Day 2: TP53, KRAS, Apoptosis in cancer Flashcards
HC05, 06, 07
Discovery p53
Research on oncogenic viruses: use cancer F9 cell lyses and healthy 3T3 cell lyses
> find host interacting protein of SV40
> SV40 contains and expresses LT (large-T) which binds p53
> Immuno-capacitate is puled down with p53 after binding
> 53 kDA protein pulled down: p53
Name the expressed proteins in SV40 and HPV which can bind p53 (tumor suppressor)
-SV40: large-T (LT)
-HPV (in cervical cancer): expresses E7 and E6
> E6 can directly bind p53 and inactivate it
Name important tumor suppressors (6)
-p53
-APC
-p16
-p14-ARF
-VHL
-TGFBR2 (TGFb recepeptor 2)
> p53 is most important
Universal tumor suppressor mutation in cancers
p53 (needed for cancer cells to thrive)
> a high percentage of patients in most cancers contain TP53 mutation, a lot in CRC and lung cancer
Why is the frequency of TP53 mutations in cervical cancer low?
Mostly induced by HPV
> expresses E6 which binds and inactivated p53, no mutation needed!
Mice with p53-/-, p53+/- and p53+/+ and survival
p53+/+: 100% survival
p53+/-: okay survival but susceptible to spontaneous tumor formation
p53-/-: progressive decline to 0% survival
Which mutations are the drivers in pancreatic cancer?
KRAS
Induce KRAS mutations in mice results in mice which do quite well, why?
p53 tumor suppressor
> K,P,Cre mice have real quick decline in survival
Why is p53 so often mutated in cancer?
There is enormous selective pressure on the gene
Why can other animals like elephants suffer less from p53 deficiency?
More copies of the gene, humans only have 2 copies
A lot of p53 is correlated with the … phenotype
Aged (old)
Domains of p53 protein
-Sequence specific DNA binding domain: for the function as transcription factor
(-Proline rich domain)
-Transactivation domain
-Tetramerization domain: p53 only functions as (homo)tetramer
Most frequent mutation in p53
Missense mutation
> change of a amino acid in the amino acid sequence
> no frameshift, still 3 nucleotides in place
Why is the mutation of one allele of p53 problematic?
It functions as tetramer: 1/2 deficient p53 > 15/16 deficient p53 tetramers
> the tetramer amplifies the mutant penetrance
> mutated subunits ‘poison’ the complex
» DOMINANT NEGATIVE MUTATION
LOH of p53
after heterozygous for p53, loss of heterozygosity, which is quickly developed because of selective pressure on the one allele
Cancer genome evolution after p53 loss (+/-)
1: TP53 LOH because pressure on one allele
2: deletions of other genes
3: genome doublings (massive effect): polyploid cells, the number of chromosomes is no longer controlled by p53
4: amplifications: certain genes amplificated: for example 8 copies of KRAS or Myc
Most mutations of p53 in …. domain
DNA binding domain
Types of mutations of p53 (classes of mutations and effect on protein)
-Loss-of-function: no activation of p53 target genes: dominant negative mutation
(-Partly functional, some selected p53 targets are recognized still)
-Gain-of-function: DNA binding modulated so that the tetramer promotes expression of other target genes which benefit the tumor cells!
p53 activating signals
-Damage to DNA and deregulated growth
> Hypoxia
> Insufficient telomere length
> DNA damage through radiation
> Ribosome synthesis problems
> Low NTP pools/ DNA fragments
> Oncogene activation: oncogene signalling
> Tumor suppressor inactivation
Regulation p53 stability
Based on post-translational modifications and protein stability, not the de novo synthesis
> speed of degradation
Mdm2 polyubiquitinates N-terminus of p53 for degradation
> phosphorylation of this N-terminal domain blocks ability to ubiquitinate and affects the binding of Mdm2 to p53: protect for degradation
Mdm2 regulation
p53 binds promotor and activates transcription of Mdm2
> translation Mdm2 in cytosol
> Mdm2 binds p53 in nucleus, polyubiquitination in the cytosol
> degradation in cytosolic proteasomes
Mutated p53 results in higher levels, why?
