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MicroRNAs (miR) are a class of small (f21 nucleotide) noncoding RNAs that, in general, negatively regulate gene expression. Some miRs harboring CGIs undergo methylationmediated silencing, a characteristic of many tumor suppressor genes. To identify such miRs in liver cancer, the miRNA expression profile was analyzed in hepatocellular carcinoma (HCC) cell lines treated with 5-azacytidine (DNA hypomethylating agent) and/or trichostatin A (histone deacetylase inhibitor). The results showed that these epigenetic drugs differentially regulate expression of a few miRs, particularly miR-1-1, in HCC cells. The CGI spanning exon 1 and intron 1 of miR-1-1 was methylated in HCC cell lines and in primary human HCCs but not in matching liver tissues. The miR-1-1 gene was hypomethylated and activated in DNMT1À/À HCT 116 cells but not in DNMT3B null cells, indicating a key role for DNMT1 in its methylation. miR-1 expression was also markedly reduced in primary human hepatocellular carcinomas compared with matching normal liver tissues. Ectopic expression of miR-1 in HCC cells inhibited cell growth and reduced replication potential and clonogenic survival. The expression of FoxP1 and MET harboring three and two miR-1 cognate sites, respectively, in their respective 3 ¶-untranslated regions, was markedly reduced by ectopic miR-1. Up-regulation of several miR-1 targets including FoxP1, MET, and HDAC4 in primary human HCCs and down-regulation of their expression in 5-AzaC-treated HCC cells suggest their role in hepatocarcinogenesis. The inhibition of cell cycle progression and induction of apoptosis after re-expression of miR-1 are some of the mechanisms by which DNA hypomethylating agents suppress hepatocarcinoma cell growth. [Cancer Res 2008;68(13):5049-58]

Liver X receptors α and β (LXRα and LXRβ) are nuclear receptors with pivotal roles in the transcriptional control of lipid metabolism. Transcriptional activity of LXRs is induced in response to elevated cellular levels of cholesterol. LXRs bind to and regulate the expression of genes that encode proteins involved in cholesterol absorption, transport, efflux, excretion and conversion to bile acids. The coordinated, tissue-specific actions of the LXR pathway maintain systemic cholesterol homeostasis and regulate immune and inflammatory responses. LXRs also regulate fatty acid metabolism by controlling the lipogenic transcription factor sterol regulatory element-binding protein 1c and regulate genes that encode proteins involved in fatty acid elongation and desaturation. LXRs exert important effects on the metabolism of phospholipids, which, along with cholesterol, are major constituents of cellular membranes. LXR activation preferentially drives the incorporation of polyunsaturated fatty acids into phospholipids by inducing transcription of the remodelling enzyme lysophosphatidylcholine acyltransferase 3. The ability of the LXR pathway to couple cellular sterol levels with the saturation of fatty acids in membrane phospholipids has implications for several physiological processes, including lipoprotein production, dietary lipid absorption and intestinal stem cell proliferation. Understanding how LXRs regulate membrane composition and function might provide new therapeutic insight into diseases associated with dysregulated lipid metabolism, including atherosclerosis, diabetes mellitus and cancer.

The latent transcription factor Stat3 is activated by gp130, the common receptor for the interleukin (IL)-6 cytokine family and other growth factor and cytokine receptors. Ligand-induced dimerization of gp130 leads to activation of the Stat1, Stat3 and Shp2-Ras-Erk signaling pathways. Here we assess genetically the contribution of exaggerated Stat3 activation to the phenotype of gp130 (Y757F/Y757F) mice, in which a knock-in mutation disrupts the negative feedback mechanism on gp130-dependent Stat signaling. Compared to gp130 (Y757F/Y757F) mice, reduced Stat3 activation in gp130 (Y757F/Y757F) Stat3(+/-) mice increased their lifespan, prevented splenomegaly, normalized exaggerated hepatic acute-phase response and lymphocyte trafficking, and suppressed the growth of spontaneously arising gastric adenomas in young mice. These lesions share histological features of gastric polyps in aging mice with monoallelic null mutations in Smad4, which encodes the common transducer for transforming growth factor (TGF)-beta signaling. Indeed, hyperactivation of Stat3 desensitizes gp130 (Y757F/Y757F) cells to the cytostatic effect of TGF-beta through transcriptional induction of inhibitory Smad7, thereby providing a novel link for cross-talk between Stat and Smad signaling in gastric homeostasis.

The fatty acyl composition of phospholipids determines the biophysical character of membranes and impacts the function of membrane proteins. Here we define a nuclear receptor pathway for the dynamic modulation of membrane composition in response to changes in cellular lipid metabolism. Ligand activation of LXR preferentially drives the incorporation of polyunsaturated fatty acids into phospholipids through induction of the remodeling enzyme Lpcat3. Promotion of Lpcat3 activity ameliorates ER stress induced by saturated free fatty acids in vitro or by obesity and hepatic lipid accumulation in vivo. Conversely, Lpcat3 knockdown in liver exacerbates ER stress and inflammation. Mechanistically, Lpcat3 modulates inflammation both by regulating c-Src and JNK kinase activation through changes in membrane composition and by affecting substrate availability for inflammatory mediator production. These results outline an endogenous mechanism for the preservation of membrane homeostasis during lipid stress and identify Lpcat3 as an important mediator of LXRs effects on metabolism.

To identify microRNAs that may play a causal role in hepatocarcinogenesis, we used an animal model in which C57/BL6 mice fed choline deficient and amino acid defined (CDAA) diet develop preneoplastic lesions at 65 weeks and hepatocellular carcinomas after 84 weeks. miRNA expression profiling showed significant upregulation of miR-181b and miR-181d in the livers of mice as early as 32 weeks that persisted at preneoplastic stage. The expression of TIMP3, a tumor suppressor and a validated miR-181 target, was markedly suppressed in the livers of mice fed CDAA diet. Upregulation of hepatic TGFβ and its downstream mediators Smad 2, 3 and 4 and increase in phospho-Smad2 in the liver nuclear extract correlated with elevated miR-181b/d in mice fed CDAA diet. The levels of the precursor and mature miR-181b were augmented upon exposure of hepatic cells to TGFβ and were significantly reduced by siRNA-mediated depletion of Smad4, demonstrating the involvement of TGFβ signaling pathway in miR-181b expression. Ectopic expression and depletion of miR-181b showed that miR-181b enhanced MMP2 and MMP9 activity and promoted growth, clonogenic survival, migration and invasion of HCC cells that could be reversed by modulating TIMP3 level. Further, depletion of miR-181b inhibited tumor growth of HCC cells in nude mice. miR-181b also enhanced resistance of HCC cells to the anti-cancer drug doxorubicin. Based on these results, we conclude that upregulation of miR-181b at early stages of feeding CDAA diet promotes hepatocarcinogenesis.

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