Epithelial-Mesenchymal Epithelial-Mesenchymal Transition (EMT)Transition (EMT)

Hallmarks of CancerHallmarks of Cancer

19 February 200719 February 2007

Richard M. ShowmanRichard M. Showman

DEFINITION:DEFINITION:

An orchestrated series of events in An orchestrated series of events in which cell-cell and cell-extracellular which cell-cell and cell-extracellular matrix (ECM) interactions are altered matrix (ECM) interactions are altered to release epithelial cells from the to release epithelial cells from the surrounding tissue, the cytoskeleton surrounding tissue, the cytoskeleton is reorganized to allow movement in is reorganized to allow movement in 3 dimensions in the ECM and a new 3 dimensions in the ECM and a new transcriptional program is induced to transcriptional program is induced to maintain the mesenchymal maintain the mesenchymal phenotypephenotype

Cell TypesCell Types

Epithelial cellsEpithelial cells

Mesenchymal cellsMesenchymal cells

All animals start as epithelial cellsAll animals start as epithelial cells

NOTE: Both types can form all three germ NOTE: Both types can form all three germ layers, ectoderm, mesoderm and layers, ectoderm, mesoderm and endoderm (?)endoderm (?)

Characteristics of Epithelial CellsCharacteristics of Epithelial Cells

Typically a sheet 1 cell thickTypically a sheet 1 cell thick Individual cells abutting each otherIndividual cells abutting each other Regularly spaced cell junctions and Regularly spaced cell junctions and

adhesions between neighboring cellsadhesions between neighboring cells Tight adhesion between cells Tight adhesion between cells

resulting in inhibition of movement resulting in inhibition of movement away from the monolayeraway from the monolayer

Epithelium (cont.)Epithelium (cont.)

Enclose a 3-dimensional space withinEnclose a 3-dimensional space within Gives structural definition and Gives structural definition and

rigidityrigidity Epithelial sheet is polarizedEpithelial sheet is polarized Apical and basal surfaces often very Apical and basal surfaces often very

differentdifferent– Adheres to different substratesAdheres to different substrates– Has different functionHas different function

Epithelium (cont)Epithelium (cont)

Movement of epithelial cells is done Movement of epithelial cells is done en block with the motive force en block with the motive force usually generated within the sheet usually generated within the sheet by the sum of the cells’ shape by the sum of the cells’ shape changes.changes.

Examples: Gastrulation; NeurulationExamples: Gastrulation; Neurulation

Characteristics of Mesenchymal Characteristics of Mesenchymal CellsCells

Lack regimented structureLack regimented structure Few tight intracellular adhesionsFew tight intracellular adhesions Weak adhesions which allow for ease Weak adhesions which allow for ease

of mobilityof mobility Forms irregular structures that are Forms irregular structures that are

not uniform in composition or densitynot uniform in composition or density More extended and elongated in More extended and elongated in

shapeshape

Mesenchyme (cont.)Mesenchyme (cont.)

Lacks rigid topological specialization Lacks rigid topological specialization (no compartments)(no compartments)

Cells move as individuals, not en Cells move as individuals, not en block, often leaving a trailing region block, often leaving a trailing region behindbehind

Migration mechanistically different Migration mechanistically different and more dynamicand more dynamic

Epithelial and Mesenchymal CellsEpithelial and Mesenchymal Cells

Discovery of EMTDiscovery of EMT

First observed and defined by First observed and defined by Elizabeth Hay in late 1960’s at Elizabeth Hay in late 1960’s at HarvardHarvard

First associated with early stages of First associated with early stages of embryonic development.embryonic development.

Process is reversible w/unstable Process is reversible w/unstable intermediateintermediate

EMT Metastable MET EMT Metastable MET

EMT MarkersEMT Markers Proteins that increase in Proteins that increase in

abundanceabundance N-cadherinN-cadherin VimentinVimentin FibronectinFibronectin Snail1 (Snail)Snail1 (Snail) Snail2(Slug)Snail2(Slug) TwistTwist GoosecoidGoosecoid FOXC2FOXC2 Sox10Sox10 MMP-2MMP-2 MMP-3MMP-3 MMP9MMP9 Integrin vß6Integrin vß6

