IntroductionNAFLD is characterized by an excessive accumulation of fat in the liver. The most severe form of NAFLD, NASH, often progresses to liver cancer (1-3). Tracking with the increasing prevalence of general obesity, 30% of the US population is now estimated to have NAFLD, and 25% of these individuals will develop NASH (1, 3). Currently, there are no effective therapies to prevent the incidence and progression of NAFLD or NASH (4). This makes clarification of the detailed mechanisms of disease progression using appropriate animal models that much more urgent.Insulin signaling begins with the binding of insulin to the insulin receptor (IR). This then phosphorylates insulin receptor substrates (IRSs) and subsequently triggers the recruitment of PI3K and the activation of AKT (5-9). Deletion of IRS1 and IRS2 in the mouse liver reduces AKT activity and gives rise to insulin resistance (10). Excessive AKT activation leads to the development of NAFLD by promoting the maturation of the transcription factor SREBP1c (11,12). In its mature form, SREBP1c contributes to the induction of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), which are key enzymes in de novo lipogenesis (13). Phosphatase and tensin homolog (PTEN) is a negative regulator of AKT signaling, and several human cancers are associated with mutations or downregulation of the PTEN gene (14-16). Liver-specific PTEN-knockout mice progressively develop NAFLD, NASH, and HCC (17, 18) as a result of increased AKT signaling (19).The Hippo signaling pathway has been implicated in the suppression of tissue regeneration, the proliferation of stem cells, and the development of cancer by inhibiting the oncogenic activity of the transcriptional coactivators YAP and TAZ (20,21). In mice, liverspecific knockout of the Hippo pathway components MST1/2, SAV1, or NF2 induces the expansion of hepatic progenitor cells via YAP/TAZ activation and leads eventually to the development of liver cancer (HCC, cholangiocarcinoma [CC], or both) (22)(23)(24)(25). Despite its importance in tumorigenesis, the role of Hippo signaling in the metabolic dysregulation that precedes the development of liver cancer remains unclear.Previous studies have suggested that YAP regulates components of the AKT pathway (i.e., PI3K, PTEN, and AKT) and that the Drosophila Hippo ortholog MST1 binds and inhibits AKT directly (26)(27)(28)(29). Increased YAP expression in human liver tumors is associated with high levels of p- AKT (30,31). This suggests that crosstalk between the Hippo and AKT pathways may be important in the maintenance of functional liver homeostasis. The molecular coordination of these 2 pathways in liver tumorigenesis, however, has not been revealed. Using several mouse models, we now show Nonalcoholic fatty liver disease (NAFLD) is a major risk factor for liver cancer; therefore, its prevention is an important clinical goal. Ablation of phosphatase and tensin homolog (PTEN) or the protein kinase Hippo signaling pathway induces liver cancer via activation of AKT or the transc...

BACKGROUND & AIMS 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. METHODS 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 PCR, 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 were also 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). RESULTS Incubation of colon cancer cell lines with PGE2 led to phosphorylation of cAMP responsive element binding protein 1 (CREB1) and increased levels of YAP1 mRNA and protein and YAP1’s transcriptional activity. This led to increased transcription of the prostaglandin-endoperoxide synthase 2 gene (PTGS2 or COX2) 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-PDGH-knockout mice did not develop spontaneous tumors following colitis induction, but SAV1/15-PDGH 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. Conclusion PGE2 signaling increases expression and transcriptional activities of YAP1, leading to increased expression of COX2 and EP4 to activate a positive signaling loop. This pathway promotes proliferation of colon cancer...