Search

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid malignancy affecting adults. The NF-κB transcription factor family is activated by 2 main pathways, the canonical and the alternative NF-κB activation pathway, with different functions. The alternative NF-κB pathway leads to activation of the transcriptionally active RelB NF-κB subunit. Alternative NF-κB activation status and its role in DLBCL pathogenesis remain undefined. Here, we reveal a frequent activation of RelB in a large cohort of DLBCL patients and cell lines, independently of their activated B-cell–like or germinal center B-cell–like subtype. RelB activity defines a new subset of patients with DLBCL and a peculiar gene expression profile and mutational pattern. Importantly, RelB activation does not correlate with the MCD genetic subtype, enriched for activated B-cell–like tumors carrying MYD88L265P and CD79B mutations that cooperatively activate canonical NF-κB, thus indicating that current genetic tools to evaluate NF-κB activity in DLBCL do not provide information on the alternative NF-κB activation. Furthermore, the newly defined RelB-positive subgroup of patients with DLBCL exhibits a dismal outcome after immunochemotherapy. Functional studies revealed that RelB confers DLBCL cell resistance to DNA damage–induced apoptosis in response to doxorubicin, a genotoxic agent used in the front-line treatment of DLBCL. We also show that RelB positivity is associated with high expression of cellular inhibitor of apoptosis protein 2 (cIAP2). Altogether, RelB activation can be used to refine the prognostic stratification of DLBCL and may contribute to subvert the therapeutic DNA damage response in a segment of patients with DLBCL.

Although mortality rates have declined in recent years, the majority of cancers remain incurable and the medical challenge is evident. Recent progress in cancer genetics and genomics along with the identification of a novel generation of cancer hallmarks, that is, reprogramming of energy metabolism and evasion from immune surveillance, have led to the discovery of novel NF-κB-dependent cancer vulnerabilities. These discoveries have led to better patient stratification, and new drug combinations using cutting-edge therapies. This review aims to give an up-to-date view on the therapeutic potential of NF-κB transcription factors and the signaling pathways that control their activity in the new era of cancer therapy.

NF-κB is a major transcription factor whose activation is triggered through two main activation pathways: the canonical pathway involving disruption of IκB-α/NF-κB complexes and the alternative pathway whose activation relies on the inducible proteolysis of the inhibitory protein p100. One central step controlling p100 processing consists in the interaction of the E3 ubiquitin ligase β-TrCP with p100, thereby leading to its ubiquitinylation and subsequent either complete degradation or partial proteolysis by the proteasome. However, the interaction mechanism between p100 and β-TrCP is still poorly defined. In this work, a diphosphorylated 21-mer p100 peptide model containing the phosphodegron motif was used to characterize the interaction with β-TrCP by NMR. In parallel, docking simulations were performed in order to obtain a model of the 21P-p100/β-TrCP complex. Saturation transfer difference (STD) experiments were performed in order to highlight the residues of p100 involved in the interaction with the β-TrCP protein. These results highlighted the importance of pSer

Objective The pathophysiology of giant cell arteritis (GCA) and the mechanisms underlying vascular remodeling, are poorly understood. We aimed to compare vascular smooth muscle cells (VSMCs) from patients with GCA and controls by a proteomic and gene expression profile approach and to identify the signaling pathways involved in proliferation. Methods VSMCs were cultured from temporal artery biopsies (TABs) from patients with biopsy-proven GCA (TAB+-GCA), biopsy-negative GCA (TAB−-GCA), and diagnosis other than GCA (GCA-control). VSMCs from normal human aorta (HAoSMC) were used as controls. 2D-differential in-gel electrophoresis and Affymetrix chips were used to compare proteomes and gene expression profiles of VSMCs. Proliferation was assessed by BrdU incorporation assay. TAB+-GCA and GCA-control TABs underwent immunohistochemistry staining for endothelin-1 (ET-1) and receptors ETAR and ETBR. Results We identified 16, 30 and 2 protein spots differentially expressed between TAB+-GCA and GCA-control VSMCs, TAB+-GCA and TAB−-GCA VSMCs and TAB−-GCA and GCA-control VSMCs, respectively (fold change ≥ 1.5 and p ≤ 0.05). Among the 153 proteins differentially expressed between TAB+-GCA and HAoSMC VSMCs, many were linked with ET-1. Genes differentially expressed between TAB+-GCA and GCA-control VSMCs were involved in proliferation. ET-1 was identified as a link between genes of interest. Proliferation was reduced for TAB+-GCA VSMCs on treatment with the endothelin antagonist macitentan and its active metabolite. Patients showing transmural expression of ET-1 in temporal artery lesions received a significantly higher glucocorticoid daily dose after 6-month follow-up. Conclusion Inhibiting the proliferation with macitentan, combined with glucocorticoids, might be a promising therapeutic approach for patients with GCA.

