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Transforming growth factor (TGF)-β in conjunction with H-ras activation promotes malignant progression of MCF10A breast epithelial cells
www.elsevier.com/locate/issn/10434666 Cytokine 29 (2005) 84e91
Transforming growth factor (TGF)-b in conjunction with H-ras activation promotes malignant progression of MCF10A breast epithelial cells Eun-Sook Kim1, Mi-Sung Kim1,2, Aree Moon* College of Pharmacy, Duksung Women’s University, Seoul 132-714, Republic of Korea Received 7 July 2004; received in revised form 2 October 2004; accepted 8 October 2004
Abstract To address how transforming growth factor (TGF)-b and oncogenic H-ras signal transduction pathways interact with each other in the malignant progression of breast epithelial cells, we investigated the role of TGF-b signaling pathway in invasive and migrative properties of H-ras-transformed MCF10A human breast epithelial cells in this study. Here we show that TGF-b treatment significantly enhanced invasion and migration of H-ras MCF10A cells. H-ras-mediated activation of p38 MAPK and ERK-1/2 was stimulated by TGF-b. TGF-b increased expression of matrix metalloproteinase (MMP)-2 through transcriptional activation while TGF-b-stimulated MMP-9 up-regulation did not occur at transcription level. Activation of p38 MAPK pathway was required for TGF-b-induced cell migration, invasion and MMP-2/-9 up-regulation, indicating a critical role of p38 MAPK signaling in TGF-bpromoted tumor progression of H-ras-activated cells. ERKs signaling was also crucial for TGF-b-enhanced invasive and migrative phenotypes but the up-regulation of MMP-2/-9 was not dependent on ERKs activity. Taken together, we show that TGFb promotes H-ras-mediated cell migration and invasive phenotypes in which p38 MAPK and ERKs signaling pathways are involved. Our findings revealing how H-ras and TGF-b signal pathways interact with each other in MCF10A human breast cells may provide an insight into molecular mechanisms for contribution of TGF-b to a malignant progression of breast cancer in collaboration with activated H-ras. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: TGF-b; H-ras; Breast epithelial cells; Invasion; Migration; MMP; MAPK
1. Introduction The cytokine transforming growth factor (TGF)-b exerts diverse effects on a wide array of cellular processes ranging from proliferation, differentiation and apoptosis [1,2]. TGF-b has been identified as a potent inhibitor of the growth of normal epithelial cells while
* Corresponding author. Tel.: C82 2 901 8394; fax: C82 2 901 8386. E-mail address: [email protected] (A. Moon). 1 Both authors equally contributed to this study. 2 Present address: College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea. 1043-4666/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2004.10.001
it acts as a stimulator of tumor invasion in advanced cancer cells. It has emerged as a potent inhibitor of the progression of normal epithelial cells and endothelial cells by growth arrest in the cell cycle [3,4]. On the other hand, TGF-b can exacerbate the malignant phenotype at later stages of tumorigenesis [5,6] by inducing epithelial-to-mesenchymal transition (EMT), cell invasion and migration of epithelial tumor cells [7,8]. TGFb stimulates type IV collagenases, 72 kDa matrix metalloproteinase (MMP)-2 and/or 92 kDa MMP-9 [9], which can degrade type IV collagen, the major structural collagen of the basement membrane and thus play a critical role in tumor invasion and metastasis formation [10,11].
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Signaling of TGF-b is mediated by a heteromeric complex of two types of transmembrane serine/ threonine kinase receptors. Binding of TGF-b to the receptor complex leads the type II receptor kinase to phosphorylate and thereby activate the type I receptor kinase. The activated type I receptor then phosphorylates receptor-activated Smads (R-Smads), Smad2 and Smad3 [1]. After phosphorylation by the type I receptor kinase, the R-Smads bind to Smad4 and move into the nucleus. In the nucleus, this Smad complex associates with other transcription factors to activate transcription of target genes [12,13]. In addition to the Smad-mediated TGF-b signaling pathway, recent evidences suggest that TGF-b may signal through other pathways, e.g. mitogen-activated protein kinases (MAPKs) including c-JunN-terminal protein kinase (JNK) [14], extracellular signal-regulated kinase (ERK) [15] and p38 MAPK [16]. Ras proteins are activated by multiple extracellular stimuli and are involved in regulatory biological processes from the outside of the cell to its interior through a complex array of downstream effectors, thereby controlling a variety of cellular responses such as proliferation, apoptosis, adhesion, and cytokine/matrix production [17e19]. Ras expression has been suggested as a marker for tumor aggressiveness of breast cancer [20e22]. It has been demonstrated that TGF-b collaborates with oncogenic Ras and brings about metastatic and invasive phenotypic changes in Ras-transformed mammary epithelial cells [23]. This process requires cooperation of Ras-MAPK and TGF-b signaling pathways contributing to tumor invasion [24,25]. We have previously shown that active mutant of HrasG12/D12, but not N-rasG12/D12, induces invasive and migrative phenotypes in MCF10A human breast epithelial cells [26,27]. Our previous study also showed that TGF-b treatment induced migrative and invasive phenotypes, important phenotypic conversion during tumor progression, in preneoplastic MCF10A cells [28]. To address how H-ras and TGF-b signal transduction pathways interact with each other in malignant breast
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cell behavior, we investigated the TGF-b signaling pathway and its role in invasive and migrative properties of H-ras MCF10A cells. Here, we show that TGFb stimulates H-ras-mediated cell migration and invasive phenotypes, which involves activation of p38 MAPK and ERKs pathways. We also provide evidence that TGF-b-enhanced MMP-2 and MMP-9 expression depends only on p38 MAPK signaling but is independent of ERKs activity.
2. Results 2.1. TGF-b stimulates invasion and migration of H-ras MCF10A cells To investigate if TGF-b enhanced malignant cell behavior of highly invasive H-ras MCF10A cells, we examined invasive and migrative phenotypes of these cells. TGF-b significantly increased the number of invaded (Fig. 1A) and migrated (Fig. 1B) cells of H-ras MCF10A, suggesting that TGF-b contributes to tumor progression by converting H-ras-transformed MCF10A to a more malignant breast cancer cell line. 2.2. TGF-b stimulates H-ras-mediated activation of ERK-1/2 and p38 MAPK Our previous study revealed that H-ras induced activation of p38 MAPK and ERK-1/2 but not that of JNK in MCF10A cells [27]. We examined the effect of TGF-b on the H-ras-induced activation of MAPK family members. H-ras MCF10A cells were treated with various concentrations of TGF-b for 1 h and the activation of JNK-1, ERK-1/2 and p38 MAPK was determined by immunoblot analysis using antibodies specific for the phosphorylated forms of these MAPKs. As shown in Fig. 2A, TGF-b enhanced the activation of ERKs and p38 MAPK in a concentration-dependent manner with 0.1 ng/ml being sufficient to increase phosphorylation of
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Fig. 1. TGF-b enhances invasive and migrative phenotypes of H-ras MCF10A cells. Cells were treated with TGF-b (10 ng/ml) and subjected to in vitro invasion assay (A) and in vitro migration assay (B). The number of invaded/migrated cells per field was counted (!400) in 13 fields. The results represent mean G SE of triplicates. *Statistically different from control at p ! 0.05.
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Fig. 2. TGF-b activates ERK-1/2 and p38 MAPK in H-ras MCF10A cells. Cells were incubated with either increasing amounts of TGF-b for 1 h (A) or with fixed concentration of TGF-b (5 ng/ml) for various periods of time (B). The levels of activated JNK-1, ERK-1/2 and p38 MAPK in H-ras MCF10A cells treated with TGF-b were determined by immunoblot analysis of whole cell lysates using phospho-specific antibodies (pJNK-1, pERK1/2 and pp38) and antibodies which detect total JNK-1, ERK-1/2 and p38 MAPK.
