kras mutant colorectal cancers -...
TRANSCRIPT
A
B
p-AKT S473
ERK
p-ERK
p-AKT T308
AKT
H630 CaR-1
sh KRAS
KRAS
SW1463 Lovo Gp5d HCT-116 LS-174TT84
KRAS/PIK3CA mutant
KRAS wild type
KRAS mutant
SW948HCT-15
p-AKT S473
ERK
p-ERK
p-AKT T308
AKT
sh KRAS
KRAS
HT55
Supplemental Figure 1. KRAS is required for activation of MEK-ERK in KRAS mutant
colorectal cancers
KRAS mutant and KRAS/PIK3CA double mutant (A) and KRAS wt (B) colorectal cancer cells
were infected with two shRNA targeting KRAS (B, C) or control shRNA. 3 days following
infection, protein lysates were prepared and probed with the indicated antibodies.
Gp5dLS-174T T84
KRAS/PIK3CA mutant
p-AKT S473
ERK
p-ERK
p-AKT T308
AKT
S6
p-S6
p-EGFR
EGFR
HCT-116 SW948HCT-15
SW837 SW1463 Lovo
KRAS mutant
p-AKT S473
ERK
p-ERK
p-AKT T308
AKT
S6
p-S6
p-EGFR
EGFR
SW620
N.D
N.D
Supplemental Figure 2. EGFR does not regulate PI3K-AKT signaling in KRAS mutant
colorectal cancers
The indicated cell lines, grouped according to mutational status, were treated with Gefitinib
(Gef) 1μM or Cetuximab (Cet) 10 μg/mL for 6 hours. Lysates were probed with the indicated
antibodies.
H630 CaR-1
p-AKT S473
ERK
p-ERK
p-AKT T308
AKT
p-EGFR
EGFR
KRAS wild type
HT55 CoCM-1 CCK-81 Difi
Supplemental Figure 3. EGFR inhibition suppresses MEK-ERK signaling pathway in KRAS
wildtype cancers
KRAS wildtype cancer cell lines were treated with 1μM of gefitinib (Gef) or 10 μg/mL of
cetuximab (Cet) for 6 hours, and lysates were probed with the indicated antibodies.
p-AKT S473
AKT
sh KRAS
Cet (-)
ERK
p-ERK
Cet (+)
SW1463
KRAS
-
SW837
Dox - + +
- + - +Cet
p-AKT S473
AKT
ERK
p-ERK (short)
KRAS
p-ERK (long)
Supplemental Figure 4. KRAS exerts a more dominant regulation on ERK signaling than
EGFR in SW1463 and SW837 cell lines. (Left) SW1463 cells were infected with one of two
independent shRNA targeting KRAS (B, C) or control shRNA. 3 days following infection, cells were
treated with vehicle or cetuximab (10 μg/mL) for 6 hours. The cells were lysed and western blots
were probed with the indicated antibodies. (Right) SW837 cells infected with lentivirus expressing
a doxcycyline-inducible KRAS shRNA were cultured in the presence or absence of doxycycline (20
ng/mL) for 72 hours. Then cells were treated with cetuximab for 6 hours. The cells were lysed and
western blots were probed with the indicated antibodies.
p-AKT S473
p-ERK
KRAS
ERK
AKT
- +Dox - +
SW620 (0.5%) SW620 (5%)
- + - + Dox
HCT-116 (0.5%) HCT-116 (5%)
- + - + - + - +
p-AKT S473
p-ERK
KRAS
ERK
AKT
Supplemental Figure 5. Restored expression of KRAS rescues the effects of KRAS
knockdown on downstream signaling.
SW620 and HCT-116 cells harboring doxycyline-inducible KRAS or control shRNA (Figure
1F) were infected with GFP-expressing lentivirus encoding KRAS(12V) or control cDNA and
sorted for low GFP fluorescence intensity by FACS. Since the KRAS shRNA targets the 3’UTR,
the KRAS expression was restored in the cells infected with the KRAS(12V) expressing
constructs. Stable polyclonal cell lines were then cultured in the presence or absence of
doxycycline (10 ng/mL) in either full serum (5% FBS) or low serum (0.5% FBS) for 72 hours.
Lysates were probed with the indicated antibodies.