It cannot bind to right DNA to upregulate Mdm2
Which molecule used in screening inhibits Mdm2
Nutlin-1 (antagonist Mdm2)
> screen for TP53 mutant cells
> Nutlin-1 + TP53 WT > decrease cell viability (p53 pathway upregulated: cell death)
> Nutlin-1 + TP53 mutant > no decrease in viability
DNA damage sensing and p53 pathway
> input: UV radiation, ionizing radiation, lack of nucleotides
DNA damage sensed by ATM and ATR
> inactivation Mdm2 by phosphorylation at binding site for p53
» ATR and ATM activate Chk1/2 which phosphorylates Mdm2
> activation p53 through phosphorylation
» ATM and Chk1/2 phosphorylate and activate p53
Phosphorylations of Mdm2
-Inactivation by phosphorylation by Chk1/2 on p53 binding site
-Activation in cytosol when translated and activated by phosphorylation by PKB/Akt via cell survival signals > translocation to nucleus and binds p53 and ubiquitination for degradation
Pathway p53 and oncogene signalling input
Ras or Myc activation will promote formation E2F (TF for p14-ARF)
> transcription p14-ARF
> ARF produced
> ARF binds Mdm2: p53 no longer degraded
> p53 accumulates in nucleoplasm
> p53 induces cell cycle arrest and apoptosis
Mechanisms of inactivating p53
-Missense mutation in DNA binding domain: prevents p53 from binding target DNA sequences and activating adjacent genes
-Viral infection: products of viral oncogenes bind and inactivate p53 in cell or stimulate p53 degradation
-Deletion p14-ARF gene: failure to inhibit Mdm2 and keep p53 degradation under control
-Multiplication Mdm2 gene in genome
-Mislocalization p53 to cytoplasm, outside nucleus: no function
-Deletion C-terminal domain p53: no tetramerization
Outputs p53
-Cell cycle arrest
> return to proliferation or senescence
-DNA repair
-Block angiogenesis
-Apoptosis
-Alter metabolism
p53 and cell cycle arrest
p53 promotes expression p21 gene
> p21 is a Cdk (cyclin dependent kinase) inhibitor protein which binds G1/S Cdk or S-Cdk to inactivate
> cell cycle arrest
Ras induced when p21+ and p21-
p21+: no effect
p21-: tumor formation
Senescence via p53
Degree of damage leads to proportional response
> when DNA damage too high, oncogene stress or telomere length low, cells cannot get out of it: senescence
> alternatively stuck in S phase and G2/M transition block
Apoptosis and p53
-p53 induces expression Puma (BH3 peptide) and Bax (pro-apoptotic for CytC release) and
-Induce expression FAS (death receptor)
HC06: Ras activation
Binding of extracellular signal input (like EGFR for EGF)
> Trans-autophosphorylation receptor tyrosine kinase
> Adaptors bind to phosphorylated tyrosine: Grb2 and Sos to the phosphorylated (by EGFR) Grb2
> Sos is a GEF
> Activation Ras by replacing GDP for GTP
Name downstream pathways of Ras
- MAPK pathway (Mek/Erk): Activation for example Myc > proliferation
- PI3K to Akt/PKB pathway > inhibit Bad and therefore inhibit apoptosis and promote survival
- Cytoskeleton pathway
Different growth factor ligands and receptors which lead to Ras (in)activation
EGF, TGFa
Receptors HER1-4
Discovery KRAS
Viruses as oncogenic agents wasn’t panning out: as genetic disease: DNA transformation from cancer cells lead to tumor formation
> Expose healthy cells to chemicals to make cancer cells and transfect normal fibroblasts with cancerous DNA > tumor formation
> discovery retrovirus associated oncogenes: Raf, H-ras, K-ras, Myc
> where on genome oncogenes? > detection foci after transfection experiment with DNA piece that contained oncogene vs cloned proto-oncogene (healthy)
> make mixes
> half-oncogene and half proto-oncogene, which of the two forms foci, in that half of the DNA resides he ondogene
> until small fragment: oncogene for seuquence analysis and proto-oncogne to determine mutation to form oncogene
Mutation in KRAS from discovery
Single codon mutated
> G12 (glycine-12)
The types of Ras mutations (HRAS, KRAS and NRAS) differentiates per tumor type. Which percentage in CRC?
45% gene mutation
In which organ highest frequency KRAS mutation when cancer?
Pancreas (90%)
Where does KRAS mutation not cause cancer?
In the blood
order of transformation:
loss APC, activation KRAS, loss 18q TSG, loss p53, DNA hypomethyation
Normal epithelium
> loss APC
Hyperplastic epithelium
> DNA hypomethylation
Early adenoma
> activation KRAS
Intermediate adenoma
> loss 18q TSG
Late adenoma
> loss p53
Carcinoma
>
Invasion and metastasis
Ras genes
-KRAS
-HRAS
-NRAS: neuroblastoma
Ras genes are the most frequent mutated oncogenes. Are there many mutations in KRAS oncogenic?
