Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cellsReport as inadecuate




Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells - Download this document for free, or read online. Document in PDF available to download.

Breast Cancer Research

, 15:R113

First Online: 27 November 2013Received: 22 May 2012Accepted: 31 October 2013DOI: 10.1186-bcr3580

Cite this article as: Hugo, H.J., Pereira, L., Suryadinata, R. et al. Breast Cancer Res 2013 15: R113. doi:10.1186-bcr3580

Abstract

IntroductionEpithelial-to-mesenchymal transition EMT promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition MET in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells.

MethodsMYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA shRNA-mediated knockdown-overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia 1% oxygen for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann–Whitney, and repeated measures two-way ANOVA tests determined statistical significance P < 0.05.

ResultsParental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells MYBsh-LA led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity.

ConclusionsThis work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.

Abbreviations2DTwo dimensional

3DThree dimensional

ANOVAAnalysis of variance

ATCCAmerican Type Culture Collection

bpBase pair

BrdUBromodeoxyuridine

BSABovine serum albumin

CATChloramphenicol acetyltransferase

ChIPChromatin immunoprecipitation

DAB3,3′-diaminobenzidine

DMEMDulbecco modified eagle medium

EGFEpidermal growth factor

EMTEpithelial-to-mesenchymal transition

EREstrogen receptor

ETPMC42-ET cells

FACSFluorescence-activated cell sorting

FCSFetal calf serum

ICCImmunocytochemistry

IFImmunofluorescence

IHCImmunohistochemistry

LAPMC42-LA cells

LN METLymph node metastatic

METMesenchymal-to-epithelial transition

MT-PCRMultiplex tandem polymerase chain reaction

PBSPhosphate-buffered saline

QRT-PCRQuantitative reverse-transcription polymerase chain reaction

RPMIRoswell Park Memorial Institute medium

shRNAShort-hairpin ribonucleic acid

SRBSulforhodamine B

WTWild type.

Electronic supplementary materialThe online version of this article doi:10.1186-bcr3580 contains supplementary material, which is available to authorized users.

Donald F Newgreen and Erik W Thompson contributed equally to this work.

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Author: Honor J Hugo - Lloyd Pereira - Randy Suryadinata - Yvette Drabsch - Thomas J Gonda - N P A Devika Gunasinghe - Cletus 

Source: https://link.springer.com/



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