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

Post-Transcriptional Regulation of Snail/EMT 1. A. Cano et al., Nat. Cell Biol. 2:76-83, B.P. Zhou et al., Nat. Cell Biol. 6:931-40, Z. Yang et al., Cancer Res. 65: , H. Peinado et al., EMBO J. 24: , J.I. Yok et al., Nat. Cell Biol. 8: , E. Langer et al., Dev. Cell March 11, 2008 Reviews 1.J.P. Thiery and J.P. Sleeman Nature Rev. Mol. Cell Biol. 7: , H. Peinado et al., Nature Rev. Cancer 7: , A. Barrallo-Gimeno and M.A. Nieto. Development 132: , J.P. Thiery. Nature Rev. Cancer 2:442-54, 2002 Breast Cancer 1.S.E. Moody et al., Cancer Cell 8: , N. Fujita et al., Cell 113:207-19, C. Xue et al., Cancer Res. 63: , A. Dhasarathy et al., Mol. Endocrinology 21: , 2007

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 OUTLINE

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 GastrulationNeural Crest Delamination EpithelialMesenchymal 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)

Lost or decreased 1.Epithelial adhesion receptors - E-cadherin, Occludin, Claudins  -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  -smooth muscle actin (myofibroblasts)  v  6 integrin 7.Motility, Invasiveness Cellular changes during EMT

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

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

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)

E-cadherin (brown) Colon Cancer

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

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 SIGNALING

+ Snail

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

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

SNAG DomainZinc Fingers 93 - SDEDSGKGSQPPS PP SPAPSSFSSTSVSSLE- 122 GSK3  nuclear export GSK3  cytoplasmic destruction S 246 Pak1 K 98 K 137 LOX 2/3 Ajuba LIM proteins: - adapters that assemble repressor complex (co-repressors) Snail Modification/Function

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

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

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