CANCER INVASION AND METASTASIS - 2007

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CANCER INVASION AND METASTASIS:THE CONCEPT OF EPITHELIAL-MESENCHYMAL TRANSITION (EMT) : 

CANCER INVASION AND METASTASIS:THE CONCEPT OF EPITHELIAL-MESENCHYMAL TRANSITION (EMT) PRESENTER: Dr. Sandeep Kumar P. MODERATOR: Prof. G.V. Manjunath

Definition - EMT : 

Definition - EMT Biologic process that allows a polarized epithelial cell, which normally interacts with basement membrane via its basal surface, to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype.

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This includes: enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of ECM components.

History of EMT : 

History of EMT First observed and defined by Elizabeth Hay in late 1960’s at Harvard First associated with early stages of embryonic development. Elizabeth Hay - “epithelial-mesenchymal transformation” – model of chick primitive streak formation. The term “transformation” has been replaced with “transition”

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Incomplete and bi-directional process Process is reversible with an unstable intermediate EMT Metastable MET

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ELIZABETH DEXTER HAY (1927-2007)

Characteristics Of Epithelial And Mesenchymal Cells : 

Characteristics Of Epithelial And Mesenchymal Cells

Epithelial Cells : 

Epithelial Cells Sheet of cells – often one cell thick Individual cells abut each other Regularly spaced cell-cell junctions and adhesions between neighbouring epithelial cells Individual cell movement cannot occur Polarised

Functions Of Epithelial Cells : 

Functions Of Epithelial Cells Formation of vast surface areas for exchange, eg. Microvilli Formation of cavities or tubular structures, eg. Intestinal and neural tubes Formation of biological compartments of different ionic compositions by selective permeability Absorption, transcytosis and vectorial transport

Mesenchymal Cells : 

Mesenchymal Cells Do not form continuous sheets nor tight intercellular adhesions Form loose networks No particular spatial arrangement No cytoplasmic polarity Potentially mobile– vimentin, smooth muscle cell actin

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Development and cancer progression - mesenchymes can be transitional in the formation of new epithelia from pre-existing epithelia, enabling migration As lasting organisation - support and nutrient supply

Epithelial Cell Plasticity Under Pathological Conditions : 

Epithelial Cell Plasticity Under Pathological Conditions

1. Wound Healing : 

1. Wound Healing Skin and cornea Epithelial proliferation & differentiation – essential Take time to develop More rapid recovery – by migratory behaviour of residential epithelial cells

2. Chronic Inflammatory Conditions : 

2. Chronic Inflammatory Conditions Chronic kidney disease Liver fibrosis 3. Cancer

Classification Of EMT : 

Classification Of EMT

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Encountered in 3 different biological settings Carry different functional consequences

Type 1 EMT : 

Type 1 EMT Implantation, embryo formation, organ development Neither causes fibrosis nor induces an invasive phenotype Can generate mesenchymal cells (primary mesenchyme) that have the potential to subsequently undergo a mesenchymal-epithelial transition to generate secondary epithelia

Type 2 EMT : 

Type 2 EMT Wound healing, tissue regeneration, organ fibrosis Begins as part of a repair mechanism Helps to reconstruct tissues following trauma and inflammatory injury Type 2 EMT is associated with inflammation & ceases once inflammation is attenuated

Type 3 EMT : 

Type 3 EMT Occurs in neoplastic cells that have previously undergone genetic & epigenetic changes Carcinoma cells undergoing a type 3 EMT may invade & metastasise and thereby generate the final, life-threatening manifestations of cancer progression

Intercellular Adhesions : 

Intercellular Adhesions

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Apical domain – characteristics depend on the functional needs of the cell Lateral domains Tight junctions - claudins, occludins Adherens junctions – Cadherins, β-catenin, α-catenin Gap junctions – connexins Desmosomes - desmoglein and desmocollin Basal domains – interacts with the basement membrane – hemidesmosome (integrins)

Adherens Junctions(zonula adherens or "belt desmosome“) : 

Adherens Junctions(zonula adherens or "belt desmosome“) Protein complexes that occur at cell-cell junctions in epithelial tissues Located more basal than tight junctions Appear as bands encircling the cell – zonula adherens Serve as a bridge connecting the actin cytoskeleton of neighboring cells through direct interaction

