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Cancer genes 1.0
Cancer genes 1.0：It is how we discovered p53, we found SV40 large T antigen can binds a protein weights 53 kDa.
编辑于2022-06-08 22:38:33- Cancer
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Cancer genes 1.0
proto-oncogenes
How can we identify oncogenes?
transfer tumour derived DNA into NIH3T3 mouse fibroblasts to find out whether the DNA can transform the cells.
Criteria for the transformation
Cells change its compartments morphologies
loss of contact inhibition
(it might due to the growth factor independence)
gain of substratum and serum independence
My question is how can I know exactly which DNA transformed the cell in a bunch of randomly extracted DNA?
The cellular derived oncogenes using the method above has many overlaps with the viral derived oncogenes
Most viral derived oncogenes have cellular homologous
Mechanisms of oncogenesis(ways the proto-oncogenes become activated into oncogenes)
1. increase protein expression level (without mdodifying the protein sequence encoded)
1. increase mRNA transcripts
the present of a strong promotor/enhancer
Could be viral derived
MMTV(mouse mammary tumour virus) & c-int.
InT may encodes 3 types of proteins
They are growth factors / growth factor receptors
can due to cellular chromosome translocation
c-myc in BL(Burkitt's Lymphoma)
C-myc gene is translocated from chromosome 8 to the IgH on the chromosome 14 resulting in abnormal c-myc expression leading to cell transformation.
the duplication of gene copies
Amplification of n-myc ---> Childhood neuroblastoma
Amplification of erbB-2 ---> Breast & ovarian carcinomas
They are identified by FISH.
decrease of the mRNA degradation
2. increase protein translation
3. decrease of protein degredation
ex. EGFR may decrease endocytosis in certain types of cancer.
2. gain of function mutations on the protein sequence encoded.
1. point mutations
Ras
Mutations in codon 12 of the ras proto-oncogene results in the loss of GTPase activity. This results in continuous activation and the conversion to an oncogene.
2. trancation
v-ErbB encodes a truncated form of EGFR lacking ectodomain, it is constantly activated ignoring the extracellular signals.
3. Gene fusion
gag gene replaces regulatory domain of Raf gene, this leads to Raf kinase domain constitutively activated
identities of Proto-oncogenes
Growth factors and Growth factor receptors (RTK)
How growth factors were discovered
People found that foetal calf serum is necessary for the cultured cells to grow and divide.
growth factors can stimulate the growth and devide of cell, and some growth factors can enhance the mobilities of the cells.
Heregulin(a growth factor within EGF family) can acts as motogen, it stimulates lamellipodium formation, and it results in cell motility.
Growth factor receptors
Belong to the family of receptor tyrosine kinase (RTK)
above indicated the structural organizations of some common RTKs
The growth factors and their receptors are tissue specific
Dimerization of growth factor receptors
growth factor binds to the receptor, and it induces the dimerization of receptors.
Integration of different growth factor receptors signalings
Down stream signaling of the growth factor receptors
Different phosphorylated residues can recruit different protein adaptors and trigure different signaling pathways
Growth factor receptors will generally activate Ras protein
mutations on Ras
or up stream of Ras or down stream of Ras are most frequently identified in all types of cancer
like the mutations in KRAS kinase down stream of Ras is frequently identified in all kinds of cancers
Will lead to enhanced cell proliferation and enhanced survival and resistance to apoptosis.
Ras has 3 isoforms that involved in cancer
Most common gain of function on Ras
3 different point mutations on its effector loop will create 3 preferencial docking sites for PI3K, Raf, and Ral-GEF seperately.
3 pathways are linked under Ras:
1. MAPK pathway induces cell growth and shape change
2. PI3K pathway induces the evading of appoptosis
Here is an alternative treatment of the membrane bounded lipid PIP2
phospholipase C will cleaves phosphodiester bond of PIP2 to yield IP3(Free part) and DAG(Membrane bounded part)
PI3K first phosphorylates PIP2 into PIP3, and PIP3 will recruits Akt/PKB, PDK1 and PDK2 will phosphorylate and activate the PIP3 bounded Akt/PKB, the activated Akt/PKB will transduce its signal to the downstream
the docking domain of AKt/PKB on PIP3 is called PH domain.
