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Molecular Basis of Carcinogenesis III, Rb is not phosphorylated, binds E2F…
Molecular Basis of Carcinogenesis III
Cell cycle regulation
Progression through cell cycle is regulated by activity of specific CDK-cyclin complexes
Activity of CDKs is dependent upon binding to specific cyclins
CDK activity is also regulated by phosphorylation
Kinases
Phosphatases
CDK expression levels remain constant throughout cell cycle
Cyclin expression levels fluctuate
Transcription + degradation
Specific complexes are active in different points in the cell cycle
Negative regulators of cell cycle are tumour suppressors
INK family
p16\(^{INK4A}\)
p15\(^{INK4B}\)
p18\(^{INK4C}\)
p19\(^{INK4D}\)
Block formation of cyclin D-CDK4/6
Inhibit those already formed
Cip/Kip family
p21\(^{cip1}\)
p27\(^{kip1}\)
p57\(^{kip2}\)
Can inhibit all CDKs
Oncogenes
Genes that drive proliferation + increase cell survival
Mutant versions in cancer cells have gained function/become hyperactive
One mutant allele usually sufficient to affect cell behaviour
Proto-oncogene
Normal function
Mutates to oncogene
Types of mutation
Viral insertion
Mutations
Chromosomal rearrangements
Translocation
Rearrangement of chromosomal material involving 2 or more chromosomes
Increased protein expression + activity
Breakpoint causes the structure of the gene to be altered
Fusion to actively transcribed gene greatly over-produces onco-protein or fusion protein is hyper-active
Gene structure is not altered but expression is
Rearrangement can either place the gene adjacent to a promoter/enhancer or further from a repressor
Burkitt Lymphoma
Originates in B cells
Cytogenetic + molecular features
Translocations juxtapose the c-myc to immunoglobulin genes (highly expressed in B cells) leading to inappropriate overexpression of oncogene
Gene amplification
Loss of degradation signals
Changes in miRNA mediated pathways
\(\geq\)100 oncogenes
Cause stimulatory proteins to be overactive (onco-proteins)
Gain of function, dominant mutations
Cells proliferate excessively (proliferative advantage)
Cancer
Amplification of oncoenes
Multiplication of the no. copies of a gene
Overexpression of its protein
Mechanism
Involve poorly-regulated DNA replication and recombination
Gene amplification can be measured using FISH (Fluorescence in situ Hybridisation)
c-MYC frequently amplified in cancer
Suggestion that exposure to ionising radiation can drive c-MYC amplification
Regulation of G1-S progression
Controlled by E2F-1 transcription factor
E2F is negatively regulated by retinoblastoma (Rb) tumour suppressor protein
Rb blocks E2F in early G1 phase
At restriction point, Rb is hyperphosphorylated + cannot bind to E2F-1
Cell is committed to to progress through G1
E2F target genes
Cdc6
C-MYC
Cyclin
DNA Pol
Cdc45
As cells progress through G1, Rb is progressively phosphorylated by 2 complexes
CyclinD-CDK4/6
CyclinE-CDK2
P27\(^{kip1}\) inhibits CyclinE-CDK2
At restriction point, CyclinE-CDK2 phosphorylates p27\(^{kip1}\) + targets it for degradation
Degraded by proteasome
Ubiquitinated
Aids in hyperphosphorylation of Rb
Checkpoint
In presence of DNA damage, cell cycle is arrested at G1-S checkpoint
Essential that cells do not enter S-phase with DNA damage
Regulated by p53 and p21 tumour suppressor proteins
Double strand break
ATM activated
ATM phosphorylates CHK2 (T68)
CHK2 phosphorylates p53 (S20)
CHK2 inactivates CDC25A phosphatase
CDC25A active form phosphorylates Rb
ATM phosphorylates p53 (S15)
p53 transcribes p21 (CDK2 inhibitor)
p21 inhibits CDK1-cyclin E complex + PCNA
ATM phosphorylates MDM2 (S395)
MDM2 unphosphorylated blocks p53
p21 deregulation in cancer
Functions as both a tumour suppressor + an oncogene
Tumour suppressor
Colon carcinomas
Strong assoc. between reduced/absent p21 + development of metastases and death
Oncogene
Breast and liver cancer
Cytoplasmic expression of p21 correlates with aggressive tumours + poor prognosis
p21 is usually present in the nucleus
Oncogenic functions are assoc. with cytoplasmic localisation
Inhibits apoptosis
Could explain paradoxical oncogenic activities
Mutations in Rb
First tumour suppressor gene
Identified in retinoblastoma
Rare childhood cancer
<5 years old
Also found in other tumour types
Sarcoma
Small-cell lung carcinoma
Loss of E2F regulation
