Sorafenib and 2-Deoxyglucose Synergistically Inhibit Proliferation of Both Sorafenib-Sensitive and -Resistant HCC Cells by Inhibiting ATP Production.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths globally. Sorafenib is the only first-line systemic drug for advanced HCC, but it has very limited survival benefits because patients treated with sorafenib either suffer from side effects or show disease progression after initial response. Thus, there is an urgent need to develop novel strategies for first-line and second-line therapies. The association between sorafenib resistance and glycolysis prompted us to screen several drugs with known antiglycolytic activity to identify those that will sensitize cells to sorafenib. We demonstrate that the combination of glycolytic inhibitor 2-deoxyglucose (2DG) and sorafenib drastically inhibits viability of sorafenib-sensitive and -resistant cells. However, the combination of other antiglycolytic drugs like lonidamine, gossypol, 3-bromopyruvate, and imatinib with sorafenib does not show synergistic effect. Cell cycle analysis revealed that the combination of 2DG and sorafenib induced cell cycle arrest at G0/G1. Mechanistic investigation suggests that the cell cycle arrest is due to depletion of cellular ATP that activates AMP-activated protein kinase (AMPK), which, in turn, inhibits mammalian target of rapamycin (mTOR) to induce cell cycle arrest. This study provides strong evidence for the therapeutic potential of the combination of sorafenib and 2DG for HCC.

Indole-3-carbinol inhibits tumorigenicity of hepatocellular carcinoma cells via suppression of microRNA-21 and upregulation of phosphatase and tensin homolog.

A major obstacle to successful treatment of hepatocellular carcinoma (HCC) is its high resistance to cytotoxic chemotherapy due to overexpression of multidrug resistance genes. Activation of the AKT pathway is known to be involved in chemoresistance in HCC; however, the underlying mechanisms modulating the AKT pathway by chemopreventive agents remain unclear. In the present study, we found that indole-3-carbinol (I3C) treatment for tumor cells repressed the AKT pathway by increasing the expression of phosphatase and tensin homolog (PTEN) in HCC xenograft tumor and HCC cell lines. qRT-PCR data showed that the expression of miR-21 and miR-221&222 was significantly reduced by I3C in HCC cells in vitro and in vivo. Reactivation of the AKT pathway via restoration of miR-21 was reversed by I3C. Ectopic expression of miR-21 mediated-accelerated wound healing was abrogated by I3C. Moreover, reducing the expression of miR-21 by anti-miR decreased the resistance of HCC cells to I3C. These results provide experimental evidences that I3C could function as a miR-21 regulator, leading to repression of the PTEN/AKT pathway and opening a new avenue for eradication of drug-resistant cells, thus potentially helping to improve the therapeutic outcome in patients diagnosed with HCC.

Reciprocal regulation of microRNA-122 and c-Myc in hepatocellular cancer: role of E2F1 and transcription factor dimerization partner 2.

UNLABELLED: c-Myc is a well-known oncogene frequently up-regulated in different malignancies, whereas liver-specific microRNA (miR)-122, a bona fide tumor suppressor, is down-regulated in hepatocellular cancer (HCC). Here we explored the underlying mechanism of reciprocal regulation of these two genes. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and northern blot analysis demonstrated reduced expression of the primary, precursor, and mature miR-122 in c-MYC-induced HCCs compared to the benign livers, indicating transcriptional suppression of miR-122 upon MYC overexpression. Indeed, chromatin immunoprecipitation (ChIP) assay showed significantly reduced association of RNA polymerase II and histone H3K9Ac, markers of active chromatin, with the miR-122 promoter in tumors relative to the c-MYC-uninduced livers, indicating transcriptional repression of miR-122 in c-MYC-overexpressing tumors. The ChIP assay also demonstrated a significant increase in c-Myc association with the miR-122 promoter region that harbors a conserved noncanonical c-Myc binding site in tumors compared to the livers. Ectopic expression and knockdown studies showed that c-Myc indeed suppresses expression of primary and mature miR-122 in hepatic cells. Additionally, Hnf-3ß, a liver enriched transcription factor that activates miR-122 gene, was suppressed in c-MYC-induced tumors. Notably, miR-122 also repressed c-Myc transcription by targeting transcriptional activator E2f1 and coactivator Tfdp2, as evident from ectopic expression and knockdown studies and luciferase reporter assays in mouse and human hepatic cells. CONCLUSION: c-Myc represses miR-122 gene expression by associating with its promoter and by down-regulating Hnf-3ß expression, whereas miR-122 indirectly inhibits c-Myc transcription by targeting Tfdp2 and E2f1. In essence, these results suggest a double-negative feedback loop between a tumor suppressor (miR-122) and an oncogene (c-Myc).

Regulation of glucose metabolism in hepatocarcinogenesis by microRNAs.

