Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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PACT-PKR Pathway Transactivates the Tumor Suppressor RhoB through C/EBPbeta in Cancer Cells with High Akt Activity

ABSTRACT: Activation of the PI3K/Akt/mTOR pathway in cancers can occur through loss of PTEN. Transcriptional profiling of pathway inhibitors identified the tumor suppressor RhoB as a gene markedly upregulated by lipid-based Akt inhibitors (LBAI). Here, we demonstrate that the C/EBPbeta full-length isoform LAP is responsible for transcriptional induction through its binding site within the RhoB proximal promoter. LBAI strongly transactivate RhoB by switching translation of C/EBPbeta from the truncated isoform LIP to LAP via PACT-mediated PKR activation in cancer cells with high Akt activity. Unlike PTEN commonly mutated, endogenous RhoB tumor-suppressive activity can be reconstituted by restoring its expression, which was noninvasively monitored by a RhoB promoter-driven luciferase reporter in living mice. LBAI administration increased luciferase activity and decreased the growth of human tumor xenografts. Increased PKR activation by LBAI leads to more robust RhoB induction and cytotoxicity than other PI3K/Akt/mTOR axis inhibitors, revealing a novel strategy for cancer therapy. H157 cells were plated 2 x 10^6 in T-75 flasks in RPMI 1640 containing 10% FBS and incubated for 24h. The medium was then changed to RPMI 1640 with 0.1% FBS and the cells were incubated overnight. The following morning, cells were treated with 10 microM of LY294002, OSU03012, PIA23, Perifosine, Miltefosine, API-2, DZ-50, or 100 nM of Wortmannin and Rapamycin for 6 hours, or an equal amount of DMSO as control. Following incubation, total RNA was extracted from cells in T-75 flasks using TRIzol reagent (Invitrogen). Two dye-swapped replicates were performed for each treatment.

ORGANISM(S): Homo sapiens

SUBMITTER: Phillip Dennis

PROVIDER: E-GEOD-28993 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Activation of the serine/threonine kinase Akt contributes to the formation, maintenance, and therapeutic resistance of cancer, which is driving development of compounds that inhibit Akt. Phosphatidylinositol ether lipid analogues (PIAs) are analogues of the products of PI3K that inhibit Akt activation, translocation, and the proliferation of a broad spectrum of cancer cell types. To gain insight into the mechanism of PIAs, time-dependent transcriptional profiling of 5 active PIAs and the PI3K inhibitor LY294002 (LY) was performed in non-small cell lung cancer (NSCLC) cells using high-density oligonucleotide arrays. Gene ontology analysis revealed genes involved in apoptosis, wounding response, and angiogenesis were upregulated by PIAs, while genes involved in DNA replication, repair and mitosis were suppressed. Genes that exhibited early differential expression were partitioned into 3 groups; those induced by PIAs only (DUSP1, KLF6, CENTD2, BHLHB2, PREX1), those commonly induced by PIAs and LY (TRIB1, KLF2, RHOB and CDKN1A), and those commonly suppressed by PIAs and LY (IGFBP3, PCNA, PRIM1, MCM3 and HSPA1B). Increased expression of the tumor suppressors RHOB (RhoB), KLF6 (COPEB) and CDKN1A (p21Cip1/Waf1) was validated as an Akt-independent effect that contributed to PIA-induced cytotoxicity. Despite some overlap with LY, active PIAs have a distinct expression signature that contributes to their enhanced cytotoxicity. H157 cells were plated at 2x10^6 in T-75 flasks in RPMI medium 1640 containing 10% FBS and incubated for 24h. The media was then changed to RPMI medium 1640 with 0.1% FBS and the cells were incubated overnight. The following morning, cells were treated with 10 microM PIA6 dissolved in DMSO for 0h, 2h, 6h or 12h, and an equal volume of DMSO was added to control samples. For the PIA comparison, 10 microM PIAs (5, 6, 7, 23, 24, 25) or 10 microM LY294002 (LY) were incubated with the cells for 6h. In the PIA comparison experiment, the same concentration of PIA7 (an inert analog consisting of only the lipid side chain) was used as a control. Following incubation, the alterations in cellular morphology were photographed, and cells from 6-well plates were harvested for immunoblot analysis. Total RNA was extracted from cells treated in T-75 flasks using TRIzol reagent (Invitrogen) and chloroform, and purified according to the RNeasy midiprep spin kit protocol (Qiagen). Oligonucleotide microarray was performed with dye-swap. Microarray chips were generated from the 34,580 longmer probe set Human Genome Oligo Set Version 3.0 (Qiagen). Protocols for cDNA labeling, hybridization, and scanning are available through the National Human Genome Research Institute microarray core.

