EMT + metastasis Flashcards

Question

Post-translational regulation of EMT

Answer 1

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

Answer 2

Remodelling of the actin cytoskeleton:

Answer 3

driving membrane protrusion Cell shape changes Maintaining cell-ECM linkages Cell contraction

Answer 4

Actiation causes activation of RhoGTPases, which causes the disassembly of adherin junctions and remodelling of the actin cytoskeleton

Answer 5

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)

Answer 6

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

Answer 7

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

Answer 8

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

Answer 9

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

Answer 10

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

Answer 11

Focal adhesions and actin cytoskeleton

Answer 12

1. extension - actin polymerisation and branching facilitates this 2. Adhesion - focal adhesion formation 3. Translocation - retraction facilitatied by increased tension/contraction 4. De-adhesion - focal adhesions break down

Answer 13

Actin-rich membrane ruffles lamellipodium

Answer 14

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

Answer 15

Lamellipodium further bacj, Filopodium right at the edge Lamellipodium are branched actin, Filopodium are parallel actin, both still have the plus and minus end

Answer 16

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)

Answer 17

Intact cell-cell junctions

Answer 18

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)

Answer 19

E - form islands, group together M - Dont group as much, dont form islands

Answer 20

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

Answer 21

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

Answer 22

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

Answer 23

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)

Answer 24

activation of MMP2

Answer 25

1. MT1-MMP monomer expression on the cell surface 2. MT1-MMP dimerization through Hpx and TM domain 3. Formation of activation complex of dimer MT1-MMP, TIMP-2 and proMMP-2 4. Propeptide cleavage by MT1-MMP free from TIMP-2 5. MMP-2 activation by free MT1-MMP subunit by cleavage followed by release of active MMP-2

Answer 26

Tissue inhibitor of metalloproteinase 2

Answer 27

MMP activation

Answer 28

Cell protrusions of the plasma membrane – filopodia-like structures - actin rich secrete MMPs and degrade extracellular matrix

Answer 29

Initiation - by Src kinase Assembly - by proteins attainh to actin Maturation - microtubules connect to the actin RW to check this is correct

Answer 30

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

Answer 31

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

Answer 32

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

Answer 33

ROS - caused by hypoxia, CAMS and CAFs RNS - caused by CAMs Endothelial precurosir cells ECM stifness Acidity (lowering of pH)

Answer 34

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

Answer 35

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

Answer 36

Upregulation of various collagen types, fibroectin, proteoglycans

Answer 37

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

Answer 38

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

Answer 39

Masson's trichome stain (blue for collagen)

Answer 40

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

Answer 41

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

Answer 42

Increased ECM deposited = increased ECM tension/regidity - this negatively correlates with patients survival

Answer 43

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

Answer 44

Tumour-associated collagen signature

Answer 45

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

Answer 46

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

Answer 47

increased integrin signalling and focal adhesion formation, enhanced PI3 kinase activity, increased cell invasion

Answer 48

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

Answer 49

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

Answer 50

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

Answer 51

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)

Answer 52

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

Answer 53

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

Answer 54

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

Answer 55

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

Answer 56

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

Answer 57

Doxorubicin RW

Answer 58

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

EMT + metastasis Flashcards

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