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ADHERENS JUNCTION

Cadherins : 

Cadherins "calcium-dependent adhesion“ Class of type-1 transmembrane proteins Dependent on calcium (Ca2+) ions to function, hence their name Important roles in cell adhesion, ensuring that cells within tissues are bound together

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CADHERIN

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Cadherin superfamily includes cadherins, protocadherins, desmogleins, and desmocollins, and more They share cadherin repeats Multiple classes of cadherin molecule, each designated with a prefix

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E-cadherins – epithelial tissue N-cadherins – neurons P-cadherins – placenta

E-Cadherin : 

E-Cadherin 5 cadherin repeats in the extracellular domain One transmembrane domain An intracellular domain Intracellular domain – highly phosphorylated region Vital to beta-catenin binding and, therefore, to E-cadherin function

Catenins('catena' means 'chain' in Latin) : 

Catenins('catena' means 'chain' in Latin) Proteins found in complexes with cadherin cell adhesion molecules. Types: alpha-catenin beta-catenin delta-catenin gamma-catenin Beta-catenin binds the cytoplasmic domain of some cadherins.

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Dual role in EMT: enhances cell–cell adhesion when bound to cadherin complexes functions as a transcriptional coactivator upon entry into the nucleus The ability of β-catenin to enhance cadherin-dependent adhesion depends on β-catenin binding to α-catenin and on α-catenin binding to the actin.

Steps In EMT : 

Steps In EMT Disintegration of cell–cell adhesions Cytoskeletal remodeling & Loss of apico-basolateral polarity Increased motility Cell–matrix adhesion

Micro-environmental Cues Induce EMT : 

Micro-environmental Cues Induce EMT EMT is induced by stimulating factors in the micro-environment, for which the responding cells express receptors These factors include: Extracellular matrix molecules a)Collagens b)Hyaluronic acid c)Fibronectin

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2. Growth factors a)TGF b)FGF c)EGF d)Scatter factor/HGF 3. Ligands of – Wnt, Notch & Akt pathways

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These act through their receptors: Integrins TGFR Receptor tyrosine kinases(RTK) Frizzled

Disintegration Of Cell–Cell Adhesions : 

Disintegration Of Cell–Cell Adhesions

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Tight junctions - disassembly is stimulated by TGF-beta Adherens junctions - Transcriptional repression of E-cadherin is mediated by members of the Snail/Slug family of transcriptional repressors Desmosomes - Disassembly of desmosomes is regulated by Slug

SNAI1 & SNAI2 Are Central Regulators : 

SNAI1 & SNAI2 Are Central Regulators Interaction of FGF, EGF, or HGF with their respective RTKs Activation of small GTPase family (Ras, Rho, Rac) Activation of transcription factors such as SNAI1(Snail) & SNAI2(Slug) by phosphorylation Translocates to the nucleus Down-regulates E-cadherin expression

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Wnt TGF-beta Notch SNAI1

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SNAI1 & SNAI2 orchestrate the modifications in the pattern of genes expressed responsible for remodelling the cytoskeleton: Up-regulation of vimentin expression Activation of contractile apparatus Modification of integrin expression

Role Of Beta-Catenin : 

Role Of Beta-Catenin Various signalling pathways eventually lead to down-regulation of E-cadherin expression Disassembly of junctional complexes Liberation of beta-catenin Translocates into the nucleus Activates the Wnt pathway E-cadherin down-regulation & activation of EMT target genes

Cytoskeletal Remodeling & Loss Of Polarity : 

Cytoskeletal Remodeling & Loss Of Polarity Polarized epithelial cells – cortical filamentous actin bundles that are connected with intracellular juxta-membranous components of cell adherens junctions Dissociation of intercellular junctions loss of polarity remodeling of the actin cytoskeleton from cortical actin to actin stress fibers Hallmark of migratory, mesenchymal cells

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TGF-beta Activate GTPases of the Rho family De novo formation of stress fibers + Formation of focal adhesions (mediate the communication of fibroblastoid cells with ECM)

Cell–Matrix Adhesion : 

Cell–Matrix Adhesion Transitioning cells need to resolve integrin-mediated cell–matrix (basal lamina) contacts Digest/dissolve adjacent basal lamina Basal lamina – collagen IV

