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Nanomolar EP4 receptor potency and expression of eicosanoid-related enzymes in normal appearing colonic mucosa from patients with colorectal neoplasia

Abstract

Background

Aberrations in cyclooxygenase and lipoxygenase (LOX) pathways in non-neoplastic, normal appearing mucosa from patients with colorectal neoplasia (CRN), could hypothetically qualify as predisposing CRN-markers.

Methods

To test this hypothesis, biopsies were obtained during colonoscopy from macroscopically normal colonic mucosa from patients with and without CRN. Prostaglandin E2 (PGE2) receptors, EP1-4, were examined in Ussing-chambers by exposing biopsies to selective EP receptor agonists, antagonists and PGE2. Furthermore, mRNA expression of EP receptors, prostanoid synthases and LOX enzymes were evaluated with qPCR.

Results

Data suggest that PGE2 binds to both high and low affinity EP receptors. In particular, PGE2 demonstrated EP4 receptor potency in the low nanomolar range. Similar results were detected using EP2 and EP4 agonists. In CRN patients, mRNA-levels were higher for EP1 and EP2 receptors and for enzymes prostaglandin-I synthase, 5-LOX, 12-LOX and 15-LOX.

Conclusions

In conclusion, normal appearing colonic mucosa from CRN patients demonstrates deviating expression in eicosanoid pathways, which might indicate a likely predisposition for early CRN development and furthermore that PGE2 potently activates high affinity EP4 receptor subtypes, supporting relevance of testing EP4 antagonists in colorectal neoplasia management.

Peer Review reports

Background

Colorectal cancer (CRC) is the third most common type of cancer worldwide and the second leading cause of cancer related deaths [1]. Adenocarcinomas constitute the majority of CRC and the carcinogenesis of this type of CRC is a multifactorial process, in which an accumulation of mutations leads to the formation of colorectal neoplasia (CRN), initially as benign adenomas and subsequently malignant adenocarcinomas [2]. Genetics and chronic colonic inflammation are known risk factors for developing CRC [3], involving altered activity of the arachidonic acid (AA) metabolism including prostaglandins. The specific mechanisms, however, are poorly understood.

Non-steroid anti-inflammatory drugs (NSAIDs), as aspirin (acetylsalicylic acid), and non-selective cyclooxygenase (COX) inhibitors ameliorate CRC development [4, 5]. NSAIDs attenuate the inflammatory response mainly by inhibiting enzyme activity of COX isozymes, COX-1 and COX-2, thus preventing conversion of AA into the prostanoids PGD2, PGE2, PGF, PGI2 and thromboxane A2 (TXA2), Fig. 1, [3].

Fig. 1
figure 1

Model of the metabolization of arachidonic acid (AA). AA is metabolized by 3 different groups of enzymes: cyclooxygenases (COX), lipoxygenases (LOX) and epoxygenases (cytochrome P450). The COX pathway consists of 2 isozymes: COX-1 and COX-2. Both isozymes metabolize AA into PGG2 and then into PGH2, which is further converted to the prostaglandins (PGs) PGD2, PGE2, PGF, PGI2 and thromboxane A2, (TXA2) by their respective synthases [3]. Each product binds to its specific membrane receptor. The CYP-450 pathway converts AA by epoxygenases and ω-hydroxylase into other downstream products, not shown. The LOX pathway consists of 3 main enzymes termed 5-LOX, 12-LOX and 15-LOX (isozymes 15-LOX-1 and 15-LOX-2). They metabolize AA into hydroperoxyl-eicosatetraenoic acids (HPETEs), which are further reduced to hydroxyeicosatetraenoic acids (HETEs). The 5-LOX enzyme differs by also metabolizing 5-HPETE into leukotriene A4 by means of 5-lipoxygenase-activating protein (FLAP). *Enzymes already investigated in our laboratory; data published. Receptors/enzymes investigated in this study are underlined with red

COX-2 expression is elevated in human adenomas as well as in adenocarcinomas, which is why COX-2 is believed to be central to CRN and CRC pathogenesis [6]. Accordingly, the protective effect of NSAIDs on CRC development is likely due to a reduced COX-activity as well as associated PGE2 production [3, 5, 7].

PGE2 elicits tumorigenic effects by binding to either of its 4 G-protein coupled surface receptors, EP1-4, Fig. 1 [8]. These effects include proliferation, migration, invasion and angiogenesis [8]. Each of the receptor subtypes has been linked to CRC tumorigenesis using knock-out mice [9,10,11]. In particular, EP4 is suspected to be of special tumorigenic importance due to its activation of several central kinases [12, 13].

For the remaining prostanoids; TXA2 is considered mainly tumorigenic, PGI2 anti-tumorigenic and PGF2 and PGD2 have uncertain tumorigenic roles [14, 15].

Recently, another AA-related pathway, the lipoxygenase (LOX) pathway, was suggested to be associated with CRC. Particularly the enzymes 5-LOX, 12-LOX and 15-LOX and its isoforms (15-LOX-1 and 15-LOX-2) appear to be involved [16, 17]. Unlike the COX pathway, the end products of LOX enzymes are hydroxyeicosatetraenoic acids (HETEs) derivates, Fig. 1. Current evidence suggests a pro-tumorigenic effect of 5-LOX and 12-LOX metabolites in CRC, whereas 15-LOX-1 and 15-LOX-2 are mainly classified as anti-tumorigenic and downregulated in CRC tissue [16, 17].

