Initiation of aberrant DNA methylation patterns and heterogeneity in precancerous lesions of human hepatocellular cancer

<p>While intratumor heterogeneity contributes to disease progression, metastasis, and resistance to chemotherapy, it also provides a route to understanding the evolution and drivers of disease. Defects in epigenetic landscapes are intimately linked to pathogenesis of a variety of human diseases, with epigenetic deregulation promoting tumorigenesis. Understanding epigenetic heterogeneity is crucial in hepatocellular carcinoma (HCC), where epigenetic alterations are frequent, early, and pathogenic events. We determined genome-wide DNA methylation and copy number variation leveraging the Infinium 450K in a series of regenerative nodules from within single patient livers. Bioinformatics strategies were used to ascertain within-patient heterogeneity, link epigenetic changes to clinical features, and determine their relevance to disease pathogenesis. Our data demonstrate that DNA methylation and copy number alterations evolve during the pre-neoplastic phase of HCC and independently segregate regenerative nodules into distinct clusters. Regenerative nodules with a high frequency of epigenetic changes have significantly lower copy number variation, suggesting that individual nodules have differential enrichment of epigenetic and genetic components, with both contributing to disease progression. Regenerative nodules were scored based on ‘epigenetic progression’ with higher scores associated with increased proliferation measured by Ki67 staining. Early events observed in epigenetically ‘aggressive’ nodules are enriched for genes involved in liver cancer. Our study demonstrates that marked epigenetic and genetic heterogeneity exists in early pre-neoplastic liver tissue within individual patients, emphasizing the potential contributions of each mechanism to driving liver disease progression, and it unveils strategies for identifying epigenetic drivers of hepatocellular carcinoma.</p

XIAP promoter predicted GLI-binding sites exhibit sequence-specific binding to nuclear proteins.

<p><i>A,</i> Putative GLI binding sites in the XIAP promoter region. Nucleotide positions were counted from the transcription start site (TSS). Potential binding sites were observed as indicated by arrows on the schematic (I and II). <i>B,</i> Nuclear protein extracts were isolated from KMCH cells. EMSA was performed using Cy5.5-labeled double-stranded oligonucleotides containing the putative GLI binding sequences within the human XIAP promoter, and competition experiments with 200-fold molar excess cold oligonucleotides as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018330#s2" target="_blank">MATERIALS AND METHODS</a>”. <i>C</i>, Total cellular protein was isolated 48 hours after transient transfection with the indicated siRNA against GLI family members. XIAP protein expression was determined by immunoblotting and the signal was quantified by densitometry as a ratio to actin expression. <i>D,</i> Chromatin immunoprecipitation using antiserum to GLI1, GLI2, GLI3, or a control IgG was performed followed by PCR using primers flanking site I (341 bp) within the <i>XIAP</i> promoter region, as indicated. As a positive control, ChIP was performed using primers flanking the <i>Bcl-2</i> promoter GLI-binding site (GLI1 and GLI2), or primers flanking the GLI3 binding site within the <i>GLI1</i> promoter.</p

High fat diet and exercise lead to a disrupted and pathogenic DNA methylome in mouse liver

<p>High-fat diet consumption and sedentary lifestyle elevates risk for obesity, non-alcoholic fatty liver disease, and cancer. Exercise training conveys health benefits in populations with or without these chronic conditions. Diet and exercise regulate gene expression by mediating epigenetic mechanisms in many tissues; however, such effects are poorly documented in the liver, a central metabolic organ. To dissect the consequences of diet and exercise on the liver epigenome, we measured DNA methylation, using reduced representation bisulfite sequencing, and transcription, using RNA-seq, in mice maintained on a fast food diet with sedentary lifestyle or exercise, compared with control diet with and without exercise. Our analyses reveal that genome-wide differential DNA methylation and expression of gene clusters are induced by diet and/or exercise. A combination of fast food and exercise triggers extensive gene alterations, with enrichment of carbohydrate/lipid metabolic pathways and muscle developmental processes. Through evaluation of putative protective effects of exercise on diet-induced DNA methylation, we show that hypermethylation is effectively prevented, especially at promoters and enhancers, whereas hypomethylation is only partially attenuated. We assessed diet-induced DNA methylation changes associated with liver cancer-related epigenetic modifications and identified significant increases at liver-specific enhancers in fast food groups, suggesting partial loss of liver cell identity. Hypermethylation at a subset of gene promoters was associated with inhibition of tissue development and promotion of carcinogenic processes. Our study demonstrates extensive reprogramming of the epigenome by diet and exercise, emphasizing the functional relevance of epigenetic mechanisms as an interface between lifestyle modifications and phenotypic alterations.</p

Cyclopamine sensitizes cells to TRAIL-induced cell death despite knockdown of Bid or Bim.

