epithelial and mesenchymal transition in invasion and metastasis

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EPITHELIAL MESENCHYMAL TRANSITION IN INVASION AND METASTASISMODERATOR : Prof. PARAMESHAPRESENTER : Dr. ASHWINI K.T

NEOPLASIA Neo + Plasia New + Growth. Tumour Swelling any swelling*Willis definition: A neoplasm is an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissue and persists in the same manner after cessation of the stimuli which evoked the changeCell division without controlIrreversible DNA damage, resulting in autonomous growth of abnormal cells

Cell population / Growth Control:Proliferation Differentiation Apoptosis * Normal

Stem cellApoptosis

Organ / Tissue

Neoplasia Benign - Malignant

CCarcinogenInitiator + PromotorInflamProto-Oncogenes (growth factors) Growth/Tumor suppressor genes. Genes controlling Apoptosis.Genes controlling DNA Repair.RASRbMYCp53

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Non neoplastic Neoplastic (Polyclonal) (Monoclonal)Growth Disorders:

Normal Adaptation Benign Malignant HyperplasiaHypertrophyAplasiaAtrophyMetaplasiaDysplasia

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Cell division Control - Carcinogenesis*

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Cancer Biology:Structure:Parenchyma Neoplastic cells.Stroma: Non neoplastic - normal DNAFeatures:Differentiation Maturation of cells.Rate of Growth Mitotic rate / Ki 67Local invasion Hemorrhage, necrosis, destructionMetastasis Distant Spread.

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Tumors are clonal (one parent)But have different mutations different shapes & features.Each new mutation adds a new feature.Malignant cellsNormalcellFourth orlater mutationThirdmutationSecondmutationFirstmutation

More new mutations with time.

National Cancer InstituteUnderstanding Cancer and Related Topics Understanding Cancer GenomicsNCI Web site: http://cancer.gov/cancertopics/understandingcancer7Each cell, when it divides, generates two identical new ones. So, when a cell acquires a mutation, it passes that mutation on to its progeny during cell growth and division. Because cells with cancer-linked mutations tend to proliferate more than normal cells, cellular candidates for additional mutations grow in number. Mutations continue to accumulate and are copied to descendant cells. If one cell finally acquires enough mutations to become cancerous, subsequent cancer cells will be derived from that one single transformed cell. So all tumors are clonal, which means that they originate from a single parent cell, whether that first mutant cell was of germline or somatic origin.

Features of Cancer cells:

Clinically each patients cancer has a different mix of features depending on quality & quantity of mutations Changes With Time...!

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Invasion and metastasis are biologic hallmarks of malignant tumorsFor tumor cells to break loose from a primary mass, enter blood vessels or lymphatics, and produce a secondary growth at a distant site, they must go through a series of step (the metastatic cascade):Divided into two phases: Invasion of the extracellular matrix (ECM)Vascular dissemination, homing of tumor cells, and colonization

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Metastasis

In situ carcinoma

Microinvasive

Vascular invasion

Metastatic deposit in brain

The metastatic cascade Sequential steps involved in the hematogenous spread of a tumor

16FIGURE 7-36 The metastatic cascade. Sequential steps involved in the hematogenous spread of a tumor.

Invasion of Extracellular MatrixTwo types of ECM: Basement membrane (BM) and Interstitial connective tissueComposition: ECM is made up of collagens, glycoproteins, and proteoglycansA carcinoma must first breach the underlying BMThen traverse the interstitial connective tissue, and Gain access to the circulation by penetrating the vascular BM

This process is repeated in reverse when tumor cell emboli extravasate at a distant site

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Invasion of Extracellular MatrixInvasion of the ECM initiates the metastatic cascade and is an active process that can be resolved into several steps Changes (loosening up) of tumor cell-cell interactions Degradation of ECM Attachment to novel ECM components Migration of tumor cells

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Sequence of events in the invasion of epithelial basement membranes by tumor cells: Tumor cells detach from each other because of reduced adhesiveness,then secrete proteolytic enzymes, degrading the basement membrane. Binding to proteolytically generated binding sites and tumor cell migration follow

19FIGURE 7-37 AD, Sequence of events in the invasion of epithelial basement membranes by tumor cells. Tumor cells detach from each other because of reduced adhesiveness, then secrete proteolytic enzymes, degrading the basement membrane. Binding to proteolytically generated binding sites and tumor cell migration follow.


20Now we are going to deal with the first step: Changes (loosening up) of tumor cell-cell interactions.

