Role of prostaglandin receptor EP2 in the regulations of cancer cell proliferation, invasion, and inflammation

Abstract

Population studies, preclinical, and clinical trials suggest a role for cyclooxygenase-2 (COX-2, PTGS2) in tumor formation and progression. The downstream prostanoid receptor signaling pathways involved in tumorigenesis are poorly understood, although prostaglandin E2 (PGE(2)), a major COX-2 metabolite which is usually upregulated in the involved tissues, presumably plays important roles in tumor biology. Taking advantage of our recently identified novel selective antagonist for the EP2 (PTGER2) subtype of PGE(2) receptor, we demonstrated that EP2 receptor activation could promote prostate cancer cell growth and invasion in vitro, accompanied by upregulation of the tumor-promoting inflammatory cytokines, such as IL-1β and IL-6. Our results suggest the involvement of prostaglandin receptor EP2 in cancer cell proliferation and invasion possibly via its inflammatory actions, and indicate that selective blockade of the PGE(2)-EP2 signaling pathway via small molecule antagonists might represent a novel therapy for tumorigenesis.

Fig. 1.

COX-2 and prostaglandin receptor EP2 signaling in cancer cells. (A) Chemical structure of EP2 antagonist TG4-155 (SID 17,515,129). (B) The Schild K_B values of TG4-155 were compared for inhibition of eight human prostanoid receptors. Schild K_B values: 34.5, 2100, 2.4, 10,000, 11,400, 10,000, 1320, and 910 nM for DP1, EP1, EP2, EP3, EP4, FP, IP, and TP receptors, respectively. (C) The expression of COX-2 and EP2 receptor in three human prostate cancer cell lines, DU145, LNCap, and PC3, was examined by Western blot analysis. A low basal level of COX-2 (72 kDa) was detected in all three types of cancer cells and the PC3 cells have a relatively high level of EP2 (52 kDa) expressed. (D) Data mining from NCI CellMiner (http://discover.nci.nih.gov). Relative mRNA levels in PC3 cells of COX-2; prostaglandin E synthase; prostanoid receptors DP1, EP1, EP2, EP3, EP4, FP, IP, and TP; and cytokines IL-1β, IL-6, IFN-γ, and TNF-α, as measured by Agilent Whole Human Genome Oligo Microarray kit (Agilent-mRNA, Agilent Technologies). (E) Butaprost induced cAMP production in PC3 cells with an EC₅₀ = 54 nM, which was substantially blocked by EP2 antagonist TG4-155 in a concentration-dependent manner. Data were normalized as percent maximum response; points represent mean ± S.E.M. (n = 4 independent experiments). (F) Schild regression analysis was performed to evaluate the potency of TG4-155 in PC3 cells. TG4-155 displayed a competitive antagonism mode of action on EP2 receptor shown by Schild plot with a K_B value 1.3 nM and a slope of 1.0.

Fig. 2.

Effects of EP2 receptor activation and inhibition on cancer cell proliferation and invasion. (A) EP2 activation by its natural ligand, PGE₂, promoted PC3 cell growth, measured by MTT cell proliferation assay; growth was attenuated by treatment with compound TG4-155 in a concentration-dependent manner. (B) EP2 selective agonist butaprost promoted PC3 cell growth, which was also attenuated by TG4-155. (C) TG4-155 blocked the PC3 cell invasion triggered by EP2 activation. Butaprost (1 µM) treatment significantly increased the number of cells crossing the filter coated with Matrigel, which was blocked by cotreatment with EP2 antagonist TG4-155 (1 µM). Bars represent the mean ± S.E.M. (n = 4 independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001 by one-way ANOVA and post hoc Bonferroni test with selected pairs.

Fig. 3.

EP2 receptor activation induces inflammatory cytokines in cancer cells. (A) PC3 cells were incubated with 1 µM butaprost for 0, 2, and 8 hours, and the levels of four proinflammatory cytokines, IL-1β, IL-6, IFN-γ, and TNF-α in the medium were measured. EP2 activation by butaprost significantly elevated IL-1β at 2 hours and IL-6 at 8 hours after butaprost stimulation, but not IFN-γ and TNF-α. (B) Cytokine induction following EP2 activation was mitigated by treatment with EP2 antagonist TG4-155 (1 µM), assessed at 2 hours and 8 hours after treatment of IL-1β and IL-6, respectively. Bars represent the mean ± S.E.M. (n = 5 independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001 by one-way ANOVA and post hoc Bonferroni test with selected pairs.

Fig. 4.

EP2 receptor mediates cancer cell activities possibly via an inflammatory mechanism. (A) Low cytotoxicity of EP2 antagonist TG4-155. Cytotoxicity of TG4-155 was tested in C6G cells with the CellTiter-Glo luminescent cell viability assay. TG4-155 did not show significant cytotoxicity with CC₅₀ = 190 µM; doxorubicin as positive control with CC₅₀ = 0.23 µM. Data are shown as mean ± S.E.M. (n ≥ 6 independent experiments). (B) Model proposed for the inflammatory action of EP2 signaling in cancer cell activities. COX-2 increases PGE₂ level in tumor tissues. PGE₂ signaling through the EP2 receptor upregulates tumor-promoting cytokines, including IL-1β and IL-6 in cancer cells, while downregulating antitumor cytokines such as IFN-γ and TNF-α in immune cells (Yamane et al., 2000; Walker and Rotondo, 2004; Li et al., 2006; Oxford et al., 2010); therefore, tumor growth and invasion are facilitated. A selective EP2 antagonist might reduce inflammation in tumor tissues, thus repress tumor growth.