EMT Pathway in Tumor Progression

Abstract

Epithelial to mesenchymal transition pathway responsible for embryogenesis, organ development, and tumor metastasis is an important pathway in tumor progression. The gaining of mesenchymal properties such as migratory, invasive phenotype and losing the epithelial properties such as apical to basal polarity. EMT is a multi-step process i.e. from the gaining of mesenchymal features to the formation of a secondary tumor microenvironment through intravasation and extravasation of circulating tumor cells (CTCs). Loss or low E-cadherin expression is the major hallmark of this pathway. Various signaling pathways are involved in the induction of EMT that regulate it directly or indirectly. TGFβ, Wnt/β-Catenin, PI3K/Akt, STAT/LIV-1 signaling and Hedgehog signaling pathway are responsible for the induction of the EMT pathway through heterotypic signaling. Whereas GSK-3β, MTA3 repressor, and drugs such as Herceptin, erlotinib, and imatinib are responsible for the downregulation of the EMT pathway in tumor cells. Transcription factors such as Snail, Twist, Slug, and Goosecoid also induce EMT during tumor progression. Targeting the pathways which induce the EMT pathway and over-activation of pathways that are downregulating the EMT pathway may elicit good treatment procedures for fighting against cancer. Using inhibitory drugs, induced miRNA expression, and ligand/trap method against an EMT-inducing pathway could be the novel approach for fighting against cancer and its progression.

  

Introduction

The transformation of polarized epithelial cells in contact with basement membrane into migratory and invasive mesenchymal cells is the EMT characteristics. It is having resistance to the apoptotic pathway and produce increase amount of fibronectin and collagen depositions. It is involved in embryogenesis, embryo implantation, organ development e.g. neural tube. The features of EMT pathway are the destruction of the basement membrane, local invasion, intravasation, survival of tumor cells in the bloodstream and extravasation. Various proteins, signaling pathway, and transcription factors help in inducing the EMT pathway. The MET (Mesenchymal to Epithelial Transition) is an antagonistic pathway to EMT. Besides regulating organ development, cancer progression and metastasis, it is also associated with tissue regeneration and organ fibrosis (produce an excessive amount of collagen-rich ECM in myofibroblast). The hallmarks of EMT resembles with hallmarks of tumor cells such as excessive cell proliferation, angiogenesis, expression of mesenchymal markers (alpha-SMA, vimentin, FSP-1, desmin, etc.). Vimentin is intermediate filament comprises the cytoskeleton along with tubulin-based microtubules and actin-based microfilaments. The mesenchymal markers associated with entering of EMT induced cells into invasive metastatic cascade i.e. intravasation, blood stream transport, extravasation, the formation of microenvironment (micrometastasis), colonization and growth into macrometastasis.

Role in Embryogenesis –

EMT is induced in epithelial cells during embryogenesis period for the development of different organs such as neurocoele which further form the cranial neural crest cells. During development, epidermal layer invaginates towards hollow space. With the loss of epithelial markers and adhesive molecule, EMT induced cells to loose adhesion and bud off to form a circle like structure that further give rise to cylindrically shaped neurocoele and helps in the neural tube and cranial neural crest cell (responsible for craniofacial development) formation. Cranial cells and muscles, odontoblasts, ganglia, melanocytes, cardiac septum, urogenital tract, skeleton muscles and tendons, embryonic notochord formation.

Epithelial cells and mesenchymal cell features - 

Major characteristics of tumor cells during EMT induction –

•      Loss of E-Cadherin expression

•      Disrupts ECM formation

•      Increase collagen and fibronectin depositions

•      Loss of cell to cell adhesion

•      Loss of epithelial properties and gain of mesenchymal properties

•      Self-sufficiency in growth signals

•      Resistance to anti-growth signals

•      Limitless replicative potential (High mitotic rate)

•      Evasion of apoptosis

•      Sustained angiogenesis

Multiple steps involved in induction of EMT –

There are various levels of the induction of EMT in tumor metastasis. Cancer cell recruit macrophage (undergo +ve feedback loop) and other stromal cells secreting CSF. Macrophage also releases EGF which induce the releasing of more CSF and Cathepsin B to cleave E-Cadherin attachments to the epithelium. More EGF along with TNF-α is produced to stop E-Cadherin expression and to increase N-Cadherin transcription. Stromal cells release MMPs (Matrix Metallo Proteases) to degrade the basement membrane, chemoattractant factors are released such as HGF (Hepatocyte Growth Factor). HGF (inducing mitogenic activity) attracts the tumor cells along chemical gradient causing actin rearrangement via Ras-like GTPases. Cancer cell produces broad flat lamellipodia in the direction of the HGF (forming adhesion at the front and loosing old ones). N-Cadherin allows tumor cells to interact with other stromal cells. ECM degradation helps in finding these cells their way to blood vessels for intravasation. When a cancer cell adheres to endothelial cells of a blood vessel, it up-regulates src kinase pathway which phosphorylates β-catenin causing intracellular connection between two endothelial cells to fail and allow the cancer cell to slip through. This increases tumor cell permeability and allowing the cancer cell to enter blood vessels, the process known as intravasation.  Attachment of platelets to migratory cell and trapping them in microvessels aid circulating tumor cells (CTCs) to avoid immune recognition, this aids extravasation and increase survival rate in smaller vessels.

