everything Flashcards
(17 cards)
1
Q
General iron metabolism
A
- Absorbed through epithelial lining cells of small intestine
- Ferritin binds two Fe 3+
- TFR1 trans located by endocytosis
- Acidic = fe released
- STEAP3 = reduction of iron or fe 2+
- DMT1 transports to Liable iron pool
- Used in mitochondria, DNA synthesis, storage or efflux
- Transported out by FPN1
- Hepcidin negatively regulates FPN1 causing its degradation
2
Q
how is iron uptake effected in cancer
A
- Increased expression of TFR1 – proven by use of antibodies against it
- Increased steap 3 expression
- LCN2 released which is a small sidophore that binds iron and is transported back in via LCN2 receptor.
3
Q
how is iron efflux effected in cancer
A
- FPN1 expressed less
* Hepcidin expression is increased causing FPN1 degradation
4
Q
iron storage in cancer
A
- C-myc protooncogene increased to inhibit ferritin to leave fe free for utilisation in liable iron pool
- HRAS gene expression does a similar thing.
5
Q
ribonucleotide reductase
A
- Required for catalysis of deoxyribonucleotides
- R2 subunit required for S/G2 (synthesis of DNA)
- p53R2 subunit is used in conditions of repair in G0/G1
- Both have an iron binding site
- Chelators inhibit these and therefore an effective method
6
Q
Cyclins and CDKs
A
- DFO inhibits D1 cyclins in pancreatic and prostate cancers
- Iron chelators can decrease expression of CDK2 and CDK4 although is a cell type depependent and can vary under different experimental conditions
- Iron chelators can inhibit C1, 2 and 3 and to a lesser extent cyclins A and B
7
Q
p53 tumour supressor
A
- DFO shown to increase its expression at the post translational level
- P53 has crucial role at G1/S phase and is activated under damage and stress to initiate repair or apoptosis
- DFO caused increase in nuclear p53 and its binding
- Regulates by reducing p53 binding to haem groups which would usually result in the degradation and export of p53
8
Q
CDK inhibitor p21 - helps cancer
A
- P21wafw/cici negatively regulates the cell cycle by binding G1/S CDK-cyclin complexes leaving them inactive
- Allows Retinoblastoma protein (Rb) to bind E2F1 (Transcription factor) preventing G1/S transition meaning cell arrest.
- Expression of p21 is needed at low levels to allow CDK-cyclin D complexes to form
- P21 has also been shown to inhibit apoptosis through inhibiting caspase 3.
- DFO up regulates MCF-7 mRNA of p21 but decreases the protein expression.
- MCF-7 has abnormally high p21 levels meaning they can suppress apoptosis
- DFO causes post transcriptional regulation of P21 to stop protein production and therefore proliferation
9
Q
c-MYC - helps cancer
A
- Not a typical cell cycle regulator but can enable cells entering it/ accelerating it.
- Induction of c-myc is required for cell cycle response to mitogenic signals
- Positively regulates D/CDK4/6 and represses P21.
- DFO has decreased its expression in leukemic blood cells, prostate and pancreatic cancers
- The mechanism could involve inhibition of STAT3 pathway and subsequently inhibiting downstream targets cyclin D1, c-myc and Bcl-2
- Involved in many pathways crosstalk such as MAPK, TGF-B, Wnt and signal transduction and activation of STAT pathways.
10
Q
N-myc downstream regulated gene 1 (NDRG1)- suppresses cancer
A
- Metastasis suppressor gene that encodes a protein with range of significant cell functions including cell cycle regulation, differentiation, angiogenesis, maintenance of myelin sheath of schwann cells and mast cell maturation, microtubule organisation and mitosis.
- Pleiotropic nature is cell type dependent
- Lower in various cancers including breast
- Its lower expressed in cancers that have increased penetration – proof of metastasis inhibition
- Iron depletion upregulated NDRG1
- This occurs via hypoxia inducible factor 1a dependent and independent mechanisms
- Under low iron eIF3a increases its expression (HIF-1a independent pathway)
- NDRG1 is phosphorylated at by serum and glucocorticoid-induced kinase 1 (SGK-1) making it a substrate for glycogen synthase kinase 3 beta (GSK3B) which then phosphorylates it.
- DFO causes increased accumulation of NDRG1 and in phosphorylated form.
