Project description:Introduction: Hepatocellular carcinoma (HCC) is one of the most aggressive solid tumors and oncogenic pathways (e.g Akt, IGF signaling) are often activated in high proliferating HCC. Among several genetic alterations, epigenetic changes seem to be involved in the development and progression of HCC. DNA hypermethylation of promoter regions is almost always associated with transcriptional silencing and can lead to inactivation of tumor suppressor genes (TSG) in cancer cells. Aim: (1) To identify genes differently methylated in a subclass of HCC associated with proliferation and, (2) To correlate methylation changes with activation of molecular pathways. Methods: gDNA of 20 HCC, 8 cirrhotic and 8 normal liver samples was extracted and the methylation status was detected by the Illumina HumanMethylation27 BeadChip and immunohistochemistry of p-AKT, pIGF IR, p-S6 was analyzed. Results: Unsupervised clustering clearly classified normal livers, cirrhosis and HCC in 3 different groups. 961 genes were significantly hypermethylated in HCC compared to cirrhotic and 3942 genes showed hypermethylation in cirrhotic compared to normal liver tissue. 163 genes showed stepwise significant hypermethylation from normal to cirrhotic to HCC, including well described (p16, SOCS2, SFRP5, RBP1) and potential (SRD5A2, PCDH8, IGF-1R, UCHL1) TSGs, and the miR-10a. 133 genes were specifically hypermethylated in HCC. Among them the transcription factors GATA2, DLX1, and KLF14, all significantly inversely correlated to gene expression (p=0.003, p=0.03, and p=0.007, respectively). The methylation status of SOCS2 (p=0.025) and DLX1 (p=0.025) was significantly correlated to phosphorylation of IGFR1. Samples with RBP1 hypermethylation showed significantly higher AFP serum levels (p=0.018). Conclusion: Whole genome methylation analysis markedly classifies normal, cirrhotic and HCC samples. 8 TSGs play a key role in this stepwise progression of hypermethylation in the development of HCC and could be a promising point of action in anticancer therapy. Genomic DNA extracted from fresh frozen tissue specimens and cell lines was hybridized to genome-wide mthylation beadarray after bisulphite treatment. Keywords: DNA methylation, hepatocellular carcinoma, tissue, cell line

Project description:Introduction: Hepatocellular carcinoma (HCC) is one of the most aggressive solid tumors and oncogenic pathways (e.g Akt, IGF signaling) are often activated in high proliferating HCC. Among several genetic alterations, epigenetic changes seem to be involved in the development and progression of HCC. DNA hypermethylation of promoter regions is almost always associated with transcriptional silencing and can lead to inactivation of tumor suppressor genes (TSG) in cancer cells. Aim: (1) To identify genes differently methylated in a subclass of HCC associated with proliferation and, (2) To correlate methylation changes with activation of molecular pathways. Methods: gDNA of 20 HCC, 8 cirrhotic and 8 normal liver samples was extracted and the methylation status was detected by the Illumina HumanMethylation27 BeadChip and immunohistochemistry of p-AKT, pIGF IR, p-S6 was analyzed. Results: Unsupervised clustering clearly classified normal livers, cirrhosis and HCC in 3 different groups. 961 genes were significantly hypermethylated in HCC compared to cirrhotic and 3942 genes showed hypermethylation in cirrhotic compared to normal liver tissue. 163 genes showed stepwise significant hypermethylation from normal to cirrhotic to HCC, including well described (p16, SOCS2, SFRP5, RBP1) and potential (SRD5A2, PCDH8, IGF-1R, UCHL1) TSGs, and the miR-10a. 133 genes were specifically hypermethylated in HCC. Among them the transcription factors GATA2, DLX1, and KLF14, all significantly inversely correlated to gene expression (p=0.003, p=0.03, and p=0.007, respectively). The methylation status of SOCS2 (p=0.025) and DLX1 (p=0.025) was significantly correlated to phosphorylation of IGFR1. Samples with RBP1 hypermethylation showed significantly higher AFP serum levels (p=0.018). Conclusion: Whole genome methylation analysis markedly classifies normal, cirrhotic and HCC samples. 8 TSGs play a key role in this stepwise progression of hypermethylation in the development of HCC and could be a promising point of action in anticancer therapy.

