HM15912, a Novel Long-Acting Glucagon-Like Peptide-2 Analog, Improves Intestinal Growth and Absorption Capacity in a Male Rat Model of Short Bowel Syndrome.

Extensive bowel resection caused by various diseases that affect the intestines, such as Crohn's disease, volvulus, and cancer, leads to short bowel syndrome (SBS). Teduglutide is the only approved glucagon-like peptide-2 (GLP-2) drug for SBS; however, it requires daily administration. A novel GLP-2 analog with a prolonged duration of action to reduce dosing frequency and promote a greater efficacy may provide patients with a better quality of life. In the present study, the sustained exposure of HM15912 was characterized in normal male rats. The efficacy of HM15912 on intestinal growth and absorption capacity was also evaluated in normal male mice, rats, and SBS rats. HM15912 exhibited a remarkably extended half-life (42.3 hours) compared with teduglutide (0.6 hours) in rats. Despite somewhat lower in vitro potency on GLP-2 receptor than human GLP-2 or teduglutide, this longer-lasting mode of action promotes HM15912 to be more effective in terms of small intestinal growth than existing GLP-2 analogs even with a less frequent dosing interval of as little as once a week in rodents, including SBS rats. Furthermore, the small intestinal weight was approximately doubled, and the D-xylose absorption was significantly increased after pre-treatment of existing GLP-2 analogs on the market or under clinical development followed by HM15912 in rodents. These results indicate that HM15912 possesses a significant small bowel trophic effect driven by continuously increased exposure, supporting that HM15912 may be a novel treatment option with greater efficacy and the longest dosing interval among existing GLP-2 analogs for SBS with intestinal failure. SIGNIFICANCE STATEMENT: HM15912, a novel long-acting glucagon-like peptide-2 (GLP-2) analog, has a significant small bowel hypertrophic effect in rodents with a reduced frequency of administration compared to the existing GLP-2 analogs on the market or currently under clinical development. This study supports the possibility that HM15912 could be administered much less frequently than other long-acting GLP-2 analogs for patients with short bowel syndrome.

EZH2 Promotes Cholangiocarcinoma Development and Progression through Histone Methylation and microRNA-Mediated Down-Regulation of Tumor Suppressor Genes.

Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tree. Although studies have implicated enhancer of Zeste homolog 2 (EZH2) in CCA growth, the role of EZH2 in CCA development has not been investigated, and the mechanism for EZH2-regulated gene expression in CCA remains to be further defined. The current study used a mouse model of CCA induced by hydrodynamic tail vein injection of Notch1 intracellular domain and myristoylated-AKT plasmids. Mice with liver-specific EZH2 knockout displayed reduced CCA development. In a xenograft model, EZH2 knockdown significantly decreased CCA progression. Administration of the EZH2 inhibitor GSK126 decreased CCA tumor burden in mice. Accordingly, EZH2 depletion or inhibition reduced the growth and colony formation capability of CCA cells. Analysis of high-throughput data identified a set of 12 tumor-inhibiting genes as targets of EZH2 in CCA. The experimental results suggest that EZH2 may down-regulate these tumor-inhibiting genes through methylation of lysine 27 on histone H3 (H3K27) in the gene louses and through regulation of specific miRNAs. High mobility group box 1 was shown to facilitate the methyltransferase activity of EZH2, which is implicated in the regulation of CCA cell growth. The study shows that EZH2 promotes CCA development and progression through a complicated regulatory network involving tumor-inhibiting genes, miRNAs, and high mobility group box 1, which support targeting EZH2 as a potentially effective strategy for CCA treatment.

