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Epithelial Mesenchymal Transitions (EMT) In Cancer Metastasis Greg Longmore, February 19, 2008 Reviews 1. 2. 3. 4. J.P. Thiery and J.P. Sleeman Nature Rev. Mol. Cell Biol. 7:131-142, 2006 H. Peinado et al., Nature Rev. Cancer 7:415-428, 2007 A. Barrallo-Gimeno and M.A. Nieto. Development 132:3150-61, 2005 J.P. Thiery. Nature Rev. Cancer 2:442-54, 2002 Post-Transcriptional Regulation of Snail/EMT 1. 2. 3. 4. 5. 6. A. Cano et al., Nat. Cell Biol. 2:76-83, 2000 B.P. Zhou et al., Nat. Cell Biol. 6:931-40, 2004 Z. Yang et al., Cancer Res. 65:3179-84, 2005 H. Peinado et al., EMBO J. 24:3446-58, 2005 J.I. Yok et al., Nat. Cell Biol. 8:1389-406, 2006 E. Langer et al., Dev. Cell March 11, 2008 Breast Cancer 1. 2. 3. 4. S.E. Moody et al., Cancer Cell 8:197-209, 2005 N. Fujita et al., Cell 113:207-19, 2003 C. Xue et al., Cancer Res. 63:3386-94, 2003 A. Dhasarathy et al., Mol. Endocrinology 21:2907-18, 2007 OUTLINE 1. Cancer Metastasis 2. EMT - MET - definitions 3. In Normal Development 4. In Adult Pathology 5. Signals that Induce EMT 6. Snail Family - Transcriptional regulators of EMT 7. Clinical - Breast Cancer 1. Primary tumors (10%) rarely kill, metastases do (90%) 2. Primary tumor size often predicts for metastasis 3. Some tumors don’t metastasize (skin SCC, brain glioblastoma) while other do frequently (melanoma) 4. Some tumors have a propensity for specific tissue metastasis (breast, prostate - bone), while others are excluding from tissues - when considering blood flow as a single variable 5. “micrometastases” at diagnosis - breast, colon - worse outcomes 6. Organ fibrosis is a significant risk factor for the development of aggressive cancers (hepatic cirrhosis, lung fibrosis) 7. The metastatic process (Fig.) Cancer Metastasis INVASION EMT MET EMT in Development Gastrulation Epithelial Neural Crest Delamination Mesenchymal EMT in the Adult - epithelia wound healing (skin) - tissue fibrosis in response to injury (lung, kidney, liver) - epithelial cancer metastasis Skin wound healing Slug expression Epithelial Mesenchymal Transition (EMT) Altered Cell Morphology Breakdown of Intercellular Junctions Increased Cell Motility / Invasiveness Mesenchymal Epithelial Transition (MET) Cellular changes during EMT Lost or decreased 1. Epithelial adhesion receptors - E-cadherin, Occludin, Claudins 2. -catenin, -catenin frequently translocates to nucleus (Wnt) 3. Circumferential F-actin fibers 4. Epithelial cytokeratins 5. Apico-basal polarity Acquired 1. Intermediate filament protein - Vimentin 2. Matrix metalloproteinases secreted, produced 3. Fibronectin secretion 4. N-cadherin 5. -smooth muscle actin (myofibroblasts) 6. v6 integrin 7. Motility, Invasiveness Epithelial Cells Apical Surface Tight junction Adherens junction Desmosome Gap junction focal adhesions Basolateral Surface Epithelial cell-cell adhesive complexes: general organization transmembrane receptor outside cytoplasmic plaque proteins “scaffolding / adapter proteins” inside signal transduction Cytoskeletal elements proliferation cell fate Adherens Junctions: Cadherins - catenins - Actin polarity E-cadherin and Cancer pathogenesis “A metastasis tumor suppressor gene?” 1. Mouse models - TAG-insulinomas 2. Germline mutations in CDH1 strongly predispose individuals to gastric cancer and breast cancer 3. Somatic inactivating mutations in CDH1 in gastric cancers and infiltrative lobular breast cancers 4. But in the majority of cancers where CDH1 expression is lost mutations are rare or absent (? Epigenetics or trans-acting factors) Colon Cancer E-cadherin (brown) Does EMT occur in vivo? transformed human mammary cells implanted in a mouse Other Data Lung Fibrosis model: - -gal transgenic mice + TGFgenerate -gal + myofibroblasts PyV-mT, FSP1.TK mice - less invasion and Metastasis following treatment with GCV SIGNALING Extrinsic Signals that Induce EMT: - Tumor-derived (autocrine), Stromal Cell-derived (paracrine) FGF, TGF-, EGF, HGF (scatter factor), Wnt, TNF- E-cadherin cleavage (MMPs) E-cadherin endocytosis Intracellular Pathways: - PI3K - Ras - MAPK, GSK3, NF- B, p38, Smads, STAT3 Rac1b - ROS (MMP-3) Transcriptional regulation: - E2a/E47, FOXC2, SIP1, Snail, Slug, Twist QuickTime™ and a decompressor are needed to see this picture. + Snail The Snail family of transcriptional repressors SNAG Domain Zinc Fingers Snail 264aa Slug 269aa Smuc 292aa Scratch 348aa FGF Wnt Neural crest Neural crest Gastrulation Heart dev’pt Limb dev’pt Tumor metastasis Tumor metastasis TGF EGF BMP Skin Mammary dev’pt Neural crest Palate fusion gastrulation L/R asymmetry Tissue fibrosis Tumor metastasis Heart dev’pt Tumor metastasis Snail or Slug MTA3 GSK3-mediated phosphorylation Estrogens BUT, There is only a modest inverse relationship between Snail and E-cadherin expression (IHC, mRNA) in many metastatic cancers With possibly one exception - breast cancer (see later) Snail Modification/Function GSK3 cytoplasmic destruction GSK3 nuclear export 93 - SDEDSGKGSQPPSPPSPAPSSFSSTSVSSLE- 122 SNAG Domain K98 K137 Zinc Fingers S246 Pak1 LOX 2/3 Ajuba LIM proteins: - adapters that assemble repressor complex (co-repressors) Inhibit GSK3 - increase Snail - decrease E-cadherin - metastasis QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. How a Wnt signal cooperates with Snail to influence metastasis Wnt - Axin2(mRNA) - GSK3 nuclear-cytoplasm - Snail nuclear - EMT/Invasion QuickTime™ and a decompressor are needed to see this picture. QuickTime™ and a decompressor are needed to see this picture. Remember Wnt also inhibits GSK3 - stabilizes Snail, and - results in nuclear translocation of -catenin Snail or Slug functions Epithelial Markers Proliferation Cyclin D E-cadherin CDK4 Claudins Occludins Rb phosph p21 Desmoplakin Cytokeratins Mesenchymal Cell shape changes markers Cell movements, invasion Fibronectin Vitronectin Vimentin RhoB MMPs Survival PI3K activity ERK activity Caspases P53 BID