Several theories in form of “field effects” and “mutator pathways” for primary tumor-induced changes in near and distant gene expression have been forwarded over the last 70 years [18,19,20]. It remains unsolved whether tumor-adjacent imbalances in eicosanoid-related enzymes and/or receptors are inherited initiating factors, a predisposition, rather than consequences of a nearby tumor’s neoplastic “field effect”.

Here we hypothesize that genetically inherited constructs in eicosanoid signaling might be an individual early CRC tumorigenic predisposition detectable in macroscopically normal appearing tissue. Accordingly, we examined eicosanoid-related enzymes and receptors in non-neoplastic colonic mucosa both from patients with and without CRN. Specifically, we characterized function and expression of the EP receptor subtypes and examined the expression levels of prostaglandin D2 synthase (PTGDS), prostaglandin I2 synthase (PTGIS) and the PGF2α- reductase AKR1B1 (an aldo–keto reductase), all as indicators for altered levels of their respective prostanoids [21]. Finally, we determined expression levels of 5-, 12-, and 15-LOX enzymes. Both the actual and former eicosanoid-related entities, studied for function and expression by us, are labeled in Fig. 1.

Methods

Study population

White Danish patients (45–80 years of age) referred for colonoscopy on suspicion of colorectal disease (e.g. positive fecal occult blood test or persistent abdominal discomfort), were screened for participation. Exclusion criteria included history of inflammatory bowel disease, conditions of intestinal malabsorption (e.g. coeliac disease and lactose intolerance), familiar risk of CRC (hereditary nonpolyposis colorectal cancer and familial adenomatous polyposis), pregnancy and/or continuous treatment with NSAID, anti-coagulant or phosphodiesterase inhibitor. Furthermore, incomplete examination of the entire colon resulted in exclusion.

Patients were divided into 2 groups based on endoscopic findings and medical history: patients with present or history of CRN defined as either sessile serrate lesions (all types), high and low grade tubular adenomas, villous adenomas, tubule-villous adenomas and adenocarcinomas were termed CRN patients and patients without present nor history of CRN termed and served as controls, CTRL patients. A total of 73 patients were enrolled, hereof 53 CRN patients (Male/Female = 27/26) of which 5 were diagnosed with CRC (one patient had T3N1M0, while the others had T1N0M0) and remaining 20 were CTRL patients (Male/Female = 8/12). Mean age was 63 (50–78) in CRN patients and 61 (46–76) in CTRL patients. Twenty-eight patients in the CRN group and 5 patients in the CTRL group were regularly using medications e.g. anti-diabetics, anti-estrogens, anti-epileptics, anti-hypertensives, asthma inhalers, bisphosphonate, methotrexate, proton pump inhibitors, thyroid hormones, triptans, selective serotonin reuptake inhibitors, statins and xanthine oxidase inhibitors. An expected imbalance between patient groups was observed for comorbidities and medications. This diversity could have a potential impact on the obtained results.

Ethics

The study protocol was approved by the Scientific Ethical Committee of Copenhagen (H-3-2013-107) and the Danish Data Protection Agency (BBH-2013-024, I-Suite no: 02342). The study was conducted in accordance with the Helsinki declaration. All participating patients gave written informed consent.

Chemicals

SC 51322, PF 04418948, L-798,106, L-161,982, amiloride, theophylline, indomethacin, acetazolamide, bumetanide, ouabain as well as salts for Ringer’s solution were purchased from Sigma-Aldrich (Brøndby, Denmark). GW627368X, TCS 2510, and Sulprostone were purchased Santa Cruz Biotechnology (Texas, USA). ONO-DI004 and ONO-AE1-259 were kindly provided by Ono Pharmaceuticals Co., Ltd. (Osaka, Japan). All other chemicals were of analytical grade.

Selection of receptor agonists and antagonists was based on a thorough search of available literature, with a preference for compounds tested on human tissue.

Biopsy extraction

All endoscopies and biopsy extractions took place at the Endoscopic Unit of Digestive Disease Center K, Bispebjerg Hospital, Nielsine Nielsens Vej 41K, 2400 Copenhagen NV, Denmark. Six endoscopic biopsies were obtained from each patient using standard biopsy forceps (Boston Scientific, Radial Jaw 4, large capacity). Biopsies were taken from macroscopically normal appearing sigmoid mucosa on retraction of the endoscope; about 30 cm orally from the anal verge and at least 10 cm from macroscopically abnormal appearing tissue.

Four biopsies allocated for functional studies, were immediately placed in an iced bicarbonate Ringer solution containing (in mM): Na+ (140), Cl (117), K+ (3.8), PO4 (2.0), Mg2+ (0.5), Ca2+ (1.0), and HCO3 [25], and transferred to the laboratory. The remaining biopsies were snap frozen in liquid nitrogen and stored at − 80 °C until further examination.

Experimental methods

Two experimental methods were employed: functional studies in modified air suction Ussing (MUAS) chambers measuring short circuit current (SSC) and quantitative real-time polymerase chain reaction (qPCR).

Functional studies in MUAS-chambers

Four biopsies were mounted and oxygenated in MUAS-chambers after extraction as described by Larsen et al. [22] generally within 45 min after extraction. Biopsies were bathed on both sides with 10 mL Ringer, supplemented with 5.5 mM D-glucose. Temperature was maintained at 37.2 °C by water jackets. An automated voltage-clamp device continuously recorded SCC and slope conductance [22].

Experiments began after a stable basal SCC was obtained within 10 min after proper mounting. All experiments were initiated by addition of amiloride (20 µM, mucosal side) to inhibit electrogenic sodium absorption mediated through epithelial sodium channels and followed by theophylline (400 µM, serosal side) to inhibit phosphodiesterase-dependent cyclic adenosine monophosphate (cAMP) degradation. Finally, to eliminate endogenous prostaglandin synthesis, indomethacin (13 µM, serosal side) was added and incubated for 40 min.

