CANCER; Lecture 7, 8 and 9 - Apoptosis, Oncogenes and Tumour Suppressors, Angiogenesis Flashcards
Why do we need programmed cell death?
Removes: Harmful cells (e.g. cells with viral infection, DNA damage) Developmentally defective cells (e.g. B lymphocytes expressing antibodies against self-antigens) Excess/unnecessary cells: Embryonic development e.g. brain to eliminate excess neurons; liver regeneration; sculpting of digits and organs Obsolete organs (e.g. mammary epithelium at the end of lactation) Exploitation - chemotherapeutic killing of cells
What is the difference between cell death -> necrosis vs apoptosis?
Necrosis - unregulated cell death associated with trauma, cellular disruption and an inflammatory response Apoptosis (programmed cell death) - regulated cell death; controlled disassembly of cellular contents without disruption; no inflammatory response
What occurs in necrosis?
Plasma membrane becomes permeable; cell swelling and rupture of cell membranes; release of proteases leading to autodigestion and dissolution of the cell; localised inflammation
What are the 2 phases of apoptosis?
Latent phase and execution phase
What occurs in the latent phase of apoptosis?
Death pathways activated but cells appear morphologically the same
What occurs in the execution phase of apoptosis?
Loss of microvilli and intercellular junctions; cell shrinkage; loss of plasma membrane asymmetry (phosphatidylserine lipid appears in outer leaflet); chromatin and nuclear condensation; DNA fragmentation; formation of membrane blebs; fragmentation into membrane-enclosed apoptotic bodies. NB: PLASMA MEMBRANE REMAINS INTACT = NO INFLAMMATION
What happens to the apoptotic bodies?
Taken up by macrophages
What does DNA modification in apoptosis lead to?
Fragmentation of DNA ladders and formation of more ends which are labelled by adding a fluorescently-tagged base in a TUNEL assay
What are the 4 types of cell death?
Necrosis, apoptosis, apoptosis-like PCD and necrosis-like PCD -> cells often die of something in between necrosis and apoptosis as it is a graded response
What is apoptosis-like PCD?
Some (not all) features of apoptosis -> display of phagocytotic recognition molecules before plasma membrane lysis
What is necrosis-like PCD?
Variable features of apoptosis before cell lysis (aborted apoptosis)
What are the mechanisms of apoptotic cell death?
Executioners (caspases); initiating death programme (death receptors and mitochondria); Bcl-2 family; stopping the death programme
What are caspases?
Cysteine-dependent aspartate-directed proteases -> cut proteins after aspartate residue, executioners of apoptosis, activated by proteolysis and take part in cascade of activation
What are the 2 types of caspases?
- Effector -> start as single chain polypeptide with 2 subunits, which are released by proteolytic cleavage during maturation.
- Initiator -> same 2 subunits in effectors with an extra targeting subunit (protein-protein interacting domain), targeting subunit directs them to a particular location -> CARD and DED are the targeting subunits
How does caspases maturation occur?
- Procaspases are single chain polypeptides which become activated by proteolytic cleavage to form large and small subunits (initiator also cleaved to release targeting subunit) ->
- carried out by caspases.
- After cleavage, folding of 2 large and 2 small chains to form active L2S2 heterotetramer
What are caspase cascades?
Once apoptosis is triggered, initiator caspases cleave and activate effector capsases
What is the role of effector caspases?
Execute apoptotic programme by 2 ways
What are the 2 mechanisms of caspase activation?
Death by design (receptor mediated pathways); death by default (mitochondrial death pathway)
What are the receptors that are activated via the death by design pathway?
- Death receptors ->
- extracellular cysteine-rich domain, single transcellular domain and cytoplasmic tail (with death domain) ->
- only activated when encounter secreted or transmembrane trimeric ligand (TNF-a, Fas) = death ligands
Which adapter proteins are present in the death by design pathway?
- FADD = positive regulator (required for the death pathway to become activated) which promotes cell death;
- FLIP = negative regulator (inhibits death pathway and allow regulation)
How does signalling occur through Death receptors using Fas/Fas-ligand?
- Fas = death receptor which is upregulated if apoptosis is required ->
- Fas ligand binds to Fas receptor on surface of cytotoxic T-cells, where FasR undergoes trimeristation, bringing the 3 cytoplasmic DD together ->
- recruit FADD by own DD, causing recruitment and oligomerisation of procaspase 8 through its DED to FADD’s DED ->
- Forms Death-inducing signalling comlex
What is oligomerisation?
Chemical process linking monomeric compounds to form dimers, trimers, tetramers or longer chain molecules
What does the DISC formation result in?
Cross-activation of procaspase 8, where they cleave each other within complex, so active caspase 8 is released, cleaving effector caspases to execute death programme
How does procaspase 8 undergo oligomerisation?
What inhibits death receptor activation of procaspase 8?