1-Dissociation of cells from one another(loosening up) of tumor cell-cell interactionsAs a result of alterations in intercellular adhesion molecules

21Dissociation of cells from one another is often the result of alterations in intercellular adhesion molecules. Normal cells are neatly glued to each other and their surroundings by a variety of adhesion molecules.[111] Cell-cell interactions are mediated by the cadherin family of transmembrane glycoproteins. E-cadherins mediate homotypic adhesions in epithelial tissue, thus serving to keep the epithelial cells together and to relay signals between the cells; intracellularly the E-cadherins are connected to -catenin and the actin cytoskeleton. In several epithelial tumors, including adenocarcinomas of the colon and breast, there is a down-regulation of E-cadherin expression. Presumably, this down-regulation reduces the ability of cells to adhere to each other and facilitates their detachment from the primary tumor and their advance into the surrounding tissues. E-cadherins are linked to the cytoskeleton by the catenins, proteins that lie under the plasma membrane (see Fig. 7-33 ). The normal function of E-cadherin is dependent on its linkage to catenins. In some tumors E-cadherin is normal, but its expression is reduced because of mutations in the gene for catenin.

Normal cells are bound together by adhesion moleculesCell-cell interactions are mediated by the cadherin family of transmembrane glycoproteins Intracellularly the E-cadherins are connected to -catenin and the actin cytoskeleton

22Normal cells are neatly glued to each other and their surroundings by a variety of adhesion molecules.[111] Cell-cell interactions are mediated by the cadherin family of transmembrane glycoproteins. E-cadherins mediate homotypic adhesions in epithelial tissue, thus serving to keep the epithelial cells together and to relay signals between the cells; intracellularly the E-cadherins are connected to -catenin and the actin cytoskeleton.

23This image is shown to highlight the cell to cell interaction by cadherin, beta-catenin and cytoskeleton.

Tumors with down-regulated E-cadherin expressionSeen in several epithelial tumors, including adenocarcinomas of the colon and breast This down-regulation reduces the ability of cells to adhere to each other and facilitates their detachment from the primary tumorThe normal function of E-cadherin is dependent on its linkage to cateninsIn some tumors E-cadherin is normal, but its expression is reduced because of mutations in the gene for catenin

24In several epithelial tumors, including adenocarcinomas of the colon and breast, there is a down-regulation of E-cadherin expression. Presumably, this down-regulation reduces the ability of cells to adhere to each other and facilitates their detachment from the primary tumor and their advance into the surrounding tissues. E-cadherins are linked to the cytoskeleton by the catenins, proteins that lie under the plasma membrane (see Fig. 7-33 ). The normal function of E-cadherin is dependent on its linkage to catenins. In some tumors E-cadherin is normal, but its expression is reduced because of mutations in the gene for catenin.

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26Next step in invasion of ECM is degradation of ECM.

2-Local degradation of the basement membrane and interstitial connective tissueElaboration of proteases byTumor cells themselves orStromal cells [induced by tumor cells]Many different families of proteasesMatrix metalloproteinases (MMPs) Cathepsin D & Urokinase plasminogen activator

27The second step in invasion is local degradation of the basement membrane and interstitial connective tissue. Tumor cells may either secrete proteolytic enzymes themselves or induce stromal cells (e.g., fibroblasts and inflammatory cells) to elaborate proteases. Many different families of proteases, such as matrix metalloproteinases (MMPs), cathepsin D, and urokinase plasminogen activator, have been implicated in tumor cell invasion. MMPs regulate tumor invasion not only by remodeling insoluble components of the basement membrane and interstitial matrix but also by releasing ECM-sequestered growth factors. Indeed, cleavage products of collagen and proteoglycans also have chemotactic, angiogenic, and growth-promoting effects.[112] For example, MMP9 is a gelatinase that cleaves type IV collagen of the epithelial and vascular basement membrane and also stimulates release of VEGF from ECM-sequestered pools. Benign tumors of the breast, colon, and stomach show little type IV collagenase activity, whereas their malignant counterparts overexpress this enzyme. Concurrently, the concentrations of metalloproteinase inhibitors are reduced so that the balance is tilted greatly toward tissue degradation. Indeed, overexpression of MMPs and other proteases has been reported for many tumors. However, recent in vivo imaging experiments have shown that tumor cells can adopt a second mode of invasion, termed ameboid migration.[113] In this type of migration the cell squeezes through spaces in the matrix instead of cutting its way through it. This ameboid migration is much quicker, and tumor cells seem to be able to use collagen fibers as high-speed railways in their travels. Tumor cells, in vitro at least, seem to be able to switch between the two forms of migration, perhaps explaining the disappointing performance of MMP inhibitors in clinical trials.

