Cancer results from mutations in genes regulating cell growth

• Two classes of genes are involved:

• 1) - positive regulators promote cancer by hyperactivity (one allele is enough)

• 2) - negative regulators, promote cancer by loss of activity (two allele must be mutated)

Oncogenes

Suppressor genes

• The first tumor suppressor- (retinoblastoma)

• The most frequent mutated tumor suppressor -

• p53 was first cloned at the weizmann institute and is

mutated in > 50% of all cancer.

• 25,000 papers published on p53 in 22 years

How p53 functions as a tumor suppressor?

Rb

p53

Evidence that p53 is a tumor suppressor (II)

• Transformation of primary rat embryo fibroblasts (REF) with a combination oftwo different oncogenes (e.g. myc+ras) gives rise to transformed foci.

• Wild type p53 (wtp53) can suppress the transformation of REF by oncogenes.

• Mutant forms of p53, found in tumors, can not suppress transformation (in fact,they enhance transformation).

• Conclusion: p53 is a tumor suppressor.

myc+ras myc+ras+ wtp53 myc+ras+ mutp53

Additional evidence that p53 is a tumor suppressor

• 1. Li-Fraumeni syndrome. An inherited syndrome of familial cancer. Affected fami ly memberstend to develop cancer at early age (below 40). The most common tumors are breast cancer and softtissue sarcoma, but other types of cancer are also frequent. In the majority of families (about 80%),affected individuals carry one mutated germline allele of p53 (in all somatic cells of their body). In thetumors of the LFS patients, the normal allele is either lost or mutated, and only the mutant allele isretained. As a result, the tumor cells do not express any wt p53.

• 2 Re-introduction of wt p53 into human cancer-derived, p53-negative cells. Parental (non-corrected) cells form tumors in nude mice. Stable cell lines can be obtained which express low levels ofwt p53. In some cases, the cells grow more slowly in culture and are non-tumorigenic in nude mice. Inother cases the cells grow normally in culture, but still are non-tumorigenic in mice. Similarly, re-introduction of wt p53 in p53-negative mouse lymphoma cells causes a reduction of tumorigenicity insyngeneic mice.

• 3. p53 knock-out mice. The mice appear healthy upon birth (with some specific exceptions- apercentage of female embryos die because of a defect in neural tube closure). Mice develop more or lessnormally, with no gross developmental defects after birth. However, within very few months, all micedevelop cancer and die. The predominant type of cancer is lymphoma of T cell origin.

Parental M1

M1-p53Val135 clones

at 32oC

Induction of cell death by active p53

Activation of temperature sensitive p53 prevents transformation

ts p53 in mutant

conformation

ts p53 in wild type conformation

Myc + Ras +no p53 temperature sensitive p53

II III IV VI

Transactivation Specific DNA binding

N C

2001001 393300

NLS1Proline rich TetramerisationProtein-protein interactions,Transcriptional repressionapotosis

Negative regulation of DNA binding

(Ko LJ & Prives C, Genes & Dev. 10:1054-1072, 1996)

Most of p53 mutations are found in the conserved regionsof the central DNA binding domain

(Cho Y et al., Science 265:346-55, 1994)

Transactivation

Growth arrest

Other activities (C terminal = TFIIH binding?)(N terminal = SH3 binding?)

Apoptosis

Bax, IGF-BP3, Fas, killer/DR5, Noxa, PIG3, p53AIP1, PIDD, Puma

p21/Waf1

Activated p53

Other genesetc, etc, etc

DNA damage, oncogene activation

Functional p53

Growth arrest, apoptosis

Loss of p53 function

Three Experimental Systems

1. Primary and Secondary Targets of p53

2. Target genes related to apoptosis

3. Comparison of p53 and p73

A chip of 10,000 genes is more than ten thousand northern blots

Filter hybridizationFilter hybridization

p21

PCNALIG1

MDM2

Representation of 7000 genes on 1 cmRepresentation of 7000 genes on 1 cm22 chip chip

DIRECT AND INDIRECT TARGETS

CHX INHIBITS PROTEIN SYNTHESIS AND PREVENTS ACTIVATION OF SECONDARY TARGETS

Effect of Cycloheximide on H1299 val135 Cells

38 24

Only ˜10% of the genes changed expression in all 3

repeats (Coller et al. 2000)

Primary p53 target genes in presence of CHX

Clustering of 259 genes upregulated without CHX at least three times > 2.5 fold

a. 9 genes (false?)

