Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Comparative transcription profiling upon FBLN5 overexpression in transformed COS-7 and normal CV-1 cells

ABSTRACT: A novel class of genes has recently been identified as anticancer genes, which upon ectopic overexpression selectively destroy the transformed tumour cells, while leaving the untransformed normal cells unharmed. Examples include TRAIL, PAR4 and Orctl3. We have isolated 16 novel anticancer genes of human origin through a gain of function, forward genetic screen in mammalian cells. Among these, FBLN5 was chosen for further investigation. FBLN5 is a secreted ECM glycoprotein the physiological functions of which remain largely elusive, however it is downregulated in numerous malignancies and Fibln5-/- mice exhibited elastic fibre abnormalities including cutis laxa. When transfected in the normal CV-1 cells and their SV-40 transformed counterpart COS-7 cells, FBLN5 caused extensive cell death in latter and not in the former. In order to investigate such differential effects of FBLN5, we performed transcriptional profiling of COS-7 and CV-1 cells upon FBLN5 transfection where wild type COS-7 and CV-1 cells were used as controls. We conducted exon-level expression profiles of > 540,000 transcripts including coding mRNA, long non-coding RNA, microRNA, novel transcripts and also the alternative splice variants. Briefly, FBLN5 caused downregulation of MYC regulated genes in COS-7 cells while opposite was observed in CV-1 cells. Also, FBLN5 caused upregulation of cell death related genes in COS-7 cells but not in CV-1 cells.

ORGANISM(S): Chlorocebus aethiops

SUBMITTER: Motasim Masood

PROVIDER: E-MTAB-11449 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Abstract: Chemogenomic fitness assays were combined with a transcriptome analysis to understand both the mode of action and the mechanisms of resistance to Chitosan oligosaccharides (COS). COS are deacetylated chitin compounds, with antimicrobial properties, that are presumed to act by disrupting the cell membrane. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified encode membrane proteins and members of the protein degradation/ proteosome pathway. The transcriptomes of wild-type and five suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and furthermore, treatment with environmental stressors does not provide resistance to COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the RAS signal transduction pathway. Down-regulated transcripts included those encoding protein folding components, and respiratory chain proteins. Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to amphotericin B, fluconazole and terbinafine as the wild-type (vector control). The gene targets of COS identified in this study suggest that COSM-bM-^@M-^Ys mechanism of action is different from other commonly studied fungicides, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens. We selected the 5 overexpressing strains based on the characteristics of the genes. ARL1, encoding a GTPase and is involved in membrane trafficking, was selected since it was observed in the HIP-HOP assay as a sensitive homozygous deletion strain and in the MSP assay as multicopy suppressor. The rest of selected overexpressing strains were BCK2 and MSG5, involved in cell integrity pathways, ERG24 in ergosterol synthesis, and RBA50 in transcription. BCK2 is a Ser-Thr rich protein with protein kinase C activity that acts in signal transduction. Overexpression of BCK2 can rescue defects in yeast cwh43M-NM-^T, which displays several cell wall defects [29]. BCK2 overexpression also can suppress a cell lysis defect of mpk1M-NM-^T and pck1M-NM-^T [30]. MSG5 is also involved in signal transduction. This gene encodes a protein phosphatase involved in cell cycle control through the dephosphorylation of MAPK and is indispensable for restricting the signaling by the cell integrity pathway in yeast [31]. The inhibition of MAPK signaling leads to inhibition of cell differentiation and cell division [32]. The functions of ARL1 and ERG24 and their potential roles in chitosan resistance will be described in more detail in the Discussion. To gain a further understanding on the mode of action and mechanisms of resistance to COS, we performed a transcriptome analysis of the above mentioned five overexpressing strains known to increase resistance to COS-5.44. Each overexpressing strain and the wild type (vector control) were treated with COS-5.44 and RNAs isolated from both treated and untreated cells. The RNAs were converted to labeled cDNA and hybridized to NimbleGen expression microarrays (see Methods). Three cDNA biological replicates either with or without a 60 minutes exposure to COS-5.44 for each of the five overexpressing strains as well as an untransformed wild type BY4743 cells (vector control; for a total of 36 samples) were hybridized to NimbleGen 4X72k microarrays (Roche NimbleGen, Inc. Design ID A6186-00-01, TI4932 60mer expr X4).

Project description:Vascular endothelial growth factor is a multifunctional cytokine playing important roles in angiogenesis, tumor progression and metastasis. Alternative splicing results in the production of several different isoforms of VEGF. We have previously generated human breast cancer cells overexpressing VEGF165 or VEGF189 isoforms (referred to as the V165 and V189 clones, respectively) and showed that VEGF189-transfected cells were less tumorigenic. In this study, we used bioluminescence imaging to analyze the metastasis capacity of breast cancer cell lines (MDA-MB-321) overexpressing VEGF isoforms in nude mice. V165, V189 and control cV clones were transfected with a luciferase plasmid to generate bioluminescent clones (the V165-B, V189-B and cV clones, respectively). These clones were then injected into the left heart ventricle of nude mice. Analysis of the location of metastases shows that V189-B cells induced fewer metastases in lung and bone than V165-B and cV-B cells. Moreover, there was a delay on metastasis appearance in mice receiving VEGF V189-B clone, consistent with increase survival in these mice. We investigated the possible mechanisms underlying these effects and found that VEGF189 increased adhesion and decreased cell invasion and survival in vitro. In the other side, transcriptomic analyses shown expression of genes, implicated in breast cancer and metastatic dissemination, in VEGF165 overexpressing cells. After in silico analyses of genes differentially expressed between V189 and V165 cells,we used Q-PCR assays to quantify mRNA levels of some gene of interest in 120 breast tumors from patients with or without metastasis and/or specially lung metastasis. We found that genes have prognostical significance and /or showed an influence on relapse-free survival. These data provide the first evidence that different VEGF isoforms have different effects on breast cancer cell line invasion and colonization. Human MDA-MB-231 breast cancer cells were used to generate stable transfected clones overexpressing VEGF165 isoform, VEGF189 isoform, or control vector (referred to as 42ctl8, 13ctl3 and PCIctl3, respectively). Analysis of the metastasis capacity of these clones in nude mice.

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Comparative transcription profiling upon FBLN5 overexpression in transformed COS-7 and normal CV-1 cells

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