Oncogenic dependency on ß-catenin in liver cancer cell lines correlates with pathway activation.

Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/ß-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (ß-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of ß-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on ß-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on ß-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) ß-catenin were significantly more sensitive to ß-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active ß-catenin. Finally, significant therapeutic benefit of ß-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. ß-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of ß-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant ß-catenin signaling in the maintenance of oncogenic phenotype in HCC.

Anti-miR-21 Suppresses Hepatocellular Carcinoma Growth via Broad Transcriptional Network Deregulation.

UNLABELLED: Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti-miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti-miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti-miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. IMPLICATIONS: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention.

Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells.

Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.

Selective inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule.

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

High-fat diet increases and the polyphenol, S17834, decreases acetylation of the sirtuin-1-dependent lysine-382 on p53 and apoptotic signaling in atherosclerotic lesion-prone aortic endothelium of normal mice.

Our purpose was to determine if high-fat diet and treatment with a polyphenol regulate the acetylation of lysine-382 of p53, the site regulated by sirtuin-1, and apoptosis in the endothelium of the atherosclerotic lesion-prone mouse aortic arch. In cultured endothelial cells, 2 atherogenic stimuli, hydrogen peroxide and tumor necrosis factor-α, increased the acetylation of p53 lysine-382, and caspase-3 cleavage, an indicator of apoptotic signaling. The polyphenol, S17834, significantly prevented these changes. In low-density lipoprotein receptor-deficient mice, a high-fat diet increased, and treatment with S17834 attenuated early atherosclerotic lesions on the lesser curvature of the aortic arch. In wild-type C57BL6 mice fed the same diet, no atherosclerotic lesions were observed in this lesion-prone area, but p53 acetylation and caspase-3 cleavage increased in the endothelium. In high-fat fed mice, S17834 increased sirtuin-1 protein in the lesion-prone endothelium and prevented both the increase in p53 acetylation and caspase-3 cleavage without affecting blood lipids. These results indicate that high-fat diet increases and S17834 decreases the acetylation of p53 in lesion-prone aortic endothelial cells of normal mice independently of blood lipids, suggesting that the polyphenol may regulate endothelial cell p53 acetylation and apoptosis via local actions.

S-glutathiolation of p21ras by peroxynitrite mediates endothelial insulin resistance caused by oxidized low-density lipoprotein.

OBJECTIVE: To understand the mechanism by which oxidants are linked to insulin resistance, bovine aortic endothelial cells were exposed to oxidized low-density lipoproteins (oxLDL) or peroxynitrite. METHODS AND RESULTS: OxLDL transiently increased phosphorylation of Erk and Akt within 5 minutes, but 60 minutes later, resulted in decreased insulin-induced Akt phosphorylation. OxLDL promoted a 2- to 5-fold increase in oxidant generation as measured by dihydrorhodamine or dihydroethidium oxidation that was ascribed to peroxynitrite. Exogenous peroxynitrite (25 to 100 micromol/L) or oxidized glutathione mimicked the effects of oxLDL. OxLDL increased the S-glutathiolation of p21ras, and adenoviral transfection with either a mutant p21ras (C118S) lacking the predominant site of S-glutathiolation or a dominant-negative mutant restored insulin-induced Akt phosphorylation. The requirement for oxidant-mediated S-glutathiolation and activation of p21ras in mediating insulin resistance was further implicated by showing that insulin signaling was restored by Mek inhibitors or by overexpression of glutaredoxin-1. Furthermore, oxLDL increased Erk-dependent phosphorylation of insulin receptor substrate-1 serine-616 that was prevented by inhibiting oxidant generation, Erk activation, or by the p21ras C118S mutant. CONCLUSIONS: This study provides direct evidence for a novel molecular mechanism by which oxidants can induce insulin resistance via S-glutathiolation of p21ras and Erk-dependent inhibition of insulin signaling.

The thromboxane A2 receptor antagonist S18886 prevents enhanced atherogenesis caused by diabetes mellitus.

