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01-07-2012 | Translational Research and Biomarkers

Transient but Not Stable ZEB1 Knockdown Dramatically Inhibits Growth of Malignant Pleural Mesothelioma Cells

Authors: Mihoko Horio, MD, Mitsuo Sato, MD, PhD, Yoshihiro Takeyama, MD, PhD, Momen Elshazley, MD, MSc, Ryo Yamashita, MD, Tetsunari Hase, MD, Kenya Yoshida, MD, Noriyasu Usami, MD, PhD, Kohei Yokoi, MD, PhD, Yoshitaka Sekido, MD, PhD, Masashi Kondo, MD, PhD, Shinya Toyokuni, MD, PhD, Adi F. Gazdar, MD, John D. Minna, MD, Yoshinori Hasegawa, MD, PhD

Published in: Annals of Surgical Oncology | Special Issue 3/2012

Abstract

Background

The role of ZEB1, a master epithelial-to-mesenchymal transition gene, in malignant pleural mesothelioma (MPM) is unclear.

Methods

The expression of ZEB1, E-cadherin, vimentin, and epithelial cell adhesion molecule (EpCAM) in 18 MPM cell lines and a normal pleural mesothelial cell line MeT-5A was determined by quantitative real-time polymerase chain reaction and Western blot testing. RNA interference–mediated transient and/or stable knockdown of ZEB1 and EpCAM was performed. Microarray expression analysis was performed with a TORAY-3D gene chip. Growth was evaluated by colorimetric proliferation and colony formation assays. Luciferase reporter assay was performed to access the effects of ZEB1 knockdown on EpCAM promoter activity.

Results

Most MPM cell lines exhibited mesenchymal phenotype and expressed ZEB1. Transient ZEB1 knockdown suppressed growth in all four cell lines studied (ACC-MESO-1, H2052, Y-MESO-8A, Y-MESO-29) while stable ZEB1 knockdown suppressed growth only in Y-MESO-29. Genome-wide gene expression analysis revealed that EpCAM was the most prominently up-regulated gene by both transient and stable ZEB1 knockdown in ACC-MESO-1, with more marked up-regulation in stable knockdown. We hypothesized that EpCAM up-regulation counteracts the stable ZEB1 knockdown-induced growth inhibition in ACC-MESO-1. Transient EpCAM knockdown suppressed growth dramatically in ACC-MESO-1 cells expressing shZEB1 but only modestly in those expressing shGFP, supporting our hypothesis. Luciferase reporter assay showed that ZEB1 knockdown resulted in increased EpCAM promoter activity. EpCAM was also up-regulated in Y-MESO-29 expressing shZEB1, but this EpCAM up-regulation did not counteract ZEB1knockdown-induced growth suppression, suggesting that the counteracting effects of EpCAM may be cellular context dependent.

Conclusions

RNA interference-mediated ZEB1 knockdown may be a promising therapeutic strategy for MPM, but one has to consider the possibility of diminished growth inhibitory effects of long-term ZEB1 knockdown, possibly as a result of EpCAM up-regulation and/or other gene expression changes resulting from ZEB1 knockdown.

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Title: Transient but Not Stable ZEB1 Knockdown Dramatically Inhibits Growth of Malignant Pleural Mesothelioma Cells
Authors: Mihoko Horio, MD
Mitsuo Sato, MD, PhD
Yoshihiro Takeyama, MD, PhD
Momen Elshazley, MD, MSc
Ryo Yamashita, MD
Tetsunari Hase, MD
Kenya Yoshida, MD
Noriyasu Usami, MD, PhD
Kohei Yokoi, MD, PhD
Yoshitaka Sekido, MD, PhD
Masashi Kondo, MD, PhD
Shinya Toyokuni, MD, PhD
Adi F. Gazdar, MD
John D. Minna, MD
Yoshinori Hasegawa, MD, PhD
Publication date: 01-07-2012
Publisher: Springer-Verlag
Published in: Annals of Surgical Oncology / Issue Special Issue 3/2012
Print ISSN: 1068-9265
Electronic ISSN: 1534-4681
DOI: https://doi.org/10.1245/s10434-011-2142-0

At a glance: The STEP trials

Springer Medicine

Transient but Not Stable ZEB1 Knockdown Dramatically Inhibits Growth of Malignant Pleural Mesothelioma Cells

Abstract

Background

Methods

Results

Conclusions

At a glance: The STEP trials

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

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