Amphiregulin contributes to the transformed phenotype of human hepatocellular carcinoma cells

Hepatocellular carcinoma is a major cause of cancer-related deaths. Current treatments are not effective, and the identification of relevant pathways and novel therapeutic targets are much needed. Increasing evidences point to the activation of the epidermal growth factor receptor (EGFR) as an important mechanism in the development of hepatocarcinoma. We previously described that amphiregulin (AR), a ligand of the EGFR, is not expressed in healthy liver but is up-regulated during chronic liver injury, the background on which most liver tumors develop. Now, we have studied the expression and role of AR in human hepatocarcinoma. AR expression and function was studied in human liver tumors and cell lines. AR is expressed in human hepatocellular carcinoma tissues and cell lines and behaves as a mitogenic and antiapoptotic growth factor for hepatocarcinoma cells. We provide several lines of evidence, including AR silencing by small interfering RNAs and inhibition of amphiregulin by neutralizing antibodies, showing the existence of an AR-mediated autocrine loop that contributes to the transformed phenotype. Indeed, interference with endogenous AR production resulted in reduced constitutive EGFR signaling, inhibition of cell proliferation, anchorage-independent growth, and enhanced apoptosis. Moreover, knockdown of AR potentiated transforming growth factor-beta and doxorubicin-induced apoptosis. Conversely, overexpression of AR in SK-Hep1 cells enhanced their proliferation rate, anchorage-independent growth, drug resistance, and in vivo tumorigenic potential. These observations suggest that AR is involved in the acquisition of neoplastic traits in the liver and thus constitutes a novel therapeutic target in human hepatocarcinoma

FOSL1 promotes cholangiocarcinoma via transcriptional effectors that could be therapeutically targeted

Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention

The epidermal growth factor receptor ligand amphiregulin is a negative regulator of hepatic acute-phase gene expression

BACKGROUND/AIMS: The modulation of the hepatic acute-phase reaction (APR) that occurs during inflammation and liver regeneration is important for allowing normal hepatocellular proliferation and the restoration of homeostasis. Activation of acute-phase protein (APP) gene expression by interleukin-6 (IL-6)-type cytokines is thought to be counteracted by growth factors released during hepatic inflammation and regeneration. The epidermal growth factor receptor (EGFR) ligand amphiregulin (AR) is readily induced by inflammatory signals and plays a nonredundant protective role during liver injury. In this paper, we investigated the role of AR as a modulator of liver APP gene expression. METHODS: Expression of APP genes was measured in the livers of AR(+/+) and AR(-/-)mice during inflammation and regeneration and in cultured liver cells treated with AR and oncostatin M (OSM). Crosstalk between AR and OSM signalling was studied. RESULTS: APP genes were overexpressed in the livers of AR(-/-) mice during inflammation and hepatocellular regeneration. In cultured AR-null hepatocytes and human hepatocellular carcinoma (HCC) cells after AR knockdown, APP gene expression is enhanced. AR counteracts OSM-triggered signal transducer and activator of transcription 3 signalling in hepatocytes and attenuates APP gene transcription. CONCLUSIONS: Our data support the relevance of EGFR-mediated signalling in the modulation of cytokine-activated pathways. We have identified AR as a key regulator of hepatic APP gene expression during inflammation and liver regeneration

TREM-2 plays a protective role in cholestasis by acting as a negative regulator of inflammation