No, some specific ones are oncogenic
Pathways Ras
PI3K pathway:
> Inhibition Bad (apoptosis evaded)
> mTOR stimulation: stimulate protein synthesis for cell growth
> cell proliferation
Mek/Erk
> Protein synthesis
> Chromatin remodeling
Ral-GEFs
> Cytoskeleton: Cdc42 for filopodia and Rac for lamellipodia are both inhibited»_space; cell movement
Ras activation
Ras inactive: bound by GDP
> GEF activated by upstream stimulatory factor
> GEF activates Ras: GDP for GTP exchange
Ras active: downstream signalling
> GAP activation
> GAP induces GTPase property: GTP hydrolysis by Ras and GDP-bound
GEF function
Guanine nucleotide exchange factor
> GEF takes out GDP
> in the cell there is a lot of GTP (high GTP/GDP), and GTP will be bound by Ras spontaneously (high affinity binding)
> conformational change > switch domains active
GAP function
GAP interacts with Ras, induce hydrolysis by intrinsic GTPase activity
Which property of Ras is affected by an oncogenic mutation
Interaction domain for interaction with GAP
> no hydrolysis GTP
> always active
> continuously phosphorylating activity
(Ras is an on/off switch, mostly inactive, but now always active!)
Conformational change by Ras
Change in Switch I and Switch II domains when either GTP or GDP bound
> Interact with different downstream proteins
> switch domains interact with downstream proteins and are altered (activated) when GTP bound. When GDP bound, they cannot interact.
Oncogenic signalling through KRAS: Erk signalling
Ras > Raf > Mek > Erk1/2 (kinase cascade)
- Activation gene programs
> for protein synthesis, chromatin remodeling: make more cells
- linear signalling route
Ras and Raf mutations are …
mutually exclusive, do not occur together: in same pathway, no selective pressure
Interaction Ras with Raf via …
Switch I domain of Ras
Cancers with Ras WT have…
BRAF mutation
> when KRAS mutation, BRAF WT
> pathway already overactivated
PI3K route of KRAS oncogenic signalling (thus overactive)
PI3K phosphorylates PIP2 to make PIP3 > activate Akt/PKB which bind to PIP3 with PH domains and PDK1 phosphorylates Akt
PI: phosphatidyl inositol
Effect PTEN deficiency on pAkt (phosphorylated, active, Akt)
PTEN deficient
> less dephosphorylation PIP3 (is blocked now)
> more activation and more pAkt
Ral signalling (KRAS oncogenic)
Cytoskeletal effects
> GEF mediated signalling
Why mutual exclusivity of oncogenic hits in KRAS route?
Linearity in signalling
Why is targeting the binding of GTP with K-Ras hard?
High affinity binding and there is a lot of GTP in the cell
> but: no other apparant binding pocket (no noncompetitive inhibition possible)
> Competitive hard because high affinity
> Ras inhibitors need affinity
KRASG12C inhibitors have clinical success, what is it?
Inhibitors that occupy the induced switch II pocket of KRASG12C
> covalent bond to Cys12 (but not in most KRAS mutants, then the Glycine-12 changed to other amino acid)
» disulfide bridges
Farnesyl transferase inhibitors
KRAS interaction with the PM disrupted because hypothesis to associate via farnesyl group (very lipophilic, stuck in PM)
How to skip KRAS in inhibiting it?
Combined up- and downstream targeting
> inhibit Mek for example and FGFR1
A recent breakthrough is a noncovalent inhibitor for KRASG12D. Why this mutant?
Has the second highest hydrolysis rate among KRAS mutants
> highest rate of GAP-mediated GTP hydrolysis
> like MRTX1133
MRTX1133 function
Bind to GDP-bound form of KRASG12D with super high affinity
> mess up interaction with downstream targets
> only when KRAS in GDP state, which is unfortunately rare in cancer
> results in conformational change of switch I and II even when activated afterwards
> does not inhibit KRAS altogether, only the KRASG12D mutant
Increasing doses of KRAS inhibitors results in
better response
HC07: In tissue there is balance between life and death. Why is apoptosis important?
In rapid renewing tissues like intestinal epithelium and in patterning and development of separating digits for example.
How many cells die per day in human adult?