b. All 38 primary

c. 87% of primary

p53 - Upregulated Genes in H1299-Val135 system

GENE/ PROTEIN NAME

p21waf1

MIC1 - member of TGF-b family

MDM2

PCNA

GADD45

Mitochondrial Stress 70 (Mortalin2)

p57KIP2-CDK inhibitor 1C

PIG3-p53 induced gene 3

FAS/APO1

BAX-Bcl2 associated X protein

BAK1- Bcl2 antagonist/killer 1

38.0

10.0

8.3

3.9

3.0

1.5

11.0

3.8

3.7

1.5

RATIO FUNCTION

CELL CYCLE

APOPTOSIS

P53 - Upregulated Genes in H1299-Val135 system

GENE/ PROTEIN NAME

DDB2-Nucleotide Excision Repair

LIG1 - DNA ligase 1

ERCC5 - DNA Excision Repair related

TDG - G/T mismatch DNA Glycosylase

RPA1 - Replication Factor A Protein 1

MAPK14

MAP4K5 Activaes Jun N-term Kinase

MAP2K1 - MEK1

MYD88 - Myeloid differentiation

Retinoic Acid Receptor Beta

FKBP4

HOXD3 - Homeobox protein

CSPG2 - Chondroitin sulfate proteoglycan 2

6.5

2.3

1.9

1.8

1.6

3.8

1.6

1.5

5.4

4.6

3.1

1.8

2.0

RATIO FUNCTION

DNA REPAIR

KINASE

ECM

DEVELOP.

IMMUNOPHIL.

RECEPTOR

DEVELOP.

p53- DRIVEN APOPTOSIS

A different cell line (M1) that undergoes apoptosis by p53 at 32

- It plays an essential role during developmental as well as adult stages by allowing tissue remodeling, tissue renewal and maintenance of tissue homeostasis

APOPTOSIS :

- Apoptosis is a genetically controlled program of cell death, also referred to as cell suicide or Programmed Cell Death (PCD).

- It is an evolutionary conserved mechanism.

- It ultimately leads to elimination of undesired cells•either superfluous•or potentially harmful when damaged

Pro apop

Anti apop

APOPTOSIS IN LTR6 CELLS AT 32C.

Sub G1

SCATTER PLOTS OF 404 GENES THAT WERE REGULATED BY P53 IN LTR6 CELLS

A

2h M1

9h L

TR

6

12h

LT

R6

2h M1

2h L

TR

6

12h

M1

2h M1

2h M1

DC

B

A

B

C

D

E

F

12 2 6 9 12 2 2 6 9 12 12 2 6 9 12 M1 LTR6 M1 LTR6 M1 LTR6 Time (hr)

Cell typeControl M1 2hr M1 12hr LTR6 2hr

Clustering of 404 genes based on expression kinetics at 32C

Kinetics of transcriptional activation

0

20

40

60

80

100

120

Cluster D Cluster E Cluster C

Rel

ativ

e ra

tio

M1 2 6 9 12 M1 2 6 9 12 M1 2 6 9 12

ACCESSION NO.

X63717

U82987

U00115

U16811

MOUSE

ACCESSION NO.

AB021961

M83649

U82532

J04953

Z16410

AW060710

X74504

AF064071

Fas/APO-1 cell surface antigen

Bcl-2 binding component 3 (bbc3) PUMA

Bcl-6

Bak

p53

Fas antigen/TNFR6

TNFR18

Gelsolin

Btg1

EST=PIG8 (Etoposide induced)

T10 mRNA/human sentrin/SUMO-1

Apaf-1

9.8

30.5

6.8

7.4

57.8

54.5

11.3

2.9

9.0

6.2

4.3

7.2

RATIO OF EXPRESSION (12h)

HUMAN

ACCESSION NO.A1909620

X89101

A1923712

X04412

X61123

R11732

U83117

AL135220

Apoptosis related genes upregulated by p53

H1299 Val Cells

LTR 6 Cells

Northern Analysis of LTR6 Cells

Apaf-1 mRNA induced at 32 C by

ts-p53

APAF-1 promoter contains p53 target at -604

RRRCWWGYYY N{0-13 bp} RRRCWWGYYY

APAF-1 AGACATGTCT GGAGACCCTAGGA cGACAAGCCC

BAX tcACAAGTTa G AGACAAGCCT

GADD45 GAACATGTCT AAGCATGCTg

MDM2 GGtCAAGTTg GGACAcGTCC

400800bp

Others Ab x’sX x’sAOligo A A A A A A B C Dp53 + + + + mut + + +

Oligo A: AGACATGTCTGGAGACCCTAGGACGACAAGCCC Oligo B: AGACATGTCT CGACAAGCCCOligo C: AGACATGTCTGGAGAC CGACAAGCCCOligo D: AGAaATGTCTGGAGACCCTAGGACGAaAAGCCC

p53 Binding to APAF-1 Target by Gel Shift Analysis

IAPs

Smac

Bcl-2

Apoptosis

Apaf-1

Caspase-9

Cyt c

Bax

Apoptotic stimuli

/dATP

Caspase-3

Cellular targets

p53

p53

PUMA

p53 family members

Comparison of p73 and p53 induced genes

Scatter plot

Rb family(active)

Rb

E2F(inactive)

cyclin E, D, cdk 2,4,6 p21

cdk inactivation

G1 arrest

p21/Waf1

Activated p53

G2 arrest

14.3.3σGADD4 5

p21 cyclin B cdc 2

/Cip1

CDK4cycD

RB

E2F X

RB

P

E2F

Active

cy cD

CDK4p 1 6

p 1 6 Melanoma

Breast

Retina, Lung

G1 S