Unknown,Transcriptomics,Genomics,Proteomics

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Involvement of transcription factor early growth response 4 (EGR4) in lung cancer bone metastasis

ABSTRACT: EGR4 (early growth response 4), a transcription factor, was previously shown to be up-regulated in lung cancer bone metastasis, compared with its expression in metastasis in other organs, using a multiorgan metastasis model of human small-cell lung cancer cells (SBC-5) in NK cell-depleted SCID mice (Kakiuchi S et al. Mol Cancer Res 2003). Here we show that EGR4 was essential for survival cancer cells, and migration ability. Next, in searching of downstream genes regulated by EGR4, we analyzed the changes in transcriptomes of SBC-5 cells at different time-points (24, 48 and 72 hr) when knockdown of EGR4 by siRNA. Lots of genes were identified either up-regulated or down-regulated in siEGR4 samples compared with control siRNA. Among these genes, there were genes with roles in metastasis or bone biology. The fact that EGR4 knockdown led to up-regulation or suppression of these genes also indicated dual roles of EGR4 in transcriptional regulation, which is consistent with previous reports: EGR4 may repress its own promoter (Zipfel PF et al. Biochim Biophys Acta. 1997) or activate certain inflammatory genes (Decker EL et al. Nucleic Acids Res. 2003; Wieland GD et al. J Cell Sci. 2005). SBC-5 cells were cultured at 1x10^6 cells/2 ml in 3.5 cm plate. After seeding 24 hours, cells were transfected either siEGR4 or siEGFP (for control), using Lipofectamin. At 24, 48 and 72 hours after starting of siRNA, we extracted RNA from these cells, and subjected these RNAs to microarray analysis. The labeling, amplification, hybridization and microarray data analysis were described previously (Dat LT et al. Int J Oncol, 40: 1455-1469 2012).

ORGANISM(S): Homo sapiens

SUBMITTER: Le Dat

PROVIDER: E-GEOD-40558 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Using a multiorgan metastasis model of human NSCLC cells (ACC-LC319/bone2) in NK cell-depleted SCID mice, we attempted to identify genes involved in the organ-selective nature of lung cancer-metastasis process, especially bone metastasis. Genome-wide gene expression profile of 15 metastatic lesions from three organs (bone, lung and liver) in this mouse model revealed lot of genes that seemed to reflect the organ specificity of metastatic cells. Among bone-specifically-expressed genes, dozen of genes involved in cell adhesion, cytoskeleton/cell motility, extracellular matrix remodeling, and cell-cell signaling. The upregulation of 11 genes, some of them were either previously reported to be involved in metastatic characteristics, were confirmed by quantitative RT-PCR. The high ability for bone metastasis human lung adenocarcinoma cell line ACC-LC319/bone2 was established in our laboratory from the parental cell line ACC-LC319 (a kind gift from Dr T. Takahashi, Nagoya University, Nagoya, Japan). ACC-LC319 cells with low capacity of bone metastatic ability were selected in vivo for two cycles in bone metastasis mouse models. After two cycles of selection, the derivative cell line designated as ACC-LC319/bone2 showed increased bone metastatic ability compared to parental cell line (Otsuka S., Oncol Res 2009). In this multiorgan metastasis model, SCID mice were depleted NK cells, and two days later, these mice were intravenously injected 1x10^6 cells. The mice were followed up for clinical symptoms and signs of metastases, and were sacrificed on day 34 after tumor inoculation. Metastastic tissues in lung, liver and bone were collected and snap frozen in liquid nitrogen then keep at -80C until cryosection. Laser microbeam microdissection was used to capture pure population of cancer cells in each organ as well as mouse normal tissues in corresponding organ (i.e. the surrounding normal tissues with no microscopic metastasis). Totally, there were 19 samples, including one sample for metastases in each organ (bone, lung and liver) in five mice (i.e., 15 samples); one for each normal mouse tissue (i.e.,three samples); and one for the in vitro cell line (ACC-LC319/bone 2). DNA microarray were performed using Agilent platform, and the gene expression profile of metastases in bone were compared with the gene expression profile of metastases in other two organs (lung, liver). Those genes whose expression in mouse normal tissues was above the cut off value were excluded. The expression of genes in the in vitro cells was used as the universal normalization for the in vivo genes expression.

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Involvement of transcription factor early growth response 4 (EGR4) in lung cancer bone metastasis

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