RAC1B Induces SMAD7 via USP26 to Suppress TGFβ1-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)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.

RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGFβ1 in the Suppression of Cell Motility

“…Recently, we demonstrated in pancreatic epithelial cells that RAC1B promoted the expression of CDH1 (encoding E-cadherin, ECAD) and other epithelial genes, while inhibiting the expression of mesenchymal genes and EMT [15,16]. Mechanistically, RAC1B-dependent protection from mesenchymal conversion and acquisition of a motile phenotype was due to suppression of tumorpromoting MEK-ERK2 signaling [15,16] and interference with TGFβ1 signaling via downregulation of the TGFβ type I receptor ALK5 [4] and induction of the inhibitory Smad, SMAD7 [17]. We also observed that RAC1B upregulated SMAD3 which in its non-activated form exhibited an anti-migratory effect in pancreatic cancer cells [18] presumably due to its ability to promote the expression of ECAD, via transcriptional induction of miR-200 [19], or biglycan (BGN), a pericellular proteoglycan and potent TGFβ inhibitor [18].…”

The Small GTPase RAC1B: A Potent Negative Regulator of-and Useful Tool to Study-TGFβ Signaling

“…Activated ALK5 is known to recruit SMAD7 in a complex with the E3 ligase Smurf2 to promote its internalization and eventual proteasomal degradation in order to terminate TGFβ signaling. Earlier, we demonstrated that the suppressive effect of RAC1B on ALK5 was dependent on SMAD7, and further that RAC1B upregulated protein expression of SMAD7 via intermittent induction of USP26 (see Section 4.2.1 ) [ 84 ].…”

“…SMAD7 . In a recent study, we have shown that i) RAC1B promotes the expression of SMAD7 and ii) SMAD7 mediates the suppressive effect of RAC1B on ALK5 protein and its associated kinase activity [ 84 ]. We further revealed that upregulation of SMAD7 by RAC1B requires the rapid transcriptional induction of USP26 [ 84 ].…”

“…In a recent study, we have shown that i) RAC1B promotes the expression of SMAD7 and ii) SMAD7 mediates the suppressive effect of RAC1B on ALK5 protein and its associated kinase activity [ 84 ]. We further revealed that upregulation of SMAD7 by RAC1B requires the rapid transcriptional induction of USP26 [ 84 ]. The involvement of USP26 strongly suggests that RAC1B increases SMAD7 protein stability by reducing the rate of proteasomal degradation; however, a direct demonstration of RAC1B-induced SMAD7 deubiquitination in pancreatic cells needs experimental verification.…”

Rho Family GTPases in Cancer

“…Two articles by Dr. Hendrick Ungefroren’s group contribute to the role of the constitutively active Rac1B splice variant in transforming growth factor (TGFβ) signaling [ 8 , 9 ]. The authors elucidate the mechanisms by which Rac1B acts to inhibit TGFβ-1-dependent cell migration.…”