Chronic lymphocytic leukemia (CLL) is characterized by a clonal accumulation of mature neoplastic B cells that are resistant to apoptosis. Aiolos, a member of the Ikaros family of zinc-finger transcription factors, plays an important role in the control of mature B lymphocyte differentiation and maturation. In this study, we showed that Aiolos expression is up-regulated in B-CLL cells. This overexpression does not implicate isoform imbalance or disturb Aiolos subcellular localization. The chromatin status at the Aiolos promoter in CLL is defined by the demethylation of DNA and an enrichment of euchromatin associated histone markers, such as the dimethylation of the lysine 4 on histone H3. These epigenetic modifications should allow its upstream effectors, such as nuclear factor-κB, constitutively activated in CLL, to gain access to promoter, resulting up-regulation of Aiolos. To determine the consequences of Aiolos deregulation in CLL, we analyzed the effects of Aiolos overexpression or down-regulation on apoptosis. Aiolos is involved in cell survival by regulating the expression of some Bcl-2 family members. Our results strongly suggest that Aiolos deregulation by epigenetic modifications may be a hallmark of CLL.

International audience; MPhi and DC are key elements in the control of tissue homeostasis and response to insult. In this work, we demonstrate that MPhi and DC are the major producers of the phenylalanine catabolizing enzyme IL-4-induced gene 1 (IL4I1) under inflammatory conditions. IL4I1 was first described in B cells, which indeed can produce IL4I1 in vitro, although at much lower levels. In vivo, IL4I1 is highly expressed by MPhi and DC of Th1 granulomas (sarcoidosis, tuberculosis) but poorly detected in Th2 granulomas (schistosomiasis). In vitro, expression of the enzyme is induced in mononuclear phagocytes by various pro-inflammatory stimuli through the activation of the transcription factors NF-kappaB and/or STAT1. B cells also express IL4I1 in response to NF-kappaB-activating stimuli such as CD40L; however, in contrast to myeloid cells, B cells are insensitive to IFN-gamma but respond to stimulation of the IL-4/STAT6 axis. As we show that the expression of IL4I1 by a monocytic cell line inhibits T-cell proliferation and production of IFN-gamma and inflammatory cytokines, we propose that IL4I1 participates in the downregulation of Th1 inflammation in vivo.

The family of NF-kappaB transcription factors plays a key role in diverse biological processes, such as inflammatory and immune responses, cell survival and tumor development. A new NF-kappaB activation pathway has been recently uncovered, the so-called alternative NF-kappaB activation pathway. It has been shown that this pathway mainly controls the activity of RelB, a member of the NF-kappaB family, and its activation requires IKKalpha, one of the two catalytic subunits of the IKK complex. Recent studies suggest that constitutive activation of the alternative pathway may be involved in tumor development, but most surprisingly it is also reported that IKKalpha exerts a protective function against uncontrolled cell proliferation and cancer development. This review discusses recent progresses in the understanding on how the alternative pathway may participate in cancer development, focusing more specifically on RelB and IKKalpha.

Mutations in the gene encoding the transcriptional modulator methyl-CpG binding protein 2 (MeCP2) are responsible for the neurodevelopmental disorder Rett syndrome which is one of the most frequent sources of intellectual disability in women. Recent studies showed that loss of Mecp2 in astrocytes contributes to Rett-like symptoms and restoration of Mecp2 can rescue some of these defects. The goal of this work is to compare gene expression profiles of wild-type and mutant astrocytes from Mecp2(308/y) mice (B6.129S-MeCP2tm1Heto/J) by using Affymetrix mouse 2.0 microarrays. Results were confirmed by quantitative real-time RT-PCR and by Western blot analysis. Gene set enrichment analysis utilizing Ingenuity Pathways was employed to identify pathways disrupted by Mecp2 deficiency. A total of 2152 genes were statistically differentially expressed between wild-type and mutated samples, including 1784 coding transcripts. However, only 257 showed fold changes1.2. We confirmed our data by replicative studies in independent primary cultures of cortical astrocytes from Mecp2-deficient mice. Interestingly, two genes known to encode secreted proteins, chromogranin B and lipocalin-2, showed significant dysregulation. These proteins secreted from Mecp2-deficient glia may exert negative non-cell autonomous effects on neuronal properties, including dendritic morphology. Moreover, transcriptional profiling revealed altered Nr2f2 expression which may explain down- and upregulation of several target genes in astrocytes such as Ccl2, Lcn2 and Chgb. Unraveling Nr2f2 involvement in Mecp2-deficient astrocytes could pave the way for a better understanding of Rett syndrome pathophysiology and offers new therapeutic perspectives.