<p><i>A,</i> Immunoblot analysis was performed on whole cell lysates obtained from KMCH cells stably transfected with the specific shRNA targeting <i>Bid</i> or <i>Bim,</i> using indicated antisera. Actin was used as a loading control. <i>B</i>, KMCH cells stably transfected with Bid- or Bim-shRNA and untransfected parental KMCH cells were treated with Fas agonistic antibody CH-11 (100 µg/mL) for 5 hours followed by DAPI staining. Cells with apoptotic morphology were counted. Mean ± SEM, ***p<0.001 compared to parental cells treated with CH-11. <i>C,</i> In parallel, caspase-3/7 activity was measured in cells treated as in panel <i>B</i>. Mean ± SEM, ***p<0.001. <i>D</i>, KMCH cells stably transfected with Bid- or Bim-shRNA were pretreated with vehicle or cyclopamine (5 µM) for 24 hours, and then treated with TRAIL (5 ng/mL) for 5 hours followed by DAPI staining. Cells with apoptotic morphology were counted. Mean ± SEM, ***p<0.001 compared to cells treated with TRAIL alone. <i>E</i>, KMCH stable cell lines were treated as in panel <i>D</i>, and after 5 hours caspase-3/7 activity was measured. Mean ± SEM, ***p<0.001 compared to cells treated with TRAIL alone. <i>F</i>, Whole cell lysates from KMCH, HuCCT-1, and Mz-ChA-1 cells treated with vehicle or cyclopamine (5 µM) for 24 hours were analyzed by immunoblot using anti-Bcl-2, Mcl-1, or Bcl-x antisera. Actin was used as a loading control. <i>G</i>, Whole cell lysates were obtained from shBid-, shBim- and untransfected parental KMCH cells treated with vehicle or cyclopamine (5 µM) for 24 hours and were analyzed by immunoblot using anti-Bcl-2, Mcl-1, or Bcl-x antisera. Actin was used as a loading control.</p

Knockdown of XIAP sensitizes human cholangiocarcinoma cells to TRAIL cytotoxicity.

<p>KMCH cells were transfected with scrambled siRNA or specific siRNA for <i>cIAP-1</i> or <i>XIAP</i>. <i>A</i>, Whole cell lysates were prepared for immunoblotting from KMCH cells 48 hours after siRNA transfection. <i>B</i>, Forty-eight hours after transfection as in panel <i>A</i>, cells were treated with human recombinant TRAIL (5 ng/mL) or medium for 5 hours. Cells were then stained with DAPI and cells with apoptotic nuclear morphology were counted and expressed as a percentage of total nuclei. <i>C,</i> In parallel, cells were transfected and treated with TRAIL as in panel <i>B</i>, and after 5 hours caspase-3/7 activity was measured. Mean ± SEM, *p<0.05; **p<0.01; ***p<0.001.</p

Brivanib inhibits liver fibrosis induced by thioacetamide.

<p>(A) Histological analysis of livers from placebo and brivanib-treated (25 and 50 mg/kg) groups at 4 weeks after initiation of thioacetamide (TAA). Pictures of Sirius red and Masson's trichrome staining are taken at 100×, magnification. (B) The number of bands of bridging fibrosis per high power field were counted in images obtained at 100× magnification after Sirius red staining. There was a significant decrease in the number of bands in both the 25 mg/kg and 50 mg/kg brivanib groups, compared to placebo. (C) The hepatic level of <i>collagen Iα1</i> mRNA was measured by real time PCR in placebo, 25 mg/kg and 50 mg/kg brivanib groups (n = 6 per group) at 4 weeks after the initiation of TAA. There was a substantial reduction in <i>collagen Iα1</i> mRNA at both brivanib dose levels. (D) Western immunoblotting for α-SMA showing decreased whole liver α-SMA after treatment with brivanib.</p

Heat stress induced, ligand-independent MET and EGFR signalling in hepatocellular carcinoma

<p><b>Purpose:</b> The aims of the present study were 2-fold: first, to test the hypothesis that heat stress induces MET and EGFR signalling in hepatocellular carcinoma (HCC) cells and inhibition of this signalling decreases HCC clonogenic survival; and second, to identify signalling pathways associated with heat stress induced MET signalling.</p> <p><b>Materials and Methods:</b> MET⁺ and EGFR⁺ HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control ± HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry. IACUC approved partial laser thermal or sham ablation was performed on orthotopic N1S1 and AS30D HCC tumours and liver/tumour assessed for phospho-MET and phospho-EGFR immunostaining.</p> <p><b>Results:</b> Heat-stress induced rapid MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation at the HCC tumour ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and EGFR phosphorylation and inhibition of MET decreased HCC clonogenic survival following heat stress. Pathway analysis of quantitative phosphoproteomic data identified downstream pathways associated with heat stress induced MET signalling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signalling did not block AKT signalling.</p> <p><b>Conclusions:</b> Heat-stress induced MET and EGFR signalling is distinct from growth factor mediated signalling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism.</p

XIAP promoter predicted GLI-binding sites exhibit sequence-specific binding to nuclear proteins.

