The ERK- MAP Kinase-PEA3-MMP-1 axis is operative in Oesophageal Adenocarcinoma

Yeng Ang

Consultant Gastroenterologist, Salford Royal University NHS FT and Honorary Reader, GI Science, Institute of Inflammation and Repair and Molecular Cancer

University of ManchesterAug 2015

The ERK MAP kinase-PEA3/ETV4-MMP-1 axis is operative in oesophageal adenocarcinoma

• Keld R, Guo B, Downey P, Gulmann C, Ang YS, Sharrocks AD.

• Mol Cancer. 2010 Dec 9;9:313. doi: 10.1186/1476-4598-9-313.

• Some recent data with further work

The problems with Oesophageal Adenocarcinoma

• Rising incidence

• Pre malignant stage poorly understood

• Late presentation

• Inadequate treatment options

• Poor prognosis

The PEA3 transcription factor sub family are all remarkably similar

Acidic Domain

Acidic Domain

Acidic Domain

ER81

ERM

PEA3 ETS

ETS

ETS

Ct

Ct

Ct 484

458

510

Amino acids

K K K K

K K K K

K K K K

GGAA/T

GGAA/T

GGAA/T

95% sequence homology

Known targets genes for PEA3 and ER81 transcription factors

Cell Surface Growth Factor Receptor

Mediators of Tissue Invasion

Cell Cycle Regulator

Self Sustained / Auto-regulation

Mediators of Angiogenesis

HER-2

Cyclin D3

Matrix Metalloproteases

COX II and VEGF

PEA3

PEA3

G1

Mitosis

S

G2

RAS

MEK

ERK

PEA3

HER-2

P

P

P

P

MMP-1

Metastases

Aims

ER81P

1. Is the PEA3 subfamily associated with Oesophageal Adenocarcinomas?

2. Can cell line models be used to investigate PEA3 subfamily regulation and targets?

3. Can parallels be drawn from cell lines to patients?

Cell membrane

Nuclear membrane

DNA

In Oesophageal Adenocarcinoma Tissue

PEA3 protein is expressed in Oesophageal Adenocarcinomas

3/27 2/24 22/57Number of cases

H and E PEA3 Antibody

Ne

ga

tive

Sq

ua

mo

us

Mu

cosa

N

eg

ativ

eA

de

no

carc

ino

ma

P

osi

tive

Ad

en

oca

rcin

om

a

0.15

0.25

0.35

0.45

0.55

B

A PEA3

ER81

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

Rela

tive PEA3

mRN

A ex

pres

sion

Normal oesophageal tissue

Barrett’s oesophagus

Oesophageal adenocarcinoma

0.9

1.9

2.9

3.9

4.9

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Rela

tive ER

81 m

RNA

expr

essi

on

Barrett’snormaloesophageal tissue

Adenocarcinomanormal oesophageal tissue

PEA3 and ER81 mRNA expression is associated with oesophageal adenocarcinoma

p = 0.005

p = 0.007

31/41 (77%)positive PEA3 or ER81 expression

PEA3

ER81

In Cell Line Models

MMP13*

MMP9*

RT-PCR and MAP Kinase activity in cell lines

PEA3

ER81

MMP1

MMP7

GAPDH

Col

on C

ance

r

SW

480

Kid

ney

293

TO

esop

hage

al

SC

C O

E21

Oes

opha

geal

Ade

no O

E33

HE

T1A

Nor

mal

Oes

opha

gus

Oes

opha

geal

Ade

no F

LO1

pERK

ERK2

RAS

MEK

ERK

PEA3

P

P

P

P

MMP-1

HER-2

PEA3 siRNA treatment leads to a reduction in both ER81 and MMP1 mRNA

RAS

MEK

ERK

PEA3

P

P

P

P

MMP-1PEA3P

ER81

siRNA PEA3

Genes amplified by RT-PCR

PEA3 knock down

00.20.40.60.8

11.21.41.61.8

22.2

Control PEA3 ER81 MMP-1 MMP-7 VEGF COX2 GAPDH

Rel

ativ

e g

ene

exp

ress

ion

Matrigel Invasion Assay

PEA3 knockdown inhibits cancer cell invasion.

