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MicroRNA expression analysis of mammospheres cultured from human breast cancers

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Abstract

Purpose

There is accumulating evidence suggests that tumors are initiated and maintained by a small fraction of tumor-initiating cells (TICs). TICs can be enriched by mammospheres culturing without surface markers. MicroRNAs participated in many important processes of life including regulating tumorigenicity of TICs. However, roles of miRNAs in TICs of breast cancer are still unknown.

Methods

We compared mammospheres formation of four breast cancer cell lines, cultured mammospheres from breast cancers specimens of three patients and compared microRNAs profiling of mammospheres cultured from breast cancer specimens with differentiated progenies.

Results

Three of the four breast cancer cell lines showed the ability of mammospheres formation. The proportions of CD24cells in mammospheres were increased significantly in the three cell lines. The expression of genes associated with stem cells and chemoresistance increased significantly after mammospheres formation. Breast cancer cells isolated from patients’ specimens survived in nonadherent culture conditions generated mammospheres with ability of self-renewal and bilineage potential. MicroRNA expression profiling of mammospheres compared with differentiated progenies isolated and propagated from the three patients identified 17 microRNAs. And the target genes of these miRNAs are involved in several key signaling pathways.

Conclusions

The results suggested that mammospheres were enriched in TICs and proved a valuable model for studies of breast cancer TICs in vitro, microRNAs played critical roles in maintenance of stemness properties of mammospheres and provided novel insights into breast cancer therapy.

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Abbreviations

TICs:

Tumor-initiating cells

iPSCs:

Induced pluripotent stem cells

References

  • Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100:3983–3988

    Article  PubMed  CAS  Google Scholar 

  • Asiedu MK, Ingle JN, Behrens MD, Radisky DC, Knutson KL (2011) TGF-beta/TNF (alpha)-mediated epithelial-mesenchymal transition generates breast cancer stem cells with a claudin-low phenotype. Cancer Res 71:4707–4719

    Article  PubMed  CAS  Google Scholar 

  • Bussolati B, Grange C, Sapino A, Camussi G (2009) Endothelial cell differentiation of human breast tumor stem/progenitor cells. J Cell Mol Med 13:309–319

    Article  PubMed  CAS  Google Scholar 

  • Curley MD, Therrien VA, Cummings CL, Sergent PA, Koulouris CR, Friel AM, Roberts DJ, Seiden MV, Scadden DT, Rueda BR, Foster R (2009) D133 expression defines a tumor initiating cell population in primary human ovarian cancer. Stem Cells 27:2875–2883

    PubMed  CAS  Google Scholar 

  • Curtin JC, Lorenzi MV (2010) Drug discovery approaches to target Wnt signaling in cancer stem cells. Oncotarget 1:563–577

    PubMed  Google Scholar 

  • Dey D, Saxena M, Paranjape AN, Krishnan V, Giraddi R, Kumar MV, Mukherjee G, Rangarajan A (2009) Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture. PLoS One 4:e5329

    Article  PubMed  Google Scholar 

  • Dong J, Huang S, Caikovski M, Ji S, McGrath A, Custorio MG, Creighton CJ, Maliakkal P, Bogoslovskaia E, Du Z, Zhang X, Lewis MT, Sablitzky F, Brisken C, Li Y (2011) ID4 regulates mammary gland development by suppressing p38MAPK activity. Development 138:5247–5256

    Article  PubMed  CAS  Google Scholar 

  • Garcia DM, Baek D, Shin C, Bell GW, Grimson A, Bartel DP (2011) Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other miRNAs. Nat Struct Mol Biol 18:1139–1146

    Article  PubMed  CAS  Google Scholar 

  • Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1:555–567

    Article  PubMed  CAS  Google Scholar 

  • Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP (2007) MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 27:91–105

    Article  PubMed  CAS  Google Scholar 

  • Honeth G, Bendahl PO, Ringnér M, Saal LH, Gruvberger-Saal SK, Lövgren K, Grabau D, Fernö M, Borg A, Hegardt C (2008) The CD44+/CD24 phenotype is enriched in basal-like breast tumors. Breast Cancer Res 10:R53

    Article  PubMed  Google Scholar 

  • Huang DW, Sherman BT, Lempicki RA (2009a) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protoc 4:44–57

    Article  CAS  Google Scholar 

  • Huang DW, Sherman BT, Lempicki RA (2009b) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37:1–13

    Article  Google Scholar 

  • Hwang-Verslues WW, Kuo WH, Chang PH, Pan CC, Wang HH, Tsai ST, Jeng YM, Shew JY, Kung JT, Chen CH, Lee EY, Chang KJ, Lee WH (2009) Multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers. PLoS One 4:e8377

    Article  PubMed  Google Scholar 

  • Kanehisa M, Goto S, Furumichi M, Tanabe M, Hirakawa M (2010) KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res 38(Database issue):D355–D360

    Article  PubMed  CAS  Google Scholar 

  • Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, Hilsenbeck SG, Pavlick A, Zhang X, Chamness GC, Wong H, Rosen J, Chang JC (2008) Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst 100:672–679

