Jeanin E. van Hooft, MD, PhD Gastroenterologist Academic Medical Centre
The ERK- MAP Kinase-PEA3-MMP-1 axis is operative in Oesophageal Adenocarcinoma Yeng Ang Consultant...
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Transcript of The ERK- MAP Kinase-PEA3-MMP-1 axis is operative in Oesophageal Adenocarcinoma Yeng Ang Consultant...
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
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
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
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
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
The ERK-PEA3-MMP-1 axis also appears operative in cancer tissue
RAS
MEK
ERK
PEA3
P
P
P
P
MMP-1
HER-2
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