- Short Report
- Open Access
Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells
- Rui Neves1,
- Christina Scheel2,
- Sandra Weinhold1,
- Ellen Honisch3,
- Katharina M Iwaniuk1,
- Hans-Ingo Trompeter1,
- Dieter Niederacher3,
- Peter Wernet1,
- Simeon Santourlidis†1 and
- Markus Uhrberg†1Email author
© Uhrberg et al; licensee BioMed Central Ltd. 2010
Received: 22 June 2010
Accepted: 3 August 2010
Published: 3 August 2010
The miR-200c/141 cluster has recently been implicated in the epithelial to mesenchymal transition (EMT) process. The expression of these two miRNAs is inversely correlated with tumorigenicity and invasiveness in several human cancers. The role of these miRNAs in cancer progression is based in part on their capacity to target the EMT activators ZEB1 and ZEB2, two transcription factors, which in turn repress expression of E-cadherin. Little is known about the regulation of the mir200c/141 cluster, whose targeting has been proposed as a promising new therapy for the most aggressive tumors.
We show that the miR-200c/141 cluster is repressed by DNA methylation of a CpG island located in the promoter region of these miRNAs. Whereas in vitro methylation of the miR-200c/141 promoter led to shutdown of promoter activity, treatment with a demethylating agent caused transcriptional reactivation in breast cancer cells formerly lacking expression of miR-200c and miR-141. More importantly, we observed that DNA methylation of the identified miR-200c/141 promoter was tightly correlated with phenotype and the invasive capacity in a panel of 8 human breast cancer cell lines. In line with this, in vitro induction of EMT by ectopic expression of the EMT transcription factor Twist in human immortalized mammary epithelial cells (HMLE) was accompanied by increased DNA methylation and concomitant repression of the miR-200c/141 locus.
The present study demonstrates that expression of the miR-200c/141 cluster is regulated by DNA methylation, suggesting epigenetic regulation of this miRNA locus in aggressive breast cancer cell lines as well as untransformed mammary epithelial cells. This epigenetic silencing mechanism might represent a novel component of the regulatory circuit for the maintenance of EMT programs in cancer and normal cells.
Epithelial to mesenchymal transition (EMT) is considered an essential early step in tumor metastasis formation by controlling the detachment of invasive cancer cells from the primary tumor . Interestingly, EMT is also seen as a facilitator of tissue remodeling during embryonic development. The phenotypical changes and the gain of invasive capacity occurring during EMT are consequences of a cascade of events ultimately leading to downregulation of cell-to-cell adhesion proteins such as E-cadherin. Recently, specific microRNAs (miRNAs), namely members of the miRNA-200 family including miR-200c and miR-141, have been implicated in this process [2–4].
MiRNAs are evolutionary conserved small RNAs, able to modulate gene expression by inhibiting the protein translation process and/or degrading the respective target messenger RNA . They have been shown to participate in many cellular processes including tumorigenesis and specific miRNAs have been assigned either oncogenic or tumor suppressor roles . With respect to the EMT process, observations suggest that members of the miRNA-200 family (especially the two clustered miRNAs miR-200c and miR-141) play a prominent role as metastasis suppressor genes by preventing the expression of zinc finger E-box binding homeobox 1 (ZEB1), which in turn promotes EMT and the switch to an invasive phenotype [4, 7–10]. Importantly, loss of expression of miRNA-200 family members correlates with EMT in various tumor entities such as breast , renal , and ovarian  cancer and thus seems to be a conserved pathway promoting metastasis formation.
During tumorigenesis and EMT, also epigenetic mechanisms, in particular DNA methylation, play a decisive role and contribute to the regulation of key factors involved in this process. While hypermethylation is observed in regulatory regions of many tumor suppressor genes leading to their transcriptional silencing (e.g. E-cadherin ), on the global level genome-wide DNA demethylation is observed .
In order to more closely characterize the miR-200c/141 promoter, luciferase reporter gene assays of the genomic region comprising the putative promoter region including both miRNAs (spanning the region between nucleotide -707 - +501 as defined in Fig. 1A) were performed. Indeed, strong promoter activity was detected in the respective region (Fig. 1B). Furthermore, to determine whether the putative RNA polymerase II (RNA Pol II) promoter is sufficient to enable proper downstream processing of both miRNAs, Northern blot analyses were performed. As shown in Fig. 1C, mature forms of both miRNAs were over-expressed in a time-dependent manner after transfection of Hela cells that express low levels of these two miRNAs.