P100, which is encoded by NF-kappa B2, inhibits Rel dimers. It can also be processed into p52, one of the DNA binding sub-units of NF-kappa B/Rel factors. Several p100 C-terminal truncations that result from gene rearrangements are associated with lymphomagenesis. Here, we characterized a new p100 mutant that we termed p100HB. It originates from a point-mutation that generates a premature stop-codon, and thus the protein lacks the last 125 amino acids. We have detected p100HB in several human tumor cell lines. The truncated protein is mainly unprocessed, and although it still binds Rel dimers, it has reduced inhibitory potency compared to p100 and translocates into the nucleus. Thus, p100HB may be associated with deregulated NF-kappa B/Rel functions.

Clear cell renal cell carcinomas (RCC) frequently display inactivation of von Hippel-Lindau (VHL) gene leading to increased level of hypoxia-inducible factors (HIF). In this study, we investigated the potential role of HIF2α in regulating RCC susceptibility to natural killer (NK) cell–mediated killing. We demonstrated that the RCC cell line 786-0 with mutated VHL was resistant to NK-mediated lysis as compared with the VHL-corrected cell line (WT7). This resistance was found to require HIF2α stabilization. On the basis of global gene expression profiling and chromatin immunoprecipitation assay, we found ITPR1 (inositol 1,4,5-trisphosphate receptor, type 1) as a direct novel target of HIF2α and that targeting ITPR1 significantly increased susceptibility of 786-0 cells to NK-mediated lysis. Mechanistically, HIF2α in 786-0 cells lead to overexpression of ITPR1, which subsequently regulated the NK-mediated killing through the activation of autophagy in target cells by NK-derived signal. Interestingly, both ITPR1 and Beclin-1 silencing in 786-0 cells inhibited NK-induced autophagy and subsequently increased granzyme B activity in target cells. Finally, in vivo ITPR1 targeting significantly enhanced the NK-mediated tumor regression. Our data provide insight into the link between HIF2α, the ITPR1-related pathway, and natural immunity and strongly suggest a role for the HIF2α/ITPR1 axis in regulating RCC cell survival. Cancer Res; 74(23); 6820–32. ©2014 AACR.

International audience; Development of novel therapies is critical for T-cell acute leukae-mia (T-ALL). Here, we investigated the effect of inhibiting the MAPK/MEK/ERK pathway on T-ALL cell growth. Unexpectedly, MEK inhibitors (MEKi) enhanced growth of 70% of human T-ALL cell samples cultured on stromal cells independently of NOTCH activa-tion and maintained their ability to propagate in vivo. Similar results were obtained when T-ALL cells were cultured with ERK1/ 2-knockdown stromal cells or with conditioned medium from MEKi-treated stromal cells. Microarray analysis identified interleu-kin 18 (IL-18) as transcriptionally up-regulated in MEKi-treated MS5 cells. Recombinant IL-18 promoted T-ALL growth in vitro, whereas the loss of function of IL-18 receptor in T-ALL blast cells decreased blast proliferation in vitro and in NSG mice. The NFKB pathway that is downstream to IL-18R was activated by IL-18 in blast cells. IL-18 circulating levels were increased in T-ALL-xeno-grafted mice and also in T-ALL patients in comparison with controls. This study uncovers a novel role of the pro-inflammatory cytokine IL-18 and outlines the microenvironment involvement in human T-ALL development.

