Epithelial Mesenchymal Transitions (EMT) In Cancer Metastasis

1. Epithelial Mesenchymal Transitions (EMT) In Cancer Metastasis Greg Longmore, February 19, 2008

2. Reviews 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. A. Cano et al., Nat. Cell Biol. 2:76-83, 2000 2. B.P. Zhou et al., Nat. Cell Biol. 6:931-40, 2004 3. Z. Yang et al., Cancer Res. 65:3179-84, 2005 4. H. Peinado et al., EMBO J. 24:3446-58, 2005 5. J.I. Yok et al., Nat. Cell Biol. 8:1389-406, 2006 6. E. Langer et al., Dev. Cell March 11, 2008 Breast Cancer 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

3. OUTLINE • Cancer Metastasis • EMT - MET - definitions • In Normal Development 4. In Adult Pathology 5. Signals that Induce EMT • Snail Family - Transcriptional regulators of EMT 7. Clinical - Breast Cancer

4. Primary tumors (10%) rarely kill, metastases do (90%) • Primary tumor size often predicts for metastasis • Some tumors don’t metastasize (skin SCC, brain glioblastoma) while other do frequently (melanoma) • 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 • “micrometastases” at diagnosis - breast, colon - worse outcomes • Organ fibrosis is a significant risk factor for the development of aggressive cancers (hepatic cirrhosis, lung fibrosis) • The metastatic process (Fig.)

5. Cancer Metastasis INVASION EMT MET

7. Gastrulation Neural Crest Delamination Epithelial Mesenchymal EMT in Development EMT in the Adult • epithelia wound healing (skin) • tissue fibrosis in response to injury (lung, kidney, liver) • epithelial cancer metastasis

8. Skin wound healing Slug expression

9. Epithelial Mesenchymal Transition (EMT) Altered Cell Morphology Breakdown of Intercellular Junctions Increased Cell Motility / Invasiveness Mesenchymal Epithelial Transition (MET)

10. Cellular changes during EMT Lost or decreased Epithelial adhesion receptors - E-cadherin, Occludin, Claudins -catenin, -catenin frequently translocates to nucleus (Wnt) Circumferential F-actin fibers Epithelial cytokeratins Apico-basal polarity Acquired Intermediate filament protein - Vimentin Matrix metalloproteinases secreted, produced Fibronectin secretion N-cadherin -smooth muscle actin (myofibroblasts) v6 integrin Motility, Invasiveness

11. Epithelial Cells Apical Surface Tight junction Adherens junction Desmosome Gap junction focal adhesions Basolateral Surface

12. transmembrane receptor outside cytoplasmic plaque proteins “scaffolding / adapter proteins” signal transduction inside Cytoskeletal elements polarity proliferation cell fate Epithelial cell-cell adhesive complexes: general organization Adherens Junctions: Cadherins - catenins -Actin

13. E-cadherin and Cancer pathogenesis “A metastasis tumor suppressor gene?” • Mouse models - TAG-insulinomas • 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)

14. Colon Cancer E-cadherin (brown)

15. Does EMT occur in vivo? transformed human mammary cells implanted in a mouse Other Data Lung Fibrosis model: - -gal transgenic mice + TGFgenerate -gal + myofibroblasts PyV-mT, FSP1.TK mice - less invasion and Metastasis following treatment with GCV

16. 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

17. + Snail

18. The Snail family of transcriptional repressors SNAG Domain Zinc Fingers Snail 264aa Slug 269aa Smuc 292aa Scratch 348aa

19. FGF Wnt EGF BMP TGF Neural crest Gastrulation Limb dev’pt Tumor metastasis Neural crest Heart dev’pt Tumor metastasis Skin Palate fusion Tissue fibrosis Heart dev’pt Tumor metastasis Mammary dev’pt gastrulation Tumor metastasis Neural crest L/R asymmetry Snail or Slug MTA3 GSK3-mediated phosphorylation Estrogens

20. 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)

21. Snail Modification/Function GSK3 cytoplasmic destruction GSK3 nuclear export 93 - SDEDSGKGSQPPSPPSPAPSSFSSTSVSSLE- 122 K98 K137 S246 SNAG Domain Zinc Fingers Pak1 LOX 2/3 Ajuba LIM proteins: - adapters that assemble repressor complex (co-repressors)

22. Inhibit GSK3 - increase Snail - decrease E-cadherin - metastasis

23. How a Wnt signal cooperates with Snail to influence metastasis Wnt - Axin2(mRNA) - GSK3 nuclear-cytoplasm - Snail nuclear - EMT/Invasion • Remember Wnt also inhibits GSK3 • stabilizes Snail, and • results in nuclear translocation of -catenin

24. Proliferation Mesenchymal markers Cellshape changes Cell movements, invasion Survival Fibronectin Vitronectin Vimentin Cyclin D CDK4 Rb phosph p21 RhoB MMPs PI3K activity ERK activity Caspases P53 BID Snail or Slug functions Epithelial Markers E-cadherin Claudins Occludins Desmoplakin Cytokeratins