Project description:BACKGROUND & AIMS: C/EBPbeta is involved in numerous process as carcinogenesis but its role is still not clear due to the existence of an active form (LAP) and an inhibitory form (LIP) of this transcription factor. The main goals of the present research were (i) the identification of genes inversely regulated by LAP and LIP i-e the genuine C/EBPbeta molecular signature in the Hep3B human hepatoma cell line (ii) a better understanding of LAP and LIP respective role in hepatic cells survival and proliferation (iii) the search of the C/EBPbeta signature among hepatocellular carcinomas. METHODS: Using Tet-off expression system we engineered Hep3BLAP and Hep3BLIP cells, in which LAP and LIP were over-expressed respectively. Then, using both expression profiling (DNA arrays) and ChIP-on-chip analysis, we identified genes inversely and/or directly regulated by each of the C/EBPbeta isoforms. The expression levels of these genes regulated by LAP/LIP were compared in controls and HCCs patients. RESULTS: We identified 676 genes inversely regulated by LAP and LIP and among these, 45 are direct targets. Using functional studies, we displayed the opposite role of LAP and LIP in staurosporine-induced cell death and the implication of LAP in the repression of Hep3B cells proliferation. Finally we identified a subgroup of HCCs with a deregulation of 165 genes belonging to C/EBPbeta signature and coding for proteins involved in chemoresistance and metastasis formation. CONCLUSIONS: Our study increases knowledge on LAP and LIP functions and provides first evidence that their molecular signature in the HCCs could predict tumor evolution. Total genomic DNA were extracted from 3 Hep3BLAP expressing LAP and were labelled Cy3 fluorochrome. Genomic DNA were extracted from 3 Hep3BLAP expressing LAP, were immunoprecipited with anti-CEBPbeta antibody and were labelled with Cy5 fluorochrome. Each sample was hybridized on an Agilent two-color microarray G4489A (Human Promoter ChIP-on-Chip Set 244K).

Project description:Gene expression profiling of tissues derived from different genetic backgrounds can provide insight to potential phenotype-related changes in gene expression. We have compared wildtype C57BL/6 (melan-Ink4a) melanocyte cell gene expression to that of, âbrownâ Tyrp1b/ b (melan-Ink4a-b) and âchocolateâ Rab38 cht/cht (melan-cht) melanocyte derived lines. We observed alterations in gene expression consistent with a physiological response of melan-Ink4a-b cells to endoplasmic reticulum protein-folding impairment. Additionally, a set of loci was identified that are expressed in a similar manner to known melanocyte genes. Importantly, the collection of 254 genes is enriched for expression in the multiple melanocyte lines relative to control cell populations. This set contains Tyr, Tyrp1, Dct, Si, Melna, Slc24a5, Slc45a2, Slc24a4, Mitf, and Sox10, in addition to genes not previously attributed to pigmentation and genes with unknown function. Further evaluation of one gene in this cohort, Gpnmb, demonstrated an expression pattern similar to Dct, Si and Mitf. This expression is vastly reduced in MitfMi mutants embryos, indicating that it is dependent on Mitf for in vivo expression. Consistent with this notion, a highly-conserved MITF binding site resides directly upstream of human GPNMB, and deletion of this sequence dramatically reduces in vitro enhancer activity. Keywords: NIH embryo fibroblast cell line, melanocyte cell line, Ink4a, Tyrp1b, Rab38 A 32k expression arrays were generated by printing Oligo nucleotides from Operon Mouse oligo set (MV3) onto epoxy slides. Total RNA from immortalized melanocyte cell lines derived from C57BL/6J Ink4a-/- (melan-Ink4a-1), C57BL/6J Ink4a-/-; Tyrp1b/b (melan-Ink4a-b) and 2 separate C57BL/6J Ink4a-/-; RaB38cht/cht lines (melan-cht4), (melan-cht5) and NIH3T3 were labeled with Cy5/3 and co-hybridized with a total RNA universal reference (Stratagene, Inc) labeled with Cy3/5. Each comparison was done in Triplicate including one dye swap.

