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

Opposing roles of netrin-1 and the dependence receptor DCC in cancer cell invasion, tumor growth and metastasis

“…In coherence with our data, it has been demonstrated that the A2B-R is induced in hypoxia, inflammatory bowel diseases and promotes an angiogenic phenotype, as measured by endothelial tube formation (Kong et al, 2006). Thus, we found that the PLC signaling cascade activated by the physiological agonist adenosine is the critical invasion effector acting through Rho/ROK-independent mechanisms and the Ca 2 þ -dependent myosin light chain kinase that is believed to promote the contractility of the actin cytoskeleton, as shown previously in our laboratory (Nguyen et al, 2005). Both adenosine and the A2B-R are linked to cAMP pathways and to the recruitment of DCC receptors from an intracellular pool to the cell surface, in a netrin-dependent manner (Bouchard et al, 2004).…”

“…Inhibition of Rho-GTPases (with C3T exoenzyme) and ROK (with Y27632) obliterated the invasive responses promoted by netrin-1 and NECA, but not adenosine in HCT8/S11 cells ( Figure 2a). As expected (Nguyen et al, 2005), the adenosine proinvasive pathways linked to Gaq are not interrupted by pharmacological inhibitors and molecular interventions targeting the Rho-ROK axis, including C3T, Y27936, the RhoA antagonist DC-RhoD (G26V-RhoD) and the interfering mutants DN-RhoA (T19N-RhoA) and DN-ROK (DRB/PH). In contrast, DN-Rac1 (T17N-Rac1) and DN-Cdc42 (T17N-Cdc42), as well as the phosphatidylinositol 3-kinase (PI3K) kinase inhibitor wortmannin, abolished the invasive responses promoted by the proinvasive agents netrin-1, NECA and adenosine.…”

“…As with parental HCT8/S11 cells, control vector transfected HCT8/S11-pcDNA3 cells were noninvasive and external addition of netrin or NECA simulated collagen type I invasion. The invasive phenotype exhibited by the HCT8/S11-netrin cells (Supplementary data 1) was reversed by the inhibitors of the Rho-ROK axis and PI3K (Kotelevets et al, 1998;Barbier et al, 2001;Nguyen et al, 2005). Conversely, restoration of wt-DCC in DCC-deficient HCT8/S11 cells obliterated the proinvasive activity of netrin-1, NECA and other proinvasive agents, including TGFa and trefoil factors.…”

“…Conversely, stimulation with netrin and NECA was insensitive to U73122, while this PLC inhibitor abrogated the adenosine signals. As Rho GTPases and Rho-kinase ROK are required for the activation of the actin cytoskeleton and epithelial cell movements and invasiveness (Nguyen et al, 2005), we next neutralized these small GTPases by functionblocking pharmacological inhibitors and interfering mutants. Inhibition of Rho-GTPases (with C3T exoenzyme) and ROK (with Y27632) obliterated the invasive responses promoted by netrin-1 and NECA, but not adenosine in HCT8/S11 cells ( Figure 2a).…”

“…Only one clone was positive from 12 G418-resistant colonies tested after transfection with each DCC variant (wt, Stop-1291 and D1290N). HCT8/S11 cells stably transfected by the dominant-negative forms of the RhoA GTPases (DNRhoA, DNRac and DNCdc42), Rho-kinase ROK (DNROK) and the constitutively activated form of the RhoA antagonist RhoD (DCRhoD) have been characterized elsewhere (Nguyen et al, 2005). The kidney cancer cell line MDCKts.src established after stable transfection of a thermosensitive variant of v-src and the PCAA/C1 adenomatous colon cell line derived from a patient with familial adenomatous polyposis were maintained in DMEM supplemented with 10-20% FCS.…”