A phosphate called PTEN here provides a negative feedback mechanism, it dephosphorylates PIP3 back into PIP2 and inhibits the downstream signals.
PTEN is a tumour suppressor protein.
The loss of function mutations on PTEN are involved in many kinds of cancer.
In human tumours, the transcription of PTEN often get inhibited through the methylation on its promotor region.
3. Ral-GEF pathway induces the change of cell mobilities
Picture
Incidences of oncogenesis
1. Over expression of Growth factor and its receptors
Most tumours over express EGFR
1. mostly increase transcriptional level
2. some may be amplification of genes
3. some may decrease Protein degredation/decrease endocytosis
endocytosis regulates the life time of cell surface proteins
Huntingtin-interacting protein-l (HIPl) facilitates endocytosis, cancer cells over express mutated HIPI that can inhibits endocytosis.
Cyclin G associated kinase (GAK) promotes endocytosis, cancer cells may suppress its expression, this will leads to endocytosis decrease by 50 folds.
c-Cpl ubiquitinates ligands binding EGFR, ubiquitination on EGFR will faciliates its endocytosis and degredation
cellular proteins (Sts-l , Sts-2) and viral oncoproteins v-Cbl can inhibit this ubiquitination mediated endocytosis.
4. viral encoded growth factor homologous
v-sis viral derived oncogene of siman sarcoma virus is the homologous of the B chain of platelet-derived groth factor protein(PDGF)
Consequences
Increase surface level of Growthfactor receptors will lead to hyper-responsiveness to growth factors
This allows cancer cells can grow in low growth factors enviroment.
2. Mutations causes constitutive activations of growth factor receptors
1. caused by point mutations
2. caused by trancation mutations
1. viro oncoproteins can be constitutively activated EGFR
v-ErbB encodes a EGF-R homologous but lack part of the ectodomain, which can release mitogenic signals constitutively
3. caused by gene fusion mutations (induce dimerization)
part of N terminus Muscle tropomyosin replaces part of the N terminus extracellular domain of Trk, the resulting oncoprotein is constantly dimerized and constitutively activated, and it locates only in cytosol.
Othercases of constitutively activated onco-receptors by fusion induced dimerizations
Consequence
constivtutive activations will leads to ligand-independent constant activation
3. Disruptions of negative-feedback mechanisms that attenuate proliferative signaling.
4. Autocrine and paracrine stimulation of cell growth.
1. cancer cell may secretes growth signals that effect on its own.
This leads to self-sufficiency in growth factors, which finally reduces its dependence on the growth factors in its micro enviroment.
table
The symptom is similar with the over expression of growth factor and its receptors.
Actually it is just the over expression of itself's growth factor, a particular case of 1
A summurized table
Oncogenic growth factor receptors and their growth factors
Oncogenic growth factors
therapeutic targets
Monoclonal antibodies against growth factor receptors.
Kinase inhibitors inhibits key signaling kinases.
Picture
Cell adhersion receptors (receptors that are physically attched with extracellular matrix components) and related protein factors
Integrins
hetero dimeric structural organization
α plus β subunit。
Outside in signaling
When ectodomains bind to specific components in EM, intermediary proteins will link the cytoplasmic domain of beta subunit to the cytoskeleton(actin fibires). at the same time, the cytoplasmic domains of beta subunit can attract variaty of signaling molecules.
The signaling adaptors of integrins
FAK(focal adhesion kinase)
It can activates most pathways activated by growth factor receptors.
inside out signaling
cytoplasmic signals can control the binding affinities of integrins for their ECM ligands.
this may lead to the breaking existing contacts and forging new ones in their place.
roles in cell motility
inside out signalings induce integrin to forge new linkages with the ECM.
When cell needs to loosen its tehers at the front, signalings will cause release contact with the substratum.
E-cadherin
Intermediate proteins link cadherin's intracellular domain with cytoskeleton(actin filament).
3 Intermediate proteins are involved, their spacial interactions are indicated above.
p120
β-catenin
The accumulation of β-catenin is very common in cancer.
It may promotes cell proliferation by its transcriptional factor activity.
