NF-κB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression
Abstract
The transcription factor NF-κB is activated in a range of human cancers and is thought to promote tumorigenesis, mainly due to its ability to protect transformed cells from apoptosis. To investigate the role of NF-κB in epithelial plasticity and metastasis, we utilized a well-characterized in vitro/in vivo model of mammary carcinogenesis that depends on the collaboration of the Ha-Ras oncoprotein and TGF-β. We show here that the IKK-2/IκBα/NF-κB pathway is required for the induction and maintenance of epithelial-mesenchymal transition (EMT). Inhibition of NF-κB signaling prevented EMT in Ras-transformed epithelial cells, while activation of this pathway promoted the transition to a mesenchymal phenotype even in the absence of TGF-β. Furthermore, inhibition of NF-κB activity in mesenchymal cells caused a reversal of EMT, suggesting that NF-κB is essential for both the induction and maintenance of EMT. In line with the importance of EMT for invasion, blocking of NF-κB activity abrogated the metastatic potential of mammary epithelial cells in a mouse model system. Collectively, these data provide evidence of an essential role for NF-κB during distinct steps of breast cancer progression and suggest that the cooperation of Ras- and TGF-β–dependent signaling pathways in late-stage tumorigenesis depends critically on NF-κB activity.
Acknowledgments
We are grateful to M. Jechlinger for providing EpRas and EpRasXT cells; B. Anic and G. Litos for excellent technical assistance; K. Stangl and P. Garin-Chesa for help with histology; the Institute of Molecular Pathology BioOptics Department for support with immunofluorescence microscopy; and K. Scharffetter-Kochanek, P. Petzlbauer, C. Hoeller, S. Maschler, and M. Herlyn for scientific discussions and useful comments. This work was in part funded by the Genome Research in Austria (GEN-AU) program (to M.A. Huber) and by the German Science Foundation (DFG SFB497/B1 to B. Baumann; SFB451/A9 to T. Wirth).
Footnotes
Nonstandard abbreviations used: benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (Z-VAD-FMK); constitutively active (CA); electrophoretic mobility-shift assay (EMSA); epithelial-mesenchymal transition (EMT); IκB kinase (IKK); inhibitor κB (IκB); NF-κB essential modulator (NEMO); TGF-β1–activated kinase 1 (TAK1); trans-dominant (TD).
Conflict of interest: The authors have declared that no conflict of interest exists.
References
- 1. Orlowski RZ, Baldwin Jr. AS. NF-κB as a therapeutic target in cancer. Trends Mol. Med.2002;8:385–389.[PubMed]
- 2. Karin M, Cao Y, Greten FR, Li ZWNF-κB in cancer: from innocent bystander to major culprit. Nat. Rev. Cancer.2002;2:301–310.[PubMed]
- 3. Ghosh S, May MJ, Kopp EBNF-κB and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol.1998;16:225–260.[PubMed]
- 4. Karin M, Ben-Neriah YPhosphorylation meets ubiquitination: the control of NF-κB activity. Annu. Rev. Immunol.2000;18:621–663.[PubMed]
- 5. Gilmore TDMultiple mutations contribute to the oncogenicity of the retroviral oncoprotein v-Rel. Oncogene.1999;18:6925–6937.[PubMed]
- 6. Cahir-McFarland ED, Izumi KM, Mosialos GEpstein-Barr virus transformation: involvement of latent membrane protein 1-mediated activation of NF-κB. Oncogene.1999;18:6959–6964.[PubMed]
- 7. Sun SC, Ballard DWPersistent activation of NF-κB by the tax transforming protein of HTLV-1: hijacking cellular IκB kinases. Oncogene.1999;18:6948–6958.[PubMed]
- 8. Rayet B, Gelinas CAberrant rel/nfkb genes and activity in human cancer. Oncogene.1999;18:6938–6947.[PubMed]
- 9. Bargou RC, et al Constitutive nuclear factor-κB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells. J. Clin. Invest.1997;100:2961–2969.
- 10. Cabannes E, Khan G, Aillet F, Jarrett RF, Hay RTMutations in the IκBα gene in Hodgkin’s disease suggest a tumour suppressor role for IκBα Oncogene.1999;18:3063–3070.[PubMed]
- 11. Boyer B, Valles AM, Edme NInduction and regulation of epithelial-mesenchymal transitions. Biochem. Pharmacol.2000;60:1091–1099.[PubMed]
- 12. Thiery JPEpithelial-mesenchymal transitions in tumour progression. Nat. Rev. Cancer.2002;2:442–454.[PubMed]
- 13. Thiery JPEpithelial-mesenchymal transitions in development and pathologies. Curr. Opin. Cell Biol.2003;15:740–746.[PubMed]
- 14. Petersen OW, et al Epithelial to mesenchymal transition in human breast cancer can provide a nonmalignant stroma. Am. J. Pathol.2003;162:391–402.
