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NF-κB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2

Abstract

The transcription factor nuclear factor kappa B (NF-κB) is constitutively active in both cancer cells and stromal cells of breast cancer; however, the precise role of activated NF-κB in cancer progression is not known. Using parental MCF10A cells and a variant that expresses the myoepithelial marker p63 stably overexpressing the constitutively active p65 subunit of NF-κB (MCF10A/p65), we show that NF-κB suppresses the expression of epithelial specific genes E-cadherin and desmoplakin and induces the expression of the mesenchymal specific gene vimentin. P65 also suppressed the expression of p63 and the putative breast epithelial progenitor marker cytokeratin 5/6. MCF10A/p65 cells were phenotypically similar to cells undergoing epithelial to mesenchymal transition (EMT). MCF10A/p65 cells failed to form characteristic acini in three-dimensional Matrigel. Analysis of parental and MCF10A/p65 cells for genes previously shown to be involved in EMT revealed elevated expression of ZEB-1 and ZEB-2 in MCF10A/p65 cells compared to parental cells. In transient transfection assays, p65 increased ZEB-1 promoter activity. Furthermore, MCF10A cells overexpressing ZEB-1 showed reduced E-cadherin and p63 expression and displayed an EMT phenotype. The siRNA against ZEB-1 or ZEB-2 reduced the number of viable MCF10A/p65 but not parental cells, suggesting the dependence of MCF10A/p65 cells to ZEB-1 and ZEB-2 for cell cycle progression or survival. MCF10A cells chronically exposed to tumor necrosis factor alpha (TNFα), a potent NF-κB inducer, also exhibited the EMT-like phenotype and ZEB-1/ZEB-2 induction, both of which were reversed following TNFα withdrawal.

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Abbreviations

EMT:

epithelial to mesenchymal transition

IL-1:

interleukin-1

MnSOD:

manganese superoxide dismutase

NF-κB:

nuclear factor kappaB

NLS:

nuclear localization signal

TGFβ:

transforming growth factor beta

TNF:

tumor necrosis factor

References

  • Adriance MC, Inman JL, Petersen OW, Bissell MJ . (2005). Myoepithelial cells: good fences make good neighbors. Breast Cancer Res 7: 190–197.

    Article  CAS  Google Scholar 

  • Akimaru H, Hou DX, Ishii S . (1997). Drosophila CBP is required for dorsal-dependent twist gene expression. Nat Genet 17: 211–214.

    Article  CAS  Google Scholar 

  • Barbareschi M, Pecciarini L, Cangi MG, Macri E, Rizzo A, Viale G et al. (2001). p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Am J Surg Pathol 25: 1054–1060.

    Article  CAS  Google Scholar 

  • Bhat-Nakshatri P, Newton TR, Goulet Jr R, Nakshatri H . (1998). NF-kappaB activation and interleukin 6 production in fibroblasts by estrogen receptor-negative breast cancer cell-derived interleukin 1alpha. Proc Natl Acad Sci USA 95: 6971–6976.

    Article  CAS  Google Scholar 

  • Bhat-Nakshatri P, Sweeney CJ, Nakshatri H . (2002). Identification of signal transduction pathways involved in constitutive NF-kappaB activation in breast cancer cells. Oncogene 21: 2066–2078.

    Article  CAS  Google Scholar 

  • Bhowmick NA, Chytil A, Plieth D, Gorska AE, Dumont N, Shappell S et al. (2004). TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Science 303: 848–851.

    Article  CAS  Google Scholar 

  • Biswas DK, Cruz AP, Gansberger E, Pardee AB . (2000). Epidermal growth factor-induced nuclear factor kappa B activation: a major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells. Proc Natl Acad Sci USA 97: 8542–8547.

    Article  CAS  Google Scholar 

  • Biswas DK, Shi Q, Baily S, Strickland I, Ghosh S, Pardee AB et al. (2004). NF-kappa B activation in human breast cancer specimens and its role in cell proliferation and apoptosis. Proc Natl Acad Sci USA 101: 10137–10142.

    Article  CAS  Google Scholar 

  • Biswas DK, Singh S, Shi Q, Pardee AB, Iglehart JD . (2005). Crossroads of estrogen receptor and NF-kappaB signaling. Sci STKE 2005: pe27.

    PubMed  Google Scholar 

  • Boecker W, Moll R, Dervan P, Buerger H, Poremba C, Diallo RI et al. (2002). Usual ductal hyperplasia of the breast is a committed stem (progenitor) cell lesion distinct from atypical ductal hyperplasia and ductal carcinoma in situ. J Pathol 198: 458–467.