0

0.2

0.4

0.6

0.8

1

OE 33 si RNA Contr ol OE 33 si RNA P E A3

Aver age r elative invasive capaci ty

0

0.2

0.4

0.6

0.8

1

OE 33 HE T 1A

Re

lativ

e c

ell

inva

sio

n

p = 0.02

RAS

MEK

ERK

PEA3

P

P

P

P

MMP-1

siRNA PEA3

OE33 cancer cells

1

0.8

0.6

0.4

0.2

0

1

0.8

0.6

0.4

0.2

0

Re

lativ

e c

ell

inva

sio

n

p = 0.01

0

0.4

0.8

1.2

HE T 1A siRNA scr ambled HE T 1A siRNA P E A3R

ela

tive

ce

ll in

vasi

on

1.2

0.8

0.4

0

HET1A epithelial cells

Cancer cells Epithelial cells siRNA Control siRNA PEA3 siRNA PEA3 siRNA Control

PEA3 knockdown inhibits cancer cell proliferation

*P = 0.04

*

0

2

4

6

8

10

12

0 20 40 60 80 100

Live adherent cells

Fo

ld in

cre

ase

in c

ell

nu

mb

er

RAS

MEK

ERK

PEA3

P

P

P

P

?

siRNA PEA3

siRNA PEA3siRNA control

ControlsiRNA

PEA3siRNA

Hours from transfection

* p = 0.04

*

PEA3 and ER81 expression is dependant on MAP Kinase

RAS

MEK

ERK

X

P

P

P

P

PEA3

U0126

40

40

0 2 6 8 10 12Hours treated U0126

pERK

ERK2

XP

MMP-1

OE33 cell behaviour alters with ERK inhibition

RAS

MEK

ERK

X

P

P

P

PEA3

U0126

XP

MMP-1

P

Rescue experiments support the ERK-PEA3-MMP-1 axis

RAS

MEK

ERK

PEA3

P

P

P

P

MMP-1

PMA

RAS

MEK

ERK

PEA3

P

P

P

P

MMP-1

siRNA PEA3

Luciferase

Mouse PEA3P P

OE33 FLO-1

Parallels in tissue specimens

The ERK-PEA3-MMP-1 axis also appears operative in cancer tissue

RAS

MEK

ERK

PEA3

P

P

P

P

MMP-1

HER-2

Dual combination of ERK and PEA3 is associated with advanced disease staging

RAS

MEK

ERK

PEA3

HER-2

P

P

P

P

MMP-1

MetastasesCyclin?

Proliferation

ER81PEA3?

Sustained Activation?

Conclusions

In oesophageal adenocarcinoma cells, PEA3:

• Is regulated by ERK MAP kinase• Regulates the expression of MMP1 and ER81.• Alters cell invasion and proliferation.

In oesophageal adenocarcinoma tissue, PEA3:

• Is frequently expressed.• In combination with ERK, PEA3 is associated with MMP-1.• Appears to be associated advanced disease.

Future Direction

What this research adds:

Identifies an important component of the mechanism of metastatic progression in oesophageal adenocarcinoma

Identification of a prognostic indicator and drug targets

ERK MAP kinase signalling pathway could be a favourable target,

Patient selection with a dependant transcription proteins may yield more favourable outcomes

Acknowledgements

Wigan Infirmary Beaumont Hospital, Dublin

Richard Keld Christian GulmannChris Sutton Paul DownyDavid WalshJames Harrison Christie and Wythenshawe HospitalsAnn AndertonChristine Peel Catherine WestAnn Marie Gore Ian Welch

Karim SillahJoely IrlamSue Pritchard

Manchester University Helen ValentineSimon Galloway

Riahcrd KeldAndrew SharrocksBaoqiang Guo Hope HospitalJohn McLaughlin Laura FormellaStaff and students in Lab 2051 Stephen Hayes

John Vickers