Glioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study, we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, and HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signaling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration, which was accompanied by the sharp reduction in neural stem cell transcription factor expression (ASCL1, OLIG2, and SOX2), while HEY1/2, KLF9, and SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. In xenotransplantation experiments, contrasting with the infiltrative and poorly vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly disseminating but highly vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties. STEM CELLS 2015;33:21-34

Glioblastoma multiform (GBM) are devastating brain tumors containing a fraction of multipotent stem-like cells which are highly tumorigenic. These cells are resistant to treatments and are likely to be responsible for tumor recurrence. One approach to eliminate GBM stem-like cells would be to force their terminal differentiation. During development, neurons formation is controlled by neurogenic transcription factors such as Ngn1/2 and NeuroD1. We found that in comparison with oligodendrogenic genes, the expression of these neurogenic genes is low or absent in GBM tumors and derived cultures. We thus explored the effect of overexpressing these neurogenic genes in three CD133(+) Sox2(+) GBM stem-like cell cultures and the U87 glioma line. Introduction of Ngn2 in CD133(+) cultures induced massive cell death, proliferation arrest and a drastic reduction of neurosphere formation. Similar effects were observed with NeuroD1. Importantly, Ngn2 effects were accompanied by the downregulation of Olig2, Myc, Shh and upregulation of Dcx and NeuroD1 expression. The few surviving cells adopted a typical neuronal morphology and some of them generated action potentials. These cells appeared to be produced at the expense of GFAP(+) cells which were radically reduced after differentiation with Ngn2. In vivo, Ngn2-expressing cells were unable to form orthotopic tumors. In the U87 glioma line, Ngn2 could not induce neuronal differentiation although proliferation in vitro and tumoral growth in vivo were strongly reduced. By inducing cell death, cell cycle arrest or differentiation, this work supports further exploration of neurogenic proteins to oppose GBM stem-like and non-stem-like cell growth.

Simultaneous discovery of members of the annexin family of calcium and phospholipid binding proteins by several groups is intimately linked to the possibility that these proteins may be controlled by phosphorylation. Indeed, annexin I and annexin II have been identified as major substrates for the tyrosine kinase activity associated with epidermal growth factor receptor (EGF-R) and for the retrovirus encoded protein tyrosine kinase pp60v-arc. Both annexins are also in vitro and/or in situ substrates for platelet derived growth factor (PDGF), insulin and hepatocyte growth factor/scatter factor (HGF/SF) receptor tyrosine kinases. In addition, to serve as substrates for tyrosine protein kinases some annexins are cellular targets for serine threonine protein kinases such as protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA). Although the role of annexin phosphorylation has not been studied in detail, it is thought to influence their vesicle aggregation and phospholipid binding properties. Some annexins are also potent inhibitors of various serine/threonine and tyrosine kinases. The physiological functions of the annexins have still not been clearly defined. Therefore the identification of the ability of these proteins to undergo phosphorylation may be helpful in assigning them a precise biological role.

Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G 1 to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type 1A receptor (CHO-AT 1A ). We recently reported that in these cells, Ang II induced cyclin D1 promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/ extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Bé ré ziat, G., and Rothhut, B. (2000) J. Biol. Chem. 275, 26349 -26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) ؊136 and ؊96 and between bp ؊29 and ؉139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the ؊136 to ؊96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21 ras , Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the ؊29 to ؉139 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the ؊136 to ؊96 bp region of the cyclin D1 promoter and by activation of the ؊29 to ؉139 bp region, both in a p21 ras /Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.The control of mammalian cell proliferation by extracellular signals in G 1 to S phase progression of the cell cycle is largely mediated by serine/threonine cyclin-dependent kinases CDK4 1 and CDK6, which interact with specific D-type cyclins. The CDK-D-type cyclin complexes induce phosphorylation of the retinoblastoma protein (pRb), thereby releasing the transcription factor E2F, which is required for the transcription of S phase-specific genes (1-4). Activation by mitogenic stimuli of D-type cyclins during the G 1 phase appears to be an essential and rate-limiting step in G 1 to S phase progression of the cell cycle (5-7). The cyclin D1 gene expression seems to be essentially regulated at the transcription level. The promoter region of the cyclin D1 gene contains multiple potential cis-regulatory elements including binding sites for AP1, E2F, Oct, Egr-1...

