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
What faciliates cell movement?
Focal adhesions and actin cytoskeleton
Steps to cell movement:
- extension - actin polymerisation and branching facilitates this
- Adhesion - focal adhesion formation
- Translocation - retraction facilitatied by increased tension/contraction
- De-adhesion - focal adhesions break down
Describe the leading edge of a migrating cancer cell
Actin-rich membrane ruffles
lamellipodium
How does the actin cytoskeleton work?
A linear actin filament (F-actin) is made up of many individual globular actin (G-actin) monomers
Each molecule of actin is bound to either ATP or ADP
ATP-G-actin is added to the growing (plus) end of the filament and ADP-G-actin is disassembled from the retreating (minus) end of the actin filament
Explain actin cytoskeleton organisation at the leading edge of a migrating cell
Lamellipodium further bacj, Filopodium right at the edge
Lamellipodium are branched actin, Filopodium are parallel actin, both still have the plus and minus end
Explain actin cytoskeleton regulators and cell migration
At the uropod (cell end where migration has come from) - RhoA activation -> ROCK -> pMLC -> increased myosin contractility (actin here is rich in myosin-II -> cross-links actin and contributes to contractility)
At the leading end of the cell:
Rac1 -> WAVE
Cdc42 -> WASP
both lead to Arp2/3 activation that promotes branched actin (therefore lamellipodium formation)
Cdc42 also leads to mDia (Formin) activation, which is an actin nucleator that promotes actin polymerisation (Filopodia formation)
How can collective cell migration be identified
Intact cell-cell junctions
Types of cell migration
Collective migration: cell-cell and cell ECM interactions (with some leading cells to cause movment)
Mesenchymal cell migration: utelises interactions with the ECM to migrate
Amoeboid migration: doesn’t depend on ECM, finds path though chemoattractants
Scaffold cell-dependant migration (cell-cell dependant interactions)
difference in breast epithelial cells compared to mesenchymal
E - form islands, group together
M - Dont group as much, dont form islands
Mesenchymal vs. collective cell migration
Mesenchymal & collective – both path-generating
Collective – retains cadherin cell-cell junctions, while only exhibiting focalised cell-matrix adhesions and ECM degradation in leader cells
RW
Table of amoeboid (collective) vs mesencymal
M Vs. A
Migration stratagy: Path generating Path finding
Mechanisms for. Proteolytic ECM. Mprphalogical
overcomig tissue barriers: Degradation. Adaptation
Composition of
cell-ECM interactions: Focalised Diffuse(integrin-R)
integrin-Rs clustered non-clustered
MMP features
Zn2+ dependent proteases secreted by cells into extracellular space
secreted as an inactive pro-form and following activation in the extracellular space they cleave extracellular matrix proteins and/or activate latent TGFβ1
Regulate migration, invasion, proliferation, differentiation, angiogenesis
The action of MT1-MMP in cancer cells
MT1-MMP directly degrades ECM (e.g. basal lamina), allowing cells to invade into underlying connective tissue layer
MT1-MMP can activate MMP-2 within connective tissue layer, thus further facilitating cancer cells ability to migrate and invade surrounding tissue
MT1-MMP (membrane bound) – transmembrane metalloprotease → directly remodels extracellular matrix (fibronectin, collagen, laminin, vitronectin)
Whats a natural inhibator MMP
TIMP-2
In terms of MMPs, what is indirect remodelling through?
activation of MMP2
Steps of remodelling and MMP-2 activation
- MT1-MMP monomer expression on the cell surface
- MT1-MMP dimerization through Hpx and TM domain
- Formation of activation complex of dimer MT1-MMP, TIMP-2 and proMMP-2
- Propeptide cleavage by MT1-MMP free from TIMP-2
- MMP-2 activation by free MT1-MMP subunit by cleavage followed by release of active MMP-2
What does TIMP-2 stand for?