- Unsure of its use but in pancreatic cancer cells its phosphorylation is essential for tumour suppressive action of cxc chemokine expression and nuclear factor light chain enhancer of activated B cells (NF-KB) signalling pathway.
- Evidence its localized to centromere and the cleavage furrow in dividing cells implying roles in microtubule organisation, cell cycle and mitosis.
- Prostate cancers cleave NDG1 so truncated NDRG1 cannot act.
- Alters expression of transcription factors and genes involved in ribosome and protein synthesis and reduces cathepsin C – used for invasion
- Reduces thtpa expression that’s used for energy metabolism
- Up regulates CDK inhibitor P21.
- Acts on PI3K/AKT, MAPK and TGF-Beta pathways.
11
Q
PI3K/AKT/mTOR signalling
A
- key regulator of mammalian cell proliferation, growth and survival
- Binding of insulin or insulin-like growth factors to a receptor tyrosine kinase (RTK), results in direct interaction between the RTK and regulatory subunits of class I PI3Ks
- This enables the PI3K catalytic subunits to exhibit their kinase activity and catalyze the phosphorylation of (PIP2) to (PIP3)
- This activates AKT as it allows association with PDKs and now AKT is phosphorylated.
- PIP3 is regulated by tumour suppressor phosphatases
- Activated AKT causes multiple signaling cascades with important consequecnes for cell survival, proliferation and growth
- PI3K/AKT is an anti-apoptotic pathway by phosphorylating Bcl-2 proteins like BAD which forms non functional dimer with BclXL
- Promotes proliferation by inhibiting GSK3Beta – a protein that degrades cyclin D1.
- Regulate TFs including CREB protein, NF-KB and fordhead TF – all premote proliferation and survival.
- Also targets serine/threonine kinase mTOR.
- Could occur by interaction between mTOR and p-AKT or pAKT inhibiton of TSC2 – this inhibits Rheb and this activates mTOR.
- mTOR activates p70S6 kinase and eIF4E binding proteins.
- These enhance mRNA recruitment to ribosomes
- mTOR pathway also regulates Bcl-2 proteins involved in apoptosis and c-MYC – promoting cell survival, proliferation and oncogenesis
- PTEN inactivation occurs in breast cancer. This is associated with AKT/PI3K activation.
- Mutation in the PI3K catalytic subunit occurs in breast cancer which causes activation.
- Hyperactivation of PI3K/AKT is targeted for cancer therapy. These include inhibitors of its downstream effects.
- Including dual PI3K-mTOR inhibitors, PI3K inhibitors, AKT inhibitors, and mTOR complex catalytic site inhibitors
- The could overcome resistance to therapies that target RTKs
- Iron chelators target PI3K/AKT/PTEN in prostate cancers (DFO)
- DFO increased tumour suppressor PTEN and NDRG1
- NDRG1 attenuates pAKT and p-mTOR
- NDRG1 reduces expression of PI3K subunits
- DFO inhibits cyclin D1 expression
12
Q
Ras/Raf/MEK/ERK signalling
A
- These are the four major MAPK pathways
- These are activated by Growth factors, cytokines, JNK, p38 and ERK5 (environmental stress)
- (MAPK) They regulate proliferation, survival, differentiation, migration and angiogenesis
- activated upon binding receptor tyrosine kinases (hormones to hormone receptors)
- this causes extracellular conformation and dimerization of two receptor internal domains.
- Auto phosphorylation occurs
- So now GRB2 binds via SH2 domain. Then 2 SH3 domains interact with SOS which activates Ras by GDP -> GTP
- Activated ras recruits and activates either MAPKKK or MEK which then phosphorylate MAPKs.
- Activates MAPK can enter nucleus to regulate transcription factors like c-myc, CREBs and forkhead TF. This promotes cells growth and proliferation.
- Ras/Raf/MEK/ERK can also inhibit apoptosis phosphorylating Bim and BAD therefore activating the anti-apoptotic roles of Bcl-2 and Bcl-xL
- ERK has roles in motility and metastasis as it can activate MMP-2/9 and myosin light chain kinase MLCK
- EGFR overexpression seen in ½ carcinomas and ErbB2 in 30% of breast cancers.
- Mutations in Ras in multiple cancers.
