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Modulation of Cytokine Gene Expression by EGF-R Inhibition

Modulation of Cytokine Gene Expression by EGF-R Inhibition. Craig Woodworth, Department of Biology, Clarkson University. Background. HPV-16 E6 and E5 genes stimulate expression and activation of the EGF-R, respectively

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Modulation of Cytokine Gene Expression by EGF-R Inhibition

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

  2. Background • 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

  3. Does PD153035 inhibit immortalization of keratinocytes or expression of cytokines? PD 153035 4-[(3-Bromophenyl)amino]-6,7-dimethoxyquinazoline an extremely potent and specific inhibitor of the tyrosine kinase activity of the EGF-R (IC50 = 25pM)

  4. 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

  5. EGF-R inhibitor PD153035 inhibits growth of cervical carcinoma cells in monolayer culture PD153035 EGF-R AG1295 PDGF-R tumor-1 100 100 tumor-2 tumor-3 75 75 tumor-4 Percent growth 50 50 25 25 0 0 0 0.1 0.3 1 3 10 0 0.03 .01 0.3 1 3 Conc. (mM) Conc. (mM)

  6. EGF-R Inhibition blocksimmortalization by HPV-16 E6/E7 untreated 160 0.1 mM PD153035 140 1.0 mM PD153035 120 100 Colonies / dish 80 60 40 20 0 Exp1 Exp2 Exp3

  7. Organotypic culture of cervical cells results in stratified squamous epithelia 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 inhibits invasionof carcinoma cells into collagen rafts 100 untreated 80 0.3 mM 3.0 mM 60 Cells invading gel 40 20 0 Tumor 1 Tumor 2 Tumor 3

  11. PD153035 inducesproinflammatory cytokines

  12. PD153035 alters expression of genes that regulate apoptosis

  13. PD153035 alters expressionof cell cycle genes

  14. Summary • 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

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

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