The Histone Methyltransferase G9a Promotes Cholangiocarcinogenesis Through Regulation of the Hippo Pathway Kinase LATS2 and YAP Signaling Pathway.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a highly malignant epithelial tumor of the biliary tree with poor prognosis. In the current study, we present evidence that the histone-lysine methyltransferase G9a is up-regulated in human CCA and that G9a enhances CCA cell growth and invasiveness through regulation of the Hippo pathway kinase large tumor suppressor 2 (LATS2) and yes-associated protein (YAP) signaling pathway. APPROACH AND RESULTS: Kaplan-Meier survival analysis revealed that high G9a expression is associated with poor prognosis of CCA patients. In experimental systems, depletion of G9a by small interfering RNA/short hairpin RNA or inhibition of G9a by specific pharmacological inhibitors (UNC0642 and UNC0631) significantly inhibited human CCA cell growth in vitro and in severe combined immunodeficient mice. Increased G9a expression was also observed in mouse CCA induced by hydrodynamic tail vein injection of notch intracellular domain (NICD) and myr-Akt. Administration of the G9a inhibitor UNC0642 to NICD/Akt-injected mice reduced the growth of CCA, in vivo. These findings suggest that G9a inhibition may represent an effective therapeutic strategy for the treatment of CCA. Mechanistically, our data show that G9a-derived dimethylated H3K9 (H3K9me2) silenced the expression of the Hippo pathway kinase LATS2, and this effect led to subsequent activation of oncogenic YAP. Consequently, G9a depletion or inhibition reduced the level of H3K9me2 and restored the expression of LATS2 leading to YAP inhibition. CONCLUSIONS: Our findings provide evidence for an important role of G9a in cholangiocarcinogenesis through regulation of LATS2-YAP signaling and suggest that this pathway may represent a potential therapeutic target for CCA treatment.

Yes-Associated Protein in Kupffer Cells Enhances the Production of Proinflammatory Cytokines and Promotes the Development of Nonalcoholic Steatohepatitis.

BACKGROUND AND AIMS: Yes-associated protein (YAP) plays an important role in hepatocarcinogenesis, although the potential role of YAP in non-neoplastic liver diseases remains largely unknown. We report herein that YAP in Kupffer cells (KCs) enhances the production of proinflammatory cytokines and promotes the development of nonalcoholic steatohepatitis (NASH). Our data show that the expression of YAP is significantly increased in KCs of wild-type mice fed a high-fat diet (HFD). APPROACH AND RESULTS: We generated mice with macrophage/monocyte-specific deletion of YAP (YAPϕKO ) or Toll-like receptor 4 (TLR4; TLR4ϕKO ), and animals were fed an HFD or treated with lipopolysaccharide (LPS). Our data showed that YAPϕKO mice fed an HFD exhibited lower serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels and less hepatic inflammation when compared to their littermate controls. LPS treatment induced accumulation of YAP in KCs in vitro and in mice, which was prevented by macrophage/monocyte-specific deletion of TLR4 (TLR4ϕKO ). LPS transcriptionally activates YAP through activator protein 1 in macrophages/KCs. LPS-induced YAP further enhances expression of proinflammatory cytokines (including monocyte chemoattractant protein 1, tumor necrosis factor alpha, and interleukin 6) through YAP association with the TEA domain-binding motif in the promoter region of inflammatory cytokines. Forced overexpression of active YAP (YAP5SA) in KCs enhanced the production of proinflammatory cytokines. Treatment of HFD-fed mice with verteporfin inhibited KC activation, reduced liver inflammation, and decreased serum ALT/AST levels. Analyses of liver tissues from NASH patients reveal that YAP is increased in KCs and that level of YAP in human liver tissues is positively correlated with expression of proinflammatory cytokines. CONCLUSIONS: This study describes an important role of YAP in KCs for regulation of liver inflammation in NASH. Our findings suggest that inhibition of YAP may represent an effective therapeutic strategy for NASH treatment.

A Transforming Growth Factor-ß and H19 Signaling Axis in Tumor-Initiating Hepatocytes That Regulates Hepatic Carcinogenesis.