ERKs and p38 MAPK. Phosphorylated level of JNK-1 was not affected by TGF-b treatment. The effects of TGF-b on ERKs and p38 MAPK vary in kinetics. A kinetic study showed that ERKs were activated at 30 min after TGF-b (5 ng/ml) treatment and the activated levels remained until 180 min (Fig. 2B). In contrast, 5 ng/ml TGF-b markedly increased the active form of p38 MAPK within 5 min, maximally activated at 30 min after the treatment. These data demonstrate that TGF-b stimulates H-ras-mediated activation of ERK-1/2 and p38 MAPK pathways possibly through different mechanisms. 2.3. TGF-b induces MMP-2 and MMP-9 activities in H-ras MCF10A cells Invasive phenotype is often associated with increased expression of MMP-2 and/or MMP-9 [29]. We have previously shown that H-ras activation of both p38 MAPK and ERKs induces MMP-2 and MMP-9 expression [27]. To examine the regulation of MMP2/-9 enzyme activities by TGF-b in H-ras MCF10A cells, gelatin zymogram assay was performed on the conditioned medium of cells treated with TGF-b for 48 h. As shown in Fig. 3A, TGF-b markedly induced gelatinolytic activities of secreted MMP-2 and MMP-9
in a dose-dependent manner. Active form of MMP-2 (62 kDa) was detected in H-ras MCF10A cells treated with 1 and 10 ng/ml TGF-b, indicating that TGFb treatment not only upregulated MMP-2 but also induced the activation of MMP-2 in H-ras MCF10A cells. To investigate direct gene transcription as a potential mechanism for the TGF-b-stimulated increase of MMP2 and MMP-9 activities observed, promoter assay was performed on the H-ras MCF10A cells transfected with a luciferase reporter plasmid construct under the transcriptional control of the wild type MMP-2 [30] or MMP-9 [31] promoters. Treatment of H-ras MCF10A cells with TGF-b enhanced the MMP-2 promoter activity in a dose-dependent manner while MMP-9 promoter activity was not significantly increased by TGF-b (Fig. 3B). The data indicate that TGF-b-induced MMP-2 up-regulation involves transcriptional activation, whereas the up-regulation of MMP-9 expression by TGF-b was not through transcriptional response. 2.4. Activation of ERKs and p38 MAPK is required for TGF-b-induced invasion and migration In order to examine the role of ERKs and p38 MAPK in TGF-b-stimulated invasive phenotype, we
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Fig. 3. TGF-b induces up-regulation of MMP-2 and MMP-9 in H-ras MCF10A cells. (A) Gelatin zymogram assay was performed on the cells treated with various concentrations of TGF-b for 48 h. Relative band intensities were determined by using an Image analyzer. (B) Luciferase assay was performed to detect promoter activities of MMP-2 and MMP-9 in TGF-b-treated H-ras MCF10A cells. The luciferase activity in 1 mg of cell lysate was normalized to b-galactosidase activity. The data represent three independent experiments performed in triplicates (mean G S.E. of triplicates). * Statistically different from control at p ! 0.05.
treated the H-ras MCF10A cells with PD98059 or SB203580, specific inhibitors of ERKs and p38 MAPK pathways, respectively. Enhanced invasive phenotype by TGF-b was almost completely blocked by treatment of cells with 25 mM PD98059 or 50 mM SB203580, indicating that the induction of invasion by TGF-b was dependent on ERKs and p38 MAPK pathways (Fig. 4A). We then tested the effect of the MAPK inhibitors on TGF-b-mediated migration of H-ras MCF10A cells. TGF-b-stimulated H-ras MCF10A cell migration was significantly inhibited by treatment with PD98059 or SB203580. The data suggest that both ERKs and p38 MAPK pathways are critical for the TGF-b-stimulated invasive and migrative phenotypes of H-ras MCF10A cells.
2.5. TGF-b-stimulated up-regulation of MMP-2 and MMP-9 is dependent on p38 MAPK activity We elucidated the specific roles of ERK-1/2 and p38 MAPK in mediating the enhancing effect of TGF-b on MMP-2/-9 expression in H-ras MCF10A cells. As shown in Fig. 5A, up-regulatory effect of TGF-b on enzymatic activities and expression level of these MAPKs was inhibited by blocking p38 MAPK pathway by SB203580 in a dose-dependent manner. Treatment of 50 mM SB203580 almost entirely abolished the TGF-binduced up-regulation of secreted enzymatic activities and expression levels of MMP-2 and MMP-9 in a comparable extent. In contrast, blocking the ERK-1/ 2 pathway by PD98059 did not markedly inhibit the
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Fig. 4. Activation of ERK and p38 MAPK is required for TGF-b-induced invasion and migration. Cells were treated with TGF-b (10 ng/ml) in the absence or presence of 25 mM PD98059 or 50 mM SB203580 for 17 h and subjected to in vitro invasion assay (A) and in vitro migration assay (B). The number of invaded/migrated cells per field was counted (!400) in 13 fields. The results represent mean G SE of triplicates. **Statistically different from cells treated with TGF-b alone at p ! 0.01.
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Fig. 5. TGF-b-mediated up-regulation of MMP-2 and MMP-9 is dependent on p38 MAPK activity. Cells were treated with 10 ng/ml TGF-b in the absence or presence of various concentrations of SB203580 (A) or PD98059 (B) for 48 h. Gelatinolytic activities and the expression levels of secreted MMP-2 and MMP-9 were determined by gelatin zymogram assay (left) and immunoblot analysis (right), respectively. Relative band intensities were determined by using an Image analyzer.
TGF-b-stimulated enhancement of activities and expression of MMP-2 and MMP-9 (Fig. 5B). The results demonstrate that TGF-b-mediated up-regulation of MMP-2 and MMP-9 in H-ras MCF10A cells is dependent on the activation of p38 MAPK signaling pathway but not that of ERK pathway.