Tissue inhibitor of metalloproteinase 2
What do levels of TIMP-2 regulate
MMP activation
Sites of ECM degradation - invadopodia formation
Cell protrusions of the plasma membrane – filopodia-like structures - actin rich
secrete MMPs and degrade extracellular matrix
Mechanisms of metastasis
Initiation - by Src kinase
Assembly - by proteins attainh to actin
Maturation - microtubules connect to the actin
RW to check this is correct
Define the TME
defined as all the non-transformed elements residing within or in the vicinity of the tumour:
Cancer cells
Immune cells
Cancer-associated fibroblasts
Vasculature
Extracellular matrix
Normal stroma features
cell types and ECM that support the function of any particular organ
fibroblasts, adipocytes, macrophages, pericytes
provide growth factors, cytokines, and extracellular matrix components
Not cancerous themselves, but support tumour growth, influence therapeutic intervention, modulate gene expression
Features of tumour stroma
normal stroma - essential for maintenance of epithelial tissue and regulates tissue homeostasis
communication between stroma and epithelial cells by direct cell-cell contact or secreted factors
during cancer → stroma becomes ‘reactive’ or ‘activated’ - like a wound response to danger signal
consists of the non-malignant cells of the tumour and extracellular matrix
may act as a physical barrier preventing spread of tumour or therapeutic intervention
Or may facilitate metastasis by providing growth factors, secreting ECM, or degrading ECM
Factors in the tumour microenvironment influencing cell motility/metastasis
ROS - caused by hypoxia, CAMS and CAFs
RNS - caused by CAMs
Endothelial precurosir cells
ECM stifness
Acidity (lowering of pH)
Define ECM
molecules secreted by cells that provide structural support and biochemical interactions
composed of proteins, glycoproteins, proteoglycans, and polysaccharides – give structura; support ans organisation
Some proteins interact with cell surface receptors
Types of ECM components:
molecules secreted by cells that provide structural support and biochemical interactions
composed of proteins, glycoproteins, proteoglycans, and polysaccharides – give structura; support and organisation
Some proteins interact with cell surface receptors
What happens to the ECM in cancer
Upregulation of various collagen types, fibroectin, proteoglycans
Types of ECM rich environments within tissues
Basement membrane (basal lamina) - provides separation between different tissues:
- more compact and less porous
- underlying epithelial & endothelial cells, acts as barrier
- type IV collagen, laminin, fibronectin
Interstitial matrix (between cells):
- highly negatively charged, hydrated, and provides tensile strength to tissues (able to handle large amounts of stress before it breaks)
- fibrillar collagen (type I), proteoglycans, fibronectin, tenascin-C
Explain desmoplasia
also called desmoplastic response:
Secondary to the formation of the cancer
Forms around tumour and consists of cancer-associated fibroblasts (myofibroblasts – have muscle cell characteristics, e.g. alpha-smooth muscle actin), they can remodel the ECM
Usually associated with malignant tumours (poor prognosis)
Growth of hard, fibrous tissue - rich in collagen and other types of extracellular matrix as well as fibroblast cell types
Name of the stain used to show increased collegen in desmoplasia
Masson’s trichome stain (blue for collagen)
Mechanisms of ECM function in cancer
Barrier to therapy
Signallig to cancer cells
Biomechanical force
ECM fragments can also signal, and act as a co-receptor activating signal
Act as cell migration tracks for cancer cells to metastisize
Mammary gland transformation and influence of ECM stiffening:
Epithelial cells become disrupted and apical basal polarity is lost, normal gland morphology is lost and ECM is remodelled as tumour progression continued, creates lined fibrils
Influence of ECM Rigidity (tension)
Increased ECM deposited = increased ECM tension/regidity - this negatively correlates with patients survival
Stromal cell remodelling of the ECM:
TACS= tumour associated collegen signatures
Normal ECM (TACS-1):
Basement membrane, normal fibroblasts, epithelial cells
Predisposed (TACS-2):
Pre-alligned collagen, Protease and other factors, cancer cells, CAFs
Desmoplasmic ECM (TACS-3):
stiffness from collagen, reduced elasticity, intravasation of cancer cells
What does TACS stand for?
Tumour-associated collagen signature
Hypoxia and tumour metastasis
Hypoxia Inducible Factor 1α (Hif1α) transcription factor – regulated by intracellular oxygen levels → as levels of oxygen decrease, Hif1α protein is not degraded (due to not being hydroxylated) by proteasome and accumulates in the cell
This leads to overexpression of plasma membrane receptors (e.g. Met receptor) or angiogenic factors (VEGF), also leads to increased sensitivity to growth factors such as HGF (ligand for MetR)
leads to:
ECM degradation, EMT, chemotaxis - seek O2 rich regions, angiogenesis
How much stiffer is breast cancer tissue than normal tissue
10x
Features of LOX
copper dependent amine-oxidase
upregulated in a variety of tumours in response to hypoxia (regulated by Hif1α)
Oxidizes peptidyl-lysine residues, resulting in reactive aldehydes, leading to inter and intra molecular covalent crosslinks
cross-links collagen fibers and other ECM molecules (elastin)