Project description:Background & Aims. Hepatocellular carcinoma (HCC), the most prevalent form of liver cancer, is growing in incidence but treatment options remain limited, particularly for late stage disease. As liver cirrhosis is the principal risk state for HCC development, markers to detect early HCC within this patient population are urgently needed. Perturbation of epigenetic marks, such as DNA methylation (5mC), is a hallmark of human cancers, including HCC. Identification of regions with consistently altered 5mC levels in circulating cell free DNA (cfDNA) during progression from cirrhosis to HCC could therefore serve as markers for development of minimally-invasive screens of early HCC diagnosis and surveillance. Methods. To discover DNA methylation derived biomarkers of HCC in the background of liver cirrhosis, we profiled genome-wide 5mC landscapes in patient cfDNA using the Infinium HumanMethylation450k BeadChip Array. We further linked these findings to primary tissue data available from TCGA and other public sources. Using biological and statistical frameworks, we selected CpGs that robustly differentiated cirrhosis from HCC in primary tissue and cfDNA followed by validation in an additional independent cohort. Results. We identified CpGs that segregate patients with cirrhosis, from patients with HCC within a cirrhotic liver background, through genome-wide analysis of cfDNA 5mC landscapes. Lasso regression analysis pinpointed a panel of probes in our discovery cohort that were validated in two independent datasets. A panel of five CpGs (cg04645914, cg06215569, cg23663760, cg13781744, and cg07610777) yielded AUROCs of 0.9525, 0.9714, and 0.9528 in cfDNA discovery and tissue validation cohorts 1 and 2, respectively. Conclusions. 5mC markers derived from cfDNA robustly identify HCC within a cirrhotic liver background indicating that further validation is warranted. Our finding that 5mC markers derived from primary tissue did not perform well in cfDNA, compared to those identified directly from cfDNA, reveals potential advantages of starting with cfDNA to discover high performing markers for liquid biopsy development.

Project description:Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide, reflecting the need for a better understanding of hepatocarcinogenesis. In this study, we examined the genome-wide expression profiles of both miRNAs and mRNAs from paired tumor and adjacent non-tumor tissues from a cohort of 96 HCC patients in Hong Kong where hepatitis B virus (HBV) is endemic. The comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that it is an important regulator for normal mitochondrial metabolism. A decreased level of miR-122 leads to an increase in expression of miR-122 seed-matched genes, a loss of mitochondrial metabolic function, and a decrease in the expression of many miR-122 secondary targets. These secondary targets are prognostic markers for HCC patients. Transcriptome profiling data from an additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. To test the HCC findings in normal mouse liver, we used an antagomir against miR-122 and identified altered gene expression profiling by microarray analysis in mouse in vivo experiments. The mouse data recapitulated the human HCC findings. Our anti-miR122 data further provided a direct link between increases in the mRNA levels of miR-122 controlled genes and impairments of mitochondrial metabolism. In summary, we find miR-122 loss may be detrimental to mitochondrial-related metabolisms in sustaining critical liver function and contribute to the morbidity and mortality of liver cancer patients. Mouse liver profiling: In one treatment regimen, four animals were given four doses of 100mg ASO/kg body weight, administered every other day, and sacrificed one day after the last dose. Four animals treated for four weeks were treated twice weekly with 50mg ASO/kg body weight, and sacrificed two days after the last dose. Mice were sacrificed in the morning, and liver was removed for further analysis. Samples from oligonucleotide-treated mice were referenced against samples from saline-treated mice in fluor-reversed pairs. Human liver profiling: For human HCC patient samples, 192 samples -- 96 resected tumor and 96 adjacent non-tumor liver tissues -- were collected from 96 patients who had undergone hepatectomy for curative treatment of HCC at Queen Mary Hospital, Pokfulam, Hong Kong between 1990 and 2007. Informed consents were obtained from patients regarding the use of the liver specimens for research. Samples were hybridized to Affymetrix arrays for mRNA profiling and were analyzed by quantitative PCR for microRNA profiling.

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MiR-579-3p controls hepatocellular carcinoma formation by regulating the PI3K-AKT pathway in chronically inflamed liver (mRNA expression)

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