<p><i>A,</i> Putative GLI binding sites in the XIAP promoter region. Nucleotide positions were counted from the transcription start site (TSS). Potential binding sites were observed as indicated by arrows on the schematic (I and II). <i>B,</i> Nuclear protein extracts were isolated from KMCH cells. EMSA was performed using Cy5.5-labeled double-stranded oligonucleotides containing the putative GLI binding sequences within the human XIAP promoter, and competition experiments with 200-fold molar excess cold oligonucleotides as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018330#s2" target="_blank">MATERIALS AND METHODS</a>”. <i>C</i>, Total cellular protein was isolated 48 hours after transient transfection with the indicated siRNA against GLI family members. XIAP protein expression was determined by immunoblotting and the signal was quantified by densitometry as a ratio to actin expression. <i>D,</i> Chromatin immunoprecipitation using antiserum to GLI1, GLI2, GLI3, or a control IgG was performed followed by PCR using primers flanking site I (341 bp) within the <i>XIAP</i> promoter region, as indicated. As a positive control, ChIP was performed using primers flanking the <i>Bcl-2</i> promoter GLI-binding site (GLI1 and GLI2), or primers flanking the GLI3 binding site within the <i>GLI1</i> promoter.</p

Brivanib decreases the transcription of growth factors and their receptors in carbon tetrachloride.

<p>Hepatic levels of growth factors and growth factor receptor mRNA were measured by real time PCR in sham and CCl₄ mice treated with no brivanib or with brivanib 25 mg/kg, 50 mg/kg, or 100 mg/kg. In all of the sham experiments, higher concentrations of brivanib decreased the mRNA levels of the growth factors and their receptors. (A) mRNA levels of <i>PDGFB</i> are not affected by brivanib treatment in CCl₄. (B) mRNA levels of <i>PDGFRB</i> decrease as the concentration of brivanib increases in CCl₄. (C) mRNA levels of <i>TGFB1</i> decrease as the concentration of brivanib increases in CCl₄. (D) mRNA levels of <i>TGFBR2</i> are lower in brivanib-treated CCl₄ mice compared to no brivanib. (E) mRNA levels of <i>FGF2</i> are not affected by brivanib treatment in CCl₄. (F) mRNA levels of <i>FGFR2</i> are lower in brivanib-treated CCl₄ mice compared to no brivanib. (G, H) mRNA levels of <i>VEGFA</i> and <i>VEGFR2</i> decrease as the concentration of brivanib increases in CCl₄ mice.</p

XIAP knockdown sensitizes cells to TRAIL-induced cell death despite Bid inhibition.

<p><i>A,</i> KMCH cells were incubated for 24 hours in medium or cyclopamine (5 µM) followed by treatment with either vehicle (DMSO, final concentration 0.1% v/v) or the Bid inhibitor, BI-6C9 (10 µM) for 1 hour. Cells were subsequently treated with Fas agonistic antibody CH-11 (100 µg/mL) for an additional 5 hours (where appropriate, BI-6C9 remained in the medium during CH-11 treatment). Cells with apoptotic nuclear morphology were quantified as referenced in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018330#s2" target="_blank">Materials and Methods</a>. Mean +/− SEM; ***p<0.001; n.s. indicates p>0.10 by ANOVA with Bonferroni correction. <i>B,</i> KMCH cells were incubated overnight with or without cyclopamine followed by BI-6C9 as in panel <i>A</i>, except that apoptosis was induced by TRAIL (5 ng/mL) for 5 hours (where appropriate, BI-6C9 remained in the medium during TRAIL treatment) and apoptotic nuclear morphology was quantified. Mean +/− SEM; ***p<0.001; n.s. indicates p>0.10 by ANOVA with Bonferroni correction. <i>C,</i> KMCH cells transiently transfected with XIAP siRNA or scrambled siRNA (48 hours) were pretreated with BI-6C9 (10 µM) or DMSO vehicle (final concentration 0.1% v/v) for 1 hour. Next, the cells were treated with TRAIL (5 ng/mL) for 5 hours (where appropriate, BI6C9 remained in the medium during TRAIL treatment). Cells with apoptotic nuclear morphology were quantified. Mean ± SEM, *p<0.05; n.s. indicates p>0.10 by ANOVA with Bonferroni correction.</p