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Supplementary Table S1

Immune resistance may arise from both genetic instability and tumor heterogeneity. Microenvironmental stresses such as hypoxia and various resistance mechanisms promote carcinoma cell plasticity. AXL, a member of the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, is widely expressed in human cancers and increasingly recognized for its role in cell plasticity and drug resistance. To investigate mechanisms of immune resistance, we studied multiple human lung cancer clones derived from a model of hypoxia-induced tumor plasticity that exhibited mesenchymal or epithelial features. We demonstrate that AXL expression is increased in mesenchymal lung cancer clones. Expression of AXL in the cells correlated with increased cancer cell–intrinsic resistance to both natural killer (NK)– and cytotoxic T lymphocyte (CTL)–mediated killing. A small-molecule targeting AXL sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte–mediated killing. Mechanistically, we showed that attenuation of AXL-dependent immune resistance involved a molecular network comprising NF-κB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Higher ICAM1 and ULBP1 tumor expression correlated with improved patient survival in two non–small cell lung cancer (NSCLC) cohorts. These results reveal an AXL-mediated immune-escape regulatory pathway, suggest AXL as a candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC.

This file contains supplementary Figures

Supplemental video 1. 786-0 cells described in Figure 3B were recorded by time-lapse video microscopy

Supplementary Figures S1-S5. Figure S1. Differential susceptibility of 786-0, PRC3, and WT7 cell lines to NK-mediated lysis. Figure S2. Role of ANGPTL4 and ADM in RCC cell susceptibility to NK-mediated lysis. Figure S3. VHL and NK ligand expression in RCC cells. Figure S4. Silencing of HIF-2α and NK ligand expression in 786-0 cells. Figure S5. VHL status, targeting of HIF-2α or silencing of ITPR1 does not regulate autophagy in 786-0 cells.

Supplemental video 2. 786-0 cells described in Figure 3B were recorded by time-lapse video microscopy

Ultrahigh-resolution NMR has recently attracted considerable attention in the field of complex samples analysis. Indeed, the implementation of broadband homonuclear decoupling techniques has allowed us to greatly simplify crowded

Cancer cells outgrowing in distant organs of metastasis rewire their metabolism to fuel on the available nutrients. While this is often considered an adaptive pressure limiting metastasis formation, some nutrients available at the metastatic site naturally or through changes in organ physiology may inherently promote metastatic growth. We find that the lung, a frequent site of metastasis, is a lipid-rich environment. Moreover, we observe that pathological conditions such as pre-metastatic niche formation and obesity further increase the availability of the fatty acid palmitate in the lung. We find that targeting palmitate processing inhibits spheroid growthin vitroand metastasis formation in lean and obese mice. Mechanistically, we discover that breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a (CPT1a)-dependent manner. Lysine acetyltransferase 2a (KAT2a), whose expression is promoted by palmitate availability, relies on the available acetyl-CoA to acetylate the NF-κB subunit p65. This favors nuclear location of p65 and activates a pro-metastatic transcriptional program. Accordingly, deletion of KAT2a phenocopies CPT1a silencingin vitroas well asin vivoand patients with breast cancer show co-expression of both proteins in metastases across palmitate-rich metastatic sites. In conclusion, we find that palmitate-rich environments foster metastasis growth by increasing p65 acetylation resulting in elevated NF-κB signaling.

