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  • Original Article
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Zeb1 is required for TrkB-induced epithelial-mesenchymal transition, anoikis resistance and metastasis

Abstract

Anoikis (detachment-induced apoptosis) prevents the survival of cells at inappropriate sites of the body and can therefore act as a barrier to metastasis. In a function-based genome-wide screen, we have previously identified the neurotrophic tyrosine kinase receptor TrkB as a potent suppressor of anoikis. Consistently, activated TrkB oncogenically transforms non-malignant epithelial cells and causes them to invade and produce metastatic tumors in vivo. Overexpression of activated TrkB also results in morphological transformation, resembling epithelial-mesenchymal transition (EMT). E-cadherin, an important EMT regulator, and two E-cadherin repressors, Twist and Snail, are critical for these TrkB functions. As Snail has been shown to induce Zeb1, another E-cadherin repressor, we hypothesized that Zeb1 could be a TrkB target, too. We show here that Zeb1 is required for TrkB-induced EMT in epithelial cells, as RNAi-mediated knockdown of Zeb1 reverted the morphological changes induced by TrkB. Furthermore, Zeb1 is involved in TrkB-induced anoikis resistance, migration and invasion. In vivo, knockdown of Zeb1 strongly reduced TrkB-induced metastasis. Finally, epistasis experiments showed that Zeb1 acts downstream of Twist and Snail. We conclude that Zeb1 is required for several TrkB-induced effects in vitro and in vivo, including metastasis.

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References

  • Adachi Y, Takeuchi T, Nagayama T, Ohtsuki Y, Furihata M . (2009). Zeb1-mediated T-cadherin repression increases the invasive potential of gallbladder cancer. FEBS Lett 583: 430–436.

    Article  CAS  PubMed  Google Scholar 

  • Aigner K, Dampier B, Descovich L, Mikula M, Sultan A, Schreiber M et al. (2007a). The transcription factor ZEB1 (deltaEF1) promotes tumour cell dedifferentiation by repressing master regulators of epithelial polarity. Oncogene 26: 6979–6988.

    Article  CAS  PubMed  Google Scholar 

  • Aigner K, Descovich L, Mikula M, Sultan A, Dampier B, Bonne S et al. (2007b). The transcription factor ZEB1 (deltaEF1) represses Plakophilin 3 during human cancer progression. FEBS Lett 581: 1617–1624.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Batlle E, Sancho E, Franci C, Dominguez D, Monfar M, Baulida J et al. (2000). The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2: 84–89.

    Article  CAS  PubMed  Google Scholar 

  • Bendoraite A, Knouf EC, Garg KS, Parkin RK, Kroh EM, O′Briant KC et al. (2010). Regulation of miR-200 family microRNAs and ZEB transcription factors in ovarian cancer: evidence supporting a mesothelial-to-epithelial transition. Gynecol Oncol 116: 117–125.

    Article  CAS  PubMed  Google Scholar 

  • Bracken CP, Gregory PA, Kolesnikoff N, Bert AG, Wang J, Shannon MF et al. (2008). A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition. Cancer Res 68: 7846–7854.

    Article  CAS  PubMed  Google Scholar 

  • Brodeur GM . (2003). Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer 3: 203–216.

    Article  CAS  PubMed  Google Scholar 

  • Brummelkamp TR, Bernards R, Agami R . (2002). Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2: 243–247.

    Article  CAS  PubMed  Google Scholar 

  • Burk U, Schubert J, Wellner U, Schmalhofer O, Vincan E, Spaderna S et al. (2008). A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells. EMBO Rep 9: 582–589.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cano A, Perez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG et al. (2000). The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2: 76–83.

    Article  CAS  PubMed  Google Scholar 

  • Christofori G . (2006). New signals from the invasive front. Nature 441: 444–450.

    Article  CAS  PubMed  Google Scholar 

  • Chua HL, Bhat-Nakshatri P, Clare SE, Morimiya A, Badve S, Nakshatri H . (2007). NF-kappaB 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.

    Article  CAS  PubMed  Google Scholar 

  • Desmet CJ, Peeper DS . (2006). The neurotrophic receptor TrkB: a drug target in anti-cancer therapy? Cell Mol Life Sci 63: 755–759.

    Article  CAS  PubMed  Google Scholar 

  • Dionne CA, Camoratto AM, Jani JP, Emerson E, Neff N, Vaught JL et al. (1998). Cell cycle-independent death of prostate adenocarcinoma is induced by the trk tyrosine kinase inhibitor CEP-751 (KT6587). Clin Cancer Res 4: 1887–1898.

    CAS  PubMed  Google Scholar 

  • Dohadwala M, Yang SC, Luo J, Sharma S, Batra RK, Huang M et al. (2006). Cyclooxygenase-2-dependent regulation of E-cadherin: prostaglandin E(2) induces transcriptional repressors ZEB1 and snail in non-small cell lung cancer. Cancer Res 66: 5338–5345.