Biopsies from 47 patients were treated with PGE2 and selective EP receptor agonists to investigate receptor function, Table 1. A single agonist was added in increasing concentrations (1 nM to 5 µM, serosal side) to each MUAS-chamber. The final agonist concentration step was followed by the addition of 5 µM PGE2, to elicit a maximal PGE2-induced response.

Table 1 Selected agonists and antagonists and applied antagonist concentrations for functional MUAS chamber experiments

Biopsies from 26 patients were treated with selective EP receptor antagonists, Table 1. A combination of 3 antagonists was added to each MUAS chamber (serosal side), to single out and investigate the remaining non-inhibited EP receptor subtype. After antagonist incubation (45 min), cumulative doses of PGE2 were added (3 nM to 1 µM, serosal side). The EP4 receptor was also examined with another selective antagonist, GW627368X (GW-X, 5 µM, serosal side).

Experiments were terminated by the addition of acetazolamide, a carbonic anhydrase inhibitor (250 µM, serosal side), to measure HCO3/H+-secretion, followed by bumetanide (25 µM, serosal side), to inhibit Na–K–Cl cotransporters and chloride secretion, and finally the Na+/K+-ATPase inhibitor ouabain (0.2 mM, serosal side) to assess and ensure tissue viability and data quality.

Quantitative real-time PCR

RNA isolation

Twenty biopsies, 10 from CRN and 10 from CTRL patients, were matched according to gender and used for further qPCR investigations. RNA was extracted from the biopsies using RNeasy Mini Kit (Qiagen, Copenhagen, Denmark). Following extraction, RNA samples were placed on ice and quantified using a Nanodrop Spectrophotometer (LabTech International) in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines (MIQE guidelines) [23].

qPCR analysis

RNA was reverse transcribed to cDNA using the nanoScript2 (Primerdesign Ltd., U.K.) according to the manufacturer’s protocol. Quantitative analysis of specific genes of interest within our cDNA samples was determined using Precision-iC SYBR green mastermix (Primerdesign Ltd.) with the CFX96 Real-Time PCR Detection System (Bio-Rad, Denmark). Duplicate reactions were performed in 20 μL volumes containing 10 μL Precision-iC SYBR green master mix, 300 nM primer (Primerdesign Ltd.), 15 ng cDNA and made up to 20 μL with nuclease-free water. The following cycling conditions were used: initial activation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min and data was collected during each cycling phase. Melt curve analysis, to ensure each primer set amplified a single, specific product, completed the protocol. Quantification cycle (Cq) values were determined using Bio-Rad CFX96 Manager 3.0 software and the single threshold mode.

The geNorm reference gene selection kit (Primerdesign Ltd.) was used to identify the most stable reference genes and to determine optimal number of reference genes required for reliable normalization of qPCR data in these tissue samples [24]. ß-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were validated as the most stable reference genes in samples. The expression levels of genes of interest are expressed relative to the mean Cq value of the reference genes in each sample.

Primers were designed, synthesized and quality controlled by Primerdesign Ltd., Additional file 1: Table S1. The sequences for the reference genes ß-actin and GAPDH are commercially sensitive and therefore unavailable.

Data analyses

The present study is exploratory and therefore not statistically powered for specific endpoints. If identical experiments were performed on several biopsies from the same patient, a mean value of parameter results was used. A comparison of parameter values between patient groups was performed by an unpaired t-test when standard deviations were equal, and a Welch’s t-test if unequal. Furthermore, normality was tested for data. Data are presented as mean ± SEM.

To assess agonists and receptors, data obtained from dose–response curves were analyzed with either a single-Michaelis–Menten model (srm) or a two-Michaelis–Menten receptor/site model (trm) using Sigmaplot 13.0 for Windows, Systat Software Inc. (USA/Canada). Outcome data were maximum SCC responses (RMax) and EC50 of these analyses.

All other statistics were performed using RStudio (Boston, USA), or GraphPad Prism (San Diego, USA) version 8 for the qPCR analysis. P-values < 0.05 were considered significant.

Results

High and low affinity EP receptors and nanomolar EP4 receptor potency

PGE2 stimulation increased SCC in both patient groups, even at concentrations as low as 1 nM, Additional file 1: Figs. S1 and S2. The EP4 agonist produced a similar sensitivity, demonstrating high potency in the low nanomolar range, Additional file 1: Fig. S1. Concentrations of 30 nM or higher were necessary to induce SCC increases when stimulating with the other selective EP-agonists, Additional file 1: Fig. S1. Moreover, 4 out of 22 biopsies exposed to the selective EP1 agonist showed no increase in SCC.

When applying Michaelis–Menten models (srm and trm) to data, a trm provided a better fit than the srm in most analyses of data from experiments with PGE2, and agonists for EP2 and EP4 receptor subtypes, Fig. 2. Accordingly, at least 2 types of EP receptors appear activated, a high and a low affinity receptor, with different EC50s separated by a factor up to 200 in single experiments, Fig. 3. Average separation factors of the receptors were 64 for PGE2 stimulation and 15 for the EP4 agonist, Fig. 3. In experiments using either the EP1 agonist or the EP3 agonist, trm equations did not fit convincingly. Mean EC50 values from both srm and trm analyses are summarized in Fig. 3. Using the srm, CRN patients demonstrated a higher EC50 related to stimulation with the EP4 agonist compared to CTRLs, Fig. 3A.

