22 research outputs found
Chronic Idiopathic and Chronic Autoimmune Urticaria: Clinical and Immunopathological Features of 68 Subjects
Skin tests with autologous serum elicit an immediate wheal-and-flare response in about 30 â 50% of chronic idiopathic urticaria subjects, suggesting that an autoimmune mechanism might be involved in the pathogenesis of this disease. The aim of the present work, involving 68 subjects with chronic idiopathic urticaria, was to distinguish between the serum-positive and serum-negative cases and highlight the clinical differences between the two groups on the basis of the Breneman scale score. We also tried to correlate the finding of a positive response to the autologous serum skin test with other autoimmune diatheses or fully developed autoimmune disorders. Our results did not demonstrate any significant differences between the two groups with regard to mean age, sex distribution, angioedema and mucosal/cutaneous atopy. However, all subjects with positive autologous serum skin test presented more severe clinical features than serumnegative subjects. We found no differences between the two groups in the incidence of autoimmune disease. Key words: chronic urticaria; autologous serum skin test; autoimmunity
RĂŽle de la forme hypoxique VDAC1, VDAC1-âC, dans lâexpression du cil primaire et la progression tumorale
Voltage-dependent anion channel1 (VDAC1) is a porin of the mitochondrial outer membrane that plays a very important role in the regulation of cellular metabolism and apoptosis. As a consequence of hypoxia (i.e. decrease of oxygen availability), a new form of VDAC1, VDAC1-âC, is produced from a microfusion between abnormally enlarged mitochondria and endolysosomes. Through this mechanism, the endolysosomal asparagine endopeptidase Legumain (LGMN) mediates the cleavage of VDAC1 at specific sites. Clear cell Renal Cell Carcinoma (ccRCC) is the most common form of kidney cancer characterized by loss or mutation of VHL tumor suppressor gene, occurring in both sporadic and hereditary ccRCCs. VHL inactivation is responsible for the normoxic stabilization of hypoxia-inducible transcription factors (HIFs) and thus a dysregulation of the hypoxic pathway. Moreover, ccRCC is usually characterized by loss of the primary cilium (PC) a non-motile sensory organelle of cell surface, making this cancer a model of ciliopathy. The primary cilium serves a number of key functions, among which cell signalization and cell cycle regulation are of special interest in tumorigenesis. In vitro, we demonstrated the presence of VDAC1-âC in RCC4 cells, stabilizing both HIF1-α and HIF2-α. VDAC1-âC was not detected in RCC4 cells re-expressing the wt pVHL (RCC4+pVHL), without HIFs stabilization. The absence of VDAC1-âC in these cells was correlated with a low expression of LGMN and with the increase of the number of ciliated cells compared to RCC4 cell line. The silencing of VDAC1 or of LGMN in RCC4 cells abolished the presence of VDAC1-âC and significantly increased both the number of ciliated cells and their invasive potential. In parallel, we demonstrated that a restrict group of patients, in a cohort of 19 ccRCC patients, were characterized by the absence of VDAC1-âC, low expression of LGMN and an increase in ciliated cells.We found a gene signature based on GLI and IFT20 genes, which are markers of PC activity and formation respectively, whose overexpression (GLI1+/IFT20+) reflected the increase of PC expression. This classification, based on VDAC1, LGMN and the genes GLI1 and IFT20, was reinforced from the study of a TCGA cohort of 375 non-metastatic ccRCC. 