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Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells

BACKGROUND: Transforming growth factor-β (TGF-β) plays a paradoxical role in cancer: it suppresses proliferation at early stages but promotes metastasis at late stages. This cytokine is upregulated in cholangiocarcinoma and is implicated in cholangiocarcinoma invasion and metastasis. Here we investi...

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Autores principales: Sritananuwat, Phaijit, Sueangoen, Natthaporn, Thummarati, Parichut, Islam, Kittiya, Suthiphongchai, Tuangporn
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615482/
https://www.ncbi.nlm.nih.gov/pubmed/28959141
http://dx.doi.org/10.1186/s12935-017-0454-2
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author Sritananuwat, Phaijit
Sueangoen, Natthaporn
Thummarati, Parichut
Islam, Kittiya
Suthiphongchai, Tuangporn
author_facet Sritananuwat, Phaijit
Sueangoen, Natthaporn
Thummarati, Parichut
Islam, Kittiya
Suthiphongchai, Tuangporn
author_sort Sritananuwat, Phaijit
collection PubMed
description BACKGROUND: Transforming growth factor-β (TGF-β) plays a paradoxical role in cancer: it suppresses proliferation at early stages but promotes metastasis at late stages. This cytokine is upregulated in cholangiocarcinoma and is implicated in cholangiocarcinoma invasion and metastasis. Here we investigated the roles of non-Smad pathway (ERK1/2) and Smad in TGF-β tumor promoting and suppressing activities in intrahepatic cholangiocarcinoma (ICC) cells. METHODS: TGF-β1 effects on proliferation, invasion and migration of ICC cells, KKU-M213 and/or HuCCA-1, were investigated using MTT, colony formation, in vitro Transwell and wound healing assays. Levels of mRNAs and proteins/phospho-proteins were measured by quantitative (q)RT-PCR and Western blotting respectively. E-cadherin localization was examined by immunofluorescence and secreted MMP-9 activity was assayed by gelatin zymography. The role of ERK1/2 signaling was evaluated by treating cells with TGF-β1 in combination with MEK1/2 inhibitor U0126, and that of Smad2/3 and Slug using siSmad2/3- and siSlug-transfected cells. RESULTS: h-TGF-β1 enhanced KKU-M213 cell invasion and migration and induced epithelial-mesenchymal transition as shown by an increase in vimentin, Slug and secreted MMP-9 levels and by a change in E-cadherin localization from membrane to cytosol, while retaining the cytokine’s ability to attenuate cell proliferation. h-TGF-β1 stimulated Smad2/3 and ERK1/2 phosphorylation, and the MEK1/2 inhibitor U0126 attenuated TGF-β1-induced KKU-M213 cell invasion and MMP-9 production but moderately enhanced the cytokine growth inhibitory activity. The latter effect was more noticeable in HuCCA-1 cells, which resisted TGF-β-anti-proliferative activity. Smad2/3 knock-down suppressed TGF-β1 ability to induce ERK1/2 phosphorylation, Slug expression and cell invasion, whereas Slug knock-down suppressed cell invasion and vimentin expression but marginally affected ERK1/2 activation and MMP-9 secretion. These results indicate that TGF-β1 activated ERK1/2 through Smad2/3 but not Slug pathway, and that ERK1/2 enhanced TGF-β1 tumor promoting but repressed its tumor suppressing functions. CONCLUSIONS: Inhibiting ERK1/2 activation attenuates TGF-β1 tumor promoting effect (invasion) but retains its tumor suppressing role, thereby highlighting the importance of ERK1/2 in resolving the TGF-β paradox switch. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12935-017-0454-2) contains supplementary material, which is available to authorized users.
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spelling pubmed-56154822017-09-28 Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells Sritananuwat, Phaijit Sueangoen, Natthaporn Thummarati, Parichut Islam, Kittiya Suthiphongchai, Tuangporn Cancer Cell Int Primary Research BACKGROUND: Transforming growth factor-β (TGF-β) plays a paradoxical role in cancer: it suppresses proliferation at early stages but promotes metastasis at late stages. This cytokine is upregulated in cholangiocarcinoma and is implicated in cholangiocarcinoma invasion and metastasis. Here we investigated the roles of non-Smad pathway (ERK1/2) and Smad in TGF-β tumor promoting and suppressing activities in intrahepatic cholangiocarcinoma (ICC) cells. METHODS: TGF-β1 effects on proliferation, invasion and migration of ICC cells, KKU-M213 and/or HuCCA-1, were investigated using MTT, colony formation, in vitro Transwell and wound healing assays. Levels of mRNAs and proteins/phospho-proteins were measured by quantitative (q)RT-PCR and Western blotting respectively. E-cadherin localization was examined by immunofluorescence and secreted MMP-9 activity was assayed by gelatin zymography. The role of ERK1/2 signaling was evaluated by treating cells with TGF-β1 in combination with MEK1/2 inhibitor U0126, and that of Smad2/3 and Slug using siSmad2/3- and siSlug-transfected cells. RESULTS: h-TGF-β1 enhanced KKU-M213 cell invasion and migration and induced epithelial-mesenchymal transition as shown by an increase in vimentin, Slug and secreted MMP-9 levels and by a change in E-cadherin localization from membrane to cytosol, while retaining the cytokine’s ability to attenuate cell proliferation. h-TGF-β1 stimulated Smad2/3 and ERK1/2 phosphorylation, and the MEK1/2 inhibitor U0126 attenuated TGF-β1-induced KKU-M213 cell invasion and MMP-9 production but moderately enhanced the cytokine growth inhibitory activity. The latter effect was more noticeable in HuCCA-1 cells, which resisted TGF-β-anti-proliferative activity. Smad2/3 knock-down suppressed TGF-β1 ability to induce ERK1/2 phosphorylation, Slug expression and cell invasion, whereas Slug knock-down suppressed cell invasion and vimentin expression but marginally affected ERK1/2 activation and MMP-9 secretion. These results indicate that TGF-β1 activated ERK1/2 through Smad2/3 but not Slug pathway, and that ERK1/2 enhanced TGF-β1 tumor promoting but repressed its tumor suppressing functions. CONCLUSIONS: Inhibiting ERK1/2 activation attenuates TGF-β1 tumor promoting effect (invasion) but retains its tumor suppressing role, thereby highlighting the importance of ERK1/2 in resolving the TGF-β paradox switch. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12935-017-0454-2) contains supplementary material, which is available to authorized users. BioMed Central 2017-09-26 /pmc/articles/PMC5615482/ /pubmed/28959141 http://dx.doi.org/10.1186/s12935-017-0454-2 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Primary Research
Sritananuwat, Phaijit
Sueangoen, Natthaporn
Thummarati, Parichut
Islam, Kittiya
Suthiphongchai, Tuangporn
Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells
title Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells
title_full Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells
title_fullStr Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells
title_full_unstemmed Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells
title_short Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells
title_sort blocking erk1/2 signaling impairs tgf-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells
topic Primary Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615482/
https://www.ncbi.nlm.nih.gov/pubmed/28959141
http://dx.doi.org/10.1186/s12935-017-0454-2
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