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Emerging evidence suggests a link between tumor hypoxia and immune suppression. In this study, we investigated the role of hypoxia-induced Nanog, a stemness-associated transcription factor, in immune suppression. We observed that hypoxia-induced Nanog correlated with the acquisition of stem cell–like properties in B16-F10 cells. We further show that Nanog was selectively induced in hypoxic areas of B16-F10 tumors. Stable short hairpin RNA–mediated depletion of Nanog, combined with melanocyte differentiation Ag tyrosinase-related protein-2 peptide-based vaccination, resulted in complete inhibition of B16-F10 tumor growth. Nanog targeting significantly reduced immunosuppressive cells (regulatory T cells and macrophages) and increased CD8+ T effector cells in tumor bed in part by modulating TGF-β1 production. Additionally, Nanog regulated TGF-β1 under hypoxia by directly binding the TGF-β1 proximal promoter. Collectively, our data establish a novel functional link between hypoxia-induced Nanog and TGF-β1 regulation and point to a major role of Nanog in hypoxia-driven immunosuppression.

Hypoxia upregulates the core pluripotency factors NANOG, SOX2, and OCT4, associated with tumor aggressiveness and resistance to conventional anticancer treatments. We have previously reported that hypoxia-induced NANOG contributed in vitro to tumor cell resistance to autologous-specific CTL and in vivo to the in situ recruitment of immune-suppressive cells. In this study, we investigated the mechanisms underlying NANOG-mediated tumor cell resistance to specific lysis under hypoxia. We demonstrated the tumor-promoting effect of hypoxia on tumor initiation into immunodeficient mice using human non–small lung carcinoma cells. We next showed a link between NANOG and autophagy activation under hypoxia because inhibition of NANOG decreased autophagy in tumor cells. Chromatin immunoprecipitation and luciferase reporter assays revealed a direct binding of NANOG to a transcriptionally active site in a BNIP3L enhancer sequence. These data establish a new link between the pluripotency factor NANOG and autophagy involved in resistance to CTL under hypoxia.

It is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.

In response to stress, cells start transcriptional and transcription-independent programs that can lead to adaptation or death. Here, we show that multiple inducers of autophagy, including nutrient depletion, trigger the activation of the IKK (IkappaB kinase) complex that is best known for its essential role in the activation of the transcription factor NF-kappaB by stress. Constitutively active IKK subunits stimulated autophagy and transduced multiple signals that operate in starvation-induced autophagy, including the phosphorylation of AMPK and JNK1. Genetic inhibition of the nuclear translocation of NF-kappaB or ablation of the p65/RelA NF-kappaB subunit failed to suppress IKK-induced autophagy, indicating that IKK can promote the autophagic pathway in an NF-kappaB-independent manner. In murine and human cells, knockout and/or knockdown of IKK subunits (but not that of p65) prevented the induction of autophagy in response to multiple stimuli. Moreover, the knockout of IKK-beta suppressed the activation of autophagy by food deprivation or rapamycin injections in vivo, in mice. Altogether, these results indicate that IKK has a cardinal role in the stimulation of autophagy by physiological and pharmacological stimuli.

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.

Rapid childhood T-ALL growth in xenograft models correlates with mature phenotype and NF-κB pathway activation but not with poor prognosis

TNFα is a potent cytokine that plays a critical role in numerous cellular processes, particularly immune and inflammatory responses, programmed cell death, angiogenesis, and cell migration. Thus, understanding the molecular mechanisms that mediate TNFα-induced cellular responses is a crucial issue. It is generally accepted that global DNA binding activity of the NF-κB avian reticuloendotheliosis viral (v-rel) oncogene related B (RelB) subunit is not induced upon TNFα treatment in fibroblasts, despite its TNFα-induced nuclear accumulation. Here, we demonstrate that RelB plays a critical role in promoting fibroblast migration upon prolonged TNFα treatment. We identified the two kinases IκB kinase α (IKKα) and IκB kinase β (IKKβ) as RelB interacting partners whose activation by TNFα promotes RelB phosphorylation at serine 472. Once phosphorylated on serine 472, nuclear RelB dissociates from its interaction with the inhibitory protein IκBα and binds to the promoter of critical migration-associated genes, such as the matrix metallopeptidase 3 (MMP3). Further, we show that RelB serine 472 phosphorylation status controls MMP3 expression and promigration activity downstream of TNF receptors. Our findings provide new insights into the regulation of RelB activity and reveal a novel link between selective NF-κB target gene expression and cellular response in response to TNFα.

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.

