles in tumor development and progression of many types of human cancers, in these human gastric and breast cancer cell lines (Figure 3A). EGFR, ErbB2 and ErbB3 were variably expressed. We compared the responsiveness of these cell lines to MIC-1 with regard to ERK-1/2 and AKT activation with ErbB2 protein expression (Figure 3B). We did a non-parametric Spearman rank order correlation analysis. Spearman correlation coefficient, R, for MIC-1-induced activation of ERK-1/2 and Akt was 0.5667 (P = 0.1206) and 0.7875 (P = 0.01722), respectively. Interestingly, two cell lines, SNU-216 and SK-BR-3 cells, which evidenced profound Akt and ERK-1/2 activation in response to MIC-1 treatment, expressed ErbB2 at a higher level than was observed with other cell lines.


Figure 3
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  		Fig. 3. MIC-1 is able to stimulate tyrosine phosphorylation of ErbB2. (A) Western blot analysis of EGFR, ErbB2 and ErbB3 expression in various human gastric and breast cancer cell lines. (B) Non-parametric Spearman rank order correlation analysis of ErbB2 protein expression with MIC-1-induced activation of ERK-1/2 (left) and Akt (right). The Spearman correlation coefficient R is indicated in the graph. (C and D) Tyrosine phosphorylation of EGFR, ErbB2 and ErbB3 by MIC-1. SNU-216 (C) and SK-BR-3 (D) cells were serum-starved for 12 h, and then stimulated with 20 ng/ml of rMIC-1 for 5 min. Each of the three ErbBs was immunoprecipitated (IP) from cell lysates, and the immunoprecipitates were immunoblotted with an anti-phosphotyrosine antibody. The same membranes were stripped and reblotted with EGFR, ErbB2 or ErbB3 antibody. (E) Effect of AG825 on MIC-1-induced phosphorylation of ErbB2 and ErbB3. Serum-starved SK-BR-3 cells were treated with AG825 (50 µM) or vehicle control (dimethylsulfoxide) for 1 h, and then stimulated with 20 ng/ml of rMIC-1 for 5 min. ErbB2 and ErbB3 were immunoprecipitated (IP) from cell lysates, and the immunoprecipitates were immunoblotted with an anti-phosphotyrosine antibody. The same membranes were stripped and reblotted with ErbB2, or ErbB3 antibody.

 
We then attempted to determine whether MIC-1 induced the phosphorylation of EGFR family members in SNU-216 and SK-BR-3 cells (Figure 3C and D). The cells were treated for 5 min with 20 ng/ml of rMIC-1, and then the levels of phosphotyrosine content of EGFR, ErbB2 and ErbB3 were determined via immunoprecipitation-coupled western blot analysis. MIC-1 significantly induced the tyrosine phosphorylation of EGFR, ErbB2 and ErbB3 in both cell lines. Furthermore, AG825 completely abolished MIC-1-induced tyrosine phosphorylation of ErbB2 and ErbB3 (Figure 3E).

In order to verify the involvement of ErbB2 in the MIC-1-induced activation of Akt and ERK-1/2, we attempted to determine whether the siRNA-mediated downregulation of EGFR, ErbB2 and ErbB3 affected MIC-1-induced Akt and ERK-1/2 activation in SK-BR-3 cells (Figure 4A). SK-BR-3 cells were transfected with siRNA against EGFR, ErbB2 or ErbB3, after which the level of MIC-1-induced Akt and ERK-1/2 phosphorylation was analyzed via western blot analysis. The transfection of EGFR siRNA did not significantly suppress the MIC-1-induced activation of ERK-1/2 and Akt. The transfection of ErbB3 siRNA significantly suppressed the MIC-1-induced Akt activation, but not MIC-1-induced ERK-1/2 activation. Consistent with the results of a study conducted with AG1478 and AG825, transfection with ErbB2 siRNA resulted in a significant suppression of the phosphorylation of both Akt and ERK-1/2. As we previously reported that MIC-1 induced the invasiveness of SNU-216 gastric cancer cells via ERK-1/2 activation (17), we attempted to determine whether the siRNA-mediated downregulation of ErbB2 affects the MIC-1-induced invasiveness of SK-BR-3 cells. As had been expected, the siRNA-mediated downregulation of ErbB2 in SK-BR-3 cells induced a significant abrogation of MIC-1-induced invasiveness of those cells (Figure 4B). Furthermore, the MIC-1-induced invasiveness of SK-BR-3 cells was significantly inhibited by wortmannin, PD98059 and Akt inhibitor (Figure 4C). Collectively, these results indicate that MIC-1 could induce Akt and ERK-1/2 activation via ErbB2 phosphorylation, which may perform a critical function in MIC-1-induced Akt and ERK-1/2 activation.


