Gene expression-based chemical genomics identifies potential therapeutic drugs in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an aggressive tumor with a poor prognosis. Currently, only sorafenib is approved by the FDA for advanced HCC treatment; therefore, there is an urgent need to discover candidate therapeutic drugs for HCC. We hypothesized that if a drug signature could reverse, at least in part, the gene expression signature of HCC, it might have the potential to inhibit HCC-related pathways and thereby treat HCC. To test this hypothesis, we first built an integrative platform, the "Encyclopedia of Hepatocellular Carcinoma genes Online 2", dubbed EHCO2, to systematically collect, organize and compare the publicly available data from HCC studies. The resulting collection includes a total of 4,020 genes. To systematically query the Connectivity Map (CMap), which includes 6,100 drug-mediated expression profiles, we further designed various gene signature selection and enrichment methods, including a randomization technique, majority vote, and clique analysis. Subsequently, 28 out of 50 prioritized drugs, including tanespimycin, trichostatin A, thioguanosine, and several anti-psychotic drugs with anti-tumor activities, were validated via MTT cell viability assays and clonogenic assays in HCC cell lines. To accelerate their future clinical use, possibly through drug-repurposing, we selected two well-established drugs to test in mice, chlorpromazine and trifluoperazine. Both drugs inhibited orthotopic liver tumor growth. In conclusion, we successfully discovered and validated existing drugs for potential HCC therapeutic use with the pipeline of Connectivity Map analysis and lab verification, thereby suggesting the usefulness of this procedure to accelerate drug repurposing for HCC treatment.     

Epigenetic dosage identifies two major and functionally distinct β cell subtypes

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Gene expression profiling identifies eleven DNA repair genes down-regulated during mouse neural crest cell migration

Neural Crest Cells (NCCs) are transient multipotent migratory cells that derive from the embryonic neural crest which is itself derived from the margin of the neural tube. DNA repair genes are expressed in the early stages of mammalian development to reduce possible replication errors and genotoxic damage. Some birth defects and cancers are due to inappropriate or defective DNA repair machinery, indicating that the proper functioning of DNA repair genes in the early stages of fetal development is essential for maintaining DNA integrity. We performed a genome-wide expression analysis combining laser capture microdissection (LCM) and high-density oligo-microarray of murine NCCs at pre-migratory embryonic days 8.5 (E8.5), and at E13.5, as well as on neural crest-derived cells from the adrenal medulla at postnatal day 90. We found 11 genes involved in DNA repair activity (response to DNA damage stimulus, DNA damage checkpoint, base-excision repair, mismatch repair), over-expressed in the early stages of mouse embryo development. Expression of these 11 genes was very low or undetectable in the differentiated adrenal medulla of the adult mouse. Amongst the 11 genes, 6 had not been previously reported as being over-expressed during mouse embryonic development. High expression of DNA repair genes in enriched NCCs during early embryonic development may contribute to maintaining DNA integrity whilst failure of some of these genes may be associated with the onset of genetic disease and cancer. Our model of enriched murine NCCs and neural crest-derived cells can be used to elucidate the key roles of genes during normal embryonic development and in cancer pathogenesis.     

Gene expression profile and mutational analysis of DNA mismatch repair genes in carcinoma prostate in Indian population

DNA mismatch repair (MMR) plays a role in promoting genetic stability by repairing DNA replication errors, inhibiting recombination between nonidentical DNA sequences, and participating in responses to DNA damage. Although the role of MMR in prostate carcinogenesis remains unclear, MMR deficiency in Carcinoma Prostate (Pca) could prove to be clinically significant. Thus, the present study investigated the gene expression profile of six major MMR genes, viz. hMLH1, hMSH2, hPMS1, hPMS2, hMSH3, and hMSH6, and polymorphism in hMLH1 and hMSH2 in Pca in Indian population. Further, correlation with clinicopathological parameters was evaluated to establish their role as a potential prognostic marker. A significant downregulation of hMLH1, hMSH2, and hPMS2 expression was observed in Pca compared to benign prostatic hyperplasia (BPH). A greater loss of hPMS2 protein in poorly differentiated tumors was demonstrated, which was in concordance with a significant inverse correlation of hPMS2 gene expression with the Gleason score indicating its significance as a marker for Pca progression. An important association of hMLH1-93G>A polymorphism with the risk of Pca was also identified. The results of the present study suggest that an altered MMR has important biological and clinical significance in Pca in Indian population.     

KSHV-infected PEL cell lines exhibit a distinct gene expression profile

We analyzed the gene expression profiles of lymphocyte-originated tumor cell lines - primary effusion lymphoma (PEL) cell lines, T-cell leukemia (TCL) cell lines, Burkitt lymphoma (BL) cell lines - and two sets of normal peripheral blood mononuclear cells (PBMCs) - in order to determine characteristic gene expression profiles for each of the former three groups. And we found that these cell lines showed respective typical gene expression profiles and classified into clear four groups, PEL, TCL, BL, and normal PBMCs. Two B lymphocyte-originated tumor cell lines, PEL and BL cell lines, clearly exhibited distinct gene expression profiles, respectively. Even though there was only one line that was co-infected with both Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), KSHV seemed to govern the gene expression profile of the co-infected line. These data suggested not only that established typical tumor cell lines show a distinct gene expression profile but also that this profile may be governed by certain viruses.     

