Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells

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

Research output: Contribution to journalArticle

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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.

Original languageEnglish
JournalAnnals of Surgical Oncology
Volume19
Issue numberSUPPL. 3
DOIs
Publication statusPublished - 01-07-2012

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Growth
Up-Regulation
Cell Line
RNA Interference
Luciferases
Epithelial Cell Adhesion Molecule
Malignant Mesothelioma
Gene Expression
Epithelial-Mesenchymal Transition
Vimentin
Cadherins
Microarray Analysis
Oligonucleotide Array Sequence Analysis
Genes
Real-Time Polymerase Chain Reaction
Western Blotting
Genome
Phenotype

All Science Journal Classification (ASJC) codes

  • Surgery
  • Oncology

Cite this

Horio, M., Sato, M., Takeyama, Y., Elshazley, M., Yamashita, R., Hase, T., ... Hasegawa, Y. (2012). Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells. Annals of Surgical Oncology, 19(SUPPL. 3). https://doi.org/10.1245/s10434-011-2142-0
Horio, Mihoko ; Sato, Mitsuo ; Takeyama, Yoshihiro ; Elshazley, Momen ; Yamashita, Ryo ; Hase, Tetsunari ; Yoshida, Kenya ; Usami, Noriyasu ; Yokoi, Kohei ; Sekido, Yoshitaka ; Kondo, Masashi ; Toyokuni, Shinya ; Gazdar, Adi F. ; Minna, John D. ; Hasegawa, Yoshinori. / Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells. In: Annals of Surgical Oncology. 2012 ; Vol. 19, No. SUPPL. 3.
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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.",
author = "Mihoko Horio and Mitsuo Sato and Yoshihiro Takeyama and Momen Elshazley and Ryo Yamashita and Tetsunari Hase and Kenya Yoshida and Noriyasu Usami and Kohei Yokoi and Yoshitaka Sekido and Masashi Kondo and Shinya Toyokuni and Gazdar, {Adi F.} and Minna, {John D.} and Yoshinori Hasegawa",
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Horio, M, Sato, M, Takeyama, Y, Elshazley, M, Yamashita, R, Hase, T, Yoshida, K, Usami, N, Yokoi, K, Sekido, Y, Kondo, M, Toyokuni, S, Gazdar, AF, Minna, JD & Hasegawa, Y 2012, 'Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells', Annals of Surgical Oncology, vol. 19, no. SUPPL. 3. https://doi.org/10.1245/s10434-011-2142-0

Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells. / Horio, Mihoko; Sato, Mitsuo; Takeyama, Yoshihiro; Elshazley, Momen; Yamashita, Ryo; Hase, Tetsunari; Yoshida, Kenya; Usami, Noriyasu; Yokoi, Kohei; Sekido, Yoshitaka; Kondo, Masashi; Toyokuni, Shinya; Gazdar, Adi F.; Minna, John D.; Hasegawa, Yoshinori.

In: Annals of Surgical Oncology, Vol. 19, No. SUPPL. 3, 01.07.2012.

Research output: Contribution to journalArticle

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

PY - 2012/7/1

Y1 - 2012/7/1

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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