Project description:Methylation profilingof isolated hepatocytes after DNMT1 knock down. DNMT1 KO versus control livers at week 4, 8 and 20. 18 samples analyzed
Project description:Classification of human liver cancer into biologically distinct subgroups suggests origin from different hepatic lineage cells. To clarify contribution of lineage stage in liver oncogenesis, we transduced H-Ras and SV40LT into hepatic progenitor cells (HPC), hepatoblasts (HB) and terminally differentiated adult hepatocytes (AH). Regardless of origin, all transformed cell types acquired common cancer stem cell characteristics in vitro and in vivo. However, expression analysis distinguished tumors of different cellular origin underscoring the contribution of lineage/stage-dependent genetic changes in malignant transformation. Notably, AH-derived tumors showed specific enrichment of c-Myc target genes. These data establish that any hepatic lineage cell can be a target for transformation and acquire common cancer stem cell traits via activation of diverse cell type specific pathways. Profiling of individual tumors derived by Ras/SV40 transformation of hepatic progenitor cell (oval cells), hepatoblasts (fetal) and adult hepatocytes
Project description:Transcriptomic profiling; Determination of the transcriptomic similarity between Egfr fl/fl and Met fl/fl progenitor cells isolated from excised livers (n=3, each) Profiling of hepatic progenitor cells
Project description:Determine the effect and specificity of HDAC2 siRNA compared to SAHA inhibition of HDAC2 in hepatocellular carcinoma (HCC) Profile of treated cells compared to control Transcriptomic profiling
Project description:Transcriptomic profiling Background Cholangiocarcinoma accounts for 5-10% of primary hepatic cancers. The etiology is unclear and patients are often diagnosed without risk factors. Resection is the only curative treatment although patients frequently remain undiagnosed until advanced stage of disease. Methods To construct molecular classification of cholangiocarcinoma, we profiled the transcriptomes of 104 freshly-frozen tumors and 59 matched non-cancerous livers obtained from Australia, Europe and the United States. We also performed mutational analysis of KRAS, EGFR and BRAF, and used laser-capture microdissection to obtain independent gene expression profiles for epithelial and stromal compartments in a subset of tumors. The selected target genes were validated by western blotting and immunohistochemistry. Results Transcriptomic profiling classified cholangiocarcinoma into two distinct subclasses defined by survival (P<0.0007) and early recurrence (P<0.001). Applying leave-one-out cross-validation, we optimized the prognostic classifier to 238 genes which were positively enriched in the epithelial tumor compartment. A deregulated HER2 network was associated with the epithelial compartment which also showed a frequent overexpression of Ki67, EGFR, MET and pRPS6 whereas inflammatory cytokines were enriched in tumor stroma specifically in patients with poor prognosis. KRAS mutations were found in 24.6% of patients with poor disease outcome. Conclusion Our study presents new insights into pathogenesis of cholangiocarcinoma and stratification of the patients according to survival and recurrence. Identification of a subgroup of patients among the poor prognostic cohort characterized by KRAS mutations and oncogenic-addiction may provide a novel therapeutic opportunity for this treatment-refractory malignancy. Profiling of individual cholangiocarcinomas and non-cancerous matched surrounding livers using normal bile ducts as reference
Project description:Reversal of DNA hypermethylation and associated gene silencing is an emerging cancer therapy approach. In this context we have addressed the impact of epigenetic alterations and local microenvironment on the functional and transcriptional reprogramming of hepatic cancer stem cells. (CSCs) using the DNMT1 inhibitor Zebularine (ZEB). We show for the first time that cellular context is a critical determinant in the response to DNMT1 inhibition resulting in either a long term epigenetically driven malignant reprogramming or an effective antitumor therapeutic reprogramming. Furthermore, permanent reduction of DNMT1 protein level renders the HCC cell lines insensitive to both DNMT1 inhibition and cellular context. These results emphasize the importance of decoding the mechanisms involved in therapeutic application of DNA demethylating agents. Huh7 and PLC treated with ZEB, and Huh7 depleted for DNMT1
Project description:Global transcriptiome changes between SP and NSP cells of liver cancer cell lines w and w/o Zebularine treatment Huh7,WRL68, KMCH FACS sorted in SP and NSP w and w/o Zebularine and subjected to illumina microarray analysis.