Cells respond to stress by activating cytoplasmic mechanisms as well as transcriptional programs that can lead to adaptation or death. Autophagy represents an important cytoprotective response that is regulated by both transcriptional and transcription-independent pathways. NFkappaB is perhaps the transcription factor most frequently activated by stress and has been ascribed with either pro- or anti-autophagic functions, depending on the cellular context. Our results demonstrate that activation of the IKK (IkappaB kinase) complex, which is critical for the stress-elicited activation of NFkappaB, is sufficient to promote autophagy independent of NFkappaB, and that IKK is required for the optimal induction of autophagy by both physiological and pharmacological autophagic triggers.

Proteins with seven transmembrane segments (7TM) define a superfamily of receptors (7TM receptors) sharing the same topology: an extracellular N-terminus, three extramembranous loops on either side of the plasma membrane, and a cytoplasmic C-terminal tail. Upon ligand binding, cytoplasmic portions of the activated receptor interact with heterotrimeric G-coupled proteins to induce various second messengers. A small group, recently recognized on the basis of homologous primary amino acid sequences, comprises receptors to hormones of the secretin/vasoactive intestinal peptide/glucagon family, parathyroid hormone and parathyroid hormone-related peptides, growth hormone-releasing factor, corticotropin-releasing factor, and calcitonin. A cDNA, extracted from a neuroectodermal cDNA library, was predicted to encode a new 886-amino-acid protein with three distinct domains. The C-terminal third contains the seven hydrophobic segments and characteristic residues that allow the protein to be readily aligned with the various hormone receptors in the family. Six egf-like modules, at the N-terminus of the predicted mature protein, are separated from the transmembrane segments by a serine/threonine-rich domain, a feature reminiscent of mucin-like, single-span, integral membrane glycoproteins with adhesive properties. Because of its unique characteristics, this putative egf module-containing, mucin-like hormone receptor has been named EMR1. Southern analysis of a panel of somatic cell hybrids and fluorescence in situ hybridization have assigned the EMR1 gene to human chromosome 19p13.3.

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.

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.

Despite recent progress in the treatment of acute myeloid leukemia (AML), the prognosis of this rather heterogeneous disease remains poor and novel chemotherapeutics that specifically target leukemic cells must be developed. To address this need at the preclinical level, we implemented a high content imaging-based screen for the identification of small agents that induce AML cell death in vitro. Among a panel of 1040 Food and Drug Administration-approved agents, we identified pyrithione zinc (PZ) and ouabain (OUA) as potential antileukemic compounds. Both PZ and OUA efficiently induced cell death associated with apoptotic chromatin condensation and inhibition of nuclear factor-κB survival signaling, leading to reduced expression of antiapoptotic proteins, in several AML cell lines. PZ- and OUA-induced cell death was associated with the permeabilization of the outer mitochondrial membrane and led to the release of cytochrome c followed by caspase activation. Both PZ and OUA exerted significant anticancer effects in vivo, on human AML cells xenografts as well as ex vivo, on CD34(+) (but not CD34(-)) malignant myeloblasts from AML patients. Altogether, our results suggest that PZ and OUA may exhibit antileukemic effects by inducing the apoptotic demise of AML cells.

NF-κB transcription factors are pivotal players in controlling inflammatory and immune responses, as well as cell proliferation and apoptosis. Aberrant regulation of NF-κB and the signaling pathways that regulate its activity have been involved in various pathologies, particularly cancers, as well as inflammatory and autoimmune diseases. NF-κB activation is tightly regulated by the IκB kinase (IKK) complex, which is composed of two catalytic subunits IKKα and IKKβ, and a regulatory subunit IKKγ/NEMO. Although IKKα and IKKβ share structural similarities, IKKα has been shown to have distinct biological functions. However, the molecular mechanisms that modulate IKKα activity have not yet been fully elucidated. To understand better the regulation of IKKα activity, we purified IKKα-associated proteins and identified ABIN-2. Here, we demonstrate that IKKα and IKKβ both interact with ABIN-2 and impair its constitutive degradation by the proteasome. Nonetheless, ABIN-2 enhances IKKα- but not IKKβ-mediated NF-κB activation by specifically inducing IKKα autophosphorylation and kinase activity. Furthermore, we found that ABIN-2 serine 146 is critical for the ABIN-2-dependent IKKα transcriptional up-regulation of specific NF-κB target genes. These results imply that ABIN-2 acts as a positive regulator of NF-κB-dependent transcription by activating IKKα.

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.

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 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.

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.

FLI1 is a common mouse viral integration region in virus-induced leukemias and lymphomas. Using an evolutionarily conserved mouse probe and Southern hybridization to (rodent x human) somatic cell hybrid DNAs, the human homolog of FLI1 has been shown to lie on a fragment of chromosome 11 flanked on the centromeric side by the acute lymphoblastic leukemia-associated t(4;11)(q21;q23) translocation breakpoint and on the telomeric side by the Ewing- and neuroepithelioma-associated t(11;22) (q24;q12) breakpoint.