promotes ECM stiffening and tumour invasion
What leads to ECM stiffening
increased integrin signalling and focal adhesion formation, enhanced PI3 kinase activity, increased cell invasion
The Warburg effect
First described by Otto Heinrich Warburg in 1924
It was observed that cancer cells produce energy by a high rate of glycolysis (they rely on higher levels of glycolysis)
Production of lactic acid by tumour cells due to anaerobic glycolysis rather than oxidative phosphorylation for energy production – cancer cells rely on glycolysis even if oxygen is available
High lactate leads to a high proton concentration → therefore an acidic environment
Cancer Imaging – based on cancer cell metabolic activity:
Positron emission tomography (PET) with 2-deoxy-2-[fluorine-18]fluoro-D-glucose (18F-FDG) (a glucose analogue that doesn’t get broken down) – measures gamma radiation from radioactive isotope
Exploits cancer cells increased glucose uptake and glycolytic activity
observes metabolic abnormalities before phenotypic changes
Sensitive technique
Can be used to monitor patient’s response to chemotherapy based on metabolic activity (responders and non-responders) - before any reduction in tumour size occurs
Example of PET scan after cancer treatment
Patient with Hodgkin’s lymphoma - treated with Sirolimus (mTOR inhibator) and vorinostat (Histone deacetylase inhibator) for three cycles, PET scans taken befor and after
showed large decrease in glycolytic activity indivcationg cancer cell decrease
Subbiah et al 2014
Hypoxia and an acidic microenvironment
External pH of solid tumours is acidic due to increased metabolism of glucose as well as poor fluid and gas exchange (perfusion)
Activates stress pathways (Reactive oxygen species), inflammatory response – inducing chromosomal instability
stimulates the secretion of lysosomal proteases (cysteine cathepsins) and active collagenases (matrix metalloproteinases)
Fibrobalsts in cancer
Mesenchymal stromal cells, exhibit cell plasticity – different phenotypes depending on tissue of origin/function
Normal fibroblasts – reside within connective tissue (interstitial matrix) and synthesise and remodel the extracellular matrix; quiescent (non-dividing) under normal circumstances, just provide a supporting role
Cancer associated fibroblasts
Originate from activation of normal fibroblasts by tumour derived growth factors or may result from endothelial to mesenchymal transition (EndMT)
TGFβ1, PDGF, bFGF
Triggers conversion of fibroblasts, immune cells, pericytes, smooth muscle cells, adipocytes (many normal cell types can turn into CAFs)
Increased proliferation rate, enhanced ECM production and ECM remodeling – enhance tumourigenesis
CAF features
CAFs facilitate transformation – may infiltrate the tumour or reside at the tumour margins
CAFs:
Express high levels of alpha-smooth muscle actin, fibroblast specific protein 1, extracellular matrix (e.g. periostin, tenascin-C), fibroblast activation protein (FAP)
FAP – type II integral membrane serine protease; restricted to reactive fibroblasts; detected on the surface of fibroblasts in stroma surrounding >90% of epithelial cancers; may have role in matrix digestion, immunosuppression e.g. of NK cells
secrete growth factors – promote paracrine signaling
Example: Stroma derived factor 1 (SDF-1α)/CXCL12 interacts with CXCR4 (receptor) on tumour cells and promotes cell migration
SDF-1/CXCR4 signaling:
Fibroblasts react to local environment e.g. higher levels of TGFb1, causing change of a cell into a CAF, this then drives growth factors e.g. SDF-1, this causes autocrine signalling loop driving differentiation/activation of fibroblasts, and a paracrine signaling pathway (SDF-1 binds to CXCR4 receptor) causing increase cell proliferation and motility of cancer cell itself (by increasing signaling pathways e.g. ERK1/2, PI3K/Akt) SEE DIAGRAM
Other functions of CAFs
Tumour angiogenesis:
releasing growth factors (VEGF, FGF)
recruiting endothelial precursor cells
Extracellular Matrix (ECM):
-Remodeling ECM architecture, exposing cryptic binding sites (allowing for alternative interactions to occur)
–promotes cancer cell migration
-ECM stiffening
–modulate intercellular adhesion and cell contractility – disrupts tissue organisation, promotes cell migration
Tumour-related inflammation:
-Recruiting immune cells (monocytes, macrophages)
-Modulate function of immune cells
–Pro-inflammatory signalling
–Inhibiting Natural Killer cell and CD8+ cytotoxic T-lymphocyte function
Tumour stroma and its influence on cancer therapy
Ligands derived from activated fibroblasts may signal to receptor (e.g. receptor tyrosine kinases) on tumour cells (paracrine signalling) – leading to therapeutic resistance (e.g. pro-survival signalling)
Fibroblasts and extracellular matrix may provide a physical barrier or an avenue for metastasis
Impaired drug delivery –
- Density of the tumour stroma
- Lack of vasculature
- inefficient blood flow
Name an autofluorescent chemotherapeutic drug for treatment of pancreatic ductal carcinoma
Doxorubicin
RW
Immune system in the TME
Infiltration of immune cells, such as macrophages, contributes to cancer progression – promoting survival and immunosuppressive environment
Release of pro-inflammatory cytokines, such as TNFα, may also promote survival and resistance to chemotherapy
