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Downstream EGFR Protein Phosphorylation and Gefitinib Inhibition in Non-Small
Cell Lung Cancer Cells Detected With Multiplex Phosphoprotein Assays
Life Science Group
2000 Alfred Nobel Drive
Hercules, CA 94547 USA
Qian Gao, Abraham Bautista Jr., and Sophie Allauzen
Introduction
Table 1. Magnitude of gefitinib inhibitory effect on six phosphoprotein targets in
three NSCLC cell lines. Numbers represent the fold decline in phosphorylation measured
between EGF and EGF + gefitinib lysates, calculated using MFI from the Bio-Plex
phosphoprotein assay.
Downstream Target Detection
2,000
Untreated
p-MEK1 (Ser217/221)
16,000
ERK1/2
EGF
Gefitinib + EGF
Untreated
EGF
Gefitinib + EGF
0
Untreated
EGF
p70 S6K
H-1734
Wild type
H-1975
No gefitinib response
16,000
15,000
8,000
8,000
10,000
4,000
4,000
5,000
Untreated
EGF
Gefitinib + EGF
H-1734
Wild type
0
Untreated
EGF
Gefitinib + EGF
0
Untreated
EGF
Gefitinib + EGF
1,000
NSCLC cell lines ­
Untreated
EGF
Gefitinib + EGF
MFI
3,000
H-1734
Wild type
0
Untreated
EGF
Gefitinib + EGF
2,500
0
Untreated
MFI
0
6.3
3.3
4.0
H-1975
1.4
0.9
0.8
1.0
1.0
0.8
H-1650
15.0
45.0
20.4
4.7
11.5
18.9
Among three NSCLC cell lines, the wild type (H-1734) showed moderate inhibition of
phosphorylation on the downstream targets by gefitinib. The cell line H-1650, mutated by a
deletion from L747 to P753, showed dramatic phosphorylation inhibition on all tested targets.
The Bio-Plex assay measured signal decreases between EGF- and gefitinib/EGF-treated
samples from 4.7-fold to 45-fold (Table 1). However, cell line H-1975, which carried the double
point mutation (L858R and T790M), did not respond to the drug with the exception of p-Akt,
which showed a slight signal decrease between EGF- and gefitinib/EGF-treated samples
(1.4-fold). No other targets showed inhibition by gefitinib. These results correspond with studies
showing that the second point mutation (T790M) reverses the drug response because of the
substitution of methionine for threonine at position 790, resulting in steric hindrance of the drug
binding to EGFR.
EGF
Gefitinib + EGF
Lynch TJ et al., Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung
cancer to gefitinib, N Engl J Med 350, 2129–2139 (2004)
2,000
1,000
Untreated
EGF
Gefitinib + EGF
30,000
H-1734
Wild type
0
Untreated
EGF
Gefitinib + EGF
25,000
MFI
Fig. 2. Protocol to detect the six phosphoprotein targets downstream of EGFR that are shown in Figure 1.
Experiments were performed on three NSCLC lines using both the Bio-Plex phosphoprotein assay and western blot analysis.
15,000
10,000
5,000
Results
0
p-EGFR Detection on NSCLC Cell Lines
H-1975
No gefitinib response
Untreated
EGF
H-1975
No gefitinib response
25,000
20,000
35,000
5.6
4,000
2,000
p-p70 S6K (Thr 421/Ser 424)
Western blotting with
individual antibodies
H-1734
Wild type
4.3
0
Untreated
EGF
Gefitinib + EGF
EGF 75 ng/ml for 20 min
Cell lysate preparation; 10 µg of protein was run with:
Bio-Plex multiplex
phosphoprotein assays
8.0
6,000
3,000
1,500
500
EGF 75 ng/ml for 20 min
H-1734
H-1650
High gefitinib response
8,000
4,000
1,000
Gefitinib 3 µm for 3 hr
H-1975
No gefitinib response
5,000
2,000
Untreated
p-p90RSK
Iressa is a trademark of AstraZeneca UK Limited Ltd. xMAP is a trademark of Luminex Corporation. The Bio-Plex suspension
array system includes fluorescently labeled microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc. by the
Luminex Corporation.
p-GSK-3a/b (Ser21/Ser9)
Cell culturing and starvation with serum-free medium overnight
p-p70 S6K
Kobayashi S et al., An alternative inhibitor overcomes resistance caused by a mutation of the epidermal growth factor
receptor, Cancer Res 65, 7096–7101 (2005)
500
Fig. 1. Schematic of the signal transduction pathway downstream of EGFR. We used Bio-Plex phosphoprotein assays
to detect the six phosphoprotein targets highlighted at bottom. The drug gefitinib inhibits EGFR stimulation.
