Journal article
Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells
Breast Cancer Research, Vol.15(6), pp.1-19
2013
PMCID: PMC3979034
PMID: 24283570
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.
Details
- Title
- Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells
- Authors
- Honor J Hugo (Author) - St Vincents Institute of Medical ResearchLloyd Pereira (Author) - The University of MelbourneRandy Suryadinata (Author) - St Vincents Institute of Medical ResearchYvette Drabsch (Author) - The University of QueenslandThomas J Gonda (Author) - The University of QueenslandN P A Devika Gunasinghe (Author) - The University of MelbourneCletus Pinto (Author) - The University of MelbourneEliza TL Soo (Author) - The University of MelbourneBryce JW van Denderen (Author) - The University of MelbournePrue Hill (Author) - The University of MelbourneRobert G Ramsay (Author) - The University of MelbourneBoris Sarcevic (Author) - The University of MelbourneDonald F Newgreen (Author) - Murdoch Children's Research InstituteErik W Thompson (Author) - Queensland University of Technology
- Publication details
- Breast Cancer Research, Vol.15(6), pp.1-19
- Publisher
- BioMed Central Ltd.
- Date published
- 2013
- DOI
- 10.1186/bcr3580
- ISSN
- 1465-542X; 1465-5411
- PMID
- 24283570; PMC3979034
- Copyright note
- This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
- Organisation Unit
- University of the Sunshine Coast, Queensland; School of Health and Behavioural Sciences - Legacy
- Language
- English
- Record Identifier
- 99518707402621
- Output Type
- Journal article
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- Domestic collaboration
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- Web Of Science research areas
- Oncology
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