Proteins that decrease in Proteins that decrease in abundanceabundance

E-cadherenE-cadheren DesmoplakinDesmoplakin CytokeratinCytokeratin OccludinOccludin Proteins whose activity Proteins whose activity

increasesincreases ILKILK GSK-3ßGSK-3ß RhoRho Proteins that accumulateProteins that accumulate

in the nucleusin the nucleus ß-cateninß-catenin Smad-2/3Smad-2/3 NF- ßNF- ß Snail1 (Snail)Snail1 (Snail) Snail2 (Slug)Snail2 (Slug) TwistTwist

TransitionsTransitions

Events Comprising EMTEvents Comprising EMT Specification to differentiate into a type of Specification to differentiate into a type of

cell that will go through EMTcell that will go through EMT.. Specification Specification toward a mesenchymal phenotype toward a mesenchymal phenotype initiates many important changes in gene initiates many important changes in gene expression and protein function that must expression and protein function that must all work in concert for a developmental all work in concert for a developmental EMT to occur correctly. This will direct the EMT to occur correctly. This will direct the subsequent steps and may require subsequent steps and may require stopping cell division so that the stopping cell division so that the cytoskeleton can be used to drive the cell cytoskeleton can be used to drive the cell shape changes and motility needed for shape changes and motility needed for EMT. EMT.

EMTEMT

Temporal and spatial patterning of the Temporal and spatial patterning of the progress of the EMT within the area progress of the EMT within the area destined to undergo EMTdestined to undergo EMT.. Patterning is Patterning is important in that large areas of epithelium important in that large areas of epithelium destined to undergo EMT usually do so destined to undergo EMT usually do so progressively from a restricted zone. This progressively from a restricted zone. This allows both a necessary maintenance of allows both a necessary maintenance of physiological and mechanical continuity of physiological and mechanical continuity of the remaining epithelium and the spatial the remaining epithelium and the spatial regulation of morphogenesis. regulation of morphogenesis.

EMTEMT Move, or be moved, to the site of EMT, Move, or be moved, to the site of EMT,

generally through epithelial generally through epithelial morphogenesismorphogenesis.. Movement of cells to the Movement of cells to the correct position is not always a correct position is not always a requirement, as they may initially lie there requirement, as they may initially lie there to begin with (sea urchins), but in other to begin with (sea urchins), but in other cases it is clearly required, as in the chick cases it is clearly required, as in the chick or mouse primitive streak or the urodele or mouse primitive streak or the urodele amphibian, where large areas of amphibian, where large areas of epithelium are moved to a local site of epithelium are moved to a local site of ingression. The mechanism behind these ingression. The mechanism behind these movements is poorly understood in nearly movements is poorly understood in nearly all cases. all cases.

EMTEMT Alteration or disruption of the basal laminaAlteration or disruption of the basal lamina..

Ingressing cells often move past or through a Ingressing cells often move past or through a basal lamina, which may mechanically impede basal lamina, which may mechanically impede their ingression and therefore must be disrupted their ingression and therefore must be disrupted prior to ingression, presumably by the ingressing prior to ingression, presumably by the ingressing cells. The mechanism behind this is again poorly cells. The mechanism behind this is again poorly understood. Matrix metaloproteases are thought understood. Matrix metaloproteases are thought to be important in, among other things, to be important in, among other things, remodeling or degrading the extracellular matrix remodeling or degrading the extracellular matrix during organogenesis, later tissue remodeling during organogenesis, later tissue remodeling events, and cancer and perhaps cell migration events, and cancer and perhaps cell migration during gastrulation but evidence for a role in during gastrulation but evidence for a role in primary developmental EMTs is lacking so far.primary developmental EMTs is lacking so far.

EMTEMT Change in cell shape, generally by an apical actin-myosin Change in cell shape, generally by an apical actin-myosin

contractile mechanism and/or changes in adhesioncontractile mechanism and/or changes in adhesion.. Ingressing cells often but not always go through a bottle-Ingressing cells often but not always go through a bottle-shaped stage, which may have two functions: by shaped stage, which may have two functions: by constricting their apices cells may displace much of their constricting their apices cells may displace much of their intracellular contents basally and initiate movement out of intracellular contents basally and initiate movement out of the epithelium. Perhaps more important, apical the epithelium. Perhaps more important, apical constrictions reduce the amount of non-adhesive apical constrictions reduce the amount of non-adhesive apical membrane and circumferential, apical junctions that must membrane and circumferential, apical junctions that must finally be broken upon ingressing. It also reduces the size of finally be broken upon ingressing. It also reduces the size of the hole left in the epithelium. It is generally thought that the hole left in the epithelium. It is generally thought that apical constriction is driven by an actin-myosin-based apical constriction is driven by an actin-myosin-based contraction, while the apical membrane is reduced by contraction, while the apical membrane is reduced by endocytosis. Changes in adhesion may also contribute to endocytosis. Changes in adhesion may also contribute to cell shape change on EMT. Cell behaviors in echinoderm cell shape change on EMT. Cell behaviors in echinoderm gastrulation are consistent with the possibility that cells gastrulation are consistent with the possibility that cells round up by loss of basolateral adhesion round up by loss of basolateral adhesion

EMTEMT De-epithelializeDe-epithelialize.. We define de-epithelialization as the loss of We define de-epithelialization as the loss of

the coherent contact between neighbors that characterizes the coherent contact between neighbors that characterizes a particular epithelium, and the eventual loss of an apical a particular epithelium, and the eventual loss of an apical membrane domain. This involves a loss of the extensive membrane domain. This involves a loss of the extensive circumferential apical junctions, specifically the circumferential apical junctions, specifically the circumapical tight and adherens junctions, in the case of circumapical tight and adherens junctions, in the case of epithelia that are physiologically and mechanically very epithelia that are physiologically and mechanically very impermeant and coherent, but it can also involve loss of the impermeant and coherent, but it can also involve loss of the junctions accounting for the apical coherence of less junctions accounting for the apical coherence of less coherent and resistive epitheloid sheets, a state of coherent and resistive epitheloid sheets, a state of ‘epithelialness’ that is poorly characterized. How these ‘epithelialness’ that is poorly characterized. How these processes occur is not understood. The evidence suggests processes occur is not understood. The evidence suggests that targeted endocytosis of epithelial junctions and that targeted endocytosis of epithelial junctions and adhesion molecules may be important and the apical adhesion molecules may be important and the apical membrane may eventually be completely eliminated by membrane may eventually be completely eliminated by endocytosis. endocytosis.

EMTEMT IngressIngress.. We define ingression simply as the We define ingression simply as the

withdrawal of the ingressing cell's apex from the withdrawal of the ingressing cell's apex from the epithelial layer and into the deep layer. It differs epithelial layer and into the deep layer. It differs from de-epithelialization in that a cell could de-from de-epithelialization in that a cell could de-epithelialize and not move out of the sheet. epithelialize and not move out of the sheet. Normal ingression is associated with de-Normal ingression is associated with de-epithelialization and adoption of basal epithelialization and adoption of basal mesenchymal characteristics, including an active mesenchymal characteristics, including an active motility and strong traction on deep tissues or motility and strong traction on deep tissues or structures, to pull the cell out of the epithelium. structures, to pull the cell out of the epithelium. The cell might also be squeezed out of the The cell might also be squeezed out of the remaining epithelium by virtue of the fact that remaining epithelium by virtue of the fact that loss of apical coherence is likely to stimulate loss of apical coherence is likely to stimulate wound healing wound healing

EMTEMT Differentiate cell behavior and organization characteristic of a Differentiate cell behavior and organization characteristic of a

mesenchymal phenotypemesenchymal phenotype.. This process begins prior to de- This process begins prior to de-epithelialization, continues through ingression, and is not yet complete epithelialization, continues through ingression, and is not yet complete in recently ingressed cells. Ingressed cells often retain markers of their in recently ingressed cells. Ingressed cells often retain markers of their apices shortly after ingression, such as remnants of tight junctions. Cells apices shortly after ingression, such as remnants of tight junctions. Cells must continue the process of turning off epithelial characters and must continue the process of turning off epithelial characters and turning on mesenchymal characters. This requires a major turning on mesenchymal characters. This requires a major reorganization of the cell, including completely dismantling the apical reorganization of the cell, including completely dismantling the apical junctional ‘scaffold’ that is thought to regulate discrimination between junctional ‘scaffold’ that is thought to regulate discrimination between apical and basal–lateral (e.g. by vesicular traffic, and organization of the apical and basal–lateral (e.g. by vesicular traffic, and organization of the cytoskeleton.) This, with the removal of the apical membrane, results in cytoskeleton.) This, with the removal of the apical membrane, results in the loss of the cell's apical–basal polarity. The basal–lateral membrane the loss of the cell's apical–basal polarity. The basal–lateral membrane also must be remodeled, including the removal of epithelial adhesive also must be remodeled, including the removal of epithelial adhesive molecules, perhaps by endocytosis, and replacement by mesenchymal-molecules, perhaps by endocytosis, and replacement by mesenchymal-type adhesion molecules (cadherins, for example) and matrix receptors type adhesion molecules (cadherins, for example) and matrix receptors (integrins). The cytoskeleton must be remodeled, from what we imagine (integrins). The cytoskeleton must be remodeled, from what we imagine is a static, structural epithelial configuration to a dynamic, migratory is a static, structural epithelial configuration to a dynamic, migratory configuration, a process that involves change from epithelial configuration, a process that involves change from epithelial cytokeratins to mesenchymal vimentins, and probably substantial cytokeratins to mesenchymal vimentins, and probably substantial changes in regulation of actin polymerization, microtubule dynamics and changes in regulation of actin polymerization, microtubule dynamics and myosin function to allow protrusive activity, all poorly understood myosin function to allow protrusive activity, all poorly understood phenomena in embryonic EMTs. phenomena in embryonic EMTs.

Steps of EMTSteps of EMT First, inductive or other First, inductive or other

specification events occur, specification events occur, committing the cell to an EMT committing the cell to an EMT (dark green), highlighted cell, A). (dark green), highlighted cell, A). Generally but not always, the cell Generally but not always, the cell undergoes a constriction of its undergoes a constriction of its apical region (small thick arrows, apical region (small thick arrows, B,C), a process which probably B,C), a process which probably involves either a circumferential involves either a circumferential contractile cytoskeleton (B′) or a contractile cytoskeleton (B′) or a contractile cytoskeletal meshwork contractile cytoskeletal meshwork spanning the apices (B″). spanning the apices (B″). Coincident with the apical Coincident with the apical constriction, the cell often begins constriction, the cell often begins to elongate the apical–basal axis to elongate the apical–basal axis as cytoplasm is pushed basally as cytoplasm is pushed basally (small skinny arrows, B,C). The cell (small skinny arrows, B,C). The cell also begins to break down the also begins to break down the basal lamina (magenta, A–C). basal lamina (magenta, A–C).

Steps of EMTSteps of EMT Other changes may include Other changes may include

formation of protrusions at the formation of protrusions at the basal ends (gray, C,D), down-basal ends (gray, C,D), down-regulation of epithelial cell regulation of epithelial cell adhesion and cell–extracellular adhesion and cell–extracellular matrix adhesion receptors, and matrix adhesion receptors, and expression of mesenchymal expression of mesenchymal adhesion molecules adhesion molecules (basolateral spots, C,D). (basolateral spots, C,D). Epithelial cell adhesion Epithelial cell adhesion molecules are down-regulated, molecules are down-regulated, and as the apical region of the and as the apical region of the cell shrinks, the apical cell shrinks, the apical junctions decrease in junctions decrease in circumference and in strength, circumference and in strength, and eventually the cell pulls and eventually the cell pulls itself, or is pulled or pushed itself, or is pulled or pushed beneath the surface and out of beneath the surface and out of the epithelium (C–E).the epithelium (C–E).

Steps of EMTSteps of EMT Epithelial cell adhesion Epithelial cell adhesion

molecules are down-regulated, molecules are down-regulated, and as the apical region of the and as the apical region of the cell shrinks, the apical cell shrinks, the apical junctions decrease in junctions decrease in circumference and in strength, circumference and in strength, and eventually the cell pulls and eventually the cell pulls itself, or is pulled or pushed itself, or is pulled or pushed beneath the surface and out of beneath the surface and out of the epithelium (C–E). In some the epithelium (C–E). In some cases the apical membrane is cases the apical membrane is thrown into microvilli or thrown into microvilli or microfolds as the apical region microfolds as the apical region of the cell decreases in area, of the cell decreases in area, and membrane may be and membrane may be internalized (C′). Molecules or internalized (C′). Molecules or whole junctions of the whole junctions of the junctional complex may also be junctional complex may also be removed from the cell surface removed from the cell surface and internalized as vesicles (Cand internalized as vesicles (C′). ′).

