Proneoplastic effects of PGE2 mediated by EP4 receptor in colorectal cancer.
BACKGROUND
Prostaglandin E2 (PGE2) is the major product of Cyclooxygenase-2 (COX-2) in colorectal cancer (CRC). We aimed to assess PGE2 cell surface receptors (EP 1-4) to examine the mechanisms by which PGE2 regulates tumour progression.


METHODS
Gene expression studies were performed by quantitative RT-PCR. Cell cycle was analysed by flow cytometry with cell proliferation quantified by BrdU incorporation measured by enzyme immunoassay. Immunohistochemistry was employed for expression studies on formalin fixed paraffin embedded tumour tissue.


RESULTS
EP4 was the most abundant subtype of PGE2 receptor in HT-29 and HCA7 cells (which show COX-2 dependent PGE2 generation) and was consistently the most abundant transcript in human colorectal tumours (n = 8) by qRT-PCR (ANOVA, p = 0.01). G0/G1 cell cycle arrest was observed in HT-29 cells treated with SC-236 5 microM (selective COX-2 inhibitor) for 24 hours (p = 0.02), an effect abrogated by co-incubation with PGE2 (1 microM). G0/G1 arrest was also seen with a specific EP4 receptor antagonist (EP4A, L-161982) (p = 0.01). Treatment of HT-29 cells with either SC-236 or EP4A caused reduction in intracellular cAMP (ANOVA, p = 0.01). Early induction in p21WAF1/CIP1 expression (by qRT-PCR) was seen with EP4A treatment (mean fold increase 4.4, p = 0.04) while other genes remained unchanged. Similar induction in p21WAF1/CIP1 was also seen with PD153025 (1 microM), an EGFR tyrosine kinase inhibitor, suggesting EGFR transactivation by EP4 as a potential mechanism. Additive inhibition of HCA7 proliferation was observed with the combination of SC-236 and neutralising antibody to amphiregulin (AR), a soluble EGFR ligand. Concordance in COX-2 and AR localisation in human colorectal tumours was noted.


CONCLUSIONS
COX-2 regulates cell cycle transition via EP4 receptor and altered p21WAF1/CIP1 expression. EGFR pathways appear important. Specific targeting of the EP4 receptor or downstream targets may offer a safer alternative to COX-2 inhibition in the chemoprevention of CRC.
Doherty
Glen A
GA
Molecular Medicine Group, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland. glen_doherty@hotmail.com
Byrne
Sinead M
SM
Molloy
Eamonn S
ES
Malhotra
Vikrum
V
Austin
Sandra C
SC
Kay
Elaine W
EW
Murray
Frank E
FE
Fitzgerald
Desmond J
DJ
D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D011230:Precancerous Conditions; D018078:Receptors, Prostaglandin E / Q000378:metabolism*; D058309:Receptors, Prostaglandin E, EP4 Subtype; D020133:Reverse Transcriptase Polymerase Chain Reaction
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; C545650:PTGER4 protein, human; D018078:Receptors, Prostaglandin E; D058309:Receptors, Prostaglandin E, EP4 Subtype; D051546:Cyclooxygenase 2; D066246:ErbB Receptors; D015232:Dinoprostone