Project description:We have developed a method for rapidly identifying early, regionally-expressed molecules: FACS-Assisted Microdissection of Photolabeled cells (FAM-P). For our first FAM-P project, we injected embryos with purified Kaede protein, a stony coral protein that fluoresces at 514 nm (green) prior to- and 582 nm (red) after photoconversion at 405 nm. We used the scanning laser of a confocal microscope to photolabel late blastula-stage embryos along 4-6 tiers of marginal cells, comprising the mesoderm and endoderm germ layer precursors. This procedure left the neurectoderm precursor cells marked by green fluorescence and the mesendoderm precursor cells marked by red fluorescence. Embryonic cells were dissociated and subjected to FACS, separating red mesendoderm precursors from green neurectoderm precursors. RNA was extracted and linearly amplified once, then dye-labeled and co-hybridized to a microarray with over 30,000 oligos representing more than 20,000 unique zebrafish genes. In validation of our strategy, many genes known to have elevated expression in the late blastula margin (e.g., squint, fgf8, lim1 and gata5) and several genes known to be specific to the early neurectoderm (e.g., sox31 and otx2) were independently identified by this approach. In addition to known genes, our method has identified dozens of previously uncharacterized genes that are enriched in the pre-mesendoderm or pre-neurectoderm. Keywords: 40% epiboly-stage embryos, injected with purified Kaede protein, differentially photolabeled, disaggregated and FACS sorted 34k expression arrays were generated by printing Oligo nucleotides from three different sets (MWG, Compugen and Operon) onto epoxy slides. Total RNA from Kaede protein-injected and FAM-P treated zebrafish embryos was amplified using an Ambion kit (Cat#1753), labeled with Cy dyes and hybridized overnight to the oligo chip in a MAUI chamber. The experiment comparing mesendoderm was performed four times (twice with WIK/AB hybrid strain embryos, once with EK strain embryos and once with AB strain embryos) each time including a dye swap, for a total of eight (four forward and four dye-swap) hybridizations. Three control cRNA probes were also generated from AB-strain embryos, namely: (1) whole 40% epiboly stage embryos, (2) FACS-sorted green cells from dissociated 40% epiboly embryos that were Kaede-injected but NOT photolabeled and (3) FACS-sorted red cells from dissociated 40% epiboly embryos that were Kaede-injected AND photolabeled. These probes were each generated three times, each time including a dye swap and co-hybridized as follows: (1) whole embryo cRNA vs green cell cRNA (three forward and three dye-swap hybridiizations) and (2) green cell cRNA vs. red cell cRNA (three forward and three dye-swap hybridizations).

Project description:C/EBPβ plays a major role in numerous biological processes but unfortunately its precise role is not still clear and conflicting studies showed that this transcription factor could have contradictory functions. These latter arise from the complexity of mechanisms regulating C/EBPβ activity. Indeed, C/EBPβ encodes an intronless gene that generates a single mRNA that is alternatively translated into two major isoforms of 35kDa (liver-enriched activator protein: LAP) and 20kDa (liver-enriched inhibitory protein: LIP). LAP is the active isoform of this transcription factor whereas LIP, a truncated isoform negatively regulate C/EBPβ-LAP-mediated gene expression. The main goal of our research was to understand how LAP and LIP isoforms governe C/EBPβ cellular functions with the identification of new genes and pathways regulated by these isoforms in the human Hep3B hepatoma cell line. For this purpose an original in vitro system characterized by a target genes induction with the activatory C/EBPß isoform LAP and a target genes repression with the inhibitory C/EBPß isoform (LIP) was used to identify the genuine C/EBPβ molecular signature. Using a cDNA microarray which provides a complete coverage of the liver transcriptome, we identified 676 genes inversely regulated by LAP and LIP. These selected genes are involved in many biological processes as the hepatic metabolism (cholesterol), detoxification, induction of apoptosis and negative regulation of the cell proliferation. According to the involvement of C/EBPβ in hepatic carcinogenesis we focused on cell cycle regulation and apoptosis. Through functional studies, we proved for the first time that LIP plays in favor of the survival of the Hep3B cells whereas LAP makes the cells more sensitive to staurosporine-induced cell death. Moreover, a lot of studies demonstrated that the anti-proliferative action of C/EBPβ mainly depends on RB protein. By studying the rate of Hep3B cells proliferation which overexpress LAP, we brought to the fore that this isoform would be able to induce a repression of the Hep3B cells line proliferation - a RB- and p53- negative cell line. Thus, LAP seems able to induce the repression of proliferation by a different metabolic way from the RB one. Keywords: comparison of cells expressing LAP or LIP RNA were extracted from 5 Hep3BLAPexpressing LAP, 5 Hep3BLAP control without expression of LAP, 5 Hep3BLIP expressing LIP and 5 Hep3BLIP control without epression of LIP. Each sample was hybridized once in 5 different nylon membranes.

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PACT-PKR Pathway Transactivates the Tumor Suppressor RhoB through C/EBPbeta in Cancer Cells with High Akt Activity

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