    Article  Google Scholar 

  • Brantley DM, Chen CL, Muraoka RS, Bushdid PB, Bradberry JL, Kittrell F et al. (2001). Nuclear factor-kappaB (NF-kappaB) regulates proliferation and branching in mouse mammary epithelium. Mol Biol Cell 12: 1445–1455.

    Article  CAS  Google Scholar 

  • Cao Y, Bonizzi G, Seagroves TN, Greten FR, Johnson R, Schmidt EV et al. (2001). IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development. Cell 107: 763–775.

    Article  CAS  Google Scholar 

  • Charafe-Jauffret E, Ginestier C, Monville F, Finetti P, Adelaide J, Cervera N et al. (2006). Gene expression profiling of breast cell lines identifies potential new basal markers. Oncogene 25: 2273–2284.

    Article  CAS  Google Scholar 

  • Clarke CL, Sandle J, Parry SC, Reis-Filho JS, O'Hare MJ, Lakhani SR . (2004). Cytokeratin 5/6 in normal human breast: lack of evidence for a stem cell phenotype. J Pathol 204: 147–152.

    Article  CAS  Google Scholar 

  • Clarkson RW, Heeley JL, Chapman R, Aillet F, Hay RT, Wyllie A et al. (2000). NF-kappaB inhibits apoptosis in murine mammary epithelia. J Biol Chem 275: 12737–12742.

    Article  CAS  Google Scholar 

  • Cogswell PC, Guttridge DC, Funkhouser WK, Baldwin Jr AS . (2000). Selective activation of NF-kappaB subunits in human breast cancer: potential roles for NF-kappaB2/p52 and for Bcl-3. Oncogene 19: 1123–1131.

    Article  CAS  Google Scholar 

  • Comijn J, Berx G, Vermassen P, Verschueren K, van Grunsven L, Bruyneel E et al. (2001). The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. Mol Cell 7: 1267–1278.

    Article  CAS  Google Scholar 

  • Debnath J, Mills KR, Collins NL, Reginato MJ, Muthuswamy SK, Brugge JS . (2002). The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell 111: 29–40.

    Article  CAS  Google Scholar 

  • Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 92: 9363–9367.

    Article  CAS  Google Scholar 

  • Dontu G, El-Ashry D, Wicha MS . (2004). Breast cancer, stem/progenitor cells and the estrogen receptor. Trends Endocrinol Metab 15: 193–197.

    Article  CAS  Google Scholar 

  • Eger A, Aigner K, Sonderegger S, Dampier B, Oehler S, Schreiber M et al. (2005). DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells. Oncogene 24: 2375–2385.

    Article  CAS  Google Scholar 

  • Elloul S, Elstrand MB, Nesland JM, Trope CG, Kvalheim G, Goldberg I et al. (2005). Snail, Slug, and Smad-interacting protein 1 as novel parameters of disease aggressiveness in metastatic ovarian and breast carcinoma. Cancer 103: 1631–1643.

    Article  CAS  Google Scholar 

  • Ghosh S, Karin M . (2002). Missing pieces in the NF-kappaB puzzle. Cell 109 (Suppl): S81–S96.

    Article  CAS  Google Scholar 

  • Gordon LA, Mulligan KT, Maxwell-Jones H, Adams M, Walker RA, Jones JL . (2003). Breast cell invasive potential relates to the myoepithelial phenotype. Int J Cancer 106: 8–16.

    Article  CAS  Google Scholar 

  • Guaita S, Puig I, Franci C, Garrido M, Dominguez D, Batlle E et al. (2002). Snail induction of epithelial to mesenchymal transition in tumor cells is accompanied by MUC1 repression and ZEB1 expression. J Biol Chem 277: 39209–39216.

    Article  CAS  Google Scholar 

  • Huber MA, Azoitei N, Baumann B, Grunert S, Sommer A, Pehamberger H et al. (2004). NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression. J Clin Invest 114: 569–581.

    Article  CAS  Google Scholar 

  • Ip MM, Shoemaker SF, Darcy KM . (1992). Regulation of rat mammary epithelial cell proliferation and differentiation by tumor necrosis factor-alpha. Endocrinology 130: 2833–2844.

    Article  CAS  Google Scholar 

  • Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N et al. (2005). Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial–mesenchymal transition. J Cell Biol 171: 1023–1034.

    Article  CAS  Google Scholar 

  • Janda E, Lehmann K, Killisch I, Jechlinger M, Herzig M, Downward J et al. (2002). Ras and TGF[beta] cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J Cell Biol 156: 299–313.

    Article  CAS  Google Scholar 

  • Jonkers J, Berns A . (2004). Oncogene addiction: sometimes a temporary slavery. Cancer Cell 6: 535–538.