    Article  CAS  PubMed  Google Scholar 

  • Douma S, Van Laar T, Zevenhoven J, Meuwissen R, Van Garderen E, Peeper DS . (2004). Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB. Nature 430: 1034–1039.

    Article  CAS  PubMed  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  PubMed  Google Scholar 

  • Ernfors P, Lee KF, Jaenisch R . (1994). Mice lacking brain-derived neurotrophic factor develop with sensory deficits. Nature 368: 147–150.

    Article  CAS  PubMed  Google Scholar 

  • Frisch SM, Francis H . (1994). Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124: 619–626.

    Article  CAS  PubMed  Google Scholar 

  • Geiger TR, Peeper DS . (2005). The neurotrophic receptor TrkB in anoikis resistance and metastasis: a perspective. Cancer Res 65: 7033–7036.

    Article  CAS  PubMed  Google Scholar 

  • Geiger TR, Peeper DS . (2007). Critical role for TrkB kinase function in anoikis suppression, tumorigenesis, and metastasis. Cancer Res 67: 6221–6229.

    Article  CAS  PubMed  Google Scholar 

  • Geiger TR, Peeper DS . (2009). Metastasis mechanisms. Biochim Biophys Acta 1796: 293–308.

    CAS  PubMed  Google Scholar 

  • Genetta T, Ruezinsky D, Kadesch T . (1994). Displacement of an E-box-binding repressor by basic helix-loop-helix proteins: implications for B-cell specificity of the immunoglobulin heavy-chain enhancer. Mol Cell Biol 14: 6153–6163.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Graham TR, Zhau HE, Odero-Marah VA, Osunkoya AO, Kimbro KS, Tighiouart M et al. (2008). Insulin-like growth factor-I-dependent up-regulation of ZEB1 drives epithelial-to-mesenchymal transition in human prostate cancer cells. Cancer Res 68: 2479–2488.

    Article  CAS  PubMed  Google Scholar 

  • Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G et al. (2008). The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 10: 593–601.

    Article  CAS  PubMed  Google Scholar 

  • Grooteclaes ML, Frisch SM . (2000). Evidence for a function of CtBP in epithelial gene regulation and anoikis. Oncogene 19: 3823–3828.

    Article  CAS  PubMed  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  PubMed  Google Scholar 

  • Gupta GP, Massague J . (2006). Cancer metastasis: building a framework. Cell 127: 679–695.

    Article  CAS  PubMed  Google Scholar 

  • Huber MA, Kraut N, Beug H . (2005). Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol 17: 548–558.

    Article  CAS  PubMed  Google Scholar 

  • Klein R, Smeyne RJ, Wurst W, Long LK, Auerbach BA, Joyner AL et al. (1993). Targeted disruption of the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death. Cell 75: 113–122.

    Article  CAS  PubMed  Google Scholar 

  • Kudo-Saito C, Shirako H, Takeuchi T, Kawakami Y . (2009). Cancer metastasis is accelerated through immunosuppression during Snail-induced EMT of cancer cells. Cancer Cell 15: 195–206.

    Article  CAS  PubMed  Google Scholar 

  • Kupferman ME, Jiffar T, El-Naggar A, Yilmaz T, Zhou G, Xie T et al. (2010). TrkB induces EMT and has a key role in invasion of head and neck squamous cell carcinoma. Oncogene 29: 2047–2059.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liotta LA, Kohn E . (2004). Anoikis: cancer and the homeless cell. Nature 430: 973–974.

    Article  CAS  PubMed  Google Scholar 

  • Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY et al. (2008). The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133: 704–715.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Meredith Jr JE, Fazeli B, Schwartz MA . (1993). The extracellular matrix as a cell survival factor. Mol Biol Cell 4: 953–961.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miknyoczki SJ, Lang D, Huang L, Klein-Szanto AJ, Dionne CA, Ruggeri BA . (1999). Neurotrophins and Trk receptors in human pancreatic ductal adenocarcinoma: expression patterns and effects on in vitro invasive behavior. Int J Cancer 81: 417–427.

    Article  CAS  PubMed  Google Scholar 

  • Nakagawara A, Azar CG, Scavarda NJ, Brodeur GM . (1994). Expression and function of TRK-B and BDNF in human neuroblastomas. Mol Cell Biol 14: 759–767.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Palmer HG, Larriba MJ, Garcia JM, Ordonez-Moran P, Pena C, Peiro S et al. (2004). The transcription factor SNAIL represses vitamin D receptor expression and responsiveness in human colon cancer. Nat Med 10: 917–919.

    Article  CAS  PubMed  Google Scholar 

  • Park SM, Gaur AB, Lengyel E, Peter ME . (2008). The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev 22: 894–907.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Peinado H, Olmeda D, Cano A . (2007). Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 7: 415–428.

    Article  CAS  PubMed  Google Scholar 

  • Pena C, Garcia JM, Silva J, Garcia V, Rodriguez R, Alonso I et al. (2005). E-cadherin and vitamin D receptor regulation by SNAIL and ZEB1 in colon cancer: clinicopathological correlations. Hum Mol Genet 14: 3361–3370.

