Proneoplastic effects of PGE₂mediated by EP4 receptor in colorectal cancer

HT-29 cells were purchased from the ATCC (Rockville, MD) and maintained in McCoy's 5 A medium containing 1.5 mM L-glutamine, 10% FBS, penicillin 100 U/ml and streptomycin 100 μg/ml. HCA7 cells were kindly donated by Susan Kirkland (ICRF, London, UK) and were cultured in DMEM with 10% FBS, supplemented with 1 mM sodium pyruvate and 100 μg/ml kanamycin. SC236, a selective COX-2 inhibitor was a gift from Dr. Peter Isakson (Searle, Skokie, IL). PGE₂ was purchased from Cayman (St. Louis, MO). L-161982 (EP4A), a selective antagonist of the EP4 receptor was a kind gift of Merck Frosst, Canada [19]. PD153035 (EGFR tyrosine kinase inhibitor) was purchased from Calbiochem (La Jolla, CA). Wortmannin was purchased from Sigma Aldrich (Dublin, Ireland).

Total RNA was isolated from cells and tissue following homogenisation in RNA lysis buffer (Qiagen Ltd. GmbH, Germany) supplemented with 1% β-mercaptoethanol. Extraction was performed using RNeasy™ Mini Kits (Qiagen Ltd. GmbH, Germany). Total RNA (1 μg) was reverse transcribed using Moloney Murine Leukaemia Virus (MMLV) reverse transcriptase (Promega, Madison, WI) according to the manufacturer's instructions. Gene expression was quantified by RT-PCR using SYBR Green Universal Master Mix (Roche Diagnostics Corp., Indianapolis, IN). Reactions were carried out in a 96 well format in the ABI 7700 Sequence Detector (Perkin Elmer/Applied Biosystems, UK). Results were then normalized to 18S rRNA amplified from the same cDNA mix. Sequences of the primer pairs used are listed below.

EP1- F ATG GTG GGC CAG CTT GTC

EP1- R GCC ACC AAC ACC AGC ATT G

EP2- F TGC CTT TCA CGA TTT TTG CA

EP2- R TTA ATT GAT AAA AAC CTA AGA GCT TGG A

EP3- F TCT CCG CTC CTG ATA ATG ATG TT

EP3- R TCT GCT TCT CCG TGT GTG TCT T

EP4-F CGA CCT TCT ACA CGC TGG TAT G

EP4-R CCG GGC TCA CCA ACA AAG T

Amphireg-F CTC GGG AGC CGA CTA TGA CTA

Amphireg-R GCT TAA CTA CCT GTT CAA CTC TGA CTG A

CyclinD1-F CTG GAG GTC TGC GAG GAA CA

CyclinD1-R TGC AGG CGG CTC TTT TTC

CDK4-F TGT TGT CCG GCT GAT GGA

CDK4-R AAA CAC AGG GTT ACC TTG ATC TC

CDK6-F CAA CTA GGA AAA ATC TTG GAC GTG AT

CDK6-R TTG GTT GGG CAG ATT TTG AAT

p21-F GCA GAC CAG CAT GAC AGA TTT CTA

p21-R GCG GAT TAG GGC TTC CTC TT

p27-F CCT GCA ACC GAC GAT TCT TC

p27-R TCT TAA TTC GAG CTG TTT ACG TTT GA

Samples of formalin fixed, paraffin embedded tissue were deparaffinised and rehydrated in Xylene and Methanol. Detection of COX-2. Endogenous peroxidase activity was quenched with 0.3% H₂O₂ in Methanol. Specimens were blocked in 1.5% normal serum and then incubated with antibody to COX-2 (Rabbit polyclonal anti-human COX-2, Cayman Chemical Co.) diluted 1/200, followed by secondary antibody and ABC complex from Vectastain Elite kit (Vector Laboratories, Burlingame, CA). Sections were exposed to diaminobenzidine (DAB) (Sigma Aldrich, Dublin, Ireland) and counterstained with hematoxylin (Sigma Aldrich, Ireland) and mounted using DPX (BDH, Poole, UK). Detection of Amphiregulin. Antigen retrieval was performed by heating slides in 10 mM citrate buffer (pH6.0) for 4 minutes in a pressure cooker. Blocking was performed with goat serum for 30 minutes. Slides were then incubated with primary antibody to amphiregulin (rabbit polyclonal to Amphiregulin by Abcam, Cambridge, MA) diluted 1/200 in ChemMate™ antibody diluent (DakoCytomation, Galway, Ireland) for 1 hour and staining completed using the ChemMate™ DAKO Envision™ detection kit (DakoCytomation, Galway, Ireland) visualisation, counterstaining and mounting were performed as outlined above.