Dox-KRAS
KRAS-withdrawal
48hr
p-ERKp-AKT
B
p-ERK
ERK
AKT
p-AKT S473
p-AKT T308
24 48
Dox –
withdrawal
(hr)
A
Supplemental Figure 6. KRAS knockdown does not suppress PI3K signaling
(A, B) Tet-op-mutant Krasmice were induced to develop lung tumors with doxycycline. The
doxycycline was removed and lungs were harvested 24 and 48 hours later. The lungs were
assessed by western blot analyses (A) and immunohistochemical analyses (B) with the
indicated antibodies. Scale bar, 100μM.
p-RSK
RSK
p-ERK
ERK
Gp5d SW1463
p-S6
S6
SW837
Supplemental Figure 7. MEK-ERK signaling regulates S6 phosphorylation in multiple KRAS
mutant cancer cell lines. Cells were treated with AZD6244 1μM for 6 hours. Protein lysates were
blotted with the indicated antibodies.
Gp5d T84LS-174T
p-AKT S473
ERK
p-ERK
p-AKT T308
AKT
S6
p-S6
KRAS/PIK3CA mutant
HCT-15 SW948
SW620 SW1463
p-AKT S473
ERK
p-ERK
p-AKT T308
AKT
S6
p-S6
Lovo
KRAS mutant
p-AKT S473
ERK
p-ERK
p-AKT T308
AKT
S6
p-S6
T84
p-EGFR
EGFR
B
A
Supplemental Figure 8. Mechanisms of PI3K
activation in KRAS mutant colorectal cancers
(A) Cells were treated with either DMSO (-) or the
indicated drug(s) for 6 hours. Drug concentrations are the
same as in Figure 4A. Protein lysates were immunoblotted
and probed with the indicated antibodies.
(B) PI3K suppression requires IGF-IR and EGFR inhibition
in the presence of MEK inhibitors in T84 cells. Cells were
treated with either DMSO (-) or the indicated drug
combinations for 6 hours. Drugs were used at 1μM.
Protein lysates were probed with indicated antibodies.
SW1463
GP5d
SW620SW837
KRAS mutant
KRAS/PIK3CA mutant
B
A
Sub-G
1
popula
tion (
%)
0
5
10
15
20
25
30
T84LS-174T HCT-116
Supplemental Figure 9. Decreased cell viability is induced by combined inhibition of
IGF-IR (or MET) and MEK in KRAS mt colorectal cancers
(A) Cell viability data for the individual KRASmutant and KRAS/PIK3CA mutant cell lines
using the indicated drugs and combinations. Each concentration was assessed in sixplicate,
and the averages and standard deviations are shown. All cell lines except LS-174T showed
synergy with IGF-IR and MEK inhibitors.
(B) A combination of MET and MEK inhibitors is the most effective in Lovo cells. (left) Lovo
cells were assessed by a 72-hour survival assay in increasing concentrations of the indicated
drugs and combinations. Combined inhibition of MET and MEK showed synergy comparing
to single treatments. (right) Lovo cells were incubated in the presence of the indicated
drug(s), and the percentage of cells with sub-G0/G1 DNA content was determined by
propidium iodine staining and fluorescence-activated cell sorting analyses.
0
0.5
1
1.5
2
2.5
3
3.5
4
0 1 2 3>50% <50%
IC50 (mM
)Gef/NVP
AZD/NVP
p < 0.001
Supplemental Figure 10. The effects of inhibitor combinations on the activation MEK/ERK
and PI3K/AKT pathways correlates with their efficacy. The levels of phosphorylated ERK and
AKT after treatment with gefitinib and NVP-AEW541 or AZD6244 and NVP-AEW541 were
quantified for each cell line examined (raw data shown in Figure 4A and Supplemental Figure 8A).
The average of suppression of AKT and ERK phosphorylation was calculated in each cell lines.
Using a cutoff of 50% reduction of phosphorylation, cells were plotted versus IC50s.
Average reduction of p-AKT and
p-ERK following treatment
*
Supplemental Figure 11. Tumor volume at the end of the treatment. Tumor volumes at the
beginning and the end of the experiment shown in Figure 5 are presented as the average + SEM.
Combination treatment showed significant reduction in tumor volume (* indicates p <0.05 by
paired t-test).
