Proteomic analysis of tyrosine phosphorylation during human liver transplantation

BACKGROUND: Ischemia-reperfusion (I/R) causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. RESULTS: Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH(2)/SH(3 )adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. CONCLUSION: Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes

Graft calcifications and dysfunction following liver transplantation

BACKGROUND: The molecular events, following ischemia and reperfusion (I/R) of the liver during transplantation are largely unknown. There is evidence that apoptotic and necrotic events may take place, and occasionally result in primary graft dysfunction. We herein report two cases, where significant I/R injury correlated with the development of liver calcification and primary liver dysfunction. CASE PRESENTATION: Both patients with clinical and biochemical evidence of primary graft dysfunction demonstrated calcification at light and electron microscopy levels. In addition, one patient had macroscopic evidence of calcification on cross-sectional imaging. Both patients died secondary to the sequelae of the graft dysfunction. CONCLUSIONS: Severe I/R-induced injury to the liver, clinically leads to graft dysfunction. This is due to advanced apoptotic and/or necrotic events at the hepatocyte level that may, on the most severe form, lead to calcification. The study of microcalcification at the early posttransplant period could provide insight in the events taking place following significant ischemia/reperfusion-induced injury to the graft

Identification of a novel BET bromodomain inhibitor-sensitive, gene regulatory circuit that controls Rituximab response and tumour growth in aggressive lymphoid cancers.: CYCLON-induced Rituximab resistance

International audienceImmuno-chemotherapy elicit high response rates in B-cell non-Hodgkin lymphoma but heterogeneity in response duration is observed, with some patients achieving cure and others showing refractory disease or relapse. Using a transcriptome-powered targeted proteomics screen, we discovered a gene regulatory circuit involving the nuclear factor CYCLON which characterizes aggressive disease and resistance to the anti-CD20 monoclonal antibody, Rituximab, in high-risk B-cell lymphoma. CYCLON knockdown was found to inhibit the aggressivity of MYC-overexpressing tumours in mice and to modulate gene expression programs of biological relevance to lymphoma. Furthermore, CYCLON knockdown increased the sensitivity of human lymphoma B cells to Rituximab in vitro and in vivo. Strikingly, this effect could be mimicked by in vitro treatment of lymphoma B cells with a small molecule inhibitor for BET bromodomain proteins (JQ1). In summary, this work has identified CYCLON as a new MYC cooperating factor that autonomously drives aggressive tumour growth and Rituximab resistance in lymphoma. This resistance mechanism is amenable to next-generation epigenetic therapy by BET bromodomain inhibition, thereby providing a new combination therapy rationale for high-risk lymphoma

Long-term analysis of the RiBVD phase II trial reveals the unfavorable impact of <i>TP53</i> mutations and hypoalbuminemia in older adults with mantle cell lymphoma; for the LYSA group

Between 2011 and 2012, a phase II trial evaluated the use of the RiBVD (rituximab, bendamustine, velcade and dexamethasone) combination as first-line treatment for mantle cell lymphoma (MCL) patients over the age of 65. We have now re-examined the classic prognostic factors, adding an assessment of TP53 mutation status. Patients (N=74; median age 73 years) were treated with the RiBVD combination. Median progression-free survival (mPFS) was 79 months and median overall survival (mOS) was 111 months. TP53 mutation status was available for 54/74 (73%) patients. TP53 mutations (TP53mt) were found in 12 patients (22.2%). In multivariate analysis, among the prognostic factors (PF) evaluated, only TP53mt and an albumin level (Alb) 3.6 g/dL were independently associated with a shorter mPFS. A hazard ratio (HR) of 3.16 (1.3-9.9, P=0.014) was obtained for TP53mt versus TP53 wild-type (wt), and 3.6 (1.39-9.5, P=0.009) for Alb <3.6 g/dL versus Alb ≥3.6 g/dL. In terms of mOS, multivariate analysis identified three PF: TP53mt (HR: 5.9 [1.77-19.5, P=0.004]), Alb <3.6 g/dL (HR: 5.2 [1.46- 18.5, P=0.011]), and ECOG=2 (HR: 3.7 [1.31-10.6, P=0.014]). Finally, a score combining TP53 status and Alb distinguished three populations based on the presence of 0, 1, or 2 PF. For these populations, mPFS was 7.8 years, 28 months, and 2.5 months, respectively. Our prolonged follow-up confirmed the efficacy of the RiBVD regimen, comparing it favorably to other regimens. TP53mt and hypoalbuminemia emerge as strong PF that can be easily integrated into prognostic scores for older adult patients with MCL

