TY - JOUR
T1 - Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells
AU - Horio, Mihoko
AU - Sato, Mitsuo
AU - Takeyama, Yoshihiro
AU - Elshazley, Momen
AU - Yamashita, Ryo
AU - Hase, Tetsunari
AU - Yoshida, Kenya
AU - Usami, Noriyasu
AU - Yokoi, Kohei
AU - Sekido, Yoshitaka
AU - Kondo, Masashi
AU - Toyokuni, Shinya
AU - Gazdar, Adi F.
AU - Minna, John D.
AU - Hasegawa, Yoshinori
N1 - Funding Information:
ACKNOWLEDGMENT We thank Drs. Thomas Brabletz for providing pSUPER.retro-ZEB1 and GFP vectors and Osamu Maeda for giving us technical advice on luciferase reporter assay. This work was supported by Grant-in-Aid for Scientific Research (C) 20590919 (to M.S.), Grant-in-Aid for Scientific Research (C) 20590918 (to M.K.), and Grant-in-Aid for Scientific Research (B) 21390257 (to Y.H.) from the Japan Society for the Promotion of Science, Global COE program at Nagoya University Graduate School of Medicine, which is funded by Japan’s Ministry of Education, Culture, Sports, Science and Technology, NCI Special Program of Research Excellence in Lung Cancer (SPORE P50CA70907), and DOD PROSPECT (to J.D.M. and A.F.G.).
PY - 2012/7
Y1 - 2012/7
N2 - 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.
AB - 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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U2 - 10.1245/s10434-011-2142-0
DO - 10.1245/s10434-011-2142-0
M3 - Article
C2 - 22086445
AN - SCOPUS:84864972062
SN - 1068-9265
VL - 19
SP - S634-S645
JO - Annals of Surgical Oncology
JF - Annals of Surgical Oncology
IS - SUPPL. 3
ER -