In cancer, specific epigenetic changes are believed to be early events leading to subsequent changes in gene expression . Given the reported role of miRNA-200c and miRNA-141 in metastasis formation [16, 17] and, more recently, in tumorigenesis, development and stem cells homeostasis [14, 24] we speculated that this locus might be subject to epigenetic regulation. To explore this, we used the MDA-MB-231 breast cancer cells (mesenchymal-like and highly metastatic cell line derived from a pleural effusion of an invasive ductal breast carcinoma) that under normal culture conditions express only residual amounts of these miRNAs [4, 21]. We treated MDA-MB-231 cells with the DNA demethylating agent 5-AZA-CdR. The agent leads to irreversible inhibition of DNMT1, which is the maintenance DNA methyltransferase that copies methylation patterns to the newly synthesized DNA strand during DNA replication . Notably, 5-AZA-CdR is a highly cytotoxic agent that in many cases leads to stalled cell proliferation and accelerated cell death during in vitro culture. In order to diminish these problems, in the present work we used mild dosages of 5-AZA-CdR (0,2 μM and 1 μM) (Additional file 1: Materials and Methods). This enabled successful propagation of cell culture experiments for periods of more than 30 days.
To exclude the possibility that activation of the miR-200c/141 cluster observed by chemical demethylation was mainly due to an indirect effect, e.g. activation of third party transcription factors, we next explored if expression of the miRNA cluster could be directly inhibited by DNA methylation. In order to reduce unspecific background signals we used an expression vector harboring a CpG-free luciferase transcriptional unit. This construct was methylated in-vitro using the DNA methylase Sss1 before being introduced into Hela cells. Indeed, after in-vitro methylation, promoter activity was strongly silenced (Fig. 2B) and together, these observations suggest a direct role of DNA methylation in transcriptional regulation of the miR-200c/141 cluster.
Consistent with previous observations , only breast cancer lines with an epithelial phenotype exhibited expression of the two miRNAs (Fig. 3B). Of note, the expression levels in the epithelial cell lines were substantially higher than the levels we could reach by demethylation of the mesenchymal MDA-MB-231 line, which is again consistent with incomplete demethylation of the miR-200c/141 promoter during 5-AZA-CdR treatment (Additional file 1: Supplementary Figure S2).
During preparation of this manuscript, a correlation between the expression levels of miR-200c/141 and the degree of DNA methylation of the promoter-associated CpG island was also reported by Vrba and colleagues . Similar to the present study, the authors demonstrate a correlation of DNA methylation levels with invasive phenotype and the capacity of 5-AZA-CdR to reactivate the expression of the formerly silenced miRNAs in invasive breast cancer cell lines. The present study goes beyond the correlative analysis by providing evidence that expression of the miR-200c/141 locus is indeed partly controlled by DNA methylation. Firstly, in vitro methylation experiments showing how DNA methylation of the miR200c/141 locus shuts down expression of miR-200c and miR-141 provides a functional link between DNA methylation of the promoter and expression of the miR-200c/141 locus. Secondly, ectopic expression of the EMT inducer Twist led to a limited increase of DNA methylation in the miR200c/141 promoter, which was nevertheless accompanied by complete shut down of miRNA expression. The latter data indicate that even limited levels of DNA methylation can cause transcriptional silencing of the miRNA locus. Importantly, the effect of Twist on DNA methylation levels shown in our study further stresses the functional relevance of epigenetic changes in the miR200c/141 locus and suggests a potential role for epigenetic regulation of EMT.
Although the present work supports the idea that changes in DNA methylation of this particular locus might be involved in EMT, it remains to be determined if the initial trigger to shutdown the miR-200c/141 promoter during the EMT process is given by changes in DNA methylation levels or binding of repressors (as ZEB1, ZEB2, or Twist) to the promoter or if several of these repressor mechanisms act simultaneously and synergistically. In the latter case, DNA methylation of miRNAs in conjunction with ZEB1 expression would then support transition to a mesenchymal phenotype. Interestingly, in clones established after experimental knockdown of ZEB1 in MDA-MB-231 cells, others observed an upregulation of miR-200c/141 expression . This opens the possibility that ZEB1 might be necessary for maintaining DNA methylation of the miR-200c/141 promoter. In this regard, it is known that ZEB1 interacts with CtBP , that in turn interacts with components of the Polycomb complex . As these complexes promote DNA methylation via interaction with DNMTs , ZEB1 could indeed enforce DNA methylation of the miR-200c/141 promoter. These questions surely deserve further investigation.