3. Discussion Ras-transformed cells exhibit a limited growth inhibitory response to TGF-b [32] but may respond to TGF-b with invasive activity and metastatic behavior [23,33]. In cooperation with activated Ras, TGF-b can induce a complete EMT in both mammary and keratinocyte-derived tumors [34,35], and it can drive metastasis of epitheloid tumors [36]. TGF-b has been shown to activate Ras in TGF-b-sensitive intestinal and epithelial cells [37,38]. Here, we show that TGF-b contributes to tumor progression by enhancing cell invasiveness and migration through the collaboration with H-ras signaling pathway in human breast epithelial cells. Our previous study showed that H-ras activated
ERKs and p38 MAPK but not JNK in MCF10A cells [27]. Similarly, this study demonstrates that TGF-b enhances activation of ERKs and p38 MAPK but not that of JNK in H-ras MCF10A cells, suggesting a possibility that the cellular effect of TGF-b is mediated by H-ras. It would be worthwhile to further investigate if the TGF-b-stimulated activation of ERKs and p38 MAPK pathways is a direct effect of TGF-b or an indirect response through the consequence of H-ras activation by TGF-b. Involvement of the p38 MAPK pathway in TGF-bstimulated EMT, cell migration and gene expression has been implicated in various cell systems [16,39,40]. Using a mutant TGF-b type I receptor, it has been shown that TGF-b receptor-activated p38 MAPK mediates Smadindependent TGF-b responses [41]. Our results showed that p38 MAPK, which was required for TGF-b-induced invasion, migration and MMP-2 up-regulation, was rapidly activated by TGF-b (Fig. 2B). This rapid activation of p38 MAPK by TGF-b suggests that it might be through a direct post-translational modification rather than by Smad-dependent responses. In contrast, ERKs were activated by TGF-b with a relatively slow kinetics,
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indicating that it might be delayed, indirect effects, possibly resulting from Smad-dependent transcriptional response. On contrary to our observation, a rapid activation of ERK-1 was detected in proliferating cultures of epithelial cells [15]. We are currently investigating the involvement of Smads in TGF-b-stimulated activation of p38 MAPK and ERKs pathways which are critical for malignant cancer cell phenotype of MCF10A human breast epithelial cells promoted by TGF-b. TGF-b regulates MMP expression in a cell typespecific manner. Enhanced expression of MMP-2 but not MMP-9 by TGF-b was reported in pancreatic cancer cells [42], whereas TGF-b was shown to induce MMP-9 in transformed keratinocytes [43,44]. Recent evidences show that induction of MMP-13 expression by TGF-b was blocked by SB203580, but not by PD98059 in human gingival fibroblasts [45]. Critical role of p38 MAPK-and MMP-dependent pathway in TGF-b-induced osteoblast elongation was demonstrated [46]. In this study, we demonstrated that TGF-b enhanced H-ras-induced upregulation of MMP-2 and MMP-9 in MCF10A cells. Of note, although TGF-b induced secreted gelatinolytic activities of MMP-2 and MMP-9 at comparable levels (Fig. 3A), it increased MMP-2 promoter activity more efficiently than MMP-9 (Fig. 3B). A marked induction of MMP-2 promoter activity by TGF-b indicates that TGF-b-induced MMP-2 up-regulation involved transcriptional activation. In contrast, MMP-9 promoter activity was not significantly induced by the same treatment, suggesting that the increased expression of MMP-9 by TGF-b was through other mechanism(s) including increased mRNA stability as previously shown in human prostate cancer cells [47]. Our results demonstrate a critical role of p38 MAPK signaling in TGF-b-promoted tumor progression of Hras-activated breast epithelial cells. ERKs activity was also required for the invasive and migrative phenotypes promoted by TGF-b (Fig. 4), though TGF-b-enhanced up-regulation of MMP-2 and MMP-9 was not dependent on ERKs pathway (Fig. 5B). The results suggest that for the completion of invasion and migration of Hras MCF10A cells, not only matrix-degrading proteinase activities of MMP-2 and MMP-9 but also other intracellular events through activation of ERKs signaling pathways are essential. Ras expression has been shown to be related to the invasiveness of breast cells since elevated levels of the ras protein have been found in 60e70% of human primary breast carcinomas [20], suggesting Ras expression as a marker for tumor aggressiveness of breast cancer [20e23]. In this study, we reveal how H-ras and TGF-b signal pathways interact with each other in malignant breast cell behavior. Our findings may provide an insight into molecular mechanisms for contribution of TGF-b to a malignant progression of breast cancer in collaboration with activated H-ras.
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4. Materials and methods 4.1. Materials Dulbecco’s modified Eagle’s medium (DMEM), penicillinestreptomycin, L-glutamine and trypsineEDTA were purchased from Gibco BRL (Grand Island, NY).TGF-b, epidermal growth factor (EGF), cholera enterotoxin, amphotericin B, insulin, hydrocortisone, SB203580 and PD98059 were purchased from SigmaeAldrich (St. Louis, MO). 4.2. Cell lines and culture conditions The development and characterization of the H-ras MCF10A cells have been previously described [26]. Cells were cultured in DMEM/F12 supplemented with 5% horse serum, 0.5 mg/ml hydrocortisone, 10 mg/ml insulin, 20 ng/ml EGF, 0.1 mg/ml cholera enterotoxin, 100 units/ ml penicillinestreptomycin, 2 mM L-glutamine and 0.5 mg/ml amphotericin B. 4.3. Immunoblot analysis Equal amounts of protein extracts in SDS-lysis buffer were subjected to 12% SDS-PAGE analysis and electrophoretically transferred to a nitrocellulose membrane. Anti-JNK, anti-phosphorylated JNK, anti-ERK-1/2, anti-phosphorylated ERK-1/2, anti-p38 and anti-phosphorylated p38 antibodies were purchased from Cell Signaling Tech (Beverly, MA). MMP-2 and MMP-9 antibodies were from Santa Cruz Biotech (Santa Cruz, CA). Enhanced chemiluminescence (ECL, AmershamPharmacia, Buckinghamshire, UK) system was used for detection. Relative band intensities were determined by quantitation of each band with an Image analyzer (Vilber Lourmat, France). 4.4. In vitro invasion assay In vitro invasion assay was performed using 24-well transwell unit with polycarbonate filters (Corning Costar, Cambridge, MA) as previously described [26]. The lower side of the filter was coated with type I collagen, and the upper side was coated with Matrigel (Collaborative Research, Lexington, KY). Lower compartment was filled with serum-free media containing 0.1% BSA. Cells were placed in the upper part of the transwell unit, incubated for 17 h, fixed with methanol and stained with hematoxylin for 10 min followed briefly by eosin. The invasive phenotypes were determined by counting the cells that migrated to the lower side of the filter with microscopy at !400. Thirteen fields were counted for each filter and each sample was assayed in triplicate.
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4.5. In vitro migration assay using transwell In vitro migration assay was performed using a 24well transwell unit with polycarbonate filters as previously described [27]. Experimental procedures were the same as the in vitro invasion assay described above except that the filter was not coated with Matrigel for the migration assay. 4.6. Gelatin zymogram assay Cells were cultured in serum-free DMEM/F12 medium containing chemicals for 48 h. Conditioned medium was collected and centrifuged at 3000 rpm for 10 min to remove cell debris. The protein concentration was measured using BCA protein assay reagents (Pierce, Rockford, IL). Gelatinolytic activity of the conditioned medium was determined by gelatin zymogram assay as previously described [26,27]. Areas of gelatinase activity were detected as clear bands against the blue-stained gelatin background. 4.7. Promoter assay (luciferase/b-galactosidase assay) Cells were seeded in a 6-well plate at 1 ! 106 cells/ well and transiently transfected with 4 mg MMP-2 promoter-luciferase construct and MMP-9 promoterluciferase construct (kindly provided by Dr. Benveniste at University of Alabama, Birmingham, AL) and 0.13 mg b-galactosidase expression plasmid pMDV-lacZ. The transfectants were treated with TGF-b for 24 h. The luciferase activity in 1 mg of cell lysate was normalized to b-galactosidase activity and total protein was determined by BCA protein assay kit (Pierce, Rockford, IL). Luciferase and b-galactosidase activities were assayed using luciferase assay kit (Promega, Medison, WI) and Galacto-Light Kit (Tropix Inc, Bedford, MA) and measured with a Luminometer (Tuner Designs, Sunnyvale, CA). Acknowledgements This work was supported by the Korea Food and Drug Administration Grant KFDA-04092-LIF-002. References [1] Massague J. TGF-beta signal transduction. Annu Rev Biochem 1998;67:753–91. [2] Whitman M. Smads and early developmental signaling by the TGFbeta superfamily. Genes Dev 1998;12:2445–62. [3] Alexandrow MG, Moses HL. Transforming growth factor beta and cell cycle regulation. Cancer Res 1995;55:1452–7. [4] Taipale J, Saharinen J, Keski-Oja J. Extracellular matrixassociated transforming growth factor-beta: role in cancer cell growth and invasion. Adv Cancer Res 1998;75:87–134.