How it is regulated
Wnt -> Frizzled -> Dishevelled - axin -| inactive SK-3β ---|-> β-catenin
this finally promotes cell prolifereation
axin -> GSK-3β(glycogen synthase kinase-3β) +p-|(targets it for degredation) β-catenin
Apc (Adenomatous polyposis coli) here is part of the protein complex: {axin][GSK-3β][APC][β-catenin][Wtx}
it is critical for the successfuly capture of β-catenin, that is then phosphorylated and targeted to degredation by β-catenin.
Onco-proteins in and around β-catenin
Constitutively activated β-catenin.
APC mtants(fail to bind and down regulates β-catenin levels).
α-catenin
tumour suppressor/onco proteins around cell cycle regulations
pRb
pRb A molecular checkpoint for the cell cycle restriction point
pRb -| E2F -o-> Proteins to path restriction points
It is phosphorylated by CDKs
Cyclin D-CDK4/6 +p pRb -|-> E2F -o-> DNA polymerase and other proteins for DNA synthesis
interpretation: pRb is the checkpoint to certain stage of cell cycle, because it normally inhibits cell cycle, and it needs to be induced by CDK2-CyclinE.
It also contains a design of positive feed back loop
E2F -o-> Clyclin E, CDK-2, E2F
CyclinE-CDK-2 +p pRb -|-> E2F -o-> ~~~~
So we call it a positive feed back loop
The phosphorylation of pRb becomes CyclinD independent.
Pictures
in cancer, pRb is often inactivated by lose of function mutations.
TGFβ
TGFβ is the receptor for antigrowth signals, TGFβ activation inhibits the cell cycle progression
Type I TGFβ signaling
TGFβ -> SMADs --o-> p15, p21
Here
p21 is also transcirptional activated by p53
p15 has a full name of p15INK4b
p15 -| CyclinD-CDK4-6 --> path of restriction point
p21 -| CyclinE-CDK2 --> path of restriction point
TGFβ -> SMADs -o-| c-Myc -| Miz -o-> p15ink4b,p21zip -| cyclin-CDKs -> path of restriction points.
p.s.
an error in the picture, p15ink 4b and p21zip has the same roles with p15 and p21, i.e. they inhibit cyclines to arrest cell cycles
TGFbeta
Type II TGFβ signaling
in cancer, TGFβ is often inactibated by lose of function mutations.
SMADs(the protein adaptors at down stream of TGFβ) is often inactivated by lose of function mutations.
smad2- in colon cancer
smad4- in pancrease cancer
c-Myc
cMyc is another cell cycle check point protein, it is a transcroptional factor that can either inhibits or activates cell cycle progression depends on its interacting partners
c-Myc can dimerizes with
c-Myc(form homodimer)
Max
Myc-Max is a transcriptional activator
Myc-Max -o-> Cyclin D2, E2F1, 2,3 , CDK4
results: cell will path G1 checkpoint
Miz
Myc-Miz can be thought as a transcriptional repressor
Miz is a transcriptional activator, and Myc binds and inhibits Miz.
Myc -| Miz -o-> p15ink4b, p21zip
Myc-Miz -o-| p15ink4b, p21zip -| cyclincdks
results: cell will not path restriction point
viral oncogenes
Introduction
Viruses cause 15% cancer world wides, most liver and cervical cancers are caused by viruses.
classes of viruses can induce malignant transformation
5 classes of DNA virus and 1 class of RNA virus
All the malignant transformations are done in non-permissive cells.
It means that the virus failed to replicate and release themselves, otherwise they will lysis and kill the cell.
All transformation requires incorporation of viral DNA into the genome.
DNA virus derived tumour suppressor genes inhibitors
Evolusionary explanasion
DNA viruses requires push cells into S phase to use eukaryotic DNA polymerase for its genome replications.
Most DNA virus oncogenes are "early genes".
early DNA virus genes promote cell cycle progressions.
generaly, a early protein called large T antigen will bind to DNA and promote late gene transcription.
large T antigen is also a helicase for virus genome replications.
late DNA virus genes encode structural proteins.
p53 inhibitor
SV40 large T antigen(Tag)
It is how we discovered p53, we found SV40 large T antigen can binds a protein weights 53 kDa.
