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Phosphorylation of two tyrosines in the death domain of CD95 is a critical mechanism in determining the receptor's choices between cell death and survival signals. Recently, site-specific monoclonal antibodies against phosphorylated tyrosines of CD95 have been generated and used to successfully detect each phosphorylated death domain tyrosine of CD95 directly and separately by immunoblotting. Here we provide detailed protocols and useful tips for a successful site-specific detection of phosphorylated death domain tyrosine of CD95 following a protein separation by sizes (conventional SDS-PAGE) and by degrees of phosphorylation (phospho-protein affinity, mobility shift SDS-PAGE).

International audience; Phosphorylation of two tyrosines in the death domain of CD95 is a critical mechanism in determining the receptor's choices between cell death and survival signals. Recently, site-specific monoclonal antibodies against phosphorylated tyrosines of CD95 have been generated and used to successfully detect each phosphorylated death domain tyrosine of CD95 directly and separately by immunoblotting. Here we provide detailed protocols and useful tips for a successful site-specific detection of phosphorylated death domain tyrosine of CD95 following a protein separation by sizes (conventional SDS-PAGE) and by degrees of phosphorylation (phospho-protein affinity, mobility shift SDS-PAGE).

International audience; This study describes the molecular signaling involved in the different cell death modes of triple-negative breast cancer cells induced by hexadecylphosphocholine (HePC/miltefosine), a clinically relevant anticancer alkylphosphocholine. We found that the HePC treatment triggers cell-type-dependent apoptotic and non-apoptotic cell death processes. Moreover, the expression level of the apoptosis activator Fas, and Fas/Fas ligand signaling capacity are not attributing factors for the preference toward apoptosis. Using Fas siRNA and overexpression approaches we establish that Fas is not a pro-apoptotic factor but a contributor to cell protection in HePC-apoptosis-sensitive cells. The insight in the multi-modal anticancer capability of HePC in triple-negative breast cancer cells may facilitate the targeted design of therapeutic strategies against triple-negative breast cancers.

Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive “Fas apoptosis” to “Fas multisignals” paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases., Signalling by the tumor necrosis factor receptor (TNFR) superfamily member Fas can promote either survival or death of a cell, but the mechanism underlying this choice is unclear. This study reveals that the outcome of Fas signalling (death versus survival) is determined by the tyrosine phosphorylation status of its death domain., Author Summary The versatility of the tumor necrosis factor receptor superfamily members in cell fate regulation is well illustrated by the dual signaling generated by one of the most extensively studied members of the family, Fas (CD95/TNFSFR6). Upon binding its ligand, Fas is able to elicit both pro-death and pro-survival signals. Until now, we have lacked mechanistic knowledge about when and how one signaling output of Fas is favored over the other. We demonstrate here that the outcome of Fas signaling is determined by the phosphorylation status of two tyrosine residues (Y232 and Y291) within the death domain. Dephosphorylation of Fas tyrosines by SHP-1 tyrosine phosphatase turns on the pro-apoptotic signal whereas the tyrosine phosphorylation by Src family kinases (SFKs) turns off the pro-apoptotic signal and turns on the pro-survival signal. Furthermore, we provide evidence that Fas tyrosine phosphorylation status may vary among different cancer types and influence the response to anti-cancer treatments. This information reveals an opportunity to use the screening of Fas tyrosine phosphorylation, a newly discovered direct molecular indicator of Fas functional output, to aid the design of Fas-related cancer therapies.

Fas ligand (FasL) is a type II transmembrane protein belonging to the tumor necrosis factor family. Its binding to the cognate Fas receptor triggers the apoptosis that plays a pivotal role in the maintenance of immune system homeostasis. The cell death-inducing property of FasL has been associated with its extracellular domain, which can be cleaved off by metalloprotease activity to produce soluble FasL. The fate of the remaining membrane-anchored N-terminal part of the FasL molecule has not been determined. Here we show that post-translational processing of overexpressed and endogenous FasL in T-cells by the disintegrin and metalloprotease ADAM10 generates a 17-kDa N-terminal fragment, which lacks the receptor-binding extracellular domain. This FasL remnant is membrane anchored and further processed by SPPL2a, a member of the signal peptide peptidase-like family of intramembrane-cleaving proteases. SPPL2a cleavage liberates a smaller and highly unstable fragment mainly containing the intracellular FasL domain (FasL ICD). We show that this fragment translocates to the nucleus and is capable of inhibiting gene transcription. With ADAM10 and SPPL2a we have identified two proteases implicated in FasL processing and release of the FasL ICD, which has been shown to be important for retrograde FasL signaling.

