Abstract
In the mammary gland, the stromal extracellular matrix (ECM) undergoes dramatic changes during development and in tumorigenesis. For example, normal adult breast tissue is largely devoid of the ECM protein fibronectin (FN) whereas high FN levels have been detected in the stroma of breast tumors. FN is an established marker for epithelial-mesenchymal transition (EMT), which occurs during development and has been linked to cancer. During EMT, epithelial cell adhesion switches from cell-cell contacts to mainly cell-ECM interactions, raising the possibility that FN may have a role in promoting this transition. Using MCF-10A mammary epithelial cells, we show that exposure to exogenous FN induces an EMT response including upregulation of the EMT markers FN, Snail, N-cadherin, vimentin, the matrix metalloprotease MMP2, α-smooth muscle actin and phospho-Smad2, as well as acquisition of cell migratory behavior. FN-induced EMT depends on Src kinase and extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase signaling but not on the immediate early gene EGR-1. FN initiates EMT under serum-free conditions; this response is partially reversed by a transforming growth factor (TGF)β-neutralizing antibody, suggesting that FN enhances the effect of endogenous TGFβ. EMT marker expression is upregulated in cells on a fragment of FN containing the integrin-binding domain but not other domains. Differences in gene expression between FN and Matrigel are maintained with addition of a subthreshold level of TGFβ1. Together, these results show that cells interacting with FN are primed to respond to TGFβ. The ability of FN to induce EMT shows an active role for the stromal ECM in this process and supports the notion that the increased levels of FN observed in breast tumors facilitate tumorigenesis.
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References
Wiseman BS, Werb Z . Stromal effects on mammary gland development and breast cancer. Science 2002; 296: 1046–1049.
Shekhar MP, Pauley R, Heppner G . Host microenvironment in breast cancer development: extracellular matrix-stromal cell contribution to neoplastic phenotype of epithelial cells in the breast. Breast Cancer Res 2003; 5: 130–135.
Ghajar CM, Bissell MJ . Extracellular matrix control of mammary gland morphogenesis and tumorigenesis: insights from imaging. Histochem Cell Biol 2008; 130: 1105–1118.
Li ML, Aggeler J, Farson DA, Hatier C, Hassell J, Bissell MJ . Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells. Proc Natl Acad Sci USA 1987; 84: 136–140.
Klinowska TC, Soriano JV, Edwards GM, Oliver JM, Valentijn AJ, Montesano R et al. Laminin and beta1 integrins are crucial for normal mammary gland development in the mouse. Dev Biol 1999; 215: 13–32.
Boudreau N, Sympson CJ, Werb Z, Bissell MJ . Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 1995; 267: 891–893.
Slade MJ, Coope RC, Gomm JJ, Coombes RC . The human mammary gland basement membrane is integral to the polarity of luminal epithelial cells. Exp Cell Res 1999; 247: 267–278.
Christensen L . The distribution of fibronectin, laminin and tetranectin in human breast cancer with special attention to the extracellular matrix. APMIS Suppl 1992; 26: 1–39.
Helleman J, Jansen MP, Ruigrok-Ritstier K, van Staveren IL, Look MP, Meijer-van Gelder ME et al. Association of an extracellular matrix gene cluster with breast cancer prognosis and endocrine therapy response. Clin Cancer Res 2008; 14: 5555–5564.
Ioachim E, Charchanti A, Briasoulis E, Karavasilis V, Tsanou H, Arvanitis DL et al. Immunohistochemical expression of extracellular matrix components tenascin, fibronectin, collagen type IV and laminin in breast cancer: their prognostic value and role in tumour invasion and progression. Eur J Cancer 2002; 38: 2362–2370.
Kadar A, Tokes AM, Kulka J, Robert L . Extracellular matrix components in breast carcinomas. Semin Cancer Biol 2002; 12: 243–257.
Koukoulis GK, Howeedy AA, Korhonen M, Virtanen I, Gould VE . Distribution of tenascin, cellular fibronectins and integrins in the normal, hyperplastic and neoplastic breast. J Submicrosc Cytol Pathol 1993; 25: 285–295.
Yao ES, Zhang H, Chen YY, Lee B, Chew K, Moore D et al. Increased beta1 integrin is associated with decreased survival in invasive breast cancer. Cancer Res 2007; 67: 659–664.
Geiger B, Yamada KM . Molecular architecture and function of matrix adhesions. Cold Spring Harb Perspect Biol 2011; 3: 1–21.
Nelson CM, Bissell MJ . Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu Rev Cell Dev Biol 2006; 22: 287–309.
Debnath J, Brugge JS . Modelling glandular epithelial cancers in three-dimensional cultures. Nat Rev Cancer 2005; 5: 675–688.
Williams CM, Engler AJ, Slone RD, Galante LL, Schwarzbauer JE . Fibronectin expression modulates mammary epithelial cell proliferation during acinar differentiation. Cancer Res 2008; 68: 3185–3192.
Sandal T, Valyi-Nagy K, Spencer VA, Folberg R, Bissell MJ, Maniotis AJ . Epigenetic reversion of breast carcinoma phenotype is accompanied by changes in DNA sequestration as measured by AluI restriction enzyme. Am J Pathol 2007; 170: 1739–1749.
Kalluri R . EMT: when epithelial cells decide to become mesenchymal-like cells. J Clin Invest 2009; 119: 1417–1419.
Kalluri R, Weinberg RA . The basics of epithelial-mesenchymal transition. J Clin Invest 2009; 119: 1420–1428.
Duband JL, Blavet C, Jarov A, Fournier-Thibault C . Spatio-temporal control of neural epithelial cell migration and epithelium-to-mesenchyme transition during avian neural tube development. Dev Growth Differ 2009; 51: 25–44.