Functions of transforming growth factor-ß (TGF-ß) in the liver vary depending on specific cell types and their temporal response to TGF-ß during different stages of hepatocarcinogenesis (HCG). Through analysis of tumor tissues from hepatocellular carcinoma (HCC) patients, we were able to cluster hepatic epithelial cell-derived TGF-ß gene signatures in association with distinct clinical prognoses. To delineate the role of hepatic epithelial TGF-ß signaling in HCC development, we used an experimental system in which tumor-initiating hepatocytes (TICs) were isolated from TGF-ß receptor II floxed mice (Tgfbr2fl/fl ) and transplanted into syngeneic C57BL/6J mice by splenic injection. Recipient mice were then administered Cre-expressing adenovirus (Ad-Cre) to inactivate Tgfbr2 in transplanted TICs. After latency, Tgfbr2-inactivated TICs formed larger and more tumor nodules in recipient livers compared to TICs without Tgfbr2 inactivation. In vitro analyses revealed that treatment of cultured TICs with TGF-ß inhibited expression of progenitor cell factors (including SRY (sex determining region Y)-box 2 [Sox2]). RNA sequencing (RNA-seq) analysis identified H19 as one of the most up-regulated long noncoding RNA (lncRNA) in association with Tgfbr2 inactivation in TICs. Tgfbr2 inactivation by Ad-Cre led to a 5-fold increase of H19 expression in TICs. Accordingly, TGF-ß treatment reduced H19 expression. We observed that forced overexpression of Sox2 in TICs increased transcription of H19, whereas knockdown of Sox2 decreased it. Furthermore, depletion of H19 reduced the progenitor property of TICs in vitro and decreased their tumorigenic potential in vivo. Finally, we observed a low level of H19 mRNA expression in human HCC tissues from patients with the epithelial TGF-ß gene signature in association with favorable prognosis. Conclusion: Our findings describe a TGF-ß and H19 signaling axis by Sox2 in TICs that importantly regulates HCG.

Adult Hepatocytes Are Hedgehog-Responsive Cells in the Setting of Liver Injury: Evidence for Smoothened-Mediated Activation of NF-κB/Epidermal Growth Factor Receptor/Akt in Hepatocytes that Counteract Fas-Induced Apoptosis.

Although hedgehog (Hh) signaling pathway is inactive in adult healthy liver, it becomes activated during acute and chronic liver injury and, thus, modulates the reparative process and disease progression. We developed a novel mouse model with liver-specific knockout of Smoothened (Smo LKO), and animals were subjected to Fas-induced liver injury in vivo. Results showed that Smo deletion in hepatocytes enhances Fas-induced liver injury. Activation of Hh signaling in hepatocytes in the setting of Fas-induced injury was indicated by the fact that Jo2 treatment enhanced hepatic expression of Ptch1, Smo, and its downstream target Gli1 in control but not Smo LKO mice. Primary hepatocytes from control mice showed increased Hh signaling activation in response to Jo2 treatment in vitro. On the other hand, the Smo KO hepatocytes were devoid of Hh activation and were more susceptible to Jo2-induced apoptosis. The levels of NF-κB and related signaling molecules, including epidermal growth factor receptor and Akt, were lower in Smo KO livers/hepatocytes than in control livers/hepatocytes. Accordingly, hydrodynamic gene delivery of active NK-κB prevented Jo2-induced liver injury in the Smo LKO mice. Our findings provide important evidence that adult hepatocytes become responsive to Hh signaling through up-regulation of Smo in the setting of Fas-induced liver injury and that such alteration leads to activation of NF-κB/epidermal growth factor receptor/Akt, which counteracts Fas-induced hepatocyte apoptosis.

Epigenetic Silencing of miRNA-34a in Human Cholangiocarcinoma via EZH2 and DNA Methylation: Impact on Regulation of Notch Pathway.

Aberrant expression and regulation of miRNAs have been implicated in multiple stages of tumorigenic processes. The current study was designed to explore the biological function and epigenetic regulation of miR-34a in human cholangiocarcinoma (CCA). Our data show that the expression of miR-34a is decreased significantly in CCA cells compared with non-neoplastic biliary epithelial cells. Forced overexpression of miR-34a in CCA cells inhibited their proliferation and clonogenic capacity in vitro, and suppressed tumor xenograft growth in severe combined immunodeficiency mice. We identified three key components of the Notch pathway, Notch1, Notch2, and Jagged 1, as direct targets of miR-34a. Our further studies show that down-regulation of miR-34a is caused by Enhancer of zeste homolog 2 (EZH2)-mediated H3 lysine 27 trimethylation as well as DNA methylation. Accordingly, treatment with the EZH2 inhibitor, selective S-adenosyl-methionine-competitive small-molecule (GSK126), or the DNA methylation inhibitor, 5-Aza-2'-deoxycytidine, partially restored miR-34a levels in human CCA cells. Immunohistochemical staining and Western blot analyses showed increased EZH2 expression in human CCA tissues and cell lines. We observed that GSK126 significantly reduced CCA cell growth in vitro and intrahepatic metastasis in vivo. Our findings provide novel evidence that miR-34a expression is silenced epigenetically by EZH2 and DNA methylation, which promotes CCA cell growth through activation of the Notch pathway. Consequently, these signaling cascades may represent potential therapeutic targets for effective treatment of human CCA.

