Prostaglandin E2 Activates YAP and a Positive-Signaling Loop to Promote Colon Regeneration After Colitis but Also Carcinogenesis in Mice

Background & AimsProstaglandin E2 (PGE2) is mediator of inflammation that regulates tissue regeneration, but its continual activation has been associated with carcinogenesis. Little is known about factors in the PGE2 signaling pathway that contribute to tumor formation. We investigated whether yes-associated protein 1 (YAP1), a transcriptional co-activator in the Hippo signaling pathway, mediates PGE2 function.MethodsDLD-1 and SW480 colon cancer cell lines were transfected with vectors expressing transgenes or small hairpin RNAs and incubated with recombinant PGE2, with or without pharmacologic inhibitors of signaling proteins, and analyzed by immunoblot, immunofluorescence, quantitative reverse-transcription polymerase chain reaction, transcriptional reporter, and proliferation assays. Dextran sodium sulfate (DSS) was given to induce colitis in C57/BL6 (control) mice, as well as in mice with disruption of the hydroxyprostaglandin dehydrogenase 15 gene (15-PGDH–knockout mice), Yap1 gene (YAP-knockout mice), and double-knockout mice. Some mice also were given indomethacin to block PGE2 synthesis. 15-PGDH knockout mice were crossed with mice with intestine-specific disruption of the salvador family WW domain containing 1 gene (Sav1), which encodes an activator of Hippo signaling. We performed immunohistochemical analyses of colon biopsy samples from 26 patients with colitis-associated cancer and 51 age-and sex-matched patients with colorectal cancer (without colitis).ResultsIncubation of colon cancer cell lines with PGE2 led to phosphorylation of cyclic adenosine monophosphate–responsive element binding protein 1 and increased levels of YAP1 messenger RNA, protein, and YAP1 transcriptional activity. This led to increased transcription of the prostaglandin-endoperoxide synthase 2 gene (PTGS2 or cyclooxygenase 2) and prostaglandin E–receptor 4 gene (PTGER4 or EP4). Incubation with PGE2 promoted proliferation of colon cancer cell lines, but not cells with knockdown of YAP1. Control mice developed colitis after administration of DSS, but injection of PGE2 led to colon regeneration in these mice. However, YAP-knockout mice did not regenerate colon tissues and died soon after administration of DSS. 15-PGDH–knockout mice regenerated colon tissues more rapidly than control mice after withdrawal of DSS, and had faster recovery of body weight, colon length, and colitis histology scores. These effects were reversed by injection of indomethacin. SAV1-knockout or 15-PGDH–knockout mice did not develop spontaneous tumors after colitis induction, but SAV1/15-PGDH double-knockout mice developed polyps that eventually progressed to carcinoma in situ. Administration of indomethacin to these mice prevented spontaneous tumor formation. Levels of PGE2 correlated with those of YAP levels in human sporadic colorectal tumors and colitis-associated tumors.ConclusionsPGE2 signaling increases the expression and transcriptional activities of YAP1, leading to increased expression of cyclooxygenase 2 and EP4 to activate a positive signaling loop. This pathway promotes proliferation of colon cancer cell lines and colon tissue regeneration in mice with colitis. Constitutive activation of this pathway led to formation of polyps and colon tumors in mice. Prostaglandin E2 (PGE2) is mediator of inflammation that regulates tissue regeneration, but its continual activation has been associated with carcinogenesis. Little is known about factors in the PGE2 signaling pathway that contribute to tumor formation. We investigated whether yes-associated protein 1 (YAP1), a transcriptional co-activator in the Hippo signaling pathway, mediates PGE2 function. DLD-1 and SW480 colon cancer cell lines were transfected with vectors expressing transgenes or small hairpin RNAs and incubated with recombinant PGE2, with or without pharmacologic inhibitors of signaling proteins, and analyzed by immunoblot, immunofluorescence, quantitative reverse-transcription polymerase chain reaction, transcriptional reporter, and proliferation assays. Dextran sodium sulfate (DSS) was given to induce colitis in C57/BL6 (control) mice, as well as in mice with disruption of the hydroxyprostaglandin dehydrogenase 15 gene (15-PGDH–knockout mice), Yap1 gene (YAP-knockout mice), and double-knockout mice. Some mice also were given indomethacin to block PGE2 synthesis. 15-PGDH knockout mice were crossed with mice with intestine-specific disruption of the salvador family WW domain containing 1 gene (Sav1), which encodes an activator of Hippo signaling. We performed immunohistochemical analyses of colon biopsy samples from 26 patients with colitis-associated cancer and 51 age-and sex-matched patients with colorectal cancer (without colitis). Incubation of colon cancer cell lines with PGE2 led to phosphorylation of cyclic adenosine monophosphate–responsive element binding protein 1 and increased levels of YAP1 messenger RNA, protein, and YAP1 transcriptional activity. This led to increased transcription of the prostaglandin-endoperoxide synthase 2 gene (PTGS2 or cyclooxygenase 2) and prostaglandin E–receptor 4 gene (PTGER4 or EP4). Incubation with PGE2 promoted proliferation of colon cancer cell lines, but not cells with knockdown of YAP1. Control mice developed colitis after administration of DSS, but injection of PGE2 led to colon regeneration in these mice. However, YAP-knockout mice did not regenerate colon tissues and died soon after administration of DSS. 15-PGDH–knockout mice regenerated colon tissues more rapidly than control mice after withdrawal of DSS, and had faster recovery of body weight, colon length, and colitis histology scores. These effects were reversed by injection of indomethacin. SAV1-knockout or 15-PGDH–knockout mice did not develop spontaneous tumors after colitis induction, but SAV1/15-PGDH double-knockout mice developed polyps that eventually progressed to carcinoma in situ. Administration of indomethacin to these mice prevented spontaneous tumor formation. Levels of PGE2 correlated with those of YAP levels in human sporadic colorectal tumors and colitis-associated tumors. PGE2 signaling increases the expression and transcriptional activities of YAP1, leading to increased expression of cyclooxygenase 2 and EP4 to activate a positive signaling loop. This pathway promotes proliferation of colon cancer cell lines and colon tissue regeneration in mice with colitis. Constitutive activation of this pathway led to formation of polyps and colon tumors in mice.