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

1. Inhibition of Epidermal Growth Factor Receptor Function in Cervical Carcinoma Cells Alters Expression of Genes Involved in Invasion, Apoptosis, Inflammation, and Cell Cycle Regulation 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

2. The Epidermal Growth Factor Receptor (EGF-R) is a Membrane Tyrosine Kinase EGF-R ErbB-2 ErbB-3 ErbB-4 b-cellulin epiregulin amphiregulin EGF TGF-a HB-EGF tyrosine kinase domain links to signaling pathways

3. Binding of EGF Induces Dimerization, Tyrosine Phosphorylation and Signaling EGF EGF-R ErbB-2 ErbB-3 ErbB-4 b-cellulin epiregulin amphiregulin TGF-a HB-EGF tyrosine phosphorylation proliferation  motility  angiogenesis  differentiation  apoptosis (cell death)  P P

4. The EGF-R as a Cofactorfor HPV-Associated Cancer • 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

5. Does Inhibition of EGF-R Function Alter Growth, Differentiation, or Gene Expression of Cervical Carcinoma Cells? PD 153035 4-[(3-Bromophenyl)amino]-6,7-imethoxyquinazoline a potent and specific inhibitor of the tyrosine kinase activity of the EGF-R (IC50 = 25pM)

6. PD153035 Inhibits Tyrosine Phosphorylationof the EGF-R in a Dose-Dependent Manner CXT2 CXT3 mM 0 0.1 0.3 1.0 3.0 0 0.1 0.3 1.0 3.0 mM P-Tyr P-Tyr EGF-R EGF-R

7. Organotypic Culture to Promote Cell-Cell and Cell-Matrix Interactions Construct rafts composed of collagen and fibroblasts Allow fibroblasts to contract raft for 2 days Add normal human cervical cells or cervical cancer cells to the surface of raft Raise rafts on steel mesh grids and maintain at the air-liquid interface After 10 days scrape epithelia from raft and purify RNA, or fix the raft for histology

8. Carcinoma Cells Form Dysplastic Epitheliaand Invade the Underlying Collagen Normal cervical cells CXT2 carcinoma cells

9. EGF-R Inhibitor PD153035 Blocks Invasion untreated 0.3 mM 3.0 mM

10. EGF-R Inhibitor PD153035 Blocks Invasionin a Dose-Dependent Manner 100 untreated 0.3 mM 80 3.0 mM 60 Cells invading gel 40 20 0 Tumor 1 Tumor 2 Tumor 3

11. Identification of Genes DifferentiallyExpressed After PD153035 Treatment microarray protocol microarray results

12. Inhibition of the EGF-R Alters Expressionof Several Clusters of Genes decreased increased attachment and motility inflammation Immune response cell cycle

13. PD153035 Alters Expression of Genes that Regulate Attachment and Motility

14. PD153035 Increases Expression of RNAs for Cytokines and Chemokines

15. Verification of Selected Microarray Results Using Real Time RT-PCR No treatment 0.1 mM PD153035 5 0.3 mM PD153035 4 3 Fold increase 2 1 0 IL6 IL7 CCL3 CCL3 RELA CXCR6 CCL11 DAPK1 IL1 alpha CX3CL1 NFKBIA IL1 beta IKB alpha

16. Summary • 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

17. Acknowledgements Matthias Nees University of Heidelberg Evan Michael University of Michigan Laura Smith Clarkson University Sarah Allen Mandy Heitzke April Krumnow