Fig. 2
figure 2

Dose–response curves of (A) EP2 agonist ONO-AE1-259 and (B) PGE2 and EP4 agonist, TCS 2510, experiments. X-axis: ligand concentrations scaled logarithmically. Y-axis: changes in SCC. A: Large dots (black) show increases in SCC as a response to increasing EP2 agonist concentrations. The unbroken line in cyan resembles single receptor model (srm) fitting, while the long dotted line in blue resembles two receptor model (trm) fitting. B: Triangles (black) indicate increases in SCC as a response to increasing PGE2 concentrations. Large dots (black) show increases in SCC as a response to increasing EP4 agonist concentrations. Dotted and long dotted lines (in blue colors) resemble single (srm) and two receptor model (trm) fitting for PGE2 respectively. The unbroken and the medium dotted lines (in red colors) show trm and srm respectively for EP4 agonist. The trm fits data points more closely

Fig. 3
figure 3

Calculated mean EC50 values of PGE2 and EP receptor agonists using (A) single receptor model (srm) equations and of (B) high and (C) low affinity receptors following PGE2 and EP receptor agonists stimulation using two receptor model equations (trm). Numbers under the graph show N/n, N = number of patients, n = number of biopsies, NA = not applicable due to insufficient N/n. Data are presented as means ± SEM. *p < 0.05

Maximum SCC responses (RMax) computed from srm and trm are shown in Fig. 4. As PGE2 stimulates all EP receptors, RMax was highest for PGE2 followed by the selective EP4 agonist eliciting approximately 50% and 75% of the PGE2 response in CTRL and CRN patients, respectively. The remaining EP-agonists had RMax means ranging between 20 and 30% of the PGE2 response. Finally, RMax was significantly increased for low affinity receptors in EP4 agonist studies (trm) in CRN patients, Fig. 4B.

Fig. 4
figure 4

Calculated mean RMax values displayed as µA·cm−2 from (A) single receptor models and (B) two receptor models upon biopsy stimulation with PGE2 or a selective EP receptor agonist. Numbers under the graph show N/n, N = number of patients, n = number of biopsies, NA = not applicable due to insufficient N/n. Data are presented as means ± SEM. *p < 0.05

Selective EP antagonists are unsuitable for determining EP receptor subtypes

Forty-one biopsies from 26 patients were exposed to EP antagonist cocktails, intended to inhibit all but one of the 4 EP receptor subtypes, followed by increasing PGE2 concentrations. To our surprise, we recorded sizable SCC increases upon ensuing PGE2 stimulation, even in the low nanomolar range, regardless of antagonist combination as well as in the presence of all 4 EP receptor antagonists, data not shown. These data indicate a lack of irreversible and/or competitive inhibition by all the 4 selective EP antagonists. Thus, with the present study design and protocol, none of the employed selective antagonists acted as expected.

Competitive antagonism between EP4 receptor antagonist GW-X and PGE2

Additional experiments were performed with only the selective EP4 antagonist GW-X, added prior to stimulation with PGE2. Figure 5 A shows the rightward shift induced by GW-X on PGE2 dose–response curves. The effect of GW-X demonstrates a competitive inhibition of PGE2 in the low nanomolar concentration range. Moreover, high PGE2 concentrations elicited about the same maximal increase in SCC regardless of GW-X addition, further supporting simple competitive antagonism between GW-X and PGE2. An agonist-based Cheng-Prusoff analysis of the PGE2-GW-X interactions resulted in an IC50 of 210 nM for GW-X, see Additional file 1: Data S1 and Fig. 5 B. To run a t-test for reliable judgement of differences in mean EC50s for GW-X between patient groups, more experiments are required.

Fig. 5
figure 5

Dose–response curves of PGE2 stimulation with and without EP4 antagonist GW627368X (GW-X) and calculated mean EC50 values. A: X-axis: PGE2 concentrations scaled logarithmically. Y-axis: changes in SCC. Triangles (black) show increases in SCC as a response to PGE2 doses without the addition of GW-X. Big dots (black) show increases in SCC in the presence of EP4 antagonist GW-X followed by PGE2 stimulation. The small dotted and the unbroken line (blue colors) resemble single (srm) and two receptor model (trm) fitting. Long dotted line (red) shows srm for experiments with GW-X, trm could not be calculated. B: Mean EC50 (nM) values of PGE2 and EP4 agonist TCS 2510 following inhibition with GW627368X (GW-X), calculated from single receptor model (srm) equations. Numbers under the graph show N/n, N = number of patients, n = number of biopsies. Data are presented as means ± SEM

EP1 and EP2 receptor subtypes are upregulated in CRN patients

mRNA expression levels of EP1 and EP2 were elevated in CRN patients compared to CTRLs, Fig. 6. EP3 and EP4 mRNA expression showed a trend of elevation in CRN patients.

Fig. 6
figure 6

Expression levels of investigated enzymes and receptors. Expression of EP1, EP2 5-LOX, 12-LOX, 15-LOX as well as PTGIS are significantly higher in CRN patients. Expression levels are relative to ß-actin and GAPDH. Data are presented as means ± SEM. *p < 0.05 and **p < 0.01

Enzymes related to the COX and LOX pathways are upregulated in CRN patients

All investigated LOX enzymes (5-LOX, 12-LOX, and 15- LOX) demonstrated elevated levels of mRNA in CRN patients compared to CTRLs, Fig. 6. Moreover, the expression of PTGIS was significantly upregulated in the CRN group, whereas expression levels of PTGDS and ARK1B1 were unaltered, Fig. 6.