[5] Reiss M, Barcellos-Hoff MH. Transforming growth factor-beta in breast cancer: a working hypothesis. Breast Cancer Res Treat 1997;45:81–95. [6] Akhurst RJ, Balmain A. Genetic events and the role of TGF beta in epithelial tumour progression. J Pathol 1999;187:82–90. [7] Portella G, Cumming SA, Liddell J, Cui W, Ireland H, Akhurst RJ, et al. Transforming growth factor beta is essential for spindle cell conversion of mouse skin carcinoma in vivo: implications for tumor invasion. Cell Growth Differ 1998;9: 393–404. [8] McEarchern JA, Kobie JJ, Mack V, Wu RS, Meade-Tollin L, Arteaga CL, et al. Invasion and metastasis of a mammary tumor involves TGF-beta signaling. Int J Cancer 2001;91:76–82. [9] Lin SW, Lee MT, Ke FC, Lee PP, Huang CJ, Ip MM, et al. TGFbeta1 stimulates the secretion of matrix metalloproteinase 2 (MMP2) and the invasive behavior in human ovarian cancer cells, which is suppressed by MMP inhibitor BB3103. Clin Exp Metastasis 2000;18:493–9. [10] Tryggvason K, Hoyhtya M, Pyke C. Type IV collagenases in invasive tumors. Breast Cancer Res Treat 1993;24:209–18. [11] Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, et al. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 1994;370:61–5. [12] Zhang Y, Derynck R. Regulation of Smad signalling by protein associations and signalling crosstalk. Trends Cell Biol 1999;9:274–9. [13] Massague J. How cells read TGF-beta signals. Nat Rev Mol Cell Biol 2000;1:169–78. [14] Atfi A, Djelloul S, Chastre E, Davis R, Gespach C. Evidence for a role of Rho-like GTPases and stress-activated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK) in transforming growth factor beta-mediated signaling. J Biol Chem 1997;272: 1429–32. [15] Hartsough MT, Mulder KM. Transforming growth factor beta activation of p44mapk in proliferating cultures of epithelial cells. J Biol Chem 1995;270:7117–24. [16] Hanafusa H, Ninomiya-Tsuji J, Masuyama N, Nishita M, Fujisawa J, Shibuya H, et al. Involvement of the p38 mitogenactivated protein kinase pathway in transforming growth factorbeta-induced gene expression. J Biol Chem 1999;274:27161–7. [17] Bourne HR, Sanders DA, McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature 1991;349:117–27. [18] Marshall MS. Ras target proteins in eukaryotic cells. FASEB J 1995;9:1311–8. [19] Tanaka Y, Nakayamada S, Fujimoto H, Okada Y, Umehara H, Kataoka T, et al. H-Ras/mitogen-activated protein kinase pathway inhibits integrin-mediated adhesion and induces apoptosis in osteoblasts. J Biol Chem 2002;277:21446–52. [20] Clair T, Miller WR, Cho-Chung YS. Prognostic significance of the expression of a ras protein with a molecular weight of 21,000 by human breast cancer. Cancer Res 1987;47:5290–3. [21] Watson DM, Elton RA, Jack WJ, Dixon JM, Chetty U, Miller WR. The H-ras oncogene product p21 and prognosis in human breast cancer. Breast Cancer Res Treat 1991;17:161–9. [22] Clark GJ, Der CJ. Aberrant function of the Ras signal transduction pathway in human breast cancer. Breast Cancer Res Treat 1995;35:133–44. [23] Oft M, Peli J, Rudaz C, Schwarz H, Beug H, Reichmann E. TGFbeta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells. Genes Dev 1996;10:2462–77. [24] de Caestecker MP, Piek E, Roberts AB. Role of transforming growth factor-beta signaling in cancer. J Natl Cancer Inst 2000;92:1388–402. [25] Derynck R, Akhurst RJ, Balmain A. TGF-beta signaling in tumor suppression and cancer progression. Nat Genet 2001;29:117–29.
E.-S. Kim et al. / Cytokine 29 (2005) 84e91 [26] Moon A, Kim MS, Kim TG, Kim SH, Kim HE, Chen YQ, et al. H-ras, but not N-ras, induces an invasive phenotype in human breast epithelial cells: a role for MMP-2 in the H-ras-induced invasive phenotype. Int J Cancer 2000;85:176–81. [27] Kim MS, Lee EJ, Kim HR, Moon A. p38 Kinase is a key signaling molecule for H-Ras-induced cell motility and invasive phenotype in human breast epithelial cells. Cancer Res 2003;63:5454–61. [28] Kim ES, Kim MS, Moon A. TGF-b-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERKs signaling in MCF10A human breast epithelial cells. Int J Oncol 2004;25:1375–82. [29] Ura H, Bonfil RD, Reich R, Reddel R, Pfeifer A, Harris CC, et al. Expression of type IV collagenase and procollagen genes and its correlation with the tumorigenic, invasive, and metastatic abilities of oncogene-transformed human bronchial epithelial cells. Cancer Res 1989;49:4615–21. [30] Bian J, Sun Y. Transcriptional activation by p53 of the human type IV collagenase (gelatinase A or matrix metalloproteinase 2) promoter. Mol Cell Biol 1997;17:6330–8. [31] Ma Z, Qin H, Benveniste EN. Transcriptional suppression of matrix metalloproteinase-9 gene expression by IFN-gamma and IFN-beta: critical role of STAT-1alpha. J Immunol 2001;167:5150–9. [32] Schwarz LC, Gingras MC, Goldberg G, Greenberg AH, Wright JA. Loss of growth factor dependence and conversion of transforming growth factor-beta 1 inhibition to stimulation in metastatic H-ras-transformed murine fibroblasts. Cancer Res 1988;48:6999–7003. [33] Yin JJ, Selander K, Chirgwin JM, Dallas M, Grubbs BG, Wieser R, et al. TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. J Clin Invest 1999;103:197–206. [34] Akhurst RJ, Derynck R. TGF-beta signaling in cancer a doubleedged sword. Trends Cell Biol 2001;11:S44–51. [35] Oft M, Akhurst RJ, Balmain A. Metastasis is driven by sequential elevation of H-ras and Smad2 levels. Nat Cell Biol 2002;4:487–94. [36] Muraoka RS, Dumont N, Ritter CA, Dugger TC, Brantley DM, Chen J, et al. Blockade of TGF-beta inhibits mammary tumor cell viability, migration, and metastases. J Clin Invest 2002;109: 1551–9.
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[37] Mulder KM, Morris SL. Activation of p21ras by transforming growth factor beta in epithelial cells. J Biol Chem 1992;267:5029–31. [38] Yan Z, Winawer S, Friedman E. Two different signal transduction pathways can be activated by transforming growth factor beta 1 in epithelial cells. J Biol Chem 1994;269:13231–7. [39] Bakin AV, Rinehart C, Tomlinson AK, Arteaga CL. p38 Mitogen-activated protein kinase is required for TGFbetamediated fibroblastic transdifferentiation and cell migration. J Cell Sci 2002;115:3193–206. [40] Hedges JC, Dechert MA, Yamboliev IA, Martin JL, Hickey E, Weber LA, et al. A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem 1999;274:24211–9. [41] Yu L, Hebert MC, Zhang YE. TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses. EMBO J 2002;21:3749–59. [42] Ellenrieder V, Hendler SF, Ruhland C, Boeck W, Adler G, Gress TM. TGF-beta-induced invasiveness of pancreatic cancer cells is mediated by matrix metalloproteinase-2 and the urokinase plasminogen activator system. Int J Cancer 2001;93:204–11. [43] Johansson N, Ala-aho R, Uitto V, Grenman R, Fusenig NE, Lopez-Otin C, et al. Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase. J Cell Sci 2000;113:227–35. [44] Santibanez JF, Guerrero J, Quintanilla M, Fabra A, Martinez J. Transforming growth factor-beta1 modulates matrix metalloproteinase-9 production through the Ras/MAPK signaling pathway in transformed keratinocytes. Biochem Biophys Res Commun 2002;296:267–73. [45] Ravanti L, Hakkinen L, Larjava H, Saarialho-Kere U, Foschi M, Han J, et al. Transforming growth factor-beta induces collagenase-3 expression by human gingival fibroblasts via p38 mitogenactivated protein kinase. J Biol Chem 1999;274:37292–300. [46] Karsdal MA, Fjording MS, Foged NT, Delaisse JM, Lochter A. Transforming growth factor-beta-induced osteoblast elongation regulates osteoclastic bone resorption through a p38 mitogenactivated protein kinase-and matrix metalloproteinase-dependent pathway. J Biol Chem 2001;276:39350–8. [47] Sehgal I, Thompson TC. Novel regulation of type IV collagenase (matrix metalloproteinase-9 and -2) activities by transforming growth factor-beta1 in human prostate cancer cell lines. Mol Biol Cell 1999;10:407–16.