48 patients (GLI1+/IFT20+)/375, about 12% of patients, had a poor prognosis in terms of overall survival and disease free survival. We found that an epithelial to mesenchymal transition (EMT) signature and maintenance of glycolytic metabolism were at the basis of the increase in tumor aggressiveness. Moreover, we found that patients with i) an increased expression of PC, ii) the absence of VDAC1-âC and iii) a GLI1+/IFT20+ signature were more resistant to sunitinib, the current standard of care treatment for metastatic ccRCC, and we confirmed the same results in vitro, in ccRCC cell lines. However, since the immunophenoscore was in favor to GLI1+/IFT20+ group of patients, immunotherapy could be a particularly beneficial treatment. To understand the VDAC1-âC -dependent restraint of PC expression, RAS-transformed mouse embryonic fibroblasts (MEF) cell lines knockout for VDAC1 (Vdac1-/-) expressing i) human VDAC1 wt (VDAC1+/+), ii) a non- cleavable form of VDAC1 (VDAC1Mut) and iii) only the truncated form of VDAC1 (VDAC1-STOP) have been engineered. We first determined that VDAC1-âC is involved in the increase of glycolysis and respiration and that VDAC1-âC confers the capability to metabolize more metabolites. We also demonstrated that VDAC1-âC inhibits PC formation, thus participating to the ciliopathic phenotype of tumors. Finally, we demonstrated that the presence of VDAC1-âC can also occur in normoxia upon silencing of the iron-sulfur biosynthetic machinery in mitochondria or with impaired iron homeostasis, attributing a new role to VDAC1 in the context of iron deprivation.Thus, my research demonstrated a new function of VDAC1 and, in particular, of VDAC1-âC in both hypoxic and normoxic contexts.Le voltage-dependent anion channel 1 (VDAC1) est une porine exprimĂ©e au niveau de la membrane externe des mitochondries et qui joue un rĂŽle trĂšs important dans la rĂ©gulation du mĂ©tabolisme cellulaire et de l'apoptose. En condition dâhypoxie (c'est-Ă -dire de diminution de la disponibilitĂ© en oxygĂšne), une nouvelle forme de VDAC1, VDAC1-ÎC, est produite par la microfusion entre mitochondries et endolysosomes. Par ce mĂ©canisme, lâasparagine endopeptidase endolysosomale Legumain (LGMN) induit le clivage de VDAC1 sur des sites spĂ©cifiques.Le carcinome Ă cellules claires des cellules rĂ©nales (ccRCC) est la forme la plus courante de cancer du rein caractĂ©risĂ©e par la perte ou la mutation du gĂšne suppresseur de tumeur vhl, aussi bien dans les ccRCC sporadiques que dans les ccRCC hĂ©rĂ©ditaires. L'inactivation de pVHL est responsable de la stabilisation en normoxie des facteurs de transcription Hypoxia-Inducible Factor (HIF) ainsi que de la dĂ©rĂ©gulation de la voie hypoxique. De plus, les ccRCC sont gĂ©nĂ©ralement caractĂ©risĂ©s par la perte du cil primaire (PC), un organite sensoriel non mobile prĂ©sent Ă la surface cellulaire, et faisant de ce cancer un modĂšle de ciliopathie. Le cil primaire remplit plusieurs fonctions clĂ©s, parmi lesquelles la signalisation et la rĂ©gulation du cycle cellulaire revĂȘtent un intĂ©rĂȘt particulier pour la tumorigenĂšse.In vitro, nous avons Ă©tabli que lâabsence de VDAC1-ÎC dans les lignĂ©es cellulaires ccRCC correspondait Ă une multiplication du nombre de cellules ciliĂ©es, Ă une faible expression de la LGMN et Ă une augmentation de leur potentiel invasif.En parallĂšle, nous avons dĂ©montrĂ©, in vivo, qu'un groupe restreint de patients, dans une cohorte de 19 patients ccRCC, Ă©tait caractĂ©risĂ© par l'absence de VDAC1-ÎC, une faible expression de LGMN et une augmentation des cellules ciliĂ©es. Nous avons caractĂ©risĂ© une signature gĂ©nique basĂ©e sur les gĂšnes GLI1 et IFT20, qui sont respectivement des marqueurs de l'activitĂ© et de la formation du PC. Leur surexpression (IFT20+/GLI1+) reflĂšte l'augmentation de l'expression du PC. Cette classification, basĂ©e sur VDAC1, LGMN, et sur les gĂšnes GLI1 et IFT20, a Ă©tĂ© renforcĂ©e Ă partir dâune cohorte de 375 ccRCC non mĂ©tastatiques du TCGA. 48 patients (GLI1+/IFT20+)/375, soit 12% des patients, avaient un mauvais pronostic. Nous avons constatĂ© que la signature de transition Ă©pithĂ©liale Ă mĂ©senchymateuse (EMT) et le maintien du mĂ©tabolisme glycolytique Ă©taient Ă la base de l'augmentation de l'agressivitĂ© de la tumeur. De plus, nous avons constatĂ© que les patients avec i) une expression accrue de PC, ii) l'absence de VDAC1-ÎC et iii) une signature GLI1+/IFT20+ Ă©taient plus rĂ©sistants au sunitinib, le traitement de rĂ©fĂ©rence actuel des ccRCC mĂ©tastatiques. Cependant, lâimmunophĂ©noscore Ă©tant en faveur du groupe de patients GLI1+/IFT20+, lâimmunothĂ©rapie pourrait ĂȘtre un traitement particuliĂšrement bĂ©nĂ©fique.Afin de mieux comprendre lâimpact de VDAC1-ÎC sur la biogenĂšse du PC, la lignĂ©e cellulaire de fibroblastes