- DFO could regulate RAS/Raf/MEK/ERK by decreasing phosphorylation of ERK1/2 in prostate cancers
- ERK controls multiple signalling pathways so is a good target. E.g. m-TOR and c-myc
13
Q
JNK/p38
A
- Two parrallel MAPK pathways which are activated by stress.
- Control pathways involved in anti-proliferation, and pro-apoptotic
- Activated by MEKK1-4, apoptosis signal-regulating kinase 1 (ASK1), TGF-Beta activated kinase 1 (TAK1) and mixed lineage kinase (MLK)
- These activate two subgroups of MAPKK: MKK4/7 and MKK3/6
- That activates JNK and p38 MAP kinases
- They regulate numerous kinases and TFs: p53, Activating transcription factor 2 (ATF2), CREB and ETS domain containing protein (Elk1) – effect transcription, proliferation and survival.
- MKK4 supression can occur in cancers to reduce JNK and p38 activation
- MKK4 is mutated in certain breast cancers at low 5% frequency
- DFO induced apoptosis by mediating activation of JNK and p38 MAPKs
- This causes phosphorylation of MKK3 and MKK6 and downstream targets AFT2 MAPK activated protein kinase 2
- DFO can also induce phosphorylation of JNK and p38 to cause activation of downstream p53 and ATF2
- DFO iron removal is mediated by Trx oxidation causing dissociation of ASK1-Trx complex which usually inhibits ASK 1 kinase so now ASK1 dependent apoptosis can occur – cell lines not specified
- Very variable between cell types
- DFO shown to increase ERK phosphorylation but in prostate it decreases it.
- Indicates cell type differences in regulation of MAPK signalling
- P38 on the other hand is more consistent with it labelled as a tumour suppressor as its suppression has large influence on Ras-induced transformation.
- P38 MAPK are major mediator of DFo induced apoptosis over other MAPKs
14
Q
TGF-beta signalling – suppressive
A
- activated when cytokine TGF-beta binds to TGF-B receptor II expressed on cell membranes
- this recruits TGF-B receptor 1 subunit which auto phosphorylate and TGF-BR1 is activated and is a kinase
- SMAD2 and SMAD3 translocate membrane to reach active TGF-BI-II complex to be phosphorylated
- This results in complex with SMAD 4 which enters nucleus to bind SMAD binding elements to regulate genes
- TGF-B is regulated by effects on cell cycle like upregulation of p15, p21 and p27kipi as well as down regulation of c-myc
- Also upregulation of pro-apoptotic BAX, Caspases and downregulation of anti-apoptotic Bcl-2
- Shown to reduce proliferation and cause cell death in cancers
- The TGF-B / SMAD signalling is negatively regulated by Ras/Raf/MEK/ERK signalling which is induced by EGF receptor signalling.
- Ras phosphorylates SMAD2 at linker region (different site to its activation)
- In normal epithelial cells RAS-mediated phosphorylation of SMAD2 just adjusts TGF-B signalling without fully inhibiting it
- In cancer cells Ras is hyper active which could fully reduce TGF-B signalling causing proliferation and cell survival – worthy target
- DFO can attenuate phosphorylation of ERK1/2 in prostate cancer cells and decrease amount of SMAD2 linker phosphorylation. Also no activation of SMAD 2 occurred. So TGF-B wasn’t activated
- So DFO reduced the SMAD2 linker phos so therefor TGF-B can activate it.
15
Q
STAT3 signalling – activation of cancer
A
- STAT family of proteins that transduce extracellular signals to regulate genes involved in growth, survival and differentiation.
- STAT3 is most characterised
- Activated by upstream cytokine-receptor interactions
- Can also be activated by growth factor RTKs and non-receptor kinases – Src and c-Abl
- Activation cause phosphorylation causing dimer, moves to nucleus to then bind STAT DNA binding elements to effect gene expression
- It promotes cell cycle progression by up regulation cyclin D1, c-myc and pim 1
- Suppression of apoptosis through Bcl-2, myeloid leukaemia cell differentiation protein Mcl-1 and cellular inhibitor of apoptosis 2 (c-IAP2) expression.
