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

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.

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

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  1. 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

  2. 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

  3. The problems with Oesophageal Adenocarcinoma • Rising incidence • Pre malignant stage poorly understood • Late presentation • Inadequate treatment options • Poor prognosis

  4. The PEA3 transcription factor sub family are all remarkably similar Amino acids K K K K PEA3 484 Acidic Domain ETS Ct GGAA/T K K K K ER81 458 Acidic Domain ETS Ct GGAA/T K K K K ERM 510 Acidic Domain ETS Ct GGAA/T 95% sequence homology

  5. Known targets genes for PEA3 and ER81 transcription factors PEA3 Cell Surface Growth Factor Receptor HER-2 Mitosis Cell Cycle Regulator Cyclin D3 G2 G1 Mediators of Tissue Invasion S Matrix Metalloproteases Mediators of Angiogenesis COX II and VEGF Self Sustained / Auto-regulation PEA3

  6. Aims Cell membrane 1. Is the PEA3 subfamily associated with Oesophageal Adenocarcinomas? HER-2 P RAS 2. Can cell line models be used to investigate PEA3 subfamily regulation and targets? P MEK Nuclear membrane P ERK 3. Can parallels be drawn from cell lines to patients? P P ER81 PEA3 DNA MMP-1 Metastases

  7. In Oesophageal Adenocarcinoma Tissue

  8. PEA3 protein is expressed in Oesophageal Adenocarcinomas H and E PEA3 Antibody Negative Squamous Mucosa Negative Adenocarcinoma Positive Adenocarcinoma Number of cases 3/27 2/24 22/57

  9. PEA3 and ER81 mRNA expression is associated with oesophageal adenocarcinoma 31/41 (77%) positive PEA3 or ER81 expression PEA3 p = 0.007 ER81 p = 0.005

  10. In Cell Line Models

  11. RT-PCR and MAP Kinase activity in cell lines HET1A Normal Oesophagus Oesophageal SCC OE21 Oesophageal Adeno OE33 Colon Cancer SW480 Oesophageal Adeno FLO1 Kidney 293T HER-2 PEA3 P RAS ER81 P MEK MMP1 P ERK MMP7 P pERK PEA3 MMP9* ERK2 MMP-1 MMP13* GAPDH

  12. PEA3 siRNA treatment leads to a reduction in both ER81 and MMP1 mRNA PEA3 knock down P RAS P MEK P ERK Genes amplified by RT-PCR siRNA PEA3 P PEA3 P PEA3 MMP-1 ER81

  13. Matrigel Invasion Assay

  14. PEA3 knockdown inhibits cancer cell invasion. p = 0.01 p = 0.02 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1.2 0.8 0.4 0 P RAS Relative cell invasion Relative cell invasion Relative cell invasion P MEK P ERK Cancer cells Epithelial cells siRNA Control siRNA PEA3 siRNA PEA3 siRNA PEA3 siRNA Control P OE33 cancer cells HET1A epithelial cells PEA3 MMP-1

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

  16. PEA3 and ER81 expression is dependant on MAP Kinase Hours treated U0126 0 2 6 8 10 12 40 pERK ERK2 40 P RAS P MEK U0126 P ERK P X P X PEA3 MMP-1

  17. OE33 cell behaviour alters with ERK inhibition P RAS P MEK U0126 P ERK P X P X PEA3 MMP-1

  18. Rescue experiments support the ERK-PEA3-MMP-1 axis OE33 FLO-1 P P RAS RAS P P MEK PMA MEK P ERK P ERK siRNA PEA3 P P PEA3 PEA3 MMP-1 MMP-1 Luciferase P P Mouse PEA3

  19. Parallels in tissue specimens

  20. The ERK-PEA3-MMP-1 axis also appears operative in cancer tissue HER-2 P RAS P MEK P ERK P PEA3 MMP-1

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

  22. Conclusions HER-2 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. P RAS P MEK P ERK P PEA3 MMP-1 Cyclin? Metastases Proliferation ER81PEA3? Sustained Activation?

  23. 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

  24. Acknowledgements Wigan Infirmary Beaumont Hospital, Dublin Richard Keld Christian Gulmann Chris Sutton Paul Downy David Walsh James Harrison Christie and Wythenshawe Hospitals Ann Anderton Christine Peel Catherine West Ann Marie Gore Ian Welch Karim Sillah Joely Irlam Sue Pritchard Manchester University Helen Valentine Simon Galloway Riahcrd Keld Andrew Sharrocks Baoqiang Guo HopeHospital John McLaughlin Laura Formella Staff and students in Lab 2051 Stephen Hayes John Vickers

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