1. Craig Woodworth, Department of Biology, Clarkson University Modulation of Cytokine Gene Expression by EGF-R Inhibition

2. HPV-16 E6 and E5 genes stimulate expression and activation of the EGF-R, respectively Expression of the EGF-R is strongly increased in papillomas and cancers of the uterine cervix Patients with the highest EGF-R expression often have a poor prognosis Targeted disruption of the EGF-R gene inhibits formation of papillomas and carcinomas in a mouse model Background

3. PD 153035 4-[(3-Bromophenyl)amino]-6,7-dimethoxyquinazoline an extremely potent and specific inhibitor of the tyrosine kinase activity of the EGF-R (IC 50 = 25pM) Does PD153035 inhibit immortalization of keratinocytes or expression of cytokines?

4. 0 CXT3CXT2 3.01.00.30.1 EGF-R P-Tyr EGF-R P-Tyr 03.01.00.30.1 MM MM PD153035 inhibits tyrosine phosphorylation of the EGF-R in a dose-dependent manner

5. 100 50 25 0 75 31.010.300.03 tumor-4 tumor-1 tumor-3 tumor-2 Percent growth PD153035 EGF-R Conc. (  M) 0 25 50 75 100 00.10.31310 AG1295 PDGF-R Conc. (  M) EGF-R inhibitor PD153035 inhibits growth of cervical carcinoma cells in monolayer culture

6. EGF-R Inhibition blocks immortalization by HPV-16 E6/E7 0 20 40 60 80 100 120 140 160 Exp1Exp2Exp3 Colonies / dish untreated 0.1  M PD153035 1.0  M PD153035

7. 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 of cervical cells results in stratified squamous epithelia

8. Normal cervical cells CXT2 carcinoma cells Carcinoma cells form dysplastic epithelia and invade the underlying collagen

9. untreated 0.3  M 3.0  M EGF-R inhibitor PD153035 blocks invasion

10. Tumor 1Tumor 2Tumor 3 Cells invading gel 0 20 40 60 80 100 3.0  M 0.3  M untreated EGF-R inhibitor PD153035 inhibits invasion of carcinoma cells into collagen rafts

11. PD153035 induces proinflammatory cytokines

12. PD153035 alters expression of genes that regulate apoptosis

13. PD153035 alters expression of cell cycle genes

14. Inhibition of EGF-R function by PD153035 blocks immortalization of human cervical cells by HPV-16 E6/E7 genes 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 EGF-R activity stimulates multiple genes that mediate inflammation Summary

15. Questions Could EGF-R inhibitors reduce the incidence of HPV infection or dysplasia? Does inhibition of EGF-R function stimulate inflammation or innate immune responses? The keratinocyte; NF-kB activation, expression of HLA class I genes, production of cytokines, HPV-16 gene expression

16. Darci Gaiotti Evan Michael Joel Geoghegan Donald Vice Eudora Jones Peter Munson Laura Hansen Stuart Yuspa Matthias Nees Acknowledgements