Localization of the death receptor Fas to specialized membrane microdomains is crucial to Fas-mediated cell death signaling. Here, we report that the post-translational modification of Fas by palmitoylation at the membrane proximal cysteine residue in the cytoplasmic region is the targeting signal for Fas localization to lipid rafts, as demonstrated in both cell-free and living cell systems. Palmitoylation is required for the redistribution of Fas to actin cytoskeleton-linked rafts upon Fas stimulation and for the raft-dependent, ezrin-mediated cytoskeleton association, which is necessary for the efficient Fas receptor internalization, death-inducing signaling complex assembly and subsequent caspase cascade leading to cell death.

International audience; In chronic myeloid leukemia (CML), evidence is supporting the role of VEGF in growth, and survival of leukemia cells. The evaluation of plasma VEGF levels in 403 CML patients randomized within SPIRIT study to received imatinib-400mg versus imatinib+cytarabine versus imatinib+interferon (IFN) versus imatinib-600mg demonstrated that VEGF is an independent factor of BCR-ABL burden. VEGF low levels at diagnosis were associated with a progression-free survival of 100% at 48 months. Under treatment, significant lowest levels were observed in imatinib+IFN arm. These results support the use of VEGF as a parameter to predict CML evolution and let us to speculate about antiangiogenic properties of IFN.

Selective compartmentalization and internalization have been shown as a means for regulating specific signals of cell surface receptors to correspond to cellular requirements and conditions. Here, we present a conserved extracellular glycosphingolipid-binding motif of Fas as one of the regulatory elements in the selection of its internalization route and consequently the signals transmitted upon ligand binding. This motif is required for clathrin-mediated internalization of Fas, which allows the transduction of its cell death signal. The loss of function of the motif drives the activated receptor to an alternative internalization route that is independent of clathrin and cholesterol-dependent rafts but dependent on ezrin, and thereby extinguishing its cell death signal while promoting its non-death functions. Through biochemical, biophysical, and genetic approaches, we present a protein/lipid-based mechanism as a key to the versatility of the signal transduction by the multifunctional Fas receptor-ligand system.Cell Death and Differentiation advance online publication, 1 August 2008; doi:10.1038/cdd.2008.115.

Increased angiogenesis in bone marrow (BM) is one of the characteristics of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder which expresses a chimeric Bcr/Abl protein. The therapeutic strategy in CML has been totally modified with the development of Imatinib Mesylate (STI571), a specific inhibitor of Bcr/Abl tyrosine kinase activity. Previously, we studied the effect of Imatinib on the Vascular Endothelial Growth Factor (VEGF), one of the most potent regulators of angiogenesis (1). Thus, we demonstrated, for the first time, the decrease in VEGF production in CML patients treated with Imatinib. It pointed out the potential prognostic value of determining the VEGF plasma level of patients in order to follow the evolution of this hematologic malignancy. Actually, we showed that low plasma VEGF levels could be one of the characteristics of complete cytogenetic remission. However, investigations with a larger number of patients were necessary to evaluate the potential of VEGF levels in the monitoring of CML patients treated with Imatinib. So, we analyzed VEGF plasma level in patients treated in the SPIRIT, the CML French Group phase III, multicenter, open-label, prospective trial. Patients are randomized between the 3 experimental arms [IM 400mg daily in combination with Peg-IFN-α2a (Peg-IFNα2a, 90 μg weekly) or with Ara-C (20 mg/m2/day, days 15–28 of 28-day cycles) or IM 600mg daily] and the reference arm, IM 400mg daily. The Endpoint are overall survival (primary), rate and duration of hematologic, cytogenetic and molecular responses and tolerability. This VEGF evaluation is based on a cohort of 110 pts [median age 51 yrs (19–81); Sokal distribution : 30% of pts low, 37% intermediate, and 33% high]. Males predominated (62%) and percentage of patients enrolled in IM 400mg, IM 600mg, IM 400 with Ara-C and IM 400mg with Peg-IFN arm is respectively 28%, 28%, 24% and 30%. At diagnosis, the median of VEGF plasma level is 945 pg/ml (range: 42.3–4001.5). After 3 months of treatment, the median of VEGF level decreases significantly (p To determine prognosis value of VEGF we correlated the results with the cytogenetic and molecular response.