Loss of hematopoietic stem cell (HSC) function and increased risk of developing hematopoietic malignancies are severe and concerning complications of anticancer radiotherapy and chemotherapy. We have previously shown that thrombopoietin (TPO), a critical HSC regulator, ensures HSC chromosomal integrity and function in response to γ-irradiation by regulating their DNA-damage response. TPO directly affects the double-strand break (DSB) repair machinery through increased DNA-protein kinase (DNA-PK) phosphorylation and nonhomologous end-joining (NHEJ) repair efficiency and fidelity. This effect is not shared by other HSC growth factors, suggesting that TPO triggers a specific signal in HSCs facilitating DNA-PK activation upon DNA damage. The discovery of these unique signaling pathways will provide a means of enhancing TPO-desirable effects on HSCs and improving the safety of anticancer DNA agents. We show here that TPO specifically triggers Erk and nuclear factor κB (NF-κB) pathways in mouse hematopoietic stem and progenitor cells (HSPCs). Both of these pathways are required for a TPO-mediated increase in DSB repair. They cooperate to induce and activate the early stress-response gene, Iex-1 (ier3), upon DNA damage. Iex-1 forms a complex with pERK and the catalytic subunit of DNA-PK, which is necessary and sufficient to promote TPO-increased DNA-PK activation and NHEJ DSB repair in both mouse and human HSPCs.

Tumor necrosis factor alpha (TNFalpha) is a potent proinflammatory cytokine that plays an important role in immunity and inflammation, and in the control of cell proliferation, differentiation and apoptosis. TNFalpha is also the founding member of a still growing family of cytokines with diverse bioregulatory functions. Considerable progress has been made in understanding the molecular mechanisms that mediate TNFalpha-induced cellular responses. Binding of TNFalpha to its two receptors, TNFR1 and TNFR2, results in recruitment of signal transducers that activate at least three distinct effectors. Through complex signaling cascades and networks, these effectors lead to the activation of caspases and two transcription factors, AP-1 and NF-kappaB. Similar signaling mechanisms are likely to be used by other members of the TNF family. This review focuses on proteins that transduce the signals generated at TNF receptors to nuclear targets such as AP-1 and NF-kappaB.

The IKKalpha and IKKbeta catalytic subunits of IkappaB kinase (IKK) share 51% amino-acid identity and similar biochemical activities: they both phosphorylate IkappaB proteins at serines that trigger their degradation. IKKalpha and IKKbeta differ, however, in their physiological functions. IKKbeta and the IKKgamma/NEMO regulatory subunit are required for activating NF-kappaB by pro-inflammatory stimuli and preventing apoptosis induced by tumour necrosis factor-alpha (refs 5,6,7,8,9,10,11). IKKalpha is dispensable for these functions, but is essential for developing the epidermis and its derivatives. The mammalian epidermis is composed of the basal, spinous, granular and cornified layers. Only basal keratinocytes can proliferate and give rise to differentiated derivatives, which on full maturation undergo enucleation to generate the cornified layer. Curiously, keratinocyte-specific inhibition of NF-kappaB, as in Ikkalpha-/- mice, results in epidermal thickening but does not block terminal differentiation. It has been proposed that the epidermal defect in Ikkalpha-/- mice may be due to the failed activation of NF-kappaB. Here we show that the unique function of IKKalpha in control of keratinocyte differentiation is not exerted through its IkappaB kinase activity or through NF-kappaB. Instead, IKKalpha controls production of a soluble factor that induces keratinocyte differentiation.

Monogenic interleukin-10 (IL-10) and IL-10 receptor (IL-10R) deficiencies cause very early onset severe inflammatory bowel disease. Here, we report that 5 patients with an IL-10R1 (n = 1) or IL-10R2 (n = 4) deficiency developed B-cell non-Hodgkin lymphoma between the ages of 5 and 6 years (which was recurrent in 1 patient). These lymphomas had some of the characteristics of diffuse large B-cell lymphomas and contained monoclonal, Epstein-Barr virus-negative germinal center B cells. The tumors displayed a remarkably homogeneous signature, with original activation of the nuclear factor κB pathway and a decrease in intratumor T-cell infiltration. Hence, IL-10R deficiency is associated with a high risk of developing B-cell lymphoma. Our results revealed an unexpected role of the IL-10R pathway in lymphomagenesis.