    Article  PubMed  CAS  Google Scholar 

  • Li Y, Vandenboom TG 2nd, Wang Z, Kong D, Ali S, Philip PA, Sarkar FH (2010) miR-146a suppresses invasion of pancreatic cancer cells. Cancer Res 70:1486–1495

    Article  PubMed  CAS  Google Scholar 

  • Li Q, Bian S, Hong J, Kawase-Koga Y, Zhu E, Zheng Y, Yang L, Sun T (2011) Timing specific requirement of microRNA function is essential for embryonic and postnatal hippocampal development. PLoS One 6:e26000

    Article  PubMed  CAS  Google Scholar 

  • Liao M, Zhang CC, Zhou B, Zimonjic DB, Mani SA, Kaba M, Gifford A, Reinhardt F, Popescu NC, Guo W, Eaton EN, Lodish HF, Weinberg RA (2007) Enrichment of a population of mammary gland cells that form mammospheres and have in vivo repopulating activity. Cancer Res 67:8131–8138

    Article  PubMed  CAS  Google Scholar 

  • Lü X, Xu K, Lü H, Yin Y, Ma C, Liu Y, Li H, Suo Z (2011) CD44(+)/CD24(−) cells are transit progenitors and do not determine the molecular subtypes and clinical parameters in breast carcinomas. Ultrastruct Pathol 35:72–78

    Article  PubMed  Google Scholar 

  • Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related inflammation. Nature 454:436–444

    Article  PubMed  CAS  Google Scholar 

  • Mulholland DJ, Xin L, Morim A, Lawson D, Witte O, Wu H (2009) Lin-Sca-1+CD49fhigh stem/progenitors are tumor-initiating cells in the Pten-null prostate cancer model. Cancer Res 69:8555–8562

    Article  PubMed  CAS  Google Scholar 

  • Nahid MA, Pauley KM, Satoh M, Chan EKL (2009) MiR-146a is critical for endotoxin-induced tolerance: implication in innate immunity. J Biol Chem 284:34590–34599

    Article  PubMed  CAS  Google Scholar 

  • Nasser MW, Datta J, Nuovo G, Kutay H, Motiwala T, Majumder S, Wang B, Suster S, Jacob ST, Ghoshal K (2008) Down-regulation of micro-RNA-1 (miR-1) in lung cancer. Suppression of tumorigenic property of lung cancer cells and their sensitization to doxorubicin-induced apoptosis by miR-1. J Biol Chem 283:33394–33405

    Article  PubMed  CAS  Google Scholar 

  • Neveu P, Kye MJ, Qi S, Buchholz DE, Clegg DO, Sahin M, Park IH, Kim KS, Daley GQ, Kornblum HI, Shraiman BI, Kosik KS (2010) MicroRNA profiling reveals two distinct p53-related human pluripotent stem cell states. Cell Stem Cell 7:671–681

    Article  PubMed  CAS  Google Scholar 

  • Pan J, Hu H, Zhou Z, Sun L, Peng L, Yu L, Sun L, Liu J, Yang Z, Ran Y (2010) Tumor-suppressive mir-663 gene induces mitotic catastrophe growth arrest in human gastric cancer cells. Oncol Rep 24:105–112

    PubMed  CAS  Google Scholar 

  • Papageorgis P, Lambert AW, Ozturk S, Gao F, Pan H, Manne U, Alekseyev YO, Thiagalingam A, Abdolmaleky HM, Lenburg M, Thiagalingam S (2010) Smad signaling is required to maintain epigenetic silencing during breast cancer progression. Cancer Res 70:968–978

    Article  PubMed  CAS  Google Scholar 

  • Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, ClayPool K, Tang DG (2005) Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2 cancer cells are similarly tumorigenic. Cancer Res 65:6207–6219

    Article  PubMed  CAS  Google Scholar 

  • Png KJ, Yoshida M, Zhang XH, Shu W, Lee H, Rimner A, Chan TA, Comen E, Andrade VP, Kim SW, King TA, Hudis CA, Norton L, Hick J, Massagué J, Tavazoie SF (2011) MicroRNA-335 inhibits tumor reinitiation and is silenced through genetic and epigenetic mechanisms in human breast cancer. Genes Dev 25:226–231

    Article  PubMed  CAS  Google Scholar 

  • Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S, Pierotti MA, Daidone MG (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65:5506–5511

    Article  PubMed  CAS  Google Scholar 

  • Ricardo S, Vieira AF, Gerhard R, Leitão D, Pinto R, Cameselle-Teijeiro JF, Milanezi F, Schmitt F, Paredes J (2011) Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype. J Clin Pathol 64:937–946

    Article  PubMed  Google Scholar 

  • Sachdeva M, Mo YY (2010) MicroRNA-145 suppresses cell invasion and metastasis by directly targeting mucin 1. Cancer Res 70:378–387

