Negative regulation of TGF-β1-induced MKK6-p38 and MEK-ERK signalling and epithelial-mesenchymal transition by Rac1b

Abstract

Prompted by earlier findings that the Rac1-related isoform Rac1b inhibits transforming growth factor (TGF)-β1-induced canonical Smad signalling, we studied here whether Rac1b also impacts TGF-β1-dependent non-Smad signalling such as the MKK6-p38 and MEK-ERK mitogen-activated protein kinase (MAPK) pathways and epithelial-mesenchymal transition (EMT). Transient depletion of Rac1b protein in pancreatic cancer cells by RNA interference increased the extent and duration of TGF-β1-induced phosphorylation of p38 MAPK in a Smad4-independent manner. Rac1b depletion also strongly increased basal ERK activation - independent of the kinase function of the TGF-β type I receptor ALK5 - and sensitised cells towards further upregulation of phospho-ERK levels by TGF-β1, while ectopic overexpression of Rac1b had the reverse effect. Rac1b depletion increased an EMT phenotype as evidenced by cell morphology, gene expression of EMT markers, cell migration and growth inhibition. Inhibition of MKK6-p38 or MEK-ERK signalling partially relieved the Rac1b depletion-dependent increase in TGF-β1-induced gene expression and cell migration. Rac1b depletion also enhanced TGF-β1 autoinduction of crucial TGF-β pathway components and decreased that of TGF-β pathway inhibitors. Our results show that Rac1b antagonises TGF-β1-dependent EMT by inhibiting MKK6-p38 and MEK-ERK signalling and by controlling gene expression in a way that favors attenuation of TGF-β signalling.

RAC1B: A Guardian of the Epithelial Phenotype and Protector Against Epithelial-Mesenchymal Transition

“…The observation that RAC1B expression was more abundant in well differentiated pancreatic cancer cell lines prompted us to hypothesize that RAC1B promotes epithelial differentiation not only by the upregulation of epithelial marker genes but also by blocking the route to mesenchymal differentiation. Support for this comes from previous studies showing that RAC1B suppressed SERPINE1 (encoding plasminogen-activator inhibitor 1), MMP9 (encoding MMP9) [17], and TGFBRI (encoding the TGF-β type I receptor activin receptor-like kinase 5) [20]. Here we analyzed if the intermediate filament protein Vimentin (encoded by VIM), a marker for EMT [29] associated with pancreatic tumor budding [7], is also targeted for inhibition by RAC1B.…”

“…Whereas Rac1b has been reported to promote EMT induced by matrix metalloproteinase 3 (MMP3) in an immortalized mouse mammary epithelial cell line [15,16], our group observed in human PDAC-derived ductal epithelial cells that RAC1B potently inhibited mesenchymal differentiation induced by TGF-β1 [17]. The potential role of RAC1B as an endogenous inhibitor of (TGF-β-dependent) EMT is supported by its potent suppressive effect on basal and TGF-β1-induced cell migration (a hallmark feature of EMT) in various benign and malignant human cell lines of pancreatic and breast origin [17][18][19][20][21].…”

“…Moreover, loss of RKIP is associated with the presence of nodal and/or distant metastases as well as reduced patient survival and hence may be used as a predictor of high-grade tumor budding, tumor aggressiveness, and unfavorable outcome [48]. Remarkably, the loss of RKIP or RAC1B share several consequences in common: the appearance of morphological hallmarks of EMT and an increase in cell motility [17,47], a decrease in E-cadherin and an increase in Vimentin, SNAIL, and SLUG expression [47], as well as derepression of MEK-ERK and p38 MAPK signaling [47,49]. It therefore appears that selective loss of RKIP in the tumor buds can drive tumor progression and eventually metastatic disease.…”

“…Prompted by the association of RAC1B expression with higher epithelial differentiation in PDAC-derived cells, we addressed the question if RAC1B is causally involved in maintaining an epithelial phenotype. To do this, we inhibited RAC1B expression in Capan1, Colo357, and Panc1 cells by siRNA-mediated knockdown, targeting exon 3b of RAC1 [17,18,20,21] followed by measurement of epithelial gene expression. The RAC1B knockdown resulted in downregulation of CDH1 mRNA in all three cell lines ( Figure 2A), and of CLDN7 and EPCAM mRNA in Panc1 cells (Supplementary Figure S3, for E-cadherin protein expression in Capan1 cells see Supplementary Figure S1).…”

The role of Rac in tumor susceptibility and disease progression: from biochemistry to the clinic

“…Despite the reported ability of Rac1b to promote cellular transformation in vitro , studies in mice suggest that Rac1b alone is insufficient to promote tumor initiation, however it synergizes with other oncogenic inputs, such as the described synergistic interaction with mutant K-ras for promoting lung tumorigenesis (34). Rac1b displays distinctive signaling properties in the context of EMT, where it emerged as an endogenous inhibitor of Rac1 in TGF-β signaling in pancreatic cancer (35, 36). …”