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RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGF?1 in the Suppression of Cell Motility.

ABSTRACT: Autocrine transforming growth factor (TGF)? 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?1 via downregulation of the TGF? type I receptor, ALK5, but whether RAC1B also impacts autocrine TGF? signaling has not yet been studied. Intriguingly, RNA interference-mediated knockdown (RNAi-KD) or CRISPR/Cas-mediated knockout of RAC1B in TGF?1-secreting PDAC-derived Panc1 cells resulted in a dramatic decrease in secreted bioactive TGF?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? 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?1 which in a SMAD3-dependent manner blocks EMT-associated gene expression and cell motility.

SUBMITTER: Ungefroren H

PROVIDER: S-EPMC7760153 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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RAC1B Regulation of <i>TGFB1</i> Reveals an Unexpected Role of Autocrine TGFβ1 in the Suppression of Cell Motility.

Ungefroren Hendrik H Otterbein Hannah H Wellner Ulrich F UF Keck Tobias T Lehnert Hendrik H Marquardt Jens-Uwe JU

Cancers 20201129 12

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 <i>TGFB1</i> remain poorly characterized. In both cancer types, the small GTPase RAC1B inhibits cell motility induced by recombinant human TGFβ1 via downregulation of ...[more]

PMID: 33260366

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Project description:Pancreatic ductal adenocarcinoma (PDAC) cells are known for their high invasive/metastatic potential, which is regulated in part by the transforming growth factor β1 (TGFβ1). The involvement of at least two type I receptors, ALK5 and ALK2, that transmit downstream signals of the TGFβ via different Smad proteins, SMAD2/3 and SMAD1/5, respectively, poses the issue of their relative contribution in regulating cell motility. Real-time cell migration assays revealed that the selective inhibition of ALK2 by RNAi or dominant-negative interference with a kinase-dead mutant (ALK2-K233R) strongly enhanced the cells' migratory activity in the absence or presence of TGFβ1 stimulation. Ectopic ALK2-K233R expression was associated with an increase in the protein levels of RAC1 and its alternatively spliced isoform, RAC1b, both of which are implicated in driving cell migration and invasion. Conversely, the RNAi-mediated knockdown or CRISPR/Cas9-mediated knockout of RAC1b resulted in the upregulation of the expression of ALK2, but not that of the related BMP type I receptors, ALK3 or ALK6, and elevated the phosphorylation of SMAD1/5. PDAC is a heterogeneous disease encompassing tumors with different histomorphological subtypes, ranging from epithelial/classical to extremely mesenchymal. Upon treatment of various established and primary PDAC cell lines representing these subtypes with the ALK2 inhibitor, LDN-193189, well-differentiated, epithelial cell lines responded with a much stronger increase in the basal and TGFβ1-dependent migratory activity than poorly differentiated, mesenchymal ones. These data show that (i) ALK2 inhibits migration by suppressing RAC1/RAC1b proteins, (ii) ALK2 and RAC1b act together in a self-perpetuating the autoregulatory negative feedback loop to mutually control their expression, and (iii) the ALK2 antimigratory function appears to be particularly crucial in protecting epithelial subtype cells from becoming invasive, both spontaneously and in a TGFβ-rich tumor microenvironment.

Dataset Information

RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGF?1 in the Suppression of Cell Motility.

Publications

RAC1B Regulation of <i>TGFB1</i> Reveals an Unexpected Role of Autocrine TGFβ1 in the Suppression of Cell Motility.

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