Pre-treatment
End of treatment
0
500
1000
1500
2000
R1507 AZD6244 Comb
Volu
me (
mm
3)
p-Tyr
p85
IRS-1
(short exposure)
IRS-1
(long exposure)
KRAS mutation
BRAF mutation
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
+
-
+
-
-
+
-
+
-
+
IRS-1
ERBB3
IP
p-Tyr
p85
No. 7
IRS-1
IRS-1
A B
C
p-EGFR
p-IGFR
EGFR
IGFR
p-ERBB3
ERBB3
Supplemental Figure 12. IRS-1 associates with PI3K in human colorectal cancers
(A) Lysates from the human colorectal cancer specimens. Whole cell lysates from nine
different human colorectal cancer specimens used for the immunoprecipitations in Figure
6 were immunoblotted for the indicated antibodies. Phospho-MET was not detected in any
samples.
(B) The protein lysate from patient #7 (7th lane of Figure 6A) was immunoprecipitated with
p85 (lane 1) and IRS1 (lane 3). Supernatant from the IRS-1 IP was subsequently followed
by p85 IP (lane 2). Please note that IP of IRS-1 cleared the pTyr band associated with the
p85 IP.
(C) p85 was immunoprecipitated from additional 13 human colorectal cancer specimens
followed by western blotting with the indicated antibodies. The KRAS and BRAF mutational
status of each cancer specimen is indicated.
0
2
4
6
8
10
% a
popto
sis
SW620
Supplemental Figure 13. Concomitant inhibition of PI3K-AKT and MEK-ERK signaling does
not lead to apoptosis in SW620 cells
SW620 cells were treated with indicated drugs and combinations for 72 hours. The percent of
cells undergoing apoptosis, as measured by annexin V positivity, is shown relative to untreated
cells. The average + SD of three independent experiments is shown. GDC-0941 (GDC) is a pan-PI3
kinase inhibitor.
P-Tyr
p85
250
150
100
ERBB3
IRS1/2
A B C
p-AKT S473
ERK
p-ERK
p-AKT T308
AKT
S6
p-S6
NCI-H630
% p
-AK
T f
ollo
win
g tr
ea
tme
nt
0
20
40
60
80
100
120
p < 0.01
p < 0.01
Supplemental Figure 14. Regulation of the PI3K-AKT signaling pathway in KRAS
wildtype cancers
(A) Cell extracts from the indicated cell lines were immunoprecipitated with an antibody to
p85 and blotted with an anti-pTyr antibody.
(B) NCI-H630 cells were treated with control, gefitinib (1 μM), NVP-AEW541 (NVP) (1 μM),
a MEK inhibitor AZD6244 (AZD) (1 μM), and gefitinib and NVP-AEW541 (Gef/NVP) for 6
hours. Cell lysates were probed with the indicated antibodies.
(C) The normalized levels of phosphorylated AKT were quantified for each of the KRAS
wildtype cell lines 6 hours following the indicated treatments (blots are shown in
Supplemental Figure 3 and 15B). Each point represents the results from a single cell line.
Bars indicate mean values.
A
B
C
HT55 CaR-1 H630 CoCM-1
0
20
40
60
80
0
10
20
30
40
CoCM-1CCK-81 Difi
0
10
20
30
40
% a
popto
tic
% a
popto
tic
% a
popto
tic
p-AKT S473
ERK
p-ERK
p-AKT T308
AKT
S6
p-S6
HT55 CoCM-1H630 CaR-1
*
CCK-81 Difi
Supplemental Figure 15. Combined EGFR and IGF-IR inhibition downregulate both AKT
and ERK phosphorylation leading to apoptosis in KRAS wt cancers
(A) Cell viability data for the individual KRASwt cell lines using the indicated drugs and
combinations. Each concentration was assessed in sixplicate, and the averages and standard
deviations are shown.
(B) Cells were treated with either DMSO (-) or the indicated drug or drug combinations for 6
hours. Drugs were used at the same concentrations as in Supplemental Figure 8A. Protein lysates
were probed with the indicated antibodies. *, non-specific bands.
(C) KRAS wt cells were treated with indicated drugs and combinations for 72 hours. The percent
of cells undergoing apoptosis, as measured by annexin V positivity, is shown relative to untreated
cells. The average + SD of three independent experiments is shown.