Sophie Lopez

Effect of atorvastatin and fluvastatin on the expression of plasminogen activator inhibitor type-1 in cultured human endothelial cells

Inhibitors of HMG-CoA reductase, namely statins, improve endothelial function independently of their cholesterol-lowering effects. Plasminogen activator inhibitor type-1 (PAI-1) plays a critical role in vascular pathophysiology both at the intra- and extravascular levels. We therefore investigated the effects of atorvastatin (ATOR) and fluvastatin (FLU) on PAI-1 and also tissue-type plasminogen activator (t-PA) synthesis in 20% fetal calf serum-cultured human umbilical vein endothelial cells (HUVEC) stimulated or not by recombinant human pro-inflammatory cytokines, i.e. tumor necrosis factor alpha (TNFalpha) and interleukin 1alpha (IL-1alpha). In non-stimulated HUVEC, ATOR and FLU significantly diminished (-50% at 2.0 micromol/l) the constitutive production of PAI-1 (mRNA level and protein secretion). This effect was prevented by addition of mevalonate (100 micromol/l). In HUVEC cultivated in 20% fetal calf serum, the t-PA antigen accumulation was not significantly altered, whereas in low serum concentration (1%) a significant stimulatory effect of ATOR (+30%) and FLU (+76%) was observed. In TNFalpha-stimulated cells, ATOR and FLU had a modest down-modulating effect (-17 and -20%, respectively) on TNFalpha-induced increase in PAI-1 synthesis. No effect of statins was observed in IL-1alpha-stimulated HUVEC, suggesting that statins do not interfere with the up-regulation of PAI-1 synthesis by pro-inflammatory cytokines. However, ATOR and FLU inhibited the TNFalpha-induced decrease in t-PA release. In conclusion, these results show that statins favorably modulate the expression of fibrinolytic factors produced by human endothelial cells.

Mechanisms of STAT protein activation by oncogenic KIT mutants in neoplastic mast cells.

Mutations in the c-kit gene occur in the vast majority of mastocytosis. In adult patients as well as in the cell line derived from mast cell neoplasms, the mutations occur almost exclusively at amino acid 816 within the kinase domain of KIT. Among the downstream effectors of KIT signaling, STAT3 and STAT5 have been shown to be critical for cell proliferation elicited by the KIT-Asp816 mutant protein. However, little is known about the mechanisms of activation of STAT proteins. In this study, we identify and clarify the contribution of various STAT kinases in two widely used neoplastic mast cell lines, P815 and HMC-1. We show that STAT1, -3, and -5 proteins are activated downstream of the KIT-Asp816 mutant. All three STAT proteins are located in the nucleus and are phosphorylated on serine residues. KIT-Asp816 mutant can directly phosphorylate STATs on the activation-specific tyrosine residues in vitro. However, within cells, SRC family kinases and JAKs diversely contribute to tyrosine phosphorylation of STAT proteins downstream of the KIT mutant. Using a panel of inhibitors, we provide evidence for the implication or exclusion of serine/threonine kinases as responsible for serine phosphorylation of STAT1, -3, and -5 in the two cell lines. Finally, we show that only STAT5 is transcriptionally active in these cells. This suggests that the contribution of STAT1 and STAT3 downstream of KIT mutant is independent of their transcription factor function.

The E3 ubiquitin ligase HOIL-1 induces the polyubiquitination and degradation of SOCS6 associated proteins.

The suppressor of cytokine signaling (SOCS) proteins are thought to exert their function through the recruitment of interacting‐proteins to the ubiquitin/proteasome degradation pathway. All SOCS proteins bind an Elongin BC E3 ubiquitin ligase complex through the common Socs‐box. Here, we show that haem‐oxidized IRP2 ubiquitin ligase‐1 (HOIL‐1), another E3 ubiquitin ligase, interacts with SOCS6. The Ubl domain of HOIL‐1 and the SH2 and Socs‐box domains of SOCS6 are required for the interaction. HOIL‐1 expression stabilizes SOCS6 and induces the ubiquitination and degradation of proteins associated with SOCS6. These data suggest that SOCS proteins may interact with different E3 ubiquitin ligases in addition to a common Elongin BC E3 complex.

Effect of atorvastatin on adhesive phenotype of human endothelial cells activated by tumor necrosis factor alpha.