Discussion

In the present study, we identified several differences in normal-appearing colonic mucosa from CRN patients, supporting the hypothesis of aberrations in enzymes and receptors of the eicosanoid pathway.

Independently of CRN history, we demonstrate that EP receptors bind PGE2 with 2 different affinities indicating the presence of high and low affinity EP receptor subtypes. Furthermore, we observed similar mucosal responses to selective EP2 and EP4 receptor agonists. Assuming selectivity of these compounds towards their receptors, our data suggest presence of both a high affinity EP4 and a low affinity EP2 receptor subtype [25, 26]. High and low affinity EP receptors in human colonic mucosa have been reported previously, but not investigated further [27, 28].

Our experiments identified the EP4 receptor to be the EP receptor subtype with the highest secretory response in the colon, which is consistent with existing reports [28, 29]. Furthermore, based on experiments with the highly selective EP4 receptor agonist TC 2510 [26], our data suggest a presence of both high and low affinity EP4 receptors with associated higher mean potencies and lower mean efficiencies compared to PGE2. Meanwhile, the existence of 2 EP4 receptors was not corroborated by experiments with the selective EP4 receptor antagonist, GW-X, which was effective in human colonic mucosa previously [28]. GW-X eliminated the biphasic PGE2 dose–response curve, resulting in a single receptor dose–response curve. This may be explained as a surmountable rightward potency-shift for a single EP4 high affinity receptor, moving it closer to the potency of the low affinity receptor(s) in the presence of GW-X, maintaining a combined efficiency at high concentrations of PGE2 with no antagonist present.

Stimulation of the EP4 subtype receptor is well documented as an important immunosuppressive trigger in the CRC microenvironment [30]. Accordingly, several interventional clinical phase-1 studies with focus on CRC have been initiated with newly developed EP4 antagonists [31], and recently another trial, testing an EP4 antagonist in metastatic CRC patients, has proceeded to phase II (NCT05205330). Furthermore, another study points to a carcinogenic mechanism involving pericryptal COX-2-expressing fibroblasts, which exert paracrine control over tumor-initiating stem cells via a COX-2 and PGE2–EP4–Yap signaling pathway [32, 33].

Taken together and respecting the relative few subjects in the present study, our findings support presence of high sensitivity for PGE2 in even normal appearing colonic mucosa.

Separate additions of single selective EP antagonists did not change the ensuing PGE2-induced SCC. Whether the PGE2-induced SCC increases reflect remaining secretion of incompletely inhibited EP receptor subtype(s) or resemble PGE2-induced secretion by other prostanoid receptors cannot be ascertained. Surprisingly, employed EP receptor antagonists, except for GW-X, were not useful in the present study. Our findings have not been tested under the same in vivo conditions by others, so the results await confirmation from other laboratories.

Our mRNA expression studies revealed increased expressions of receptor subtypes EP1 and EP2 in CRN patients. We, as others, have investigated EP receptor expression levels in human colonic tissue previously [34, 35]. The mRNA expression levels for EP1, EP2 and EP3 in this study are at variance with a former study from our laboratory [34]. Since identical primers against the subtype receptors were used in the 2 studies, presently the only recognized difference in study design were the number of reference genes, as two reference genes where used in the present study, while only one was used in the study by Petersen et al. Beside this our only other explanation for the deviation in results, is a greater variance in the general population of humans undetectable in small scale studies. Thus, our results should be taken as preliminary indication and be confirmed in much larger cohorts.

We found PTGIS expression to be upregulated in CRN patients. Previous expression studies of PTGIS/PGI2 in CRC patients have been ambiguous. One study found decreased PGI2 levels using radioimmunoassay in CRC patients [36]. Conversely, Lichao et al. found weak or no staining of PTGIS in normal tissue (corresponding to our biopsies from CRN patients) in microarray expression studies, while PTGIS expression was detected in CRC patients and increased in CRC patients with liver metastasis [37]. Merging results, we hypothesize a stepwise increase relationship in PTGIS expression and the degree of colonic mucosa dysplasia and risk of liver metastasis.

All tested LOX enzymes had higher mRNA expression levels in colonic mucosa from CRN patients. For 5-LOX and 12-LOX, this is consistent with the bulk of literature. Both enzymes elicit key pro-inflammatory and pro-tumorigenic downstream functions and are upregulated in human colon adenomas and adenocarcinomas [16, 38, 39]. Our results suggest that an upregulation of the LOX pathway is already present in normal appearing colonic mucosa from CRN patients. As such, 5-LOX and/or 12-LOX, enzyme expression might possess the potential of becoming an early predictive biomarker of CRN development.

Both 15-LOX isoforms are considered anti-tumorigenic and especially 15-LOX-1 and its product 13(S)-HODE appear tumor protective and downregulated in CRC tissue [17, 39]. Our employed 15-LOX primer unfortunately did not differentiate between the 2 isoforms. In contrast to previous studies, we observed increased 15-LOX expression in the mucosa of CRN patients. Given that we only investigated normal-appearing mucosa, the observed upregulated expression of 15-LOX might be a compensatory effect before mucosal cells become neoplastic. It would be interesting to further track the expression of 15-LOX, to determine whether the expression is suppressed as the cells become carcinogenic.