    CAS  PubMed  Google Scholar 

  • Karin M, Lin A . (2002). NF-kappaB at the crossroads of life and death. Nat Immunol 3: 221–227.

    Article  CAS  Google Scholar 

  • Keyes WM, Wu Y, Vogel H, Guo X, Lowe SW, Mills AA . (2005). p63 deficiency activates a program of cellular senescence and leads to accelerated aging. Genes Dev 19: 1986–1999.

    Article  CAS  Google Scholar 

  • Kim DW, Sovak MA, Zanieski G, Nonet G, Romieu-Mourez R, Lau AW et al. (2000). Activation of NF-kappaB/Rel occurs early during neoplastic transformation of mammary cells. Carcinogenesis 21: 871–879.

    Article  Google Scholar 

  • Kumar S, Kishimoto H, Chua HL, Badve S, Miller KD, Bigsby RM et al. (2003). Interleukin-1 alpha promotes tumor growth and cachexia in MCF-7 xenograft model of breast cancer. Am J Pathol 163: 2531–2541.

    Article  CAS  Google Scholar 

  • Kuphal S, Poser I, Jobin C, Hellerbrand C, Bosserhoff AK . (2004). Loss of E-cadherin leads to upregulation of NFkappaB activity in malignant melanoma. Oncogene 23: 8509–8519.

    Article  CAS  Google Scholar 

  • Kurose K, Gilley K, Matsumoto S, Watson PH, Zhou XP, Eng C . (2002). Frequent somatic mutations in PTEN and TP53 are mutually exclusive in the stroma of breast carcinomas. Nat Genet 32: 355–357.

    Article  CAS  Google Scholar 

  • Lu T, Sathe SS, Swiatkowski SM, Hampole CV, Stark GR . (2004). Secretion of cytokines and growth factors as a general cause of constitutive NFkappaB activation in cancer. Oncogene 23: 2138–2145.

    Article  CAS  Google Scholar 

  • McKeon F . (2004). p63 and the epithelial stem cell: more than status quo? Genes Dev 18: 465–469.

    Article  CAS  Google Scholar 

  • Meng F, Liu L, Chin PC, D'Mello SR . (2002). Akt is a downstream target of NF-kappa B. J Biol Chem 277: 29674–29680.

    Article  CAS  Google Scholar 

  • Mills KR, Reginato M, Debnath J, Queenan B, Brugge JS . (2004). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is required for induction of autophagy during lumen formation in vitro. Proc Natl Acad Sci USA 101: 3438–3443.

    Article  CAS  Google Scholar 

  • Moody SE, Perez D, Pan TC, Sarkisian CJ, Portocarrero CP, Sterner CJ et al. (2005). The transcriptional repressor Snail promotes mammary tumor recurrence. Cancer Cell 8: 197–209.

    Article  CAS  Google Scholar 

  • Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet Jr RJ, Sledge Jr GW . (1997). Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth. Mol Cell Biol 17: 3629–3639.

    Article  CAS  Google Scholar 

  • Nakshatri H, Goulet Jr RJ . (2002). NF-kappaB and breast cancer. Curr Probl Cancer 26: 282–309.

    Article  Google Scholar 

  • Nieto MA . (2002). The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol 3: 155–166.

    Article  CAS  Google Scholar 

  • Nozaki S, Sledge Jr GW, Nakshatri H . (2000). Cancer cell-derived interleukin 1alpha contributes to autocrine and paracrine induction of pro-metastatic genes in breast cancer. Biochem Biophys Res Commun 275: 60–62.

    Article  CAS  Google Scholar 

  • Ozturk N, Erdal E, Mumcuoglu M, Akcali KC, Yalcin O, Senturk S et al. (2006). Reprogramming of replicative senescence in hepatocellular carcinoma-derived cells. Proc Natl Acad Sci USA 103: 2178–2183.

    Article  CAS  Google Scholar 

  • Pahl HL . (1999). Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 18: 6853–6866.

    Article  CAS  Google Scholar 

  • Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA et al. (2000). Molecular portraits of human breast tumours. Nature 406: 747–752.

    Article  CAS  Google Scholar 

  • Petersen OW, Nielsen HL, Gudjonsson T, Villadsen R, Rank F, Niebuhr E et al. (2003). Epithelial to mesenchymal transition in human breast cancer can provide a nonmalignant stroma. Am J Pathol 162: 391–402.

    Article  CAS  Google Scholar 

  • Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S et al. (2004). NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature 431: 461–466.

    Article  CAS  Google Scholar 

  • Postigo AA . (2003). Opposing functions of ZEB proteins in the regulation of the TGFbeta/BMP signaling pathway. EMBO J 22: 2443–2452.

