Prostaglandin E2 promotes intestinal tumor growth via DNA methylation

Abstract

Although aberrant DNA methylation is considered to be one of the key ways by which tumor-suppressor and DNA-repair genes are silenced during tumor initiation and progression, the mechanisms underlying DNA methylation alterations in cancer remain unclear. Here we show that prostaglandin E(2) (PGE(2)) silences certain tumor-suppressor and DNA-repair genes through DNA methylation to promote tumor growth. These findings uncover a previously unrecognized role for PGE(2) in the promotion of tumor progression.

Figure 1

PGE₂ silences certain tumor suppressor and DNA repair genes by enhancing their promoter CGI methylation in human CRC cell lines. (a) PGE₂ increased DNMT1 and DNMT3B protein expression in LS-174T, HCA7, and HT-29 cells. (b) Bisulfite PCR sequencing analysis showed that PGE₂ increased CGI methylation in the promoters of CNR1 and MGMT in LS-174 cells. For CNR1 promoter, a region (−370 to −160) that contains 24 CpGs was examined. Two representative CpGs were presented. For MGMT promoter, a region (+27 to +342) that contains 29 CpGs was examined. Six representative CpGs were presented. The asterix indicates the locations of CpGs. (c) PGE₂ downregulated the expression of CNR1 (CB1) and MGMT at both protein (upper panels) and mRNA (lower panels) levels in LS-174T cells. Error bars indicate s.d. * P < 0.05 (two-tailed unpaired Student’s t test). (d) Blockade of PTGER4 (EP4) attenuated the upregulation of DNMT1 and DNMT3B by PGE₂ in LS-174T cells. SC19220 (SC): PTGER1 (EP1) antagonist; AH6809 (AH): PTGER1-3 (EP1-3) antagonist; ONOAE-208 (ONO): PTGER4 (EP4) antagonist. (e,f) Knockdown of DNMT1 or DNMT3B by shRNAs attenuated PGE₂-induced downregulation of CNR1 (CB1) and MGMT in LS-174T cells. Knockdown efficiency was examined by Q-PCR in two clones (C1 and C2) along with a non-silencing shRNA transfected control (shCon) (upper panels). Error bars indicate s.d. * P < 0.05 (two-tailed unpaired Student’s t test). CNR1 (CB1) and MGMT protein expression was examined by western blotting in these two clones (C1 and C2) and control ShCon cells (lower panels).

Figure 2

PGE₂ promotes intestinal tumor growth via upregulating CGI methylation in Apc^Min/+ mice. (a) Treatment of Apc^Min/+ mice with PGE₂ increased Dnmt1 and Dnmt3b protein expression in the colonic tumor epithelial cells. (b,c) PGE₂ increased intestinal polyp number and size in Apc^Min/+ mice. Error bars indicate s.e.m (n = 7 for each group). * P <0.05 (Wilcoxon Rank Sum test). No.: number; SI: small intestine. (d) Treatment of three Apc^Min/+ mice with PGE₂ increased the promoter CGI methylation of Cnr1 and Mgmt in the colonic tumor epithelial cells as compared to three Apc^Min/+ mice treated with vehicle. For Cnr1 promoter, a region (−369 to −34) that contains 35 CpGs was examined. Three representative CpGs were presented. For Mgmt promoter, a region (−458 to −243) that contains 6 CpGs was examined. Two representative CpGs were presented. Asterix indicates the locations of CpGs. (e) Treatment of Apc^Min/+ mice with PGE₂ decreased the expression of Cnr1 and Mgmt at both protein levels (left panel) and mRNA levels (middle and right panels) in the colonic tumor epithelial cells. One representative result from three mice was shown. Error bars indicate s.d. * P < 0.05 (two-tailed unpaired Student’s t test). (f) Inhibition of CGI methylation by 5-Aza-dC attenuates PGE₂-induced tumor growth in male Apc^Min/+ mice. Error bars indicate s.e.m. (n = 15 for each group. Wilcoxon Rank Sum test). (g) Combination treatment with celecoxib and 5-Aza-dC more efficiently inhibited tumor growth. Error bars indicate s.e.m. (n = 12, 14, 14, and 7, respectively. Wilcoxon Rank Sum test).