EMT & Metastasis Flashcards
Define metastasis
The spread of malignant cells from the primary tumour to other, independent sites within the body.
What is angiogenesis?
The formation of a body supply (vascularisation) of a tumour, following the initial growth. Required for tumour growth to diffuse nutrients and waste in/out of the tumour.
Pro-angiogenic factors (FGF, VEGF) are secreted by the tumour microenvironment.
What is EMT?
- Epithelial to mesenchymal transition enables the cells to migrate and invade the surrounding tissue and results in metastasis.
- Dedifferentiation of epithelial cells and a loss of cell polarity.
- down regulation of epithelial genes (B-catenin, E-cad), subsequent loss of cell-cell junctions
- degradation of ECM so cells can move into the connective tissue
What are the 3 principle factors of the seed and soil hypothesis?
- Tumours are heterogeneous. A mix of many different cell types.
- Metastasis is selective for cancer cells that demonstrate a combination of particular properties (angiogenic, invasive, good growth rate).
- Success of metastasis at the secondary site depends on its ability to interact with and utilise the ‘soil’ (multiple factors within the microenvironment)
What are common secondary metastasis sites?
Brain, bone marrow, lungs and liver.
Bidirectionality = secondary sites can re-seed primary sites because the local environments are similar and conductive for tumour growth.
Leads to relapse.
What is the function of epithelial cells? What occurs in the development of cancers?
Act as a barrier to protect the connective tissue layer, separated by the basal lamina.
Can actively sort molecules between layers.
Normal epithelial cells are polarised and differentiated.
Cancer causes disruption of tissue organisation, invasion into connective tissue and loss of epithelial cell polarity.
How are epithelial cells attached and sealed?
- Tight junctions = restricts flow of molecules and water, maintain impermeable epithelial barrier.
- Adherens junctions = E-cadherin, lateral adhesion to neighbouring cells, maintains polarity. Contractile ring of actin for integrity, dimerisation of E-cadherin is Ca dependent.
- Desmosomes = linked to intermediate filaments to maintain structural integrity.
- Gap junctions, hemi-desomosomes (connect to ECM)
What are the factors that can promote EMT?
Provided by the tumour microenvironment.
- Pro-inflammatory cytokines (TNFa & IL1B) promote EMT and metastasis.
- Growth factors, fibronectin, laminin and hypoxia
- TGFb1
Function of TGFB1 in normal and cancer cells.
TGFB1 activation and cascade.
Usually a tumour suppressor, stopping proliferation.
Cancer signalling causes insensitivity to anti-growth signals (Myc that stops TGFB1 function)
TGFB1 accumulates and at high levels, becomes tumour promoting.
TGFB1 kept inactive by latent associated peptide (LAP) in the ECM.
Cleaved by (MMP, ROS, pH) in the ECM, so it can bind to receptors.
Receptors recruit R-SMAD, phosphorylated, binds SMAD-4, moves to nucleus and transcribes EMT regulators.
Structure of ZEB1/2 and how they cause EMT in cancers.
Zinc finger domain for transcription, binding to E-box sequences.
Repress epithelial markers (E-cad) and activate mesenchymal markers (vimentin, fibronectin).
ZEB1/2 is inhibited by p53 to stop tumorigenesis through miRNA200. When p53 is inactivated in cancers, ZEB1/2 is oevrexpressed and causes EMT.
Structure of SNAIL family of proteins and how they cause EMT in cancers.
SNAG domain that binds E-box sequences in DNA.
Cooperate with HDACs to suppress E-cad.
SNAIL phosphorylated by GSK3B to transport it out of the nucleus (1st) and then to degrade it by ubiquitination (2nd).
This can also be done by p53 (Mdm2 dependent).
When both are inactivated, SNAIL is over expressed = lack of inhibition of cell migration and activation of mesenchymal genes.
Structure of TWIST family of proteins.
Basic helix-loop-helix domains for DNA binding. Repress or activate target genes by binding and recruiting co-activators or co-repressors.
How is cell migration linked to actin cytoskeleton remodelling?
Driving protrusions Changing cell shape Maintaining cell-ECM linkages Cell contraction Integrins interact with components of the ECM to have downstream effects on actin.
List 3 members of the RhoGTPase family and how they are involved in migration / actin remodelling.
- RhoA - causes actin stress fibres and focal adhesion formation. mDIA (actin poly) and ROCK (contraction)
- Rac1 - forms lamellipodia, actin rich membrane ruffles and small focal adhesions. WAVE-ARP2/3 for polymerisation.
- Cdc42 - Forms filopodia (finger-like protrusions). WASP-ARP2/3 for polymerisation.
What are focal adhesions?
What are integrins?
Link ECM to actin cytoskeleton.
Actin binding proteins - vinculin, myosin
Signalling proteins - FAK, Src
Structural proteins - talin, paxillin
Integrin receptors mediate contacts for focal adhesions. Heterodimers of alpha and beta chains. Can be RGD, collagen or laminin receptors.
Steps in focal adhesion signalling.
Actin polymerisation extends the membrane, causes integrins on surface to bind to ECM proteins.
Focal adhesions are recruited to the contact site.
Stress fibres provide contractile force to detach FA at the rear of the cell for movement.
Structure of an actin filament.
F-actin filament contains G-actin monomers.
G-actin added to growing +end, disassemble from the -end.
Lamellipodia = branched actin
Filopodia = parallel fibres w/cross links
How does the ARP2/3 complex mediate actin branching and polymerisation?