Zavadil J, Bottinger EP . TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 2005; 24: 5764–5774.
Sechler JL, Rao H, Cumiskey AM, Vega-Colon I, Smith MS, Murata T et al. A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly. J Cell Biol 2001; 154: 1081–1088.
Lee JM, Dedhar S, Kalluri R, Thompson EW . The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 2006; 172: 973–981.
Kaufmann K, Bach K, Thiel G . The extracellular signal-regulated protein kinases Erk1/Erk2 stimulate expression and biological activity of the transcriptional regulator Egr-1. Biol Chem 2001; 382: 1077–1081.
Gaggioli C, Deckert M, Robert G, Abbe P, Batoz M, Ehrengruber MU et al. HGF induces fibronectin matrix synthesis in melanoma cells through MAP kinase-dependent signaling pathway and induction of Egr-1. Oncogene 2005; 24: 1423–1433.
Grotegut S, von Schweinitz D, Christofori G, Lehembre F . Hepatocyte growth factor induces cell scattering through MAPK/Egr-1-mediated upregulation of Snail. EMBO J 2006; 25: 3534–3545.
Shin S, Dimitri CA, Yoon SO, Dowdle W, Blenis J . ERK2 but not ERK1 induces epithelial-to-mesenchymal transformation via DEF motif-dependent signaling events. Mol Cell 2010; 38: 114–127.
Kasai H, Allen JT, Mason RM, Kamimura T, Zhang Z . TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT). Respir Res 2005; 6: 56.
Miettinen PJ, Ebner R, Lopez AR, Derynck R . TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors. J Cell Biol 1994; 127 (6 Pt 2): 2021–2036.
Willis BC, Borok Z . TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol 2007; 293: L525–L534.
Kim ES, Kim MS, Moon A . TGF-beta-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells. Int J Oncol 2004; 25: 1375–1382.
Schaller MD, Hildebrand JD, Shannon JD, Fox JW, Vines RR, Parsons JT . Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol 1994; 14: 1680–1688.
Schlaepfer DD, Broome MA, Hunter T . Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins. Mol Cell Biol 1997; 17: 1702–1713.
Lu P, Weaver VM, Werb Z . The extracellular matrix: a dynamic niche in cancer progression. J Cell Biol 2012; 196: 395–406.
Komiya E, Furuya M, Watanabe N, Miyagi Y, Higashi S, Miyazaki K . Elevated expression of angiomodulin (AGM/IGFBP-rP1) in tumor stroma and its roles in fibroblast activation. Cancer Sci 2012; 103: 691–699.
Ruelland A, Kerbrat P, Clerc C, Legras B, Cloarec L . Level of plasma fibronectin in patients with breast cancer. Clin Chim Acta 1988; 178: 283–287.
Martino MM, Hubbell JA . The 12th-14th type III repeats of fibronectin function as a highly promiscuous growth factor-binding domain. FASEB J 2010; 24: 4711–4721.
Smith AP, Verrecchia A, Faga G, Doni M, Perna D, Martinato F et al. A positive role for Myc in TGFbeta-induced Snail transcription and epithelial-to-mesenchymal transition. Oncogene 2009; 28: 422–430.
Nieman MT, Prudoff RS, Johnson KR, Wheelock MJ . N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression. J Cell Biol 1999; 147: 631–644.
Xu Q, Isaji T, Lu Y, Gu W, Kondo M, Fukuda T et al. Roles of N-acetylglucosaminyltransferase III in epithelial-to-mesenchymal transition induced by transforming growth factor beta1 (TGF-beta1) in epithelial cell lines. J Biol Chem 2012; 287: 16563–16574.
Martinez-Orozco R, Navarro-Tito N, Soto-Guzman A, Castro-Sanchez L, Perez Salazar E . Arachidonic acid promotes epithelial-to-mesenchymal-like transition in mammary epithelial cells MCF10A. Eur J Cell Biol 2010; 89: 476–488.
Espinosa Neira R, Salazar EP . Native type IV collagen induces an epithelial to mesenchymal transition-like process in mammary epithelial cells MCF10A. Int J Biochem Cell Biol 2012; 44: 2194–2203.
Goyal A, Martin TA, Mansel RE, Jiang WG . Real time PCR analyses of expression of E-cadherin, alpha-, beta- and gamma-catenin in human breast cancer for predicting clinical outcome. World J Surg Oncol 2008; 6: 56.
Giancotti FG, Tarone G . Positional control of cell fate through joint integrin/receptor protein kinase signaling. Annu Rev Cell Dev Biol 2003; 19: 173–206.
Debnath J, Muthuswamy SK, Brugge JS . Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 2003; 30: 256–268.
Engvall E, Ruoslahti E . Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer 1977; 20: 1–5.
Mao Y, Schwarzbauer JE . Stimulatory effects of a three-dimensional microenvironment on cell-mediated fibronectin fibrillogenesis. J Cell Sci 2005; 118 (Pt 19): 4427–4436.
Acknowledgements
We thank Jina Hong for assistance with some experiments. We thank Dr Keith Johnson (University of Nebraska Medical Center) for generously providing N-cadherin antibody. This work was supported by grants from the NIH (R01 GM059383 and R01 CA160611 to JES). JP received support from a Ruth L Kirschstein National Research Service Award (T32 CA 009528).
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Park, J., Schwarzbauer, J. Mammary epithelial cell interactions with fibronectin stimulate epithelial-mesenchymal transition. Oncogene 33, 1649–1657 (2014). https://doi.org/10.1038/onc.2013.118
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DOI: https://doi.org/10.1038/onc.2013.118
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