Competitive enzyme immunoassay (EIA) was used to determine PGE₂ levels in culture media in 96-well format by Assay Designs (Ann Arbor, MI, USA). Prostaglandin concentration was calculated using the optical density of the samples in relation to a standard curve generated by dilutions of a standard provided. Quantitative determination of cAMP concentration in cell lysates was performed using the cAMP (low pH) immunoassay by R and D Systems (Abingdon, Oxon, UK). Adherent cells were lysed in 0.1 N HCl for 10 min at 37°C and supernatants were assayed according to manufacturer's instructions in a 96 well plate. cAMP concentrations were calculated using a similar standard curve method.

Cell proliferation assays were carried out using the BrdU (colorimetric) cell proliferation ELISA (Roche Applied Science, Dublin, Ireland) according to manufacturer's instructions. Cells were seeded at 5 × 10³/well in 96 well plates and following overnight serum starvation were treated with vehicle or drug. Amphiregulin neutralisation was with anti-human neutralising AR antibody (AR-ab) from Stratech Scientific (Soham, Cambridgeshire, UK).

HT-29 cells were seeded at a density of 1 × 10⁶/well in 6 well plates and treated with drug or vehicle for 24 hours. Adherent cells were suspended using trypsin-EDTA for 3–5 min at 37°C and were fixed overnight in 75% ethanol at 4°C then washed and resuspended in a solution of PBS containing 0.1% Triton X-100, 0.05 mg/ml of DNase-free RNase and a 50 μg/ml propidium iodide (Molecular Probes, Leiden, NL) in the dark for 30 min. Cells were resuspended in PBS prior to analysis in a FACScalibur flow cytometer (Becton Dickinson, Oxford, UK) with measurement of fluorescence emission at >575 nm (FL3). Analysis of cell cycle distribution (DNA index) was performed using CellQuest™ (Becton Dickinson, Oxford, UK).

The protocol was approved by the Ethics (Medical Research) Committee of Beaumont Hospital, Dublin and all patients provided written, informed consent. Samples of colorectal tumour/normal were obtained from patients at the time of surgery and were immediately placed in RNAlater solution (Qiagen GmbH, Germany) or fixed in 10% formalin.

A one-way analysis of variance (ANOVA) was used to examine overall differences between multiple groups, with Bonferroni multiple comparisons test. Two tailed paired students T-test was used to compare the means of paired samples. The statistical packages GraphPad InStat and GraphPad Prism were used. Statistical significance was set at a P value of less than 0.05, whereas a P value less than 0.005 was considered highly significant.

EP receptor expression was assessed by qRT-PCR in the human colon cancer cell line HT-29 (n = 3) and EP4 receptor was the most abundant receptor subtype. A representative amplification plot is shown in Figure 1a with linear amplification of EP4 seen in identical template after the shortest number of cycles. The transcript for the EP2 receptor was less abundant than EP4 with significantly less transcript again for both the EP3 and EP1 receptors. A similar expression pattern of EP receptors was observed in HCA7 cells, which also generate PGE₂ in a COX-2 dependent manner.

EP receptor expression was next assayed in human tumour samples (n = 8) with a similar expression pattern in all tumours studied. The EP4 receptor was consistently the most abundant receptor transcript (shortest number of cycles to exponential amplification in identical template, ANOVA p = 0.0002, Figure 1b). While the number of cycles to amplification was significantly less for the EP4 receptor than EP1 (p = 0.001) or EP2/EP3 (p = 0.01), no significant differences were noted in the abundance of the other EP receptors relative to each other. The relative abundance of the EP4 receptor in both cancer cell lines and in human tumour tissue suggested that signalling through this receptor might therefore be important in how PGE₂ regulates tumour cell phenotypes.

While others have suggested that HT-29 cells lack the ability to generate prostaglandins through COX-2 activity [20], we confirmed that PGE₂ is generated in a COX-2 dependent fashion in HT-29 cells and with EP4 receptor activation. Significant PGE₂ generation by HT-29 cells was observed in control cells and this was reduced in a dose dependent manner by SC236, a COX-2 selective inhibitor (Figure 2a). The EP4 receptor signals by mediating changes in cAMP production via adenylate cyclase. The activity of this receptor (and thus indirectly EP4 protein expression) was demonstrated by measuring the generation of cAMP by cells with receptor modulation. Figure 2b shows the reduction in intracellular cAMP levels following both inhibition of PGE₂ production by SC-236 and by a specific antagonist of the EP4 receptor (L-161982) confirming functional EP4 activity.