10 billion
APC loss leads to hyperplasia, but not cancer immediately, explain
APC loss: hyperproliferation
> but not lowered cell death
> hyperplasia is not cancer, only when apoptosis is also inibited then cancer development (resistance)
> part of the hallmarks of cancer: resist cell death
phenotype apoptosis
- Cell shrinkage
- Membrane blebbing
- Nuclear condensation and fragmentation
- Apoptotic bodies formation
- Phagocytosis
Biochemical characteristics apoptosis
- Caspase activation
- Phosphatidyl serine (PS) exposure in outer leaflet
- Cytochrome C release
- DNA fragmentation
Apoptosis is … cell death to not make …
regulated, not make mess
Blebbing cells for apoptosis
Pinch off blebs to reuse contents
How long is DNA
2 meters
> packaging in nucleosomes, wind around histones
> more condensed, less transcription
DNA condensation in necrosis and apoptosis
Necrosis: less condensed DNA, which grabs stuff and becomes slimy
Apoptosis: DNA fragmentation activated by caspases
> observe laddering: ladder bands on gel
> avoid making complete mess
PS in PM in normal cell
Flipase keeps PS in inner leaflet actively (use ATP)
(scramblase shuffles phospholipids between leaflets, makes it random, sometimes PS in outer but fixed by Flipase)
> Annexin V can bind PS in outer leaflet through Ca2+ (for test in lab: mark Annexin V with fluorophore to mark apoptosis)
> PS is flag for apoptotic cell
> endogenous proteins have similar functions as Annexin V: detect PS and flag the cells for macrophages
Spotting Annexin V in test after radiotherapy
Tumor shrinkage and increased Annexin V spotted
Apoptosis character in function
- Clean way to get rid of cells because DNA is degraded
- Clean way to get rid of cells because dying cell is phagocytosed
- Apoptosis requires involvement of cells themselves
Caspases characteristics
- Made as inactive pro-forms in cells (zymogens)
- Proteases
- Activation through cleavage and by bringing two caspases in close proximity: dimerization
- Caspases can cleave other proteins and other caspases at aspartate (Asp/D) amino acids
- Induce DNA fragmentation
DNA fragmentation via caspases
Active executioner caspase (3) cleaves inhibitor on ICAD > CAD (caspase activated DNAase) fragments DNA
Activation caspase routes
-Intrinsic (via mitochondria, cyt C release)
-extrinsic (need active signalling receptors to receive extracellular signals: death receptors
Extrinsic apoptosis route
Death receptors like TNFR or TRAIL receptor or FasR (CD95 receptor)
> work as trimers, trimer ligand binds: trimerization > recruit and activate casp-8 and casp-10 (initiators) via FADD (Fas-associated protein with Death Domain)
> Casp-8/10 activates executioner/effector caspases like casp-3
Intrinsic route: inducers
-Stress signals
-Oncogenes
-Irradiation
-Chemotherapy
-Heat
-Hypoxia
-Glucose deprivation
-Growth factor depletion
Cytochrome C release from mitochondria (used in ETC) causes apoptosis ….
irreversibly
Formation apoptosome and function
Cyt C, procasp-9 and Apaf subunits
> apoptosome active: casp-9 activation
> cleave procasp-3 to casp-3
Can a combination of intrinsic and extrinsic apoptosis occur?
Yes
Bcl-2 hypervariable domains
BH1-4
Bcl-2 family
- Bcl-2 likes: anti-apoptotic: inhibit Bax/Bak which form pore to release Cyt C from mitochondrion
> Bcl-2, Bcl-xL, Bcl-w, Mcl1 - BH3-only peptides bind and inhibit Bcl-2 likes
> Bid, Bim, Bad, Puma, Noxa, BMF
TP53 and apoptosis
P53 increases Bax transcription and BH3 transcription (Noxa, Puma)
> apoptosis induced
> BH3s promote Bax and bind and inhibit Bcl-2
Bcl-2 only blocks apoptosis when…
the expression is high enough to counteract the pro-apoptotic activity: out-express the BH3-only peptides and Bax/Bak expression
Apoptosis in cancer: avoid it mechanisms
- Mutate death receptors
- Bax deletion
- Bcl2 overexpression
- P53 mutation
- Caspase deletion/mutation
- FLIP or IAP overexpression
> FLIP inhibits activation Casp-8 by FADD
> IAP inhibits Casp-3
-BH3-only deletion
Therapies for anti-apoptotic behaviour in cancer
-TRAIL therapy: ligand for death receptor induction
> does not work well in patients
> mutation TRAIL-R would circumvent this
-Bcl-2 targeting
> inhibitor Bcl-2
> Site in anti-apoptotic proteins and look where BH3-only peptides engage and bind: binding pocket around Arg-139
> BH3-mimetics: designed to mimic BH3 proteins and the small molecule binds to the Bcl-2 and binds way better and inhibits the anti-apoptotic proteins like Bcl-2
