Transcript Tumor Suppressor Genes

Tumor Suppressor Genes
Dr. W. Edward Mercer
Departments of Microbiology/Immunology,
Biochemistry/Molecular Biology
Jefferson Medical College,
Kimmel Cancer Center
Thomas Jefferson University
http://www4.kimmelcancercenter.org/courses/mercer/documentlistREVIEWonly.php
Cancer a Malady of Genes
Tumor Suppressor Genes
Gatekeepers and Caretakers
“Gatekeepers” are the genes that directly
control cell birth and cell death.
RB, p53, PTEN, APC, BRCA1 and BRCA2
“Caretakers” do not directly control cell birth or
cell death but rather control the rate of mutations
of other genes, including gatekeeper genes.
XP-A, ATM, hMSH1, hMLH2, hPMS1,hPMS2, WRN-H
CELL CYCLE CHECKPOINTS
CYCLIN A/ cdk2
S-phase checkpoint
S
G1/S checkpoint
CYCLIN E / cdk2
p53
G1
G2
G2/M checkpoint
CYCLIN D / cdk4,5,6
RB1
p53
M
CYCLIN B/ cdc2
Cyclin-dependent kinases (CDKs) play an important role in Cell Cycle transitions
Importance of the cell cycle and
apoptosis in tissue homeostasis.
TISSUE HOMEOSTASIS
The balance between cell birth and cell death!
Homeostasis Lost
Hyperplasia
TUMOR
Tumor growth kinetics is different
for gatekeepers and caretakers
When a gatekeeper gene
is altered through mutation, the
rate of cell birth exceeds that of cell death,
and a tumor is initiated.
When a caretaker gene is altered, the
cell accumulates mutations at a high rate
and the process of tumorigenesis is accelerated.
A raise in mutation rate may make tumorigenesis faster.
The emergence and survival of a tumor is most likely a form
“DARWINIAN MICROEVOLUTION”
Survival of the fittest!!!
Most Caretakers genes are inhibitors of
the cell cycle
First inhibitor of cell cycle cloned as a classical TSG
retinoblastoma gene (RB1)
Eye tumors
Second site primary tumors – osteosarcomas
Pineal glad tumors
Knudson’s 2-hit mutation model for retinoblastoma.
Figure 7.7 The Biology of Cancer (© Garland Science 2007)
Retinoblastoma Appearance
Retinoblastoma may be unifocal or multifocal.
About 60% of patients have unilateral RB
with a mean age of diagnosis of 24 months;
About 40% have bilateral RB
with a mean age of diagnosis of 15 months.
Secondary tumors – osteosarcomas and rhabdomyosarcomas
4.4% have secondary tumor in 10 years,
18.3% in 20 years;
26.1 % in 30 years
70% of patients have point mutations in RB1 gene;
10% -- partial deletion of RB1 gene;
20% -- causes unknown; appearance is the same
Structure of the RB1 gene
RB1 gene covers 180-kb in 13q14 region
The 27 exons of RB1
range in size from 31 to 1,889 base pairs.
The translated product of RB1 (p105-RB1)
consists of 928 amino acids.
About 80-85% of mutations result in a
premature termination codon.
Mutations are scattered throughout
exon 1 to exon 25 of the RB1 gene and its promoter region.
About 80% of de novo germline mutations are paternal in origin.
The reason for this is unknown
Structure of RB1 protein
DNA tumor virus oncoproteins: SV40 large T, HPV E7
and Ad E1A also bind the A/B pocket and block RB
function in cell cycle regulation
The RB gene Family TSG,s
Human pRB/p105
Human p107
Human pRb2/p130
P.P. Claudio et al., Genome Biology, 2002
All three members of pRB family have similar domain structure
and genomic organization
Cell-cycle dependent phosphorylation of Rb
Hyper-phosphorylated Rb
p
p
Rb
p
p
p
p
Restriction point
p
Rb
p
phase
p
G1
phase
G2
phase
M
G0
Hypo-phosphorylated Rb
Rb
S
Phosphorylation of Rb
allows cells to transit the
restriction point and
enter S phase
Quiescent cells
p
p
p
Rb
http://www3.kumc.edu/jcalvet/PowerPoint/23
phase
p
p
p
Rb
p
p
p
p
Rb
p
Molecular Mechanisms of RB1
How does RB1 control cell cycle progression?
RB1 protein sequesters a transcription factor
called E2F.
The E2F family of transcription factors are
required for transcriptional activation of
genes needed for DNA metabolism/synthesis.
Genes such as thymidine synthatase, dihydrofolate
reductase, DNA polymerase alpha, and others
DNA synthesis
not allowed
DNA synthesis
allowed
E2F1
P
P
Р
E2F1
Р
P
P
Р
P
P
pRB
P
P
Р
pRB
P
P
Р
Р
PР
P
PР
P
Cyclin D/CDK4
Cyclin complexes
are not active
pRB less phosphorylated
E2F1 bound to pocket
Cyclin Е/СDK2
Cyclin complexes
are active
pRB more phosphorylated
E2F1 is free to promote cell cycle
THE p53 TUMOR SUPPRESSOR PROTEIN
Most Frequently Inactivated Gene in Cancer
To Date: Over 30,000 Journal Citations
p53 is involved in multiple cellular
processes/pathways
Apoptosis
Cell cycle arrest
p53
p53
DNA repair
Negative regulation
Senescence
TP53 functions as a tumor suppressor.