Transforming growth factor (TGF)-b in conjunction with H-ras activation promotes malignant progression of MCF10A breast epithelial cells Eun-Sook Kim1, Mi-Sung Kim1,2, Aree Moon* College of Pharmacy, Duksung Women’s University, Seoul 132-714, Republic of Korea Received 7 July 2004; received in revised form 2 October 2004; accepted 8 October 2004
Abstract To address how transforming growth factor (TGF)-b and oncogenic H-ras signal transduction pathways interact with each other in the malignant progression of breast epithelial cells, we investigated the role of TGF-b signaling pathway in invasive and migrative properties of H-ras-transformed MCF10A human breast epithelial cells in this study. Here we show that TGF-b treatment significantly enhanced invasion and migration of H-ras MCF10A cells. H-ras-mediated activation of p38 MAPK and ERK-1/2 was stimulated by TGF-b. TGF-b increased expression of matrix metalloproteinase (MMP)-2 through transcriptional activation while TGF-b-stimulated MMP-9 up-regulation did not occur at transcription level. Activation of p38 MAPK pathway was required for TGF-b-induced cell migration, invasion and MMP-2/-9 up-regulation, indicating a critical role of p38 MAPK signaling in TGF-bpromoted tumor progression of H-ras-activated cells. ERKs signaling was also crucial for TGF-b-enhanced invasive and migrative phenotypes but the up-regulation of MMP-2/-9 was not dependent on ERKs activity. Taken together, we show that TGFb promotes H-ras-mediated cell migration and invasive phenotypes in which p38 MAPK and ERKs signaling pathways are involved. Our findings revealing how H-ras and TGF-b signal pathways interact with each other in MCF10A human breast cells may provide an insight into molecular mechanisms for contribution of TGF-b to a malignant progression of breast cancer in collaboration with activated H-ras. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: TGF-b; H-ras; Breast epithelial cells; Invasion; Migration; MMP; MAPK
1. Introduction The cytokine transforming growth factor (TGF)-b exerts diverse effects on a wide array of cellular processes ranging from proliferation, differentiation and apoptosis [1,2]. TGF-b has been identified as a potent inhibitor of the growth of normal epithelial cells while
* Corresponding author. Tel.: C82 2 901 8394; fax: C82 2 901 8386. E-mail address: [email protected] (A. Moon). 1 Both authors equally contributed to this study. 2 Present address: College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea. 1043-4666/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2004.10.001
it acts as a stimulator of tumor invasion in advanced cancer cells. It has emerged as a potent inhibitor of the progression of normal epithelial cells and endothelial cells by growth arrest in the cell cycle [3,4]. On the other hand, TGF-b can exacerbate the malignant phenotype at later stages of tumorigenesis [5,6] by inducing epithelial-to-mesenchymal transition (EMT), cell invasion and migration of epithelial tumor cells [7,8]. TGFb stimulates type IV collagenases, 72 kDa matrix metalloproteinase (MMP)-2 and/or 92 kDa MMP-9 [9], which can degrade type IV collagen, the major structural collagen of the basement membrane and thus play a critical role in tumor invasion and metastasis formation [10,11].
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Signaling of TGF-b is mediated by a heteromeric complex of two types of transmembrane serine/ threonine kinase receptors. Binding of TGF-b to the receptor complex leads the type II receptor kinase to phosphorylate and thereby activate the type I receptor kinase. The activated type I receptor then phosphorylates receptor-activated Smads (R-Smads), Smad2 and Smad3 [1]. After phosphorylation by the type I receptor kinase, the R-Smads bind to Smad4 and move into the nucleus. In the nucleus, this Smad complex associates with other transcription factors to activate transcription of target genes [12,13]. In addition to the Smad-mediated TGF-b signaling pathway, recent evidences suggest that TGF-b may signal through other pathways, e.g. mitogen-activated protein kinases (MAPKs) including c-JunN-terminal protein kinase (JNK) [14], extracellular signal-regulated kinase (ERK) [15] and p38 MAPK [16]. Ras proteins are activated by multiple extracellular stimuli and are involved in regulatory biological processes from the outside of the cell to its interior through a complex array of downstream effectors, thereby controlling a variety of cellular responses such as proliferation, apoptosis, adhesion, and cytokine/matrix production [17e19]. Ras expression has been suggested as a marker for tumor aggressiveness of breast cancer [20e22]. It has been demonstrated that TGF-b collaborates with oncogenic Ras and brings about metastatic and invasive phenotypic changes in Ras-transformed mammary epithelial cells [23]. This process requires cooperation of Ras-MAPK and TGF-b signaling pathways contributing to tumor invasion [24,25]. We have previously shown that active mutant of HrasG12/D12, but not N-rasG12/D12, induces invasive and migrative phenotypes in MCF10A human breast epithelial cells [26,27]. Our previous study also showed that TGF-b treatment induced migrative and invasive phenotypes, important phenotypic conversion during tumor progression, in preneoplastic MCF10A cells [28]. To address how H-ras and TGF-b signal transduction pathways interact with each other in malignant breast
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cell behavior, we investigated the TGF-b signaling pathway and its role in invasive and migrative properties of H-ras MCF10A cells. Here, we show that TGFb stimulates H-ras-mediated cell migration and invasive phenotypes, which involves activation of p38 MAPK and ERKs pathways. We also provide evidence that TGF-b-enhanced MMP-2 and MMP-9 expression depends only on p38 MAPK signaling but is independent of ERKs activity.
2. Results 2.1. TGF-b stimulates invasion and migration of H-ras MCF10A cells To investigate if TGF-b enhanced malignant cell behavior of highly invasive H-ras MCF10A cells, we examined invasive and migrative phenotypes of these cells. TGF-b significantly increased the number of invaded (Fig. 1A) and migrated (Fig. 1B) cells of H-ras MCF10A, suggesting that TGF-b contributes to tumor progression by converting H-ras-transformed MCF10A to a more malignant breast cancer cell line. 2.2. TGF-b stimulates H-ras-mediated activation of ERK-1/2 and p38 MAPK Our previous study revealed that H-ras induced activation of p38 MAPK and ERK-1/2 but not that of JNK in MCF10A cells [27]. We examined the effect of TGF-b on the H-ras-induced activation of MAPK family members. H-ras MCF10A cells were treated with various concentrations of TGF-b for 1 h and the activation of JNK-1, ERK-1/2 and p38 MAPK was determined by immunoblot analysis using antibodies specific for the phosphorylated forms of these MAPKs. As shown in Fig. 2A, TGF-b enhanced the activation of ERKs and p38 MAPK in a concentration-dependent manner with 0.1 ng/ml being sufficient to increase phosphorylation of
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Fig. 1. TGF-b enhances invasive and migrative phenotypes of H-ras MCF10A cells. Cells were treated with TGF-b (10 ng/ml) and subjected to in vitro invasion assay (A) and in vitro migration assay (B). The number of invaded/migrated cells per field was counted (!400) in 13 fields. The results represent mean G SE of triplicates. *Statistically different from control at p ! 0.05.
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Fig. 2. TGF-b activates ERK-1/2 and p38 MAPK in H-ras MCF10A cells. Cells were incubated with either increasing amounts of TGF-b for 1 h (A) or with fixed concentration of TGF-b (5 ng/ml) for various periods of time (B). The levels of activated JNK-1, ERK-1/2 and p38 MAPK in H-ras MCF10A cells treated with TGF-b were determined by immunoblot analysis of whole cell lysates using phospho-specific antibodies (pJNK-1, pERK1/2 and pp38) and antibodies which detect total JNK-1, ERK-1/2 and p38 MAPK.