Figure 4
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  		Fig. 4. Effect of siRNA-mediated downregulation of EGFR, ErbB2 or ErbB3 on MIC-1-induced phosphorylation of Akt and ERK-1/2. (A) Suppression of ErbB2 expression inhibits MIC-1-induced activation of Akt and ERK-1/2. Control (Co), EGFR (E1), ErbB2 (E2) or ErbB3 (E3)-targeted siRNA was transfected into SK-BR-3 cells. Cells transfected with each siRNA were serum-starved for 12 h and then stimulated with 20 ng/ml of rMIC-1 for 5 min. The levels of Akt, phosphorylated Akt, ERK-1/2, phosphorylated ERK-1/2, EGFR, ErbB2 and ErbB3 were determined by the western blot analysis. (B) Top, suppression of ErbB2 expression by ErbB2 siRNA inhibits MIC-1-induced invasiveness. SK-BR-3 cells transfected with control siRNA (Co-siRNA) or ErbB2-targeted siRNA (E2) were plated in the upper chamber of culture well inserts, and then treated with the 20 ng/ml of rMIC-1. Cells that migrate through the pores in the filter were fixed, stained and counted in five random fields visualized by microscopy (x100). Data represent average of three independent experiments performed in triplicate; bars, SD of triplicate samples from three independent experiments; *, statistically significant (P < 0.001, Student's t-test). Bottom, the same siRNA transfected cells as (B) were stimulated with 20 ng/ml of rMIC-1 for 5 min, and the levels of Akt, phosphorylated Akt, ERK-1/2, phosphorylated ERK-1/2 and ErbB2 were determined by the western blot analysis. (C) Inhibitory effect of wortmannin, Akt inhibitor and PD98059 on the invasiveness of SK-BR-3 cells induced by MIC-1. SK-BR-3 cells were plated in the upper chamber, and then pretreated with wortmannin (Wort, 500 nM), Akt inhibitor (Akt, 10 µM), PD98059 (PD, 10 µM) or vehicle (dimethylsulfoxide) prior to the stimulation with 20 ng/ml of rMIC-1. Data represent average of three independent experiments performed in triplicate; bars, SD of triplicate samples from three independent experiments; *, statistically significant (P < 0.001, Student's t-test).

 
MIC-1 induces HIF-1{alpha} expression
The PI3K/Akt/mTOR signaling pathway has been demonstrated to regulate the translation of proteins, such as HIF-1{alpha}, via the phosphorylation of p70S6K and 4E-BP1 (35,36), and the ERK-1/2 signaling pathway has been shown to regulate protein translation via mTOR (37). In order to further verify that MIC-1 activates Akt and ERK-1/2, we attempted to determine whether MIC-1 could activate mTOR, a downstream target of Akt. SK-BR-3 cells were treated with 20 ng/ml of rMIC-1 for the indicated times, and the level of mTOR phosphorylation was then determined via western blot (Figure 5). mTOR phosphorylation was determined to be induced at 2 min and remained until 30 min after MIC-1 stimulation (Figure 5A). This activation occurred in a dose-dependent manner (Figure 5B). In order to confirm the MIC-1-induced activation of mTOR, we evaluated the MIC-1-induced phosphorylation of p70S6K and 4E-BP1 via western blot analysis (Figure 5A). The levels of p70S6K and 4E-BP1 phosphorylation increased significantly in response to MIC-1 treatment. Next, we determined whether MIC-1-induced phosphorylation of mTOR was inhibited by AG1478, AG825, wortmannin or PD98059 in SK-BR-3 cells (Figure 5C). These inhibitors significantly suppressed MIC-1-induced phosphorylation of mTOR, suggesting that both PI3K and ERK-1/2 pathways could play a critical role in MIC-1-induced mTOR activation. Interestingly, the siRNA-mediated downregulation of ErbB3 induced a significant abrogation of MIC-1-induced phosphorylation of mTOR (Figure 5D).