We studied the effect of atorvastatin on the adhesive phenotype of human endothelial cells (HUVEC) stimulated by tumor necrosis factor (TNF)-&agr;. Surface expression of adhesion molecules on HUVEC was examined by flow cytometry and confocal microscopy, and adhesion of monocytes (human THP-1 cell line) was measured in vitro under flow conditions. In TNF-&agr;–activated HUVEC, atorvastatin significantly enhanced surface expression of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, E-selectin, and fractalkine, when compared with TNF-&agr; stimulation alone. This enhancement was reversed by mevalonate or geranylgeranyl pyrophosphate (GGPP) and was mimicked by an inhibitor of geranylgeranylation. The enhancing effect of atorvastatin was restricted to TNF-&agr;-inducible adhesion molecule and was the reflect of an increased protein synthesis (mRNA and protein) and not of a reduced shedding. Confocal microscopy examination showed that atorvastatin also altered the surface distribution of adhesion molecules. Adhesion of human THP-1 cells on TNF-&agr;–activated HUVEC was significantly reduced by atorvastatin (−42% at 1 &mgr;M). Mevalonate or GGPP restored the TNF-&agr;-induced adhesive potential. These results show that atorvastatin, by inhibiting prenylation of G proteins, enhances the TNF-&agr;-induced expression of adhesion molecules at the endothelial cell surface and also alters their surface distribution which may account for the reduced binding of monocytes.

FES kinases are required for oncogenic FLT3 signaling

The closely related non-receptor tyrosine kinases FEline Sarcoma (FES) and FEs Related (FER) are activated by cell surface receptors in hematopoietic cells. Despite the early description of oncogenic viral forms of fes, v-fes, and v-fps, the implication of FES and FER in human pathology is not known. We have recently shown that FES but not FER is necessary for oncogenic KIT receptor signaling. Here, we report that both FES and FER kinases are activated in primary acute myeloid leukemia (AML) blasts and in AML cell lines. FES and FER activation is dependent on FLT3 in cell lines harboring constitutively active FLT3 mutants. Moreover, both FES and FER proteins are critical for FLT3-internal tandem duplication (ITD) signaling and for cell proliferation in relevant AML cell lines. FER is required for cell cycle transitions, whereas FES seems necessary for cell survival. We concluded that FES and FER kinases mediate essential non-redundant functions downstream of FLT3-ITD.

NACA is a positive regulator of human erythroid-cell differentiation

We have previously identified the transcript encoding NACA (the α chain of the nascent-polypeptide-associated complex) as a cytokine-modulated specific transcript in the human TF-1 erythroleukemic cell line. This protein was already known to be a transcriptional co-activator that acts by potentiating AP-1 activity in osteoblasts, and is known to be involved in the targeting of nascent polypeptides. In this study, we investigate the role of NACA in human hematopoiesis. Protein distribution analyses indicate that NACA is expressed in undifferentiated TF-1 cells and in human-cord-blood-derived CD34+ progenitor cells. Its expression is maintained during in vitro erythroid differentiation but, in marked contrast, its expression is suppressed during their megakaryocytic or granulocytic differentiation. Ectopic expression of NACA in CD34+ cells under culture conditions that induce erythroid-lineage differentiation leads to a marked acceleration of erythroid-cell differentiation. Moreover, ectopic expression of NACA induces erythropoietin-independent differentiation of TF-1 cells, whereas downregulation of NACA by RNA interference abolishes the induction of hemoglobin production in these cells and diminishes glycophorin-A (GPA) expression by CD34+ progenitors cultured under erythroid differentiation conditions. Altogether, these results characterize NACA as a new factor involved in the positive regulation of human erythroid-cell differentiation.

Effect of atorvastatin on plasminogen activator inhibitor type-1 synthesis in human monocytes/macrophages.

&NA; The fibrinolytic inhibitor plasminogen activator inhibitor type 1 (PAI‐1) plays a role in the development of atherothrombosis and is produced by macrophages that infiltrate the atherosclerotic vessel wall. Because statins are effective in reducing atherosclerosis, we investigated if they modulate the synthesis of PAI‐1 in human monocytes/macrophages. To this end, we studied the effect of atorvastatin in different models of monocyte/macrophage differentiation, such as differentiated human promyelocytic cell line HL‐60 and human peripheral blood monocyte‐derived macrophages. HL‐60 cells were differentiated along monocyte lineage by phorbol myristate acetate (PMA) or a mixture of transforming growth factor‐&bgr; type 1 (TGF‐&bgr;1)/1&agr;,25‐dihydroxyvitamin D3 (D3). In these conditions, PAI‐1 synthesis was strongly induced and atorvastatin upregulated this synthesis, especially during TGF‐&bgr;1/D3‐induced differentiation. Recombinant human tumor necrosis factor‐&agr;(TNF‐&agr;) strongly upregulated PAI‐1 synthesis in PMA‐ or TGF‐&bgr;1/D3‐differentiated cells, and the potentiating effect of atorvastatin was of the same order as in the absence of TNF‐&agr;. Mevalonate reversed the enhancing effect of atorvastatin. In mature human monocyte‐derived macrophages, atorvastatin, alone or in combination with TNF‐&agr;, TGF‐&bgr;1, or PMA, did not exert any significant effect on PAI‐1 synthesis. Basal production of urokinase (uPA), which was below detection limits in HL‐60 cells and very low in human monocyte‐derived macrophages, was not altered by atorvastatin. These results show that atorvastatin upregulates PAI‐1 synthesis during the early stages of monocyte/macrophage differentiation, but has no effect on PAI‐1 and uPA synthesis in mature human monocyte‐derived macrophages. Atorvastatin did not significantly interact with the upregulating action of TNF‐&agr; on PAI‐1 synthesis during differentiation.