ERKs and p38 MAPK. Phosphorylated level of JNK-1 was not affected by TGF-b treatment. The effects of TGF-b on ERKs and p38 MAPK vary in kinetics. A kinetic study showed that ERKs were activated at 30 min after TGF-b (5 ng/ml) treatment and the activated levels remained until 180 min (Fig. 2B). In contrast, 5 ng/ml TGF-b markedly increased the active form of p38 MAPK within 5 min, maximally activated at 30 min after the treatment. These data demonstrate that TGF-b stimulates H-ras-mediated activation of ERK-1/2 and p38 MAPK pathways possibly through different mechanisms. 2.3. TGF-b induces MMP-2 and MMP-9 activities in H-ras MCF10A cells Invasive phenotype is often associated with increased expression of MMP-2 and/or MMP-9 [29]. We have previously shown that H-ras activation of both p38 MAPK and ERKs induces MMP-2 and MMP-9 expression [27]. To examine the regulation of MMP2/-9 enzyme activities by TGF-b in H-ras MCF10A cells, gelatin zymogram assay was performed on the conditioned medium of cells treated with TGF-b for 48 h. As shown in Fig. 3A, TGF-b markedly induced gelatinolytic activities of secreted MMP-2 and MMP-9
in a dose-dependent manner. Active form of MMP-2 (62 kDa) was detected in H-ras MCF10A cells treated with 1 and 10 ng/ml TGF-b, indicating that TGFb treatment not only upregulated MMP-2 but also induced the activation of MMP-2 in H-ras MCF10A cells. To investigate direct gene transcription as a potential mechanism for the TGF-b-stimulated increase of MMP2 and MMP-9 activities observed, promoter assay was performed on the H-ras MCF10A cells transfected with a luciferase reporter plasmid construct under the transcriptional control of the wild type MMP-2 [30] or MMP-9 [31] promoters. Treatment of H-ras MCF10A cells with TGF-b enhanced the MMP-2 promoter activity in a dose-dependent manner while MMP-9 promoter activity was not significantly increased by TGF-b (Fig. 3B). The data indicate that TGF-b-induced MMP-2 up-regulation involves transcriptional activation, whereas the up-regulation of MMP-9 expression by TGF-b was not through transcriptional response. 2.4. Activation of ERKs and p38 MAPK is required for TGF-b-induced invasion and migration In order to examine the role of ERKs and p38 MAPK in TGF-b-stimulated invasive phenotype, we
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Fig. 3. TGF-b induces up-regulation of MMP-2 and MMP-9 in H-ras MCF10A cells. (A) Gelatin zymogram assay was performed on the cells treated with various concentrations of TGF-b for 48 h. Relative band intensities were determined by using an Image analyzer. (B) Luciferase assay was performed to detect promoter activities of MMP-2 and MMP-9 in TGF-b-treated H-ras MCF10A cells. The luciferase activity in 1 mg of cell lysate was normalized to b-galactosidase activity. The data represent three independent experiments performed in triplicates (mean G S.E. of triplicates). * Statistically different from control at p ! 0.05.
treated the H-ras MCF10A cells with PD98059 or SB203580, specific inhibitors of ERKs and p38 MAPK pathways, respectively. Enhanced invasive phenotype by TGF-b was almost completely blocked by treatment of cells with 25 mM PD98059 or 50 mM SB203580, indicating that the induction of invasion by TGF-b was dependent on ERKs and p38 MAPK pathways (Fig. 4A). We then tested the effect of the MAPK inhibitors on TGF-b-mediated migration of H-ras MCF10A cells. TGF-b-stimulated H-ras MCF10A cell migration was significantly inhibited by treatment with PD98059 or SB203580. The data suggest that both ERKs and p38 MAPK pathways are critical for the TGF-b-stimulated invasive and migrative phenotypes of H-ras MCF10A cells.
2.5. TGF-b-stimulated up-regulation of MMP-2 and MMP-9 is dependent on p38 MAPK activity We elucidated the specific roles of ERK-1/2 and p38 MAPK in mediating the enhancing effect of TGF-b on MMP-2/-9 expression in H-ras MCF10A cells. As shown in Fig. 5A, up-regulatory effect of TGF-b on enzymatic activities and expression level of these MAPKs was inhibited by blocking p38 MAPK pathway by SB203580 in a dose-dependent manner. Treatment of 50 mM SB203580 almost entirely abolished the TGF-binduced up-regulation of secreted enzymatic activities and expression levels of MMP-2 and MMP-9 in a comparable extent. In contrast, blocking the ERK-1/ 2 pathway by PD98059 did not markedly inhibit the
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Fig. 5. TGF-b-mediated up-regulation of MMP-2 and MMP-9 is dependent on p38 MAPK activity. Cells were treated with 10 ng/ml TGF-b in the absence or presence of various concentrations of SB203580 (A) or PD98059 (B) for 48 h. Gelatinolytic activities and the expression levels of secreted MMP-2 and MMP-9 were determined by gelatin zymogram assay (left) and immunoblot analysis (right), respectively. Relative band intensities were determined by using an Image analyzer.
TGF-b-stimulated enhancement of activities and expression of MMP-2 and MMP-9 (Fig. 5B). The results demonstrate that TGF-b-mediated up-regulation of MMP-2 and MMP-9 in H-ras MCF10A cells is dependent on the activation of p38 MAPK signaling pathway but not that of ERK pathway.
3. Discussion Ras-transformed cells exhibit a limited growth inhibitory response to TGF-b [32] but may respond to TGF-b with invasive activity and metastatic behavior [23,33]. In cooperation with activated Ras, TGF-b can induce a complete EMT in both mammary and keratinocyte-derived tumors [34,35], and it can drive metastasis of epitheloid tumors [36]. TGF-b has been shown to activate Ras in TGF-b-sensitive intestinal and epithelial cells [37,38]. Here, we show that TGF-b contributes to tumor progression by enhancing cell invasiveness and migration through the collaboration with H-ras signaling pathway in human breast epithelial cells. Our previous study showed that H-ras activated
ERKs and p38 MAPK but not JNK in MCF10A cells [27]. Similarly, this study demonstrates that TGF-b enhances activation of ERKs and p38 MAPK but not that of JNK in H-ras MCF10A cells, suggesting a possibility that the cellular effect of TGF-b is mediated by H-ras. It would be worthwhile to further investigate if the TGF-b-stimulated activation of ERKs and p38 MAPK pathways is a direct effect of TGF-b or an indirect response through the consequence of H-ras activation by TGF-b. Involvement of the p38 MAPK pathway in TGF-bstimulated EMT, cell migration and gene expression has been implicated in various cell systems [16,39,40]. Using a mutant TGF-b type I receptor, it has been shown that TGF-b receptor-activated p38 MAPK mediates Smadindependent TGF-b responses [41]. Our results showed that p38 MAPK, which was required for TGF-b-induced invasion, migration and MMP-2 up-regulation, was rapidly activated by TGF-b (Fig. 2B). This rapid activation of p38 MAPK by TGF-b suggests that it might be through a direct post-translational modification rather than by Smad-dependent responses. In contrast, ERKs were activated by TGF-b with a relatively slow kinetics,
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indicating that it might be delayed, indirect effects, possibly resulting from Smad-dependent transcriptional response. On contrary to our observation, a rapid activation of ERK-1 was detected in proliferating cultures of epithelial cells [15]. We are currently investigating the involvement of Smads in TGF-b-stimulated activation of p38 MAPK and ERKs pathways which are critical for malignant cancer cell phenotype of MCF10A human breast epithelial cells promoted by TGF-b. TGF-b regulates MMP expression in a cell typespecific manner. Enhanced expression of MMP-2 but not MMP-9 by TGF-b was reported in pancreatic cancer cells [42], whereas TGF-b was shown to induce MMP-9 in transformed keratinocytes [43,44]. Recent evidences show that induction of MMP-13 expression by TGF-b was blocked by SB203580, but not by PD98059 in human gingival fibroblasts [45]. Critical role of p38 MAPK-and MMP-dependent pathway in TGF-b-induced osteoblast elongation was demonstrated [46]. In this study, we demonstrated that TGF-b enhanced H-ras-induced upregulation of MMP-2 and MMP-9 in MCF10A cells. Of note, although TGF-b induced secreted gelatinolytic activities of MMP-2 and MMP-9 at comparable levels (Fig. 3A), it increased MMP-2 promoter activity more efficiently than MMP-9 (Fig. 3B). A marked induction of MMP-2 promoter activity by TGF-b indicates that TGF-b-induced MMP-2 up-regulation involved transcriptional activation. In contrast, MMP-9 promoter activity was not significantly induced by the same treatment, suggesting that the increased expression of MMP-9 by TGF-b was through other mechanism(s) including increased mRNA stability as previously shown in human prostate cancer cells [47]. Our results demonstrate a critical role of p38 MAPK signaling in TGF-b-promoted tumor progression of Hras-activated breast epithelial cells. ERKs activity was also required for the invasive and migrative phenotypes promoted by TGF-b (Fig. 4), though TGF-b-enhanced up-regulation of MMP-2 and MMP-9 was not dependent on ERKs pathway (Fig. 5B). The results suggest that for the completion of invasion and migration of Hras MCF10A cells, not only matrix-degrading proteinase activities of MMP-2 and MMP-9 but also other intracellular events through activation of ERKs signaling pathways are essential. Ras expression has been shown to be related to the invasiveness of breast cells since elevated levels of the ras protein have been found in 60e70% of human primary breast carcinomas [20], suggesting Ras expression as a marker for tumor aggressiveness of breast cancer [20e23]. In this study, we reveal how H-ras and TGF-b signal pathways interact with each other in malignant breast cell behavior. Our findings may provide an insight into molecular mechanisms for contribution of TGF-b to a malignant progression of breast cancer in collaboration with activated H-ras.