The NF-κB family of transcription factors has emerged as a key player in the pathogenesis of multiple myeloma (MM). NF-κB is activated by at least two major signaling pathways. The classical pathway results in the activation of mainly RelA containing dimers, whereas the alternative pathway leads to the activation of RelB/p52 and RelB/p50 heterodimers. Activating mutations in regulators of the alternative pathway have been identified in 17% of MM patients. However, the status of RelB activation per se and its role in the regulation of cell survival in MM has not been investigated. Here, we reveal that 40% of newly diagnosed MM patients have a constitutive RelB DNA-binding activity in CD138(+) tumor cells, and we show an association with increased expression of a subset of anti-apoptotic NF-κB target genes, such as cIAP2. Furthermore, we demonstrate that RelB exerts a crucial anti-apoptotic activity in MM cells. Our findings indicate that RelB activation is key for promoting MM cell survival through the upregulation of anti-apoptotic proteins. Altogether, our study provides the framework for the development of new molecules targeting RelB in the treatment of MM.

International audience; The alternative nuclear factor-kappaB (NF-κB) -activation pathway proceeds via inducible p100 processing, leading to the activation of RelB-containing dimers. This pathway is aberrantly activated in several types of tumors; however, a direct role for RelB in the control of cell proliferation is still largely unexplored. Here, we demonstrate that RelB provides cell proliferation-inhibitory signals in murine fibroblasts. In agreement with these results, RelB ectopic expression inhibits xenograft tumor growth in vivo, whereas RelB knockdown enhances it. Significantly, we show that RelB inhibits cell proliferation and tumor growth in a p53-dependent manner. Mechanistic studies indicate that RelB regulates the transcription of the p53 tumor-suppressor gene through direct recruitment to the p53 promoter, thus increasing both p53 protein levels and expression of p53 target genes such as p21. Our findings define a novel link between NF-κB and growth-inhibitory pathways involving the RelB-dependent transcriptional upregulation of p53. Furthermore, they suggest that inhibition of RelB in some tumor types that retain wild-type p53 may diminish rather than improve therapeutic responses.

BACKGROUND:\ud Isolated limb perfusion with TNF-α and melphalan is used with remarkable efficiency to treat unresectable limb sarcomas. Here we tested the ability of TNF-α to directly induce apoptosis of sarcoma cells. In addition, we investigated the impact of p53 in the regulation of such effect.\ud \ud METHODOLOGY/PRINCIPAL FINDINGS:\ud We first analysed the ability of TNF-α to induce apoptosis in freshly isolated tumour cells. For this purpose, sarcoma tumours (n = 8) treated ex vivo with TNF-α were processed for TUNEL staining. It revealed substantial endothelial cell apoptosis and levels of tumour cell apoptosis that varied from low to high. In order to investigate the role of p53 in TNF-α-induced cell death, human sarcoma cell lines (n = 9) with different TP53 and MDM2 status were studied for their sensitivity to TNF-α. TP53(Wt) cell lines were sensitive to TNF-α unless MDM2 was over-expressed. However, TP53(Mut) and TP53(Null) cell lines were resistant. TP53 suppression in TP53(Wt) cell lines abrogated TNF-α sensitivity and TP53 overexpression in TP53(Null) cell lines restored it. The use of small molecules that restore p53 activity, such as CP-31398 or Nutlin-3a, in association with TNF-α, potentiated the cell death of respectively TP53(Mut) and TP53(Wt)/MDM2(Ampl). In particular, CP-31398 was able to induce p53 as well as some of its apoptotic target genes in TP53(Mut) cells. In TP53(Wt)/MDM2(Ampl) cells, Nutlin-3a effects were associated with a decrease of TNF-α-induced NF-κB-DNA binding and correlated with a differential regulation of pro- and anti-apoptotic genes such as TP53BP2, GADD45, TGF-β1 and FAIM.\ud \ud CONCLUSION/SIGNIFICANCE:\ud More effective therapeutic approaches are critically needed for the treatment of unresectable limb sarcomas. Our results show that restoring p53 activity in sarcoma cells correlated with increased sensitivity to TNF-α, suggesting that this strategy may be an important determinant of TNF-α-based sarcomas treatment.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Its pathogenesis is frequently linked to liver inflammation. Gain-of-function mutations in the gene encoding β-catenin are frequent genetic modifications found in human HCCs. Thus, we investigated whether inflammation was a component of β-catenin-induced tumorigenesis using genetically modified mouse models that recapitulated the stages of initiation and progression of this tumoral process. Oncogenic β-catenin signaling was found to induce an inflammatory program in hepatocytes that involved direct transcriptional control by β-catenin and activation of the NF-κB pathway. This led to a specific inflammatory response, the intensity of which determined the degree of tumor aggressiveness. The chemokine-like chemotactic factor leukocyte cell-derived chemotaxin 2 (LECT2) and invariant NKT (iNKT) cells were identified as key interconnected effectors of liver β-catenin-induced inflammation. In genetic deletion models lacking the gene encoding LECT2 or iNKT cells, hepatic β-catenin signaling triggered the formation of highly malignant HCCs with lung metastasis. Thus, our results identify inflammation as a key player in β-catenin-induced liver tumorigenesis. We provide strong evidence that, by activating pro- and antiinflammatory mediators, β-catenin signaling produces an inflammatory microenvironment that has an impact on tumoral development. Our data are consistent with the fact that most β-catenin-activated HCCs are of better prognosis.

