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

Autocrine transforming growth factor (TGF)β has been implicated in epithelial-mesenchymal transition (EMT) and invasion of several cancers including pancreatic ductal adenocarcinoma (PDAC) as well as triple-negative breast cancer (TNBC). However, the precise mechanism and the upstream inducers or downstream effectors of endogenous remain poorly characterized. In both cancer types, the small GTPase RAC1B inhibits cell motility induced by recombinant human TGFβ1 via downregulation of the TGFβ type I receptor, ALK5, but whether RAC1B also impacts autocrine TGFβ signaling has not yet been studied.

Negative Control of Cell Migration by Rac1b in Highly Metastatic Pancreatic Cancer Cells Is Mediated by Sequential Induction of Nonactivated Smad3 and Biglycan.

Expression of the small GTPase, Ras-related C3 botulinum toxin substrate 1B (RAC1B), a RAC1-related member of the Rho GTPase family, in tumor tissues of pancreatic ductal adenocarcinoma (PDAC) has been shown previously to correlate positively with patient survival, but the underlying mechanism(s) and the target genes involved have remained elusive. Screening of a panel of established PDAC-derived cell lines by immunoblotting indicated that both RAC1B and Mothers against decapentaplegic homolog 3 (SMAD3) were more abundantly expressed in poorly metastatic and well-differentiated lines as opposed to highly metastatic, poorly differentiated ones. Both siRNA-mediated RAC1B knockdown in the transforming growth factor (TGF)-β-sensitive PDAC-derived cell lines, Panc1 and PaCa3, or CRISPR/Cas-mediated knockout of exon 3b of in Panc1 cells resulted in a dramatic decrease in the expression of .

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

The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown to potently inhibit transforming growth factor (TGF)-β1-induced cell migration and epithelial-mesenchymal transition (EMT) in pancreatic and breast epithelial cells, but the underlying mechanism has remained obscure. Using a panel of pancreatic ductal adenocarcinoma (PDAC)-derived cell lines of different differentiation stages, we show that RAC1B is more abundantly expressed in well differentiated as opposed to poorly differentiated cells. Interestingly, RNA interference-mediated knockdown of RAC1B decreased expression of the epithelial marker protein E-cadherin, encoded by , and enhanced its TGF-β1-induced downregulation, whereas ectopic overexpression of RAC1B upregulated expression and largely prevented its TGF-β1-induced silencing of .

RAC1B Suppresses TGF-β-Dependent Chemokinesis and Growth Inhibition through an Autoregulatory Feed-Forward Loop Involving PAR2 and ALK5.

The small GTPase RAC1B functions as a powerful inhibitor of transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition, cell motility, and growth arrest in pancreatic epithelial cells. Previous work has shown that RAC1B downregulates the TGF-β type I receptor ALK5, but the molecular details of this process have remained unclear. Here, we hypothesized that RAC1B-mediated suppression of activin receptor-like kinase 5 (ALK5) involves proteinase-activated receptor 2 (PAR2), a G protein-coupled receptor encoded by that is crucial for sustaining ALK5 expression.

Changes in uPA, PAI-1, and TGF-β Production during Breast Cancer Cell Interaction with Human Mesenchymal Stroma/Stem-Like Cells (MSC).

The interactions of cancer cells with neighboring non-malignant cells in the microenvironment play an important role for progressive neoplastic development and metastasis. Long-term direct co-culture of human MDA-MB-231 breast cancer cells with benign human mesenchymal stroma/stem-like cells (MSC) MSC544 stably expressing mCherry and eGFP fluorescence proteins, respectively, was associated with the formation of three-dimensional (3D) tumor spheroids in vitro. The quantification of the breast tumor marker urokinase plasminogen activator (uPA) in mono-cultured MDA-MB-231 cells revealed an approximately 14-fold enhanced expression when compared to five different normal human MSC mono-cultures.

RAC1B Suppresses TGF-β1-Dependent Cell Migration in Pancreatic Carcinoma Cells through Inhibition of the TGF-β Type I Receptor ALK5.

The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown previously by RNA interference-mediated knockdown (KD) to function as a powerful inhibitor of transforming growth factor (TGF)-β1-induced cell migration and epithelial-mesenchymal transition in epithelial cells, but the underlying mechanism has remained enigmatic. Using pancreatic carcinoma cells, we show that both KD and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated knockout (KO) of RAC1B increased the expression of the TGF-β type I receptor ALK5 (activin receptor-like kinase 5), but this effect was more pronounced in CRISPR-KO cells. Of note, in KO, but not KD cells, ALK5 upregulation was associated with resensitization of to induction by TGF-β1 stimulation.

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

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.