Figure 5
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  		Fig. 5. Effect of MIC-1 on the activation of Akt/mTOR. (A) Western blot of cell lysates from SK-BR-3 cells stimulated with 20 ng/ml of rMIC-1 for indicated periods of time in serum-free medium. (B) Western blot of cell lysates from SK-BR-3 cells stimulated with indicated concentrations of rMIC-1 for 5 min in serum-free medium. (C) Inhibitory effect of AG1478, AG825, wortmannin, PD98059 and rapamycin on MIC-1-induced phosphorylation of mTOR. SK-BR-3cells were pretreated with AG1478 (1 µM), AG825 (50 µM), wortmannin (500 nM), rapamycin (10 nM), PD98059 (10 µM) or vehicle (dimethylsulfoxide) for 1 h, and then stimulated with 20 ng/ml of rMIC-1 for 5 min. The levels of phosphorylated mTOR were determined by the western blot analysis. (D) Suppression of ErbB3 expression inhibits MIC-1-induced activation of mTOR. Control (Co) or ErbB3 (E3)-targeted siRNA was transfected into SK-BR-3 cells. Cells transfected with each siRNA were serum-starved for 12 h and then stimulated with 20 ng/ml of rMIC-1 for 5 min. The levels of Akt, phosphorylated Akt, mTOR, phosphorylated mTOR, ErbB2 and ErbB3 were determined by the western blot analysis.

 
Next, we assessed the effects of MIC-1 on HIF-1{alpha} accumulation in the SK-BR-3 cells. A significant accumulation of HIF-1{alpha} protein was also detected 2 h after MIC-1 stimulation, with no concomitant effects on the HIF-1{alpha} mRNA expression level under normoxic conditions (Figure 6A). Furthermore, MIC-1 treatment significantly increased HIF-1{alpha} protein expression under both normoxic and hypoxic conditions, in a dose-dependent manner (Figure 6B and C). In order to further confirm the effect of MIC-1 on HIF-1{alpha} expression, we attempted to determine whether MIC-1 effected an increase in the expression of HIF-1 target genes, such as VEGF, in SK-BR-3 cells. Accordingly, SK-BR-3 cells were treated with 20 ng/ml of rMIC-1 for the indicated times under normoxic conditions, and the level of VEGF mRNA was then determined via reverse transcription–polymerase chain reaction. The level of VEGF was increased by MIC-1 treatment in a time- and dose-dependent manner (Figure 6D). Collectively, these results indicate that MIC-1 could increase HIF-1{alpha} protein accumulation under both normoxic and hypoxic conditions in ErbB2-overexpressing cancer cells such as SK-BR-3 cells.


Figure 6
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  		Fig. 6. Effect of MIC-1 on the activation of HIF-1{alpha}. (A) SK-BR-3 cells were serum-starved for 12 h and then treated with 20 ng/ml of rMIC-1 for the indicated period of time, and then total RNAs and nuclear extracts were prepared. The levels of HIF-{alpha} protein expression were determined by the western blot analysis. The same membrane was stripped and reblotted with a TOPO-1 antibody as a loading control. The expression levels of HIF-{alpha} and GAPDH mRNA were determined by the reverse transcription–polymerase chain reaction analysis. (B and C) SK-BR-3 cells were serum-starved for 12 h and then stimulated for 4 h with various concentrations of rMIC-1 in normoxic (B) or hypoxic (1% O2, C) condition. The levels of HIF-{alpha} expression were determined by the western blot analysis. (D) MIC-1 induces expression level of VEGF mRNA in a time- and dose-dependent manner. Top, serum-starved SK-BR-3 cells were treated with 20 ng/ml of rMIC-1 for the indicated period of time. The expression levels of VEGF mRNA were determined by the reverse transcription–polymerase chain reaction analysis. Bottom, serum-starved SK-BR-3 cells were treated with indicated concentrations of rMIC-1 or exposed to hypoxic condition (1% O2) for 12 h. The expression levels of VEGF mRNA were determined by the reverse transcription–polymerase chain reaction analysis. The bottom of each panel represents the densitometric quantification of the MIC-1-induced VEGF mRNA expression from three different experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
MIC-1 is a divergent member of the TGF-β superfamily, and its principal function remains to be precisely determined. Although there is a strong correlation between MIC-1 expression and epithelial tumors, less is currently known regarding its role and the manner in which it exerts its effects. The majority of studies conducted thus far suggest an antitumorigenic role for MIC-1. A number of reports have indicated that MIC-1 may play a role in apoptotic induction via both p53-dependent and p53-independent mechanisms. MIC-1 expression can be stimulated in cancer cell lines by the application of a variety of antitumorigenic agents, including the non-steroidal anti-inflammatory drugs, peroxisome proliferator-activated receptor-{gamma} ligands and genistein (reviewed in ref. 4). The roles for MIC-1 in antitumorigenic mechanisms have been assessed in a variety of studies linking the induction of MIC-1 expression to reduced cell proliferation and altered cell survival rates (38,39). Significant increases have been noted in serum levels of MIC-1 with tumor progression to metastatic disease in several epithelial cancers, including colon, prostate and pancreatic cancer (reviewed in ref. 4). We reported previously that elevated MIC-1 expression in gastric cancer cell lines was associated with a more invasive phenotype, and that MIC-1 induced the expression of the urokinase-type plasminogen activator and its receptor via ERK-1/2 activation (17).