- 15. Grünert S, Jechlinger M, Beug HDiverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis. Nat. Rev. Mol. Cell Biol.2003;4:657–665.[PubMed]
- 16. Hay EDAn overview of epithelio-mesenchymal transformation. Acta Anat. (Basel).1995;154:8–20.[PubMed]
- 17. Janda E, et al Ras and TGFβ cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J. Cell Biol.2002;156:299–313.
- 18. Jechlinger M, et al Expression profiling of epithelial plasticity in tumor progression. Oncogene.2003;22:7155–7169.[PubMed]
- 19. Oft M, et al TGF-β1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells. Genes Dev.1996;10:2462–2477.[PubMed]
- 20. Oft M, Heider KH, Beug HTGFβ signaling is necessary for carcinoma cell invasiveness and metastasis. Curr. Biol.1998;8:1243–1252.[PubMed]
- 21. Lehmann K, et al Raf induces TGFβ production while blocking its apoptotic but not invasive responses: a mechanism leading to increased malignancy in epithelial cells. Genes Dev.2000;14:2610–2622.
- 22. Oft M, Akhurst RJ, Balmain AMetastasis is driven by sequential elevation of H-ras and Smad2 levels. Nat. Cell Biol.2002;4:487–494.[PubMed]
- 23. Gotzmann J, et al Hepatocytes convert to a fibroblastoid phenotype through the cooperation of TGF-β1 and Ha-Ras: steps towards invasiveness. J. Cell. Sci.2002;115:1189–1202.[PubMed]
- 24. Dechend R, et al The Bcl-3 oncoprotein acts as a bridging factor between NF-κB/Rel and nuclear co-regulators. Oncogene.1999;18:3316–3323.[PubMed]
- 25. Cogswell PC, Guttridge DC, Funkhouser WK, Baldwin Jr. AS. Selective activation of NF-κB subunits in human breast cancer: potential roles for NF-κB2/p52 and for Bcl-3. Oncogene.2000;19:1123–1131.[PubMed]
- 26. Adolph KW, Liska DJ, Bornstein PAnalysis of the promoter and transcription start sites of the human thrombospondin 2 gene (THBS2).Gene.1997;193:5–11.[PubMed]
- 27. Pahl HLActivators and target genes of Rel/NF-κB transcription factors. Oncogene.1999;18:6853–6866.[PubMed]
- 28. Arsura M, et al Transient activation of NF-κB through a TAK1/IKK kinase pathway by TGF-β1 inhibits AP-1/SMAD signaling and apoptosis: implications in liver tumor formation. Oncogene.2003;22:412–425.[PubMed]
- 29. Huber MA, et al. The IKK-2/IκBα/NF-κB pathway plays a key role in the regulation of CCR3 and eotaxin-1 in fibroblasts. A critical link to dermatitis in IκBα -deficient mice. J. Biol. Chem.2002;277:1268–1275.[PubMed]
- 30. Kucharczak J, Simmons MJ, Fan Y, Gelinas CTo be, or not to be: NF-κB is the answer—role of Rel/NF-κB in the regulation of apoptosis. Oncogene.2003;22:8961–8982.[PubMed]
- 31. Wang CY, Cusack Jr. JC, Liu R, Baldwin Jr. AS. Control of inducible chemo-resistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-κB. Nat. Med.1999;5:412–417.[PubMed]
- 32. Cusack Jr. JC, Liu R, Baldwin Jr. AS. Inducible chemoresistance to 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin (CPT-11) in colorectal cancer cells and a xenograft model is overcome by inhibition of nuclear factor-κB activation. Cancer Res.2000;60:2323–2330.[PubMed]
- 33. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet Jr. RJ, Sledge Jr. GW. Constitutive activation of NF-κB during progression of breast cancer to hormone-independent growth. Mol. Cell. Biol.1997;17:3629–3639.
- 34. Sovak MA, et al Aberrant nuclear factor-κB/Rel expression and the pathogenesis of breast cancer. J. Clin. Invest.1997;100:2952–2960.
- 35. Romieu-Mourez R, et al Roles of IKK kinases and protein kinase CK2 in activation of nuclear factor-κB in breast cancer. Cancer Res.2001;61:3810–3818.[PubMed]
- 36. Romieu-Mourez R, et al Mouse mammary tumor virus c-rel transgenic mice develop mammary tumors. Mol. Cell. Biol.2003;23:5738–5754.
