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 Manchester Aug 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 Dec 9;9:313. doi: / 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 ER81 ERM PEA3 ETS Ct Amino acids KKKK KKKK KKKK 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 G1 Mitosis S G2

RAS MEK ERK PEA3 HER-2 P P P P MMP-1 Metastases Aims ER81 P 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 22/57 Number of cases H and E PEA3 Antibody Negative Squamous Mucosa Negative Adenocarcinoma Positive Adenocarcinoma

PEA3 and ER81 mRNA expression is associated with oesophageal adenocarcinoma p = p = /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 Colon Cancer SW480 Kidney 293T Oesophageal SCC OE21 Oesophageal Adeno OE33 HET1A Normal Oesophagus Oesophageal Adeno FLO1 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-1 PEA3 P ER81 siRNA PEA3 Genes amplified by RT-PCR PEA3 knock down

Matrigel Invasion Assay

PEA3 knockdown inhibits cancer cell invasion. Relative cell invasion p = 0.02 RAS MEK ERK PEA3 P P P P MMP-1 siRNA PEA3 OE33 cancer cells Relative cell invasion p = 0.01 Relative cell invasion HET1A epithelial cells Cancer cells Epithelial cells siRNA Control siRNA PEA3 siRNA PEA3 siRNA Control

PEA3 knockdown inhibits cancer cell proliferation *P = 0.04 * Live adherent cells Fold increase in cell number RAS MEK ERK PEA3 P P P P ? siRNA PEA3 siRNA PEA3siRNA control Control siRNA PEA3 siRNA Hours from transfection * p = 0.04 *

PEA3 and ER81 expression is dependant on MAP Kinase RAS MEK ERK X P P P P PEA3 U Hours treated U0126 pERK ERK2 X P MMP-1

OE33 cell behaviour alters with ERK inhibition RAS MEK ERK X P P P PEA3 U0126 X P 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 PEA3 PP OE33FLO-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 Metastases Cyclin? 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 InfirmaryBeaumont Hospital, Dublin Richard KeldChristian Gulmann Chris SuttonPaul Downy David Walsh James HarrisonChristie and Wythenshawe Hospitals Ann Anderton Christine PeelCatherine West Ann Marie GoreIan Welch Karim Sillah Joely Irlam Sue Pritchard Manchester UniversityHelen Valentine Simon Galloway Riahcrd Keld Andrew Sharrocks Baoqiang Guo Hope Hospital John McLaughlinLaura Formella Staff and students in Lab 2051Stephen Hayes John Vickers