Unknown,Transcriptomics,Genomics,Proteomics

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DUSP1/MKP1 promotes angiogenesis, invasion and metastasis in non-small cell lung cancer

ABSTRACT: DUSP1 is involved in different cellular pathways including cancer cell proliferation, angiogenesis, invasion and resistance to chemotherapy. To understand more about the cellular responses regulated by DUSP1 in NSCLC cells, we interfered DUSP1 expression in the NSCLC cell line H460 and studied the changes in gene expression differentially regulated by this phosphatase. Keywords: Expression profiling by array in cells with genetic modification The human non small lung cancer cell line H460 used in this study was from ATCC (American Type Culture Collection). The cell line was maintained in RPMI (Gibco, Invitrogen) supplemented with 10% bovine serum and transfected by Lipofectamine Plus Reagent from Life Technologies as directed by the manufacturer. The DUSP1 pSuperRetro-derived vectors were constructed as described (Chattoppadhyay et al., 2006). Clones expressing siRNA for DUSP1 were selected for their ability to grow in the presence of puromycin and kept for selection. Stable transfection was confirmed by Western blotting. RNA was extracted and quality control was checked before hybridization on Affymetrix microarrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Eva Bandres

PROVIDER: E-GEOD-19592 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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DUSP1/MKP1 promotes angiogenesis, invasion and metastasis in non-small-cell lung cancer.

Moncho-Amor V V Ibañez de Cáceres I I Bandres E E Martínez-Poveda B B Orgaz J L JL Sánchez-Pérez I I Zazo S S Rovira A A Albanell J J Jiménez B B Rojo F F Belda-Iniesta C C García-Foncillas J J Perona R R

Oncogene 20101004 6

DUSP1/MKP1 is a dual-specific phosphatase that regulates MAPKs activity, with an increasingly recognized role in tumor biology. To understand more about the involvement of DUSP1 in lung cancer, we performed gene expression analyses of parental and DUSP1-interfered H460 non-small-cell lung cancer (NSCLC) cells. Downregulation of DUSP1 induced changes in the expression levels of genes involved in specific biological pathways, including angiogenesis, MAP kinase phosphatase activity, cell-cell signa ...[more]

PMID: 20890299

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Project description:The activation of the transcription factor Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small-molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high throughput screen led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations but did not affect expression levels of genes outside of the HIF-1 pathway. Lead structure BAY 87-2243 was found to inhibit HIF-1Î± protein accumulation under hypoxic conditions in NSCLC cell line H460 but had no effect on HIF-1Î± protein accumulation and HIF target gene expression in RCC4 cells lacking VHL activity or in H460 cells after inhibition of HIF prolyl hydroxylase activity. BAY 87-2243 had no effect on HIF-Î±-mRNA levels. Antitumor activity of BAY 87-2243 and suppression of HIF-1 target gene expression in vivo was demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial production of reactive oxygen species (ROS) by blocking complex I activity but has no effect on complex III activity. Lowering of mitochondrial ROS production to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors. We used microarrays to detail the global programme of gene expression that is induced in NSCLC cell line H460 upon hypoxia (16 h incubation at 1 % pO2) and evaluated a dose-dependent effect of our HIF-1-pathway inhibitor BAY 87-2243 on genes tthat are affected by hypoxia. Specificity of BAY 87-2243 for the suppression of HIF-1-mediated gene transcription on a genome-wide scale was evaluated by microarray hybridizations using Affymetrix GeneChip Human Gene 1.0 ST arrays. RNA from normoxic H460 cells and from hypoxic H460 cells incubated with 1, 10 and 100 nM BAY 87-2243 respectively was subjected to array hybridization. Of those 30 genes that were most strongly suppressed by 100 nM BAY 87-2243 in hypoxic H460 cells compared to DMSO-treated hypoxic H460 cells, virtually all of them are induced by prior hypoxia and most of these genes have been described in the literature as HIF-1 target genes

Project description:Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of human ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in tumour cell lines. The hypothesis tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two tumour cell lines (H460 and HCT116). All four lines had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p=0.002) and 4.3±0.8% (p=0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no effect on glycolysis as measured by glucose consumption or lactate formation under oxic or anoxic conditions, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis. Use Affymetrix microarrays to examine the effecs of knockout of the gene ADPGK using Zinc Finger nucleases in two cultured cell lines: (i) HCT116 cells (three wild type cultures compared to three ADPGK-knockout cultures), (ii) H460 cells (two wild type cultures compared to two ADPGK-knockout cultures). Ten microarrays in total.

Dataset Information

DUSP1/MKP1 promotes angiogenesis, invasion and metastasis in non-small cell lung cancer

Publications

DUSP1/MKP1 promotes angiogenesis, invasion and metastasis in non-small-cell lung cancer.

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