No abstract

The matrix metalloprotease matrilysin is expressed in premalignant polyps and plays a key role in local invasion during the progression of digestive tumors. In the present work, we investigated the possible relationships between the activity of the mouse and human matrilysin promoters (Mp), endogenous matrilysin protein expression, and two early oncogenetic defects frequently observed in human colonic cancers, namely activation of the src oncogene and impairment of the Wnt/APC/beta-catenin pathway. Using transient transfection assays, we report here that src signaling and the HMG-box transcription factor LEF-1 act synergistically with the proximal (-61 to -67) AP-1 binding site to transactivate the Mp in premalignant and tumorigenic kidney and colonic epithelial cells, through beta-catenin- and axin-independent signaling pathways. This synergism involves the -109 and -194 Tcf/LEF-1 binding sites in the Mp and a physical interaction between LEF-1 and c-Jun. Furthermore, src coordinates accumulation of the c-Jun factor and matrilysin transcripts. Conversely, the c-Jun dominant negative mutant TAM67 and the src tyrosine kinase inhibitor M475271 impaired src-induced Mp activation, matrilysin protein accumulation, and invasion of type I collagen gels. This mechanism may thereby contribute to cellular invasion during the early-stage adenoma/adenocarcinoma conversion and the metastatic process of digestive tumors.

Inappropriate activation of the Wnt/APC/beta-catenin signaling pathways plays a critical role at early stages in a variety of human cancers. However, their respective implication in tumor cell invasion is still hypothetical. Here, we show that two activators of the canonical Wnt/beta-catenin transcription pathway, namely Dvl-2, the Axin 501-560 fragment binding glycogen synthase kinase -3beta (GSK-3beta), and the negative Wnt regulator wt-Axin did not alter cell invasion into type I collagen. In addition, both Dvl-2 and Axin 501-560 exerted a permissive action on the proinvasive activity of HGF and intestinal trefoil factor. Upstream activation of Wnt signaling by the Wnt-2 and Wnt-3a ligands, stable overexpression of Wnt-2, as well as GSK-3beta inhibition by lithium, SB216763, and GSK-3beta dominant negative forms (K85R and R96E) conferred the invasive phenotype through several proinvasive pathways. Induction of the matrix metalloprotease MMP-7 (matrilysin) gene and protein by Wnt-2 was abolished by inactivation of the AP-1 binding site in the promoter. Accordingly, invasion induced by Wnt-2 was prevented by soluble FRP-3 and FRP-1, sequestration of Gbetagamma subunits, depletion of the GSK-3beta protein by RNA interference, the c-Jun dominant negative mutant TAM67 and was not reversed by wt-Axin. Thus, the proinvasive activity of Wnt-2 is mediated by a noncanonical Wnt pathway using GSK-3beta and the AP-1 oncogene. Our data provide a potential clue for our understanding of the action and crosstalk between Wnt activators and other proinvasive pathways, in relation with matrix substrates and proteases in human cancers.

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.

STAT3 is frequently overexpressed and constitutively activated by tyrosine phosphorylation during malignant transformation. Despite the clear importance of STAT3 in cell proliferation and survival in diverse human cancers, its possible contribution to tumor cell adhesion, motility and invasion remains hypothetical. We therefore compared the transforming properties of STAT3wt, its constitutively activated dimeric form STAT3C, and the dominant negative mutant STAT3-Y705F in human colorectal HCT8/S11 cancer cells. Both STAT3wt and STAT3C exert a permissive action to the proinvasive activity of the scatter factor HGF in HCT8/S11 cells. In contrast, the monomeric and cytoplasmic mutant Y705F induces a constitutive invasive phenotype through Wnt/Rho-independent and EGFR/PI3-kinase-dependent pathways. Accordingly, Y705F decreases cell-cell homotypic adhesions, and increases cell motility and scattering, as well as lamellipodia-type cellular extensions. STAT3-Y705F-transfected HCT8/S11 cells display an increased tyrosine phosphorylation of the cell-cell adhesion regulator bcatenin and its dissociation from the invasion suppressor E-cadherin at cell-cell contacts. Our data imply that both invasion promoter and repressor genes are controlled by the canonical STAT3 transcription pathways. Disruption of this cascade by Y705F reveals the proinvasive potential of altered forms of STAT3 as a persistent signaling adaptor in cytokine/transforming growth factor receptor scaffolds and oncogenic pathways.