Several studies have addressed, documented, and discussed aberrant gene expression in tumor-adjacent colonic mucosa in relation to so-called field cancerization (tumor or environmental signaling) and mutator pathways based on tumor-induced mutations in DNA-repair genes, epigenetic methylation, genetic instability and tumor suppressor entities [18, 20, 40,41,42,43,44,45]. Furthermore, some aspects of such hypotheses are separated out as etiological factors termed ‘etiological field effects’ involving lifestyle, food mutagens, the gut microbiome, as well as environmental, hormonal, and genetic factors [43]. With an aspect of possible predisposition markers as in this study, only few studies have compared gene expression levels between normal colonic mucosa from control patients and macroscopically normal tumor-adjacent mucosa (> 10 cm tumor-distance), from CRN patients [34, 46, 47].

Lastly, it should be stressed, that our study is observational with a limited number of participants. Thus, our findings of aberrant enzyme and receptor expressions must be taken as indicators of possible predisposing factors, while confirmation of our observed statistically significant deviations requires much larger cohorts. In future studies, mRNA results should also be verified with other methods such as for example immunoblotting. Furthermore it would be preferable to get more cell type/molecular information per biopsy, as this is, even though well-known and accepted, a limitation to the study design.

Conclusions

Normal appearing colonic mucosa from patients with history of CRN demonstrates altered enzymatic expression of the eicosanoid pathway. Our data suggests a likely gene-based predisposition for early disease development. Furthermore, PGE2 did activate EP receptors with different affinity including a high affinity EP4 receptor with nanomolar potency to PGE2. Whether this highly sensitive EP4 receptor is tumorigenic and as such could be targeted in CRN management remains to be clarified. The observed aberrant gene expressions,

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

AA:

Arachidonic acid

AKR1B1:

Aldoketoreductase 1B1

ASA:

Acetylsalicylic acid

COX-1:

Cyclooxygenase 1

COX-2:

Cyclooxygenase 2

Cq:

Quantification cycle

CRC:

Colorectal cancer

CRN:

Colorectal neoplasia

cAMP:

Cyclic adenosine monophosphate

EP1:

Prostaglandin E2 receptor subtype 1

EP2:

Prostaglandin E2 receptor subtype 2

EP3:

Prostaglandin E2 receptor subtype 3

EP4:

Prostaglandin E2 receptor subtype 4

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

GW-X:

GW627368X

HETE:

Hydroxyeicosatetraenoic acids

HPETE:

Hydroperoxyl-eicosatetraenoic acids

MIQE Guidelines:

Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines

MUAS-chamber:

Modified Ussing air suction chamber

NSAID:

Non-steroid anti-inflammatory drug

PTGDS:

Prostaglandin D2 synthase

PGE2 :

Prostaglandin E2

PTGIS:

Prostaglandin-I2 synthase

qPCR:

Quantitative real-time polymerase chain reaction

SCC:

Short circuit current

References

  1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: Cancer J Clin. 2018;68(6):394–424.

    Google Scholar 

  2. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61(5):759–67.

    CAS  PubMed  Article  Google Scholar 

  3. Wang D, DuBois RN. An inflammatory mediator, prostaglandin E2, in colorectal cancer. Cancer J (Sudbury, Mass). 2013;19(6):502–10.

    CAS  Article  Google Scholar 

  4. Thun MJ, Namboodiri MM, Heath CW Jr. Aspirin use and reduced risk of fatal colon cancer. N Engl J Med. 1991;325(23):1593–6.

    CAS  PubMed  Article  Google Scholar 

  5. Thun MJ, Jacobs EJ, Patrono C. The role of aspirin in cancer prevention. Nat Rev Clin Oncol. 2012;9(5):259–67.

    CAS  PubMed  Article  Google Scholar 

  6. Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Ferrenbach S, DuBois RN. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology. 1994;107(4):1183–8.

    CAS  PubMed  Article  Google Scholar 

  7. Drew DA, Cao Y, Chan AT. Aspirin and colorectal cancer: the promise of precision chemoprevention. Nat Rev Cancer. 2016;16(3):173–86.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Wang D, Dubois RN. Prostaglandins and cancer. Gut. 2006;55(1):115–22.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. Watanabe K, Kawamori T, Nakatsugi S, Ohta T, Ohuchida S, Yamamoto H, et al. Role of the prostaglandin E receptor subtype EP1 in colon carcinogenesis. Can Res. 1999;59(20):5093–6.

    CAS  Google Scholar 

  10. Sonoshita M, Takaku K, Sasaki N, Sugimoto Y, Ushikubi F, Narumiya S, et al. Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc(Delta 716) knockout mice. Nat Med. 2001;7(9):1048–51.

    CAS  PubMed  Article  Google Scholar 

  11. Mutoh M, Watanabe K, Kitamura T, Shoji Y, Takahashi M, Kawamori T, et al. Involvement of prostaglandin E receptor subtype EP(4) in colon carcinogenesis. Can Res. 2002;62(1):28–32.

    CAS  Google Scholar 

  12. Fujino H. The roles of EP4 prostanoid receptors in cancer malignancy signaling. Biol Pharm Bull. 2016;39(2):149–55.

    CAS  PubMed  Article  Google Scholar 

  13. Karpisheh V, Joshi N, Zekiy AO, Beyzai B, Hojjat-Farsangi M, Namdar A, et al. EP4 receptor as a novel promising therapeutic target in colon cancer. Pathol Res Pract. 2020;216(12): 153247.

    CAS  PubMed  Article  Google Scholar 

  14. Wang D, Dubois RN. Eicosanoids and cancer. Nat Rev Cancer. 2010;10(3):181–93.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Cathcart MC, Reynolds JV, O’Byrne KJ, Pidgeon GP. The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer. Biochem Biophys Acta. 2010;1805(2):153–66.

