Validation of signaling pathway models
1
OncoSignal Validation of signaling pathway models ERα/β ERα/β ER ER HSP HSP Estrogen Estrogen receptor ERα/β ERα/β Target gene expression ER activity score (relative scale) Control ESR1 knockdown Estrogen receptor alpha (ESR1) knock- down by siRNA in ER positive MCF7 breast cancer cell line is associated with reduction of ER activity. ER pathway activity in MCF7 breast cancer cell line, ER activity stimulated by estrogen. ER pathway activity inhibited by fulvestrant. LuCaP35 prostate tumor implanted in mice. Androgen deprivation induced by castration. Lower AR activity in castrated mice compared to control mice. Philips OncoSignal qPCR kits are in development, not available for sale. OncoSignal pathway analysis is already available through our dedicated service lab for Research Purposes Only. Not for use in diagnostic procedures. ER pathway activity in MCF7 breast cancer cell line, ER activity stimulated by estrogen. ER pathway activity inhibited by tamoxifen. Breast cancer cells from ER negative cell lines (BT20, HCC1806) and the ER positive cell line MCF7 transplanted into the mammary fatpad (-FP) or intraductal (MIND) in the mouse. High ER pathway activity score only for MCF7, irrespective of transplanted location. AR pathway activity increased in in-vitro LNCaP cell line upon exposure to dihydrotestosterone (DHT) (left) and decreased in presence of anti-androgen bicalutamide (right). AR pathway activity in prostate cancer is high in androgen dependent growth, and low in castrate induced regression and castrate resistant regrowth. ER activity score (relative scale) Control 100nM tamoxifen 1nM E2 E2+100nM tamoxifen Cell line n = 3 n = 3 n = 3 n = 3 n = 3 n = 3 BT20-FP BT20-MIND HCC1806-FP HCC1806-MIND MCF7-FP MCF7-MIND ER activity score (relative scale) transplanted ER negative cell lines transplanted ER positive cell line ER activity score (relative scale) Control 1nM E2 10nM E2 E2+Fulves. AR AR AR AR HSP HSP Androgens (Dihydrotestosterone) Androgen receptor AR AR Target gene expression 0.0079 n = 5 n = 5 AR activity score (relative scale) Treatment Control Castration AR activity score (relative scale) Treatment LNCaP control LNCaP DHT 4h LNCaP DHT 16h n = 3 n = 3 n = 3 Treatment Control 1nM DHT 1nM DHT + Bicalutamide n = 3 n = 2 n = 3 AR activity score (relative scale) Treatment Androgen- dependent growth Castration- induced regression Castration- resistant regrowth n = 4 n = 4 n = 4 0.029 0.029 ER pathway High Hedgehog activity is measured in basal cell carcinoma. Left: Calibration on patient set with basal cell carcinoma, versus healthy skin. Right: Validation on independent patient set with basal cell carcinoma, versus healthy skin. High Hedgehog pathway activity is measured in the Sonic Hedgehog (SHH) subgroup of patients with medulloblastoma. GLI2 GLI2 Target gene expression SMO SMO Ptch1 HH HH GLI2/3 Cos2 Kif7 SuFu Fu Cilium 0.00052 n = 15 HH activity score (relative scale) n = 4 0.018 n = 2 n = 64 Tissue Normal skin Basal cell carcinoma 9.7e-06 n = 9 n = 52 HH activity score (relative scale) SHH subgroup No Yes HH pathway AR pathway Wnt inhibitor decreases Wnt pathway activity in colon cancer PDX mouse model. Colon cancer PDX mice treated with antiRSPO3 (Wnt inhibitor) and/or chemotherapy (irinotecan). High Wnt pathway activity in colon adenoma and carcinoma. Ref: W Verhaegh et al, Cancer Res 2014 Jun 1;74(11):2936 High Wnt pathway activity in medulloblastoma with an activating beta-catenin mutation. Two independent data sets. Target gene expression Frizzled LRP RSPO3 Dvl Dvl APC Axin Axin B-Catenin B-Catenin Tankyrase B-Catenin B-Catenin B-Catenin CK1 CK1 GSK3 GSK3 Wnt ligand TCF B-Catenin Treatment Control antibody AntiRSPO3 antibody Irinotecan Irinotecan + antiRSPO3 0.1 0.00016 n = 9 n = 3 n = 3 n = 9 Wnt activity score (relative scale) Group Normal colon Adenoma Carcinoma Wnt activity score (relative scale) < 2.2e-16 1.7e-15 n = 36 n = 45 n = 24 Wnt activity score (relative scale) 2e-06 n = 9 n = 53 2.2e-05 n = 4 n = 36 Beta-Catenin Wild type Mutated Wnt pathway EGFR inhibitor, erlotinib, inhibits PI3K pathway activity in breast cancer cell lines representing triple-negative (BT20), ER positive (MCF7), and HER2 positive breast cancer (MDA-MB-453). HER2-siRNA treatment inhibits PI3K pathway in breast cancer cell line (BT-474) by knocking down HER2 expression. mTOR inhibitor, rapamycin, inhibits PI3K pathway activity in HER2-overexpressing cell lines HB4a (luminal normal mammary cell line), C5.2 (HB4a transfected with HER2), and SKBR3 (HER2 amplification). EGFR, MEK, and PI3K inhibitors reduce PI3K pathway activity in a lung cancer cell line with mutated EGFR. Lung cancer cell line HCC827 treated with vehicle (DMSO), erlotinib (EGFR inhibitor), AZD6244 (selumetinib, inhibitor of MEK1/MEK2) or BEZ235 (dual inhibitor of PI3K/mTOR). FOXO activity score is inversely related to PI3K pathway activity. FOXO activity score is inversely related to PI3K pathway activity. FOXO activity score is inversely related to PI3K pathway activity. FOXO activity score is inversely related to PI3K pathway activity. Growth factor + receptor Translation machinery more efficient (Also for other protein pathways) FOXO – PI3K regulated target gene expression FOXO – Oxidative stress regulated target gene expression Oxidative stress IRS mSIN1 PI3K AKT PDK1 PIP2 PTEN FOXO3 PIP3 PIP3 PIP3 pAKT SGK1 S6K1 mTORC2 mTORC1 FOXO activity score (relative scale) 0.057 0.2 0.33 Treatment BT20 Control BT20 erlotinib 0.5h BT20 erlotinib 6h BT20 erlotinib 24h MDA-MB-453 control MDA-MB-453 erlotinib 24h MCF7 control MCF7 erlotinib 24h n = 4 n = 3 n = 3 n = 3 n = 3 n = 2 n = 2 n = 2 Treatment Control siRNA 4h HER2 siRNA 4h Control siRNA 24h HER2 siRNA 24h n = 6 n = 6 n = 6 n = 6 0.0022 0.0022 FOXO activity score (relative scale) FOXO activity score (relative scale) 0.0079 0.032 0.0079 Treatment HB4a - vehicle HB4a - RAP 20nM C5.2a - vehicle C5.2a - RAP 20nM SKBR3 - vehicle SKBR3 - RAP 20nM n = 5 n = 5 n = 4 n = 5 n = 5 n = 5 Treatment Control Erlotinib Selumetinib Dactolisib n = 4 n = 3 n = 3 n = 3 0.057 0.057 0.057 FOXO activity score (relative scale) PI3K/FOXO pathway TGF-ß induces TGF-ß pathway activity in primary macrophages that are primed by treatment with dexamethasone, samples from five independent donors. TGF-ß induces TGF-ß pathway activity in immortalized ovarian surface epithelial cells (IOSE) derived from normal ovarian epithelial cells incubated with TGF-ß-1. TGF-ß induces TGF-ß pathway activity in an ER negative breast cancer cell line (MDA-MB-231), both in control and mutant depleted p53 cells. Smad4 Smad2/3 Smad7 Smad4 SARA Smad2/3 Target gene expression TGF-ß activity score (relative scale) Treatment Control TGF-ß 24h Dex control Dex TGF-ß 3h Dex TGF-ß 24h n = 5 n = 5 n = 5 n = 5 n = 5 0.0079 0.0079 TGF-ß activity score (relative scale) Treatment duration 0 hr 3 hr 6 hr 12 hr n = 2 n = 2 n = 2 n = 2 TGF-ß pathway TGF-ß activity score (relative scale) 0.029 n = 4 n = 4 n = 4 n = 4 0.029 Treatment Control Control - TGF-ß Mutant-p53 Mutant-p53- TGF-ß Ref: EJ Blok et al, SABCS 2015.
Transcript of Validation of signaling pathway models
OncoSignalValidation of signaling pathway models
ERα/β ERα/β
ER
ER
HSP
HSP
Estrogen
Estrogen receptor
ERα/β ERα/β
Target geneexpression
ER
act
ivit
y sc
ore
(re
lati
ve s
cale
)
Control ESR1 knockdown
Estrogen receptor alpha (ESR1) knock-down by siRNA in ER positive MCF7 breast cancer cell line is associated with reduction of ER activity.
ER pathway activity in MCF7 breast cancer cell line, ER activity stimulated by estrogen. ER pathway activity inhibited by fulvestrant.
LuCaP35 prostate tumor implanted in mice. Androgen deprivation induced by castration. Lower AR activity in castrated mice compared to control mice.
Philips OncoSignal qPCR kits are in development, not available for sale. OncoSignal pathway analysis is already available through our dedicated service lab for Research Purposes Only. Not for use in diagnostic procedures.
ER pathway activity in MCF7 breast cancer cell line, ER activity stimulated by estrogen. ER pathway activity inhibited by tamoxifen.
Breast cancer cells from ER negative cell lines (BT20, HCC1806) and the ER positive cell line MCF7 transplanted into the mammary fatpad (-FP) or intraductal (MIND) in the mouse. High ER pathway activity score only for MCF7, irrespective of transplanted location.
AR pathway activity increased in in-vitro LNCaP cell line upon exposure to dihydrotestosterone (DHT) (left) and decreased in presence of anti-androgen bicalutamide (right).
AR pathway activity in prostate cancer is high in androgen dependent growth, and low in castrate induced regression and castrate resistant regrowth.
ER
act
ivit
y sc
ore
(re
lati
ve s
cale
)
Control 100nMtamoxifen
1nM E2 E2+100nMtamoxifen
Cell line
n = 3
n = 3
n = 3n = 3 n = 3n = 3
BT20-FP
BT20-MIND
HCC1806-FP
HCC1806-MIND
MCF7-FP
MCF7-MIND
ER
act
ivit
y sc
ore
(re
lati
ve s
cale
)
transplanted ERnegative cell lines
transplantedER positive
cell line
ER
act
ivit
y sc
ore
(re
lati
ve s
cale
)Control 1nM E2 10nM E2 E2+Fulves.
ARAR
AR
AR
HSP
HSP
Androgens(Dihydrotestosterone)
Androgen receptor
ARAR
Target geneexpression
0.0079
n = 5
n = 5
AR
act
ivit
y sc
ore
(re
lati
ve s
cale
)
Treatment
Control
Castration
AR
act
ivit
y sc
ore
(re
lati
ve s
cale
)
Treatment
LNCaP control
LNCaP DHT 4h
LNCaP DHT 16h
n = 3
n = 3
n = 3
AR
act
ivit
y sc
ore
(re
lati
ve s
cale
)
Treatment
Control
1nM DHT
1nM DHT +
Bicalutamide
n = 3
n = 2
n = 3 AR
act
ivit
y sc
ore
(re
lati
ve s
cale
)
Treatment
Androgen-
dependent
growth
Castration-
induced
regression
Castration-
resistant
regrowth
n = 4
n = 4
n = 4
0.029
0.029
ER pathwayHigh Hedgehog activity is measured in basal cell carcinoma.Left: Calibration on patient set with basal cell carcinoma, versus healthy skin.Right: Validation on independent patient set with basal cell carcinoma, versus healthy skin.
High Hedgehog pathway activity is measured in the Sonic Hedgehog (SHH) subgroup of patients with medulloblastoma.
GLI2
GLI2
Target geneexpression
SMO
SMO
Ptch
1
HH
HH
GLI2/3
Cos2 Kif7
SuFu Fu
Cilium
HH pathway
0.00052
n = 15
HH
act
ivit
y sc
ore
(re
lati
ve s
cale
)
Tissue
Normal skin
Basal cell carcinoma
n = 4
0.018
n = 2
n = 64
HH
act
ivit
y sc
ore
(re
lati
ve s
cale
)
Tissue
Normal skin
Basal cell carcinoma
9.7e-06
n = 9
n = 52
HH
act
ivit
y sc
ore
(re
lati
ve s
cale
)
SHH subgroup
No
Yes
HH pathway
AR pathwayWnt inhibitor decreases Wnt pathway activity in colon cancer PDX mouse model. Colon cancer PDX mice treated with antiRSPO3 (Wnt inhibitor) and/or chemotherapy (irinotecan).
High Wnt pathway activity in colon adenoma and carcinoma.
Ref: W Verhaegh et al, Cancer Res 2014 Jun 1;74(11):2936
High Wnt pathway activity in medulloblastoma with an activating beta-catenin mutation. Two independent data sets.
Target geneexpression
Frizzled
LR
P
RSPO3
Dvl
Dvl
APC
Axin
Axin
B-Catenin
B-Catenin
Tankyrase
B-CateninB-Catenin
B-Catenin
CK1
CK1
GSK3
GSK3
Wnt ligand
TCF
B-Catenin
Wnt pathway
Treatment
Control antibody
AntiRSPO3 antibody
Irinotecan
Irinotecan + antiRSPO3
0.10.00016
n = 9
n = 3
n = 3n = 9
Wn
t a
ctiv
ity
sco
re (
rela
tive
sca
le)
Group
Normal colon
Adenoma
Carcinoma
Wn
t a
ctiv
ity
sco
re (
rela
tive
sca
le)
< 2.2e-16
1.7e-15
n = 36n = 45
n = 24
Wn
t a
ctiv
ity
sco
re (
rela
tive
sca
le)
2e-06
n = 9
n = 53
Beta-Catenin
Wild type
Mutated
Wn
t a
ctiv
ity
sco
re (
rela
tive
sca
le)
2.2e-05
n = 4
n = 36
Beta-Catenin
Wild type
Mutated
Wnt pathway
EGFR inhibitor, erlotinib, inhibits PI3K pathway activity in breast cancer cell lines representing triple-negative (BT20), ER positive (MCF7), and HER2 positive breast cancer (MDA-MB-453).