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4. Materials and methods 4.1. Materials Dulbecco’s modified Eagle’s medium (DMEM), penicillinestreptomycin, L-glutamine and trypsineEDTA were purchased from Gibco BRL (Grand Island, NY).TGF-b, epidermal growth factor (EGF), cholera enterotoxin, amphotericin B, insulin, hydrocortisone, SB203580 and PD98059 were purchased from SigmaeAldrich (St. Louis, MO). 4.2. Cell lines and culture conditions The development and characterization of the H-ras MCF10A cells have been previously described [26]. Cells were cultured in DMEM/F12 supplemented with 5% horse serum, 0.5 mg/ml hydrocortisone, 10 mg/ml insulin, 20 ng/ml EGF, 0.1 mg/ml cholera enterotoxin, 100 units/ ml penicillinestreptomycin, 2 mM L-glutamine and 0.5 mg/ml amphotericin B. 4.3. Immunoblot analysis Equal amounts of protein extracts in SDS-lysis buffer were subjected to 12% SDS-PAGE analysis and electrophoretically transferred to a nitrocellulose membrane. Anti-JNK, anti-phosphorylated JNK, anti-ERK-1/2, anti-phosphorylated ERK-1/2, anti-p38 and anti-phosphorylated p38 antibodies were purchased from Cell Signaling Tech (Beverly, MA). MMP-2 and MMP-9 antibodies were from Santa Cruz Biotech (Santa Cruz, CA). Enhanced chemiluminescence (ECL, AmershamPharmacia, Buckinghamshire, UK) system was used for detection. Relative band intensities were determined by quantitation of each band with an Image analyzer (Vilber Lourmat, France). 4.4. In vitro invasion assay In vitro invasion assay was performed using 24-well transwell unit with polycarbonate filters (Corning Costar, Cambridge, MA) as previously described [26]. The lower side of the filter was coated with type I collagen, and the upper side was coated with Matrigel (Collaborative Research, Lexington, KY). Lower compartment was filled with serum-free media containing 0.1% BSA. Cells were placed in the upper part of the transwell unit, incubated for 17 h, fixed with methanol and stained with hematoxylin for 10 min followed briefly by eosin. The invasive phenotypes were determined by counting the cells that migrated to the lower side of the filter with microscopy at !400. Thirteen fields were counted for each filter and each sample was assayed in triplicate.
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4.5. In vitro migration assay using transwell In vitro migration assay was performed using a 24well transwell unit with polycarbonate filters as previously described [27]. Experimental procedures were the same as the in vitro invasion assay described above except that the filter was not coated with Matrigel for the migration assay. 4.6. Gelatin zymogram assay Cells were cultured in serum-free DMEM/F12 medium containing chemicals for 48 h. Conditioned medium was collected and centrifuged at 3000 rpm for 10 min to remove cell debris. The protein concentration was measured using BCA protein assay reagents (Pierce, Rockford, IL). Gelatinolytic activity of the conditioned medium was determined by gelatin zymogram assay as previously described [26,27]. Areas of gelatinase activity were detected as clear bands against the blue-stained gelatin background. 4.7. Promoter assay (luciferase/b-galactosidase assay) Cells were seeded in a 6-well plate at 1 ! 106 cells/ well and transiently transfected with 4 mg MMP-2 promoter-luciferase construct and MMP-9 promoterluciferase construct (kindly provided by Dr. Benveniste at University of Alabama, Birmingham, AL) and 0.13 mg b-galactosidase expression plasmid pMDV-lacZ. The transfectants were treated with TGF-b for 24 h. The luciferase activity in 1 mg of cell lysate was normalized to b-galactosidase activity and total protein was determined by BCA protein assay kit (Pierce, Rockford, IL). Luciferase and b-galactosidase activities were assayed using luciferase assay kit (Promega, Medison, WI) and Galacto-Light Kit (Tropix Inc, Bedford, MA) and measured with a Luminometer (Tuner Designs, Sunnyvale, CA). Acknowledgements This work was supported by the Korea Food and Drug Administration Grant KFDA-04092-LIF-002. References [1] Massague J. TGF-beta signal transduction. Annu Rev Biochem 1998;67:753–91. [2] Whitman M. Smads and early developmental signaling by the TGFbeta superfamily. Genes Dev 1998;12:2445–62. [3] Alexandrow MG, Moses HL. Transforming growth factor beta and cell cycle regulation. Cancer Res 1995;55:1452–7. [4] Taipale J, Saharinen J, Keski-Oja J. Extracellular matrixassociated transforming growth factor-beta: role in cancer cell growth and invasion. Adv Cancer Res 1998;75:87–134.
