Tumor Suppressor Genes Dr. W. Edward Mercer Departments of Microbiology/Immunology, Biochemistry/Molecular Biology Jefferson Medical College, Kimmel Cancer.

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.