Gastric cancer (GC) is one of the major causes of cancer-related mortality worldwide. The vast majority of GC cases are adenocarcinomas including intestinal and diffuse GC. The incidence of diffuse GCs, often associated with poor overall survival, has constantly increased in USA and Europe The molecular basis of diffuse GC aggressivity remains unclear. Using mRNA from diffuse and intestinal GC tumor samples of a Western cohort, this study reports the expression level of the immunomodulatory aryl-hydrocarbon receptor (AhR), and genes involved in immune suppression (PD1, PD-L1, PD-L2) and the early steps of tryptophan metabolism (IDO1, IDO2, TDO2). Strongly increased expression of IDO1 (p < 0.001) and PD1 (p < 0.003) was observed in the intestinal sub-type. The highest expression of IDO1 and PDL1 correlated with early clinical stage and absence of lymphatic invasion (×25 p = 0.004, ×3 p = 0.04, respectively). Our results suggest that kynurenine, produced by tryptophan catabolism, and AhR activation play a central role in creating an immunosuppressive environment. Correspondingly, as compared to intestinal GCs, expression levels of IDO1-TDO2 and PD-L1 were less prominent in diffuse GCs which also had less infiltration of immune cells, suggesting an inactive immune response in the advanced diffuse GC. Confirmation of these patterns of gene expression will require a larger cohort of early and advanced stages of diffuse GC samples.

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.

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.

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.

The family of NF-κB transcription factors plays a key role in diverse biological processes, such as inflammatory and immune responses, cell survival and tumor development. Beyond the classical NF-κB activation pathway, a second NF-κB pathway has more recently been uncovered, the so-called alternative NF-κB activation pathway. It has been shown that this pathway mainly controls the activity of RelB, a member of the NF-κB family. Post-translational modifications, such as phosphorylation, acetylation, methylation, ubiquitination and SUMOylation, have recently emerged as a strategy for the fine-tuned regulation of NF-κB. Our review discusses recent progress in the understanding of RelB regulation by post-translational modifications and the associated functions in normal and pathological conditions.

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.

Introduction Les etudes physiopathologiques sur l’arterite a cellules geantes (ACG) ont porte, avant tout, sur le dysfonctionnement du systeme immunitaire. Les mecanismes qui conduisent au remodelage arteriel et a l’eventuelle occlusion des vaisseaux sont mal connus et le role des cellules musculaires lisses vasculaires (CMLV) dans ce contexte reste a identifier. Nous avons identifie que l’endotheline-1 interagissait avec de nombreuses proteines dont l’expression par les CMLV se modifie au cours de l’ACG. Materiels et methodes Nous avons etudie l’expression de l’endotheline-1 (ET-1) et de ses recepteurs ETAR et ETBR par qPCR a partir de CMLV isolees d’artere temporale de patients suspects d’ACG et avons etudie leur expression proteique par immuno-histochimie. Nous avons etudie la proliferation des CMLV de patients atteints d’ACG comparativement a des CMLV d’aorte normale (HAoSMC) en presence de bosentan, d’ambrisentan, de macitentan et de son metabolite actif mais egalement de sildenafil et d’iloprost. Resultats Nous avons pu confirmer par qPCR l’augmentation d’expression de l’ARN de l’ET-1 et de l’ETBR dans les CMLV de patients atteints d’ACG comparativement a des CMLV de patients temoins ( p p Conclusion Nous avons confirme l’implication de l’ET-1 et de ses recepteurs ETAR et ETBR au cours de l’ACG. L’inhibition de la proliferation excessive des CMLV de patients atteints d’ACG par le macitentan et en particulier par son metabolite actif ouvre la voie a un usage therapeutique de cette molecule.