Molecular Requirements of the EMT pathway –

Genetic and epigenetic alterations result in cancer cell responsive to EMT, also inducing heterotypic signaling. It may also play a role in preventing senescence induced by oncogenes signaling cascade. Transcription factors such as Snail, Slug, ZEB1&2, Twist, Goosecoid, FOXC2, and HIF-1 are the regulators of EMT pathway. Intracellular signaling pathways regulating EMT pathway are ERK, MAPK, PI3K (3 -Phosphatidyl Inositol Kinase), Akt, Smad, Rho A, β-Catenin, LEF (Lymphocyte Enhancing Factor), Ras, c-Fos. Surface proteins such as β4 integrin, α5β1 integrin, α5β6 integrin help in inducing EMT pathway in tumorigenic cells. The regulation mechanism of these pathways is detailed as below.

Polarity Proteins –

PAR3, PAR6, atypical PKC (Protein Kinase C) complex is a major cell polarity regulator engine in all eukaryotes. The par complex function as a hub interacting with many other cell polarity factors. These proteins are conserved between Drosophila and mammals usually located at the basolateral junction and tight junction e.g.,. Lgl (Lethal giant larvae), Dlg (Discs large), Scribble (Scrib). Basal membrane is digested after losing epithelial polarity. On transferring imaginal disc cell (lacking these genes) in the abdomen, caused metastasizing tumors but failed to form in eye disc as it requires Ras cooperation. Lgl forms polarity complex with Par3, Par6, and PKC and mediates polarity in podocytes. Par6 is phosphorylated by TGFβ receptor type 2, its phosphorylation recruits E3 ligase Smurf to TGF complex after that Smurf degrades RhoA. 

PI3K/AKT Pathway

PI3K pathway is hyperactivated to protect the cell from cell cycle arrest and apoptosis mediated by canonical TGFβ signaling. PI3K also repress GSK-3. PI3K also interferes with TGFβ induced tumor suppression effect (interfering with FOX03a function).

TGFβ Signalling

TGFβ signaling represses Id protein and activates Snail family member, so inducing EMT. Phosphorylation of heterogeneous nuclear ribonucleoprotein 1 by active Akt. mTOR signaling helps in increasing translation and cell size. Par6 phosphorylation promotes Smurf induce proteasome degradation of RhoA whereas direct induction of RhoA promotes actin rearrangement (actin diaphanous). Rho-associated kinase (ROCK) phosphorylate myosin light chain (MLC) to activate LIM Kinase and thus inhibit cofilin. 

Autocrine factors

EGF, IGF, FGF contribute to EMT via autocrine production. PDGF signaling pathway (with STAT1, STAT3b) found to be upregulated during TGFβ mediated EMT. SHIP1, Crk, Fas cooperate with TGF-β signaling to cause EMT during metastasis. The β-catenin is upregulated after E-Cadherin repression (contributed by TCF/LEF and TGFβ). Dysregulating E-Cadherin expression could activate Wnt/β-catenin signaling.

Wnt/beta-catenin signaling

Downstream effectors of Ras and TGFβ to promote EMT. Cytoplasmic β-Catenin on disruption from complex enhances transcription of TCF/LEF and Smad target genes. Activated TFs such as Snail upregulate Akt and Bcl-xl which inhibit induced apoptosis in a cancer cell by downregulating cyclin D2.

Hedgehog Signaling –

These proteins are also associated with stem maintenance besides regulating EMT pathway. Higher mutated expression leads to metastatic ability in prostrate carcinoma cell lines. Exogenous expression of downstream transcription factors such as Gli-1 in non-metastatic cells caused enhanced migration in in-vitro and in-vivo and exhibiting metastatic features such as Snail expression and E-Cadherin repression. Cyclopamine drug, an inhibitor of this pathway completely remove migratory effects in the cells, thus showing the contribution of Hedgehog signaling in the EMT induction. This pathway also induces autocrine PDGF-R signalling in skin tumors and caused upregulation of Wnt pathway in both skin cancer and development.

