Unknown,Transcriptomics,Genomics,Proteomics

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Changes in gene expression during epithelial-mesenchymal transition in a breast cancer primary culture

ABSTRACT: Epithelial-Mesenchymal Transition (EMT) is an important process during normal development, but also co-opted by various cancer cells to enable them to invade and form metastases at distant sites. In this study, we examinated changes in gene expression during EMT in breast cancer using primary culture named BCpc. Changes in gene expression in primary cells from breast tumour specimen (named BCpc) were analyzed as two-color hybridizations using Agilent Technologies whole human genome 4x44K microarrays

ORGANISM(S): Homo sapiens

SUBMITTER: Valentina Bravatà

PROVIDER: E-GEOD-53175 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Gene expression profiling of epithelial-mesenchymal transition in primary breast cancer cell culture.

Minafra Luigi L Bravatà Valentina V Forte Giusi Irma GI Cammarata Francesco Paolo FP Gilardi Maria Carla MC Messa Cristina C

Anticancer research 20140501 5

<h4>Background/aim</h4>Epithelial-mesenchymal transition (EMT) is a process co-opted by cancer cells to invade and form metastases. In the present study we analyzed gene expression profiles of primary breast cancer cells in culture in order to highlight genes related to EMT.<h4>Materials and methods</h4>Microarray expression analysis of primary cells isolated from a specimen of a patient with an infiltrating ductal carcinoma of the breast was performed. Real-Time Quantitative Reverse Transcripti ...[more]

PMID: 24778019

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Project description:To understand how CK2M-NM-2 silencing promotes a mesenchymal phenotype in human epithelial cells, like TGFM-NM-2 does, we have employed whole genome microarray expression profiling as a discovery platform to compare genes regulated in CK2M-NM-2-depleted cells and in TGFM-NM-2-treated cells. To do so, we began by deriving individual gene expression signatures of WT-, TGFM-NM-2-treated, CK2M-NM-2-depleted or CK2M-NM-2-depleted and rescued with chicken CK2M-NM-2, whose expression is not affected by the shRNA in MCF10A cells, and cataloguing genes exhibiting at least a 1.5-fold up- or down-regulation under each experimental condition. We observed that EMT triggered by TGFM-NM-21 or CK2M-NM-2-depletion induced an overlapping set of changes in gene expression. Of ~25,000 unique genes tested, we found ~1,200 genes whose expression was significantly modulated in TGFM-NM-21-treated or CK2M-NM-2-depleted cells as compared to Mock-cells. Of the 439 genes down-regulated in TGFM-NM-21-treated cells, 74 genes (17%) were also repressed in CK2M-NM-2-depleted MCF10A cells and 45 genes (10%) were commonly up-regulated. As expected, among commonly regulated genes, several mesenchymal genes (CDH2, FN1, MYL9, VIM) were upregulated, whereas epithelial genes such as CDH1, CDH3, CLDN1, CLDN7, OCLN, KRT5, KRT6B, COL2A1 and MUC1, were inversely turned down. The expression of the EMT-inducing transcription factors, Snail and Zeb, was also significantly up-regulated in accordance with the repression of their known target genes. While showing similar phenotypes, the genetic program of TGFM-NM-21-treated or CK2M-NM-2-depleted MCF10A cells also exhibited distinct features. These results highlight that in addition to TGFM-NM-21-regulated genes, CK2M-NM-2-depletion may affect alternative pathways. The microarray analysis was performed with RNAs isolated from parental MCF10A cells and the following MCF10A-derived cells: M-NM-^TCK2M-NM-2- and TGFM-NM-21-treated cells (2ng/ml for 72h) and each from two independent cell cultures (duplicates).

Project description:Background: E-cadherin is an adherens junction protein that forms homophilic intercellular contacts in epithelial cells while also interacting with the intracellular cytoskeletal networks. It has roles including establishment and maintenance of cell polarity, differentiation, migration and signalling in cell proliferation pathways. Its downregulation is commonly observed in epithelial tumours and is a hallmark of the epithelial to mesenchymal transition (EMT). Methods: To improve our understanding of how E-cadherin loss contributes to tumorigenicity, we investigated the impact of its elimination from the non-tumorigenic breast cell line MCF10A. We performed cell-based assays and whole genome RNAseq to characterize an isogenic MCF10A cell line that is devoid of CDH1 expression due to an engineered homozygous 4bp deletion in CDH1 exon 11. Results: The E-cadherin-deficient line, MCF10A CDH1-/- showed subtle morphological changes, weaker cell-substrate adhesion, delayed migration, but retained cell-cell contact, contact growth inhibition and anchorage-dependent growth. Within the cytoskeleton, the apical microtubule network in the CDH1-deficient cells lacked the radial pattern of organization present in the MCF10A cells and F-actin formed thicker, more numerous stress fibres in the basal part of the cell. Whole genome RNAseq identified compensatory changes in the genes involved in cell-cell adhesion while genes involved in cell-substrate adhesion, notably ITGA1, COL8A1, COL4A2 and COL12A1, were significantly downregulated. Key EMT markers including CDH2, FN1, VIM and VTN were not upregulated although increased expression of proteolytic matrix metalloprotease and kallikrein genes was observed. Conclusions: Overall, our results demonstrated that E-cadherin loss alone was insufficient to induce an EMT or enhance transforming potential in the non-tumorigenic MCF10A cells but was associated with broad transcriptional changes associated with tissue remodelling. Examination of the impact of E-cadherin (CDH1) loss in an isogenic pair of breast cell lines.

Dataset Information

Changes in gene expression during epithelial-mesenchymal transition in a breast cancer primary culture

Publications

Gene expression profiling of epithelial-mesenchymal transition in primary breast cancer cell culture.

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