    Article  CAS  PubMed  Google Scholar 

  • Perez-Moreno MA, Locascio A, Rodrigo I, Dhondt G, Portillo F, Nieto MA et al. (2001). A new role for E12/E47 in the repression of E-cadherin expression and epithelial-mesenchymal transitions. J Biol Chem 276: 27424–27431.

    Article  CAS  PubMed  Google Scholar 

  • Postigo AA, Dean DC . (1999). ZEB represses transcription through interaction with the corepressor CtBP. Proc Natl Acad Sci USA 96: 6683–6688.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rosivatz E, Becker KF, Kremmer E, Schott C, Blechschmidt K, Hofler H et al. (2006). Expression and nuclear localization of Snail, an E-cadherin repressor, in adenocarcinomas of the upper gastrointestinal tract. Virchows Arch 448: 277–287.

    Article  CAS  PubMed  Google Scholar 

  • Schmalhofer O, Brabletz S, Brabletz T . (2009). E-cadherin, beta-catenin, and ZEB1 in malignant progression of cancer. Cancer Metastasis Rev 28: 151–166.

    Article  CAS  PubMed  Google Scholar 

  • Singh M, Spoelstra NS, Jean A, Howe E, Torkko KC, Clark HR et al. (2008). ZEB1 expression in type I vs type II endometrial cancers: a marker of aggressive disease. Mod Pathol 21: 912–923.

    Article  CAS  PubMed  Google Scholar 

  • Smit MA, Geiger TR, Song JY, Gitelman I, Peeper DS . (2009). A Twist-Snail axis critical for TrkB-induced epithelial-mesenchymal transition-like transformation, anoikis resistance, and metastasis. Mol Cell Biol 29: 3722–3737.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Spaderna S, Schmalhofer O, Hlubek F, Berx G, Eger A, Merkel S et al. (2006). A transient, EMT-linked loss of basement membranes indicates metastasis and poor survival in colorectal cancer. Gastroenterology 131: 830–840.

    Article  CAS  PubMed  Google Scholar 

  • Spaderna S, Schmalhofer O, Wahlbuhl M, Dimmler A, Bauer K, Sultan A et al. (2008). The transcriptional repressor ZEB1 promotes metastasis and loss of cell polarity in cancer. Cancer Res 68: 537–544.

    Article  CAS  PubMed  Google Scholar 

  • Spoelstra NS, Manning NG, Higashi Y, Darling D, Singh M, Shroyer KR et al. (2006). The transcription factor ZEB1 is aberrantly expressed in aggressive uterine cancers. Cancer Res 66: 3893–3902.

    Article  CAS  PubMed  Google Scholar 

  • Thiele CJ, Li Z, McKee AE . (2009). On Trk—the TrkB signal transduction pathway is an increasingly important target in cancer biology. Clin Cancer Res 15: 5962–5967.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thiery JP . (2002). Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2: 442–454.

    Article  CAS  PubMed  Google Scholar 

  • Vandewalle C, Van Roy F, Berx G . (2009). The role of the ZEB family of transcription factors in development and disease. Cell Mol Life Sci 66: 773–787.

    Article  CAS  PubMed  Google Scholar 

  • Vesuna F, van Diest P, Chen JH, Raman V . (2008). Twist is a transcriptional repressor of E-cadherin gene expression in breast cancer. Biochem Biophys Res Commun 367: 235–241.

    Article  CAS  PubMed  Google Scholar 

  • Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A et al. (2009). The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol 11: 1487–1495.

    Article  CAS  PubMed  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  PubMed  Google Scholar 

  • Yin T, Wang C, Liu T, Zhao G, Zha Y, Yang M . (2007). Expression of snail in pancreatic cancer promotes metastasis and chemoresistance. J Surg Res 141: 196–203.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M Voetel, S Greven, H Grimminck and all other animal caretakers for their excellent technical help with the in vivo experiments, JY Song and the animal pathology department for help with the pathological analysis and performing IHC stainings, L Brocks and L Oomen for help with the confocal images, F Van Diepen and A Pfauth for fluorescence-activated cell sorting, M Heimerikx, M Nieuwland, J de Ronde, D Sie, A Velds, I de Rink and R Kerkhoven for performing the microarray analysis and ML Yurda for statistical analysis. We thank Dr Nakshatri for providing the Zeb1 plasmid, R Weinberg for the Twist plasmid, A Munoz for the Snail plasmid and K Becker for the Snail antibody. We thank all members of the Peeper laboratory for their valuable input and T Geiger for useful discussions and help with the microarray experiments. This work was supported by grants from MAS and DSP from the Dutch Cancer Society (KWF) and a European Union FP6 grant.

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Correspondence to D S Peeper.

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Smit, M., Peeper, D. Zeb1 is required for TrkB-induced epithelial-mesenchymal transition, anoikis resistance and metastasis. Oncogene 30, 3735–3744 (2011). https://doi.org/10.1038/onc.2011.96

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