Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression, an effect amplified by interleukin-1ß.

The Notch1 and Notch4 signaling pathways regulate endothelial cell homeostasis. Inflammatory cytokines induce the expression of endothelial adhesion molecules, including VCAM1, partly by downregulating Notch4 signaling. We investigated the role of endothelial Notch1 in this IL-1ß-mediated process. Brief treatment with IL-1ß upregulated endothelial VCAM1 and Notch ligand Jagged1. IL-1ß decreased Notch1 mRNA levels, but levels of the active Notch1ICD protein remained constant. IL-1ß-mediated VCAM1 induction was downregulated in endothelial cells subjected to pretreatment with a pharmacological inhibitor of the γ-secretase, which activates Notch receptors, producing NotchICD. It was also downregulated in cells in which Notch1 and/or Jagged1 were silenced.Conversely, the forced expression of Notch1ICD in naïve endothelial cells upregulated VCAM1 per se and amplified IL-1ß-mediated VCAM1 induction. Jagged1 levels increased and Notch4 signaling was downregulated in parallel. Finally, Notch1ICD and Jagged1 expression was upregulated in the endothelium of the liver in a model of chronic liver inflammation.In conclusion, we describe here a cell-autonomous, pro-inflammatory endothelial Notch1-Jagged1 circuit (i) triggering the expression of VCAM1 even in the absence of inflammatory cytokines and (ii) enhancing the effects of IL-1ß. Thus, IL-1ß regulates Notch1 and Notch4 activity in opposite directions, consistent with a selective targeting of Notch1 in inflamed endothelium.

Hyper-activation of Notch3 amplifies the proliferative potential of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.

Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS.

BACKGROUND: Embryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts. METHODS: Embryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo. RESULTS: Here we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo. CONCLUSIONS: These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.

Src kinase activity coordinates cell adhesion and spreading with activation of mammalian target of rapamycin in pancreatic endocrine tumour cells.

Pancreatic endocrine tumours (PETs) are rare and heterogeneous neoplasms, often diagnosed at metastatic stage, for which no cure is currently available. Recently, activation of two pathways that support proliferation and invasiveness of cancer cells, the Src family kinase (SFK) and mammalian target of rapamycin (mTOR) pathways, was demonstrated in PETs. Since both pathways represent suitable targets for therapeutic intervention, we investigated their possible interaction in PETs. Western blot and immunofluorescence analyses indicated that SFK and mTOR activity correlate in PET cell lines. We also found that SFKs coordinate cell adhesion and spreading with activation of the mTOR pathway in PET cells. Live cell metabolic labelling and biochemical studies demonstrated that SFK activity enhance mTOR-dependent translation initiation. Furthermore, microarray analysis of the mRNAs associated with polyribosomes revealed that SFKs regulate mTOR-dependent translation of specific transcripts, with an enrichment in mRNAs encoding cell cycle proteins. Importantly, a synergic inhibition of proliferation was observed in PET cells concomitantly treated with SFK and mTOR inhibitors, without activation of the phosphatidylinositol 3-kinase/AKT pro-survival pathway. Tissue microarray analysis revealed activation of Src and mTOR in some PET samples, and identified phosphorylation of 4E-BP1 as an independent marker of poor prognosis in PETs. Thus, our work highlights a novel link between the SFK and mTOR pathways, which regulate the translation of mRNAs for cell cycle regulators, and suggest that crosstalk between these pathways promotes PET cell proliferation.

Differential contribution of the MTOR and MNK pathways to the regulation of mRNA translation in meiotic and postmeiotic mouse male germ cells.

Translation of stored mRNAs accounts for protein synthesis during the transcriptionally inactive stages of spermatogenesis. A key step in mRNA translation is the assembly of the initiation complex EIF4F, which is regulated by the MTOR (mammalian target of rapamycin) and MNK1/2 (MAP kinase-interacting kinase 1 and 2) pathways. We investigated the expression and activity of regulatory proteins of these pathways in male germ cells at different stages of differentiation. All translation factors analyzed were expressed in germ cells throughout spermatogenesis. However, while EIF4G and PABP1 (poly[A]-binding protein 1) were more abundant in postmeiotic cells, MTOR and its target EIF4EBP1 (4E-BP1) decreased steadily during spermatogenesis. In vivo labeling showed that pachytene spermatocytes display higher rates of protein synthesis, which are partially dependent on MTOR and MNK activity. By contrast, haploid spermatids are characterized by lower levels of protein synthesis, which are independent of the activity of these pathways. Accordingly, MTOR and MNK activity enhanced formation of the EIF4F complex in pachytene spermatocytes but not in round spermatids. Moreover, external cues differentially modulated the activity of these pathways in meiotic and haploid cells. Heat shock decreased MTOR and MNK activity in pachytene spermatocytes, whereas round spermatids were much less sensitive. On the other hand, treatment with the phosphatase inhibitor okadaic acid activated MTOR and MNK in both cell types. These results indicate that translational regulation is differentially dependent on the MTOR and MNK pathways in mouse spermatocytes and spermatids and suggest that the late stages of germ cell differentiation display constitutive assembly of the translation initiation complex.