    CAS  PubMed  Google Scholar 

  16. Pidgeon GP, Lysaght J, Krishnamoorthy S, Reynolds JV, O’Byrne K, Nie D, et al. Lipoxygenase metabolism: roles in tumor progression and survival. Cancer Metastasis Rev. 2007;26(3–4):503–24.

    CAS  PubMed  Article  Google Scholar 

  17. Shureiqi I, Chen D, Day RS, Zuo X, Hochman FL, Ross WA, et al. Profiling lipoxygenase metabolism in specific steps of colorectal tumorigenesis. Cancer Prev Res (Phila, PA). 2010;3(7):829–38.

    CAS  Article  Google Scholar 

  18. Shen L, Kondo Y, Rosner GL, Xiao L, Hernandez NS, Vilaythong J, et al. MGMT promoter methylation and field defect in sporadic colorectal cancer. J Natl Cancer Inst. 2005;97(18):1330–8.

    CAS  PubMed  Article  Google Scholar 

  19. Rebello D, Rebello E, Custodio M, Xu X, Gandhi S, Roy HK. Field carcinogenesis for risk stratification of colorectal cancer. Adv Cancer Res. 2021;151:305–44.

    PubMed  Article  Google Scholar 

  20. Natali F, Rancati G. The mutator phenotype: adapting microbial evolution to cancer biology. Front Genet. 2019;10:713.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Michaud A, Lacroix-Pépin N, Pelletier M, Veilleux A, Noël S, Bouchard C, et al. Prostaglandin (Pg) F2 alpha synthesis in human subcutaneous and omental adipose tissue: modulation by inflammatory cytokines and role of the human aldose reductase Akr1b1. PLoS ONE. 2014;9(3):1–10.

    Article  CAS  Google Scholar 

  22. Larsen R, Mertz-Nielsen A, Hansen MB, Poulsen SS, Bindslev N. Novel modified Ussing chamber for the study of absorption and secretion in human endoscopic biopsies. Acta Physiol Scand. 2001;173(2):213–22.

    CAS  PubMed  Article  Google Scholar 

  23. Johnson G, Nour AA, Nolan T, Huggett J, Bustin S. Minimum information necessary for quantitative real-time PCR experiments. Methods Mol Biol (Clifton, NJ). 2014;1160:5–17.

    CAS  Article  Google Scholar 

  24. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):RESEARCH0034.

    PubMed  PubMed Central  Article  Google Scholar 

  25. Sugimoto Y, Narumiya S. Prostaglandin E receptors. J Biol Chem. 2007;282(16):11613–7.

    CAS  PubMed  Article  Google Scholar 

  26. Billot X, Chateauneuf A, Chauret N, Denis D, Greig G, Mathieu MC, et al. Discovery of a potent and selective agonist of the prostaglandin EP4 receptor. Bioorg Med Chem Lett. 2003;13(6):1129–32.

    CAS  PubMed  Article  Google Scholar 

  27. Kjærgaard S, Damm MMB, Chang J, Riis LB, Rasmussen HB, Hytting-Andreasen R, et al. Altered structural expression and enzymatic activity parameters in quiescent ulcerative colitis: are these potential normalization criteria? Int J Mol Sci. 2020;21(5):1887.

    PubMed Central  Article  CAS  Google Scholar 

  28. Fairbrother SE, Smith JE, Borman RA, Cox HM. Ep4 receptors mediate prostaglandin E2, tumour necrosis factor alpha and interleukin 1beta-induced ion secretion in human and mouse colon mucosa. Eur J Pharmacol. 2012;694(1–3):89–97.

    CAS  PubMed  Article  Google Scholar 

  29. Kaltoft N, Tilotta MC, Witte AB, Osbak PS, Poulsen SS, Bindslev N, et al. Prostaglandin E2-induced colonic secretion in patients with and without colorectal neoplasia. BMC Gastroenterol. 2010;10:9.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  30. Hong DS, Parikh A, Shapiro GI, Varga A, Naing A, Meric-Bernstam F, et al. First-in-human phase I study of immunomodulatory E7046, an antagonist of Pge2-receptor E-type 4 (Ep4), in patients with advanced cancers. J Immunother Cancer. 2020;8(1):e000222.

    PubMed  PubMed Central  Article  Google Scholar 

  31. Ching MM, Reader J, Fulton AM. Eicosanoids in cancer: prostaglandin E2 receptor 4 in cancer therapeutics and immunotherapy. Front Pharmacol. 2020;11:819.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  32. Roulis M, Kaklamanos A, Schernthanner M, Bielecki P, Zhao J, Kaffe E, et al. Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche. Nature. 2020;580(7804):524–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Wang D, DuBois RN. Fibroblasts fuel intestinal tumorigenesis. Cell Res. 2020;30(8):635–6.

    PubMed  PubMed Central  Article  Google Scholar 

  34. Petersen CH, Mahmood B, Badsted C, Dahlby T, Rasmussen HB, Hansen MB, et al. Possible predisposition for colorectal carcinogenesis due to altered gene expressions in normal appearing mucosa from patients with colorectal neoplasia. BMC Cancer. 2019;19(1):643.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  35. Gustafsson A, Hansson E, Kressner U, Nordgren S, Andersson M, Wang W, et al. EP1–4 subtype, COX and PPAR gamma receptor expression in colorectal cancer in prediction of disease-specific mortality. Int J Cancer. 2007;121(2):232–40.

