Suppressive Role of ACVR1/ALK2 in Basal and TGFβ1-Induced Cell Migration in Pancreatic Ductal Adenocarcinoma Cells and Identification of a Self-Perpetuating Autoregulatory Loop Involving the Small GTPase RAC1b

Abstract

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.

The EGF Motif With CXDXXXXYXCXC Sequence Suppresses Fibrosis in a Mouse Skin Wound Model

“…These inflammatory cells produce various cytokines (23). In combination with TGFβ1, these cytokines induce the migration of fibroblasts, epithelial cells, and endothelial cells (24)(25)(26)(27). Thrombin and factors of the fibrinolytic system increase vascular permeability (28,29).…”

RAC1b Collaborates with TAp73α-SMAD4 Signaling to Induce Biglycan Expression and Inhibit Basal and TGF-β-Driven Cell Motility in Human Pancreatic Cancer

“…In light of the Thakur study highlighting stromal BGN as a TGF-β inhibitor and tumor suppressor in a murine model of human PDAC, we were especially interested in revealing if all three proteins do affect TGF-β signaling. Previous data from our group revealed that RAC1b negatively controls TGF-β signaling via the ALK2-SMAD1/5 arm [21] with inhibition of RAC1b in PANC-1 cells via RNAi-mediated knockdown or genomic knockout causing elevated levels of C-terminally phosphorylated SMAD1/5 (pSMAD1/5) after TGF-β1 treatment. In order to reveal whether TAp73 or BGN can mimic this effect, we transfected PANC-1 cells with siRNA to p73 or BGN and after a 1 h stimulation with TGF-β1 measured the abundance of pSMAD1/5 by phospho-immunoblotting.…”

“…RAC1b negatively controls TGF-β signaling via the ALK2-SMAD1/5 arm [21] with inhibition of RAC1b in PANC-1 cells via RNAi-mediated knockdown or genomic knockout causing elevated levels of C-terminally phosphorylated SMAD1/5 (pSMAD1/5) after TGF-β1 treatment. In order to reveal whether TAp73 or BGN can mimic this effect, we transfected PANC-1 cells with siRNA to p73 or BGN and after a 1 h stimulation with TGF-β1 measured the abundance of pSMAD1/5 by phospho-immunoblotting.…”