Background & Aims: Inflammation, particularly that mediated by bacterial components translocating from the gut to the liver and binding to toll-like receptors (TLRs), is central to cholestatic liver injury. The triggering receptor expressed on myeloid cells-2 (TREM-2) inhibits TLR-mediated signaling and exerts a protective role in hepatocellular injury and carcinogenesis. This study aims to evaluate the role of TREM-2 in cholestasis.Methods: TREM-2 expression was analyzed in the livers of pa-tients with primary biliary cholangitis (PBC) or primary scle-rosing cholangitis (PSC), and in mouse models of cholestasis. Wild-type (WT) and Trem-2 deficient (Trem-2-/-) mice were subjected to experimental cholestasis and gut sterilization. Pri-mary cultured Kupffer cells were incubated with lipopolysac-charide and/or ursodeoxycholic acid (UDCA) and inflammatory responses were analyzed.Results: TREM-2 expression was upregulated in the livers of patients with PBC or PSC, and in murine models of cholestasis. Compared to WT, the response to bile duct ligation (BDL)-induced obstructive cholestasis or alpha-naphtylisothiocyanate (ANIT)-induced cholestasis was exacerbated in Trem-2-/-mice. This was characterized by enhanced necroptotic cell death, in-flammatory responses and biliary expansion. Antibiotic treat-ment partially abrogated the effects observed in Trem-2-/-mice after BDL. Experimental overexpression of TREM-2 in the liver of WT mice downregulated ANIT-induced IL-33 expression and neutrophil recruitment. UDCA regulated Trem-1 and Trem-2 expression in primary cultured mouse Kupffer cells and damp-ened inflammatory gene transcription via a TREM-2-dependent mechanism.Conclusions: TREM-2 acts as a negative regulator of inflamma-tion during cholestasis, representing a novel potential thera-peutic target.Lay summary: Cholestasis (the reduction or cessation of bile flow) causes liver injury. This injury is exacerbated when gut-derived bacterial components interact with receptors (spe-cifically Toll-like receptors or TLRs) on liver-resident immune cells, promoting inflammation. Herein, we show that the anti-inflammatory receptor TREM-2 dampens TLR-mediated signaling and hence protects against cholestasis-induced liver injury. Thus, TREM-2 could be a potential therapeutic target in cholestasis

MiR-873-5p acts as an epigenetic regulator in early stages of liver fibrosis and cirrhosis

Glycine N-methyltransferase (GNMT) is the most abundant methyltransferase in the liver and a master regulator of the transmethylation flux. GNMT downregulation leads to loss of liver function progressing to fibrosis, cirrhosis, and hepatocellular carcinoma. Moreover, GNMT deficiency aggravates cholestasis-induced fibrogenesis. To date, little is known about the mechanisms underlying downregulation of GNMT levels in hepatic fibrosis and cirrhosis. On this basis, microRNAs are epigenetic regulatory elements that play important roles in liver pathology. In this work, we aim to study the regulation of GNMT by microRNAs during liver fibrosis and cirrhosis. Luciferase assay on the 3ʹUTR-Gnmt was used to confirm in silico analysis showing that GNMT is potentially targeted by the microRNA miR-873-5p. Correlation between GNMT and miR-873-5p in human cholestasis and cirrhosis together with miR-873-5p inhibition in vivo in different mouse models of liver cholestasis and fibrosis [bile duct ligation and Mdr2 (Abcb4)-/- mouse] were then assessed. The analysis of liver tissue from cirrhotic and cholestatic patients, as well as from the animal models, showed that miR-873-5p inversely correlated with the expression of GNMT. Importantly, high circulating miR-873-5p was also detected in cholestastic and cirrhotic patients. Preclinical studies with anti-miR-873-5p treatment in bile duct ligation and Mdr2-/- mice recovered GNMT levels in association with ameliorated inflammation and fibrosis mainly by counteracting hepatocyte apoptosis and cholangiocyte proliferation. In conclusion, miR-873-5p emerges as a novel marker for liver fibrosis, cholestasis, and cirrhosis and therapeutic approaches based on anti-miR-873-5p may be effective treatments for liver fibrosis and cholestatic liver disease

MiR-873-5p acts as an epigenetic regulator in early stages of liver fibrosis and cirrhosis

Glycine N-methyltransferase (GNMT) is the most abundant methyltransferase in the liver and a master regulator of the transmethylation flux. GNMT downregulation leads to loss of liver function progressing to fibrosis, cirrhosis, and hepatocellular carcinoma. Moreover, GNMT deficiency aggravates cholestasis-induced fibrogenesis. To date, little is known about the mechanisms underlying downregulation of GNMT levels in hepatic fibrosis and cirrhosis. On this basis, microRNAs are epigenetic regulatory elements that play important roles in liver pathology. In this work, we aim to study the regulation of GNMT by microRNAs during liver fibrosis and cirrhosis. Luciferase assay on the 3ʹUTR-Gnmt was used to confirm in silico analysis showing that GNMT is potentially targeted by the microRNA miR-873-5p. Correlation between GNMT and miR-873-5p in human cholestasis and cirrhosis together with miR-873-5p inhibition in vivo in different mouse models of liver cholestasis and fibrosis [bile duct ligation and Mdr2 (Abcb4)-/- mouse] were then assessed. The analysis of liver tissue from cirrhotic and cholestatic patients, as well as from the animal models, showed that miR-873-5p inversely correlated with the expression of GNMT. Importantly, high circulating miR-873-5p was also detected in cholestastic and cirrhotic patients. Preclinical studies with anti-miR-873-5p treatment in bile duct ligation and Mdr2-/- mice recovered GNMT levels in association with ameliorated inflammation and fibrosis mainly by counteracting hepatocyte apoptosis and cholangiocyte proliferation. In conclusion, miR-873-5p emerges as a novel marker for liver fibrosis, cholestasis, and cirrhosis and therapeutic approaches based on anti-miR-873-5p may be effective treatments for liver fibrosis and cholestatic liver disease