HER2-siRNA treatment inhibits PI3K pathway in breast cancer cell line (BT-474) by knocking down HER2 expression.
mTOR inhibitor, rapamycin, inhibits PI3K pathway activity in HER2-overexpressing cell lines HB4a (luminal normal mammary cell line), C5.2 (HB4a transfected with HER2), and SKBR3 (HER2 ampli� cation).
EGFR, MEK, and PI3K inhibitors reduce PI3K pathway activity in a lung cancer cell line with mutated EGFR. Lung cancer cell line HCC827 treated with vehicle (DMSO), erlotinib (EGFR inhibitor), AZD6244 (selumetinib, inhibitor of MEK1/MEK2) or BEZ235 (dual inhibitor of PI3K/mTOR).
FOXO activity score is inversely related to PI3K pathway activity. FOXO activity score is inversely related to PI3K pathway activity. FOXO activity score is inversely related to PI3K pathway activity. FOXO activity score is inversely related to PI3K pathway activity.
Growth factor + receptor
Translation machinery more e�cient(Also for other protein pathways)
FOXO – PI3K regulated target gene expression
FOXO – Oxidative stress regulated target gene expression
Oxidative stress
PI3K / FOXO pathway
IRS mSIN1
PI3K AKT PDK1
PIP2
PTEN
FOXO3
PIP3 PIP3 PIP3
pAKT
SGK1
S6K1
mTORC2
mTORC1
PI3K/FOXO pathway
FO
XO
act
ivit
y sc
ore
(re
lati
ve s
cale
)
0.057 0.2 0.33
Treatment
BT20 Control
BT20 erlotinib
0.5h
BT20 erlotinib
6h
BT20 erlotinib
24h
MDA-MB-453
control
MDA-MB-453
erlotinib 24h
MCF7 control
MCF7 erlotinib
24h
n = 4
n = 3
n = 3
n = 3
n = 3
n = 2
n = 2
n = 2
Treatment
Control
siRNA 4h
HER2 siRNA 4h
Control
siRNA 24h
HER2 siRNA 24hn = 6n = 6
n = 6
n = 6
0.0022 0.0022
FO
XO
act
ivit
y sc
ore
(re
lati
ve s
cale
)
FO
XO
act
ivit
y sc
ore
(re
lati
ve s
cale
)
0.0079 0.032 0.0079
Treatment
HB4a - vehicle
HB4a -
RAP 20nM
C5.2a - vehicle
C5.2a -
RAP 20nM
SKBR3 - vehicle
SKBR3 -
RAP 20nM
n = 5
n = 5
n = 4
n = 5
n = 5
n = 5
Treatment
Control
Erlotinib
Selumetinib
Dactolisib
n = 4
n = 3
n = 3
n = 3
0.057
0.057
0.057
FO
XO
act
ivit
y sc
ore
(re
lati
ve s
cale
)
PI3K/FOXO pathway
TGF-ß induces TGF-ß pathway activity in primary macrophages that are primed by treatment with dexamethasone, samples from � ve independent donors.
TGF-ß induces TGF-ß pathway activity in immortalized ovarian surface epithelial cells (IOSE) derived from normal ovarian epithelial cells incubated with TGF-ß-1.
TGF-ß induces TGF-ß pathway activity in an ER negative breast cancer cell line (MDA-MB-231), both in control and mutant depleted p53 cells.
Smad4Smad2/3
Smad7Smad4
SARA
Smad2/3
TGF-β Pathway
Target geneexpression
TGF-ß pathway
TG
F-ß
act
ivit
y sc
ore
(re
lati
ve s
cale
)
Treatment
Control
TGF-ß 24h
Dex control
Dex TGF-ß 3h
Dex TGF-ß 24h
n = 5
n = 5 n = 5
n = 5
n = 5
0.0079
0.0079
TG
F-ß
act
ivit
y sc
ore
(re
lati
ve s
cale
)
Treatment duration
0 hr
3 hr
6 hr
12 hr
n = 2
n = 2
n = 2
n = 2
TGF-ß pathway
TG
F-ß
act
ivit
y sc
ore
(re
lati
ve s
cale
)
0.029
n = 4
n = 4
n = 4
n = 4
0.029
Treatment
Control
Control - TGF-ß
Mutant-p53
Mutant-p53-
TGF-ß
Ref: EJ Blok et al, SABCS 2015.