Phosphoproteomic screen identifies potential therapeutic targets in melanoma

Therapies directed against receptor tyrosine kinases are effective in many cancer subtypes, including lung and breast cancer. We used a phosphoproteomic platform to identify active receptor tyrosine kinases that might represent therapeutic targets in a panel of 25 melanoma cell strains. We detected activated receptors including TYRO3, AXL, MERTK, EPHB2, MET, IGF1R, EGFR, KIT, HER3, and HER4. Statistical analysis of receptor tyrosine kinase activation as well as ligand and receptor expression indicates that some receptors, such as FGFR3, may be activated via autocrine circuits. Short hairpin RNA knockdown targeting three of the active kinases identified in the screen, AXL, HER3, and IGF1R, inhibited the proliferation of melanoma cells and knockdown of active AXL also reduced melanoma cell migration. The changes in cellular phenotype observed on AXL knockdown seem to be modulated via the STAT3 signaling pathway, whereas the IGF1R-dependent alterations seem to be regulated by the AKT signaling pathway. Ultimately, this study identifies several novel targets for therapeutic intervention in melanoma.     

Expression profile identifies novel genes involved in neuronal differentiation

In the presence of NGF, PC12 cells extend neuronal processes, cease cell division, become electrically excitable, and undergo several biochemical changes that are detectable in developing sympathetic neurons. We investigated the expression pattern of the apoptosis-related genes at each stage of neuronal differentiation using a cDNA microarray containing 320 apoptosis-related rat genes. By comparing the expression patterns through time-series analysis, we identified candidate genes that appear to regulate neuronal differentiation. Among the candidate genes, HO2 was selected by real-time PCR and Western blot analysis. To identify the roles of selected genes in the stages of neuronal differentiation, transfection of HO2 siRNA in PC12 cells was performed. Down-regulation of HO2 expression causes a reduction in neuronal differentiation in PC12 cells. Our results suggest that the HO2 gene could be related to the regulation of neuronal differentiation levels.     

Gene expression profile in immunologically injured liver cell of mice

To study the gene expression profiles between immunologically injured liver cell and normal liver cell of mice and to screen on a large scale the differentially expressed genes associated with the formation of liver injury,the experimental mice were randomly divided into the normal group for controlling and the immunologically liver-injured group induced by BCG and LPS.The liver mRNA of the two groups were extracted respectively and reversely-transcribed to cDNA with the incorpora-tion of different fluorescence(Cy3,Cy5) labeled dUTP as the hybridization probes.The mixed probes were hybridized to the cDNA microarray chips.The fluorescent signal results were acquired by scanner ScanArray 4000 and analyzed with software GenePix Pro 3.0.Among the 14112 target genes,293 genes were found to be significantly differentially expressed,in which 188 genes were up-regulated and 105 genes were down-regulated.Based on the analysis of biological functions of those differentially expressed genes,it was indicated that the occurrence and development of mouse liver damage induced by BCG and LPS were highly correlated with the processes of immune reac-tions,cell synthesis,metabolism,apoptosis and transportation in liver cell,which might be quite im-portant for elucidating the regulatory network of gene expression associated with the liver damage,also important for finally discovering the pathogenic mechanisms of immunological liver damage.     

Gene expression profile analysis in human hepatocellular carcinoma by cDNA microarray

We performed gene expression profiling of normal and hepatocellular carcinoma (HCC) liver tissues using a high-density microarray that contained 3,063 human cDNA. The results of a microarray hybridization experiment from eight different HCC tissues were analyzed and classified by the Cluster program. Among these differentially-expressed genes, the galectin-3, serine/threonine kinase SGK, translation factor eIF-4A, -4B, -3, fibroblast growth factor receptor, and ribosomal protein L35A were up-regulated; the mRNAs of Nip3, decorin, and the insulin-like growth factor binding protein-3 were down-regulated in HCC. The differential expression of these genes was further confirmed by an RT-PCR analysis. In addition, our data suggest that the gene expression profile of HCC varies according to the histological types.     

Gene expression profile in immunologically injured liver cell of mice

To study the gene expression profiles between immunologically injured liver cell and normal liver cell of mice and to screen on a large scale the differentially expressed genes associated with the formation of liver injury, the experimental mice were randomly divided into the normal group for controlling and the immunologically liver-injured group induced by BCG and LPS. The liver mRNA of the two groups were extracted respectively and reversely-transcribed to cDNA with the incorporation of different fluorescence (Cy3, Cy5) labeled dUTP as the hybridization probes. The mixed probes were hybridized to the cDNA microarray chips. The fluorescent signal results were acquired by scanner ScanArray 4000 and analyzed with software GenePix Pro 3.0. Among the 14112 target genes, 293 genes were found to be significantly differentially expressed, in which 188 genes were up-regulated and 105 genes were down-regulated. Based on the analysis of biological functions of those differentially expressed genes, it was indicated that the occurrence and development of mouse liver damage induced by BCG and LPS were highly correlated with the processes of immune reactions, cell synthesis, metabolism, apoptosis and transportation in liver cell, which might be quite important for elucidating the regulatory network of gene expression associated with the liver damage, also important for finally discovering the pathogenic mechanisms of immunological liver damage.