- Repressed p53 expression
- Help invasion, migration and EMT by promoting Rho GTPase-reguated cell migration and regulation of MMPs, E-cadherin, ZEB and vimentin
- Breast cancer has activated STAT3
- Iron chelators regulate STAT3 in pancreatic and prostate by inhibiting IL-6-induced activates of STAT3
- DFO inhibited stat3 activation, dimerization and gene expression of down stream targets.
- This could be due to decreased activation of kinases src and c-Abl which premote stat3 signalling
- Crosstalk between MAPK and AKT pathways is effected by iron chelators as MEK/ERK have been shown to increase or decrease STAT3 in different cancers as well as AKT/PTEN regulate it.
16
Q
The EMT
A
- Epithelial cells lose their polarity and cell-cell junctions, reorganise their cytoskeleton and induce signalling changes that reprogram gene expression and cell shape.
- Used in wound healing and embryogenesis
- Its reactivated for fibrogensis and tumourigenesis
- Repression of epithelial and activation of mesenchymal involves changes in master transcription factors like snail, slug, twist and ZEB.
- These cause down regulation of E-cadherin, occludin, claudins and maintain tight cell junctions with up regulation of N-cadherin, MMPs, vimentin and fibronectin which promote invasiveness.
- Two major pathways TGF-B and WNT
- In cancer TGF-B promotes tumour progression and metastasis by induction of EMT.
- This is a result of core mutations in TGF-B signalling cascade allowing tumours to escape its tumour suppressive mechanisms
- TGF-B has shown to increase proliferation via Ras/Raf/MEK/ERK pathway
- Rather then suppress it via normal SMAD signalling.
- SMAD-dependent TGF-B signalling in cancers promotes EMT by activation mesenchymal associated TFs Snail, slug twist and SEB1
- Other SMADs can regulate cell motility and invasiveness by up regulation of cytoskeleton genes including vimentin and fibronectin
- TGF-B can also activate Rho/ROCK1 and PI3K pathways.
- ERK MAPK represses E-cadherin and activates twist, N-cadherin and MMP
- TGF-B mediated activation of PI3K/AKT signalling induces activation of rapamycin complexes1/2 (mTORC1/2) – this aids EMT and nuclear translocation by up regulation of FRAT1 which prevents GSK3beta from binding beta-catenin destructive complex.
- NDRG1 could also inhibit Beta-catenin nuclear translocation that is mediated by Wnt signalling by reducing localisation of p21 activated kinase 4 (PAK4).
- These NDRG1 effects lead to Beta-catenin expression in membranes where it functions for adhesion and reducing localisation to nucleus where its oncogenic
- MCF-7 cells demonstrate NDRG1 interaction with Wnt signalling to supress EMT by reactivating GASK3beta promoting Beta-catenin degradation and inhibiting activation of ATF3 downstream.
- DFO shown to destabilise Beta-Catenin to then inhibit Wnt signalling.
- Enhanced iron leads to increase Wnt signalling – increasing proliferation
- DFO can increase EMT maybe by DFO-induced HIF-1 expression
17
Q
Migration and metastasis
A
- Direct cell invasion and migration of tumour is needed for metastasis.
- Involved F-actin polymerisation, its interactions with myosin II filaments and the subsequent formation of contractile actomyosin structures like stress fibres
- Small Rho-GTPase family is involved in contraction, motility and assembly of stress fibres through effectors Rho and Rac.
- These regulate targets to regulate cytoskeleton like p21 activated kinase (PAK) Rock/myosin light chain (MLC) and LIMK pathways that mediate formation and contraction of stress fibres.
- Phosphorylated MLC is a key regulator
- Iron chelators (DFO) can regulate migration and metastasis by modulation ROCK1/MLC2 cascade.they can supress expression of both ROCK1 and MLC2
- This was due to them up regulation of NDRG1 which regulates MCL2 and ROCK1 expression to prevent f-actin polymerization and stress fibre formation.
- C-src oncogene downstream effectors play important rolls in this.
- NDRG1 was shown to inhibit Src-induced phosphorylation of p130cas and prevented complex with CrkII
- NDGR1 could also decreae activation of c-Abl and subsequent CrkII phosphorylation.
- Results in inhibition of Rac1 signalling and decreased levels of phosphorylation PAK1 playing cruital roles in cytoskeleton dynamics and regulation of cell motility.
- DFO can inhibt Src and c-Abl aswell as upregulate NDRG1 it can regulate metastasis.