Tag -| p53
pRb inhibitors
pRb inhibitors share a common pRb binding motif: LxCxE
Actually pRb and E2F is found by the identification of E1A bounded proteins
A picture for overview
Here you can see some proteins are both pRb and p53 inhibitors.
Tag
Tag -| p53 -o-> p21 --| cell cycle progression.
Tag -| pRb -- E2F -o-> proteins required for cell cycle progression.
E6
E6 - E6AP +u--| p53
E1B
E1B - - p53 -o-| p21...(become a repressor)
E1A
E7 -| Rb
and it also indirectly targets it to degredation
Some DNA virus onco-proteins are cellular homologous
EBV LMP1 mimics CD40 (tumour necrosis factor receptor).
bovine papillomavirus E5 mimics growth factor for PDGF receptor.
Polyomavirus middle T mimics Src.
HHV 8 vIL-6 mimics CyclinD.
RNA virus derived viral oncogenes
Evolusionary explanasions
RNA virus do not requires eukaryotic DNA polymerase for its replication.
but integration of cDNA requires cell in the stage of Mitosis (lysis of cell membrane).
This requires the progression of the restriction point.
Also, the nature of the RNA virus replication cycles will inevitably be mutagenic and cause cancer
a brief overview of retrovirus life cycle
RNA virus oncogenicity
1. Insertional mutagenesis
Promotor insertions
Enhancer insertions
LTR of retrovirus genome may contain either a promotor or enhancer, when it locates at the correct place upstream of the cellular proto-oncogene, it can be carcogenic by increase it expression level.
These virus are called Non-Acutely Transforming Viruses
Avian Leukosis Virus
Mouse Mammary Tumour Virus
2. Oncogene transduction
The cellular ongogens might be packed together with the provirus sequence, and it would be carcinogenic for the next host.
in the case above, V-src is a truncated vertion of c-src due to lose of the C-terminus in insertion, v-src is a kinase that is constitutively activated due to the lose of intramolecular inhibition.
These virus are called Acutely Transforming Viruses (e.g. Rous Sarcoma Virus)
3. Transformation by an endogenous viral oncogene
HTLV-I encodes a HTLV tax protein, it can promotes T cell proliferation and viral transcription.
4. immune suppression and destroy survaliance of malignency
ex. HIV
a picture of summury
good visualization!
tumour supressor genes
Gate keeper genes
1. cell cycle progression inhibitors
pRb*
genetics
In hereditory cases, patients have only one copy of Rb genes, lose of heterozygosity in any of the retina cell can cause retinoblastoma
In nonhereditory cases, patients have 2 copies of Rb genes, they have to lose both copies of Rb genes in one retina cell to develop retinoblastoma.
inhibited by some DNA virus oncoprotein
such as SV40 tag -| pRb
it arrests cells in G1 phase
pRb -| E2F -o-> proteins for the path of G1 restriction check point
p53*
p53 -o-> p21 -| various cyclin-cdk complexes -> progression of cell cycle
p53 -o-> Bax -(form holes in mitochondria, and release)-> cytochrome c --> appoptosis
p53 level regulations
p16INK4a
p16 is encoded by gene INK4a
p16 -| cyclineD-CDK4/6 -| pRb --| progression of G1 restriction point
2. appoptosis promotors
3. oncogenes antaganists
PTEN
PTEN -p-| PIP3 -> Akt/PKB --> BCl-2 --> apoptosis inhibition
BCL-2 (B-cell lymphoma 2) is the founding member of the BCL-2 family of apoptosis regulator proteins encoded by the BCL2 gene.
