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.