Downregulation of prostaglandin E receptor subtype EP3 during colon cancer development

Abstract

Background and aims: Involvement of prostaglandin E(2) (PGE(2)) receptors EP(1), EP(2), and EP(4) in the formation of aberrant crypt foci (ACF) and/or intestinal polyps has been suggested. In contrast, EP(3) appears to have no influence on the early stages of colon carcinogenesis. In the present study, we examined expression of PGE(2) receptor subtypes EP(1), EP(2), EP(3), and EP(4) in normal colon mucosa and colon cancers, and assessed the contribution of EP(3) to colon cancer development.

Methods: mRNA expression of PGE(2) receptor subtypes EP(1), EP(2), EP(3), and EP(4) in normal colon mucosa and colon cancers in azoxymethane (AOM) treated mice and rats, and in humans, were examined by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real time RT-PCR, and immunohistochemical analyses. Evaluation of the role of EP(3) was performed by intraperitoneal injection of AOM, using EP(3) receptor knockout mice. Effects of EP(3) receptor activation on cell growth of human colon cancer cell lines were examined using ONO-AE-248, an EP(3) selective agonist. Moreover, EP(3) expression in colon cancer cell lines was analysed with or without 5-aza-2'-deoxycytidine (5-aza-dC) treatment.

Results: Expression levels of EP(1) and EP(2) mRNA were increased in cancer tissues. EP(4) mRNA was constantly expressed in normal mucosa and cancers. In contrast, expression of EP(3) mRNA was markedly decreased in colon cancer tissues, being 5% in mice, 9% in rats, and 28% in humans compared with normal colon mucosa, analysed by quantitative real time RT-PCR. Immunohistochemical staining demonstrated the rat EP(3) receptor protein to be expressed in epithelial cells of normal mucosa and some parts of small carcinomas but hardly detectable in large carcinomas of the colon. Colon cancer development induced by AOM in EP(3) receptor knockout mice was enhanced compared with wild-type mice, with a higher incidence of colon tumours (78% v 57%) and mean number of tumours per mouse (2.17 (0.51) v 0.75 (0.15); p<0.05). Expression of EP(3) mRNA was detected in only one of 11 human colon cancer cell lines tested. Treatment with 5 microM of an EP(3) selective agonist, ONO-AE-248, resulted in a 30% decrease in viable cell numbers in the HCA-7 human colon cancer cell line in which EP(3) was expressed. Treatment with 5-aza-dC restored EP(3) expression in CACO-2, CW-2, and DLD-1 cells but not in WiDr cells, suggesting involvement of hypermethylation in the downregulation of EP(3) to some extent.

Conclusion: The PGE(2) receptor subtype EP(3) plays an important role in suppression of cell growth and its downregulation enhances colon carcinogenesis at a later stage. Hypermethylation of the EP(3) receptor gene could occur and may contribute towards downregulating EP(3) expression to some extent in colon cancers.

Figure 1

Analyses of prostaglandin E₂ (PGE₂) receptors EP₁, EP₂, EP₃, and EP₄ mRNA expression. (A) Azoxymethane (AOM) treated mouse normal colon mucosa and colon carcinomas. Two pairs of samples (lanes 1, 2) and two independent samples (lane 3) were examine by reverse transcription-polymerase chain reaction (RT-PCR). (B) AOM treated rat normal colon mucosa and colon carcinomas. Four pairs of samples (lanes 1–4) were examined by RT-PCR. Expression levels of EP₃ receptor mRNA were markedly lower in adenocarcinomas than in normal mucosa in all cases. (C, D) Quantitative real time RT-PCR analysis revealed significant downregulation of EP₃ receptor mRNA in AOM treated mice (C) and rat (D) colon carcinomas compared with normal colon mucosa (mouse, n = 3; rat, n = 4). EP₃ receptor mRNA expression was downregulated in tumours, being 5% in the mouse and 9% in the rat of the average value of that in the respective normal colon mucosa. Values are mean (SD); *p<0.05, **p<0.01. (A–D) β-Actin was used as an internal control. PCR primers of mouse and rat EP₃ receptors were designed to target a sequence common to all EP₃ receptor variants expressed in each species.

Figure 2

Analyses of prostaglandin E₂ (PGE₂) receptors EP₁, EP₂, EP₃, and EP₄ mRNA expression in human colon tissues. (A) Reverse transcription-polymerase chain reaction (RT-PCR) analysis patterns in four typical pairs of samples (lanes 1–4) are shown. (B, C) Quantitative real time RT-PCR analysis revealed significant downregulation in EP₃ receptor mRNA. (B) EP₃ receptor mRNA was markedly decreased in seven of eight samples of adenocarcinomas compared with adjacent normal mucosa of the colon. (C) EP₃ receptor mRNA expression was downregulated in tumours, being 28% of the average value of that in adjacent normal colon mucosa. Values are mean (SD); *p<0.05. (A–C) β-Actin was used as an internal control. PCR primers of human EP₃ receptors were designed to target a sequence common to all EP₃ receptor variants expressed.

Figure 3

Immunohistochemical staining for the rat prostaglandin E₂ receptor subtype EP₃ of normal colon mucosa (A, C, and E) and colon adenocarcinoma (B, D, and F). Non-specific staining of some red blood cells and weak background staining were observed in the negative controls stained without anti-EP₃ receptor antibody (A, B) and in the negative controls stained with preabsorbed anti-EP₃ receptor antibody (C, D). With anti-EP₃ receptor antibody, immunoreactive EP₃ receptors were prominent in epithelial cells of normal colon mucosa (E) but no EP₃ receptor immunoreactivity was apparent in a colon adenocarcinoma (F). Magnification ×100.

Figure 4

Size distribution of colon tumours induced by azoxymethane in wild-type and prostaglandin E₂ receptor subtype EP₃ knockout (KO) mice. The number of tumours/mouse in each size class is expressed as mean (SEM). **Significantly different from the corresponding wild-type value (p<0.01).

Figure 5

Effect of ONO-AE-248 treatment on cell growth of DLD-1 and HCA-7 cells. (A) Expression of prostaglandin E₂ (PGE₂) receptors EP₁, EP₂, EP₃, and EP₄ was analysed by reverse transcription-polymerase chain reaction in 11 human colon cancer cell lines. (B) DLD-1 and HCA-7 cells were seeded onto 96 well plates at a density of 2×10³ cells/well, with media containing 5% fetal bovine serum, and treated with the EP₃ receptor selective agonist ONO-AE-248 on days 0-4. Then, cell numbers were measured by WST-1 assay on days 1, 3, and 5. Open symbols indicate DLD-1 and closed symbols HCA-7 cells; concentrations of ONO-AE-248 treatment are indicated (μM). Data are means (n = 6). *p<0.05, **p<0.01.

Figure 6

5-Aza-2′-deoxycytidine (5-aza-dC) treatment of CACO-2, CW-2, DLD-1, HCA-7, and WiDr colon cancer cell lines. Each cell line was treated with 1 and 2 μM 5-aza-dC three times. EP₃ receptor expression was analysed by reverse transcription-polymerase chain reaction.