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Molecular Oncology > Day 2: TP53, KRAS, Apoptosis in cancer > Flashcards

Day 2: TP53, KRAS, Apoptosis in cancer Flashcards

HC05, 06, 07

1
Q

Discovery p53

A

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

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2
Q

Name the expressed proteins in SV40 and HPV which can bind p53 (tumor suppressor)

A

-SV40: large-T (LT)
-HPV (in cervical cancer): expresses E7 and E6
> E6 can directly bind p53 and inactivate it

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3
Q

Name important tumor suppressors (6)

A

-p53
-APC
-p16
-p14-ARF
-VHL
-TGFBR2 (TGFb recepeptor 2)
> p53 is most important

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4
Q

Universal tumor suppressor mutation in cancers

A

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

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5
Q

Why is the frequency of TP53 mutations in cervical cancer low?

A

Mostly induced by HPV
> expresses E6 which binds and inactivated p53, no mutation needed!

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6
Q

Mice with p53-/-, p53+/- and p53+/+ and survival

A

p53+/+: 100% survival
p53+/-: okay survival but susceptible to spontaneous tumor formation
p53-/-: progressive decline to 0% survival

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7
Q

Which mutations are the drivers in pancreatic cancer?

A

KRAS

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8
Q

Induce KRAS mutations in mice results in mice which do quite well, why?

A

p53 tumor suppressor
> K,P,Cre mice have real quick decline in survival

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9
Q

Why is p53 so often mutated in cancer?

A

There is enormous selective pressure on the gene

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10
Q

Why can other animals like elephants suffer less from p53 deficiency?

A

More copies of the gene, humans only have 2 copies

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11
Q

A lot of p53 is correlated with the … phenotype

A

Aged (old)

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12
Q

Domains of p53 protein

A

-Sequence specific DNA binding domain: for the function as transcription factor
(-Proline rich domain)
-Transactivation domain
-Tetramerization domain: p53 only functions as (homo)tetramer

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13
Q

Most frequent mutation in p53

A

Missense mutation
> change of a amino acid in the amino acid sequence
> no frameshift, still 3 nucleotides in place

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14
Q

Why is the mutation of one allele of p53 problematic?

A

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

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15
Q

LOH of p53

A

after heterozygous for p53, loss of heterozygosity, which is quickly developed because of selective pressure on the one allele

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16
Q

Cancer genome evolution after p53 loss (+/-)

A

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

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17
Q

Most mutations of p53 in …. domain

A

DNA binding domain

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18
Q

Types of mutations of p53 (classes of mutations and effect on protein)

A

-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!

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19
Q

p53 activating signals

A

-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

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20
Q

Regulation p53 stability

A

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

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21
Q

Mdm2 regulation

A

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

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22
Q

Mutated p53 results in higher levels, why?

A

It cannot bind to right DNA to upregulate Mdm2

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23
Q

Which molecule used in screening inhibits Mdm2

A

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

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24
Q

DNA damage sensing and p53 pathway
> input: UV radiation, ionizing radiation, lack of nucleotides

A

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

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25
Q

Phosphorylations of Mdm2

A

-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

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26
Q

Pathway p53 and oncogene signalling input

A

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

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27
Q

Mechanisms of inactivating p53

A

-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

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28
Q

Outputs p53

A

-Cell cycle arrest
> return to proliferation or senescence
-DNA repair
-Block angiogenesis
-Apoptosis
-Alter metabolism

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29
Q

p53 and cell cycle arrest

A

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

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30
Q

Ras induced when p21+ and p21-

A

p21+: no effect
p21-: tumor formation

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31
Q

Senescence via p53

A

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

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32
Q

Apoptosis and p53

A

-p53 induces expression Puma (BH3 peptide) and Bax (pro-apoptotic for CytC release) and
-Induce expression FAS (death receptor)

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33
Q

HC06: Ras activation

A

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

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34
Q

Name downstream pathways of Ras

A
  • 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
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35
Q

Different growth factor ligands and receptors which lead to Ras (in)activation

A

EGF, TGFa
Receptors HER1-4

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36
Q

Discovery KRAS

A

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

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37
Q

Mutation in KRAS from discovery

A

Single codon mutated
> G12 (glycine-12)

38
Q

The types of Ras mutations (HRAS, KRAS and NRAS) differentiates per tumor type. Which percentage in CRC?

A

45% gene mutation

39
Q

In which organ highest frequency KRAS mutation when cancer?

A

Pancreas (90%)

40
Q

Where does KRAS mutation not cause cancer?

A

In the blood

41
Q

order of transformation:
loss APC, activation KRAS, loss 18q TSG, loss p53, DNA hypomethyation

A

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

42
Q

Ras genes

A

-KRAS
-HRAS
-NRAS: neuroblastoma

43
Q

Ras genes are the most frequent mutated oncogenes. Are there many mutations in KRAS oncogenic?

A

No, some specific ones are oncogenic

44
Q

Pathways Ras

A

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&raquo_space; cell movement

45
Q

Ras activation

A

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

46
Q

GEF function

A

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

47
Q

GAP function

A

GAP interacts with Ras, induce hydrolysis by intrinsic GTPase activity

48
Q

Which property of Ras is affected by an oncogenic mutation

A

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!)