    Article  CAS  Google Scholar 

  • Postigo AA, Dean DC . (2000). Differential expression and function of members of the zfh-1 family of zinc finger/homeodomain repressors. Proc Natl Acad Sci USA 97: 6391–6396.

    Article  CAS  Google Scholar 

  • Radisky DC, Bissell MJ . (2004). Cancer. Respect thy neighbor!. Science 303: 775–777.

    Article  CAS  Google Scholar 

  • Rapp UR, Gotz R, Albert S . (2006). BuCy RAFs drive cells into MEK addiction. Cancer Cell 9: 9–12.

    Article  CAS  Google Scholar 

  • Rizki A, Bissell MJ . (2004). Homeostasis in the breast: it takes a village. Cancer Cell 6: 1–2.

    Article  CAS  Google Scholar 

  • Romieu-Mourez R, Landesman-Bollag E, Seldin DC, Sonenshein GE . (2002). Protein kinase CK2 promotes aberrant activation of nuclear factor-kappaB, transformed phenotype, and survival of breast cancer cells. Cancer Res 62: 6770–6778.

    CAS  PubMed  Google Scholar 

  • Shin SR, Sanchez-Velar N, Sherr DH, Sonenshein GE . (2006). 7,12-dimethylbenz(a)anthracene treatment of a c-rel mouse mammary tumor cell line induces epithelial to mesenchymal transition via activation of nuclear factor-kappaB. Cancer Res 66: 2570–2575.

    Article  CAS  Google Scholar 

  • Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H et al. (2001). Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98: 10869–10874.

    Article  CAS  Google Scholar 

  • Sosic D, Richardson JA, Yu K, Ornitz DM, Olson EN . (2003). Twist regulates cytokine gene expression through a negative feedback loop that represses NF-kappaB activity. Cell 112: 169–180.

    Article  CAS  Google Scholar 

  • Sovak MA, Bellas RE, Kim DW, Zanieski GJ, Rogers AE, Traish AM et al. (1997). Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer. J Clin Invest 100: 2952–2960.

    Article  CAS  Google Scholar 

  • Tait L, Soule HD, Russo J . (1990). Ultrastructural and immunocytochemical characterization of an immortalized human breast epithelial cell line, MCF-10. Cancer Res 50: 6087–6094.

    CAS  PubMed  Google Scholar 

  • Tarin D, Thompson EW, Newgreen DF . (2005). The fallacy of epithelial mesenchymal transition in neoplasia. Cancer Res 65: 5996–6000.

    Article  CAS  Google Scholar 

  • Thiery JP . (2003). Epithelial–mesenchymal transitions in development and pathologies. Curr Opin Cell Biol 15: 740–746.

    Article  CAS  Google Scholar 

  • Thompson EW, Newgreen DF, Tarin D . (2005). Carcinoma invasion and metastasis: a role for epithelial–mesenchymal transition? Cancer Res 65: 5991–5995.

    Article  CAS  Google Scholar 

  • Tickle C . (1998). Worlds in common through NF-kappaB. Nature 392: 547–549.

    Article  CAS  Google Scholar 

  • Vandewalle C, Comijn J, De Craene B, Vermassen P, Bruyneel E, Andersen H et al. (2005). SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell–cell junctions. Nucleic Acids Res 33: 6566–6578.

    Article  CAS  Google Scholar 

  • Villadsen R . (2005). In search of a stem cell hierarchy in the human breast and its relevance to breast cancer evolution. Apmis 113: 903–921.

    Article  Google Scholar 

  • Welm B, Behbod F, Goodell MA, Rosen JM . (2003). Isolation and characterization of functional mammary gland stem cells. Cell Prolif 36 (Suppl 1): 17–32.

    Article  CAS  Google Scholar 

  • Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C et al. (2004). Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117: 927–939.

    Article  CAS  Google Scholar 

  • Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M et al. (2004). Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial–mesenchymal transition. Nat Cell Biol 6: 931–940.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Meei-Huey Jeng and George Sledge for providing the MCF10A(E) and MCF10A(M) cells, respectively, Tom Genetta for the ZEB-1 cDNA, Geert Berx for the ZEB-2 cDNA, and Louis Pelus for p63 cDNA. We are also grateful to Drs Attaya Suvannasankha, Zhuo Wang and Colin Crean for their critical review of our manuscript. HN is Marian J Morrison Investigator for Breast Cancer Research. This work is supported by the Department of Defense Grant DAMD17-01-1-0274 to H.N.

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Chua, H., Bhat-Nakshatri, P., Clare, S. et al. NF-κB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2. Oncogene 26, 711–724 (2007). https://doi.org/10.1038/sj.onc.1209808

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