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RAC1B Induces SMAD7 via USP26 to Suppress TGFβ1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells
The small GTPase RAC1B has been shown to act as a powerful inhibitor of the transforming growth factor (TGF)β type I receptor ALK5 and TGFβ1/ALK5-induced epithelial–mesenchymal transition and cell motility. However, the precise mechanism has remained elusive. RNAi-mediated knockdown of RAC1B in the...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352540/ https://www.ncbi.nlm.nih.gov/pubmed/32545415 http://dx.doi.org/10.3390/cancers12061545 |
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author | Ungefroren, Hendrik Kumarasinghe, Anuradha Musfeldt, Melina Fiedler, Christian Lehnert, Hendrik Marquardt, Jens-Uwe |
author_facet | Ungefroren, Hendrik Kumarasinghe, Anuradha Musfeldt, Melina Fiedler, Christian Lehnert, Hendrik Marquardt, Jens-Uwe |
author_sort | Ungefroren, Hendrik |
collection | PubMed |
description | The small GTPase RAC1B has been shown to act as a powerful inhibitor of the transforming growth factor (TGF)β type I receptor ALK5 and TGFβ1/ALK5-induced epithelial–mesenchymal transition and cell motility. However, the precise mechanism has remained elusive. RNAi-mediated knockdown of RAC1B in the pancreatic ductal adenocarcinoma (PDAC)-derived cell line Panc1 failed to alter transcriptional activity from a transfected ALK5 promoter–reporter construct. In contrast, pharmacological inhibition of the proteasome decreased the abundance of ALK5 protein in cell lines of the mesenchymal subtype (Panc1, IMIM-PC-1, and breast cancer MDA-MB-231), but not in a PDAC cell line of the epithelial subtype (Colo357). Here, we focused on the inhibitory Smad protein, SMAD7, as a potential candidate for RAC1B-mediated inhibition of cell migration. In Panc1 cells devoid of RAC1B, SMAD7 protein was dramatically reduced and these cells were refractory to TGFβ1-induced upregulation of SMAD7 protein but not mRNA expression. Intriguingly, RNAi-mediated knockdown or ectopic overexpression of SMAD7 in Panc1 cells up- or downregulated, respectively, ALK5 protein expression and mimicked the suppressive effect of RAC1B on TGFβ/SMAD3-dependent transcriptional activity, target gene expression and cell migration. Transfection of SMAD7 was further able to partially rescue cells from the RAC1B knockdown-mediated increase in migratory properties. Conversely, knockdown of SMAD7 was able to partially rescue Panc1 and MDA-MB-231 cells from the antimigratory effect of ectopically expressed RAC1B. Finally, we demonstrate that RAC1B upregulation of SMAD7 protein requires intermittent transcriptional induction of the deubiquitinating enzyme USP26. Our data suggest that RAC1B induces SMAD7 by promoting its deubiquitination and establishes this Smad as one of RAC1B’s downstream effectors in negative regulation of ALK5 and TGFβ1-induced cell migration in mesenchymal-type carcinoma cells. |
format | Online Article Text |
id | pubmed-7352540 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-73525402020-07-15 RAC1B Induces SMAD7 via USP26 to Suppress TGFβ1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells Ungefroren, Hendrik Kumarasinghe, Anuradha Musfeldt, Melina Fiedler, Christian Lehnert, Hendrik Marquardt, Jens-Uwe Cancers (Basel) Article The small GTPase RAC1B has been shown to act as a powerful inhibitor of the transforming growth factor (TGF)β type I receptor ALK5 and TGFβ1/ALK5-induced epithelial–mesenchymal transition and cell motility. However, the precise mechanism has remained elusive. RNAi-mediated knockdown of RAC1B in the pancreatic ductal adenocarcinoma (PDAC)-derived cell line Panc1 failed to alter transcriptional activity from a transfected ALK5 promoter–reporter construct. In contrast, pharmacological inhibition of the proteasome decreased the abundance of ALK5 protein in cell lines of the mesenchymal subtype (Panc1, IMIM-PC-1, and breast cancer MDA-MB-231), but not in a PDAC cell line of the epithelial subtype (Colo357). Here, we focused on the inhibitory Smad protein, SMAD7, as a potential candidate for RAC1B-mediated inhibition of cell migration. In Panc1 cells devoid of RAC1B, SMAD7 protein was dramatically reduced and these cells were refractory to TGFβ1-induced upregulation of SMAD7 protein but not mRNA expression. Intriguingly, RNAi-mediated knockdown or ectopic overexpression of SMAD7 in Panc1 cells up- or downregulated, respectively, ALK5 protein expression and mimicked the suppressive effect of RAC1B on TGFβ/SMAD3-dependent transcriptional activity, target gene expression and cell migration. Transfection of SMAD7 was further able to partially rescue cells from the RAC1B knockdown-mediated increase in migratory properties. Conversely, knockdown of SMAD7 was able to partially rescue Panc1 and MDA-MB-231 cells from the antimigratory effect of ectopically expressed RAC1B. Finally, we demonstrate that RAC1B upregulation of SMAD7 protein requires intermittent transcriptional induction of the deubiquitinating enzyme USP26. Our data suggest that RAC1B induces SMAD7 by promoting its deubiquitination and establishes this Smad as one of RAC1B’s downstream effectors in negative regulation of ALK5 and TGFβ1-induced cell migration in mesenchymal-type carcinoma cells. MDPI 2020-06-11 /pmc/articles/PMC7352540/ /pubmed/32545415 http://dx.doi.org/10.3390/cancers12061545 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Ungefroren, Hendrik Kumarasinghe, Anuradha Musfeldt, Melina Fiedler, Christian Lehnert, Hendrik Marquardt, Jens-Uwe RAC1B Induces SMAD7 via USP26 to Suppress TGFβ1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells |
title | RAC1B Induces SMAD7 via USP26 to Suppress TGFβ1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells |
title_full | RAC1B Induces SMAD7 via USP26 to Suppress TGFβ1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells |
title_fullStr | RAC1B Induces SMAD7 via USP26 to Suppress TGFβ1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells |
title_full_unstemmed | RAC1B Induces SMAD7 via USP26 to Suppress TGFβ1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells |
title_short | RAC1B Induces SMAD7 via USP26 to Suppress TGFβ1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells |
title_sort | rac1b induces smad7 via usp26 to suppress tgfβ1-dependent cell migration in mesenchymal-subtype carcinoma cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352540/ https://www.ncbi.nlm.nih.gov/pubmed/32545415 http://dx.doi.org/10.3390/cancers12061545 |
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