49
Q

Conformational change by Ras

A

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.

50
Q

Oncogenic signalling through KRAS: Erk signalling

A

Ras > Raf > Mek > Erk1/2 (kinase cascade)
- Activation gene programs
> for protein synthesis, chromatin remodeling: make more cells
- linear signalling route

51
Q

Ras and Raf mutations are …

A

mutually exclusive, do not occur together: in same pathway, no selective pressure

52
Q

Interaction Ras with Raf via …

A

Switch I domain of Ras

53
Q

Cancers with Ras WT have…

A

BRAF mutation
> when KRAS mutation, BRAF WT
> pathway already overactivated

54
Q

PI3K route of KRAS oncogenic signalling (thus overactive)

A

PI3K phosphorylates PIP2 to make PIP3 > activate Akt/PKB which bind to PIP3 with PH domains and PDK1 phosphorylates Akt
PI: phosphatidyl inositol

55
Q

Effect PTEN deficiency on pAkt (phosphorylated, active, Akt)

A

PTEN deficient
> less dephosphorylation PIP3 (is blocked now)
> more activation and more pAkt

56
Q

Ral signalling (KRAS oncogenic)

A

Cytoskeletal effects
> GEF mediated signalling

57
Q

Why mutual exclusivity of oncogenic hits in KRAS route?

A

Linearity in signalling

58
Q

Why is targeting the binding of GTP with K-Ras hard?

A

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

59
Q

KRASG12C inhibitors have clinical success, what is it?

A

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

60
Q

Farnesyl transferase inhibitors

A

KRAS interaction with the PM disrupted because hypothesis to associate via farnesyl group (very lipophilic, stuck in PM)

61
Q

How to skip KRAS in inhibiting it?

A

Combined up- and downstream targeting
> inhibit Mek for example and FGFR1

62
Q

A recent breakthrough is a noncovalent inhibitor for KRASG12D. Why this mutant?

A

Has the second highest hydrolysis rate among KRAS mutants
> highest rate of GAP-mediated GTP hydrolysis
> like MRTX1133

63
Q

MRTX1133 function

A

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

64
Q

Increasing doses of KRAS inhibitors results in

A

better response

65
Q

HC07: In tissue there is balance between life and death. Why is apoptosis important?

A

In rapid renewing tissues like intestinal epithelium and in patterning and development of separating digits for example.

66
Q

How many cells die per day in human adult?

A

10 billion

67
Q

APC loss leads to hyperplasia, but not cancer immediately, explain

A

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

68
Q

phenotype apoptosis

A
  • Cell shrinkage
  • Membrane blebbing
  • Nuclear condensation and fragmentation
  • Apoptotic bodies formation
  • Phagocytosis
69
Q

Biochemical characteristics apoptosis

A
  • Caspase activation
  • Phosphatidyl serine (PS) exposure in outer leaflet
  • Cytochrome C release
  • DNA fragmentation
70
Q

Apoptosis is … cell death to not make …

A

regulated, not make mess

71
Q

Blebbing cells for apoptosis

A

Pinch off blebs to reuse contents

72
Q

How long is DNA

A

2 meters
> packaging in nucleosomes, wind around histones
> more condensed, less transcription

73
Q

DNA condensation in necrosis and apoptosis

A

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

74
Q

PS in PM in normal cell

A

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

75
Q

Spotting Annexin V in test after radiotherapy

A

Tumor shrinkage and increased Annexin V spotted

76
Q

Apoptosis character in function

A
  • 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
77
Q

Caspases characteristics

A
  • 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
78
Q

DNA fragmentation via caspases

A

Active executioner caspase (3) cleaves inhibitor on ICAD > CAD (caspase activated DNAase) fragments DNA

79
Q

Activation caspase routes

A

-Intrinsic (via mitochondria, cyt C release)
-extrinsic (need active signalling receptors to receive extracellular signals: death receptors

80
Q

Extrinsic apoptosis route

A

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

81
Q

Intrinsic route: inducers

A

-Stress signals
-Oncogenes
-Irradiation
-Chemotherapy
-Heat
-Hypoxia
-Glucose deprivation
-Growth factor depletion

82
Q

Cytochrome C release from mitochondria (used in ETC) causes apoptosis ….

A

irreversibly

83
Q

Formation apoptosome and function

A

Cyt C, procasp-9 and Apaf subunits
> apoptosome active: casp-9 activation
> cleave procasp-3 to casp-3

84
Q

Can a combination of intrinsic and extrinsic apoptosis occur?

A

Yes

85
Q

Bcl-2 hypervariable domains

A

BH1-4

86
Q

Bcl-2 family

A
  • 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
87
Q

TP53 and apoptosis

A

P53 increases Bax transcription and BH3 transcription (Noxa, Puma)
> apoptosis induced
> BH3s promote Bax and bind and inhibit Bcl-2

88
Q

Bcl-2 only blocks apoptosis when…

A

the expression is high enough to counteract the pro-apoptotic activity: out-express the BH3-only peptides and Bax/Bak expression

89
Q

Apoptosis in cancer: avoid it mechanisms

A
  • 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
90
Q

Therapies for anti-apoptotic behaviour in cancer

A

-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

Molecular Oncology (7 decks)

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