Recent studies have highlighted the AT1 receptor as a potential therapeutic target in breast cancer, while the role of the AT2 subtype in this disease has remained largely neglected. The present study describes the generation and characterization of a new cellular model of human invasive breast cancer cells (D3H2LN-AT2) stably expressing high levels of Flag-tagged human AT2 receptor (Flag-hAT2). These cells exhibit high-affinity binding sites for AngII, and total binding can be displaced by the AT2-selective antagonist PD123319 but not by the AT1-selective antagonist losartan. Of interest, high levels of expression of luciferase and green fluorescent protein make these cells suitable for bioluminescence and fluorescence studies in vitro and in vivo. We provide here a novel tool to investigate the AT2 receptor functions in breast cancer cells, independently of AT1 receptor activation.

International audience; GH is a pleiotropic hormone that plays a major role in proliferation, differentiation, and metabolism via its specific receptor. It has been previously suggested that GH signaling pathways are required for normal liver regeneration but the molecular mechanisms involved have yet to be determined. The aim of this study was to identify the mechanisms by which GH controls liver regeneration. We performed two thirds partial hepatectomies in GH receptor (GHR)-deficient mice and wild-type littermates and showed a blunted progression in the G(1)/S transition phase of the mutant hepatocytes. This impaired liver regeneration was not corrected by reestablishing IGF-1 expression. Although the initial response to partial hepatectomy at the priming phase appeared to be similar between mutant and wild-type mice, cell cycle progression was significantly blunted in mutant mice. The main defect in GHR-deficient mice was the deficiency of the epidermal growth factor receptor activation during the process of liver regeneration. Finally, among the pathways activated downstream of GHR during G(1) phase progression, namely Erk1/2, Akt, and signal transducer and activator of transcription 3, we only found a reduced Erk1/2 phosphorylation in mutant mice. In conclusion, our results demonstrate that GH signaling plays a major role in liver regeneration and strongly suggest that it acts through the activation of both epidermal growth factor receptor and Erk1/2 pathways.

BackgroundEndothelial dysfunction has been implicated in the pathogenesis of diverse pathologies ranging from vascular and immune diseases to cancer. TNF-α is one of the mediators of endothelial dysfunction through the activation of transcription factors, including NF-κB. While HUVEC (macrovascular cells) have been largely used in the past, here, we documented an NF-κB gene signature in TNFα-stimulated microvascular endothelial cells HMEC often used in tumor angiogenesis studies.Methodology/principal findingsWe measured mRNA expression of 55 NF-κB related genes using quantitative RT-PCR in HUVEC and HMEC. Our study identified twenty genes markedly up-regulated in response to TNFα, including adhesion molecules, cytokines, chemokines, and apoptosis regulators, some of them being identified as TNF-α-inducible genes for the first time in endothelial cells (two apoptosis regulators, TNFAIP3 and TNFRSF10B/Trail R2 (DR5), the chemokines GM-CSF/CSF2 and MCF/CSF1, and CD40 and TNF-α itself, as well as NF-κB components (RELB, NFKB1 or 50/p105 and NFKB2 or p52/p100). For eight genes, the fold induction was much higher in HMEC, as compared to HUVEC. Most importantly, our study described for the first time a connection between NF-κB activation and the induction of most, if not all, of these genes in HMEC as evaluated by pharmacological inhibition and RelA expression knock-down by RNA interference. Moreover, since TNF-α is highly expressed in tumors, we further applied the NF-κB gene signature documented in TNFα-stimulated endothelial cells to human breast tumors. We found a significant positive correlation between TNF and the majority (85 %) of the identified endothelial TNF-induced genes in a well-defined series of 96 (48 ERα positive and 48 ERα negative) breast tumors.Conclusion/significanceTaken together these data suggest the potential use of this NF-κB gene signature in analyzing the role of TNF-α in the endothelial dysfunction, as well as in breast tumors independently of the presence of ERα.

