EMT + metastasis Flashcards
Define metastasis
spread of malignant cells from the primary tumour to other, independent sites within the body
Explain how metastasis is a multistep process
Carcinogenesis -> angiogenesis -> Detachment/invasion -> Intravasation -> migration -> extravasation -> micrometastasis -> macrometastasis
Features of angiogenesis:
Formation of blood supply (vascularisation) of tumour
occurs following transformation and initial growth of cells
At tumour size >1 mm, diffusion of nutrients and waste products become rate limiting for continued growth of tumour
-Angiogenesis must occur to provide support for growth of the tumour mass
Synthesis and secretion of pro-angiogenic factors (fibroblast growth factor, FGF; vascular endothelial growth factor, VEGF) by tumour cells and other non-cancerous cell types around cancer
How do cells become metastatic?
Initial growth of cancerous cells and formation of a tumour, developing a vascular supply
factors produced by cancerous cells and surrounding non-cancerous cell types stimulate morphological changes in cells
Phenotypic conversion and dedifferentiation of epithelial cells – indicative of carcinoma (epithelial derived cancer)
Characterised by epithelial mesenchymal transition (EMT) enabling cells to migrate and invade the surrounding tissue; invasion of the vascular and lymphatic system → resulting in metastasis
Who proposed the ‘seed and soil’ hypothesis and what is it?
Proposed by Stephen Paget (1889) – distribution of metastases is not by chance, instead metastases develop only when ‘seed’ and ‘soil’ are compatible
‘seeds’ - cancer cells with metastatic ability
‘soil’ - microenvironment
3 principle factors
Tumours are heterogeneous made up of cancer cells with subpopulations of host cells (e.g. epithelial, fibroblast, endothelial, leukocytes) exhibiting different properties (angiogenic, invasive, metastatic, growth rate)
Metastasis is selective for cancer cells which demonstrate a combination of these particular properties
Success of the resulting metastasis at the secondary site depends on its ability to interact with and utilise the ‘soil’, comprising multiple factors within the microenvironment
Does bi-directional movement between primary and distant tumour sites exist?
Yes
metastases have the ability to re-seed the primary tumour site
suggests that local environments within each site are similar and conducive to tumour growth
This may occur during relapse of disease, following initial treatment phase
Larger the arrow, more common spread to this place happens
Epithelial cells location/function
Epithelium sits on top of the connective tissue layer (stroma) – basal lamina (basement membrane – rich in ECM) separates the 2 compartments
Have a barrier function to protect the underlying tissue and also act to selectively sort molecules (by secretion, absorption) between the lumen and the underlying tissue
what is epithelia derived cancer?
Development of cancerous growth within the epithelium, leads to disruption of tissue organisation and eventual invasion into the connective tissue layer
colon, breast, ovary, lung, prostate, pancreas – common sites of epithelial-derived cancers
Epithelial cell structure
Polarised cells – has apical and basal domain, and differentiated
Explain the epithelial junctional complexes
Electron microscopy shows 3 sections (Fawcett 1966)
Tight junctions – zipper-like, restricts flow of molecules e.g. ZO-1 and water within the intercellular space, maintains impermeable epithelial barrier
Adherens junctions – (E-cadherin) – provides lateral adhesion between neighbouring epithelial cells – maintains actin contractile ring and epithelial polarity
Desmosomes – linked to intermediate filaments (e.g. cytokeratin), functions to maintain adhesion and tissue integrity
Describe E-cahedrin cell-cell junctions
Ca2+ dependent homodimerization of E-cadherin
linked to actin cytoskeleton
Provides structural support for tissue organisation
supports apical/basal polarity of individual epithelial cells by maintaining actin contractile ring - adhesion belt
Describe cell-cell adherens junctions
Actin contractile ring linked to cell-cell junctions maintains the adhesion belt between cells
Provides structural support to tissue and maintains columnar epithelial phenotype
What is EMTand ref first discovered
Epithelial-mesenchymal transition
First described by the lab of Elizabeth Hay, 1982 – occurs during development of embryo
Reversible phenotypic conversion of polarised (differentiated) epithelial cells to unpolarised mesenchymal cells
If you were to look under a microscope at cancer cells undergoing EMT, how would you distinguish between epithelial cells and mesenchymal cells?
Epithelial:
E-cahedrin and b-catenin and cell-cell junctions
Mesenchymal:
Loss of E-cahedrin
B-catenin is cytoplasmic
How does the TME influence EMT?