de souris embryonnaires (MEF), transformĂ©es par RAS, et invalidĂ©e pour VDAC1 (Vdac1-/-) ont Ă©tĂ© transfectĂ©es avec un vecteur exprimant i) le VDAC1 humain sauvage (wt VDAC1+/+), ii) la forme mutĂ©e et non clivable de VDAC1 (VDAC1Mut) ainsi que iii) la forme tronquĂ©e de VDAC1 (VDAC1-STOP). Nous avons ainsi dĂ©terminĂ© que VDAC1-ÎC est bien impliquĂ© directement dans une meilleure plasticitĂ© mĂ©tabolique et dans lâinhibition de la formation de PC, participant ainsi au phĂ©notype ciliopathique des tumeurs.Enfin, nous avons dĂ©montrĂ© que la prĂ©sence de VDAC1-ÎC peut Ă©galement se produire en normoxie lors de la dĂ©sactivation du mĂ©canisme de biosynthĂšse fer-soufre dans les mitochondries ou lorsque lâhomĂ©ostasie du fer est altĂ©rĂ©e, attribuant un nouveau rĂŽle Ă VDAC1 dans le contexte de carence en fer. Ainsi, mes recherches ont dĂ©montrĂ© une nouvelle fonction de VDAC1, et en particulier de VDAC1-ÎC, dans un contexte hypoxique mais Ă©galement normoxique
Role of VDAC1 hypoxic form, VDAC1-âC, in the expression of primary cilia and tumor progression
Le voltage-dependent anion channel 1 (VDAC1) est une porine exprimĂ©e au niveau de la membrane externe des mitochondries et qui joue un rĂŽle trĂšs important dans la rĂ©gulation du mĂ©tabolisme cellulaire et de l'apoptose. En condition dâhypoxie (c'est-Ă -dire de diminution de la disponibilitĂ© en oxygĂšne), une nouvelle forme de VDAC1, VDAC1-ÎC, est produite par la microfusion entre mitochondries et endolysosomes. Par ce mĂ©canisme, lâasparagine endopeptidase endolysosomale Legumain (LGMN) induit le clivage de VDAC1 sur des sites spĂ©cifiques.Le carcinome Ă cellules claires des cellules rĂ©nales (ccRCC) est la forme la plus courante de cancer du rein caractĂ©risĂ©e par la perte ou la mutation du gĂšne suppresseur de tumeur vhl, aussi bien dans les ccRCC sporadiques que dans les ccRCC hĂ©rĂ©ditaires. L'inactivation de pVHL est responsable de la stabilisation en normoxie des facteurs de transcription Hypoxia-Inducible Factor (HIF) ainsi que de la dĂ©rĂ©gulation de la voie hypoxique. De plus, les ccRCC sont gĂ©nĂ©ralement caractĂ©risĂ©s par la perte du cil primaire (PC), un organite sensoriel non mobile prĂ©sent Ă la surface cellulaire, et faisant de ce cancer un modĂšle de ciliopathie. Le cil primaire remplit plusieurs fonctions clĂ©s, parmi lesquelles la signalisation et la rĂ©gulation du cycle cellulaire revĂȘtent un intĂ©rĂȘt particulier pour la tumorigenĂšse.In vitro, nous avons Ă©tabli que lâabsence de VDAC1-ÎC dans les lignĂ©es cellulaires ccRCC correspondait Ă une multiplication du nombre de cellules ciliĂ©es, Ă une faible expression de la LGMN et Ă une augmentation de leur potentiel invasif.En parallĂšle, nous avons dĂ©montrĂ©, in vivo, qu'un groupe restreint de patients, dans une cohorte de 19 patients ccRCC, Ă©tait caractĂ©risĂ© par l'absence de VDAC1-ÎC, une faible expression de LGMN et une augmentation des cellules ciliĂ©es. Nous avons caractĂ©risĂ© une signature gĂ©nique basĂ©e sur les gĂšnes GLI1 et IFT20, qui sont respectivement des marqueurs de l'activitĂ© et de la formation du PC. Leur surexpression (IFT20+/GLI1+) reflĂšte l'augmentation de l'expression du PC. Cette classification, basĂ©e sur VDAC1, LGMN, et sur les gĂšnes GLI1 et IFT20, a Ă©tĂ© renforcĂ©e Ă partir dâune cohorte de 375 ccRCC non mĂ©tastatiques du TCGA. 48 patients (GLI1+/IFT20+)/375, soit 12% des patients, avaient un mauvais pronostic. Nous avons constatĂ© que la signature de transition Ă©pithĂ©liale Ă mĂ©senchymateuse (EMT) et le maintien du mĂ©tabolisme glycolytique Ă©taient Ă la base de l'augmentation de l'agressivitĂ© de la tumeur. De plus, nous avons constatĂ© que les patients avec i) une expression accrue de PC, ii) l'absence de VDAC1-ÎC et iii) une signature GLI1+/IFT20+ Ă©taient plus rĂ©sistants au sunitinib, le traitement de rĂ©fĂ©rence actuel des ccRCC mĂ©tastatiques. Cependant, lâimmunophĂ©noscore Ă©tant en faveur du groupe de patients GLI1+/IFT20+, lâimmunothĂ©rapie pourrait ĂȘtre un traitement particuliĂšrement bĂ©nĂ©fique.Afin de mieux comprendre lâimpact de VDAC1-ÎC sur la biogenĂšse du PC, la lignĂ©e cellulaire de fibroblastes de souris embryonnaires (MEF), transformĂ©es par RAS, et invalidĂ©e