KIT-D816V oncogenic activity is controlled by the juxtamembrane docking site Y568-Y570

Mutation of KIT receptor tyrosine kinase at residue D816 results in ligand-independent constitutive kinase activity. This mutation occurs in most patients with mastocytosis, a myeloproliferative neoplasm, and is detected at lower frequencies in acute myeloid leukemia and in germ cell tumors. Other KIT mutations occur in gastrointestinal stromal tumors (GIST) and mucosal melanoma. KIT is considered as a bona fide therapeutic target as c-kit mutations are driving oncogenes in these pathologies. However, several evidences suggest that KIT-D816V mutant is not as aggressive as other KIT mutants. Here, we show that an intracellular docking site in the juxtamembrane region of KIT maintains a negative regulation on KIT-D816V transforming potential. Sixteen signaling proteins were shown to interact with this motif. We further demonstrate that mutation of this site results in signaling modifications, altered gene expression profile and increased transforming activity of KIT-D816V mutant. This result was unexpected as mutations of the homologous sites on wild-type (WT) KIT, or on the related oncogenic FLT3-ITD receptor, impair their function. Our results support the hypothesis that, KIT-D816V mutation is a mild oncogenic event that is sufficient to confer partial transforming properties, but requires additional mutations to acquire its full transforming potential.

Modulation of PAI‐1 and proMMP‐9 syntheses by soluble TNFα and its receptors during differentiation of the human monocytic HL‐60 cell line

During phorbol ester‐induced differentiation of HL‐60 monocytic cells, tumor necrosis factorα (TNFα) synthesis and secretion are increased, which contributes to the autocrine regulation of TNFα‐responsive genes. We investigated how, during phorbol ester‐induced differentiation of HL‐60 cells, the secreted TNFα modulated plasminogen activator inhibitor type I (PAI‐1) and gelatinase B (MMP‐9) syntheses, two proteins involved in pericellular proteolysis. The differentiation‐induced release of TNFα, was abolished by the hydroxamate‐based matrix metalloproteinase (MMP) inhibitor, RU36156. RU36156 or a neutralizing anti‐TNFα significantly down‐regulated PAI‐1 synthesis exclusively during the early phases of differentiation (from promyelocyte to monocytic‐like cells), which underlined the activating role of autocrine TNFα during this time range. As cells progressed to monocyte/macrophage phenotype, they still released TNFα, but RU36156 or anti‐TNFα no longer had an effect on PAI‐1 synthesis. This lack of effect was not due to a default of TNFα signaling since PAI‐1 synthesis was still stimulated in response to exogenous TNFα. TNFα receptor RI was also actively released and was shown to reduce TNFα activity which may account for the inability of soluble TNFα to up‐regulate PAI‐1 synthesis. In later mature stage, cells became susceptible to exogenous TNFα‐induced apoptosis and rapidly lost their ability to respond to TNFα. The MMP‐9 synthesis followed similar regulation as PAI‐1. Isolated human blood monocytes‐derived macrophages behave like HL‐60‐derived macrophages. In conclusion, these results show that during leukocyte differentiation, time windows exist during which the autocrine TNFα is active and then down‐regulated by RI, which may temper a continuous up‐regulation of the synthesis of proteins involved in pericellular proteolysis. J. Cell. Physiol. 196: 346–353, 2003.

Comparative oncogenomics identifies tyrosine kinase FES as a tumor suppressor in melanoma

Identification and functional validation of oncogenic drivers are essential steps toward advancing cancer precision medicine. Here, we have presented a comprehensive analysis of the somatic genomic landscape of the widely used BRAFV600E- and NRASQ61K-driven mouse models of melanoma. By integrating the data with publically available genomic, epigenomic, and transcriptomic information from human clinical samples, we confirmed the importance of several genes and pathways previously implicated in human melanoma, including the tumor-suppressor genes phosphatase and tensin homolog (PTEN), cyclin dependent kinase inhibitor 2A (CDKN2A), LKB1, and others. Importantly, this approach also identified additional putative melanoma drivers with prognostic and therapeutic relevance. Surprisingly, one of these genes encodes the tyrosine kinase FES. Whereas FES is highly expressed in normal human melanocytes, FES expression is strongly decreased in over 30% of human melanomas. This downregulation correlates with poor overall survival. Correspondingly, engineered deletion of Fes accelerated tumor progression in a BRAFV600E-driven mouse model of melanoma. Together, these data implicate FES as a driver of melanoma progression and demonstrate the potential of cross-species oncogenomic approaches combined with mouse modeling to uncover impactful mutations and oncogenic driver alleles with clinical importance in the treatment of human cancer.