Non-parenchymal TREM-2 protects the liver from immune-mediated hepatocellular damage

Objective: Liver injury impacts hepatic inflammation in part via Toll-like receptor (TLR) signalling. Triggering receptor expressed on myeloid cells 2 (TREM-2) modulates TLR4-mediated inflammation in bone marrow (BM)-derived macrophages but its function in liver injury is unknown. Here we hypothesised that the anti-inflammatory effects of TREM-2 on TLR signalling may limit hepatic injury. Design: TREM-2 expression was analysed in livers of humans with various forms of liver injury compared with control individuals. Acute and chronic liver injury models were performed in wild type and Trem-2-/- mice. Primary liver cells from both genotypes of mice were isolated for in vitro experiments. Results: TREM-2 was expressed on non-parenchymal hepatic cells and induced during liver injury in mice and man. Mice lacking TREM-2 exhibited heightened liver damage and inflammation during acute and repetitive carbon tetrachloride and acetaminophen (APAP) intoxication, the latter of which TREM-2 deficiency was remarkably associated with worsened survival. Liver damage in Trem-2-/- mice following chronic injury and APAP challenge was associated with elevated hepatic lipid peroxidation and macrophage content. BM transplantation experiments and cellular reactive oxygen species assays revealed effects of TREM-2 in the context of chronic injury depended on both immune and resident TREM-2 expression. Consistent with effects of TREM-2 on inflammation-associated injury, primary hepatic macrophages and hepatic stellate cells lacking TREM-2 exhibited augmented TLR4-driven proinflammatory responses. Conclusion: Our data indicate that by acting as a natural brake on inflammation during hepatocellular injury, TREM-2 is a critical regulator of diverse types of hepatotoxic injury

Non-parenchymal TREM-2 protects the liver from immune-mediated hepatocellular damage

Objective: Liver injury impacts hepatic inflammation in part via Toll-like receptor (TLR) signalling. Triggering receptor expressed on myeloid cells 2 (TREM-2) modulates TLR4-mediated inflammation in bone marrow (BM)-derived macrophages but its function in liver injury is unknown. Here we hypothesised that the anti-inflammatory effects of TREM-2 on TLR signalling may limit hepatic injury. Design: TREM-2 expression was analysed in livers of humans with various forms of liver injury compared with control individuals. Acute and chronic liver injury models were performed in wild type and Trem-2-/- mice. Primary liver cells from both genotypes of mice were isolated for in vitro experiments. Results: TREM-2 was expressed on non-parenchymal hepatic cells and induced during liver injury in mice and man. Mice lacking TREM-2 exhibited heightened liver damage and inflammation during acute and repetitive carbon tetrachloride and acetaminophen (APAP) intoxication, the latter of which TREM-2 deficiency was remarkably associated with worsened survival. Liver damage in Trem-2-/- mice following chronic injury and APAP challenge was associated with elevated hepatic lipid peroxidation and macrophage content. BM transplantation experiments and cellular reactive oxygen species assays revealed effects of TREM-2 in the context of chronic injury depended on both immune and resident TREM-2 expression. Consistent with effects of TREM-2 on inflammation-associated injury, primary hepatic macrophages and hepatic stellate cells lacking TREM-2 exhibited augmented TLR4-driven proinflammatory responses. Conclusion: Our data indicate that by acting as a natural brake on inflammation during hepatocellular injury, TREM-2 is a critical regulator of diverse types of hepatotoxic injury

FOSL1 promotes cholangiocarcinoma via transcriptional effectors that could be therapeutically targeted

Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention