Overexpression of a Novel Noxo1 Mutant Increases Ros Production and Noxo1 Relocalisation.

Noxo1, the organizing element of the Nox1-dependent NADPH oxidase complex responsible for producing reactive oxygen species, has been described to be degraded by the proteasome. We mutated a D-box in Noxo1 to express a protein with limited degradation and capable of maintaining Nox1 activation. Wild-type (wt) and mutated Noxo1 (mut1) proteins were expressed in different cell lines to characterize their phenotype, functionality, and regulation. Mut1 increases ROS production through Nox1 activity affects mitochondrial organization and increases cytotoxicity in colorectal cancer cell lines. Unexpectedly the increased activity of Noxo1 is not related to a blockade of its proteasomal degradation since we were unable in our conditions to see any proteasomal degradation either for wt or mut1 Noxo1. Instead, D-box mutation mut1 leads to an increased translocation from the membrane soluble fraction to a cytoskeletal insoluble fraction compared to wt Noxo1. This mut1 localization is associated in cells with a filamentous phenotype of Noxo1, which is not observed with wt Noxo1. We found that mut1 Noxo1 associates with intermediate filaments such as keratin 18 and vimentin. In addition, Noxo1 D-Box mutation increases Nox1-dependent NADPH oxidase activity. Altogether, Nox1 D-box does not seem to be involved in Noxo1 degradation but rather related to the maintenance of the Noxo1 membrane/cytoskeleton balance.

Tau Regulates Glioblastoma Progression, 3D Cell Organization, Growth and Migration via the PI3K-AKT Axis.

The Microtubule-Associated Protein Tau is expressed in several cancers, including low-grade gliomas and glioblastomas. We have previously shown that Tau is crucial for the 2D motility of several glioblastoma cell lines, including U87-MG cells. Using an RNA interference (shRNA), we tested if Tau contributed to glioblastoma in vivo tumorigenicity and analyzed its function in a 3D model of multicellular spheroids (MCS). Tau depletion significantly increased median mouse survival in an orthotopic glioblastoma xenograft model. This was accompanied by the inhibition of MCS growth and cell evasion, as well as decreased MCS compactness, implying N-cadherin mislocalization. Intracellular Signaling Array analysis revealed a defective activation of the PI3K/AKT pathway in Tau-depleted cells. Such a defect in PI3K/AKT signaling was responsible for reduced MCS growth and cell evasion, as demonstrated by the inhibition of the pathway in control MCS using LY294002 or Perifosine, which did not significantly affect Tau-depleted MCS. Finally, analysis of the glioblastoma TCGA dataset showed a positive correlation between the amount of phosphorylated Akt-Ser473 and the expression of MAPT RNA encoding Tau, underlining the relevance of our findings in glioblastoma disease. We suggest a role for Tau in glioblastoma by controlling 3D cell organization and functions via the PI3K/AKT signaling axis.

Peptide screen identifies a new NADPH oxidase inhibitor: impact on cell migration and invasion.

The NADPH oxidase proteins catalyse the formation of superoxide anion which act as signalling molecules in physiological and pathological processes. Nox1-dependent NADPH oxidase is expressed in heart, lung, colon, blood vessels and brain. Different strategies involving Nox1 inhibition based on diphenylene iodonium derivatives are currently tested for colorectal cancer therapy. Here, after peptides screening on Nox1-dependent NADPH oxidase assay in HT-29 cells, we identify a peptide (referred to as NF02), cell-active, that potently block Nox1-dependent reactive oxygen species generation. Study of DEPMPO adduct formation by electron paramagnetic resonance showed that NF02 has no superoxide scavenging activity and no impact on cellular reactive oxygen species-producing enzymes such xanthine oxidase. NF02 was not cytotoxic, inhibited reactive oxygen species production of reconstituted Nox1/Noxo1/Noxa1 complex in HEK293 and did not decrease Nox2 dependent cellular NADPH oxidase reactive oxygen species production. Finally, NF02 inhibited cell migration and invasion of colorectal cancer cells which is consistent with the described impact of Nox1 inhibitors on cell migration. NF02 peptide is a new NADPH oxidase inhibitor specific for Nox1 over Nox2 and xanthine oxidase which might represent a useful Nox1 tool with potential therapeutic insights.

P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes.

α6ß4 integrin is the main component of hemidesmosomes (HD) that stably anchor the epithelium to the underlying basement membrane. Epithelial cell migration requires HD remodelling, which can be promoted by epidermal growth factor (EGF). We previously showed that extracellular nucleotides inhibit growth factor-induced keratinocyte migration. Here, we investigate the effect of extracellular nucleotides on α6ß4 integrin localisation in HD during EGF-induced cell migration. Using a combination of pharmacological inhibition and gene silencing approaches, we found that UTP activates the P2Y2 purinergic receptor and Gαq protein to inhibit EGF/ERK1/2-induced cell migration in keratinocytes. Using a keratinocyte cell line expressing an inducible form of the Raf kinase, we show that UTP inhibits the EGF-induced ERK1/2 pathway activation downstream of Raf. Moreover, we established that ERK1/2 activation by EGF leads to the mobilisation of α6ß4 integrin from HD. Importantly, activation of P2Y2R and Gαq by UTP promotes HD formation and protects these structures from EGF-triggered dissolution as revealed by confocal analysis of the distribution of α6ß4 integrin, plectin, BPAG1, BPAG2 and CD151 in keratinocytes. Finally, we demonstrated that the activation of p90RSK, downstream of ERK1/2, is sufficient to promote EGF-mediated HD dismantling and that UTP does not stabilise HD in cells expressing an activated form of p90RSK. Our data underline an unexpected role of P2Y2R and Gαq in the inhibition of the ERK1/2 signalling pathway and in the modulation of hemidesmosome dynamics and keratinocyte migration.