4. transcriptional factors/repressors
WT1(wilms tumour protein)
WT1 -o-| insulin like growth factors
SMADs in Type I TGFβ signaling
TGFβ -> SMAD -- SMAD4 -o-> p15, p21
p15 -| CyclinD-CDK4-6 --> path of restriction point
p21 -| CyclinE-CDK2 --> path of restriction point
TGFβ -> SMAD --| Myc-Miz --> p15, p21
Myc-Miz -o-| p15ink4b, p21zip -| p15, p21
Care taker genes
1. DNA repair mediators
BRCA1
familial breast cancer
BRCA2
familial breast cancer
HNPCC
Hereditary non-polyposis colorectal cancer
p53*(mainly transcriptional activation here)
p53's structure
desection of transcription factor p53
form homo tetramer
p53's roles
When absence of cellular stress
p53 -o-> antioxidant proteins -| ROS(reactive oxygen species)
When presence of cellular stress
p53 -o-> p21 -| various cyclin-cdk complexes(including that one to phosphorylate pRb) -> progression of cell cycle
p21 -| PCNA(proliferating cell nucleus antigen) -> DNA replication
p21 does not impair PCNA's DNA repair function
p53 -o-> Bax -(form holes in mitochondria, and release)-> cytochrome c --> appoptosis
p53 regulators
MDM2
MDM2 --| p53 -o-> MDM2
the method MDM2 --| p53
+u using ubiquitin ligase of MDM2
binds and Inhibits p53's transcription activities
transport p53 out of nucleus
This forms a feed back regulation
P53 stabilities depends on the Phosphorylation states of p53 and MDM2
p14ARF
oncogene activations(Sustained mitogenic stimulation, e.x. abberrant growth signaling from myc and ras) -> ARF (form stable complexes within nucleus)-| MDM2 --|p53
p53 -| cell cycle progression
p53 -> apoptosis
p14ARF is a tumour suppressor protein encoded in INK4a gene using another reading frame with INK4a
ATM
DNA double strand breaks -> ATM
ATM +P-| MDM2 -| p53 --| cell cycle G1 checkpoint progression
ATM +p-> p53 --| cell cycle G1 checkpoint progression
ATM mutant causes A-T(ATAXIA TELANGIECTASIA)
It is a phosphatidyl inositol 3-kinase(PI3K) like kinase (PIKK)
so it likes PI3K?
co-factor
Rad26
All the substrates for phosphorylation
themselves (autophosphorylation)
Rad26 (co-factor for themselves)
Each component of the 9-1-1 complex (PCNA-like complex)
NBS1 or the M-R-N complex (double strand breaks)
Claspin (adaptor)
MDM2 and p53 (cell cycle arrest & apoptosis)
BRCA1 (DNA repair, transcription…)
Chk1 & Chk2 kinases ( phosphorylate CDC25, cell cycle arrest)
The specific pathway it chooses depends on specific cell cycle check points
G1 checkpoint
ATM +P -| MDM2 -| p53 --| G1 checkpoint progression
ATM +p -> p53 --| G1 checkpoint progression
S checkpoint
ATM +p -> BRCA1,CHK2 --| S checkpoint progression
ATM +p -> {NBS1][MRE11][RAD50} --| S checkpoint progression
G2 checkpoint
ATM +p -> CHK2, CHK1 +p -(trapped in cutoplasma)| CDC25C -p -| CDC2 --| G2 checkpoint progression
ATR
Cell stress -> ATR +p-> Casein kinase II +p-> p63
p53 carcinogenesis
1.deletion of p53 genes
recessive
2. mutations that stabalize p53 proteins
harmless?
3. mutations that inactivate p53 proteins
4. mutant proteins can inactivate wild type proteins in other cells
Drugs targeting p53
DNA damages
100000 leisions per cell per day
that is why evolusion and cancer become very realistic
3 reponses can have when cell detects the leisions
1. Induction of repair mechanism
2. Cell cycle arrest
3. apoptosis
Specific types of DNA damages and their causes
and their repairs
for SSB, the most common strategy is BER
General schema of the damage to repair pathway.
Singnal
DNA damages
Sensors
ATM
DNA double strand breaks(DSB) -> ATM
ATM +P-| MDM2 -| p53 --| cell cycle G1 checkpoint progression
ATM +p-> p53 --| cell cycle G1 checkpoint progression
ATM +p -> chk2 +p -> DNA replication/repair proteins
chk2 +p-> p53
ATM mutant causes A-T(ATAXIA TELANGIECTASIA)
It is a phosphatidyl inositol 3-kinase(PI3K) like kinase (PIKK)
so it likes PI3K?