Signal transducer and activator of transcription (STAT) 3 is overexpressed or activated in most types of human tumors and has been classified as an oncogene. In the present study, we investigated the contribution of the STAT3s to the proinvasive activity of trefoil factors (TFF) and vascular endothelial growth factor (VEGF) in human colorectal cancer cells HCT8/S11 expressing VEGF receptors. Both intestinal trefoil peptide (TFF3) and VEGF, but not pS2 (TFF1), activate STAT3 signaling through Tyr705 phosphorylation of both STAT3α and STAT3β isoforms. Blockade of STAT3 signaling by STAT3β, depletion of the STAT3α/β isoforms by RNA interference, and pharmacologic inhibition of STAT3α/β phosphorylation by cucurbitacin or STAT3 inhibitory peptide abrogates TFF- and VEGF-induced cellular invasion and reduces the growth of HCT8/S11 tumor xenografts in athymic mice. Differential gene expression analysis using DNA microarrays revealed that overexpression of STAT3β down-regulates the VEGF receptors Flt-1, neuropilins 1 and 2, and the inhibitor of DNA binding/differentiation (Id-2) gene product involved in the neoplastic transformation. Taken together, our data suggest that TFF3 and the essential tumor angiogenesis regulator VEGF165 exert potent proinvasive activity through STAT3 signaling in human colorectal cancer cells. We also validate new therapeutic strategies targeting STAT3 signaling by pharmacologic inhibitors and RNA interference for the treatment of colorectal cancer patients.

Platelets are formed from mature megakaryocytes (MKs) and arise from the development of cytoplasmic extensions called proplatelets (PPT). Proliferation and full maturation of MKs require TPO, but it is dispensable for platelet shedding. To precisely define the role of different signaling pathway activated by TPO on proplatelet formation (PPF), chemical inhibitors of ERK (PD98059), p38 (SB 203580) and a pan PKC inhibitor (GF109203X) were added to MK derived from human CD34+ cells. As previously reported, the PKC inhibitor leads to a marked decrease in PPF and the p38 inhibitor has no effect on PPF. Addition at day 8 of an inhibitor of the ERK pathway (PD98059) to the purified CD34+CD41+ cultured cells leads to an early PPF associated to a 3-fold increase of PPT. As observed with PD98059, an overexpression of a dominant-negative (DN) form of MEK1 in primary MKs led to a 2-fold increase of PPT. Inhibition of the ERK pathway in the immature MKs surprisingly increased MK mean ploidy, (5.7N in the control versus 10.9N in presence of PD98059). Especially, the highest classes of ploidy (8N, 16N and 32N) were significantly increased. Altogether, those results suggest that the inhibition of MAP/ERK1/2 is an essential step for PPF in mature MKs but also that MAPK/ERK1/2 is a negative regulator of endomitosis. As PPF occurs in the presence of TPO, we investigated whether MAPK activation was down-regulated during MK differentiation. Western blot and flow cytometry analysis were performed and show that ERK activation induced by TPO was dramatically decreased at the late stages of megakaryocytopoiesis. In addition, inhibition of MEK prior to PPF was associated with an activation of caspase 3 in the absence of apoptosis suggesting a direct role of the ERK pathway in the regulation of proplatelet. Altogether, those results show a differential response of MAP kinase pathway to TPO depending on the maturation stage. ERK downregulation occurs when PPF begins and MEK inhibition is associated with increased PPF.