RhoA‐ and RhoD‐dependent regulatory switch of Gα subunit signaling by PAR‐1 receptors in cellular invasion

Abstract

Thrombin and proteinase-activated receptors (PAR) specifically regulate several functions that markedly enhance the transformation phenotype such as inflammation, cell proliferation, tumor growth, and metastasis. We recently reported that thrombin inhibits cellular invasion induced by src, hepatocyte growth factor (HGF), and leptin in kidney and colonic epithelial cells via predominant activation of the pertussis toxin (PTx) -sensitive G-proteins Galphao/Galphai. We provide pharmacological and biochemical evidence that in the presence of PTx, PAR-1 induced cellular invasion through Galpha12/Galpha13- and RhoA/Rho kinase (ROCK) -dependent signaling. However, inhibition of the endogenous small GTPase RhoA by the C3 exoenzyme, dominant-negative N19-RhoA, activated G26V-RhoD, and activators of the nitric oxide/cGMP pathways conferred invasive activity to PAR-1 via a signaling cascade using Galphaq, phospholipase C (PLC), Ca(2+)/calmodulin myosin light chain kinase (CaM-MLCK), and phosphorylation of MLC. We found that cellular invasion induced by the src oncogene is abrogated by inhibitors of the RhoA/ROCK pathway and is independent of PLC/CaM-MLCK signaling. Our data demonstrate that the RhoA and RhoD small GTPases are acting as a molecular switch of cellular invasion and reveal a novel critical mechanism by which PAR-1 bypass Galphao/i and RhoA inhibition via differential coupling to heterotrimeric G-proteins linked to divergent or convergent biological responses. Our data also indicate that Rho GTPases and ROCK mediate a src-dependent invasion signal in kidney and colonic cancer cells. We conclude that dynamic regulation of Rho GTPases activation and inactivation by oncogenes, growth factors, cGMP-inducing agents, and adhesion molecules can initiate convergent invasion signals controlled by the thrombin PAR-1 in cancer cells.-Nguyen, Q.-D., Faivre, S., Bruyneel, E., Rivat, C., Seto, M., Endo, T., Mareel, M., Emami, S., Gespach, C. RhoA- and RhoD-dependent regulatory switch of Galpha subunit signaling by PAR-1 receptors in cellular invasion.

Invasion and MMP expression profile in desmoid tumours

“…To test the hypothesis that invasion-stimulating soluble factors were present in desmoid CM, collagen invasion assays with HCT-8/ E11 colon cancer cells were performed. These colon cancer cells were used as a model because it was previously shown that they were noninvasive in type I collagen, but can become invasive depending on stimulation (Debruyne et al, 2002;Regnauld et al, 2002;Nguyen et al, 2002). In the presence of normal medium (DMEM), no invasion of the cells into the collagen was observed.…”

Commutators of PAR-1 signaling in cancer cell invasion reveal an essential role of the Rho–Rho kinase axis and tumor microenvironment

“…Thrombin is a potent mitogen for fibroblasts and epithelial cells, and potentiates the proliferative responses of tumor cells to classical growth factors, such as epidermal growth factor and insulin (Van Obberghen-Schilling et al, 1995). Both thrombin and proteinase-activated receptors type I (PAR-1) are expressed in invasive cancer cell lines and breast carcinoma biopsy specimens (Wojtukiewicz et al, 1995;Even-Ram et al, 1998, 2001Nguyen et al, 2002;Darmoul et al, 2003). However, the signaling pathways triggered by thrombin and PAR-1 signaling during tumor progression and invasion remain poorly documented.…”

“…Activated PAR-1 are coupled via several members of the heterotrimeric G-proteins Gao/i, Ga12/13, and Gaq to transduce a substantial network of signaling pathways (Coughlin, 2000). We have recently demonstrated that PAR-1 are functionally connected to both negative and positive invasion pathways in colon and kidney cancer cells Nguyen et al, 2002). In the collagen type I substratum, PAR-1 and the pertussis toxin (PTx)-sensitive Gao/i subunits were shown to exert a dominant invasion suppressor role toward several proinvasive pathways controlled by oncogenes and tumor-secreted growth factors .…”

“…Similar findings were reported by Kamath et al (2001) for the inhibition of invasion and migration in the highly invasive MDA-MB231 breast cancer cell line, through PAR-1-and Gai-dependent pathways. Conversely, we have shown that neutralization of Gao/i signaling by PTx led to the proinvasive activity of thrombin and PAR-1 through the Ga12/13/RhoA cascade, myosin light chain (MLC) phosphorylation, and activation of the actomyosin system (Kureishi et al, 1997;Nguyen et al, 2002). We presented evidence that the proinvasive potential of PAR-1 in collagen type I is also revealed by inhibition of RhoA GTPase by RhoD, C3 exoenzyme, the dominant negative N19-RhoA, and cGMP-elevating agents sodium nitroprusside and guanylin, acting through soluble and membrane-bound guanylate cyclases, respectively (Nguyen et al, 2002).…”

Epithelial effects of proteinase-activated receptors in the gastrointestinal tract

“…PAR-1 is also associated with tumour cell differentiation (Rudroff et al 2002) and adhesion (Rudroff et al 2001) in pancreatic cancer. PAR-1 appears to promote invasion signals in some cancer cells through activation of a Rho-A-dependent pathway (Nguyen et al 2002).…”