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Identification of transformation-related pathways in a breast epithelial cell model using a ribonomics approach.

ABSTRACT: The aberrant expression of many genes is a common feature in the malignant transformation of cells. In mammalian cells, posttranscriptional gene regulatory processes are emerging as critical determinants controlling gene expression both in physiologic and pathologic conditions. These regulatory mechanisms are directed primarily by the interaction of mRNAs with specific RNA-binding proteins (RBP). There is an emerging body of data demonstrating that two RBPs, AUF1 and HuR, can antagonistically affect the posttranscriptional fate of target mRNAs, as well as concurrently bind to common target transcripts. Employing MCT-1 oncogene-mediated transformation of immortalized breast epithelial MCF10A cells, we characterized the largely reciprocal association of these two RBPs with target mRNAs and their influence on protein expression vis-a-vis cellular transformation. Using a ribonomics approach, we identified mRNAs from cancer-related pathways whose association with AUF1 and/or HuR were altered when comparing immortalized with transformed MCF10A cells. Significantly, we were able to show that knockdown of HuR expression using RNA interference reduced anchorage-independent growth capacity in transformed MCF10A cells and decreased protein expression of a number of validated target genes. Our data show that the global alterations in binding of HuR and AUF1 with target transcripts have a critical role in posttranscriptional regulation of genes encoding proteins involved in breast epithelial cell transformation. These findings further support the feasibility of using a ribonomics approach for the identification of cancer-related pathways.

SUBMITTER: Mazan-Mamczarz K

PROVIDER: S-EPMC2692251 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Identification of transformation-related pathways in a breast epithelial cell model using a ribonomics approach.

Mazan-Mamczarz Krystyna K Hagner Patrick R PR Dai Bojie B Wood William H WH Zhang Yongqing Y Becker Kevin G KG Liu Zhenqui Z Gartenhaus Ronald B RB

Cancer research 20081001 19

The aberrant expression of many genes is a common feature in the malignant transformation of cells. In mammalian cells, posttranscriptional gene regulatory processes are emerging as critical determinants controlling gene expression both in physiologic and pathologic conditions. These regulatory mechanisms are directed primarily by the interaction of mRNAs with specific RNA-binding proteins (RBP). There is an emerging body of data demonstrating that two RBPs, AUF1 and HuR, can antagonistically af ...[more]

PMID: 18829526

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Project description:Estrogen is a risk factor for breast cancer, yet its mechanism in the initiation of this disease is not clear. Here we use a model of estrogen-mediated malignant transformation of MCF-10F cells to identify the temporal acquisition of changes in genome structure and gene expression that correspond to the progressive transformed phenotype culminating in tumorigenesis. Genomic DNA and total RNA were isolated from MCF10F, trMCF (MCF10F transformed by 70 nM 17-beta estradiol), bsMCF (trMCF selected by Boyden chamber, tumorigenic), bcMCF(clones of bsMCF), and caMCF (cells from tumors of bsMCF grown in SCID mice). The Affymetrix 100k SNP and HG-U133_Plus_2 chips were used for genotyping and gene expression analyses. Changes in chromosomal copy number and loss of heterozygosity were progressive. Gross changes were rarely observed in the trMCF10F cells; the earliest was a gain in chromosome 1p, 1p36.12-1p36.21. In the bsMCF and their sub-clones bcMCF, additional gains were seen in chromosomes 1p, 1p36.12-1pter, 5q, 5q21.1-5q35.3, and 13q, 13q21.32-13q34; losses were detected in chromosomes 4 and 8p, 8p11.1-8p23.1. In the caMCF, additional losses were seen in chromosomes 3p, 3p12.1-3p14.1, 9p, 9p22.1-9p24.3, and 18q, 18q11.2-18q23. Using microarray analysis we were able, in some cases, to narrow the regions of interest and their associated candidate cancer genes. The observation of progressive genomic changes, along with the tumor phenotype of a poorly differentiated adenocarcinoma, indicate an underlying mechanism common to breast cancer development in humans, and adds to the evidence indicating that estrogens are mutagenic and contribute early to the process of breast cancer. Experiment Overall Design: Human MCF-10F cells were transformed by 70nM 17-beta-estradiol. The transformed cells were selected by Boyden chamber. The selected cells were injected into SCID mice and also cloned and expanded. The thus grown tumors were harvested for cell culture. Genomic DNA and total RNA were isolated from cell lines in each step for 100K SNP and gene expression microarray assay.

Project description:INTRODUCTION: About 70% of breast cancers express oestrogen receptor alpha (ESR1/ERalpha) and are oestrogen-dependent for growth. In contrast with the highly proliferative nature of ERalpha-positive tumour cells, ERalpha-positive cells in normal breast tissue rarely proliferate. Because ERalpha expression is rapidly lost when normal human mammary epithelial cells (HMECs) are grown in vitro, breast cancer models derived from HMECs are ERalpha-negative. Currently only tumour cell lines are available to model ERalpha-positive disease. To create an ERalpha-positive breast cancer model, we have forced normal HMECs derived from reduction mammoplasty tissue to express ERalpha in combination with other relevant breast cancer genes. METHODS: Candidate genes were selected based on breast cancer microarray data and cloned into lentiviral vectors. Primary HMECs prepared from reduction mammoplasty tissue were infected with lentiviral particles. Infected HMECs were characterised by Western blotting, immunofluorescence microscopy, microarray analysis, growth curves, karyotyping and SNP chip analysis. The tumorigenicity of the modified HMECs was tested after orthotopic injection into the inguinal mammary glands of NOD/SCID mice. Cells were marked with a fluorescent protein to allow visualisation in the fat pad. The growth of the graft was analysed by fluorescence microscopy of the mammary glands and pathological analysis of stained tissue sections. Oestrogen dependence of tumour growth was assessed by treatment with the oestrogen antagonist fulvestrant. RESULTS: Microarray analysis of ERalpha-positive tumours reveals that they commonly overexpress the Polycomb-group gene BMI1. Lentiviral transduction with ERalpha, BMI1, TERT and MYC allows primary HMECs to be expanded in vitro in an oestrogen-dependent manner. Orthotopic xenografting of these cells into the mammary glands of NOD/SCID mice results in the formation of ERalpha-positive tumours that metastasise to multiple organs. The cells remain wild type for TP53, diploid and genetically stable. In vivo tumour growth and in vitro proliferation of cells explanted from tumours are dependent on oestrogen. CONCLUSION: We have created a genetically defined model of ERalpha-positive human breast cancer based on normal HMECs that has the potential to model human oestrogen-dependent breast cancer in a mouse and enables the study of mechanisms involved in tumorigenesis and metastasis.

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Identification of transformation-related pathways in a breast epithelial cell model using a ribonomics approach.

Publications

Identification of transformation-related pathways in a breast epithelial cell model using a ribonomics approach.

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