We thank Dr. Tânia Costa (Karolinska Institutet, Stockolm), Dr. Joana Paredes (IPATIMUP, Porto) and Dr. Sandra Costa (Minho University, Braga) for providing the BT-549, Hs578T and HBL-100 cells, respectively. Additionally, we thank also Dr. Li Ma from the Weinberg laboratory (Whitehead Institute/MIT) for critical reading of the manuscript and Dr. Kaiqin Lao (Applied Biosystems, Foster City) for kindly providing the TaqMan® MicroRNA multiplex assay. R.N. and M.U. were supported by Marie-Curie Actions (RTN-CT 2004-512253 "TRANS-NET") and the Forschungskommission of the Medical Faculty of the HHU Düsseldorf. H.I.T., P.W., and M.U. were supported by the Deutsche Forschungsgemeinschaft (FOR717/1). Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- Thiery JP: Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer. 2002, 2: 442-454. 10.1038/nrc822.PubMedView ArticleGoogle Scholar
- Park SM, Gaur AB, Lengyel E, Peter ME: The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev. 2008, 22: 894-907. 10.1101/gad.1640608.PubMed CentralPubMedView ArticleGoogle Scholar
- Korpal M, Lee ES, Hu G, Kang Y: 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. 2008, 283: 14910-14914. 10.1074/jbc.C800074200.PubMed CentralPubMedView ArticleGoogle Scholar
- Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, Vadas MA, Khew-Goodall Y, Goodall GJ: The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol. 2008, 10: 593-601. 10.1038/ncb1722.PubMedView ArticleGoogle Scholar
- Bartel DP: MicroRNAs: target recognition and regulatory functions. Cell. 2009, 136: 215-233. 10.1016/j.cell.2009.01.002.PubMed CentralPubMedView ArticleGoogle Scholar
- Esquela-Kerscher A, Slack FJ: Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer. 2006, 6: 259-269. 10.1038/nrc1840.PubMedView ArticleGoogle Scholar
- Nam EJ, Yoon H, Kim SW, Kim H, Kim YT, Kim JH, Kim JW, Kim S: MicroRNA Expression Profiles in Serous Ovarian Carcinoma. Clin Cancer Res. 2008, 14: 2690-2695. 10.1158/1078-0432.CCR-07-1731.PubMedView ArticleGoogle Scholar
- Taylor DD, Gercel-Taylor C: MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol. 2008, 110: 13-21. 10.1016/j.ygyno.2008.04.033.PubMedView ArticleGoogle Scholar
- Iorio MV, Visone R, Di Leva G, Donati V, Petrocca F, Casalini P, Taccioli C, Volinia S, Liu CG, Alder H: MicroRNA signatures in human ovarian cancer. Cancer Res. 2007, 67: 8699-8707. 10.1158/0008-5472.CAN-07-1936.PubMedView ArticleGoogle Scholar
- Hurteau GJ, Carlson JA, Spivack SD, Brock GJ: Overexpression of the microRNA hsa-miR-200c leads to reduced expression of transcription factor 8 and increased expression of E-cadherin. Cancer Res. 2007, 67: 7972-7976. 10.1158/0008-5472.CAN-07-1058.PubMedView ArticleGoogle Scholar
- Nakada C, Matsuura K, Tsukamoto Y, Tanigawa M, Yoshimoto T, Narimatsu T, Nguyen LT, Hijiya N, Uchida T, Sato F: Genome-wide microRNA expression profiling in renal cell carcinoma: significant down-regulation of miR-141 and miR-200c. J Pathol. 2008, 216: 418-427. 10.1002/path.2437.PubMedView ArticleGoogle Scholar
- Lombaerts M, van Wezel T, Philippo K, Dierssen JW, Zimmerman RM, Oosting J, van Eijk R, Eilers PH, van de WB, Cornelisse CJ: E-cadherin transcriptional downregulation by promoter methylation but not mutation is related to epithelial-to-mesenchymal transition in breast cancer cell lines. Br J Cancer. 2006, 94: 661-671.PubMed CentralPubMedGoogle Scholar
- Esteller M, Herman JG: Cancer as an epigenetic disease: DNA methylation and chromatin alterations in human tumours. J Pathol. 2002, 196: 1-7. 10.1002/path.1024.PubMedView ArticleGoogle Scholar
- Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A, Waldvogel B, Vannier C, Darling D, zur HA: The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol. 2009, 11: 1487-1495. 10.1038/ncb1998.PubMedView ArticleGoogle Scholar
- Saydam O, Shen Y, Wurdinger T, Senol O, Boke E, James MF, Tannous BA, Stemmer-Rachamimov AO, Yi M, Stephens RM: Downregulated microRNA-200a in meningiomas promotes tumor growth by reducing E-cadherin and activating the Wnt/beta-catenin signaling pathway. Mol Cell Biol. 2009, 29: 5923-5940. 10.1128/MCB.00332-09.PubMed CentralPubMedView ArticleGoogle Scholar
- Dykxhoorn DM, Wu Y, Xie H, Yu F, Lal A, Petrocca F, Martinvalet D, Song E, Lim B, Lieberman J: miR-200 enhances mouse breast cancer cell colonization to form distant metastases. PLoS One. 2009, 4: e7181-10.1371/journal.pone.0007181.PubMed CentralPubMedView ArticleGoogle Scholar
- Hu M, Xia M, Chen X, Lin Z, Xu Y, Ma Y, Su L: MicroRNA-141 Regulates Smad Interacting Protein 1 (SIP1) and Inhibits Migration and Invasion of Colorectal Cancer Cells. Dig Dis Sci. 2009Google Scholar
- Bracken CP, Gregory PA, Kolesnikoff N, Bert AG, Wang J, Shannon MF, Goodall GJ: A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition. Cancer Res. 2008, 68: 7846-7854. 10.1158/0008-5472.CAN-08-1942.PubMedView ArticleGoogle Scholar
- Burk U, Schubert J, Wellner U, Schmalhofer O, Vincan E, Spaderna S, Brabletz T: A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells. EMBO Rep. 2008, 9: 582-589. 10.1038/embor.2008.74.PubMed CentralPubMedView ArticleGoogle Scholar
- Jones PA, Takai D: The role of DNA methylation in mammalian epigenetics. Science. 2001, 293: 1068-1070. 10.1126/science.1063852.PubMedView ArticleGoogle Scholar
- Lacroix M, Leclercq G: Relevance of breast cancer cell lines as models for breast tumours: an update. Breast Cancer Res Treat. 2004, 83: 249-289. 10.1023/B:BREA.0000014042.54925.cc.PubMedView ArticleGoogle Scholar
- Saini HK, Griffiths-Jones S, Enright AJ: Genomic analysis of human microRNA transcripts. Proc Natl Acad Sci USA. 2007, 104: 17719-17724. 10.1073/pnas.0703890104.PubMed CentralPubMedView ArticleGoogle Scholar
- Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M: A mammalian microRNA expression atlas based on small RNA library sequencing. Cell. 2007, 129: 1401-1414. 10.1016/j.cell.2007.04.040.PubMed CentralPubMedView ArticleGoogle Scholar
- Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, Diehn M, Liu H, Panula SP, Chiao E: Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells. Cell. 2009, 138: 592-603. 10.1016/j.cell.2009.07.011.PubMed CentralPubMedView ArticleGoogle Scholar
- Jones PA, Taylor SM: Cellular differentiation, cytidine analogs and DNA methylation. Cell. 1980, 20: 85-93. 10.1016/0092-8674(80)90237-8.PubMedView ArticleGoogle Scholar
- Yoshiura K, Kanai Y, Ochiai A, Shimoyama Y, Sugimura T, Hirohashi S: Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. Proc Natl Acad Sci USA. 1995, 92: 7416-7419. 10.1073/pnas.92.16.7416.PubMed CentralPubMedView ArticleGoogle Scholar
- Paine TM, Soule HD, Pauley RJ, Dawson PJ: Characterization of epithelial phenotypes in mortal and immortal human breast cells. Int J Cancer. 1992, 50: 463-473. 10.1002/ijc.2910500323.PubMedView ArticleGoogle Scholar
- Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, Savagner P, Gitelman I, Richardson A, Weinberg RA: Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell. 2004, 117: 927-939. 10.1016/j.cell.2004.06.006.PubMedView ArticleGoogle Scholar
- Vrba L, Jensen TJ, Garbe JC, Heimark RL, Cress AE, Dickinson S, Stampfer MR, Futscher BW: Role for DNA methylation in the regulation of miR-200c and miR-141 expression in normal and cancer cells. PLoS One. 2010, 5: e8697-10.1371/journal.pone.0008697.PubMed CentralPubMedView ArticleGoogle Scholar
- Postigo AA, Dean DC: ZEB represses transcription through interaction with the corepressor CtBP. Proc Natl Acad Sci USA. 1999, 96: 6683-6688. 10.1073/pnas.96.12.6683.PubMed CentralPubMedView ArticleGoogle Scholar
- Chinnadurai G: Transcriptional regulation by C-terminal binding proteins. Int J Biochem Cell Biol. 2007, 39: 1593-1607. 10.1016/j.biocel.2007.01.025.PubMedView ArticleGoogle Scholar
- Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A, Bernard D, Vanderwinden JM: The Polycomb group protein EZH2 directly controls DNA methylation. Nature. 2006, 439: 871-874. 10.1038/nature04431.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.