- FLIP -> evolutionary related to caspases but lost catalytic activity, so can compete with procaspase 8 to bind to DED of FADD ->
- for binding to receptor tails/FADD via DED ->
- incorporates into receptor-procaspase complexes and interferes with transcleavage
What is the function of caspase 8?
Activates downstream effector caspases which then go on to carry out apoptotic programme
What is the mitochondrial regulation of apoptosis?
- Intrinsic pathway where cell stresses cause a loss of mitochondrial membrane potential ->
- results in release of cytochrome C and other apoptosis-inducing factors ->
- stimulate formation of apoptosome complex
What is the apoptosome?
- Made of APAF-1, Cytochrome C, ATP and Procaspase 9;
- APAF-1 has number of repeats involved in protein-protein interactions, ATPase domain and other end has CARD (also found in other initiator caspases).
- Cytochrome C binds to WD-40 repeats on APAF-1, forming heptamer, and requires ATP.
- CARD at centre of apoptosome interacts with CARD on procaspase 9 (7 different ones), binding them which means they are close enough to cross-cleave and activate each other, forming 7 caspase 9 ->
- which is then released, triggering the caspase cascade, leading to apoptosis
What are the principal mechanisms of apoptosis?
- Bid links the receptor mediated and mitochondrial death pathways;
- caspase 8 from receptor-mediated cleaves Bid, enhancing release of mitochondrial proteins, engaging intrinsic pathway.
- Bid promotes release of cytochrome C from mitochondrion, triggering mitochondrial death pathway
Why is ATP important in cell death?
Levels of ATP decide if cell death is by apoptosis (more ATP) or by necrosis (less ATP)
What are the intrinsic modulators of apoptosis?
Bcl-2 family proteins -> 3 main groups, all which contain BH3 domains.
Some contain other domains inc. transmembrane domain.
What is BH3?
Dimerisation motif (for protein-protein interaction) that allows proteins in Bcl-2 family to associate and dimerise with each other
What are the 2 categories of the Bcl-2 family?
Anti-apoptotic proteins are localised to the mitochondrial membrane and inhibit apoptosis.
Pro-apoptotic proteins move between the cytosol and the mitochondrial membrane and they promote apoptosis
What is the PI3’-Kinase signalling pathway in cell cycle and apoptosis regulation?
Growth factors activate 2 GF pathways associated with anti-apoptotic effects; ligand binding causes goes into the MAPK/ERK pathway; another phosphorylation site on TKR triggers PI3-Kinase pathway, involved in cell survival with anti-apoptotic effects
How is a new blood vessel made?
- Vasculogenesis (bone marrow progenitor cell),
- angiogenesis (sprouting) and
- arteriognesis (collateral growth dependent on shear stress and external factors like macrophages)
What is angiogenesis?
Formation of new blood vessel from pre-existing blood vessels -> most common in wound healing/menstrual cycle
How is angiogenesis regulated?
Need to destabilise the blood vessel and restabilise it -> activators and inhibitors of angiogenesis balance and regulate angiogenesis
How does hypoxia trigger angiogenesis?
- Hypoxia-inducible TF (HIF) is important in regulation of genes involved in angiogenesis
- normoxia inhibits HIF by Von Hippel-Lindau, so angiogenesis genes aren’t expressed.
- In hypoxia, VHL doesn’t bind to HIF so HIF is mobilised and can translocate to nucleus and drive the expression of genes involved in angiogenesis ->
- VEGF being the main target
What is the VEGF family?
Best known pro-angiogenic GF; 5 members -> VEGF-A/B/C/D and PIGF
How are tip cells chosen for angiogenesis?
Special endothelial tip cells lead to outgrowth of blood-vessel sprouts towards gradient of VEGF, controlling behaviour of surrounding cells via cell-cell communication
How does VEGF take part in notch signalling?
- Activates endothelial cells in capillary and increase DLL4 expression, which drives notch signalling,
- inhibits VEGFR2 in adjacent cell, so cells on either side of the tip cell recognise their role as stalk cells and divide to push tip cell forward
How does the stalk elongate and guide the tip to where it needs to go? What is the involvement of macrophages?
- Cells interact with ECM, with guidance systems in place.
- Macrophages also have an important role in vessel anastamosis as they carve out tunnels in ECM, providing avenues for subsequent capillary infiltration;
- tissue-resident macrophages are associated with angiogenic tip cells during anastamosis, to help stabilise newly formed vessels
How is the newly formed vessel stabilised and how does it undergo quiescence?
- Tip cells fuse and stalk cells separate to form parent tube.
- Stabilisation involves reforming the endothelial monolayer barrier and recruiting the neural cells, switching off active angiogenesis in the process
Which tight and adherens junctions are involved in vessels?
- Endothelial cells have junctions to form cohesive monolayer;
- proteins on membranes of both cells involved binding in homophilic way;
- VE-Cadherin is essential for vessel stabilisation and quiescence.
- Homophilic interaction between cadherins on the endothelial cells mediates adhesion between endothelial cells (important in IC signalling).