International audience; The natural killer (NK) cell receptor NKp30 is involved in the recognition of tumor and dendritic cells (DCs). Here we describe the influence of three NKp30 splice variants on the prognosis of gastrointestinal sarcoma (GIST), a malignancy that expresses NKp30 ligands and that is treated with NK-stimulatory KIT tyrosine kinase inhibitors. Healthy individuals and those with GIST show distinct patterns of transcription of functionally different NKp30 isoforms. In a retrospective analysis of 80 individuals with GIST, predominant expression of the immunosuppressive NKp30c isoform (over the immunostimulatory NKp30a and NKp30b isoforms) was associated with reduced survival of subjects, decreased NKp30-dependent tumor necrosis factor-α (TNF-α) and CD107a release, and defective interferon-γ (IFN-γ) and interleukin-12 (IL-12) secretion in the NK-DC cross-talk that could be restored by blocking of IL-10. Preferential NKp30c expression resulted partly from a single-nucleotide polymorphism at position 3790 in the 3' untranslated region of the gene encoding NKp30. The genetically determined NKp30 status predicts the clinical outcomes of individuals with GIST independently from KIT mutation.

NF-κB transcription factors are critical regulators of many biological processes such as innate and adaptive immune responses, inflammation, cell proliferation and programmed cell death. This versatility necessitates a highly complex and tightly coordinated control of the signaling pathways leading to their activation. Here, we review the role of proteolysis in the regulation of NF-κB activity, more specifically the contribution of the well-known ubiquitin–proteasome system and the involvement of proteolytic activity of caspases and calpains.

The nuclear factor κB (NF-κB) signalling pathway has been implicated in cancer development and progression, as well as in resistance to chemotherapy and radiation therapy. In this Perspective, Baud and Karin explore the therapeutic potential of targeting NF-κB in cancer, and discuss the challenges posed by this approach. Nuclear factor κB (NF-κB) transcription factors have a key role in many physiological processes such as innate and adaptive immune responses, cell proliferation, cell death, and inflammation. It has become clear that aberrant regulation of NF-κB and the signalling pathways that control its activity are involved in cancer development and progression, as well as in resistance to chemotherapy and radiotherapy. This article discusses recent evidence from cancer genetics and cancer genome studies that support the involvement of NF-κB in human cancer, particularly in multiple myeloma. The therapeutic potential and benefit of targeting NF-κB in cancer, and the possible complications and pitfalls of such an approach, are explored.

Transcription factor NF-kappaB, whose activation depends on the IKKbeta catalytic subunit of the IkappaB kinase, was assigned with both anti- and proapoptotic functions in T lymphocytes. To critically evaluate these functions, we transferred Ikkbeta-/- or wild-type (wt) fetal liver (FL) stem cells into lethally irradiated mice. Ikkbeta-/- radiation chimeras show thymic rudiments, aberrant lymphoid organs, and absence of T cells. T lymphopoiesis is rescued when Ikkbeta-/- stem cells are cotransferred with wt bone marrow, suggesting that IKKbeta may mediate its lymphopoietic function via extrinsic factors. However, almost normal development of Ikkbeta-/- T cells is observed upon removal of type 1 TNFalpha receptor, indicating that TNFalpha signaling accounts for the absence of Ikkbeta-/- T cells. Indeed, Ikkbeta-/- radiation chimeras exibit elevated circulating TNFalpha, and Ikkbeta-/- thymocytes display increased TNFalpha sensitivity.

