Abstract
To identify potential microRNA (miRNA) links between Smad3, a mediator of TGF-β (transforming growth factor-β) signaling, and E-cadherin, we characterized the miRNA profiles of two gastric cancer cell lines: SNU484-LPCX, which does not express Smad3, and SNU484-Smad3, in which Smad3 is overexpressed. We found that among differentially expressed miRNAs, miR-200 family members are overexpressed in SNU484-Smad3 cells. Subsequent studies, including analysis of the effects of silencing Smad3 in SNU484-Smad3 cells and a luciferase reporter assay, revealed that Smad3 directly binds to a Smad-binding element located in the promoter region of miR-200b/a, where it functions as a transcriptional activator. TGF-β did not affect the regulatory role of Smad3 in transcription of miR-200 and expression of epithelial–mesenchymal transition markers. We conclude that Smad3 regulates, at the transcriptional level, miR-200 family members, which themselves regulate ZEB1 and ZEB2, known transcriptional repressors of E-cadherin, at the posttranscriptional level in a TGF-β-independent manner. This represents a novel link between Smad3 and posttranscriptional regulation by miRNAs in epithelial–mesenchymal transition in gastric cancer cells.
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References
Ambros V . (2003). MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell 113: 673–676.
Bartel DP . (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281–297.
Bierie B, Moses HL . (2006). Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer. Nat Rev Cancer 6: 506–520.
Calonge MJ, Massague J . (1999). Smad4/DPC4 silencing and hyperactive Ras jointly disrupt transforming growth factor-beta antiproliferative responses in colon cancer cells. J Biol Chem 274: 33637–33643.
Chung A, Zhang H, Kong YZ, Tan JJ, Huang XR, Kopp JB et al. (2010). Advanced glycation end-products induce tubular CTGF via TGF-β-independent Smad3 signaling. J Am Soc Nephrol 21: 249–260.
Corcoran DL, Pandit KV, Gordon B, Bhattacharjee A, Kaminski N, Benos PV . (2009). Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data. PLoS One 4: e5279.
Elliott RL, Blobe GC . (2005). Role of transforming growth factor Beta in human cancer. J Clin Oncol 23: 2078–2093.
Fink SP, Swinler SE, Lutterbaugh JD, Massague J, Thiagalingam S, Kinzler KW et al. (2001). Transforming growth factor-beta-induced growth inhibition in a Smad4 mutant colon adenoma cell line. Cancer Res 61: 256–260.
Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G et al. (2008). The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 10: 593–601.
Hahm KB, Cho K, Lee C, Im YH, Chang J, Choi SG et al. (1999). Repression of the gene encoding the TGF-beta type II receptor is a major target of the EWS-FLI1 oncoprotein. Nat Genet 23: 222–227.
Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E et al. (1996). DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271: 350–353.
Han SU, Kim HT, Seong DH, Kim YS, Park YS, Bang YJ et al. (2004). Loss of the Smad3 expression increases susceptibility to tumorigenicity in human gastric cancer. Oncogene 23: 1333–1341.
Huang Q, Gumireddy K, Schrier M, le Sage C, Nagel R, Nair S et al. (2008). The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis. Nat Cell Biol 10: 202–210.
Humar B, Blair V, Charlton A, More H, Martin I, Guilford P . (2009). E-cadherin deficiency initiates gastric signet-ring cell carcinoma in mice and man. Cancer Res 69: 2050–2056.
Inoue Y, Canaff L, Hendy GN, Hisa I, Sugimoto T, Chihara K et al. (2009). Role of Smad3, acting independently of transforming growth factor-β, in the early induction of Wnt-β-catenin signaling by parathyroid hormone in mouse osteoblastic cells. J Cell Biochem 108: 285–294.
Kim BG, Li C, Qiao W, Mamura M, Kasprzak B, Anver M et al. (2006). Smad4 signalling in T cells is required for suppression of gastrointestinal cancer. Nature 441: 1015–1019.
Korpal M, Lee ES, Hu G, Kang Y . (2008). The miR-200 family inhibits epithelial-mesenchymal transition and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2. J Biol Chem 283: 14910–14914.
Kurrasch DM, Huang J, Wilkie TM, Repa JJ . (2004). Quantitative real-time polymerase chain reaction measurement of regulators of G-protein signaling mRNA levels in mouse tissues. Methods Enzymol 389: 3–15.
Levy C, Khaled M, Iliopoulos D, Janas MM, Schubert S, Pinner S et al. (2010). Intronic miR-211 assumes the tumor suppressive function of its host gene in melanoma. Mol Cell 40: 841–849.
Li A, Omura N, Hong SM, Vincent A, Walter K, Griffith M et al. (2010). Pancreatic cancers epigenetically silence SIP1 and hypomethylate and overexpress miR-200a/200b in association with elevated circulating miR-200a and miR-200b levels. Cancer Res 70: 5226–5237.
Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D et al. (2005). MicroRNA expression profiles classify human cancers. Nature 435: 834–838.
Ma L, Teruya-Feldstein J, Weinberg RA . (2007). Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449: 682–688.
Ma L, Weinberg RA . (2008). MicroRNAs in malignant progression. Cell Cycle 7: 570–572.
Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J et al. (1995). Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science 268: 1336–1338.
