Dataset Information

RAC1B Induces SMAD7 via USP26 to Suppress TGF?1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells.

ABSTRACT: The small GTPase RAC1B has been shown to act as a powerful inhibitor of the transforming growth factor (TGF)? type I receptor ALK5 and TGF?1/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 TGF?1-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 TGF?/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 TGF?1-induced cell migration in mesenchymal-type carcinoma cells.

SUBMITTER: Ungefroren H

PROVIDER: S-EPMC7352540 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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RAC1B Induces SMAD7 via USP26 to Suppress TGFβ1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells.

Ungefroren Hendrik H Kumarasinghe Anuradha A Musfeldt Melina M Fiedler Christian C Lehnert Hendrik H Marquardt Jens-Uwe JU

Cancers 20200611 6

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 ...[more]

PMID: 32545415

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Project description:Pancreatic ductal adenocarcinoma (PDAC) cells are known for their high invasive/metastatic potential, which is regulated in part by the transforming growth factor β1 (TGFβ1). The involvement of at least two type I receptors, ALK5 and ALK2, that transmit downstream signals of the TGFβ via different Smad proteins, SMAD2/3 and SMAD1/5, respectively, poses the issue of their relative contribution in regulating cell motility. Real-time cell migration assays revealed that the selective inhibition of ALK2 by RNAi or dominant-negative interference with a kinase-dead mutant (ALK2-K233R) strongly enhanced the cells' migratory activity in the absence or presence of TGFβ1 stimulation. Ectopic ALK2-K233R expression was associated with an increase in the protein levels of RAC1 and its alternatively spliced isoform, RAC1b, both of which are implicated in driving cell migration and invasion. Conversely, the RNAi-mediated knockdown or CRISPR/Cas9-mediated knockout of RAC1b resulted in the upregulation of the expression of ALK2, but not that of the related BMP type I receptors, ALK3 or ALK6, and elevated the phosphorylation of SMAD1/5. PDAC is a heterogeneous disease encompassing tumors with different histomorphological subtypes, ranging from epithelial/classical to extremely mesenchymal. Upon treatment of various established and primary PDAC cell lines representing these subtypes with the ALK2 inhibitor, LDN-193189, well-differentiated, epithelial cell lines responded with a much stronger increase in the basal and TGFβ1-dependent migratory activity than poorly differentiated, mesenchymal ones. These data show that (i) ALK2 inhibits migration by suppressing RAC1/RAC1b proteins, (ii) ALK2 and RAC1b act together in a self-perpetuating the autoregulatory negative feedback loop to mutually control their expression, and (iii) the ALK2 antimigratory function appears to be particularly crucial in protecting epithelial subtype cells from becoming invasive, both spontaneously and in a TGFβ-rich tumor microenvironment.

Dataset Information

RAC1B Induces SMAD7 via USP26 to Suppress TGF?1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells.

Publications

RAC1B Induces SMAD7 via USP26 to Suppress TGFβ1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells.

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