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-R ErbB-2 ErbB-3 ErbB-4 EGF TGF-  HB-EGF  -cellulin epiregulin amphiregulin 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 tyrosine phosphorylation EGF-R ErbB-2 ErbB-3 ErbB-4 PP TGF-  HB-EGF  -cellulin epiregulin amphiregulin 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 Cofactor for HPV-Associated Cancer

4-[(3-Bromophenyl)amino]-6,7-imethoxyquinazoline a potent and specific inhibitor of the tyrosine kinase activity of the EGF-R (IC 50 = 25pM) Does Inhibition of EGF-R Function Alter Growth, Differentiation, or Gene Expression of Cervical Carcinoma Cells? PD

0 CXT3CXT EGF-R P-Tyr EGF-R P-Tyr MM MM PD Inhibits Tyrosine Phosphorylation of 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 Epithelia and Invade the Underlying Collagen

untreated 0.3  M 3.0  M EGF-R Inhibitor PD Blocks Invasion

Tumor 1Tumor 2Tumor 3 Cells invading gel  M 0.3  M untreated EGF-R Inhibitor PD Blocks Invasion in a Dose-Dependent Manner

microarray protocolmicroarray results Identification of Genes Differentially Expressed After PD Treatment

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

symbolgene identification and description ITGA8integrin alpha 8, cell-cell interactions ITGAXintegrin alpha X, similar to alpha integrins CTNND2catenin, cadherin associated protein ITGB1integrin beta 1, fibronectin receptor SELEselectin E endothelial adhesion molecule DDR2discoidin, required for cell adhesion ACTN1alpha 1 actinin MMP1matrix metalloproteinase 1 (collagenase) PD Alters Expression of Genes that Regulate Attachment and Motility

symbolgene identification and description XCL1chemokine ligand 1, attracts leukocytes CX3CL1fractalkine, chemotactic for T cells CCL3 MIP-1  inflammatory and chemotactic CXCR6chemokine receptor 6, G protein receptor IL1R2IL1 receptor type II, decoy receptor IL-6interleukin 6, proinflammatory cytokine IL-7Interleukin 7, hematopoietic growth factor IRF5interferon regulatory factor 5 TNFSF4member of tumor necrosis factor family PD Increases Expression of RNAs for Cytokines and Chemokines

Verification of Selected Microarray Results Using Real Time RT-PCR CXCR6 CX3CL1 CCL3 IL6 IL7 IKB alpha DAPK1 CCL3 RELA IL1 beta NFKBIA CCL11 IL1 alpha Fold increase No treatment 0.1  M PD  M PD153035

Cervical cancer cells produce dysplastic epithelia and invade the underlying collagen in organotypic culture Inhibition of EGF-R tyrosine kinase activity by PD 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 MichaelUniversity of Michigan Laura SmithClarkson University Sarah Allen Mandy Heitzke April Krumnow Acknowledgements