    Article  PubMed  CAS  Google Scholar 

  • Sánchez-Tilló E, de Barrios O, Siles L, Cuatrecasas M, Castells A, Postigo A (2011) β-catenin/TCF4 complex induces the epithelial-to-mesenchymal transition (EMT)-activator ZEB1 to regulate tumor invasiveness. Proc Natl Acad Sci USA 108:19204–19209

    Article  PubMed  Google Scholar 

  • Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, Diehn M, Liu H, Panula SP, Chiao E, Dirbas FM, Somlo G, Pera RA, Lao K, Clarke MF (2009) Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells. Cell 138:592–603

    Article  PubMed  CAS  Google Scholar 

  • Song B, Wang Y, Xi Y, Kudo K, Bruheim S, Botchkina GI, Gavin E, Wan Y, Formentini A, Kornmann M, Fodstad O, Ju J (2009) Mechanism of chemoresistance mediated by miR-140 in human osteosarcoma and colon cancer cells. Oncogene 28:4065–4074

    Article  PubMed  CAS  Google Scholar 

  • Takata A, Otsuka M, Kojima K, Yoshikawa T, Kishikawa T, Yoshida H, Koike K (2011) MicroRNA-22 and microRNA-140 suppress NF-κB activity by regulating the expression of NF-κB coactivators. Biochem Biophys Res Commun 411:826–831

    Article  PubMed  CAS  Google Scholar 

  • Tili E, Michaille JJ, Adair B, Alder H, Limagne E, Taccioli C, Ferracin M, Delmas D, Latruffe N, Croce CM (2010) Resveratrol decreases the level of miR-155 by upregulating miR-663, a microRNA targeting JunB and JunD. Carcinogenesis 31:1561–1566

    Article  PubMed  CAS  Google Scholar 

  • Wang Y, Yu Y, Tsuyada A, Ren X, Wu X, Stubblefield K, Rankin-Gee EK, Wang SE (2011) Transforming growth factor-β regulates the sphere-initiating stem cell-like feature in breast cancer through miRNA-181 and ATM. Oncogene 30:1470–1480

    Article  PubMed  CAS  Google Scholar 

  • Wu Y, Liu S, Xin H, Jiang J, Younglai E, Sun S, Wang H (2011) Up-regulation of microRNA-145 promotes differentiation by repressing OCT4 in human endometrial adenocarcinoma cells. Cancer 117:3989–3998

    Article  PubMed  CAS  Google Scholar 

  • Xu N, Papagiannakopoulos T, Pan G, Thomson JA, Kosik KS (2009) MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell 137:647–658

    Article  PubMed  CAS  Google Scholar 

  • Xu B, Niu X, Zhang X, Tao J, Wu D, Wang Z, Li P, Zhang W, Wu H, Feng N, Wang Z, Hua L, Wang X (2011) MiR-143 decreases prostate cancer cells proliferation and migration and enhances their sensitivity to docetaxel through suppression of KRAS. Mol Cell Biochem 350:207–213

    Article  PubMed  CAS  Google Scholar 

  • Yip NC, Fombon IS, Liu P, Brown S, Kannappan V, Armesilla AL, Xu B, Cassidy J, Darling JL, Wang W (2011) Disulfiram modulated ROS-MAPK and NFκB pathways and targeted breast cancer cells with cancer stem cell-like properties. Br J Cancer 104:1564–1574

    Article  PubMed  CAS  Google Scholar 

  • Yu F, Jiao Y, Zhu Y, Wang Y, Zhu J, Cui X, Liu Y, He Y, Park EY, Zhang H, Lv X, Ma K, Su F, Park JH, Song E (2012a) MicroRNA 34c gene down-regulation via DNA methylation promotes self-renewal and epithelial-mesenchymal transition in breast tumor-initiating cells. J Biol Chem 287:465–473

    Article  PubMed  CAS  Google Scholar 

  • Yu X, Zhang X, Dhakal IB, Beggs M, Kadlubar S, Luo D (2012b) Induction of cell proliferation and survival genes by estradiol-repressed microRNAs in breast cancer cells. BMC Cancer 20:12–29

    Google Scholar 

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The authors of the article have no commercial associations that might pose a conflict of interest in connection with the submitted article.

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Correspondence to Zhang Fengchun.

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432_2012_1272_MOESM1_ESM.tif

Supplementary material 1 (a) Culture of breast cancers cells isolated from three specimens growing as nonadherent mammospheres. (b) Generation of mammosphere from a single cell. (c) Mammosphere formation analysis in primary, secondary and tertiary passages. The number of mammosphere/100 cells generated from single cells increased in the second and third passage (*P < 0.05). Differences between second and third passage were not significant. (TIFF 716 kb)

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Feifei, N., Mingzhi, Z., Yanyun, Z. et al. MicroRNA expression analysis of mammospheres cultured from human breast cancers. J Cancer Res Clin Oncol 138, 1937–1944 (2012). https://doi.org/10.1007/s00432-012-1272-5

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  • DOI: https://doi.org/10.1007/s00432-012-1272-5

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