Gq-coupled purinergic receptors inhibit insulin-like growth factor-I/phosphoinositide 3-kinase pathway-dependent keratinocyte migration.

Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P2Y(2) purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y(2) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

Insulin-like growth factor-I receptor, E-cadherin and alpha v integrin form a dynamic complex under the control of alpha-catenin.

Dynamic crosstalk between cell adhesion molecules, extracellular matrix and soluble informative factors is essential for cancer cell migration and invasion. Here, we investigated the mechanisms by which the E-cadherin/catenin complex and alpha v integrin can modulate insulin-like growth factor-I (IGF-I)-induced cell migration. Human colon mucosa, human colon cancer cell lines, HT29-D4 and HCT-8 derivatives that differ in their expression of alpha-catenin, were used as models. Interactions between E-cadherin, alpha v integrin and IGF-I receptor (IGF-IR) were analyzed by coimmunoprecipitation and immunolocalization experiments. The impact of these interactions on cell mobility was determined by haptotaxis assays. We report that alpha v integrin, E-cadherin and IGF-IR form a ternary complex in both cultured cancer cells and human normal colonic mucosa. alpha-Catenin regulates the scaffolding of this complex. IGF-IR ligation by IGF-I induces the disruption of the complex and the relocalization of alpha v integrin from cell-cell contacts to focal contact sites. This perturbation is correlated with the observed increase in cell migration. These results suggest that regulation of the alpha v integrin/E-cadherin/IGF-IR scaffolding is essential for the modulation of cell mobility. Its alteration could be of major importance to sustain alterations in cell adhesion that occur during cancer cell invasion and metastasis.

G alpha(q/11)-coupled P2Y2 nucleotide receptor inhibits human keratinocyte spreading and migration.

Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucleotides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y2 nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymography analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y2 receptor activation also induces a dramatic dismantling of the actin network, the loss of alpha3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of alpha(v) integrins and focal contact protein distribution. In addition, activation of P2Y2R prevents growth factor-induced phosphorylation of Erk(1,2) and Akt/PkB. The use of a specific pharmacological inhibitor (YM-254890), the depletion of G alpha(q/11) by siRNA, or the expression of a constitutively active G alpha(q/11) mutant (Q209L) show that activation of G alpha(q/11) is responsible for these ATP/UTP-induced effects. Finally, we report that ATP delays growth factor-induced wound healing of keratinocyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing.

Bcl-xL/Bax ratio is altered by IFNgamma in TNFalpha- but not in TRAIL-induced apoptosis in colon cancer cell line.

Apoptosis is a crucial mechanism to eliminate harmful cells in which growth factors and cytokines are key regulators. In HT29-D4 cells, a model of human colon carcinoma, IFNgamma presensitization is essential to induce an apoptotic response to TNFalpha whereas it only slightly enhances TRAIL-induced apoptosis. To compare the transcriptional profiles induced by TNFalpha and TRAIL and their regulation by IFNgamma, we optimized a cDNA array analysis on targeted signaling pathways and confirmed the gene expression modulations by comparative RT-PCR. Although the two TNFSF ligands induced a same strong up-expression of pro-apoptotic Bax gene, the expression of anti-apoptotic Bcl-xL gene was more strongly up-regulated in TNFalpha- than in TRAIL-stimulated cells. Thus, TRAIL but not TNFalpha induced apoptotic mitochondrial cascade as highlighted by cytochrome c release into cytosol. IFNgamma presensitization of TRAIL-stimulated cells did not induce any change in cytochrome c release, suggesting that the increase of IFNgamma/TRAIL-induced apoptosis is independent of this pathway. In contrast, IFNgamma pretreatment prevented Bcl-xL gene up-expression in TNFalpha-stimulated cells and allowed cytochrome c release. Thus, we hypothesize that the Bcl-xL/Bax ratio can block the apoptotic response in TNFalpha-stimulated cells but allows cell death initiation when it is altered by a crosstalk between IFNgamma presensitization and TNFalpha induced signalings.

Membrane rafts segregate pro- from anti-apoptotic insulin-like growth factor-I receptor signaling in colon carcinoma cells stimulated by members of the tumor necrosis factor superfamily.

In the tumor microenvironment, autocrine/paracrine loops of insulin-like growth factors (IGFs) contribute to cancer cell survival. However, we report here that IGF-I can send contradictory signals that interfere with cell death induced by different ligands of the tumor necrosis factor (TNF) superfamily. IGF-I protected human colon carcinoma cells from TNF-alpha-induced apoptosis, but it enhanced the apoptotic response to anti-Fas antibody and TNF-related apoptosis inducing ligand stimulation. This proapoptotic effect of IGF-I, observed in several but not all tested colon cancer cell lines, was mediated via the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway. Furthermore, IGF-I receptors (IGF-IR) were located in and out of membrane lipid rafts and were tyrosine autophosphorylated in response to IGF-I. However, disruption of rafts by acute cholesterol depletion shifted IGF-IR to non-raft domains, abolished the IGF-I-mediated proapoptotic effect, and inhibited the IGF-I-dependent IRS-1 and Akt recruitment into and phosphorylation/activation within lipid rafts. Replenishing cell membranes with cholesterol reversed these effects. Activation of extracellular-regulated kinase-1/2 and p38 mitogen-activated protein kinase, which convey the IGF-I anti-apoptotic effect, occurred independently of lipid rafts. Thus, we propose that segregation of IGF-IR in and out of lipid rafts may dynamically regulate the pro- and anti-apoptotic effects of IGF-I on apoptosis induced by TNF superfamily members.