Binds to F-actin and produces a new nucleation site for a branch point, always at 70 degree angle.
Creates fan-like protrusions.
Mediated by WASP/WAVE.
What is LIM kinase?
Inhibits Cofilin (an actin depolymerising agent) through phosphorylation and promotes assembly over disassembly.
What are the 4 types of cell migration?
- Collective – Keeps cell-cell adhesions and ECM contacts as cells move together.
- Mesechymal – Loss of cell-cell contacts and lots of ECM contacts, cells move individually.
- Amoeboid – Loss of cell-cell adhesion, but very little ECM contacts.
- Scaffold – Dependent on cell-cell interactions.
Mesenchymal VS Amoeboid cell migration
Mesechymal is path generating through proteolytic ECM degradation and uses clustered integrin receptors in focal adhesions to migrate.
Amoeboid is path finding through morphological adaptation to contrict through the ECM and diffuses using non-clustered integrin receptors.
What are MMPs, how do they work?
MT1-MMP/MMP2/TIMP-2 action mechanism.
Zinc dependent proteases secreted by cells in an inactive pro-peptide form.
When activated, they cleave ECM proteins and activate TGFB1.
Regulate migration, invasion and proliferation.
They are secreted by the leading cell edge.
MT1-MMP directly degrades the ECM and allows cells to invade the connective tissue layer. MT1-MMP can activate MMP-2 for cancer cell migration.
TIMP-2 is a natural inhibitor of MMP activity. MT1-MMP dimerises and then forms a complex with TIMP-2 and ProMMP-2.
Only 1 monomer of MT1-MMP is inhibited, so the other can cleave MMP2 and activate it. This can only be done at low levels of TIMP-2, as at high levels, both monomers of MT1 are inhibited.
Define the tumour microenvironment.
All the non-transformed elements residing within or in the vicinity of the tumour – immune cells, cancer associated fibroblasts, vasculature, extracellular matrix.
What is the stroma and what happens to it during cancer?
The cell types (fibroblasts, macrophages) and ECM that support normal organ function. They provide growth factors, cytokines and ECM components.
Not cancerous, but support cancer function.
Become reactive in cancer - may act as a physical barrier for cancer growth, but also therapeutic interventions.
May facilitate metastasis by providing or degrading ECM or providing growth factors.
What is the ECM comprised of?
Cells Proteins - collagen, elastin Glycoproteins - fibronectin, laminin Polysaccharides - GAGs, Proteoglycans - decorin, lumican
What is the desmoplatic response?
Secondary to cancer formation
Forms around the tumour and contains CAFs and lots of ECM.
Associated with poor prognosis and increased growth of hard, fibrous tissue.
Describe how cancer can disrupt mammary glands.
Mammary cells are polarised and surrounded by lots of collagen. They produce milk in the luminal area.
When mutated, the polarity is lost and the morphology of the whole gland is disrupted.
Tumour cells invade the luminal area and collagen remodelling helps formation of secondary tumours.
Remodelling of the ECM causes tension. What does this tension do?
Tension causes integrin clustering and increased downstream signalling and cancer progression.
EX - FAK –> Akt –> inhibits GSK3B –> No destruction complex, increase B-catenin –> Wnt signalling C-myc transcription –> inhibits PTEN so it can’t inhibit Akt.
Upregulation of cycle
What is TACS?
(Tumour associated collagen signature)
Associated with increased cancer progression when TACS-3 is aligned with bundled collagen fibres. Causes increased collagen stiffness and therefore cancer progression.
How does a hypoxic environment leads to increased cancer progression?
HIF1a is regulated by oxygen levels.
In oxygen, PHDs are activated which hydroxylate and target HIF1a for degradation by VHL ligases (proteasome).
PHDs are inactive when O2 is not present.
HIF1a accumulates and causes overexpression of receptors and angiogenic factors that enable EMT, ECM degradation and angiogenesis.
How is hypoxia related to extracellular matrix remodelling?
Tumour stroma is stiffer than normal stroma. Lysyl Oxidase (LOX) is a copper-dependent amine oxidase that promotes cross linking in collagen fibres and therefore ECM stiffness.
Regulated by HIF1a
Why do tumours have an acidic environment and how does it help them?
How can metabolic activity be used to locate tumours?
Cancer cells have a high rate of glycolysis.
Increased lactate production because they favour anaerobic respiration.
High lactate leads to acidic environment.
Causes poor fluid and gas exchange, more ROS production and secretion of MMPs.
18FDG (for PET) is glucose analogue taken up but not metabolised. Highlights reactive tumours based on metabolic activity.
What are fibroblasts?
How to CAFs arise?
Mesenchymal stromal cells
Remodel the ECM in the connective tissue layer as a normal function.
CAFs arise from activation of normal fibroblasts by tumour derived growth factors or EMT.
Increased proliferation and enhance tumorigenesis.
They stiffen the ECM, express FAP (recruits immune system for immunosuppression) and growth factors (angiogenesis).
Describe SDF-1/CXCR4 signalling.
SDF-1 ligand binds to CXCR4 receptor on CAFs to produce more ligand (autocrine signal)
Then bind to cancer cells (paracrine signal) to activate G-proteins which activate ERK1/2, Pi3K/Akt and PLCy for cell proliferation, survival and cell motility.
How do CAFs influence therapeutic intervention?
Tumour stroma produces pro-survival signals = resistance to therapy.
ECM = physical barrier
Doxorubicin (chemotherapy drug) - cannot penetrate the vasculature of the tumour due to insufficient blood flow and then density of the tumour stroma.