MMPsTumors either elaborate large quantities of MMPs or they may reduce the concentrations of MMP-inhibitors

They regulate tumor invasion by:Dissolving components of the BM & interstitial matrix Releasing ECM-sequestered growth factorsCleavage products of collagen and proteoglycans also have chemotactic, angiogenic, and growth-promoting effectsEg: MMP9 is a gelatinase that cleaves type IV collagen of the epithelial and vascular basement membrane and also stimulates release of VEGF from ECM-sequestered pools

Eg: Benign tumors of the breast, colon, and stomach show little type IV collagenase activity, whereas their malignant counterparts overexpress this enzyme

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29Now we will move on to the next mechanism: Attachment to novel ECM components

3 - Attachment to novel ECM componentsNormal epithelial cells have receptors, such as integrins, for basement membrane laminin and collagens that are polarized at their basal surfaceThese receptors help to maintain the cells in a resting, differentiated stateLoss of adhesion in normal cells leads to induction of apoptosis [tumor cells are resistant to this form of cell death]The matrix itself is modified in ways that promote invasion and metastasisEg: cleavage of the basement membrane proteins collagen IV and laminin by MMP2 or MMP9 generates novel sites that bind to receptors on tumor cells and stimulate migration

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31Now we will move on to the final mechanism: Migration of tumor cells

4 Migration of tumor cells - Locomotion Tumor cells propel themselves through the degraded basement membranes and zones of matrix proteolysisIt involves many families of receptors and signaling proteins that eventually impinge on the actin cytoskeletonCells must attach to the matrix at the leading edge, detach from the matrix at the trailing edge, and contract the actin cytoskeleton to ratchet forward - Ameboid migrationSuch movement are potentiated by tumor cellderived cytokinesCleavage products of matrix components (e.g., collagen, laminin) and some growth factors (e.g., IGFs I and II) have chemotactic activity for tumor cellsStromal cells also produce paracrine effectors of cell motilityHGFscatter factor, which bind to receptors on tumor cellsHGFscatter factor is elevated at the advancing edges of the highly invasive brain tumor glioblastoma multiforme

32HGF=hepatocytes growth factorLocomotion is the final step of invasion, propelling tumor cells through the degraded basement membranes and zones of matrix proteolysis. Migration is a complex, multistep process that involves many families of receptors and signaling proteins that eventually impinge on the actin cytoskeleton. Cells must attach to the matrix at the leading edge, detach from the matrix at the trailing edge, and contract the actin cytoskeleton to ratchet forward. Such movement seems to be potentiated and directed by tumor cellderived cytokines, such as autocrine motility factors. In addition, cleavage products of matrix components (e.g., collagen, laminin) and some growth factors (e.g., IGFs I and II) have chemotactic activity for tumor cells. Furthermore, proteolytic cleavage liberates growth factors bound to matrix molecules. Stromal cells also produce paracrine effectors of cell motility, such as hepatocyte growth factorscatter factor, which bind to receptors on tumor cells. Concentrations of hepatocyte growth factorscatter factor are elevated at the advancing edges of the highly invasive brain tumor glioblastoma multiforme, supporting their role in motility.It has become clear in recent years that the ECM and stromal cells surrounding tumor cells do not merely represent a static barrier for tumor cells to traverse but instead represent a varied environment in which reciprocal signaling between tumor cells and stromal cells may either promote or prevent tumorigenesis and/or tumor progression.[24] Stromal cells that interact with tumors include innate and adaptive immune cells (discussed later), as well as fibroblasts. A variety of studies have demonstrated that tumor-associated fibroblasts exhibit altered expression of genes that encode ECM molecules, proteases, protease inhibitors, and various growth factors. Thus, tumor cells live in a complex and ever-changing milieu composed of ECM, growth factors, fibroblasts, and immune cells, with significant cross-talk among all the components. The most successful tumors may be those that can co-opt and adapt this environment to their own nefarious ends.

Ameboid migrationIn this type of migration the cell squeezes through spaces in the matrix instead of cutting its way through itThis ameboid migration is much quickerTumor cells are capable of switching between the two forms of migration, perhaps explaining the disappointing performance of MMP inhibitors in clinical trials

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Malignant tumors have varied metastatic potentialCancer without metastatic potentialBasal cell carcinomaCancer with high malignant potentialMalignant melanoma

Why this variation?What genetic changes bring about metastatic potential?

Several THEORIES have been proposed to explain how the metastatic phenotype arises?The clonal evolution modelAs mutations accumulate in cancer cells, the tumor become heterogeneous

2. Metastasis is the result of multiple abnormalities that occur in most of the cells in a primary tumorMetastasis signatureIt may involve the cancer cells or in the microenvironment3. Background genetic variation in gene expression contributes to the generation of metastases4. Tumors derive from rare tumor stem cells, metastases require the spread of the tumor stem cells themselves

Mechanisms of metastasis development within a primary tumor:A nonmetastatic primary tumor is shown (light blue) on the left side of all diagrams. Four models are presented: A, Metastasis is caused by rare variant clones that develop in the primary tumor; B, Metastasis is caused by the gene expression pattern of most cells of the primary tumor, referred to as a metastatic signature; C, A combination of A and B, in which metastatic variants appear in a tumor with a metastatic gene signature; D, Metastasis development is greatly influenced by the tumor stroma, which may regulate angiogenesis, local invasiveness, and resistance to immune elimination, allowing cells of the primary tumor, as in C, to become metastatic.

37FIGURE 7-38 Mechanisms of metastasis development within a primary tumor. A nonmetastatic primary tumor is shown (light blue) on the left side of all diagrams. Four models are presented: A, Metastasis is caused by rare variant clones that develop in the primary tumor; B, Metastasis is caused by the gene expression pattern of most cells of the primary tumor, referred to as a metastatic signature; C, A combination of A and B, in which metastatic variants appear in a tumor with a metastatic gene signature; D, Metastasis development is greatly influenced by the tumor stroma, which may regulate angiogenesis, local invasiveness, and resistance to immune elimination, allowing cells of the primary tumor, as in C, to become metastatic.

Are there genes whose principal or sole contribution to tumorigenesis is to control metastasis? genes that function as metastasis oncogenes or metastatic suppressors are rareAt least a dozen genes lost in metastatic lesions have been confirmed to function as metastasis suppressorsTheir molecular functions are varied and not yet completely clear; however, most appear to affect various signaling pathwaysRecent work has suggested that two miRNAs, mir335 and mir126, suppress the metastasis of breast cancer, while a second set (mir10b) promotes metastasis

EPITHELIAL - MESENCHYMAL TRANSITION

Epithelial cells1. Closely adjoined2. PolarizedEpithelial Markers:E-Cadherin (adherens junctions)Claudins (tight junctions)Occludin (tight junctions)Desmoplakin (desmosomes)Cytokeratin-8, -18 and -19Mucin-1 There are 5 different types of cell junctions. They are tight junctions, adherens junctions, desmosomes, hemidesmosomes, and gap junctions.

Mesenchymal cells1. Not adjoined2. No polarity

Mesenchymal Markers:VimentinN-CadherinFibronectinVitronectinFSP1(fibroblast-specific protein 1)Smooth-muscle actinFGFR2 IIIb and IIIc splice variants

Epithelial cells can convert into mesenchymal cells by a process known as EMT, which disrupts cell-cell adhesion and cell-ECM adhesion.

* Embryogenesis and development* Wound healing* Fibrosis*Cancer

EPITHELIAL PLASTICITY IS BI-DIRECTIONALMESOTHELIOMA SYNOVIAL SARCOMA

CELLULAR MODIFICATIONS ASSOCIATED WITH EMT1. In vitro morphology and function * stellate or spindle shape * resistance to anoikis * increased migration * invasion to collagen matrix2. Down regulated proteins * E- Cadherin * Cytokeratin * Occludin * Claudin

3.Up regulated Proteins * N Cadherins * vimentin SNAI1 * Snai2 * Twist * MMPs (2,3,9) * Integrins y 6 4.Activated proteins * ILK * GSK 3 * Rho 5. Nuclear expression of proteins * catenin * Smad ( 2,3 ) * Snail1 * Snail2 * Twist

GenesSNAI1SmadZebTWIST

Pathways 1. Wnt PATHWAY

*REDUCED CELL ADHESION DOWNREGULATION OF E- CADHERIN

***SNAI1 - INTERMEDIATE SIGNALLING PATHWAY ***

2.Rac , FAK , Paxillin

INCREASED MIGRATION

** SNAI2 INTERMEDIATE SIGNALLING END POINT***

3. RAS , MAPK * CYTOSKELETON ACTIVATION * MIGRATION * FOCAL ADHESION REARRANGEMENT

**SNAI2 INTERMEDIATE SIGNALLING PATHWAY***

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4. RhoA

* STRESS FIBRES MIGRATION

EMT and Cancer Progression of most carcinomas is associated with the acquisition of mesenchymal phenotype. Cells with an EMT phenotype induced by different factors are rich sources for cancer stem-like cells. Moreover, induction of EMT in tumor cells not only promotes invasion and metastasis but also contributes to drug resistance