[5] Reiss M, Barcellos-Hoff MH. Transforming growth factor-beta in breast cancer: a working hypothesis. Breast Cancer Res Treat 1997;45:81–95. [6] Akhurst RJ, Balmain A. Genetic events and the role of TGF beta in epithelial tumour progression. J Pathol 1999;187:82–90. [7] Portella G, Cumming SA, Liddell J, Cui W, Ireland H, Akhurst RJ, et al. Transforming growth factor beta is essential for spindle cell conversion of mouse skin carcinoma in vivo: implications for tumor invasion. Cell Growth Differ 1998;9: 393–404. [8] McEarchern JA, Kobie JJ, Mack V, Wu RS, Meade-Tollin L, Arteaga CL, et al. Invasion and metastasis of a mammary tumor involves TGF-beta signaling. Int J Cancer 2001;91:76–82. [9] Lin SW, Lee MT, Ke FC, Lee PP, Huang CJ, Ip MM, et al. TGFbeta1 stimulates the secretion of matrix metalloproteinase 2 (MMP2) and the invasive behavior in human ovarian cancer cells, which is suppressed by MMP inhibitor BB3103. Clin Exp Metastasis 2000;18:493–9. [10] Tryggvason K, Hoyhtya M, Pyke C. Type IV collagenases in invasive tumors. Breast Cancer Res Treat 1993;24:209–18. [11] Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, et al. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 1994;370:61–5. [12] Zhang Y, Derynck R. Regulation of Smad signalling by protein associations and signalling crosstalk. Trends Cell Biol 1999;9:274–9. [13] Massague J. How cells read TGF-beta signals. Nat Rev Mol Cell Biol 2000;1:169–78. [14] Atfi A, Djelloul S, Chastre E, Davis R, Gespach C. Evidence for a role of Rho-like GTPases and stress-activated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK) in transforming growth factor beta-mediated signaling. J Biol Chem 1997;272: 1429–32. [15] Hartsough MT, Mulder KM. Transforming growth factor beta activation of p44mapk in proliferating cultures of epithelial cells. J Biol Chem 1995;270:7117–24. [16] Hanafusa H, Ninomiya-Tsuji J, Masuyama N, Nishita M, Fujisawa J, Shibuya H, et al. Involvement of the p38 mitogenactivated protein kinase pathway in transforming growth factorbeta-induced gene expression. J Biol Chem 1999;274:27161–7. [17] Bourne HR, Sanders DA, McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature 1991;349:117–27. [18] Marshall MS. Ras target proteins in eukaryotic cells. FASEB J 1995;9:1311–8. [19] Tanaka Y, Nakayamada S, Fujimoto H, Okada Y, Umehara H, Kataoka T, et al. H-Ras/mitogen-activated protein kinase pathway inhibits integrin-mediated adhesion and induces apoptosis in osteoblasts. J Biol Chem 2002;277:21446–52. [20] Clair T, Miller WR, Cho-Chung YS. Prognostic significance of the expression of a ras protein with a molecular weight of 21,000 by human breast cancer. Cancer Res 1987;47:5290–3. [21] Watson DM, Elton RA, Jack WJ, Dixon JM, Chetty U, Miller WR. The H-ras oncogene product p21 and prognosis in human breast cancer. Breast Cancer Res Treat 1991;17:161–9. [22] Clark GJ, Der CJ. Aberrant function of the Ras signal transduction pathway in human breast cancer. Breast Cancer Res Treat 1995;35:133–44. [23] Oft M, Peli J, Rudaz C, Schwarz H, Beug H, Reichmann E. TGFbeta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells. Genes Dev 1996;10:2462–77. [24] de Caestecker MP, Piek E, Roberts AB. Role of transforming growth factor-beta signaling in cancer. J Natl Cancer Inst 2000;92:1388–402. [25] Derynck R, Akhurst RJ, Balmain A. TGF-beta signaling in tumor suppression and cancer progression. Nat Genet 2001;29:117–29.
E.-S. Kim et al. / Cytokine 29 (2005) 84e91 [26] Moon A, Kim MS, Kim TG, Kim SH, Kim HE, Chen YQ, et al. H-ras, but not N-ras, induces an invasive phenotype in human breast epithelial cells: a role for MMP-2 in the H-ras-induced invasive phenotype. Int J Cancer 2000;85:176–81. [27] Kim MS, Lee EJ, Kim HR, Moon A. p38 Kinase is a key signaling molecule for H-Ras-induced cell motility and invasive phenotype in human breast epithelial cells. Cancer Res 2003;63:5454–61. [28] Kim ES, Kim MS, Moon A. TGF-b-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERKs signaling in MCF10A human breast epithelial cells. Int J Oncol 2004;25:1375–82. [29] Ura H, Bonfil RD, Reich R, Reddel R, Pfeifer A, Harris CC, et al. Expression of type IV collagenase and procollagen genes and its correlation with the tumorigenic, invasive, and metastatic abilities of oncogene-transformed human bronchial epithelial cells. Cancer Res 1989;49:4615–21. [30] Bian J, Sun Y. Transcriptional activation by p53 of the human type IV collagenase (gelatinase A or matrix metalloproteinase 2) promoter. Mol Cell Biol 1997;17:6330–8. [31] Ma Z, Qin H, Benveniste EN. Transcriptional suppression of matrix metalloproteinase-9 gene expression by IFN-gamma and IFN-beta: critical role of STAT-1alpha. J Immunol 2001;167:5150–9. [32] Schwarz LC, Gingras MC, Goldberg G, Greenberg AH, Wright JA. Loss of growth factor dependence and conversion of transforming growth factor-beta 1 inhibition to stimulation in metastatic H-ras-transformed murine fibroblasts. Cancer Res 1988;48:6999–7003. [33] Yin JJ, Selander K, Chirgwin JM, Dallas M, Grubbs BG, Wieser R, et al. TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. J Clin Invest 1999;103:197–206. [34] Akhurst RJ, Derynck R. TGF-beta signaling in cancer a doubleedged sword. Trends Cell Biol 2001;11:S44–51. [35] Oft M, Akhurst RJ, Balmain A. Metastasis is driven by sequential elevation of H-ras and Smad2 levels. Nat Cell Biol 2002;4:487–94. [36] Muraoka RS, Dumont N, Ritter CA, Dugger TC, Brantley DM, Chen J, et al. Blockade of TGF-beta inhibits mammary tumor cell viability, migration, and metastases. J Clin Invest 2002;109: 1551–9.
91
[37] Mulder KM, Morris SL. Activation of p21ras by transforming growth factor beta in epithelial cells. J Biol Chem 1992;267:5029–31. [38] Yan Z, Winawer S, Friedman E. Two different signal transduction pathways can be activated by transforming growth factor beta 1 in epithelial cells. J Biol Chem 1994;269:13231–7. [39] Bakin AV, Rinehart C, Tomlinson AK, Arteaga CL. p38 Mitogen-activated protein kinase is required for TGFbetamediated fibroblastic transdifferentiation and cell migration. J Cell Sci 2002;115:3193–206. [40] Hedges JC, Dechert MA, Yamboliev IA, Martin JL, Hickey E, Weber LA, et al. A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem 1999;274:24211–9. [41] Yu L, Hebert MC, Zhang YE. TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses. EMBO J 2002;21:3749–59. [42] Ellenrieder V, Hendler SF, Ruhland C, Boeck W, Adler G, Gress TM. TGF-beta-induced invasiveness of pancreatic cancer cells is mediated by matrix metalloproteinase-2 and the urokinase plasminogen activator system. Int J Cancer 2001;93:204–11. [43] Johansson N, Ala-aho R, Uitto V, Grenman R, Fusenig NE, Lopez-Otin C, et al. Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase. J Cell Sci 2000;113:227–35. [44] Santibanez JF, Guerrero J, Quintanilla M, Fabra A, Martinez J. Transforming growth factor-beta1 modulates matrix metalloproteinase-9 production through the Ras/MAPK signaling pathway in transformed keratinocytes. Biochem Biophys Res Commun 2002;296:267–73. [45] Ravanti L, Hakkinen L, Larjava H, Saarialho-Kere U, Foschi M, Han J, et al. Transforming growth factor-beta induces collagenase-3 expression by human gingival fibroblasts via p38 mitogenactivated protein kinase. J Biol Chem 1999;274:37292–300. [46] Karsdal MA, Fjording MS, Foged NT, Delaisse JM, Lochter A. Transforming growth factor-beta-induced osteoblast elongation regulates osteoclastic bone resorption through a p38 mitogenactivated protein kinase-and matrix metalloproteinase-dependent pathway. J Biol Chem 2001;276:39350–8. [47] Sehgal I, Thompson TC. Novel regulation of type IV collagenase (matrix metalloproteinase-9 and -2) activities by transforming growth factor-beta1 in human prostate cancer cell lines. Mol Biol Cell 1999;10:407–16.