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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.

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slide1

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
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

binding of egf induces dimerization tyrosine phosphorylation and signaling
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

the egf r as a cofactor for hpv associated cancer
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
slide5
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
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

organotypic culture to promote cell cell and cell matrix interactions
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
Carcinoma Cells Form Dysplastic Epitheliaand Invade the Underlying Collagen

Normal cervical cells

CXT2 carcinoma cells

egf r inhibitor pd153035 blocks invasion in a dose dependent manner
EGF-R Inhibitor PD153035 Blocks Invasionin a Dose-Dependent Manner

100

untreated

0.3 mM

80

3.0 mM

60

Cells invading gel

40

20

0

Tumor 1

Tumor 2

Tumor 3

identification of genes differentially expressed after pd153035 treatment
Identification of Genes DifferentiallyExpressed After PD153035 Treatment

microarray protocol

microarray results

inhibition of the egf r alters expression of several clusters of genes
Inhibition of the EGF-R Alters Expressionof Several Clusters of Genes

decreased

increased

attachment and motility

inflammation

Immune response

cell cycle

verification of selected microarray results using real time rt pcr
Verification of Selected Microarray Results Using Real Time RT-PCR

No treatment

0.1 mM PD153035

5

0.3 mM PD153035

4

3

Fold increase

2

1

0

IL6

IL7

CCL3

CCL3

RELA

CXCR6

CCL11

DAPK1

IL1 alpha

CX3CL1

NFKBIA

IL1 beta

IKB alpha

summary
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
acknowledgements
Acknowledgements

Matthias Nees University of Heidelberg

Evan Michael University of Michigan

Laura Smith Clarkson University Sarah Allen Mandy Heitzke April Krumnow