In this study, we have uncovered evidence for a role of MIC-1 that underlies the induction of tumor progression on the basis of the activation of Akt, ERK-1/2 and the HIF-1{alpha} transcription factor. (i) MIC-1 profoundly induces Akt and ERK-1/2 phosphorylation via the transactivation of EGFR family tyrosine kinases in ErbB2-overexpressing cells, including SNU-216 and SK-BR-3 cells, and this induction is inhibited effectively by ErbB2 tyrosine kinase inhibitors including AG1478 and AG825. (ii) MIC-1 induces the phosphorylation of EGFR family receptors, and the siRNA-mediated downregulation of ErbB2 results in a significant suppression of Akt and ERK-1/2 activation, as well as MIC-1-induced invasiveness. (iii) MIC-1 activates the expression of HIF-1{alpha} protein and the expression of its target gene via the activation of mTOR signaling pathways. Collectively, these data provide the first known evidence that the signaling pathways of MIC-1 operate as a mediator of tumor progression in human gastric and breast cancers.

Not a great deal is currently known with regard to the MIC-1-induced signaling pathways, and the receptor for MIC-1 remains to be identified. Previous reports have suggested that Akt and ERK-1/2 activation may be involved in MIC-1-induced signaling pathways in several cell lines (7,17,40). For example, MIC-1 protects cultured cardiomyocytes against ischemia/reperfusion-induced apoptosis via the activation of PI3K/Akt pathways (7). Also, MIC-1 increases basal ERK-1/2 phosphorylation and prolongs estrogen-induced ERK-1 phosphorylation in MCF-7 cells (40). The results of our studies clearly indicated that MIC-1 rapidly induces ERK-1/2 and Akt phosphorylation, which in turn activates mTOR signaling pathway, in SK-BR-3 cells and in SNU-216 cells. This activation was abolished to a significant degree by treatment with ErbB2 tyrosine kinase inhibitors including AG1478 and AG825 or by the siRNA-mediated downregulation of ERbB2. We were also able to demonstrate the MIC-1-induced tyrosine phosphorylation of EGFR, ErbB2 and ErbB3 in SK-BR-3 or SNU-216 cells, which overexpress ErbB2. Importantly, the inhibition of ErbB2 via the application of an ErbB2-specific siRNA or specific ErbB2 tyrosine kinase inhibitors was shown to significantly suppress the MIC-1-induced activation of both Akt and ERK-1/2.

All ErbB receptors are coupled to two major signaling pathways, the mitogen-activated protein kinase and PI3K/Akt pathways; however, PI3K couples directly with ErbB3 and indirectly with EGFR and ErbB2 (19). ErbB3 itself has impaired tyrosine kinase activity (41) and needs a dimerization partner to become phosphorylated and acquire signaling potential (42). Several reports have been shown that inactivation of ErbB2 leads to decreased ErbB3 tyrosine phosphorylation. For example, ErbB3 is coupled to active ErbB2, thereby activating the PI3K/Akt pathway in ErbB2-overexpressed cells without affecting the phosphorylation of ERK-1/2 (43). Loss of ErbB3 has no significant effects on the level of ERK-1/2 activation, but results in a profound modulation in AKT phosphorylation in SK-BR-3 cells, and the treatment of SK-BR-3 cells with PKI166, a tyrosine kinase inhibitor of EGFR and ErbB2, inhibits the activation of AKT and the mitogen-activated protein kinase pathway (43). Our results indicated that the siRNA-mediated downregulation of ErbB3 did not affect the level of ERK-1/2 phosphorylation, but instead resulted in a profound reduction in the level of MIC-1-induced Akt phosphorylation. Furthermore, AG1478 and AG825 effected a significant suppression of the MIC-1-induced phosphorylation of both Akt and ERK-1/2 in SK-BR-3 and SNU-216 cells. Collectively, these results provide further evidence to suggest that ErbB2 activation may perform a critical function in MIC-1-induced signaling pathways in ErbB2-overexpressed cells.