co-factor
Rad26
All the substrates for phosphorylation
themselves (autophosphorylation)
Rad26 (co-factor for themselves)
Each component of the 9-1-1 complex (PCNA-like complex)
NBS1 or the M-R-N complex (double strand breaks)
Claspin (adaptor)
MDM2 and p53 (cell cycle arrest & apoptosis)
BRCA1 (DNA repair, transcription…)
Chk1 & Chk2 kinases ( phosphorylate CDC25, cell cycle arrest)
The specific pathway it chooses depends on specific cell cycle check points
G1 checkpoint
ATM +P -| MDM2 -| p53 --| G1 checkpoint progression
ATM +p -> p53 --| G1 checkpoint progression
S checkpoint
ATM +p -> BRCA1,CHK2 --| S checkpoint progression
ATM +p -> {NBS1][MRE11][RAD50} --| S checkpoint progression
G2 checkpoint
ATM +p -> CHK2, CHK1 +p -(trapped in cutoplasma)| CDC25C -p -| CDC2 --| G2 checkpoint progression
ATR
SSB -> ATR +p -> chk1 +p -> DNA replication/repair proteins.
MRN complexes
heterotrimer of MRE11, RAD50, NBS1
Binds DSB
Homologous recombination repairs
Double strand breaks -> MRN ----(BRCAs are invoved)----> homologous recombination is perfectly done!
So BRCA has the ability to recruits eukaryotic DNA synthetic machinary
adaptors
Claspin
BRCA1
BRCA1 has multiple up regulatory functions on the DNA repair mechainary
ATM & ATR +p -> BRCA1
BRCA1 -> p53 -o-> p21 --> cell cycle arrst
p53 -> RAD51-BRCA2 -> DSB HR repair
BRCA1 -> pRb --> cell cycle arrest
BRCA1 -> DNA damage repair mehainary -> DNA damage repair
BRCA1 -> Ch1 & Ch2
by recruiting them to the damage sites
BRCA1 - DNA replication proteins
it might in form of BRCA1-associated genome surveillance complex (BASC)
BRCA2
RAD51- BRCA2 -----> Homologous Recombination Repair of double strand breaks
RAD51 bind BRCA2 first, and upstream signals dephosphorylate BRCA2, this activate BRCA2 to load RAD51 on ssDNA at the DSB, this facilitates the repair, after the repair, BRCA2 is rephosphorylated to inhibit the loading of RAD51
BRCA2 has 2 regions
BRC repeats
This region can binds many RAD51 for its loading by the TR2 region.
TR2 region
This region can load RAD51 on ssDNA of DSB, however, when this region is phosphorylated, the loading is inhibited.
effectors
chk1 kinase
chks are ser,thr kinases
initiates DNA damage induced cell cycle checkpoint
chk2 kinase
Here you can see it's a “big hand” of up regulator。。。
it up regulates the entire "motif" to reach its effect.
NBS1
induce DNA repair
c-jun
induce transcriptional responses
p53
induce appoptosis
cell cycle arrest
PARP
Poly(ADP-Ribose) Polymerase (PARP)
Professional SSB repairer, it initiates BER(base exision repair)
It is independent with the BRCA1/2 HR repair system.
cell may lose one but aquire another one.
mechanisms
SSB -> PARP +NAD -> multiple proteins for BER repair
It also induces chromatin relaxation
SSBs -> PARP-1/2 -> XRCC1 -(relaxation)-> chromatin
Drugs targetting PARP can inhibits SSBs repair, this results in SSBs degeneration into DSBs and induces appoptosis
This is a strategy of chemotherapy especially for the type of cancer lacks BRCA1/2.
because those cells fail to have DSR(HR)
responses
Cell cycle arrest
Apoptosis
DNA repair
Cell cycle checkpoints system
All cell cycle check points have corresponding DNA damage sensation pathway, and the corresponding cell cycle arrest, DNA damage reapairs linked.
Common feature of tumour suppressor genes
Carcinogenesis by the inactivation of lose of function mutations or deletions
Are often inherited in a recessive pattern, show lose of heterozygocity (LOH)
mechanisms of LOH
the second functional copy is easier to lose than we thought
how to see molecular pathways
if there is odd number of "-|", it will inhibits the final target
if there are even number of "-|", it will activates the final object.
Alternative reading frame proteins
Vocabulary
oncogenesis
mehchanisms of the proto-oncogenes become activated into oncogenes.
constitutive activation
Constantly activation of a receptor or signaling molecule due to any kinds of gain of function mutations on itself.