    CAS  PubMed  Article  Google Scholar 

  36. Rigas B, Goldman IS, Levine L. Altered eicosanoid levels in human colon cancer. J Lab Clin Med. 1993;122(5):518–23.

    CAS  PubMed  Google Scholar 

  37. Lichao S, Liang P, Chunguang G, Fang L, Zhihua Y, Yuliang R. Overexpression of PTGIS could predict liver metastasis and is correlated with poor prognosis in colon cancer patients. Pathol Oncol Res: POR. 2012;18(3):563–9.

    PubMed  Article  CAS  Google Scholar 

  38. Melstrom LG, Bentrem DJ, Salabat MR, Kennedy TJ, Ding XZ, Strouch M, et al. Overexpression of 5-lipoxygenase in colon polyps and cancer and the effect of 5-Lox inhibitors in vitro and in a murine model. Clin Cancer Res: Off J Am Assoc Cancer Res. 2008;14(20):6525–30.

    CAS  Article  Google Scholar 

  39. Tuncer S, Banerjee S. Eicosanoid pathway in colorectal cancer: recent updates. World J Gastroenterol. 2015;21(41):11748–66.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Loeb LA, Bielas JH, Beckman RA. Cancers exhibit a mutator phenotype: clinical implications. Cancer Res. 2008;68(10):3551–7 (discussion 7).

    CAS  PubMed  Article  Google Scholar 

  41. Facista A, Nguyen H, Lewis C, Prasad AR, Ramsey L, Zaitlin B, et al. Deficient expression of DNA repair enzymes in early progression to sporadic colon cancer. Genome Integr. 2012;3(1):3.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Hawthorn L, Lan L, Mojica W. Evidence for field effect cancerization in colorectal cancer. Genomics. 2014;103(2–3):211–21.

    CAS  PubMed  Article  Google Scholar 

  43. Lochhead P, Chan AT, Nishihara R, Fuchs CS, Beck AH, Giovannucci E, et al. Etiologic field effect: reappraisal of the field effect concept in cancer predisposition and progression. Modern Pathol: Off J U S Can Acad Pathol Inc. 2015;28(1):14–29.

    Article  Google Scholar 

  44. Aran D, Camarda R, Odegaard J, Paik H, Oskotsky B, Krings G, et al. Comprehensive analysis of normal adjacent to tumor transcriptomes. Nat Commun. 2017;8(1):1077.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  45. Sugai T, Yoshida M, Eizuka M, Uesugii N, Habano W, Otsuka K, et al. Analysis of the DNA methylation level of cancer-related genes in colorectal cancer and the surrounding normal mucosa. Clin Epigenetics. 2017;9:55.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  46. Mahmood B, Damm MMB, Jensen TSR, Backe MB, Dahllöf MS, Poulsen SS, et al. Phosphodiesterases in non-neoplastic appearing colonic mucosa from patients with colorectal neoplasia. BMC Cancer. 2016;16:938.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  47. Sanz-Pamplona R, Berenguer A, Cordero D, Molleví DG, Crous-Bou M, Sole X, et al. Aberrant gene expression in mucosa adjacent to tumor reveals a molecular crosstalk in colon cancer. Mol Cancer. 2014;13:46.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  48. Bindslev N. Drug-acceptor interactions: modeling theoretical tools to test and evaluate experimental equilibrium effects. 1st ed. London: Taylor & Francis Group; 2008. p. 33–57. https://doi.org/10.4324/9781315159782.

    Book  Google Scholar 

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Acknowledgements

We thank the staff at endoscopy unit of Digestive Disease Center at Bispebjerg Hospital for general support. A special thanks goes to Ono Pharmaceutical Co., Ltd. (Osaka, Japan) for providing us with ONO-DI004 and ONO-AE1-259 compounds.

Funding

This work was kindly supported by grants from Harboefonden (jr.no. 17368), Anita og Tage Therkelsen Fond (jr.no. 100039), Else og Mogens Wedell-Wedellsborg Fond (jr.no. 15-18-1), Trigon Fonden, Andersen-Isted Fonden (jr.no 2017-04) and C.C. Klestrup og hustru Henriette Klestrups Mindelegat (jr.no. 10761). TAJ was funded by Lundbeckfonden (R323-2018-3674).

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Contributions

URF was the principal investigator, took part in every aspect of this study and was major contributor in the writing of the manuscript. SKH was a major contributor in performing analyses of functional data and contributed in writing the manuscript. MABH contributed in generating functional data. TAJ contributed as an expert in performing and analyzing the expressional data. NB contributed as an expert in the functional part of the study, its study design, in data analysis and contributed in writing the manuscript. MBH served as the supervisor of the project and contributed in writing the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Ulrike Ries Feddersen.

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Ethics approval and consent to participate

The study protocol was approved by the Scientific Ethical Committee of Copenhagen (H-3-2013-107) and the Danish Data Protection Agency (BBH-2013-024, I-Suite no: 02342). The study was conducted in accordance with the Helsinki declaration. All participating patients gave written informed consent.

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Not applicable.

Competing interests

Mark Berner-Hansen is also a present employee of Zealand Pharma, Denmark. The present work was not related to this affiliation. All authors declare no competing interests.

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Supplementary Information

Additional file 1.

Supplementary study data.

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Feddersen, U.R., Hendel, S.K., Berner-Hansen, M.A. et al. Nanomolar EP4 receptor potency and expression of eicosanoid-related enzymes in normal appearing colonic mucosa from patients with colorectal neoplasia. BMC Gastroenterol 22, 234 (2022). https://doi.org/10.1186/s12876-022-02311-z

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Keywords

  • Colorectal cancer
  • EP receptors
  • mRNA expression
  • Short circuit current
  • Lipoxygenase