Unknown,Transcriptomics,Genomics,Proteomics

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Mouse frozen liver: vehicle-control vs. CCl4+DEN treated

ABSTRACT: Hepatocellular carcinoma (HCC) is a prevalent human cancer with rising incidence worldwide. Human HCC is frequently associated with chronic liver inflammation and cirrhosis, pathophysiological processes that are a consequence of chronic viral infection, disturbances in metabolism, or exposure to chemical toxicants. To better characterize the pathogenesis of HCC, we used a human disease-relevant mouse model of fibrosis-associated hepatocarcinogenesis. In this model, marked liver tumor response caused by a pro-mutagenic chemical N-nitrosodiethylamine in presence of liver fibrosis was associated with epigenetic events indicative of genomic instability. Therefore, we hypothesized that DNA copy number alterations (CNAs), a feature of genomic instability and a common characteristic of cancer, are concordant between human HCC and mouse models of fibrosis-associated hepatocarcinogenesis. We evaluated DNA CNAs and changes in gene expression in the mouse liver (normal, tumor and non-tumor cirrhotic tissues). In addition, we compared our findings to those in human HCC (tumor and non-tumor cirrhotic/fibrotic tissues). We observed that while fibrotic liver tissue is largely devoid of DNA CNAs, highly frequently occurring DNA CNAs are found in mouse tumors, which is indicative of a profound increase in chromosomal instability in HCC. When compared to CNAs in human HCC, we found that 33% of genes in these segments are similarly affected in the mouse tumors. Our results suggest that CNAs most commonly arise in neoplastic tissue rather than in fibrotic liver, and demonstrate the utility of this mouse model in replicating the molecular features of human HCC. Genomic DNA was extracted from frozen liver samples from vehicle-control and DEN+CCl4-treated mice using a DNEasy kit (Qiagen, Valencia, CA). Eighteen mouse tumor samples were included in the study, as well as 18 matched non-tumor samples taken from fibrotic, non-tumorous, surrounding liver tissue from the same mice. Liver DNA from 6 vehicle control-mice was pooled and used as the reference genome in the aCGH experiments. Copy number alterations were identified using the normalized data.

ORGANISM(S): Mus musculus

SUBMITTER: Ivan Rusyn

PROVIDER: E-GEOD-71849 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Gene-expression profiles of liver and hepatocellular carcinoma induced by diethylnitrosamine (DEN) in KLF6 +/- and wild type KLF6 mice. Inactivation of the KLF6 tumor suppressor is common in HCC due to hepatitis C virus (HCV), consistent with its anti-proliferative activity in HCC-derived cell lines and in hepatocytes of transgenic mice. We have evaluated the impact of KLF6 depletion on human HCC and experimental hepatocarcinogenesis. In patients with surgically resected HCC, those with significantly reduced tumor expression of KLF6 had a significantly decreased survival. We modeled this event in KLF6 +/- mice, which displayed significantly more tumorigenicity than KLF6 +/+ animals in response to the hepatic carcinogen DEN, associated with recapitulation of gene signatures in both surrounding tissue and tumors that are associated with aggressive human HCCs. In DNA microarrays, mdm2 mRNA expression was increased in tumors from KLF6 +/- compared to KLF6 +/+ mice, which was validated by realtime qPCR and Western blot in both human HCC and DEN-induced murine tumors. Moreover, chromosomal immunoprecipitation and co-transfection assays established the P2 intronic promoter of mdm2 as a bona fide transcriptional target repressed by KLF6. Whereas KLF6 over-expression in HCC cell lines led to reduced MDM2 levels and increased p53 protein and transcriptional activity, reduction in KLF6 by siRNA led to increased MDM2 and reduced p53. Our findings indicate that KLF6 deficiency contributes significantly to the carcinogenic milieu in human and murine HCC, and uncover a novel tumor suppressor activity of KLF6 in HCC, by linking its transcriptional repression of MDM2 to stabilization of p53. Keywords: Liver, Hepatocellular carcinoma, Expression array, Exon array, Affymetrix KLF6 +/- mice were previously generated by homologous recombination in which exon 2 was targeted using an 11-kb targeting construct, and replaced with neomycin/lacZ cassette. After selection with neomycin, the ES clones were injected into C57BL/6 mouse blastocysts and implanted into pseudo pregnant females; two lines of KLF6 +/- mice were generated from the resulting chimeric animals (Blood 107;1357, Oncogene 26;4428). Whereas KLF6 -/- mice are embryonic lethal, KLF6 +/- animals had no demonstrable abnormalities in the absence of any stressor. Male KLF6 +/- mice were bred with wild type C57BL/6 to generate mixed litters of KLF6 +/- and KLF6 +/+ animals. Progeny were genotyped using PCR-amplified tail DNA, using primers as previously described (Oncogene 26;4428). Amplified fragments were separated on a 2.5% agarose gel, revealing bands of ~200 bp (wild type KLF6) and ~100 bp (Neo), as expected. At 2 weeks of age, KLF6 +/+ and KLF6 +/- mice were injected intraperitoneally with either a single dose of diethyl nitrosamine (DEN), 5 µg/g body weight in 100 µl of saline, or vehicle alone. Vehicle and DEN-treated mice were maintained on standard chow, and then sacrificed 3, 6 or 9 months later. At the time of sacrifice the animals were weighed, and blood and liver samples were harvested for analysis and tumor quantification.

Project description:Background & Aims. Resection of hepatocellular carcinoma (HCC) tumors by partial hepatectomy (PHx) is associated with promoting hepatocarcinogenesis. We have previously reported that PHx promotes hepatocarcinogenesis in the Mdr2-knockout (Mdr2-KO) mouse, a model for inflammation-mediated HCC. Now, we explored the molecular mechanisms underlying the tumor-promoting effect of PHx in these mice. Methods. Using microarrays-based techniques, we compared genomic and transcriptomic profiles of HCC tumors developing in the Mdr2-KO mice either spontaneously or following PHx. Results. PHx accelerated HCC development in these mice by four months. PHx-induced tumors had only amplifications affecting multiple chromosomes and locating mainly near the acrocentric centromeres of murine chromosomes. Four different chromosomal regions were amplified each in at least three tumors. All tumors of untreated mice had chromosomal aberrations, including both deletions and amplifications. Comparison of gene expression profiles revealed a significantly enriched expression of oncogenes, chromosomal instability markers and E2F1 targets in the post-PHx compared to spontaneous tumors. Both tumor groups shared the same frequent amplification at chromosome 18. Here, we demonstrated that one of the regulatory genes encoded by this amplified region, Crem, was over-expressed in the nuclei of murine and human HCC cells in vivo, and that it stimulated proliferation of human HCC cells in vitro. Conclusions: PHx of a chronically inflamed liver directed tumor development to a discrete pathway characterized by amplification of specific chromosomal regions and expression of specific tumor-promoting genes. Crem is a new candidate HCC oncogene frequently amplified in this model and frequently over-expressed in human HCC. To explore the mechanisms of the accelerated HCC development by PHx, we compared liver tumors and their matched non-tumor liver tissues between 9-month-old hepatectomized and 13-14-month-old untreated Mdr2-KO mice. RNA was isolated from frozen liver tissues and subjected to gene expression profiling using GeneChip Mouse Gene 1.0 ST Array

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Mouse frozen liver: vehicle-control vs. CCl4+DEN treated

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