Abstract 478 The prognosis of Acute Myeloid Leukaemia (AML) remains globally poor. Aberrant activation of signalling pathways is frequently found in AML and leads to uncontrolled cell growth and survival. Effective targeting of these pathways with new therapeutics may result in suppression of leukemic cell proliferation and survival. In AML, constitutive PI3K activity, mainly due to the expression of the class IA PI3K p110δ isoform, is detected in 50% of patients at diagnosis (PI3K+ AML samples) (Sujobert, Bardet et al., Blood 2005; Tamburini, Elie et al., Blood 2007). The PI3K/Akt axis represents therefore an attractive therapeutic target in this disease. Accordingly, inhibition of PI3K activity with IC87114, a specific p110δ inhibitor decreases AML blast cells proliferation (Sujobert, Bardet et al., Blood 2005). However, IC87114 failed to induce significant apoptosis (Park, Chapuis et al., Leukemia 2008). The PI3K/Akt network controls different targets implicated in the regulation of cell survival. Among them, the FoxO transcription factors which include FoxO1, FoxO3a, FoxO4 and FoxO6 up regulate the expression of target genes involved in apoptosis, such Fas-L and Bim. Akt, by phosphorylating FoxO proteins at three conserved sites (T32, S253 and S315 on FoxO3a), negatively regulates their transcriptional activity by inducing their nuclear export. Given that IC87114 did not induce apoptosis, we hypothesized that FoxO proteins may escape to PI3K/Akt control in AML cells. For that purpose, we investigated their mechanisms of regulation in both primary AML cells and a MV4-11 human leukemic cell line engineered to stably overexpress a FoxO3awt-GPF fusion protein introduced by lentiviral infection. We focused especially on FoxO3a as we found, using real-time RT-PCR and WB analysis, that it was the only FoxO protein constantly expressed in primary AML cells. First, we examined the relationship between FoxO3a subcellular localization using immunofluorescence and PI3K activity in primary AML cells. As expected in PI3K+ AML cells (n=6), FoxO3a was phosphorylated on T32 and S253 and constantly localized in the cytoplasm. However, although Akt S473 and FoxO3a T32 and S253 phosphorylations were fully inhibited by IC87114, FoxO3a did not translocate into the nucleus of blast cells. Accordingly, Fas-L and Bim expression tested by qRT-PCR were not induced by IC87114. Identical results were obtained from the same analysis of the MV4-11FoxO3awt-GFP cell line. Furthermore, in PI3K- AML cells (n=5), neither Akt S473 nor FoxO3a T32 and S253 phosphorylations were detected but FoxO3a was still confined in the cytoplasm of these cells. Altogether, these results clearly demonstrate that an Akt-independent mechanism contributes to the nuclear exclusion of FoxO3a in AML cells. In addition to Akt, the IκB Kinase (IKK) may regulate FoxO3a subcellular localization by phosphorylation on S644 in AML, as it has previously been shown in breast cancer (Hu, Lee et al., Cell 2004). As the oncogenic IκB Kinase is known to be activated in almost all AML samples (Guzman, Neering et al., Blood 2001), we investigated whether IKK could maintain FoxO3a in the cytoplasm of AML cells. Interestingly, we found that inhibition of IKK with a specific IKKγ/NEMO-antagonistic peptide (Agou, Courtois et al., J Biol Chem 2004) induced a strong nuclear translocation of FoxO3a and an efficient apoptosis in both primary AML blast cells (n=5) and MV4-11FoxO3awt-GFP cell line. To confirm this result, MV4-11 cells were infected with a lentivirus expressing a FoxO3aS644A-GFP mutant protein in which the IKK phosphorylation is abrogated. We found that the FoxO3aS644A-GFP mutant protein primarily localized within the nucleus of MV4-11 cells and induced a decrease of cell proliferation and a moderate pro-apoptotic effect. We thus conclude that IKK, by phosphorylating FoxO3a on S644, leads to the constant nuclear export of FoxO3a in AML cells. Consequently, the therapeutical potential value of IKK targeted inhibition in AML could be due not only to the inhibition of NF-κB activity but also to the FoxO3a nuclear import. Moreover, the PI3K-independent FoxO3a regulation probably contributes to the low potential of specific PI3K inhibitors to induce apoptosis in AML cells. Disclosures: No relevant conflicts of interest to declare.

The oligomeric IκB kinase (IKK) is composed of three polypeptides: IKKα and IKKβ, the catalytic subunits, and IKKγ, a regulatory subunit. IKKα and IKKβ are similar in structure and thought to have similar function—phosphorylation of the IκB inhibitors in response to proinflammatory stimuli. Such phosphorylation leads to degradation of IκB and activation of nuclear factor κB transcription factors. The physiological function of these protein kinases was explored by analysis of IKKα-deficient mice. IKKα was not required for activation of IKK and degradation of IκB by proinflammatory stimuli. Instead, loss of IKKα interfered with multiple morphogenetic events, including limb and skeletal patterning and proliferation and differentiation of epidermal keratinocytes.