Introduction Les etudes physiopathologiques sur l’arterite a cellules geantes (ACG) ont porte, avant tout, sur le dysfonctionnement du systeme immunitaire. Les mecanismes qui conduisent au remodelage arteriel et a l’eventuelle occlusion des vaisseaux sont mal connus et le role des cellules musculaires lisses vasculaires (CMLV) dans ce contexte reste a identifier. Materiels et methodes Nous avons cultive des CMLV issues d’artere temporale de patients suspects d’ACG. Nous avons retenus les CMLV de 4 patients avec ACG prouvee histologiquement (TAB ± ACG), de 4 patients avec ACG sans preuve histologique (TAB--ACG) et de 4 patients ayant un autre diagnostic que l’ACG (GCA-controls) et avons analyse les proteomes par 2D-DIGE a pH 3-11. Les CMLV d’aorte normale (HAoSMC) ont ete utilise comme controle. Par ailleurs, nous avons fait une analyse du profil d’expression genique a l’aide de puces Affymetrix®. Resultats Nous avons identifie que 16, 30 et 2 proteines de CMLV respectivement etaient differemment exprimees entre les groupes TAB ± GCA et GCA-control, entre les groupes TAB ± GCA et TAB–GCA et entre les groupes TAB–GCA et GCA-control (rapport d’expression ≥ 1,5 et p ≤ 0,05). Nous avons pu montrer que parmi les 153 proteines differemment exprimees entre les CMLV du groupe TAB + -GCA et HAoSMC, de nombreuses etaient liees a la paxilline. De nombreux genes parmi ceux differemment exprimes entre les groupes TAB ± GCA et GCA-control sont impliques dans la proliferation cellulaire. La paxilline a de nouveau ete identifiee comme lien entre les genes d’interet. Conclusion Le contenu proteique et le profil d’expression genomique des CMLV se modifie au cours de l’ACG. Cela pourrait temoigner d’une capacite de proliferation accrue de ces CMLV. La paxilline pourrait constituer une cible therapeutique qui pourrait permettre d’inhiber le remodelage vasculaire au cours de l’ACG.

NF-κB transcription factors play critical role in cell proliferation, cell survival and the physiopathology of numerous cancers. Deregulation of the classical NF-κB pathway is known to be involved in at least the ABC subset of diffuse large B-cell lymphoma (DLBCL). However, the activation status of RelB NF-κB alternative pathway subunit and its role remain unclear. We have demonstrated a frequent engagement of RelB in human DLBCL-derived cell lines. RelB activation protected cells from DNA damage and apoptosis induced by doxorubicin, the main drug in DLBCL treatment. RelB also controlled the expression of the anti-apoptotic protein cIAP2. In a cohort of 66 de novo DLBCL patients, we have directly assessed the DNA binding activity of all NF-κB subunits by EMSA coupled with supershift. RelB activation was present in 66.6% of cases regardless of ABC or GCB classification and was an independent predictor of worse outcome. RelB activation status by EMSA allowed the definition of a RelB gene expression signature that was able to confirm RelB’s negative impact on patient outcome when extended to a larger cohort. Altogether, our study indicates that RelB is a potential new biomarker for DLBCL and sheds light on its important role in DLBCL cell biology.; Les facteurs de transcription de NF-κB jouent un rôle crucial dans la prolifération et la survie cellulaire, et la physiopathologie de nombreux cancers. La dérégulation de la voie classique de NF-κB est connue pour être impliquée au moins dans le sous-groupe ABC du lymphome diffus à grandes cellules B (DLBCL). Cependant, l'état d'activation de la sous-unité RelB de la voie alternative de NF-κB et son rôle restent inconnus. Nous avons démontré un engagement fréquent de RelB dans des lignées cellulaires humaines dérivées de DLBCL. L'activation RelB protège les cellules des dommages à l'ADN et de l'apoptose induites par la doxorubicine, le principal médicament utilisé dans le traitement du DLBCL. RelB a également contrôlé l'expression de la protéine anti-apoptotique cIAP2. Dans une cohorte de 66 patients atteints de DLBCL de novo, nous avons évalué directement l'activité de liaison à l'ADN de toutes les sous-unités de NF-κB par EMSA couplé à des supershifts. L'activation de RelB était présente dans 66,6% des cas, quelle que soit la classification ABC ou GCB, et constituait un prédicteur indépendant d’un mauvais pronostique. Le statut d’activation de RelB établi par l’EMSA a permis de définir une signature d’expression génique liée à RelB capable de confirmer l’impact négatif de RelB sur l’évolution des patients, lorsque étendue à une cohorte plus grande. Finalement, notre étude indique que RelB est potentiellement un nouveau biomarqueur pour le DLBCL et met en évidence son rôle important dans la biologie cellulaire de DLBCL.