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

Simple Summary Transforming growth factor (TGF)beta secreted by cancer cells and acting in an autostimulatory manner has been implicated in epithelial-mesenchymal transition and cellular invasion. However, the upstream inducers/downstream effectors, or the effects on cell migration, of endogenously produced TGF beta 1 remain poorly characterized. Here, we studied whether autocrine TGF beta is regulated by the small GTPase, RAC1B, and how it impacts cell motility. We found that RAC1B induced an increase in the levels of TGF beta 1 mRNA and the secreted bioactive protein in pancreatic carcinoma Panc1 and breast cancer MDA-MB-231 cells. Antibody-mediated neutralization of secreted TGF beta or inhibition of its synthesis increased cell migration and the activities of promigratory genes, but downregulated those of antimigratory genes, i.e., SMAD3. Restoration of SMAD3 protein expression in these cells was able to prevent the TGF beta 1 inhibition-induced rise in migratory activity, suggesting that a RAC1B-autocrine TGF beta 1-SMAD3 pathway can suppress mesenchymal transdifferentiation and cell motility. Autocrine transforming growth factor (TGF)beta 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 TGFB1 remain poorly characterized. In both cancer types, the small GTPase RAC1B inhibits cell motility induced by recombinant human TGF beta 1 via downregulation of the TGF beta type I receptor, ALK5, but whether RAC1B also impacts autocrine TGF beta signaling has not yet been studied. Intriguingly, RNA interference-mediated knockdown (RNAi-KD) or CRISPR/Cas-mediated knockout of RAC1B in TGF beta 1-secreting PDAC-derived Panc1 cells resulted in a dramatic decrease in secreted bioactive TGF beta 1 in the culture supernatants and TGFB1 mRNA expression, while the reverse was true for TNBC-derived MDA-MB-231 cells ectopically expressing RAC1B. Surprisingly, the antibody-mediated neutralization of secreted bioactive TGF beta or RNAi-KD of the endogenous TGFB1 gene, was associated with increased rather than decreased migratory activities of Panc1 and MDA-MB-231 cells, upregulation of the promigratory genes SNAI1, SNAI2 and RAC1, and downregulation of the invasion suppressor genes CDH1 (encoding E-cadherin) and SMAD3. Intriguingly, ectopic re-expression of SMAD3 was able to rescue Panc1 and MDA-MB-231 cells from the TGFB1 KD-induced rise in migratory activity. Together, these data suggest that RAC1B favors synthesis and secretion of autocrine TGF beta 1 which in a SMAD3-dependent manner blocks EMT-associated gene expression and cell motility.