Craig Woodworth, Department of Biology,Clarkson University

Modulation of Cytokine GeneExpression by EGF-R Inhibition

• 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

PD 1530354-[(3-Bromophenyl)amino]-6,7-dimethoxyquinazoline

an extremely potent and specific inhibitor of the tyrosine kinaseactivity of the EGF-R (IC50 = 25pM)

Does PD153035 inhibit immortalization of keratinocytes or expression of cytokines?

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

100

50

25

0

75

31.01 0.30 0.03

tumor-4

tumor-1

tumor-3

tumor-2

Per

cen

t g

row

th

PD153035EGF-R

Conc. (M)

0

25

50

75

100

0 0.1 0.3 1 3 10

AG1295PDGF-R

Conc. (M)

EGF-R inhibitor PD153035 inhibits growth of cervical carcinoma cells in monolayer culture

EGF-R Inhibition blocksimmortalization by HPV-16 E6/E7

0

20

40

60

80

100

120

140

160

Exp1 Exp2 Exp3

Co

lon

ies

/ d

ish

untreated0.1 M PD1530351.0 M PD153035

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

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

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3.0 M

0.3 Muntreated

EGF-R inhibitor PD153035 inhibits invasionof carcinoma cells into collagen rafts

PD153035 inducesproinflammatory cytokines

PD153035 alters expression of genes that regulate apoptosis

PD153035 alters expressionof cell cycle genes

• 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

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

Darci GaiottiEvan MichaelJoel GeogheganDonald ViceEudora Jones

Peter MunsonLaura HansenStuart YuspaMatthias Nees

Acknowledgements