Project description:Efforts to develop alternatives which can at least partially replace some of the currently used in vivo tests are ongoing. The recently ended FP6 European project carcinoGENOMICS had the goal to use the combination of toxicogenomics and in vitro cell culture models for identification of genotoxic- and non-genotoxic carcinogen-specific gene signatures. In this study is presented a part of the outcome of the project and in particular the performance of the gene classifier derived after exposure of the HepaRG cell line to prototypical hepatocarcinogens. Upon analyzing the data at a gene and a pathway level by using diverse biostatistical approaches, a clear-cut separation of the genotoxic from the non-genotoxic hepatocarcinogens and non-carcinogens was achieved (up to 88% correct prediction). The most characteristic pathway for genotoxic exposure was DNA damage. Further to show the robustness of the HepaRG model, the interlaboratory reproducibility of 3 blindly tested compounds was assessed. The results showed between 20% and 35% reproducibility. The subsequent classification of the 3 blindly tested compounds resulted in correct prediction of the genotoxicant, whereas the other two compounds were misclassified. In conclusion, the combination of transcriptomics and HepaRG in vitro cell model provides a solid basis for the detection of the genotoxic potential of unknown chemicals. HepaRG cells were exposed to 15 selected compounds for 72 hours, i.e. 5 GTX (N-nitrosomorpholine, NMP; Hydroquinone, HQO; Hydrazine dihydrochloride, HHC; 2-acetylaminofluorene, TAF; 2-amino-3-methylimidazo(4,5-f)quinoline, AMQ), 5 NGTX (Fumonisin B1, FMB; Cyclosporine A, CsA; Acetamide, ACE; Diethylhexyl Phthalate, DHP; Ethanol, ETH), 5 NC (4-acetylaminofluorene, FAF; D,L-Menthol, DLM; Benzoin, BEN; Benzyl Alcohol, BEA; Triclosan, TRI). The IC10 concentrations (reducing cell viability by 10%) were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT test) (number of replicates n=3). Further RNA samples were generated by exposure of the HepaRG cells to the MTT-determined IC10 for a period of 24h and 72h. Complementary RNA targets were prepared and hybridized according to the manufacturer's procedures on high-density oligonucleotide microarrays (i.e. Affymetrix U133 Plus 2.0 GeneChip). Finally,alaysis was performed at a gene and pathway level. In the analysis, control (DMSO) samples were re-normalized with several sample groups and new processed data were created for each analysis. Thus, some of the Samples in this Series represent a re-analysis of other Samples and CEL files are identical.

Project description:Oral squamous cell carcinoma (OSCC) is a prevalent form of cancer that develops from the epithelium of the oral cavity. OSCC is on the rise worldwide, and death rates associated with the disease are particularly high. Despite progress in understanding of the mutational and expression landscape associated with OSCC, advances in deciphering these alterations for the development of therapeutic strategies have been limited. Further insight into the molecular cues that contribute to OSCC is therefore required. Here we show that the transcriptional regulators YAP (YAP1) and TAZ (WWTR1), which are key effectors of the Hippo pathway, drive pro-tumorigenic signals in OSCC. Regions of pre-malignant oral tissues exhibit aberrant nuclear YAP accumulation, suggesting that dysregulated YAP activity contributes to the onset of OSCC. Supporting this premise, we determined that nuclear YAP and TAZ activity drives OSCC cell proliferation, survival, and migration in vitro, and is required for OSCC tumor growth and metastasis in vivo. Global gene expression profiles associated with YAP and TAZ knockdown revealed changes in the control of gene expression implicated in pro-tumorigenic signaling, including those required for cell cycle progression and survival. Notably, the transcriptional signature regulated by YAP and TAZ significantly correlates with gene expression changes occurring in human OSCCs identified by The Cancer Genome Atlas (TCGA), emphasizing a central role for YAP and TAZ in OSCC biology. Expression profiling was conducted following the repression of the transcriptional regulators TAZ and YAP (YAP/TAZ) in human SCC2 oral cancer cells. Human SCC2 oral cancer cells were transfected with control siRNA, or siRNAs targeting TAZ, YAP, or YAP/TAZ for 48 hours. Total RNA from three independent experiments carried out on separate days was isolated and purified and the samples were then profiled on Affymetrix Human Gene 2.0 Chips at the Boston University Microarray Core. The expression profiles were processed and normalized using the Robust Multi-array Average (RMA) procedure (23) based on a custom Brainarray CDF (24). For each of the siRNA experiments, signatures of genes differentially expressed between treatment and corresponding siRNA control with an FDR q-value ?0.05 and a fold change ?2 were identified as either activated (up-regulated in control) or repressed (up-regulated in treatment). The overlap between the differentially expressed gene signatures was evaluated by Fisher test. Hierarchical gene and sample clustering was performed on the top 3000 genes with highest median absolute deviation (MAD; a robust version of the variance) across 12 samples, using “ward” as the agglomeration rule, and 1 minus Pearson correlation and Euclidean as the distance measures for genes and samples, respectively.

Project description:For assessing the cancer-causing potential for humans of a chemical compound, the conventional approach is the use of the 2-year rodent carcinogenicity bioassay, thus alternatives such as in vitro toxicogenomics are highly desired. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically-stabilized cultures of primary rat hepatocytes, the human hepatoma-derived HepaRG and HepG2 cell lines and the human embryonic stem cell-derived hepatocyte-like cells hES-Heps are examined and compared. The global gene expression profiles of five commonly used liver-based in vitro systems are investigated, namely conventional cultures of primary rat hepatocytes (HepsC), Trichostatin A (TSA)-stabilized cultures of primary rat hepatocytes (HepsT), human embryonic stem cell-derived hepatocyte-like cells (hES-Hep), HepG2 and HepaRG cells. These models are exposed to 15 prototypical compounds which have been carefully chosen and belong to 3 toxic classes i.e. (i) genotoxic (GTX) (aflatoxin B1, AFB1; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK; 2-nitrofluorene, 2NF; benzo(a)pyrene, BaP; cyclophosphamide, CYCLO), (ii) non-genotoxic (NGTX) (methapyrilene hydrochloride, MPH; piperonylbutoxide , PIPB; Wy-14643, WYE; phenobarbital sodium, SPB; 12-O-tetradecanoylphorbol-13-acetate, TPA) carcinogens and (iii) non-carcinogens (NC) (nifedipine, NIF; clonidine, CND; D-mannitol, MAN; tolbutamide, TOL; diclofenac sodium, SDF). For the gene- and pathway- based analysis, the raw microarray data was re-annotated to Ensembl version 61 genome and Gene Chip Robust Multi-array Average (GC-RMA) normalized (Dai et al. 2005). This resulted in 11,187 and 18,919 probe sets for the rat and the human models, respectively. For the classification analysis, the raw microarray data was annotated using the chip description files from Affymetrix and then GC-RMA normalized. In order to eliminate batch effects, data from the different studies were half-z normalized. Half-z-normalization adjusts the logarithmic expression values of transcripts within a group such that each transcript has zero mean.

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