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

“The Cell Cycle”

DNA Damage Leads to p53-Mediated Arrest of Cell Cycle

p53 Regulatory Pathways DNA Repair Phosphorylation Acetylation Methylation Oncogene Expression c-Myc and Ras G2 Arrest (14-3-3s, Gadd45) (-) p19ARF DNA Damage UV, Irradiation, Genotoxic Compounds p53 MDM2 ATM G1 Arrest Apoptosis (Bax) (p21Waf1/Cip1) DNA Damage UV, Irradiation, Genotoxic Compounds DNA Repair

Carcinogens can Damage the p53 Gene

p53 Mutations in Human Cancers

p53 mutations in human cancer

p53 mutations in colon cancer

p53 mutations in lung cancer

p53 mutations in liver cancer

Can we identify genes regulated by mutant p53?

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

Growth Rates of cells +/- mutant p53

600 Gene Array

Gene Expression 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-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-3 Reporter Construct Promoter

Inhibition of TIMP promoter by mutant p53 Wt p53

Deletions in the TIMP-3 promoter -2500 -2000 -1000 -600 -2900 AP-1 2xNFkB 4xAP-1 p53 AP-1 2xSp1 TATA -500 -400 -300 -200 -100

Deletions eliminate mutant but not wild type p53 repression

Deletions in the TIMP-3 promoter -2500 -2000 -1000 -600 -2900 AP-1 2xNFkB 4xAP-1 p53 AP-1 2xSp1 TATA -500 -400 -300 -200 -100

Loss of p53-mediated repression

Inhibition of TIMP-3 in cells expressing mutant p53

Deletions in the TIMP-3 promoter -2500 -2000 -1000 -600 -2900 AP-1 2xNFkB 4xAP-1 p53 AP-1 2xSp1 TATA -500 -400 -300 -200 -100

Two Nuclear Factors Bind to TIMP-3 in the Presence of Mutant p53 + + + + + + TIMP3(1) TIMP3(4) -2800 * -2900

A 25-bp element causes repression by p53

RNAi-mediated inhibition of mutant p53 expression 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 p53 CMV 248 Cl.2 Colo 320 SW 837 T98G 1 2 3 4 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

p53 mRNA levels in Colo 320 siRNA expressing clones Cl.4 Cl.5 Cl.7 Cl.12 Cl.10 Cl.11 Cl.17 Cl.18 1 2 3 4 5 6 7 8 9

Inhibition of p53 leads to increase in TIMP-3 expression untransfected siRNA expressing Cl. 4 Cl.5 Cl.7 Cl.12 1 2 3 4 5 6 7 8 9 GAPDH

Cancer results from numerous genetic mutations

Model for role of mutant p53 expression in progression to metastasis

Future Goals Characterize factors that bind to the TIMP promoter Determine role of mutant p53 in regulating these factors Determine whether inhibition of mutant p53 can result in elevated TIMP and inhibition of tumor progression 4. Assess contribution of the pathway to cancer progression

ACKNOWLEDGEMENTS Troy Loging Sondra Spiegl Tricia Solito Shana Thomas