Regulation of gene expression by mutant and oncogenic forms of the p53 tumor suppressor

Regulation of gene expression by mutant andRegulation of gene expression by mutant andoncogenic forms of the p53 tumor suppressoroncogenic forms of the p53 tumor suppressor

““The Cell Cycle”The Cell Cycle”

DNA Damage Leads to p53-DNA Damage Leads to p53-Mediated Arrest of Cell CycleMediated Arrest of Cell Cycle

p53 Regulatory Pathways

MDM2

Apoptosis (Bax)

Oncogene Expressionc-Myc and Ras

DNA DamageUV, Irradiation,

Genotoxic Compounds

p19ARF

(-)

(p21Waf1/Cip1)G1 Arrest

DNA Repair

DNA DamageUV, Irradiation,

Genotoxic Compounds

PhosphorylationAcetylationMethylation

G2 Arrest (14-3-3, Gadd45)

DNA Repair

ATM

p53

Carcinogens can Damage the p53 Carcinogens can Damage the p53 GeneGene

p53 Mutations in Human Cancersp53 Mutations in Human Cancers

p53 mutations in human cancerp53 mutations in human cancer

p53 mutations in colon cancerp53 mutations in colon cancer

p53 mutations in lung cancerp53 mutations in lung cancer

p53 mutations in liver cancerp53 mutations in liver cancer

Can we identify genes Can we identify genes regulated by mutant p53?regulated by mutant p53?

Expression of mutant p53Expression of mutant p53

_ p53-null cells 10(3) expressing p53 mutant(248) from an expression vector

Growth Rates of cells +/- mutant p53Growth Rates of cells +/- mutant p53

1441209672482400

1

2

3

4

5

6

7

8

Hours

Nu

mb

er

of c

ells

(in

Mill

ion

s)

10(3)

CM248.2

CM248.6

600 Gene Array600 Gene Array

Gene Gene Expression Expression

Array Array p53 null

Mutant p53

Genes demonstrating altered expression in the presence of R248W p53

• Induced Repressed

_ Multidrug resistance gene (MDR1) Tissue inhibitor of metalloproteinases-3 (TIMP-3)_ Connexin-32 Monocyte chemotactic protein-3 (MCP-3)_ Ribosomal protein gene L37_ Ribosomal protein gene RPP-1_ Ribosomal protein gene S1_ Transcription/Termination Factor I (TTF-1)_ PW29, Calcium-binding protein_ Heatshock 86Kd protein

Some Properties of TIMP-3Some Properties of TIMP-3

_ Inhibitor of matrix metalloproteinases_ Required for proper tissue remodeling, vascularization and

matrix turnover_ Loss of expression in a mouse tumor progression model -

potential tumor suppressor_ Reduced expression observed in metastatic human colon

cancer_ Reconstituted/elevated expression of TIMP-3 leads to

reduced metastasis and reduced angiogenesis

TIMP-3Promoter

TIMP-3 Reporter Construct

Inhibition of TIMP promoter by Inhibition of TIMP promoter by mutant p53mutant p53

Deletions in the TIMP-3 promoterDeletions in the TIMP-3 promoter

p53 TATAAP-1AP-1-2900 2xNFkB 4xAP-1 2xSp1

-2500-2000-1000 -600

-500-400-300-200-100

0

1

2

3

4

5

6

7

1 2 3 4 5

Rel

ati

ve

Fo

ld R

epre

ssi

on

wt p53

mut p53

TIMP-3 -600bp -1000bp -2000bp -2500bp

Deletions eliminate mutant but not wild type p53 repression

Deletions in the TIMP-3 promoterDeletions in the TIMP-3 promoter

p53 TATAAP-1AP-1-2900 2xNFkB 4xAP-1 2xSp1

-2500-2000-1000 -600

-500-400-300-200-100

Loss of p53-mediated repressionLoss of p53-mediated repression

0

2

4

6

8

10

12

TIMP-3 -100 -200 -300 -400 -500

Rel

ativ

e F

old

Rep

ress

ion

0

10000

20000

30000

40000

50000

60000

10(3) CMV 248Cl.2

Colo 320 SW 837

Rela

tive L

ucif

era

se A

cti

vit

y

2900bp

-100bp

Inhibition of TIMP-3 in cells expressingInhibition of TIMP-3 in cells expressing mutant p53mutant p53

Deletions in the TIMP-3 promoterDeletions in the TIMP-3 promoter

p53 TATAAP-1AP-1-2900 2xNFkB 4xAP-1 2xSp1

-2500-2000-1000 -600

-500-400-300-200-100

Two Nuclear Factors Bind to TIMP-3 in the Two Nuclear Factors Bind to TIMP-3 in the Presence of Mutant p53Presence of Mutant p53

-2900

-2800

-2800

* *

mutant p53 - + + +

- + + +

TIMP3(1)

TIMP3(4)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

CMV 248 Cl.2 SW 837 10(3)

Rel

ativ

e Lu

cife

rase

Act

ivit

y

2900bp

-100bp

-100 + (1)

-100 + (4)

A 25-bp element causes repressionA 25-bp element causes repression by p53by p53

RNAi-mediated inhibition of mutant p53 RNAi-mediated inhibition of mutant p53 expressionexpression

_ RNA-guided regulation of gene expression _ conserved in most eukaryotic organisms. _ involves dsRNA inhibiting the expression of genes with complementary

nucleotide sequences. _ thought to have evolved as a form of innate immunity in defense against

viruses, _ plays major regulatory roles in development and genome maintenance.

RNAi inhibition of p53RNAi inhibition of p53

1 2 3 4 1 2 3 4 5 6 7

1 2 3 4 5 6 1 2 3 4 5 6 7 8

SW 837 T98G

CMV 248 Cl.2 Colo 320

p53 mRNA levels in Colo 320 siRNA p53 mRNA levels in Colo 320 siRNA expressing clonesexpressing clones

1 2 3 4 5 6 7 8 9

Cl.4 Cl.5 Cl.7 Cl.12 Cl.10 Cl.11 Cl.17 Cl.18

Inhibition of p53 leads to Inhibition of p53 leads to increase in TIMP-3 expressionincrease in TIMP-3 expression

1 2 3 4 5 6 7 8 9

GAPDH

untransfected siRNA expressing

Cl. 4 Cl.5 Cl.7 Cl.12

Cancer results from numerous genetic Cancer results from numerous genetic mutationsmutations

Model for role of mutant p53 expression in Model for role of mutant p53 expression in progression to metastasisprogression to metastasis

Future GoalsFuture Goals

1.1. Characterize factors that bind to the TIMP promoterCharacterize factors that bind to the TIMP promoter2.2. Determine role of mutant p53 in regulating these factorsDetermine role of mutant p53 in regulating these factors3.3. Determine whether inhibition of mutant p53 can result inDetermine whether inhibition of mutant p53 can result in

elevated TIMP and inhibition of tumor progressionelevated TIMP and inhibition of tumor progression4. 4. Assess contribution of the pathway to cancer progressionAssess contribution of the pathway to cancer progression

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

Troy LogingTroy LogingSondra SpieglSondra SpieglTricia SolitoTricia SolitoShana ThomasShana Thomas