Colorectal cancers – progress in discrete
steps: Loss of Heterozygosity
Definition of a tumor suppressor
loss one allele
mutate the other
ie: absence of normal protein
Knudsen’s two hit hypothesis
Mutations in p53 identified Science 244:217
www.p53.iarc.fr/index.html
chromosome 17
p53
Li-Fraumeni Syndrome
- an inherited predisposition to cancer
- various kinds of cancers occur:
osteosarcomas,
soft tissue sarcomas,
breast cancer
- multiple tumors in the same individual
- 70% of families inherit a mutation in p53
Science 250:1233; Nature 348:747 (1990)
Anatomy of Human p53 Gene
Functional domains of p53 protein
p53 protein binds p53 as a tetramer
p53 is a DNA damage response protein
”Guardian of the Genome”
Ultraviolet light
Ionizing radiation
Drugs (bleomycin, 5-FU, adriamycin)
Hypoxia
Oncogenes (eg. Ras, Myc, SV40-T)
Transfection and other forms of cellular stress; including
“growth factor deprivation” induces a p53 response often
resultin in apoptosis.
Post translational modifications and
increased p53 protein stability play a role.
Early events in p53 research
This is a benchmark paper because it was the first p53 inducible
gene identified and the first CDK inhibitor to be identified.
Biological Activities of p53 Protein
1. Wild type p53 is a transcription factor.
- positive and negative effects.
- most closely correlated with tumor suppression.
2. Wild type p53 is important role in cell cycle control.
-ectopic expression of wtp53 during the Go to Sphase transition blocks cells in G1.
-ectopic expression of wtp53 during the S-phase
blocks cells in G2-phase.
3. Wild type p53 can induce apoptosis.
-transcriptional activation of pro-apoptotic genes
Figure 9.4 The Biology of Cancer (© Garland Science 2007)
p53 hotspot mutations
Mutant p53 Protein in Colorectal Tumors
p53 mutations in colon cancer
Good Result!!
Progression to Cancer
Good Result!!
p21 WAF1/CIP
- a universal inhibitor of CDKs originally
- isolated in one of three ways: a p53 inducible transcript
(WAF1); interaction with CDK2 (CIP1); mRNA over
expressed in senescent cells (SD1)
- binds to CDK1, CDK2,3,4 and 6
- normal fibroblasts found in a quaternary complex,
throughout the cell cycle, with CDK and PCNA
(proliferating cell nuclear antigen)
- complex has activity in proliferating cells but is
inhibited by the addition of more p21 to the complex
WAF1 chromosome localization
Chromosome 6p
El-Deiry et al. Cell (1993) 75:817.
p21WAF1: a p53-inducible gene
p53 binding sequences in promoter
Cip-1/Kip family of CDK inhibitors
Inhibits Cdk2–cyclin E and cyclin A -- cdk2 complexes
Block G1/S transition
P21 Cip1=Waf1=Sdi1
Up-regulated in senescence
and differentiation
P27 Kip1
Regulated by growth inhibitory
cytokines and by contact inhibition
P57 Kip2
p21 in stress response and apoptosis
p21 counteracts the apoptotic process
p21 protects colorectal carcinoma and melanoma cells
from p53-induced apoptosis !!!
Heat shock
DNA damage
TNF-α
p21WAF1
Some cascades
ASK1 (MEKK5)
Kinase SAPK
(JNK)
Kinase p38
Classical apoptotic response
Inhibit activation
of pro-enzyme
Caspase 3
Can degrade p21 itself
INK4-family of CDK inhibitors
p16Ink4a
p15Ink4b
p18Ink4c
p19Ink4d
compete with D-type cyclins
for binding to the CDK subunit
The inhibitory action of the Ink4 proteins
is largely dependent on the presence of pRb in the cell.
When RB1 is damaged cyclin E expression is already
increased and inhibition of cyclin D-CDK4 complexes
does not inhibit S-phase entry
p16 Ink4a
Most studied gene in the family
because of three reasons:
-- Its mutations are common
in hereditary and sporadic melanoma samples
-- Only this gene out of whole family
fulfills all criteria for being tumor suppressor
-- Very special genomic structure of this gene
p16 INK4a gene structure
Actually two genes here
Indirect regulation
of Rb and p53
Sherr, C. (2001) Nature Reviews Molecular Cell Biology 2:731-737
INK4a in sporadic cancer
Mutations of INK4a-encoding gene in sporadic cancers are rare.
Major mechanism of inactivation of this gene in primary tumors
is a small (<200 kb) deletions of both copies of INK4a
PRIME EXAMPLES:
Pancreatic cancer:
30% deletions, 32% deletion + mutation, 13% deletion+methylation
Head and neck squamous cell carcinoma (HNSCC):
27% deletions, 11% deletion + mutation, 30% deletion + methylation
In HNSCC p16 mutations arise very early -in progression of benign hyperplasia to
carcinoma in situ
The ARF Tumor Suppressor
ARF inactivation reduces p53-dependent apoptosis
ARF, Mdm2, p53 low levels
Nucleolus
ARF
p53
Mdm2
Nucleus
Cytoplasm
Mdm2 targets p53
for degradation
ARF induction: p53 induced, activated, nucleoplasmic, and stable
Novel mechanism of preventing p53 turnover ARF
sequesters Mdm2 in the Nucleolus
ARF connects RB and p53 pathways
http://www.mskcc.org/mskcc/
shared/images/brain_cancer_illus.jpg
Table 7.1 part 1 of 2 The Biology of Cancer (© Garland Science 2007)
Translational Research
Basic Biomedical Science
Molecular Genetic and Biochemical Pathways
Insight into the molecular processes involving
cell cycle control and apoptotic pathways is
critically-important for the design of novel
therapeutic agents for cancer intervention.