Proteomic characterization of stress responses activated upon ischemia and reperfusion during human liver transplantation

During liver transplantation, donor organs experience some degree of cold ischemia/reperfusion injury (IRI). The magnitude of this preservation injury is critical for transplantation outcome since it influences the allograft early function. However, the multifactorial and complex cellular response initiated upon IRI remains unclear. Herein, we used liver biopsies collected during the early phases of organ procurement and transplantation to characterize the global patterns of protein expression and phosphorylation in order to identify new regulatory mechanisms involved in ischemia-induced graft damage.First, a targeted functional proteomic approach, which combined protein expression profiling and mass spectrometry phosphoproteins analysis, allowed us to identify IQGAP1, a Cdc42/Rac1 effector, as a potential regulator of IRI-induced actin cytoskeleton remodeling and the related bile canaliculi (BC) loss of integrity. We demonstrated that IQGAP1 expression and localization are affected upon IRI and related to actin reorganization. Furthermore, using an IRI model in hepatoma cells, we showed that IQGAP1 silencing decreases the basal level of actin polymerisation at BC periphery, reflecting a defect in BC structure, coincident with a reduced cellular resistance to IRI.Our proteomic data also led us to postulate that IRI-induced cellular damages may cause alterations of the secretory pathway, suggesting the activation of endoplasmic reticulum (ER) specific stress responses. Using semi quantitative RT-PCR and immunoblot with phospho-specific antibodies, we showed that the IRE-1 pathway, leading to both adaptive and pro-apoptotic responses, is first activated upon early ischemia and, in a second phase, upon early reperfusion. In contrast, the PERK pathway, leading to inhibition of capdependent translation, is mainly activated upon reperfusion, restrictively in sinusoidal endothelial cells, and could contribute to the exagerated sensitivity of this liver cell type to IRI.In summary, our work allowed to gain new mechanistic insights in the global regulation of the cellular response to IRI, and also led to the identification of new molecular mechanisms specifically involved in mediating liver resistance to IRI. First, IQGAP1, as a regulator of BC structure could be participate in maintaining a proper bile secretion, essential for graft post-transplant recovery. Then, the balance between pro-adaptive and pro-apoptotic responses triggered in the ER might, as well, influence liver secretory functions and, as a consequence, condition liver transplantation outcomes

La protéomique quantitative par la méthode SILAC

Les approches de protéomique quantitative basées sur la spectrométrie de masse sont parfaitement adaptées à la détection de changements au niveau protéique entre échantillons biologiques. Parmi ces techniques, la méthode SILAC (stable isotope labelling by amino acids in cell culture) a démontré un réel potentiel. Cette méthode s’avère en effet être précise et relativement simple à mettre en oeuvre pour quantifier les protéines extraites de cellules en culture. Le principe est le suivant : les cellules sont cultivées dans un milieu contenant soit des acides aminés naturels (synthèse de protéines « légères »), soit leurs équivalents isotopiquement marqués (synthèse de protéines « lourdes »). Les populations cellulaires à comparer sont mélangées et traitées comme un seul échantillon, ce qui permet de préparer les protéines d’intérêt sans risquer d’introduire des erreurs de quantification. Au cours de l’analyse par spectrométrie de masse, l’abondance relative entre les échantillons biologiques peut être calculée pour chaque protéine en comparant l’intensité des peptides légers et lourds. Comme le montrent ses applications à diverses problématiques biologiques et cliniques, la méthode SILAC est particulièrement prometteuse pour l’élucidation des mécanismes moléculaires impliqués dans les grandes fonctions cellulaires, ainsi que pour l’identification de biomarqueurs de maladies. La limitation de la méthode SILAC à la quantification de protéines issues de cellules en culture vient d’être levée suite à la description d’une souris SILAC dont toutes les protéines sont marquées isotopiquement. Ces travaux laissent présager une extension de cette stratégie analytique à l’étude différentielle de tissus et de fluides biologiques d’animaux modèles

BET Family Protein BRD4: An Emerging Actor in NFκB Signaling in Inflammation and Cancer