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TGF-beta or FGF2 Activation of proteolytic enzymes, such as matrix metalloproteases MMP2 and MMP9 Degradation of the collagen type IV Formation of focal adhesions (mediate the communication of fibroblastoid cells with ECM)

Molecular Marker Proteins Associated With EMT : 

Molecular Marker Proteins Associated With EMT EMT is characterized by: 1) loss of proteins associated with polarized epithelial phenotype – MUC1, E-cadherin, ZO-1, Desmoplakins, and Cytokeratin 1

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2) de novo synthesis of proteins associated with mesenchymal, migratory morphology of transitioning cells – Vimentin, Alpha-smooth muscle actin (a-SMA), Fibroblast specific protein 1 (FSP1)/S100A4 protein

EMT Markers : 

EMT Markers Proteins that increase in abundance N-cadherin Vimentin Fibronectin Snail1 (Snail) Snail2(Slug) Twist Goosecoid FOXC2 Sox10 MMP-2 MMP-3 MMP9 Integrin vß6 Proteins that decrease in abundance E-cadheren Desmoplakin Cytokeratin Occludin Proteins whose activity increases ILK GSK-3ß Rho Proteins that accumulate in the nucleus ß-catenin Smad-2/3 NF- ß Snail1 (Snail) Snail2 (Slug) Twist

Invasive Front Of A Squamous Cell Carcinoma : 

Invasive Front Of A Squamous Cell Carcinoma Cytokeratin Vimentin

EMT and Colorectal Cancer : 

EMT and Colorectal Cancer

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Two different sections from the same human breast cancer show regions where the vimentin is exclusively in the surrounding stroma and vascular structures (LHS, brown staining), or clearly in the tumor parenchyma.

Role Of Mesenchymal-Epithelial Transition (MET) : 

Role Of Mesenchymal-Epithelial Transition (MET)

Slide 55: 

The histological similarity of secondary, metastasis-derived tumors to the primary tumor indicates that EMT-mediated metastatic development must be followed by a reverse process to allow colonization of secondary sites. Re-express E-cadherin β-catenin becomes cytoplasmic

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MET has been recognized in a number of mesenchymal tumors: Synovial sarcomas Rare epitheloid variants of mesenchymal neoplasms in bone

MET in Wilms’ tumor : 

MET in Wilms’ tumor During development, epithelial nephrons develop via MET. Key genes, such as transcription factor Pax-2, are expressed during embryological MET but are switched off during terminal differentiation. A number of these genes are re-expressed in renal tumors.

MET in Ovarian carinogenesis : 

MET in Ovarian carinogenesis Normal ovarian surface epithelium rarely expresses E-cadherin Both primary and metastatic ovarian carcinomas express E-cadherin Overexpression of E-cadherin - induces a number of epithelial characteristics and markers associated with malignant transformation and tumor progression

Metastable Phenotype : 

Metastable Phenotype First described by Savagner Ability of cells to express attributes of both epithelial and mesenchymal phenotypes

Clinical Applications : 

Clinical Applications

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EMT – target for drug development in cancer and fibrosis BMP7 mimetics antagonize TGF-β–driven EMT in fibrotic kidney and heart Small molecule ILK inhibitors inhibit Snail1 production, induce E-cadherin expression, and inhibit invasion EMT could be used as a functional screen for novel anticancer agents

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Initiation of tumor growth at the secondary site is the rate-limiting step in metastasis. MET – can be targetted to prevent re-epithelialisation process which appears germane to metastatic relapse. Meeting on EMT : 2007 – Poland & 2008 – Cold Spring Harbor Laboratories

CONCLUSION : 

CONCLUSION

Summary : 

Summary

Conclusion : 

Conclusion Under pathological conditions – epithelial cells may assume characteristics of mesenchymal cells. EMT is reversible(MET) and does not necessarily represent a lineage shift. A defining feature of EMT is a reduction in E-cadhrin levels and a concomitant production of N-cadherin as well as nuclear expression of beta-catenin EMT-associated molecules will increase in importance as markers for prediction of prognosis and response to targeted therapy.

References : 

References RECENT ADVANCES IN HISTOPATHOLOGY, VOLUME 22, Pg. 67-80 ROBBINS’ PATHOLOGIC BASIS OF DISEASE VARIOUS INTERNET SOURCES

THANK YOU : 

THANK YOU