- 37. Huang S, Pettaway CA, Uehara H, Bucana CD, Fidler IJBlockade of NF-κB activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis. Oncogene.2001;20:4188–4197.[PubMed]
- 38. Helbig G, et al NF-κB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4. J. Biol. Chem.2003;278:21631–21638.[PubMed]
- 39. Huang S, DeGuzman A, Bucana CD, Fidler IJNuclear factor-κB activity correlates with growth, angiogenesis, and metastasis of human melanoma cells in nude mice. Clin. Cancer Res.2000;6:2573–2581.[PubMed]
- 40. Andela VB, Schwarz EM, Puzas JE, O’Keefe RJ, Rosier RNTumor metastasis and the reciprocal regulation of prometastatic and antimetastatic factors by nuclear factor kappa B. Cancer Res.2000;60:6557–6562.[PubMed]
- 41. Dajee M, et al NF-κB blockade and oncogenic Ras trigger invasive human epidermal neoplasia. Nature.2003;421:639–643.[PubMed]
- 42. Romashkova JA, Makarov SSNF-κB is a target of AKT in anti-apoptotic PDGF signalling. Nature.1999;401:86–90.[PubMed]
- 43. Kane LP, Shapiro VS, Stokoe D, Weiss AInduction of NF-κB by the Akt/PKB kinase. Curr. Biol.1999;9:601–604.[PubMed]
- 44. Ozes ON, et al NF-κB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature.1999;401:82–85.[PubMed]
- 45. Madrid LV, Mayo MW, Reuther JY, Baldwin Jr. AS. Akt stimulates the transactivation potential of the RelA/p65 Subunit of NF-κB through utilization of the IκB kinase and activation of the mitogen-activated protein kinase p38. J. Biol. Chem.2001;276:18934–18940.[PubMed]
- 46. Lopez-Rovira T, Chalaux E, Rosa JL, Bartrons R, Ventura F. Interaction and functional cooperation of NF-κB with Smads. Transcriptional regulation of the junB promoter. J. Biol. Chem.2000;275:28937–28946.[PubMed]
- 47. Seoane J, Le H-V, Shen L, Anderson SA, Massague JIntegration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell.2004;117:211–223.[PubMed]
- 48. Brunet A, et al Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell.1999;96:857–868.[PubMed]
- 49. Hu MC-T, et al IκB kinase promotes tumorigenesis through inhibition of forkhead FoxO3a. Cell.2004;117:225–237.[PubMed]
- 50. Lilienbaum A, Duc Dodon M, Alexandre C, Gazzolo L, Paulin DEffect of human T-cell leukemia virus type I tax protein on activation of the human vimentin gene. J. Virol.1990;64:256–263.
- 51. Li X, et al IKKα, IKKβ, and NEMO/IKKγ are each required for the NF-κB–mediated inflammatory response program. J. Biol. Chem.2002;277:45129–45140.
- 52. Katsel PL, Greenstein RJIdentification of overlapping AP-2/NF-κB-responsive elements on the rat cholecystokinin gene promoter. J. Biol. Chem.2001;276:752–758.[PubMed]
- 53. Ueda A, et al NF-κB and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J. Immunol.1994;153:2052–2063.[PubMed]
- 54. Liacini A, et al Induction of matrix metalloproteinase-13 gene expression by TNF-α is mediated by MAP kinases, AP-1, and NF-κB transcription factors in articular chondrocytes. Exp. Cell Res.2003;288:208–217.[PubMed]
- 55. Mettouchi A, et al The c-Jun-induced transformation process involves complex regulation of tenascin-C expression. Mol. Cell. Biol.1997;17:3202–3209.
- 56. Tian B, Brasier ARIdentification of a nuclear factor κB-dependent gene network. Recent Prog. Horm. Res.2003;58:95–130.[PubMed]
- 57. Philip S, Bulbule A, Kundou GCOsteopontin stimulates tumor growth and activation of promatrix metalloproteinase-2 through nuclear factor-κB-mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells. J. Biol. Chem.2001;276:44926–44935.[PubMed]
- 58. Israel A, et al TNF stimulates expression of mouse MHC class I genes by inducing an NF-κB-like enhancer binding activity which displaces constitutive factors. EMBO J.1989;8:3793–3800.
- 59. Israel A, Yano O, Logeat F, Kieran M, Kourilsky PTwo purified factors bind to the same sequence in the enhancer of mouse MHC class I genes: one of them is a positive regulator induced upon differentiation of teratocarcinoma cells. Nucleic Acids Res.1989;17:5245–5257.
- 60. Bitko V, Velazquez A, Yang L, Yang YC, Barik STranscriptional induction of multiple cytokines by human respiratory syncytial virus requires activation of NF-κB and is inhibited by sodium salicylate and aspirin. Virology.1997;232:369–378.[PubMed]
- 61. Ohmori Y, Fukumoto S, Hamilton TATwo structurally distinct kappa B sequence motifs cooperatively control LPS-induced KC gene transcription in mouse macrophages. J. Immunol.1995;155:3593–3600.[PubMed]