In the past decade, considerable effort has been made in elucidating the mechanism underlying the high level of aerobic glycolysis in cancer cells. While some recent studies have attempted to address this issue, the potential role of microRNAs in this process has not been explored until recently. These studies have demonstrated involvement of just five deregulated miRNAs in glucose metabolism in hepatocarcinogenesis. This review discusses the metabolic significance of these miRNAs in hepatoceullular carcinoma, their targets in glycolysis, gluconeogenesis, and pentose phosphate pathways, and provides an insight into the therapeutic potential of targeting specific miRNAs.

Methylation of the PTPRO gene in human hepatocellular carcinoma and identification of VCP as its substrate.

We have previously reported that the gene encoding protein tyrosine phosphatase receptor type-O (PTPRO) is suppressed by promoter methylation in a rat model of hepatocellular carcinoma (HCC) and it functions as tumor suppressor in leukemia and lung cancer. Here, we explored the methylation and expression of PTPRO as well as its function in human HCC. MassARRAY analysis of primary human HCC and matching liver samples (n = 24) revealed significantly higher (P = 0.004) methylation density at the promoter CGI in tumors. Combined bisulfite restriction analysis (COBRA) of another set of human HCC samples (n = 17) demonstrated that the CGI was methylated in 29% of tumors where expression of PTPRO was lower than that in corresponding matching livers. A substrate-trapping mutant of PTPRO that stabilizes the bound substrates was used to identify its novel substrate(s). VCP/p97 was found to be a PTPRO substrate by mass spectrometry of the peptides pulled down by the substrate-trapping mutant of PTPRO. Tyrosyl dephosphorylation of VCP following ectopic expression of wild-type PTPRO in H293T and HepG2 cells confirmed that it is a bona fide substrate of PTPRO. Treatment of PTPRO overexpressing HepG2 cells with Doxorubicin, a DNA damaging drug commonly used in therapy of primary HCC, sensitized these cells to this potent anticancer drug that correlated with dephosphorylation of VCP. Taken together, these results demonstrate methylation and downregulation of PTPRO in a subset of primary human HCC and establish VCP as a novel functionally important substrate of this tyrosine phosphatase that could be a potential molecular target for HCC therapy.

Stat3-mediated activation of microRNA-23a suppresses gluconeogenesis in hepatocellular carcinoma by down-regulating glucose-6-phosphatase and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha.

UNLABELLED: Considerable effort has been made in elucidating the mechanism and functional significance of high levels of aerobic glycolysis in cancer cells, commonly referred to as the Warburg effect. Here we investigated whether the gluconeogenic pathway is significantly modulated in hepatocarcinogenesis, resulting in altered levels of glucose homeostasis. To test this possibility, we used a mouse model (mice fed a choline-deficient diet) that develops nonalcoholic steatohepatitis (NASH), preneoplastic nodules, and hepatocellular carcinoma (HCC), along with human primary HCCs and HCC cells. This study demonstrated marked reduction in the expressions of G6pc, Pepck, and Fbp1 encoding the key gluconeogenic enzymes glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, fructose-1,6-phosphatase, respectively, and the transcription factor Pgc-1α in HCCs developed in the mouse model that correlated with reduction in serum glucose in tumor-bearing mice. The messenger RNA (mRNA) levels of these genes were also reduced by ≈80% in the majority of primary human HCCs compared with matching peritumoral livers. The expression of microRNA (miR)-23a, a candidate miR targeting PGC-1α and G6PC, was up-regulated in the mouse liver tumors as well as in primary human HCC. We confirmed PGC-1α and G6PC as direct targets of miR-23a and their expressions negatively correlated with miR-23a expression in human HCCs. G6PC expression also correlated with tumor grade in human primary HCCs. Finally, this study showed that the activation of interleukin (IL)-6-Stat3 signaling caused the up-regulation of miR-23a expression in HCC. CONCLUSION: Based on these data, we conclude that gluconeogenesis is severely compromised in HCC by IL6-Stat3-mediated activation of miR-23a, which directly targets PGC-1α and G6PC, leading to decreased glucose production.

AP-1 elements and TCL1 protein regulate expression of the gene encoding protein tyrosine phosphatase PTPROt in leukemia.

We previously demonstrated that the gene encoding PTPROt, the truncated form of protein tyrosine phosphatase receptor type O expressed predominantly in hematopoietic cells, is a candidate tumor suppressor and is down-regulated in chronic lymphocytic leukemia (CLL). Here, we show that PTPROt expression is significantly reduced in CD19(+) spleen B cells from Eµ-T cell leukemia 1 (TCL1) transgenic mice relative to the wild-type mice. Strikingly, as much as a 60% decrease in PTPROt expression occurs at 7 weeks independently of promoter methylation. To elucidate the potential mechanism for this early suppression of PTPROt in these mice, we explored the role of activating protein-1 (AP-1) in its expression. We first demonstrate that AP-1 activation by 12-O-tetradecanoylphorbol-13-acetate induces PTPROt expression with concurrent recruitment of c-fos and c-jun to its promoter. The PTPROt promoter is also responsive to over- and underexpression of AP-1, confirming the role of AP-1 in PTPROt expression. Next, we demonstrate that TCL1 can repress the PTPROt promoter by altering c-fos expression and c-jun activation state. Finally, using primary CLL cells we have shown an inverse relationship between TCL1 and PTPROt expression. These findings further substantiate the role of TCL1 in PTPROt suppression and its importance in the pathogenesis of CLL.