Project description:Transcriptomic changes in human liver cancer cell lines caused by the demethylating drug zebularine. Epigenomic changes such as aberrant hypermethylation and subsequent atypical gene silencing are characteristic features of human cancer. Here, we report a comprehensive characterization of epigenomic modulation caused by zebularine, an effective DNA methylation inhibitor, in human liver cancer. Using transcriptomic and epigenomic profiling, we identified a zebularine signature that classified liver cancer cell lines into two major subtypes with different drug-responses. In drug-sensitive cell lines, zebularine caused inhibition of proliferation coupled with increased apoptosis, whereas drug-resistant cell lines were associated with upregulation of oncogenic networks (e.g. E2F1, MYC, and TNF) driving liver cancer growth in vitro and in mice. Assessment of zebularine-based therapy in xenograft mouse models demonstrated potent therapeutic effects against tumors established from zebularine-sensitive but not zebularine-resistant liver cancer cells leading to increased survival and decreased pulmonary metastasis. Integration of zebularine gene expression and demethylation response signatures differentiated patients with HCC according to their survival and disease recurrence and identified a subclass of patients within the poor survivors likely to benefit from therapeutic agents that target the cancer epigenome. Each cell line was mock treated or treated with 100uM and 200uM zebularine for 7 days, respectively *** This Series represents the gene expression component of the study.
Project description:Recent studies suggested that embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) may represent different pluripotent states as defined by gene expression profiles and differentiation potential. Here we addressed a contribution of a lineage stage-specific donor cell memory in modulating the functional properties of iPSCs. iPSCs were generated from hepatic lineage cells at an early (hepatoblast-derived, HB-iPSCs) and end stage (adult hepatocyte, AH-iPSCs) of hepatocyte differentiation as well as from mouse fetal fibroblasts (MEF-iPSCs) using a lentiviral vector encoding four pluripotency-inducing factors Oct4, Sox2, Klf4, and c-Myc. All resulting iPS cell lines acquired iPSCs phenotype as judged by the accepted criteria including morphology, expression of pluripotency markers, silencing of transducing factors, capacity of multilineage differentiation in teratoma assay and normal diploid karyotype. However, hepatoblasts were more susceptible to reprogramming than either AH or MEF, and HB-iPSCs were more efficient in directed differentiation towards hepatocytic lineage as compared to AH-iPSCs, MEF-iPSCs or mESCs. Extensive comparative transcriptome analyses of the early passage iPSCs, donor cells and mESCs revealed that despite global similarities in gene expression patterns between generated iPSCs and mESCs, HB-iPSCs retained a transcriptional memory (7 up- and 20 down-regulated genes) typical of the original cells. Continuous passaging of HB-iPSCs abolished most of these differences including a superior capacity of hepatic re-differentiation. These results suggest that retention of lineage stage-specific donor memory in iPSCs may facilitate differentiation into donor cell type. The identified gene set may be helpful to improve hepatic differentiation for therapeutic application in liver disease modeling. A total of 200 ng RNA from four independent biological replicates of MACS-sorted mESC and iPSC were linearly amplified according to manufactures’ specification (Ambion, Austin, Tx,). For in vitro transcription (IVT), reactions were incubated for 16 h at 37ºC. The efficiency of the single round amplification was measured by NanoDrop (ND1000, Thermo Scientific). Hybridization, washing, detection (Cy3-streptavidin, Amersham Biosciences, GE Healthcare), and scanning were performed on an illumina iScan system (Illumina) using reagents and following protocols supplied by the manufacturer. The biotinylated cRNA (750 ng/sample) was hybridized on Sentrix beadchips human Ref-8v3 for 18 h at 58ºC while rocking (5 rpm).