Inhibition of hedgehog signaling ameliorates hepatic inflammation in mice with nonalcoholic fatty liver disease.

UNLABELLED: Hedgehog (Hh) signaling plays a critical role in liver development, regeneration, injury repair, and carcinogenesis. Activation of Hh signaling has been observed in patients with nonalcoholic fatty liver diseases (NAFLD); however, the pathobiological function and regulatory mechanism of hepatic Hh signaling in the pathogenesis of NAFLD remain to be further defined. This study was designed to examine the effect and mechanism of hepatic Hh signaling in high-fat diet-induced NAFLD by using pharmacological Smoothened (Smo) inhibitors (GDC-0449 and LED225) and liver-specific Smo knockout mice. Administration of Smo inhibitors to high-fat diet-fed wild-type mice significantly reduced the numbers of activated macrophages and decreased the expression of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1ß, monocyte chemoattractant protein 1, and interleukin-6) as assessed by F4/80 immunohistochemistry and quantitative reverse-transcription polymerase chain reaction, respectively. The Smo inhibitors were noted to have variable effects on hepatic fat accumulation. Liver-specific deletion of Smo also reduced macrophage activation and inhibited proinflammatory cytokine expression, while it did not significantly alter fat accumulation in the liver. Mechanistically, we found that activation of glioma-associated oncogene 1 by Hh signaling in primary hepatocytes increased the production of osteopontin, which subsequently enhanced the macrophage-mediated proinflammatory response through paracrine signaling. CONCLUSION: Hepatocyte Hh signaling can promote liver inflammation through osteopontin-mediated macrophage activation; this mechanism importantly contributes to the progression of NAFLD.

Active glycolytic metabolism in CD133(+) hepatocellular cancer stem cells: regulation by MIR-122.

Although altered metabolic pathway is an important diagnostic maker and therapeutic target in cancer, it is poorly understood in cancer stem cells (CSCs). Here we show that the CD133 (+) hepatocellular CSCs have distinct metabolic properties, characterized by more active glycolysis over oxidative phosphorylation, compared to the CD133 (-) cells. Inhibition of PDK4 and LDHA markedly suppresses CD133 (+) stemness characteristics and overcome resistance to sorafenib (current chemotherapeutic agent for hepatocellular cancer). Addition of glucose or lactate to CD133 (-) cells promotes CSC phenotypes, as evidenced by increased CD133 (+) cell population, elevated stemness gene expression and enhanced spheroid formation. Furthermore, the liver-specific miRNA, miR-122, inhibits CSC phenotypes by regulating glycolysis through targeting PDK4. Our findings suggest that enhanced glycolysis is associated with CD133 (+) stem-like characteristics and that metabolic reprogramming through miR-122 or PDK4 may represent a novel therapeutic approach for the treatment of hepatocellular cancer.

Curcumin stimulates proliferation, stemness acting signals and migration of 3T3-L1 preadipocytes.

In the present study, the potential of curcumin to stimulate proliferation, stemness acting signals and migration of 3T3-L1 preadipocytes and the associated molecular mechanisms were investigated. Low concentrations of curcumin stimulated cell proliferation, whereas high concentrations were cytotoxic to 3T3-L1 cells. In particular, application of 0.02 µM of curcumin for 24 h resulted in significantly increased cell proliferation and was determined to be the optimal treatment for this study. In a colony-forming cell assay, cells treated with 0.02 µM of curcumin showed an approximately 1.5-fold increase in colony formation. Curcumin treatment up-regulated the proliferation-related marker proteins coupled with increased cell growth, telomerase activity and overexpression of stemness acting signals, which was associated with activation of the phosphoinositide 3-kinase (PI3K) pathway. In addition, curcumin significantly inactivated p38 mitogen-activated protein kinases (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinases (SARK/JNK), coupled with inhibition of p53 and p21 tumor suppressor gene products. In addition, curcumin significantly increased cell migration through activation of migration-associated transcription factors. Therefore, these results clearly show that activation of cell proliferation by curcumin is associated with improved stem cell potency in 3T3-L1 preadipocytes.