Inhibition of Epidermal Growth Factor Receptor Function in Cervical Carcinoma Cells Alters Expression of Genes Involved in Invasion, Apoptosis, Inflammation, and Cell Cycle Regulation

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Inhibition of Epidermal Growth Factor Receptor
Function in Cervical Carcinoma Cells Alters
Expression of Genes Involved in Invasion,
Apoptosis, Inflammation, and Cell Cycle Regulation
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
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The Epidermal Growth Factor Receptor (EGF-R) is a
Membrane Tyrosine Kinase
EGF-R ErbB-2 ErbB-3 ErbB-4
b-cellulin epiregulin amphiregulin
EGF TGF-a HB-EGF
tyrosine kinase domain links to signaling
pathways
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Binding of EGF Induces Dimerization, Tyrosine
Phosphorylation and Signaling
EGF
EGF-R ErbB-2 ErbB-3 ErbB-4
b-cellulin epiregulin amphiregulin
TGF-a HB-EGF
tyrosine phosphorylation
proliferation ? motility ? angiogenesis
? differentiation ? apoptosis (cell death) ?
P
P
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The EGF-R as a Cofactorfor HPV-Associated Cancer

• 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

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Does Inhibition of EGF-R Function Alter Growth,
Differentiation, or Gene Expression of Cervical
Carcinoma Cells?
PD 153035
4-(3-Bromophenyl)amino-6,7-imethoxyquinazoline
a potent and specific inhibitor of the tyrosine
kinase activity of the EGF-R (IC50 25pM)
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PD153035 Inhibits Tyrosine Phosphorylationof the
EGF-R in a Dose-Dependent Manner
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Organotypic Culture to Promote Cell-Cell and
Cell-Matrix Interactions
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Carcinoma Cells Form Dysplastic Epitheliaand
Invade the Underlying Collagen
Normal cervical cells
CXT2 carcinoma cells
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EGF-R Inhibitor PD153035 Blocks Invasion
untreated
0.3 mM
3.0 mM
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EGF-R Inhibitor PD153035 Blocks Invasionin a
Dose-Dependent Manner
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Identification of Genes DifferentiallyExpressed
After PD153035 Treatment
microarray protocol
microarray results
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Inhibition of the EGF-R Alters Expressionof
Several Clusters of Genes
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PD153035 Alters Expression of Genes that Regulate
Attachment and Motility
symbol gene identification and description
ITGA8 integrin alpha 8, cell-cell interactions
ITGAX integrin alpha X, similar to alpha integrins
CTNND2 catenin, cadherin associated protein
ITGB1 integrin beta 1, fibronectin receptor
SELE selectin E endothelial adhesion molecule
DDR2 discoidin, required for cell adhesion
ACTN1 alpha 1 actinin
MMP1 matrix metalloproteinase 1 (collagenase)
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PD153035 Increases Expression of RNAs for
Cytokines and Chemokines
symbol gene identification and description
XCL1 chemokine ligand 1, attracts leukocytes
CX3CL1 fractalkine, chemotactic for T cells
CCL3 MIP-1a, inflammatory and chemotactic
CXCR6 chemokine receptor 6, G protein receptor
IL1R2 IL1 receptor type II, decoy receptor
IL-6 interleukin 6, proinflammatory cytokine
IL-7 Interleukin 7, hematopoietic growth factor
IRF5 interferon regulatory factor 5
TNFSF4 member of tumor necrosis factor family
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Verification of Selected Microarray Results Using
Real Time RT-PCR
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Summary

• 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 the EGF-R up regulates expression
  of genes that mediate attachment and
  inflammation, and down regulates many genes that
  stimulate growth

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Acknowledgements
Matthias Nees University of Heidelberg Evan
Michael University of Michigan Laura
Smith Clarkson University Sarah Allen
Mandy
Heitzke
April Krumnow