BACKGROUND: S18886 is an orally active thromboxane A2 (TXA2) receptor (TP) antagonist in clinical development for use in secondary prevention of thrombotic events in cardiovascular disease. We previously showed that S18886 inhibits atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice by a mechanism independent of platelet-derived TXA2. Atherosclerosis is accelerated by diabetes and is associated with increased TXA(2) and other eicosanoids that stimulate TP. The purpose of this study was to determine whether S18886 lessens the enhanced atherogenesis in diabetic apoE(-/-) mice. METHODS AND RESULTS: Diabetes mellitus was induced in apoE(-/-) mice with streptozotocin and was treated or not with S18886 (5 mg.kg(-1).d(-1)). After 6 weeks, aortic lesion area was increased >4-fold by diabetes in apoE(-/-) mice, associated with similar increases in serum glucose and cholesterol. S18886 largely prevented the diabetes-related increase in lesion area without affecting the hyperglycemia or hypercholesterolemia. S18886 prevented deterioration of endothelial function and endothelial nitric oxide synthase expression, as well as increases in intimal markers of inflammation associated with diabetes. In human aortic endothelial cells in culture, S18886 also prevented the induction of vascular cell adhesion molecule-1 and prevented the decrease in endothelial nitric oxide synthase expression caused by high glucose. CONCLUSIONS: The TP antagonist inhibits inflammation and accelerated atherogenesis caused by diabetes, most likely by counteracting effects on endothelial function and adhesion molecule expression of eicosanoids stimulated by the diabetic milieu.

Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite.

AMP-activated kinase (AMPK) is a fuel-sensing enzyme present in most mammalian tissue. In response to a decrease in the energy state of a cell AMPK is phosphorylated and activated by still poorly characterized upstream events. Exposure of bovine aortic endothelial cells (BAEC) to chemically synthesized ONOO- acutely and significantly increased phosphorylation of c-Src, PDK1, AMPK, and its downstream target, acetyl-CoA carboxylase (ACC), without affecting cellular AMP. This novel pathway for AMPK activation was confirmed by the use of pharmacological inhibitors and dominant-negative mutants. Exposure of BAEC to hypoxia-reoxygenation (H/R) caused a biphasic increase in AMPK and ACC phosphorylation, which was prevented by adenoviral overexpression of superoxide dismutase (SOD) or inhibition of nitric-oxide synthase (NOS) implicating a role of ONOO- formed during H/R. Furthermore, dominant-negative mutants of c-Src or kinase-defective PDK1 also blocked H/R-induced AMPK activation indicating that, as with addition of exogenous ONOO-, both c-Src and PI 3-kinase are upstream of AMPK. Moreover, H/R, like ONOO-, significantly increased co-immunoprecipitation of AMPK with c-Src, suggesting that ONOO- favors physical association of AMPK with upstream kinases. Taken together, our results indicate a novel pathway by which H/R via ONOO- activates AMPK in a c-Src-mediated, PI 3-kinase-dependent manner, and suggest that ONOO--induced activation of AMPK might thereby regulate metabolic enzymes, such as ACC.

Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase.

Peroxynitrite (ONOO(-)), a nitric oxide-derived oxidant, uncouples endothelial nitric oxide synthase (eNOS) and increases enzymatic production of superoxide anions (O(2)()) (Zou, M. H., Shi, C., and Cohen, R. A. (2002) J. Clin. Invest. 109, 817-826). Here we studied how ONOO(-) influences eNOS activity. In cultured bovine aortic endothelial cells (BAEC), ONOO(-) increased basal and agonist-stimulated Ser(1179) phosphorylation of eNOS, whereas it decreased nitric oxide production and bioactivity. However, ONOO(-) strongly inhibited the phosphorylation and activity of Akt, which is known to phosphorylate eNOS-Ser(1179). Moreover, expression of an Akt dominant-negative mutant did not prevent ONOO(-)-enhanced eNOS-Ser(1179) phosphorylation. In contrast to Akt, ONOO(-) significantly activated 5'-AMP-activated kinase (AMPK), as evidenced by its increased Thr(172) phosphorylation as well as increased Ser(92) phosphorylation of acetyl-coenzyme A carboxylase, a downstream target of AMPK. Associated with the increased release of O(2)(), ONOO(-) significantly increased the co-immunoprecipitation of eNOS with AMPK. Further, overexpression of the AMPK-constitutive active adenovirus significantly enhanced ONOO(-) up-regulated eNOS-Ser(P)(1179). In contrast, overexpression of a dominant-negative AMPK mutant attenuated the ONOO(-)-enhanced eNOS-Ser(1179) phosphorylation as well as O(2)() release. We conclude that ONOO(-) inhibits Akt and increases AMPK-dependent Ser(1179) phosphorylation of eNOS resulting in enhanced O(2)() release.

Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite.

Nitric oxide (NO) is produced by NO synthase (NOS) in many cells and plays important roles in the neuronal, muscular, cardiovascular, and immune systems. In various disease conditions, all three types of NOS (neuronal, inducible, and endothelial) are reported to generate oxidants through unknown mechanisms. We present here the first evidence that peroxynitrite (ONOO(-)) releases zinc from the zinc-thiolate cluster of endothelial NOS (eNOS) and presumably forms disulfide bonds between the monomers. As a result, disruption of the otherwise SDS-resistant eNOS dimers occurs under reducing conditions. eNOS catalytic activity is exquisitely sensitive to ONOO(-), which decreases NO synthesis and increases superoxide anion (O(2)(.-)) production by the enzyme. The reducing cofactor tetrahydrobiopterin is not oxidized, nor does it prevent oxidation of eNOS by the same low concentrations of OONO(-). Furthermore, eNOS derived from endothelial cells exposed to elevated glucose produces more O(2)(.-), and, like eNOS purified from diabetic LDL receptor-deficient mice, contains less zinc and fewer SDS-resistant dimers. Hence, eNOS exposure to oxidants including ONOO(-) causes increased enzymatic uncoupling and generation of O(2)(.-) in diabetes, contributing further to endothelial cell oxidant stress. Regulation of the zinc-thiolate center of NOS by ONOO(-) provides a novel mechanism for modulation of the enzyme function in disease.

High glucose via peroxynitrite causes tyrosine nitration and inactivation of prostacyclin synthase that is associated with thromboxane/prostaglandin H(2) receptor-mediated apoptosis and adhesion molecule expression in cultured human aortic endothelial cells.

Loss of the modulatory role of the endothelium may be a critical initial factor in the development of diabetic vascular diseases. Exposure of human aortic endothelial cells (HAECs) to high glucose (30 or 44 mmol/l) for 7-10 days significantly increased the release of superoxide anion in response to the calcium ionophore A23187. Nitrate, a breakdown product of peroxynitrite (ONOO(-)), was substantially increased in parallel with a decline in cyclic guanosine monophosphate (GMP). Using immunochemical techniques and high-performance liquid chromatography, an increase in tyrosine nitration of prostacyclin (PGI(2)) synthase (PGIS) associated with a decrease in its activity was found in cells exposed to high glucose. Both the increase in tyrosine nitration and the decrease in PGIS activity were lessened by decreasing either nitric oxide or superoxide anion, suggesting that ONOO(-) was responsible. Furthermore, SQ29548, a thromboxane/prostaglandin (PG) H(2) (TP) receptor antagonist, significantly reduced the increased endothelial cell apoptosis and the expression of soluble intercellular adhesion molecule-1 that occurred in cells exposed to high glucose, without affecting the decrease in PGIS activity. Thus, exposure of HAECs to high glucose increases formation of ONOO(-), which causes tyrosine nitration and inhibition of PGIS. The shunting of arachidonic acid to the PGI(2) precursor PGH(2) or other eicosanoids likely results in TP receptor stimulation. These observations can explain several abnormalities in diabetes, including 1) increased free radicals, 2) decreased bioactivity of NO, 3) PGI(2) deficiency, and 4) increased vasoconstriction, endothelial apoptosis, and inflammation via TP receptor stimulation.