Induction of EMT Generates Stem-Like Cells

Mani SA, et al. The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells. Leukemia initiating cells are CD34+CD38- cells.Colon cancer initiating cells are CD133+ cells.Brain cancer initiating cells are CD133+ cells.Prostate cancer initiating cells are CD44+21+ cells.Breast cancer initiating cells are CD44+ CD24- cells. EMT phenotype EMT induction

miRNAs Link EMT to Stem-Like Cells in Human CancersmiR-200 family and ZEB1/2miR-200a * Knockdown of Akt-1 decreases the expression of miR-200 family including miR 200a, and increases mammosphere forming ability in breast cancermiR-200b * miR-200b inhibits expression of ZEB1, ZEB2, Lin28B and Notch1 in prostate cancer * miR-200b targets Suz12 and contributes to cancer stem cells maintenance in breast cancermiR-200c * miR-200c inhibits expression of ZEB1, ZEB2 and Bmi1 in breast cancer; * miR-200c inhibits expression of ZEB1, Sox2, Bmi1 and KLF4 in pancreatic cancermiR-183 * ZEB1 represses miR-183 expression, which increases the expression of Bmi1 and KLF4 in pancreatic cancermiR-203 * ZEB1 represses miR-203 expression, which increases the expression of Bmi1 and KLF4 in pancreatic cancer

Several miRNAs have been identified as either oncogenes (miR-1792, miR-155, miR-21) or tumor suppressors (miR-15a, miR-16a, let-7) and some human tumor types can be classified by miRNA signatures. The miR-200 family of miRNAs consists of five members (miR-200a,200b, miR-200c, 141, 429) that have been demonstrated to have a role in EMT through regulation with the ZEB transcription factors and regulation of E-cadherin and vimentin expression. The most striking effect of miR-200 expression was a change in protein constituents in the media resulting from protein secretion and shedding with downregulation of extracellular matrix, peptidases and cell adhesion proteins. Proteins upregulated with miR-200 restoration were associated primarily with cytoskeletal regulation and cell adhesion

- Cancer Research (2011) Dec 15; 71(24): 76707682

WOUND HEALING Mainly in skin and cornea Wounding of epidermis

Epithelial stem cells are activated .

The adherence junctions disassemble and E- Cadherin expression is down regulated , vimentin expression is up regulated

cytoskeleton is activated

Cell start to migrate .*SNAI2 PLAYS AN IMPORTANT ORCHESRATING ROLE IN THIS PROCESS *

EMT and FibrosisFibrosis is characterized by the presence of an excess of fibrous connective tissue in an organ, and in particular by an excessive deposition of collagen I.Renal fibrosis, for example, has been associated with the activation of interstitial fibroblasts, which give rise to collagen secreting myofibroblasts. In addition, myofibroblasts can also originate from renal tubular epithelial and endothelial cells that undergo EMT. High Snail1 expression and evidence of EMT has also been found in the kidneys of patients with renal fibrosis (Boutet et al, 2006).

Cancer and Fibrosis are (Induced by) Inflammation In the context of a chronic inflammatory condition, TGF1and hypoxia activate EMT that converges in the activation of NFB, which is also induced by the inflammatory cytokines and oxidative stress.

Importance of EMT concept in the diagnosis and treatment of cancer1. Specific (ant)agonists for key molecules in the process of invasion and metastasis Targeted therapy .Eg: Cystatin C to inhibit EMT in breast cancer .

TWO problems - find solutions in the application of new knowledge * the borderline between non invasive and invasive neoplasia * prediction of tumor cell behaviour -- IMMUNOHISTOCHEMISTRY used to detect expression of molecular markers of invasion.

References :1. Kumar,Abbas,Fausto . Robbins and Cotran Pathologic basis of disease,9th ed. Elsevier .2. Recent advances in histopathology vol -22 3. Kalluri R , Weinberg RA. The basics of epithelial mesenchymal transition . The journal of clinical investigation .2009;119(6):1420-28.4. Boutet A, De Frutos CA, Maxwel PH , Mayol et al . Snai1 activation disrupts tissue homeostasis and induces fibrosis in the adult kidney. The EMBO journal . 2006;25(23):5603-5613.5.Internet sources .

epithelial and mesenchymal transition in invasion and metastasis

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