SC-236 increased the number of cells in the G₀/G₁ phase of the cell cycle over a range of doses. This G₀/G₁ arrest was much more marked with higher doses of the inhibitor (see Figure 3a). Interestingly, the effects of the inhibitor were not 'rescued' by co-incubation with exogenous prostaglandin at the higher dose. However, at doses of SC-236 in the low micromolar range (sufficient to abolish >90% of PGE₂ production), a G₀/G₁ cell cycle arrest was observed which was reversed by co-incubation with exogenous PGE₂ (Figure 3b). Cell cycle arrest was also seen on incubation with L-161982 suggesting that activation of the EP4 receptor by endogenous PGE₂ plays a role in the regulation of cell cycle progression in HT-29 cells.

The ability of selective EP4 receptor inhibition to modulate changes in the expression of cell cycle regulation genes was assessed to evaluate potential mechanisms for the observed phenotype. The expression of Cyclin D1, the cyclin dependent kinases CDK4 and CDK6 and finally the CDK inhibitors p21^WAF1/CIP1 and p27^KIP1 were evaluated. The results are summarised in Figure 4a. A significant induction in p21^WAF1/CIP1 expression was seen after 4 hours with EP4 receptor antagonism (mean change 4.4 fold, p = 0.04). Expression of the other cell cycle regulation genes remained unchanged. Similar induction in p21^WAF1/CIP1 expression was observed with PD153035, an EGFR tyrosine kinase inhibitor (p = 0.001), an effect not seen with the PI-3 kinase inhibitor wortmannin, suggesting transactivation of EGFR by EP4 as the likely mechanism for the induction of p21^WAF1/CIP1.

EGFR transactivation appears important in PGE₂ signalling and our results suggested a role in regulation of cell cycle progression genes and thus proliferation. PGE₂ mediates EGFR activation by the release of EGFR ligands. Amphiregulin (AR) is known to be the most abundant EGFR ligand in HCA7 cells [21], which exhibit PGE₂ dependent proliferation and therefore constitute an excellent in-vitro model to examining interplay between prostaglandins and EGFR.

The effect on HCA7 cell proliferation of a neutralising antibody to AR (ARab) both alone and in combination with SC-236 was evaluated. Effects of SC-236 on cell proliferation were again evident (33% mean reduction in proliferation, p = 0.001). At low concentration (0.1 – 1.0 mg/ml) the AR neutralizing antibody (ARab) had no effect (not shown), however at higher concentrations (10 mg/ml) a small effect (20% mean reduction, p = 0.05) on cell proliferation was noted (Figure 5). The effect of combined treatment with COX-2 inhibitor and ARab was greater than that of either ARab (p = 0.001) or SC-236 (p = 0.05) alone, resulting in a greater than 50% reduction in proliferation relative to control.

The relationship between COX-2 and AR expression in human CRC was next examined. The expression of amphiregulin (AR) transcript was quantified in 10 tumour/normal pairs by qRT-PCR and correlated with expression of COX-2 in the same samples. AR expression was greater in tumour relative to normal in 7 of 10 patients (Mean fold difference = 4.2, p = 0.07 for paired t-test). A non-significant positive correlation was observed (Pearson r = 0.50, p = 0.13) between the tumour/normal differences for COX-2 and AR in the samples assayed. Closer inspection revealed higher than anticipated levels of AR expression in the normal colon (explaining the failure to observe a significant correlation). In an effort to better understand the relationship, immunohistochemistry on paired tumour and normal mucosa from an additional 20 patients was performed to evaluate expression of AR and COX-2. COX-2 immunoreactivity was virtually absent in normal colonic mucosa. AR expression was detectable in all of the normal samples, with a characteristic pattern of expression, being observed in the cytoplasm of the surface columnar epithelial cells while absent or reduced in crypt epithelial cells (Figure 6a). Differential expression of AR along the colonic crypt has been described previously [22]. COX-2 and AR expression in tumour tissue showed greater variability. Six of twenty cases (30%) showed absent or low level (<5% of cells positive) for both COX-2 and AR. In the remainder of patients, significant immunoreactivity for both molecules was observed and a concordance was observed in the localisation of positive staining within tumour specimens (representative images are presented in Figure 6b). COX-2 expression was observed exclusively in the cytoplasm of tumour epithelial cells. AR expression was mostly cytoplasmic, but focal areas of positive nuclear staining were also observed (Figure 6c).