Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

TFPI alpha and beta regulate mRNAs involved in cancer biology and in the immune system

ABSTRACT: Emerging evidences indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s) mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIa or TFPIb cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA) expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. Overexpression of TFPIa or TFPIb resulted in a 6.4- and 17-fold increase in TFPI protein levels, respectively. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIa or TFPIb, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion) and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor (EGFR), small GTPases, and the NFkB and JAK/STAT cascades. Integrated mRNA-miRNA analyses showed that up to 46 and 252 genes differentially expressed in cells overexpressing TFPIa or TFPIb, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and miRNAs involved in processes facilitating cancer cell growth and immunologic response, probably by signal transduction involving the EGFR pathway. Total RNA was isolated from SK-BR-3 breast cancer cells stably transfected with TFPIalpha or TFPIbeta cDNA, or with an empty vector for control. RNA was obtained from three independent isolations, yielding three biological replicates of each of the three samples. Mean expression data from cells overexpressing TFPIalpha or TFPIbeta were compared to mean data from cells transfected with an empty vector.

ORGANISM(S): Homo sapiens

SUBMITTER: Nina Iversen

PROVIDER: E-GEOD-30037 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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TFPI alpha and beta regulate mRNAs and microRNAs involved in cancer biology and in the immune system in breast cancer cells.

Stavik Benedicte B Skretting Grethe G Olstad Ole Kristoffer OK Sletten Marit M Dehli Vigeland Magnus M Sandset Per Morten PM Iversen Nina N

PloS one 20121005 10

Emerging evidence indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s) mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIα or TFPIβ cDNA were t ...[more]

PMID: 23071754

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Project description:Emerging evidences indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s) mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIa or TFPIb cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA) expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. Overexpression of TFPIa or TFPIb resulted in a 6.4- and 17-fold increase in TFPI protein levels, respectively. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIa or TFPIb, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion) and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor (EGFR), small GTPases, and the NFkB and JAK/STAT cascades. Integrated mRNA-miRNA analyses showed that up to 46 and 252 genes differentially expressed in cells overexpressing TFPIa or TFPIb, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and miRNAs involved in processes facilitating cancer cell growth and immunologic response, probably by signal transduction involving the EGFR pathway.

Project description:Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of EGFR occurs in ~50% of basal-like breast cancer and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown. Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the ‘near normal’ MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. These transfected lines were used to investigate the effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo. We also carried out copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines. Both activating mutations of EGFR increased the proliferative ability of MCF10A and MCF10CA1a cell lines, and increased the anchorage-independent growth and sensitivity to gefitinib in MCF10A cells. In addition, the EGFR-E746-A750 deletion increased the migratory and invasive abilities of the MCF10CA1a cell line in vitro, and greatly increased the formation and growth of MCF10CA1a tumours in vivo. Compared to MCF10A cells, MCF10CA1a cells had large regions of gain on chromosome 9 and the long arm of chromosome 3, deletion in the short arm of chromosome 7, and mutations in many genes implicated in cancer. The EGFR E746-A750 deletion mutation, and to a lesser extent G719S, greatly enhance the oncogenic properties of MCF10A cell line, and increase sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased sensitivity to gefitinib, perhaps because of additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer cancer, accompanying mutations in PIK3CA may confer gefitinib resistance.

Project description:Epithelial-mesenchymal transition (EMT) has recently been recognized as a key element of cell invasion, migration, metastasis, and drug resistance in several types of cancer, including non-small cell lung cancer (NSCLC). Our aim was to clarify microRNA (miRNA) -related mechanisms underlying EMT followed by acquired resistance to epidermal growth factor receptor tyrosine-kinase inhibitor (EGFR-TKI) in NSCLC. MiRNA expression profiles were examined before and after transforming growth factor-beta1 (TGF-M-NM-21) exposure in four human adenocarcinoma cell lines with or without EMT. Correlation between expressions of EMT-related miRNAs and resistance to EGFR-TKI gefitinib was evaluated. MiRNA array and quantitative RT-PCR revealed that TGF-M-NM-21 significantly induced overexpression of miR-134, miR-487b, and miR-655, which belong to the same cluster located on chromosome 14q32, in lung adenocarcinoma cells with EMT. MAGI2 (membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2), a predicted target of these miRNAs and a scaffold protein required for PTEN (phosphatase and tensin homolog), was diminished in A549 cells with EMT after the TGF-M-NM-21 stimulation. Overexpression of miR-134 and miR-487b promoted the EMT phenomenon and affected the drug resistance to gefitinib, whereas knockdown of these miRNAs inhibited the EMT process and reversed TGF-M-NM-21-induced resistance to gefitinib. Our study demonstrated that the miR-134/487b/655 cluster contributed to the TGF-M-NM-21-induced EMT phenomenon and affected the resistance to gefitinib by directly targeting MAGI2, whose suppression subsequently caused loss of PTEN stability in lung cancer cells. The miR-134/miR-487b/miR-655 cluster may be new therapeutic targets in advanced lung adenocarcinoma patients, depending on the EMT phenomenon. miRNA expression profiles before and after TGF-M-NM-21 exposure were assessed in the four lung adenocarcinoma cell lines, A549, LC2/ad, PC3, and, PC9 by TaqMan miRNA arrays. Relative ratios of miRNAs in cells after TGF-M-NM-21 exposure were calculated when compared with the cells before TGF-M-NM-21 exposure.

Dataset Information

TFPI alpha and beta regulate mRNAs involved in cancer biology and in the immune system

Publications

TFPI alpha and beta regulate mRNAs and microRNAs involved in cancer biology and in the immune system in breast cancer cells.

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