Tumour microenvironment provides factors and different cell types that promote cancer cell dedifferentiation and metastasis
What factors promote metasiasis and EMT
TNFa and IL-1b : pro-inflammatory cytokines that promote remodelling, EMT, invasivness
Secretion of growth factors and ECM - promote cell proliferation, EMT, cell migration/invasion
Features of Transforming growth factor beta1 (TGFβ1)
Secreted growth factor, usually has a tumour supressor function
Some oncogenes e.g. Myc, when mutated, allows cells to bypass the checkpoint control of TGFb1
This causes accumulation of TGFb1 and causes it to have a tumour promoting function, promotes EMT invasion/motiliy and inirectly effects angiogenesis and immunosupression
Haynes 2011
learn diagram
Differences in actin between epithelial and mesenchymal
Epithelial:
Cortical actin ring
M:
Actin stress fibres
(can see this in the microscope when you add TGFb1 to E cells - see these fibres - Margaret 2008)
Explain what latent TGFb1 is
TGFβ1 kept in latent (inactive) form in complex with latent associated peptide (LAP) derived from N-terminal region of TGFβ1 precursor
active/mature TGFβ1 derived from C-terminus, activated by factors - MMPs, ROS, acidic pH, ECM
Heterodimerisation of type I and II receptors, leads to a kinase cascade
Recruitment of R-SMAD (Smad2/3) and phosphorylation
Translocation into nucleus and modulation of gene expression
Factors of epithelial cells
E-cadherin
cytokeratin
ZO-1
Desmoplakin
Laminin
Low cell motility, cell-cell adhesion
Factors of mesenchymal cells
N-cadherin
Vimentin (intermediate filament)
fibronectin
α5β1 integrin receptor
Twist
Slug
Snail
Alpha-smooth muscle actin
High cell motility, cell-ECM adhesion, ECM production and deposition
How is EMT regulated?
EMT is a result of transcriptional reprogramming:
Wnt
Transforming growth factor β1 (TGFβ1)
Notch
Epidermal Growth Factor (EGF)
Hepatocyte Growth Factor (HGF)
Tumour necrosis factor α (TNFα)
cause changes in TFs - ZEB1/2, Snail, Twist
leads to either EMT (high vimentin, low E-cad) or MET (Low vimentin, high E-cad)
Role of ZEB
ZEB1 – activates DNA repair pathways, promotes cell survival → in an EMT-independent and dependent manner
p53 inhibits ZEB1 expression via microRNA 200 – upon p53 deletion, ZEB1 becomes active and is able to induce EMT; p53 represses Snail expression
Regulation on EMT by miRNAs
microRNA 200 negatively regulates expression of ZEB1, thus inhibiting EMT
Post-translational regulation of EMT
Snail can be phosphorylated at 1st site by GSK3β which leads to its translocation out of the nucleus (e.g. in absence of Wnt signalling)
In the cytoplasm, Snail subsequently gets phosphorylated by GSK3β at 2nd site, leading to its ubiquitination and targeting to the proteasome for degradation
Alternatively, wild-type p53 induces the mdm2-dependent ubiquitination and degradation of Snail
Downregulation of Snail leads to an inhibition of cell migration and invasion
Whats a key feature of promoting mesenchymal cell migration?
Remodelling of the actin cytoskeleton:
Functions of the actin cytoskeleton
driving membrane protrusion
Cell shape changes
Maintaining cell-ECM linkages
Cell contraction
When the TGFb1 receptor gets activated, apart from production of EMT regulator genes, what else happens?
Actiation causes activation of RhoGTPases, which causes the disassembly of adherin junctions and remodelling of the actin cytoskeleton
briefly describe the Rho GTPase cycle:
Rho with bound GDP = inactive
GEF changes GDP for GTP, Rho with bound GTP is actuve and activates effectors and downstream pathways
GAP switches GTP for GDP (making it inactuve)
GDI (guanine nucleotide dissociation inhibatir) keeps Rho+GDP bound by binding itself to it (reversable)
Give examples of constituativly active GTPases and how expression causes changes in the actin cytoskeleton
All seen microscopically (Hall 1998)
RhoA activation: Actin stress fibres and focal adhesion
Rac1 activation: Lamellipodia formation - > actin rich membrane ruffles/small focal adhesion contacts
Cdc42 activation: Filopodia formation -> finger-like actin protrusions
Rho family effectors and function:
Rho family members - bound GTP to be active
Effector proteins interact with GTP-bound Rho protein
Kinases and actin binding proteins modulate actin
e.g.s of Rho family:
Rho - contractile phenotyping
Rac - Actin polymerisation, actin branching
Cdc42 - Actin polymerisation, filopodia
Features of focal adhesions
multiprotein complex at the plasma membrane interface – this interaction with extracellular matrix (ECM) mediated by integrin receptors on cell surface
actin-binding proteins [α-actinin, vinculin, myosin], signalling proteins [p130Cas, Src, focal adhesion kinase (FAK)], structural proteins [paxillin, talin], integrin receptor
provides tensile strength, cell shape, and facilitates membrane protrusion and cell migration – promotes cancer metastasis
Integrin receptor family:
Heterodimeric receptors comprising an alpha chain and beta chain – link internal actin cytoskeleton to ECM (except α6β4)
Different combinations interact with specific extracellular matrix proteins → provide links from inside to outside of cell
(combination of alha and beta components provides specificity
Explain focal adhesion signalling
Diamerised integrins, interacting extracellularly with the ECM, have attached intracellularly a series of molecules:
vinculin and talin – actin binding proteins
paxillin – adaptor protein
FAK - structural support and signalling platform
Src – tyrosine kinase, signals to Ras (MAPK cascade) and causes Rho GTPase activation
This leads to actin cytoskeleton remodelling and focal adhesion formation