STAT3and LIV-1

Cytokine induces the JAK/STAT pathway as it binds to the receptor. JAK adds phosphate group on the receptor. This attracts the STAT (Signal Transducers and Activator of Transcription) which is also phosphorylated and form dimer. Dimer moves into the nucleus where it binds to the DNA as co-activator and induces the transcription of EMT-inducing genes. Src activation directly activates STAT3 during EMT, tumor progression and metastasis. LIV-1 (target gene of STAT3) responsible for nuclear localization of Snail, Downregulation of E-Cadherin (during gastrulation). LIV-1 responsible for estrogen regulation showed enhanced expression in ER+ve breast cancer.

 MTA3 Repressor

Mta3 repressor a part of Mi-2/NuRD transcription corepressor complex, shown to repress Snail expression in adult mammary epithelium through HDAC. Also induced by estrogen signalling.  Its loss in low ER expression causes high expression of Snail. While on re-expression of MTA3, Snail is again repressed. RNAi knockdown of MTA3 in ER+ve cell showed enhanced expression of Snail and E-Cadherin repression.

     

Transcription factors driving EMT –

Snail, Slug, Twist, ZEB1&2, bHLH (basic helix loop helix) expression also regulate the fate of EMT pathway. Their expression leads to the repression of E-Cadherin, acquisition of mesenchymal properties. SIP-1 binds with Snail to E-Cadherin promoter. MMP-3 facilitate the EMT by inducing genomic instability via Rac1 and ROS. Micro RNA such as miR200 and miR205 inhibit repression of E-Cadherin and ZEB1, ZEB2. Loss of miR200 correlated with vimentin expression and decreased levels of E-Cadherin. Also, the miR21 helps in inducing TGFβ mediated EMT and it is upregulated in many cancers.

E-Cadherin repression and its regulation –

Histone deacetylation (HDAC1 and HDAC2)
Somatic mutation (such as in lobular breast carcinoma)     
Downregulation of gene expression (hyper promoter methylation) 

      Transcription repression is regulated by    

1. Snail, Slug (Zing finger TFs)  
2. SIP1/ZEB2  
3. bHLH such as E12/E47 (E2A gene product) and interact with 'Id' (antagonist effect of E2A).

Conclusion –

EMT pathway is necessary for malignancy in tumor progression as well as in embryonic development wound healing and inflammatory response. We came to know about the EMT pathway which is not a single step process but rather regulated by a combination of the various signalling pathway. These pathways have a different role either positive or negative (or both). Repression of E-cadherin is a major hallmark for EMT pathway, and then with the cell-cell junction dissolution, the mesenchymal expression is activated. The E-cadherin repression is regulated by various pathway either directly or indirectly. TGF-beta induces the EMT pathway by activating both Smad and non-canonical signalling, thus regulating E-Cadherin repression. By Smad regulation, activated Smad6/7 work as an inhibitor of SMAD2/3 induced apoptotic pathway and through non-canonical pathways i.e. along with different factors induce EMT pathway. PI3K/AKT pathway also induces the EMT pathway either through mTOR pathway or by inhibiting TGF-beta mediated apoptotic pathway. Hedgehog pathway is also responsible for the EMT induction through Gli1 activity. Wnt/β-catenin signaling with the help of TGFβ activation induce the EMT. Whereas MTA3 repressor downregulates EMT process by repressing Snail expression. GSK-3β also downregulate the EMT induction by downregulating Snail expression and blocking β-Catenin activity by its phosphorylation or transcriptional repression. Transcription factors such as Snail, Twist, Slug, Goosecoid also induce EMT during tumor progression.

From the clinical perspective of tumor malignancy or for the cancer treatment purposes, these pathways can be targeted, and their niche in the body could interfere. Targeting the pathways which induces the EMT pathway and over activation of pathways which are downregulating EMT pathway may elicit proper treatment procedures for fighting against cancer. Using inhibitory drugs, induced miRNA expression, ligand/trap method against EMT-inducing pathway could be the novel approach for fighting against cancer and its progression.  

Reference

Angadi PV, Kale AD. Epithelial-mesenchymal transition - A fundamental mechanism in cancer progression: Indian J Health Sci Biomed Res 2015; 8:77-84

Margit A Huber, Norbert Kraut, Hartmut Beug. Molecular requirements for epithelial–mesenchymal transition during tumor progression: Current Opinion in Cell Biology Volume 17, Issue 5, October 2005, Pages 548–558

Samy Lamouille, Jian Xu, and Rik Derynck. Molecular mechanisms of epithelial–mesenchymal transition: Nat Rev Mol Cell Biol. 2014 Mar; 15(3): 178–196.

Sei Kuriyama, Roberto Mayor. Molecular analysis of neural crest migration: Philosophical Transaction of the Royal Society (Biological Sciences).

Silvia Juliana Serrano - Gomez, Mazvita Maziveyi and Suresh K. Alahari. Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications: Biomed Central (Molecular Cancer)