- Important in contact inhibition and promote survival of endothelial cells.
- Pericytes recruited producing proteins involve in stabilising the the vessel
How do pericytes stabilise the vessels?
Mural cells (smooth muscle cells and pericytes) which are involved in formation of normal vasculature in response to VEGF.
Pericytes produce Angiopoietin 1 which is involved in Notch
What is the Angiopoietin-Tie 2 ligand-receptor system?
Summarise sprouting angiogenesis
Why do tumours need angiogenesis?
Angiogenic switch -> point where the tumour gets too large for diffusion to be sufficient so some cells in tumour send angiogenic signals
What are the characteristics of tumour blood vessels?
Not properly formed due to imbalance of signals, so haemorrhage is common
Which agents target the VEGF pathway?
What is the use of Avastin?
What are the side effects of Avastin?
What are the strategies for anti-angiogenic therapies?
- Helps normalise tumour blood vessels, but if very aggressive therapy, then can damage ability to deliver other drugs to the tumour ->
- AIM: normalise tumour blood vessels to reduce hypoxia and improve efficiency of drug delivery
What are the methods of unconventional resistance to VEGF blockade?
- Tumour adopts evasive strategy and adapts to bypass specific angiogenic blockade. Intrinsic/pre-exisiting difference ->
- tumour wasn’t sensitive to angiogenic blockade anyways
What is vasculogenic mimicry?
Describes plasticity of aggressive cancer cells forming de novo vascular networks and is associated with the malignant phenotype and poor clinical outcome
What is the future therapy for cancer?
What is PI3-K?
Lipid kinase involved in growth control and cell survival -> 3 subunits: Targeting, adapter and catalytic subunits
What does PI3-K do?
- Phosphorylates PIP2 to PIP3,
- which then is recognised by adapter subunit of PKB/Akt ->
- then recruited to cell membrane and is activated with anti-apoptotic effects ->
- phosphorylates and inactivates Bad (Bcl-2 family)
What is the function of PKB/Akt?
How is apoptosis regulated by Bcl-2 family proteins?
- Bax and Bak are held in inactive heterodimers (by BH3) to anti-apoptotic Bcl-2/xL proteins ->
- cell survival and proliferation are promoted. GF absent = PI3-K pathway inactive, so Bad isn’t phophorylated and is released from heterodimer,
- which then goes to mitochondrial membrane,
- where through BH3 domain displaces the pro-apoptotic Bcl-2 members,
- which form a pore in mitochondrial membrane, allowing escape of cytochrome C into cytosol, inducing apoptosis
What is PTEN and how does it regulate apoptosis?
Lipid phosphatase that counteracts PKB production, reducing regulation of cell survival and promoting apoptosis
What are the main characteristics of cancer cells?
What is the cell cycle and why are the checkpoints important?
How are oncogenes activated?
- Gene amplification can occur due to problems with a polymeraase protein, which will lead to overproduction of product.
- Chimeric genes -> genes formed from combination of >1 coding sequences to produce new genes; problem if one is promoter leading to upregulation of other gene portion (Burkitt’s lymphoma) -> can produce abnormal proteins (philadelphia chromosomes in CML)
What is the philadelphia chromosome?
Formed by translocation of chromosome segments from 9 and 22 -> ABL (from 9) and BCR (from 22) are translocated, forming BCR-ABL fusion genes, leading to cancer development
Which part of the signal transduction pathways are targeted as critical gene targets?
Can lead to downstream activation of signalling pathways such that they no longer respond to upstream stimuli
What is the activity of mutant Ras?
Mutant Ras fails to dephosphorylate GTP so Ras remains active, increasing cell proliferation via the MAPK/ERK pathway
What are some examples of oncogenes and what are their associated human cancers?
What is retinoblastoma and what is the cause of it?
What are the functional classes of tumour suppressor genes?
What are some examples of the tumour suppressor genes and human tumours?
What is p53?
- When bound to MDM2 it is inactive;
- important for regulation of p53 target genes and protein-protein interactons.
- Activated by cell stress.
- Mutants of p53 act in dominant manner and mutation of a single copy is sufficient to get dysregulation of activity.
- Phosphorylation (triggered by cell stress) of p53 destabilises it so that is isn’t degraded as quickly and can exert effects
What is the APC tumour suppressor gene and how does it work?
- APC is involved in cell adhesion and signalling and people with mutation develop benign adenomatous polyps in colon.
- APC participates in the WNT signalling pathway to alter transcription and growth, helping to control beta-catenin activity, preventing uncontrolled growth
Summarise the route to cancer
Summarise the development of colo-rectal cancer
- APC gene in normal epithelium is mutated and becomes inactive leading to hyperproliferation of epithelium.
- DNA hypomethylation combined with K-ras mutation makes polyps into adenomas.
- P53 mutation results in carcinoma, which can then metastasise
Describe the differences between oncogenes and tumour suppressor genes