H-1734 — wild type (low response to gefitinib)
H-1975 — L858R/T790M mutations (no response to gefitinib)
H-1650 — del L747-P753 mutations (high response to gefitinib)
p-GSK-3a/b
References
1,500
500
200
Cell Proliferation
2,000
1,000
400
Experimental Design
p-ERK1/2
Bio-Plex phosphoprotein assays are useful tools in studying signal transduction pathways and
revealing the phosphorylation status of multiple targets in anticancer drug discovery.
2,500
1,500
600
H-1650
High gefitinib response
3,000
2,000
800
0
H-1975
No gefitinib response
2,500
1,000
GSK-3a/b
p-MEK1
We measured tyrosine phosphorylation of EGFR and six downstream phosphoproteins in
three NSCLC cell lines with and without gefitinib treatment. For all tested cell lysates, the MFI
of the Bio-Plex assay correlated very well with the band intensity on western blot. The multiplex
assay needed only 10 µg of cell lysate protein for each sample. Western blot analysis needed
60 µg of protein per sample because each target was probed with individual anti-phosphospecific antibody.
H-1650
High gefitinib response
20,000
12,000
1,200
EGFR downstream
targets analyzed in
this study
p-Akt
Gefitinib + EGF
PDK1
Akt
p90RSK
0
12,000
p-ERK1/2 (Thr202/Tyr204)
MEK1
4,000
1,000
NSCLC
Cell Line
Conclusions
0
P13K
8,000
3,000
200
EGFR
B-Raf
12,000
4,000
400
H-1650
High gefitinib response
16,000
5,000
600
0
H-1975
No gefitinib response
6,000
800
Gefitinib
EGF
H-1734
Wild type
1,000
MFI
EGFR Activation and Inhibition by Gefitinib
p-Akt (Ser 473)
MFI
The purpose of this study was to detect the effects of gefitinib (marketed as Iressa by
AstraZeneca Pharmaceuticals) on the phosphorylation of downstream targets of EGFR (Figure
1) and to show the application of Bio-Plex® phosphoprotein assays to drug discovery based
on signal transduction pathways. Gefitinib, an inhibitor of epidermal growth factor receptor
(EGFR), is used to treat non-small cell lung cancer (NSCLC). The response of NSCLC to
gefitinib is related to mutations occurring within the EGFR kinase domain. Because Bio-Plex
phosphoprotein assays (xMAP technology) can detect multiple phosphoprotein targets from a
single cell lysate sample, they are a useful tool to reveal the phosphorylation state of targeted
proteins along their signal transduction pathways. We used Bio-Plex phosphoprotein assays
to probe the phosphorylation state of three NSCLC cell lines treated by gefitinib followed by
epidermal growth factor (EGF) stimulation (Figure 2). The NSCLC cell lines we used include a
wild-type (H-1734) and two mutated strains (H-1650 and H-1975). H-1650 contains a deletion
mutation (del L747-P753) and responds to gefitinib. H-1975 has the double point mutations
L858R and T790M. The cellular response to gefitinib caused by the L858R mutation is reversed
by the T790M mutation. The six EGFR downstream targets in this study are phosphorylated:
p-Akt, p-MEK1, p-ERK1/2, p-GSK-3a/b, p-p70 S6K, and p-p90RSK. Results of the multiplex
phosphoprotein assays were compared with individual western blot results (Figures 3 and 4).
Gefitinib + EGF
25,000
20,000
20,000
15,000
15,000
10,000
10,000
5,000
5,000
0
Untreated
EGF
H-1650
High gefitinib response
Gefitinib + EGF
0
Untreated
EGF
Gefitinib + EGF
H-1650
High gefitinib response
30,000
25,000
p-p90RSK (Thr359/Ser363)
20,000
2,500
15,000
10,000
2,000
5,000
1,500
MFI
MFI
Bio-Plex
phosphoprotein
assay detects
all p-Tyr
on EGFR
F
EG
F
ef
itin
ib
+
EG
G
at
e
Un
tre
+
ib
itin
ef
d
F
EG
F
EG
G
at
e
Un
tre
+
ib
itin
d
F
EG
F
EG
G
ef
Un
tre
at
e
d
0
Western
blot detects
p-Tyr1068
on EGFR
Fig. 3. The Bio-Plex assay compared well with western blot band intensity in detection of phosphorylated EGFR.
Gefitinib inhibits the EGFR pathway but its effect varies among three cell cultures (H-1734, H-1975, H-1650). MFI = mean
fluorescence intensity. Error bars indicate standard deviations.
H-1734
Wild type
2,000
4,000
3,000
1,000
2,000
500
500
Untreated
EGF
Gefitinib + EGF
0
H-1650
High gefitinib response
5,000
1,500
1,000
0
H-1975
No gefitinib response
1,000
Untreated
EGF
Gefitinib + EGF
0
Untreated
EGF
Gefitinib + EGF
Fig. 4. The Bio-Plex assay compared well with western blot in detecting six phosphoprotein targets downstream of EGFR. Gefitinib inhibits the EGFR pathway but its effect varies among three cell cultures (H-1734, H-1975, H-1650).
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