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Open Access Highly Accessed Research article

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

Honor J Hugo1, Lloyd Pereira23, Randy Suryadinata4, Yvette Drabsch125, Thomas J Gonda115, N P A Devika Gunasinghe106, Cletus Pinto16, Eliza TL Soo16, Bryce JW van Denderen18, Prue Hill9, Robert G Ramsay23, Boris Sarcevic48, Donald F Newgreen7 and Erik W Thompson16*

  • * Corresponding author: Erik W Thompson rik@svi.edu.au

  • † Equal contributors

Author Affiliations

1 Invasion and Metastasis Unit, St Vincent’s Institute, 9 Princes St, Fitzroy 3065, Victoria, Australia

2 Differentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne 3002, Victoria, Australia

3 Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Australia

4 Cell Cycle and Cancer Unit, St Vincent’s Institute, Melbourne, Australia

5 University of Queensland Diamantina Institute, Translational Research Institute, 37 Kent St, Woolloongabba 4102, Queensland, Australia

6 University of Melbourne Department of Surgery, St. Vincent’s Hospital, 41 Victoria Pde, Fitzroy 3065, Victoria, Australia

7 Embryology Laboratory, Murdoch Children’s Research Institute, Royal Children’s Hospital, 50 Flemington Rd, Parkville 3052, Victoria, Australia

8 University of Melbourne Department of Medicine, St. Vincent’s Hospital, Melbourne, Australia

9 University of Melbourne Department of Pathology and St. Vincent’s Pathology, Melbourne, Australia

10 Department of Anatomy, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka

11 School of Pharmacy, University of Queensland, PACE, 20 Cornwall Street, Woolloongabba 4102, Queensland, Australia

12 Present address: Edinburgh Cancer Research UK Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, EH42XR Edinburgh, United Kingdom

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Breast Cancer Research 2013, 15:R113  doi:10.1186/bcr3580

Published: 27 November 2013

Abstract

Introduction

Epithelial-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.

Methods

MYB, 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).

Results

Parental 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.

Conclusions

This 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.