NFκB (Nuclear Factor-κ-light-chain-enhancer of activated B cells) signaling elicits global transcriptional changes by activating cognate promoters and through genome-wide remodeling of cognate regulatory elements called “super enhancers”. BET (Bromodomain and Extra-Terminal domain) protein family inhibitor studies have implicated BET protein member BRD4 and possibly other BET proteins in NFκB-dependent promoter and super-enhancer modulation. Members of the BET protein family are known to bind acetylated chromatin to facilitate access by transcriptional regulators to chromatin, as well as to assist the activity of transcription elongation complexes via CDK9/pTEFb. BET family member BRD4 has been shown to bind non-histone proteins and modulate their activity. One such protein is RELA, the NFκB co-activator. Specifically, BRD4 binds acetylated RELA, which increases its transcriptional transactivation activity and stability in the nucleus. In aggregate, this establishes an intimate link between NFκB and BET signaling, at least via BRD4. The present review provides a brief overview of the structure and function of BET family proteins and then examines the connections between NFκB and BRD4 signaling, using the inflammatory response and cancer cell signaling as study models. We also discuss the potential of BET inhibitors for relief of aberrant NFκB signaling in cancer, focusing on non-histone, acetyl-lysine binding functions

Nck-dependent Activation of Extracellular Signal-regulated Kinase-1 and Regulation of Cell Survival during Endoplasmic Reticulum Stress

In response to stress, the endoplasmic reticulum (ER) signaling machinery triggers the inhibition of protein synthesis and up-regulation of genes whose products are involved in protein folding, cell cycle exit, and/or apoptosis. We demonstrate that the misfolding agents azetidine-2-carboxylic acid (Azc) and tunicamycin initiate signaling from the ER, resulting in the activation of Jun-N-terminal kinase, p44(MAPK)/extracellular signal-regulated kinase-1 (ERK-1), and p38(MAPK) through IRE1α-dependent mechanisms. To characterize the ER proximal signaling events involved, immuno-isolated ER membranes from rat fibroblasts treated with ER stress inducers were used to reconstitute the activation of the stress-activated protein kinase/mitogen-activate protein kinase (MAPK) pathways in vitro. This allowed us to demonstrate a role for the SH2/SH3 domain containing adaptor Nck in ERK-1 activation after Azc treatment. We also show both in vitro and in vivo that under basal conditions ER-associated Nck represses ERK-1 activation and that upon ER stress this pool of Nck dissociates from the ER membrane to allow ERK-1 activation. Moreover, under the same conditions, Nck-null cells elicit a stronger ERK-1 activation in response to Azc stress, thus, correlating with an enhanced survival phenotype. These data delineate a novel mechanism for the regulation of ER stress signaling to the MAPK pathway and demonstrate a critical role for Nck in ER stress and cell survival

The sVEGFR1-i13 splice variant regulates a β1 integrin/VEGFR autocrine loop involved in the progression and the response to anti-angiogenic therapies of squamous cell lung carcinoma

International audienceBACKGROUND:While lung adenocarcinoma patients can somewhat benefit from anti-angiogenic therapies, patients with squamous cell lung carcinoma (SQLC) cannot. The reasons for this discrepancy remain largely unknown. Soluble VEGF receptor-1, namely sVEGFR1-i13, is a truncated splice variant of the cell membrane-spanning VEGFR1 that has no transmembrane or tyrosine kinase domain. sVEGFR1-i13 is mainly viewed as an anti-angiogenic factor which counteracts VEGF-A/VEGFR signalling in endothelial cells. However, its role in tumour cells is poorly known.METHODS:mRNA and protein status were analysed by Real-Time qPCR, western blotting, ELISA assay, proximity ligation assay or immunohistochemistry in human tumour cell lines, murine tumourgrafts and non small cell lung carcinoma patients samples.RESULTS:We show that anti-angiogenic therapies specifically increase the levels of sVEGFR1-i13 in SQLC cell lines and chemically induced SQLC murine tumourgrafts. At the molecular level, we characterise a sVEGFR1-i13/β1 integrin/VEGFR autocrine loop which determines whether SQLC cells proliferate or go into apoptosis, in response to anti-angiogenic therapies. Furthermore, we show that high levels of both sVEGFR1-i13 and β1 integrin mRNAs and proteins are associated with advanced stages in SQLC patients and with a poor clinical outcome in patients with early stage SQLC.CONCLUSIONS:Overall, these results reveal an unexpected pro-tumoural function of sVEGFR1-i13 in SQLC tumour cells, which contributes to their progression and escape from anti-angiogenic therapies. These data might help to understand why some SQLC patients do not respond to anti-angiogenic therapies