Abstract 2770 Poster Board II-746 Background: AML and MDS are associated with increased in BM vascularity and increased levels of various angiogenic factors including VEGF, which is implicated in the proliferation and survival of leukemic cells and whose autocrine production may contributes to leukemia self-renewal and resistance to apoptotic and maturation stimuli (Kerbauy DB, 2007). Higher VEGF plasma levels have been observed in MDS with excess blasts (Brunner B, 2002). The humanized MoAb against VEGF, Bevacizumab (BEV) has proved an effective treatment targeting VEGF. Methods: The GFM conducted a multicentric phase II trial testing the efficacy and tolerance of BEV in MDS with excess of marrow blasts and the impact of BEV on angiogenesis (ClinicalTrials.gov Identifier: NCT00565656). BEV was administered at 5 mg/kg IV every 2 weeks for 12 weeks. Patients with no response could receive, in the absence of major toxicities, 4 additional cycles with dose escalation of BEV to 10 mg/kg every 2 weeks. Results: 20 patients (pts) were included of whom 17 were currently evaluable for efficacy: 14M/3F, median age 70 years (62–84), RAEB-1 (8), RAEB-2 (9), IPSS INT-1 (6), INT-2 (9), and high (2). No patient stopped treatment before week 12 because of side effects and no treatment related SAE was reported. Preliminary results on haematological adverse events have shown mild toxicity. Four pts with grade 0–2 neutropenia and 3 pts with grade 0–2 anemia have progressed in a grade 3–4 during treatment. One of 17 evaluable pts was responder (RBC transfusion independence in a pt requiring 2 RBC concentrates/month), during 2 months. Bone marrow microvessel density, VEGF plasma level, VEGF mRNA expression, HIF-1a expression were determined before the treatment to characterise angiogenesis in the pts included and after treatment to evaluate the impact of Bevacizumab on this angiogenesis. Median plasma VEGF was 56.6 pg/ml (range: 13.2–151.7 pg/ml) at inclusion. After 12 weeks of treatment VEGF plasma level was significantly decreased to 30.4 pg/ml (range: 13.9–53.1 pg/ml). This decrease was observed in all pts and has been confirmed for bone marrow VEGF mRNA in the first analysed patients (n=4) by RTQPCR. Further results of this study will be presented. Conclusion: In our study, BEV was well tolerated but had limited efficacy. On the other hand, in colorectal cancer, BEV alone has limited efficacy and mainly plays a therapeutic role by enhancing the efficacy of chemotherapy. The in vivo decrease of plasma VEGF with BEV in this study, and its limited toxicity, suggest that it could be associated in higher risk with agents having different mechanisms of action, especially hypomethylating agents, to produce synergistic effects while maintaining tolerable safety profiles. Brunner B et al. Blood levels of angiogenin and vascular endothelial growth factor are elevated in myelodysplastic syndromes and in acute myeloid leukemia. J Hematother Stem Cell Res. 2002 Feb;11(1):119–25 Dias S et al. Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration. J Clin Invest. 2000 Aug;106(4):511–21. Kerbauy DB, Deeg HJ. Apoptosis and antiapoptotic mechanisms in the progression of myelodysplastic syndrome. Exp Hematol. 2007 Nov;35(11):1739–46. Disclosures: No relevant conflicts of interest to declare.