Transcript Document

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
The Epidermal Growth Factor Receptor
(EGF-R) is a Membrane Tyrosine Kinase
EGF-R
ErbB-2
ErbB-3
ErbB-4
EGF
TGF-a
HB-EGF
b-cellulin
epiregulin
amphiregulin
tyrosine kinase domain
links to signaling pathways
Binding of EGF Induces Dimerization,
Tyrosine Phosphorylation and Signaling
EGF
EGF-R
ErbB-2
ErbB-3
ErbB-4
TGF-a
HB-EGF
tyrosine
phosphorylation
P
b-cellulin
epiregulin
amphiregulin
proliferation 
motility 
angiogenesis 
P
differentiation 
apoptosis (cell death) 
The EGF-R as a Cofactor
for 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
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)
PD153035 Inhibits Tyrosine Phosphorylation
of the EGF-R in a Dose-Dependent Manner
CXT2
0
CXT3
0.1 0.3 1.0 3.0 mM
0
P-Tyr
P-Tyr
EGF-R
EGF-R
0.1 0.3 1.0 3.0 mM
Organotypic Culture to Promote
Cell-Cell and Cell-Matrix Interactions
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
Carcinoma Cells Form Dysplastic Epithelia
and Invade the Underlying Collagen
Normal cervical cells
CXT2 carcinoma cells
EGF-R Inhibitor PD153035 Blocks Invasion
untreated
0.3 mM
3.0 mM
EGF-R Inhibitor PD153035 Blocks Invasion
in a Dose-Dependent Manner
Cells invading gel
100
untreated
0.3 mM
3.0 mM
80
60
40
20
0
Tumor 1
Tumor 2
Tumor 3
Identification of Genes Differentially
Expressed After PD153035 Treatment
microarray protocol
microarray results
Inhibition of the EGF-R Alters Expression
of Several Clusters of Genes
decreased
increased
inflammation
attachment and motility
Immune response
cell cycle
PD153035 Alters Expression of Genes
that Regulate Attachment and Motility
symbol
gene identification and description
ITGA8
ITGAX
CTNND2
ITGB1
SELE
DDR2
ACTN1
MMP1
integrin alpha 8, cell-cell interactions
integrin alpha X, similar to alpha integrins
catenin, cadherin associated protein
integrin beta 1, fibronectin receptor
selectin E endothelial adhesion molecule
discoidin, required for cell adhesion
alpha 1 actinin
matrix metalloproteinase 1 (collagenase)
PD153035 Increases Expression of RNAs
for Cytokines and Chemokines
symbol
XCL1
CX3CL1
CCL3
CXCR6
IL1R2
IL-6
IL-7
IRF5
TNFSF4
gene identification and description
chemokine ligand 1, attracts leukocytes
fractalkine, chemotactic for T cells
MIP-1a, inflammatory and chemotactic
chemokine receptor 6, G protein receptor
IL1 receptor type II, decoy receptor
interleukin 6, proinflammatory cytokine
Interleukin 7, hematopoietic growth factor
interferon regulatory factor 5
member of tumor necrosis factor family
Verification of Selected Microarray
Results Using Real Time RT-PCR
Fold increase
5
4
3
2
1
0
No treatment
0.1 mM PD153035
0.3 mM PD153035
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 dosedependent manner
• Inhibition of the EGF-R up regulates expression
of genes that mediate attachment and
inflammation, and down regulates many genes
that stimulate growth
Acknowledgements
Matthias Nees
University of Heidelberg
Evan Michael
University of Michigan
Laura Smith
Sarah Allen
Mandy Heitzke
April Krumnow
Clarkson University