PDGF PDGFR ruf es junctions Loss of NCAM Loss of gap Membrane N-cad β -cat FAK p120-Cat Fyn Rho GAP p190 Microtentacles P Cdc42 assembly and turnover Rac1 RhoA remodeling ECM remodelling Glu-tubulin A Cdc42 Six1 B Rac1 Invasion Filopodia Migration Lamellipodia RhoA

B, Akt

Rho GAP p190 RI p120-Cat β Examples of up- regulated T Vimentin SMA N- NCAM Fibronectin Laminins miR-661 MMPs ubulin tyrosine ligase ubulin tyrosine Examples of down- genes regulated T ZO1/ZO3 Crumbs3 Connexin43 E-cadherin -1 VE-cadherin Cytokeratins Collagen I, II -NF- κ esistance to senescence Other EMT-associated effects Other EMT-associated R Resistance to apoptosis Therapy resistance Stemness Basement membrane degradation LEI Other EMT-inducing pathways Other EMT-inducing I Endothelin-A receptor-PI3K TNF- α acid Hyaluronic COX2-PGE2 AMF PTH(rP)R Bile acids Nicotine UV irradiation SCF Axl-Gas6 E-cadherin degradation Hakai

Actin TGF- β junction signaling Adherens Pyk2 Increased CAS p130 disassembly tight junctions degradation Endocytosis Dissociation of DDR1 FAK MMPs ZO3 ZO1 ZO2 Afadin c-src FAK P Focal Rap1 adhesion formation RTK JAM TGF- β TGF- β Focal Wnt Nectin Frizzled adhesion Collagen 1 formation RI RII β β P P T β RI T β RII T FAK FAK DAB2 LRP5/6 ILK c-src Par6 Par3 BMPR1 β -cat α -cat P -cat aPKC β -cat β MESENCHYMAL Smurf1 BMPs p120-Cat -cat RhoA RhoA Ub Ub β -cat β Rac1b Dishevelled P E

Proteasome CK1 P AKT BMPR2 Snail1/2 GSK3 β NF- κ B E47 Patched P Hedgehog oxygen species Reactive FOXQ1 Karlsruhe Institute of Technology, Postfach 3640, 76021 Karlsruhe, Germany Karlsruhe Institute of Technology, 4 Cancer Science Institute, 117456 Singapore Cancer Science Institute, 117456 Singapore Smad2/3 Smad2/3 2 Kaiso ER p120-Cat KLF8 Zeb1/2 (see box A) Smoothened Smoothened miR-200 miR-205 MTA3 E-cadherin and other downregulated genes downregulated Transcription Gli1 MAPK RTK Snail1/2 PAK1 factors Growth E2.2 PI3K Ras HIF 1/2 Hypoxia GSC LoxL2 Twist (see box B) LIV1 AKT STAT3 IL-6R Upregulated genes Upregulated NF- κ B CSL IL-6 NOTCH NOTCH JAG2 2,3 α -Actinin GSK3 β Laminin 5 Plectin Cytokeratin Desmoplakin F- 4 β α -cat ZO3 ZO1 ZO2 6 X α

Afadin p120-Cat DAP21P β -cat JAM cell Nectin

T β RI E-cadherin

DDR1 Desmocollin Desmocollin migration Collective Par6

Gap junction

Tight junction Par3 and Jean Paul Thiery Par6 Par3 Rho GAP 3,4 p190 Claudin aPKC RhoE RhoA β -cat Adherens ROCK Occludin EPITHELIAL P Dynein/ dynactin complex Ninein EB1 miR-205 Clip170 Rac1 light light RhoA chain chain Cdc42 Myosin Myosin Apicobasal microtubules Zeb1/2 p53 FOXA1/2 E-cadherin miR-200 IQGAP activities Balanced cytoskeleton BASEMENT MEMBRANE Snail1/2 Stabilized actin IMCB and Experimental Therapeutics Centre, Biopolis A*STAR, 138673 Singapore; 138673 Singapore; Biopolis A*STAR, IMCB and Experimental Therapeutics Centre, University of Heidelberg, Medical Faculty Mannheim, D-68167 Germany; SnapShot: The Epithelial-Mesenchymal Transition SnapShot: Epithelial-Mesenchymal The Sleeman Jonathan P. 3 1

162 Cell 145, April 1, 2011 ©2011 Elsevier Inc. DOI 10.1016/j.cell.2011.03.029 See online version for legend and references. SnapShot: The Epithelial-Mesenchymal Transition Jonathan P. Sleeman3,4 and Jean Paul Thiery1,2 1IMCB and Experimental Therapeutics Centre, Biopolis A*STAR, 138673 Singapore 2Cancer Science Institute, 117456 Singapore 3University of Heidelberg, Medical Faculty Mannheim, D-68167 Mannheim, Germany 4Karlsruhe Institute of Technology, Postfach 3640, 76021 Karlsruhe, Germany

This SnapShot portrays important regulatory pathways and major cellular events that are activated during the transition from an epithelial to a mesenchymal morphology during development and disease. The cell on the left represents the epithelial state, whereas the central cell depicts transcriptional regulatory networks that orchestrate the process of epithelial-to-mesenchymal transition (EMT). The cell on the right illustrates some of the consequences of the activity of these networks that endow formerly epithelial cells with mesenchymal characteristics. Note that this overview does not take into account cell-type-specific regulation of EMT, and that not all illustrated mechanisms are obligate for EMT to occur. The temporal regulation of the EMT process is also not considered in this SnapShot.