Steps of EMTSteps of EMT We envision two ways of We envision two ways of

removing the cell from the removing the cell from the epithelium. The apical epithelium. The apical junctional complex breaks, the junctional complex breaks, the contiguity of the cell with contiguity of the cell with epithelium is broken, and it epithelium is broken, and it leaves the epithelium leaves the epithelium (ingression) and a hole in its (ingression) and a hole in its place (C″). Alternatively, the place (C″). Alternatively, the adjacent cells might bridge adjacent cells might bridge over the ingressing cell, form a over the ingressing cell, form a junctional complex above it, junctional complex above it, and provide physiological and and provide physiological and mechanical contiguity while mechanical contiguity while the cell ingresses (C ). the cell ingresses (C ). Disarrayed patches of junctions Disarrayed patches of junctions are often found on freshly are often found on freshly ingressed cells (C″,C ). ingressed cells (C″,C ).

Steps of EMTSteps of EMT Other cytoskeletal Other cytoskeletal

changes also occur. changes also occur. Vimentin containing Vimentin containing intermediate filaments intermediate filaments are formed in favor of the are formed in favor of the cytokeratin intermediate cytokeratin intermediate filaments of epithelial filaments of epithelial cells, and the regulation cells, and the regulation of the cytoskeleton, of the cytoskeleton, protrusive activity, and protrusive activity, and contact and guidance contact and guidance behavior is altered to the behavior is altered to the mesenchymal pattern by mesenchymal pattern by as yet poorly understood as yet poorly understood mechanisms. mechanisms.

Typical pattern of embryonic Typical pattern of embryonic development in animalsdevelopment in animals

NOTE #1: Speaking here of NOTE #1: Speaking here of Metazoans. This process does not Metazoans. This process does not occur in single celled organisms, occur in single celled organisms, fungi or plants, the latter two being fungi or plants, the latter two being unable to move their cells because of unable to move their cells because of the presence of a cell wallthe presence of a cell wall

Animal Development - IAnimal Development - I

Early cleavage results in a ball of Early cleavage results in a ball of cells which, on cue, form tight cells which, on cue, form tight desmosomal junctions and usually a desmosomal junctions and usually a hollow space, the blastocoel.hollow space, the blastocoel.

Thus the initial structure is an Thus the initial structure is an epithelium folded into a ball.epithelium folded into a ball.

Animal Development - IIAnimal Development - II

The second phase is the formation of The second phase is the formation of a Triploblastic embryo.a Triploblastic embryo.

Three primary germ layersThree primary germ layers– EctodermEctoderm– MesodermMesoderm– EndodermEndoderm

Process is called GastrulationProcess is called Gastrulation

GastrulationGastrulation

Two processes involved Two processes involved

Epithelial sheet deforms as a unit to Epithelial sheet deforms as a unit to form the archenteron or primitive gutform the archenteron or primitive gut

A small number of cells at the base A small number of cells at the base or vegetal plate loose contact with or vegetal plate loose contact with neighbors, tear loose for Basal neighbors, tear loose for Basal lamina and crawl into blastocoellamina and crawl into blastocoel

Sea Urchin EMTSea Urchin EMT

Amphibian EMTAmphibian EMT

Surface and Cross SectionSurface and Cross Section

Chicken EMTChicken EMT

Chordate Neurulation EMTChordate Neurulation EMT

EMT in TissuesEMT in Tissues Epithelium IEpithelium I induces an induces an

EMT processEMT process in in epithelium epithelium IIII ( (black arrowsblack arrows) through the ) through the secretion of secretion of inducersinducers ((purple dotspurple dots). The ). The epithelium II-derived epithelium II-derived mesenchymal populationmesenchymal population ((greengreen) is recruited by ) is recruited by epithelium Iepithelium I ( (green-green-toto--blueblue--graded arrowsgraded arrows) and ) and differentiatesdifferentiates ( (blue cellsblue cells) ) according to the molecular according to the molecular information arising from information arising from the inducing tissuethe inducing tissue ( (red red dotsdots). ).

EMT and CancerEMT and Cancer

Occurrence of EMT during tumor Occurrence of EMT during tumor progression allows benign tumors to progression allows benign tumors to infiltrate surrounding tissue and infiltrate surrounding tissue and ultimately metastasize to distant ultimately metastasize to distant sitessites

We see EMT stages in pathological We see EMT stages in pathological staging of tumorsstaging of tumors

EMT in Tumor ProgressionEMT in Tumor Progression

EMT of NBT II Cells and Mouse EMT of NBT II Cells and Mouse GastrulationGastrulation

TGF beta and Chick HeartTGF beta and Chick Heart

Sarcomas and CarcinomasSarcomas and Carcinomas

EMT and Colorectal CancerEMT and Colorectal Cancer

EMT Signaling PathwaysEMT Signaling Pathways