Uncontrolled proliferation
Two-hit hypothesis
2 mutations needed for tumourigenesis
Hereditary retinoblastoma
2nd allele is lost by
somatic
mutation in one of the retinoblasts
1st defective allele is
inherited
via a germ cell present in all somatic cells
Non-hereditary retinoblastoma
Both
normal Rb alleles are lost by
somatic mutation
in one of the retinoblasts
HPV
95% cervical cancers assoc. with HPV
Produced E7 viral oncoprotein
Binds to Rb, blocking Rb-E2F1 interaction
Up-regulation of of synthesis of S phase proteins (required for viral DNA replication)
Increases host cell proliferation
Targets Rb for degradation
Mediated by proteasome
p53 tumour suppressor
Mutated in >50% tumours
Remaining are likely to have mutations in other proteins/pathways that reduce p53 function
Transcription factor
Target genes
Cell death
Cell cycle
DNA repair pathways
Tetrameric form of p53 is active as a TF
Regulated by multiple post translational modifications
Regulation
Constantly produced in normal cells
Binds to negative regulator (MDM2)
E3 ligase enzyme that attaches ubiquitin molecules to p53
Ubquitylated proteins are degraded by proteasome
Keeps p53 levels low in normal cells
Also a p53 target gene
Negative feedback loop
Allows p53 signalling to end rapidly once DNA damage is repaired
Activation
DNA damage activates signalling cascades that converge on p53
ATM
ATR
CHK1
CHK2
Phosphorylation of p53 and MDM2 block the p53-MDM2 interaction
p53 levels increase and it forms transcriptionally active tetramer
Binds to response elements in target gene promoters
Cell cycle arrest
Apoptosis
DNA repair
Inhibition of angiogenesis
HPV
E6 oncoprotein from HPV binds p53 and targets it for proteasome-mediated degradation
Stops p53 effect on transcription
Transcriptional activation by p53
Increase in p21
Inhibits CDKs needed for proliferation
Prevents hyperphosphorylation of Rb
Increase in GADD45
Inhibits PCNA + CDK2
Promotes DNA repair by repair proteins
Increase in Bax
Intrinsic apoptosis
CytoC release
Increase in thrombospondin
Inhibits angiogenesis
Transcriptional repression by p53
Mechanism unclear but likely due to indirect repression (due to genes activated by p53)
Could also be due to recruitment of Sin3a (HDAC that deacetylates histones resulting in tightly-packed chromatin (heterochromatin)
Reduced Bcl 2
Bcl2 normally inhibits apoptosis
Apoptosis initiated
Reduced Jun + fos mRNA
Reduced Jun + fos protein
Reduced AP-1
Reduced proliferation
Dominant-negative effect
One mutated p53 can cause tumours
To function as a TF, p53 must form a tetramer
Presence of one mutant p53 in the tetramer will negatively affect function
p53 mutations are often in DNA-binding domain (DBD)
c-MYC
Myc-proteins
Basic helix-loop-helix (bHLH) family of nuclear transcription factors
Regulate gene expression
Form homodimers with themselves
Form heterodimers with other family members
myc-max bind to E boxes (CACGTG) in promoters of target genes
Drive expression of a large no. genes that favour proliferation
Myc-max
Cyclin D2
CDK4
E2FA, E2F2, E2F3
Cul1
Degrades p27
Myc-miz1
p15
p21
Function
In Burkitt Lymphoma
c-Myc under regulatory control of IgH
Resulting in over-expression of oncogene and protein
Drives proliferation
Increased cyclin D2
G\(_1\)-CDK activation (cyclin D2-CDK4/6)
Increased p27 degradation
G\(_1\)/S-CDK activation (cyclin E-CDK2)
Induces expression of hTERT
Telomeres
Repetitive non-coding DNA (TTAGGG\(_n\))
Shortened each time DNA replicates
Eventually senescence
'The end replication problem'
Protect against loss of important coding regions
hTERT encodes telomerase
Extends telomeres
Telomerase expression very low/absent in adult somatic cells
Most cancer cells express telomerase
Immortalised
c-MYC increases expression of hTERT
N-MYC amplification
Occurs in ~40% neuroblastomas
Sometimes >100 x amplification (high)
Indicative of response to routine therapy
Patients with N-MYC amplification
Inferior overall survival compared to those without
Molecularly targeted therapy
Chromosome translocation produces BCR-ABL fusion protein
Seen in 20% patients with CML
ABL is tyrosine kinase constitutively active in fusion protein
Drives proliferation
Philadelphia chromosome
Imatinib inhibits BCR-ABL fusion protein
ABL not active
Rb is not phosphorylated, binds E2F
Induces expression
Induces repression