The Epithelial Phenotype Polarized epithelial cells are typified by tight junctions, adherens junctions, desmosomes, and gap junctions. Junctional complexes not only act as mediators of polarized cell-cell contacts but also serve as anchor points for the actin cytoskeleton. Adherens junctions can additionally anchor apicobasal microtubule arrays, and through E-cadherin-DDR1 interactions are also involved in collective cell migration. Organization of the actin cytoskeleton, microtubule arrays, and cell-cell junctions is tightly coordinated in a mechanism that probably involves IQGAP, but the details remain to be investigated. Balanced regulation of the activities of RhoA (stress fibers), Cdc42 (filopodia), and Rac1 (lamellipodia) stabilizes the actin cytoskeleton and maintains the epithelial phenotype. Epithelial cells are tethered to the underlying basement membrane, for example through . The repression of EMT-inducing transcriptional regulators (for example, through microRNAs), as well the activity of positively acting factors such as FOXA1/2, ensures that expression of key junctional such as E-cadherin is maintained. Suppression of GSK3β also helps to maintain the epithelial phenotype.

Transcriptional Activation of EMT A variety of extracellular stimuli have the potential to induce EMT. A complex network of positively and negatively acting signal transduction mechanisms converge on the nucleus to downregulate genes required for the epithelial phenotype and to upregulate genes that specify mesenchymal characteristics. GSK3β and NF-κB play central roles in coordinating these pathways. Members of the Snail family of transcriptional regulators, namely Snail1 and Snail2, have emerged as a key regulatory node. The zinc finger transcription factors Zeb1 and Zeb2 also make a pivotal contribution to this regulation. EMT-inducing signals promote their expression, regulate their stability, and/or alter their subcellular location.

Loss of Epithelial and Acquisition of Mesenchymal Characteristics Key targets of the pathways that induce EMT include the adherens junction components E-cadherin and β-. In addition to being transcriptionally downregulated and epigenetically switched off, E-cadherin can be proteolytically cleaved and targeted to endosomes for degradation. Proteosomal degradation of β-catenin destabilizes adherens junctions, whereas loss of E-cadherin can increase the free pool of β-catenin that can then enter the nucleus and modulate transcription. An important consequence of EMT- inducing transcriptional modulation as well as other pro-EMT processes is the loss of the junctional complexes that typify polarized epithelial cells. Enhanced activation of the GTPases Cdc42 and Rac1 and suppression of RhoA favor the formation of lamellipodia and filopodia, migration, and invasion. A variety of mechanisms promote the assembly and turnover of focal adhesions. Extensive cytoskeleton remodeling occurs, including switching from a predominantly cytokeratin to a vimentin-rich net- work. Detyrosination of tubulin promotes microtentacle formation. Proteolytic are produced that together with increased expression of extracellular matrix components serve to remodel the microenvironment surrounding the cells. Other properties endowed on cells undergoing EMT include resistance to apoptosis, senescence, and therapeutics and the acquisition of stemness characteristics.

Acknowledgments

This work was supported by a grant to J.P.S. from the European Union under the auspices of the FP7 collaborative project TuMIC, contract no. HEALTH-F2-2008-201662.

References

Casas, E., Kim, J., Bendesky, A., Ohno-Machado, L., Wolfe, C.J., and Yang, J. (2011). Snail2 is an essential mediator of Twist1-induced epithelial mesenchymal transition and me- tastasis. Cancer Res. 71, 245–254.

Chang, C.J., Chao, C.H., Xia, W., Yang, J.Y., Xiong, Y., Li, C.W., Yu, W.H., Rehman, S.K., Hsu, J.L., Lee, H.H., et al. (2011). p53 regulates epithelial-mesenchymal transition and stem cell properties through modulating miRNAs. Nat. Cell Biol. 13, 317–323.

Hanahan, D., and Weinberg, R.A. (2011). Hallmark of cancers: the next generation. Cell 144, 646–674.

Hidalgo-Carcedo, C., Hooper, S., Chaudhry, S.I., Williamson, P., Harrington, K., Leitinger, B., and Sahai, E. (2011). Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the regulators Par3 and Par6. Nat. Cell Biol. 13, 49–58.

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

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

Thiery, J.P., and Sleeman, J.P. (2006). Complex networks orchestrate epithelial-mesenchymal transitions. Nat. Rev. Mol. Cell Biol. 7, 131–142.

Thiery, J.P., Acloque, H., Huang, R.Y., and Nieto, M.A. (2009). Epithelial-mesenchymal transitions in development and disease. Cell 139, 871–890.

Whipple, R.A., Matrone, M.A., Cho, E.H., Balzer, E.M., Vitolo, M.I., Yoon, J.R., Ioffe, O.B., Tuttle, K.C., Yang, J., and Martin, S.S. (2010). Epithelial-to-mesenchymal transition promotes tubulin detyrosination and microtentacles that enhance endothelial engagement. Cancer Res. 70, 8127–8137.

Yilmaz, M., and Christofori, G. (2009). EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev 28, 15-33.

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