GeP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer

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GeP100 links epidermal growth factor receptor
signallingto Arf6 activation to induce breast
cancer invasion

• Jin Hua
• Rnomics
• Apr. 10, 2008

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The aim

• Identify signalling pathways
• that are specifically used in tumour
  invasion, but rarely used in normal cells.
• It is important for targeted cancer therapy.

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Action mechanism of Ras superfamily proteins
GEF (guanine nucleotide-exchanging factor )
GAP (GTPase-activating protein )
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Overexpression of EGFR
?
Arf6 (RAS superfamily) AMAP1 (a ArfGAP)
Essential role
Invasive breast cancer
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Searching the ArfGEF responsible for tumor
invasion
ArfGEF candidates (15 genes) Proteins bearing
Sec7 domain, a putative ArfGEF domain
Expression of ArfGEF mRNAs and their proteins in
MDA-MB-231 cells.
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GEP100 is responsible for the Matrigel invasion
activity of MDA-MB-231 cells.
Rescue analysis
Knockdown of ArfGEF
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EGF stimulation has been implicated in Arf6
activation
Are GEP100 and Arf6 located downstream of EGFR
in the induction of invasive Activities?
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GEP100 and Arf6 are located downstream of EGFR
in the induction of invasive activities
Arf6 activities
Matrigel invasion activities
The glutathione S-transferase (GST)golgi-localize
d, ?-ear-containing, Arf-binding protein (GGA)
pulldown
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GEP100 specifically associates withEGFR to
activate Arf6, upon EGF stimulation
IP Endogenous proteins
Mapping interaction domain
(PI pre-immune serum)
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Arf6 is activated by GEP100 and ligand activated
EGFR in the reconstituted system
Cos-7 cell
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The GEP100 and EGFR complex is mediated
interaction through the Tyr1068-or
Tyr1086-phosphorylation sites of EGFR
In vitro co-precipitation
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Immunohistochemical analyses
ductal carcinoma in situ specimens
(DCIS) invasive ductal carcinoma specimens (IDC)
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• Study system Invasion assay
• Knockdown of GEP100 (MDA-MB-231)
• GEP100 is located downstream of EGFR
• Direct interaction, mapping interaction domain
• Overexpression of GEP100 (MCF7)

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Co-expression of GEP100 and Arf6 evokes
EGF-dependent invasiveness in MCF7 cells
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E-cadherin-mediated cellcell adhesion
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In vitro

• Study system Invasion assay
• Knockdown of GEP100 (MDA-MB-231)
• GEP100 is located downstream of EGFR
• Direct interaction, mapping interaction domain
• Overexpression of GEP100 (MCF7)
• In vivo tumor metastasis (mouse mammary-tumour
  cell-line 4T1/luc)

In vivo
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Silencing of GEP100 blocks Matrigel invasion
activity of 4T1/luc cells
mouse mammary-tumour cell-line 4T1/luc
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Silencing of GEP100 blocks lung metastasis in mice
Metastasis assay fat pad injection lung cancer
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Silencing of GEP100 blocks lung metastasis in mice
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Summary
In vitro

• Study system Invasion assay
• Knockdown of GEP100 (MDA-MB-231)
• GEP100 is located downstream of EGFR
• Direct interaction, mapping interaction domain
• Overexpression of GEP100 (MCF7)
• In vivo tumor metastasis (mouse mammary-tumour
  cell-line 4T1/luc)

In vivo
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Model