Stimulus-response (S-R) coupling in platelets requires an intermediary other than an elevation in cytosolic free calcium ([Ca2+]i). While an increase in [Ca2+]i is essential in S-R coupling, effecting phosphorylation of myosin of relative molecular mass (Mr) 20,000 (20 K), platelet activation is also associated with phosphorylation of a 40K protein, which can occur in the absence of changes in [Ca2+]i. The 40K protein is the substrate for protein kinase C (PKC). Mounting evidence suggests that activation of PKC by diacylglycerol is the other signal involved in S-R coupling. Although phosphorylation of the 40K protein is associated with certain platelet functional responses, no precise role has been accredited to it. Recently, we and others have described several proteins (collectively known as lipocortin) which inhibit phospholipase A2 (PLA2). One of the most conspicuous proteins of this group is a 40K peptide whose inhibitory activity can be suppressed by prior phosphorylation. We hypothesized that the 40K protein described in platelets may possess anti-PLA2 activity and that phosphorylation by PKC, suppressing its inhibitory activity, may represent the mechanism underlying mobilization of arachidonic acid, the precursor of prostaglandins. The results of the present study strongly support this hypothesis.

Camptothecin and doxorubicin belong to a family of anticancer drugs that exert cytotoxic effects by triggering apoptosis in various cell types. However there have only been few investigations showing that matricellular proteins like thrombospondin-1 (TSP-1) could be involved in the underlying mechanism of this cytotoxicity. In this report, using Hoechst reagent staining, reactive oxygen species production and caspase-3 activity measurement, we determined that both camptothecin and doxorubicin induced apoptosis in human thyroid carcinoma cells (FTC-133). On the one hand, we demonstrated that camptothecin and doxorubicin inhibited TSP-1 expression mainly occurring at the transcriptional level. On the other hand, drug-induced apoptosis determined by western blot analysis for PARP cleavage and caspase-3 activity measurement, was significantly decreased in presence of exogenous TSP-1. In order to identify the sequence responsible for this effect, we used the CD47/IAP-binding peptide 4N1 (RFYVVMWK), derived from the C-terminal domain of TSP-1, and known to play a role in apoptosis. Thus, in presence of 4N1, camptothecin and doxorubicin-induced pro-apoptotic activity was considerably inhibited. These findings suggest that induction of apoptosis by camptothecin or doxorubicin in FTC-133 cells is greatly dependent by a down-regulation of TSP-1 expression and shed new light on a possible role for TSP-1 in drug resistance.

Elastin peptides (EPs) produced during cancer progression bind to the elastin binding protein (EBP) found at the surface of dermal fibroblasts, leading to the expression of collagenase-1 gene. The production of this enzyme involved in stromal reaction is caused by the sustained activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway via cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K). However, the mechanism of these signaling events remains unknown. We show that -elastin (E), a commonly used EP, induces maximum phosphorylation of mitogen-activated protein kinase/extracellular signalregulated kinase (MEK)1/2 and ERK1/2 after 30 min. The simultaneous inhibition of PKA and PI3K, by N-(2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide (H89) and 2-(4-morpholynil)-8-phenyl-4H-1-bemzopyran-4-one (LY294002), respectively, blocked MEK1/2 and ERK1/2 phosphorylation, as did lactose, an EBP antagonist. E induced Raf-1 phosphorylation and activation in a PI3K-dependent manner. In our system, the PI3K p110␥ is expressed and activated by ␤␥-derived subunits from a pertussis toxin-sensitive G protein after fibroblast stimulation. Pertussis toxin also blocks the Raf-1/MEK1/2/ERK1/2 phosphorylation cascade. In addition, we found that B-Raf is expressed in dermal fibroblasts and activated in a PKA-dependent manner after E treatment, thereby integrating PKA signals to MEK1/2. It is noteworthy that Ras involvement was excluded because ERK1/2 activation by E was not blocked in RasN17-transfected fibroblasts. Together, our results identify a novel Rasindependent ERK1/2 activation system in which p110␥/Raf-1/ MEK1/2 and PKA/B-Raf/MEK1/2 cooperate to activate ERK1/2. Thus, p110␥ and B-Raf seem to be important modulators of dermal fibroblasts physiology and should now qualify as therapeutic targets in strategies aiming at limiting elastin degradation contribution to cancer progression.

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