Massague J, Chen YG . (2000). Controlling TGF-beta signaling. Genes Dev 14: 627–644.
Neves R, Scheel C, Weinhold S, Honisch E, Iwaniuk KM, Trompeter HI et al. (2010). Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells. BMC Res Notes 3: 219.
Park K, Kim SJ, Bang YJ, Park JG, Kim NK, Roberts AB et al. (1994). Genetic changes in the transforming growth factor beta (TGF-beta) type II receptor gene in human gastric cancer cells: correlation with sensitivity to growth inhibition by TGF-beta. Proc Natl Acad Sci USA 91: 8772–8776.
Park SM, Gaur AB, Lengyel E, Peter ME . (2008). The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev 22: 894–907.
Parsons R, Myeroff LL, Liu B, Willson JK, Markowitz SD, Kinzler KW et al. (1995). Microsatellite instability and mutations of the transforming growth factor beta type II receptor gene in colorectal cancer. Cancer Res 55: 5548–5550.
Pohl M, Radacz Y, Pawlik N, Schoeneck A, Baldus SE, Munding J et al. (2010). SMAD4 mediates mesenchymal-epithelial reversion in SW480 colon carcinoma cells. Anticancer Res 30: 2603–2613.
Roberts AB, Tian F, Byfield SD, Stuelten C, Ooshima A, Saika S et al. (2006). Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis. Cytokine Growth Factor Rev 17: 19–27.
Roberts AB, Wakefield LM . (2003). The two faces of transforming growth factor beta in carcinogenesis. Proc Natl Acad Sci USA 100: 8621–8623.
Saika S, Kono-Saika S, Ohnishi Y, Sato M, Muragaki Y, Ooshima A et al. (2004). Smad3 signaling is required for epithelial-mesenchymal transition of lens epithelium after injury. Am J Pathol 164: 651–663.
Schetter AJ, Leung SY, Sohn JJ, Zanetti KA, Bowman ED, Yanaihara N et al. (2008). MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. JAMA 299: 425–436.
Schmierer B, Hill CS . (2007). TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol 8: 970–982.
Sen GL, Reuter JA, Webster DE, Zhu L, Khavari PA . (2010). DNMT1 maintains progenitor function in self-renewing somatic tissue. Nature 463: 563–567.
Takagi Y, Koumura H, Futamura M, Aoki S, Ymaguchi K, Kida H et al. (1998). Somatic alterations of the SMAD-2 gene in human colorectal cancers. Br J Cancer 78: 1152–1155.
Tavazoie SF, Alarcon C, Oskarsson T, Padua D, Wang Q, Bos PD et al. (2008). Endogenous human microRNAs that suppress breast cancer metastasis. Nature 451: 147–152.
Ventura A, Jacks T . (2009). MicroRNAs and cancer: short RNAs go a long way. Cell 136: 586–591.
Vincent T, Neve EP, Johnson JR, Kukalev A, Rojo F, Albanell J et al. (2009). A SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-beta mediated epithelial-mesenchymal transition. Nat Cell Biol 11: 943–950.
Vrba L, Jensen TJ, Garbe JC, Heimark RL, Cress AE, Dickinson S et al. (2010). Role for DNA methylation in the regulation of miR-200c and miR-141 expression in normal and cancer cells. PLoS One 5: e8697.
Wheeler JM, Kim HC, Efstathiou JA, Ilyas M, Mortensen NJ, Bodmer WF . (2001). Hypermethylation of the promoter region of the E-cadherin gene (CDH1) in sporadic and ulcerative colitis associated colorectal cancer. Gut 48: 367–371.
Wiklund ED, Bramsen JB, Hulf T, Dyrskjot L, Ramanathan R, Hansen TB et al. (2011). Coordinated epigenetic repression of the miR-200 family and miR-205 in invasive bladder cancer. Int J Cancer 128: 1327–1334.
Yanagisawa K, Uchida K, Nagatake M, Masuda A, Sugiyama M, Saito T et al. (2000). Heterogeneities in the biological and biochemical functions of Smad2 and Smad4 mutants naturally occurring in human lung cancers. Oncogene 19: 2305–2311.
Yang F, Chung A, Huang XR, Lan HY . (2009). Angiotensin II induces connective tissue growth factor and collagen I expression via transforming growth factor-β-dependent and -independent Smad pathways: the role of Smad3. Hypertension 54: 877–884.
Zhu E, Zhao F, Xu G, Hou H, Zhou L, Li X et al. (2010). mirTools: microRNA profiling and discovery based on high-throughput sequencing. Nucleic Acids Res 38: W392–W397.
Acknowledgements
We thank Dr GJ Goodall for kindly providing the DNA constructs. This work was supported by the National Research Foundation grant funded by the Korea government (MEST) to S Hong (KRF-2008-313-C00676 and no. 2009-0081756), JY Cha and SM Ahn (no. 2009-0081789) and by the generous funding from the department of gastroenterology, Gachon University Gil Hospital to D Park.
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Ahn, SM., Cha, JY., Kim, J. et al. Smad3 regulates E-cadherin via miRNA-200 pathway. Oncogene 31, 3051–3059 (2012). https://doi.org/10.1038/onc.2011.484
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DOI: https://doi.org/10.1038/onc.2011.484
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