Rac1b negatively regulates TGF-β1-induced cell motility in pancreatic ductal epithelial cells by suppressing Smad signalling

Abstract

Transforming growth factor (TGF)-β1 promotes progression of pancreatic ductal adenocarcinoma (PDAC) by enhancing epithelial-mesenchymal transition, cell migration/invasion, and metastasis, in part by cooperating with the small GTPase Rac1. Prompted by the observation of higher expression of Rac1b, an alternatively spliced Rac1 isoform, in pancreatic ductal epithelial cells and in patients with chronic pancreatitis vs. PDAC, as well as in long-time vs. short-time survivors among PDAC patients, we asked whether Rac1b might negatively affect TGF-β1 prometastatic function. Interestingly, the non-malignant pancreatic ductal epithelial cell line H6c7 exhibited a higher ratio of active Rac1b to total Rac1b than the TGF-β1-responsive PDAC cell lines Panc-1 and Colo357. Notably, siRNA-mediated silencing of Rac1b increased TGF-β1/Smad-dependent migratory activities in H6c7, Colo357, and Panc-1 cells, while ectopic overexpression of Rac1b in Panc-1 cells attenuated TGF-β1-induced cell motility. Depletion of Rac1b in Panc-1 cells enhanced TGF-β1/Smad-dependent expression of promoter-reporter genes and of the endogenous Slug gene. Moreover, Rac1b depletion resulted in a higher and more sustained C-terminal phosphorylation of Smad3 and Smad2, suggesting that Rac1b is involved in Smad2/3 dephosphorylation/inactivation. Since pharmacologic or siRNA-mediated inhibition of Smad3 but not Smad2 was able to alleviate the Rac1b siRNA effect on TGF-β1-induced cell migration, our results suggests that Rac1b inhibits TGF-β1-induced cell motility in pancreatic ductal epithelial cells by blocking the function of Smad3. Moreover, Rac1b may act as an endogenous inhibitor of Rac1 in TGF-β1-mediated migration and possibly metastasis. Hence, it could be exploited for diagnostic/prognostic purposes or even therapeutically in late-stage PDAC as an antimetastatic agent.

Ionizing radiation induces a motile phenotype in human carcinoma cells in vitro through hyperactivation of the TGF-beta signaling pathway

“…Since activins preferably activate Smad2, the IR-induced upregulation of p-Smad3C, p-p38 MAPK as well as the response of the 3TPlux reporter are consistent with TGF-b being responsible for this induction. This is in accordance with results showing that both Smad3 and p38 mediate the pro-migratory TGF-b effect in PDAC cells [22].…”

“…The real-time cell analysis migration assays were performed with xCELLigence Ò technology (OLS, Bremen, Germany) in a chemokinesis setup as described in detail earlier [21,22]. Briefly, experiments were performed with modified 16-well plates consisting of an upper and a lower chamber separated by a porous membrane (8-lm pores).…”

Interaction of E3 Ubiquitin Ligase MARCH7 with Long Noncoding RNA MALAT1 and Autophagy-Related Protein ATG7 Promotes Autophagy and Invasion in Ovarian Cancer

“…TGF-β facilitates the progression and metastasis of tumors in advanced cancer [3][4][5][6]. Recent studies have showed that TGF-β induced autophagy in normal bovine mammary epithelial BME-UV1 cells and some cancer cells [7].…”

The role of small GTPases of the Rho/Rac family in TGF‐β‐induced EMT and cell motility in cancer

“…Both MMP3 and Rac1b are expressed in PDAC cells, and their expression was found to be associated with all tumor stages, whereby the subcellular distribution of Rac1b in PDAC correlates with the patient outcome (Mehner et al, ). Since Rac1b negatively regulates TGF‐β1‐induced cell migration in pancreatic cells, it is conceivable that it also controls TGF‐β1‐dependent EMT in a negative fashion (Ungefroren et al, ).…”

“…In the course of analyzing the role of Rac1 in TGF‐β‐induced cell migration and invasion, expression of Rac1b was noted in various PDAC cell lines by qPCR and immunoblot analysis, as well as in ductal cells in PDAC tissue from patients using immunohistochemistry (Ungefroren et al, ). In the light of the high structural similarity of Rac1 and Rac1b, both proteins were expected to be functionally equivalent and to both promote TGF‐β1‐induced cell migration.…”