How might MIC-1 activate ErbB2 tyrosine kinase? Thus far, the receptor for MIC-1 has yet to be identified. Unlike other members of this receptor family (EGFR, ErbB3 and ErbB4) that bind to specific ligands and form heterodimers with ErbB2, ErbB2 does not appear to have a ligand (20). In fact, ErbB2 appears to be the preferred partner of the other ligand-bound ErbBs (44). MIC-1 may not be a ligand for EGFR or ErbB3 (14). ErbB2 has been shown to be transactivated by several factors (25–28). For example, CXCL-12, the receptor of which is a member of a family of G-protein-coupled receptors, transactivates ErbB2 in MBA-MB-361 and SK-BR-3 human breast cancer cells, via Src kinase activation (27). Prolactin, the receptor of which is a member of the cytokine receptor superfamily characterized by the lack of an intrinsic kinase domain, also activates ErbB2 via Janus kinase Jak2 (25). Therefore, it can be speculated that MIC-1 may transactivate ErbB2 tyrosine kinase via an unknown mechanism after binding to its own receptor. The mechanism underlying this activation should be described in detail, but not until the MIC-1 receptor has been identified.

MIC-1-induced activation of ERK-1/2 was abolished to a significant degree by treatment with a PI3K inhibitor, wortmannin. Recent several studies have shown the presence of cross-talk between PI3K and ERK-1/2 signaling pathways. For example, placental growth factor caused a time-dependent transient increase in phosphorylation of ERK-1/2, which was completely inhibited by wortmannin in THP-1 cells (45). Another report has shown that PI3K is required for insulin-stimulated but not for EGF-stimulated activation of ERK-1/2 in several cancer cells (46). Taken together, our results suggested that PI3K could play a role in MIC-1-induced ERK-1/2 activation in ErbB2-overexpressed cells.

Considering the pleiotropic effects of MIC-1 in tumors (4), our results indicate that MIC-1 may positively affect tumor progression. Recent studies have demonstrated that HIF is upregulated in a broad range of cancers, including those that overexpress ErbB2, and its expression has been correlated with tumor grade and vascularity (29). ErbB2 signaling increases the rate of HIF-1{alpha} synthesis that is dependent on PI3K/Akt and mTOR pathways (47). Our results show that MIC-1 transactivates ErbB2 in ErbB2-overexpressing cancer cells, the activation of which may upregulate HIF-1{alpha} protein expression and the expression of its target genes, such as VEGF, via the activation of mTOR pathway in the absence of hypoxic stimulation. This MIC-1-induced HIF-1 expression may allow the ErbB2-overexpressing tumors to retain a certain growth advantage, thereby enhancing their tumor progression ability. It is worthy of note that the results of the current study are also relevant to our understanding of the roles of MIC-1, a TGF-β superfamily cytokine, within a cellular context. ErbB2 is amplified and is recognized as an indicator of poor prognosis in many cancers, including breast and gastric cancers (21,22). TGF-β is considered to function as a tumor suppressor during the early stages of cancer, and as a growth/metastasis enhancer in the later stages of cancer (48). In a similar fashion, MIC-1 may operate as a tumor promoter, at least in certain ErbB2-overexpressing cancer cells.

In conclusion, our results indicate a novel mechanism of MIC-1-induced Akt and ERK-1/2 activation, occurring via ErbB2 transactivation. Any potential activation of EGFR family tyrosine kinases by MIC-1 in ErbB2-overexpressing cells is likely to promote the ability of tumor cells to activate oncogenic signaling, most notably Akt and ERK-1/2 signaling. The finding that MIC-1 regulates HIF-1{alpha} expression via ErbB2 transactivation opens a new avenue for the investigation of the close links between MIC-1 signaling, the EGFR family and tumor progression, and possibly metastasis.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
21C Frontier Functional Human Genome Project from Korea Ministry of Science and Technology, M106KB010010.


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received August 15, 2007; revised January 6, 2008; accepted January 26, 2008.


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