Structure-activity relationships for 4-anilinoquinoline derivatives as inhibitors of the DNA methyltransferase enzyme DNMT1.

A series of 4-anilinoquinoline derivatives related to the known inhibitor SGI-1027, containing side chains of varying pK(a), were prepared by acid-catalysed coupling of the pre-formed side chains with 4-chloroquinolines. The compounds were evaluated for their ability to reduce the level of DNMT1 protein in HCT116 human colon carcinoma cells by Western blotting. With a very strongly basic N-methylpyridinium side chain, only NHCO-linked compounds were effective, whereas less strongly basic ((diaminomethylene)hydrazono)ethyl or 3-methylpyrimidine-2,4-diamine side chains allowed both NHCO- and CONH-linked compounds to show activity. In contrast, the pK(a) of the quinoline unit had little apparent influence on activity.

Cationic lipid nanoparticles for therapeutic delivery of siRNA and miRNA to murine liver tumor.

miR-122, a liver-specific tumor suppressor microRNA, is frequently down-regulated in hepatocellular carcinoma (HCC). LNP-DP1, a cationic lipid nanoparticle formulation, was developed as a vehicle to restore deregulated gene expression in HCC cells by miR-122 delivery. LNP-DP1 consists of 2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), egg phosphatidylcholine, cholesterol and cholesterol-polyethylene glycol. In vitro, LNP-DP1-mediated transfection of a miR-122 mimic to HCC cells down-regulated miR-122 target genes by >95%. In vivo, siRNAs/miRNAs encapsulated in LNP-DP1 were preferentially taken up by hepatocytes and tumor cells in a mouse HCC model. The miR-122 mimic in LNP-DP1 was functional in HCC cells without causing systemic toxicity. To demonstrate its therapeutic potential, LNP-DP1 encapsulating miR-122 mimic was intratumorally injected and resulted in ~50% growth suppression of HCC xenografts within 30 days, which correlated well with suppression of target genes and impairment of angiogenesis. These data demonstrate the potential of LNP-DP1-mediated microRNA delivery as a novel strategy for HCC therapy. FROM THE CLINICAL EDITOR: In this study, LNP-DP1 -a cationic lipid nanoparticle formulation -is reported as a vehicle to restore deregulated gene expression in hepatic carcinoma cells by siRNA and miRNA delivery using a mouse model. Further expansions to this study may enable transition to clinical trials of this system.

Correction: Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor ß 1 (TGF-ß 1) Axis in Hepatitis C Virus-Expressing Hepatocytes.

[This corrects the article DOI: 10.1371/journal.pone.0046526.].

Anti-microRNA-222 (anti-miR-222) and -181B suppress growth of tamoxifen-resistant xenografts in mouse by targeting TIMP3 protein and modulating mitogenic signal.

We have shown earlier that miR-221 and -222 are up-regulated in tamoxifen-resistant MCF-7 (OHT(R)) cells and Her2-positive human breast tumors when compared with Her2 negative tumors. In this study, we report markedly enhanced expression of miR-181b in OHT(R) cells and endocrine-resistant tumors. Further, anti-miR-222 or -181b in combination with tamoxifen suppressed growth of tamoxifen-resistant xenografts in mice. Luciferase reporter assay and expression analysis showed that TIMP3, a tissue metalloproteinase inhibitor, is a common target of miR-221/222 and -181b. In situ hybridization and immunohistochemical analysis demonstrated reciprocal relationships between TIMP3 and miR-221/222/181b expression in primary human breast carcinomas. Ectopic expression of TIMP3 inhibited growth of the OHT(R) cells, and its depletion in MCF-7 cells reduced sensitivity to tamoxifen in vitro and in vivo. EGF-induced MAPK and AKT phosphorylation were significantly higher in OHT(R) cells and miR-221/222-overexpressing MCF-7 cells than in control cells, which suggests modulation of mitogenic signaling by TIMP3 and the miRs. On the contrary, phosphoMAPK and phosphoAKT levels were diminished in TIMP3-overexpressing OHT(R) cells and increased in TIMP3-depleted MCF-7 cells. Low levels of estrogen or tamoxifen elicited similar differences in phosphoMAPK levels in these cells. Reduced levels of TIMP3 facilitated growth of tamoxifen-resistant cells by alleviating its inhibitory effect on ADAM10 and ADAM17, which are critical for OHT(R) cell growth. In conclusion, miR-221/222 and -181b facilitate growth factor signaling in tamoxifen-resistant breast cancer by down-regulating TIMP3, and corresponding anti-miRs can be used to render these tumors responsive to tamoxifen.