miR-200 enhances mouse breast cancer cell colonization to form distant metastases
Derek M Dykxhoorn, Yichao Wu, Huangming Xie, Fengyan Yu, Ashish Lal, Fabio Petrocca, Denis Martinvalet, Erwei Song, Bing Lim, Judy Lieberman, Derek M Dykxhoorn, Yichao Wu, Huangming Xie, Fengyan Yu, Ashish Lal, Fabio Petrocca, Denis Martinvalet, Erwei Song, Bing Lim, Judy Lieberman
Abstract
Background: The development of metastases involves the dissociation of cells from the primary tumor to penetrate the basement membrane, invade and then exit the vasculature to seed, and colonize distant tissues. The last step, establishment of macroscopic tumors at distant sites, is the least well understood. Four isogenic mouse breast cancer cell lines (67NR, 168FARN, 4TO7, and 4T1) that differ in their ability to metastasize when implanted into the mammary fat pad are used to model the steps of metastasis. Only 4T1 forms macroscopic lung and liver metastases. Because some miRNAs are dysregulated in cancer and affect cellular transformation, tumor formation, and metastasis, we examined whether changes in miRNA expression might explain the differences in metastasis of these cells.
Methodology/principal findings: miRNA expression was analyzed by miRNA microarray and quantitative RT-PCR in isogenic mouse breast cancer cells with distinct metastatic capabilities. 4T1 cells that form macroscopic metastases had elevated expression of miR-200 family miRNAs compared to related cells that invade distant tissues, but are unable to colonize. Moreover, over-expressing miR-200 in 4TO7 cells enabled them to metastasize to lung and liver. These findings are surprising since the miR-200 family was previously shown to promote epithelial characteristics by inhibiting the transcriptional repressor Zeb2 and thereby enhancing E-cadherin expression. We confirmed these findings in these cells. The most metastatic 4T1 cells acquired epithelial properties (high expression of E-cadherin and cytokeratin-18) compared to the less metastatic cells.
Conclusions/significance: Expression of miR-200, which promotes a mesenchymal to epithelial cell transition (MET) by inhibiting Zeb2 expression, unexpectedly enhances macroscopic metastases in mouse breast cancer cell lines. These results suggest that for some tumors, tumor colonization at metastatic sites might be enhanced by MET. Therefore the epithelial nature of a tumor does not predict metastatic outcome.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
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References
- Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297.
- Dykxhoorn DM, Chowdhury D, Lieberman J. RNA interference and cancer: endogenous pathways and therapeutic approaches. Adv Exp Med Biol. 2008;615:299–329.
- Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature. 2005;433:769–773.
- Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A. 2005;102:13944–13949.
- Eis PS, Tam W, Sun L, Chadburn A, Li Z, et al. Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci U S A. 2005;102:3627–3632.
- Kluiver J, Poppema S, de Jong D, Blokzijl T, Harms G, et al. BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas. J Pathol. 2005;207:243–249.
- Asangani IA, Rasheed SA, Nikolova DA, Leupold JH, Colburn NH, et al. MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene. 2008;27:2128–2136.
- Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 2005;65:7065–7070.
- Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 2005;65:6029–6033.
- Zhu S, Wu H, Wu F, Nie D, Sheng S, et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res. 2008;18:350–359.
- Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature. 2007;449:682–688.
- Segura MF, Hanniford D, Menendez S, Reavie L, Zou X, et al. Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor. Proc Natl Acad Sci U S A. 2009;106:1814–1819.
- Gebeshuber CA, Zatloukal K, Martinez J. miR-29a suppresses tristetraprolin, which is a regulator of epithelial polarity and metastasis. EMBO Rep 2009
- Tavazoie SF, Alarcon C, Oskarsson T, Padua D, Wang Q, et al. Endogenous human microRNAs that suppress breast cancer metastasis. Nature. 2008;451:147–152.
- Yu F, Yao H, Zhu P, Zhang X, Pan Q, et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell. 2007;131:1109–1123.
- Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev Med. 2007;58:267–284.
- Dalerba P, Clarke MF. Cancer stem cells and tumor metastasis: first steps into uncharted territory. Cell Stem Cell. 2007;1:241–242.
- Wicha MS. Cancer stem cells and metastasis: lethal seeds. Clin Cancer Res. 2006;12:5606–5607.
- Al-Hajj M. Cancer stem cells and oncology therapeutics. Curr Opin Oncol. 2007;19:61–64.
- Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, et al. Cancer stem cells–perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006;66:9339–9344.
- Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer. 2002;2:442–454.
- Berx G, Raspe E, Christofori G, Thiery JP, Sleeman JP. Pre-EMTing metastasis? Recapitulation of morphogenetic processes in cancer. Clin Exp Metastasis. 2007;24:587–597.
- Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet. 2008;40:499–507.
- Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 2007;449:557–563.
- Baum B, Settleman J, Quinlan MP. Transitions between epithelial and mesenchymal states in development and disease. Semin Cell Dev Biol. 2008;19:294–308.
- Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer. 2007;7:415–428.
- Moreno-Bueno G, Cubillo E, Sarrio D, Peinado H, Rodriguez-Pinilla SM, et al. Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a distinct role for Snail, Slug, and E47 factors in epithelial-mesenchymal transition. Cancer Res. 2006;66:9543–9556.
- Wells A, Yates C, Shepard CR. E-cadherin as an indicator of mesenchymal to epithelial reverting transitions during the metastatic seeding of disseminated carcinomas. Clin Exp Metastasis. 2008;25:621–628.
- Aslakson CJ, Miller FR. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res. 1992;52:1399–1405.
- Bracken CP, Gregory PA, Kolesnikoff N, Bert AG, Wang J, et al. A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition. Cancer Res. 2008;68:7846–7854.
- Gregory PA, Bracken CP, Bert AG, Goodall GJ. MicroRNAs as regulators of epithelial-mesenchymal transition. Cell Cycle. 2008;7:3112–3118.
- Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol. 2008;10:593–601.
- Burk U, Schubert J, Wellner U, Schmalhofer O, Vincan E, et al. 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.
- Parker C, Rampaul RS, Pinder SE, Bell JA, Wencyk PM, et al. E-cadherin as a prognostic indicator in primary breast cancer. Br J Cancer. 2001;85:1958–1963.
- 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.
- 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.
- Peter ME. Let-7 and miR-200 microRNAs: Guardians against pluripotency and cancer progression. Cell Cycle. 2009;8
- Liu G, Min H, Yue S, Chen CZ. Pre-miRNA loop nucleotides control the distinct activities of mir-181a-1 and mir-181c in early T cell development. PLoS ONE. 2008;3:e3592. doi: .
- Moll R, Divo M, Langbein L. The human keratins: biology and pathology. Histochem Cell Biol. 2008;129:705–733.
- Hashizume R, Koizumi H, Ihara A, Ohta T, Uchikoshi T. Expression of beta-catenin in normal breast tissue and breast carcinoma: a comparative study with epithelial cadherin and alpha-catenin. Histopathology. 1996;29:139–146.
- Gonzalez MA, Pinder SE, Wencyk PM, Bell JA, Elston CW, et al. An immunohistochemical examination of the expression of E-cadherin, alpha- and beta/gamma-catenins, and alpha2- and beta1-integrins in invasive breast cancer. J Pathol. 1999;187:523–529.
- Lipponen P, Saarelainen E, Ji H, Aaltomaa S, Syrjanen K. Expression of E-cadherin (E-CD) as related to other prognostic factors and survival in breast cancer. J Pathol. 1994;174:101–109.
- Kartenbeck J, Haselmann U, Gassler N. Synthesis of junctional proteins in metastasizing colon cancer cells. Eur J Cell Biol. 2005;84:417–430.
- Gabbert HE, Mueller W, Schneiders A, Meier S, Moll R, et al. Prognostic value of E-cadherin expression in 413 gastric carcinomas. Int J Cancer. 1996;69:184–189.
- Chaffer CL, Dopheide B, McCulloch DR, Lee AB, Moseley JM, et al. Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder. Clin Exp Metastasis. 2005;22:115–125.
- Chaffer CL, Brennan JP, Slavin JL, Blick T, Thompson EW, et al. Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis: role of fibroblast growth factor receptor-2. Cancer Res. 2006;66:11271–11278.
- Bracken CP, Gregory PA, Khew-Goodall Y, Goodall GJ. The role of microRNAs in metastasis and epithelial-mesenchymal transition. Cell Mol Life Sci 2009
- Huang Q, Gumireddy K, Schrier M, le Sage C, Nagel R, et al. The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis. Nat Cell Biol. 2008;10:202–210.
- Yan LX, Huang XF, Shao Q, Huang MY, Deng L, et al. MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA. 2008;14:2348–2360.
- Wickramasinghe NS, Manavalan TT, Dougherty SM, Riggs KA, Li Y, et al. Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells. Nucleic Acids Res 2009
- Iorio MV, Visone R, Di Leva G, Donati V, Petrocca F, et al. MicroRNA signatures in human ovarian cancer. Cancer Res. 2007;67:8699–8707.
- Nam EJ, Yoon H, Kim SW, Kim H, Kim YT, et al. MicroRNA Expression Profiles in Serous Ovarian Carcinoma. Clin Cancer Res. 2008;14:2690–2695.
- Zhang L, Huang J, Yang N, Greshock J, Megraw MS, et al. microRNAs exhibit high frequency genomic alterations in human cancer. Proc Natl Acad Sci U S A. 2006;103:9136–9141.
- Kim SW, Kim JW, Kim YT, Kim JH, Kim S, et al. Analysis of chromosomal changes in serous ovarian carcinoma using high-resolution array comparative genomic hybridization: Potential predictive markers of chemoresistant disease. Genes Chromosomes Cancer. 2007;46:1–9.
- Meng F, Henson R, Lang M, Wehbe H, Maheshwari S, et al. Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology. 2006;130:2113–2129.
- Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 2009;19:92–105.
- Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411:494–498.
- Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet. 2003;33:401–406.
- Chang K, Elledge SJ, Hannon GJ. Lessons from Nature: microRNA-based shRNA libraries. Nat Methods. 2006;3:707–714.
- Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 1993;41:1599–1604.
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