Craig D. Woodworth, Evan Michael, Laura Smith, and Matthias Nees. Department of Biology, Clarkson

University, Potsdam, NY, USA, and Department of Pediatric Oncology, Hematology & Immunology, University of Heidelberg, Heidelberg, Germany

Inhibition of Epidermal Growth Factor Receptor Function in Cervical Carcinoma

Cells Alters Expression of Genes Involved in Invasion, Apoptosis, Inflammation, and Cell

Cycle Regulation

EGF-RErbB-2ErbB-3ErbB-4

EGFTGF-HB-EGF

-cellulinepiregulinamphiregulin

tyrosine kinase domain links to signaling pathways

The Epidermal Growth Factor Receptor (EGF-R) is a Membrane Tyrosine Kinase

proliferation motility angiogenesis

differentiation apoptosis (cell death)

EGF

tyrosinephosphorylation

EGF-RErbB-2ErbB-3ErbB-4

P P

TGF-HB-EGF

-cellulinepiregulinamphiregulin

Binding of EGF Induces Dimerization, Tyrosine Phosphorylation and Signaling

• HPV-16 E6 and E5 genes stimulate expression and activation of the epidermal growth factor receptor (EGF-R), respectively

• Expression of the EGF-R is increased in papillomas and cancers of the uterine cervix, and patients with the highest EGF-R expression often have a poor prognosis

• Targeted disruption of the EGF-R gene in a mouse model inhibits formation of papillomas and carcinomas from HPV-immortalized keratinocytes

The EGF-R as a Cofactorfor HPV-Associated Cancer

4-[(3-Bromophenyl)amino]-6,7-imethoxyquinazoline a potent and specific inhibitor of the tyrosine kinase

activity of the EGF-R (IC50 = 25pM)

Does Inhibition of EGF-R Function Alter Growth, Differentiation, or Gene Expression

of Cervical Carcinoma Cells?

PD 153035

0

CXT3CXT2

3.01.00.30.1

EGF-R

P-Tyr

EGF-R

P-Tyr

0 3.01.00.30.1M M

PD153035 Inhibits Tyrosine Phosphorylationof the EGF-R in a Dose-Dependent Manner

Construct rafts composed of collagen and fibroblasts

Allow fibroblasts to contract raft for 2 days

Raise rafts on steel mesh grids and maintain at the air-liquid interface

Add normal human cervical cells or cervical cancer cells to the surface of raft

After 10 days scrape epithelia from raft and purify RNA, or fix the raft for histology

Organotypic Culture to Promote Cell-Cell and Cell-Matrix Interactions

Normal cervical cells

CXT2 carcinoma cells

Carcinoma Cells Form Dysplastic Epitheliaand Invade the Underlying Collagen

untreated

0.3 M

3.0 M

EGF-R Inhibitor PD153035 Blocks Invasion

Tumor 1 Tumor 2 Tumor 3

Cel

ls i

nva

din

g g

el

0

20

40

60

80

100

3.0 M

0.3 Muntreated

EGF-R Inhibitor PD153035 Blocks Invasionin a Dose-Dependent Manner

microarray protocol microarray results

Identification of Genes DifferentiallyExpressed After PD153035 Treatment

Inhibition of the EGF-R Alters Expressionof Several Clusters of Genes

Immune response

attachment and motility

cell cycle

inflammation

increased

decreased

symbol gene identification and description

ITGA8 integrin alpha 8, cell-cell interactions

ITGAX integrin alpha X, similar to alpha integrins

CTNND2 catenin, cadherin associated protein

ITGB1 integrin beta 1, fibronectin receptor

SELE selectin E endothelial adhesion molecule

DDR2 discoidin, required for cell adhesionACTN1 alpha 1 actininMMP1 matrix metalloproteinase 1 (collagenase)

PD153035 Alters Expression of Genes that Regulate Attachment and Motility

symbol gene identification and description

XCL1 chemokine ligand 1, attracts leukocytes

CX3CL1 fractalkine, chemotactic for T cells

CCL3 MIP-1inflammatory and chemotactic

CXCR6 chemokine receptor 6, G protein receptor

IL1R2 IL1 receptor type II, decoy receptor

IL-6 interleukin 6, proinflammatory cytokine

IL-7 Interleukin 7, hematopoietic growth factor

IRF5 interferon regulatory factor 5

TNFSF4 member of tumor necrosis factor family

PD153035 Increases Expression of RNAs for Cytokines and Chemokines

Verification of Selected Microarray Results Using Real Time RT-PCR

0

1

2

3

4

5

CXCR6

CX3CL1

CCL3IL

6IL

7

IKB a

lpha

DAPK1

CCL3

RELA

IL1

beta

NFKBIA

CCL11

IL1

alpha

Fo

ld in

crea

se

No treatment0.1 M PD1530350.3 M PD153035

• Cervical cancer cells produce dysplastic epithelia and invade the underlying collagen in organotypic culture

• Inhibition of EGF-R tyrosine kinase activity by PD153035 decreases invasion in a dose-dependent manner

• Inhibition of the EGF-R up regulates expression of genes that mediate attachment and inflammation, and down regulates many genes that stimulate growth

Summary

Matthias Nees University of Heidelberg

Evan Michael University of Michigan

Laura Smith Clarkson University Sarah Allen Mandy Heitzke April Krumnow

Acknowledgements