tumor suppressor genes dr. w. edward mercer departments of microbiology/immunology,...
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Tumor Suppressor GenesTumor Suppressor GenesTumor Suppressor GenesTumor Suppressor GenesDr. W. Edward MercerDr. W. Edward Mercer
Departments of Microbiology/Immunology,Departments of Microbiology/Immunology,Biochemistry/Molecular BiologyBiochemistry/Molecular Biology
Jefferson Medical College, Jefferson Medical College, Kimmel Cancer Center Kimmel Cancer Center
Thomas Jefferson UniversityThomas Jefferson University
Dr. W. Edward MercerDr. W. Edward MercerDepartments of Microbiology/Immunology,Departments of Microbiology/Immunology,
Biochemistry/Molecular BiologyBiochemistry/Molecular BiologyJefferson Medical College, Jefferson Medical College,
Kimmel Cancer Center Kimmel Cancer Center
Thomas Jefferson UniversityThomas Jefferson University
http://www4.kimmelcancercenter.org/courses/mercer/documentlistREVIEWonly.php
Cancer a Malady of GenesCancer a Malady of Genes
Tumor Suppressor GenesGatekeepers and Caretakers
Tumor Suppressor GenesGatekeepers and Caretakers
“Caretakers” do not directly control cell birth or cell death but rather control the rate of mutations
of other genes, including gatekeeper genes.
“Gatekeepers” are the genes that directly control cell birth and cell death.
RB, p53, PTEN, APC, BRCA1 and BRCA2
XP-A, ATM, hMSH1, hMLH2, hPMS1,hPMS2, WRN-H
CELL CYCLE CHECKPOINTS
G1
S
G2
MCYCLIN B/ cdc2
CYCLIN A/ cdk2
CYCLIN E / cdk2
CYCLIN D / cdk4,5,6
Cyclin-dependent kinases (CDKs) play an important role in Cell Cycle transitions
RB1
p53
p53
G1/S checkpoint
G2/M checkpoint
S-phase checkpoint
Importance of the cell cycle and apoptosis in tissue homeostasis.
TISSUE HOMEOSTASIS
The balance between cell birth and cell death!
Homeostasis Lost
TUMOR
Hyperplasia
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!!!
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 tumorsSecond site primary tumors – osteosarcomasPineal 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
G1
S
G2M
phase
phase
phase
phase
Rbp
p
p
pRb
p
p
p
p
Rbp
p
p
p
Rbp
p
p
p
G0Quiescent cells
Rbp
p
Restriction point
Rbp
p
Phosphorylation of Rb allows cells to transit the restriction point and enter S phase
Hyper-phosphorylated Rb
Hypo-phosphorylated Rb
http://www3.kumc.edu/jcalvet/PowerPoint/23
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
Р
pRB
E2F1
PРP
PРP
E2F1
pRB
Cyclin D/CDK4 Cyclin Е/СDK2
PРP
PРP
PРP
PРP
PРP
DNA synthesis not allowed
DNA synthesis allowed
Cyclin complexes are not active
Cyclin complexes are active
pRB less phosphorylated pRB more phosphorylated
E2F1 bound to pocket E2F1 is free to promote cell cycle
Most Frequently Inactivated Gene in Cancer
To Date: Over 30,000 Journal Citations
THE p53 TUMOR SUPPRESSOR PROTEIN
p53p53p53
Apoptosis
Cell cycle arrest
DNA repair
Negative regulation
Senescence
p53 is involved in multiple cellular processes/pathways
p53 is involved in multiple cellular processes/pathways
TP53 functions as a tumor suppressor.
Colorectal cancers – progress in discrete steps: Loss of Heterozygosity
Definition of a tumor suppressorloss one allelemutate the other
ie: absence of normal protein Knudsen’s two hit hypothesis
Mutations in p53 identified Science 244:217www.p53.iarc.fr/index.html
TP53 functions as a tumor suppressor.
Colorectal cancers – progress in discrete steps: Loss of Heterozygosity
Definition of a tumor suppressorloss one allelemutate the other
ie: absence of normal protein Knudsen’s two hit hypothesis
Mutations in p53 identified Science 244:217www.p53.iarc.fr/index.html
p53
chromosome 17
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”
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 S-phase 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!!
Good Result!!
Progression to Cancer
- 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
p21 WAF1/CIP
WAF1 chromosome localization
El-Deiry et al. Cell (1993) 75:817.
Chromosome 6p
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
P27 Kip1
P57 Kip2
Up-regulated in senescence and differentiation
Regulated by growth inhibitory cytokines and by contact inhibition
p21 in stress response and apoptosis
p21 counteracts the apoptotic process
p21 protects colorectal carcinoma and melanoma cells from p53-induced apoptosis !!!
DNA damage
TNF-α
Heat shock
Some cascades
Kinase SAPK (JNK)
Kinase p38
Classical apoptotic response
ASK1 (MEKK5)
Caspase 3
Inhibit activation of pro-enzyme
Can degrade p21 itself
p21WAF1
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 proteinsis 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
Sherr, C. (2001) Nature Reviews Molecular Cell Biology 2:731-737
Actually two genes hereIndirect regulation
of Rb and p53
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
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
PRIME EXAMPLES:
In HNSCC p16 mutations arise very early -- in progression of benign hyperplasia to carcinoma in situ
The ARF Tumor SuppressorARF inactivation reduces p53-dependent apoptosis
ARFNucleolusMdm2
Nucleus
Cytoplasm
p53
ARF, Mdm2, p53 low levels
ARF induction: p53 induced, activated, nucleoplasmic, and stable
Novel mechanism of preventing p53 turnover ARF sequesters Mdm2 in the Nucleolus
Mdm2 targets p53 for degradation
http://www.mskcc.org/mskcc/shared/images/brain_cancer_illus.jpg
ARF connects RB and p53 pathways
Table 7.1 part 1 of 2 The Biology of Cancer (© Garland Science 2007)
Translational ResearchBasic 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.