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