pour VDAC1 (Vdac1-/-) ont Ă©tĂ© transfectĂ©es avec un vecteur exprimant i) le VDAC1 humain sauvage (wt VDAC1+/+), ii) la forme mutĂ©e et non clivable de VDAC1 (VDAC1Mut) ainsi que iii) la forme tronquĂ©e de VDAC1 (VDAC1-STOP). Nous avons ainsi dĂ©terminĂ© que VDAC1-ÎC est bien impliquĂ© directement dans une meilleure plasticitĂ© mĂ©tabolique et dans lâinhibition de la formation de PC, participant ainsi au phĂ©notype ciliopathique des tumeurs.Enfin, nous avons dĂ©montrĂ© que la prĂ©sence de VDAC1-ÎC peut Ă©galement se produire en normoxie lors de la dĂ©sactivation du mĂ©canisme de biosynthĂšse fer-soufre dans les mitochondries ou lorsque lâhomĂ©ostasie du fer est altĂ©rĂ©e, attribuant un nouveau rĂŽle Ă VDAC1 dans le contexte de carence en fer. Ainsi, mes recherches ont dĂ©montrĂ© une nouvelle fonction de VDAC1, et en particulier de VDAC1-ÎC, dans un contexte hypoxique mais Ă©galement normoxique.Voltage-dependent anion channel1 (VDAC1) is a porin of the mitochondrial outer membrane that plays a very important role in the regulation of cellular metabolism and apoptosis. As a consequence of hypoxia (i.e. decrease of oxygen availability), a new form of VDAC1, VDAC1-âC, is produced from a microfusion between abnormally enlarged mitochondria and endolysosomes. Through this mechanism, the endolysosomal asparagine endopeptidase Legumain (LGMN) mediates the cleavage of VDAC1 at specific sites. Clear cell Renal Cell Carcinoma (ccRCC) is the most common form of kidney cancer characterized by loss or mutation of VHL tumor suppressor gene, occurring in both sporadic and hereditary ccRCCs. VHL inactivation is responsible for the normoxic stabilization of hypoxia-inducible transcription factors (HIFs) and thus a dysregulation of the hypoxic pathway. Moreover, ccRCC is usually characterized by loss of the primary cilium (PC) a non-motile sensory organelle of cell surface, making this cancer a model of ciliopathy. The primary cilium serves a number of key functions, among which cell signalization and cell cycle regulation are of special interest in tumorigenesis. In vitro, we demonstrated the presence of VDAC1-âC in RCC4 cells, stabilizing both HIF1-α and HIF2-α. VDAC1-âC was not detected in RCC4 cells re-expressing the wt pVHL (RCC4+pVHL), without HIFs stabilization. The absence of VDAC1-âC in these cells was correlated with a low expression of LGMN and with the increase of the number of ciliated cells compared to RCC4 cell line. The silencing of VDAC1 or of LGMN in RCC4 cells abolished the presence of VDAC1-âC and significantly increased both the number of ciliated cells and their invasive potential. In parallel, we demonstrated that a restrict group of patients, in a cohort of 19 ccRCC patients, were characterized by the absence of VDAC1-âC, low expression of LGMN and an increase in ciliated cells.We found a gene signature based on GLI and IFT20 genes, which are markers of PC activity and formation respectively, whose overexpression (GLI1+/IFT20+) reflected the increase of PC expression. This classification, based on VDAC1, LGMN and the genes GLI1 and IFT20, was reinforced from the study of a TCGA cohort of 375 non-metastatic ccRCC. 48 patients (GLI1+/IFT20+)/375, about 12% of patients, had a poor prognosis in terms of overall survival and disease free survival. We found that an epithelial to mesenchymal transition (EMT) signature and maintenance of glycolytic metabolism were at the basis of the increase in tumor aggressiveness. Moreover, we found that patients with i) an increased expression of PC, ii) the absence of VDAC1-âC and iii) a GLI1+/IFT20+ signature were more resistant to sunitinib, the current standard of care treatment for metastatic ccRCC, and we confirmed the same results in vitro, in ccRCC cell lines. However, since the immunophenoscore was in favor to GLI1+/IFT20+ group of patients, immunotherapy could be a particularly beneficial treatment. To understand the VDAC1-âC -dependent restraint of PC expression, RAS-transformed mouse embryonic fibroblasts (MEF) cell lines knockout for VDAC1 (Vdac1-/-) expressing i) human VDAC1 wt (VDAC1+/+), ii) a non- cleavable form of VDAC1 (VDAC1Mut) and iii) only the truncated form of VDAC1 (VDAC1-STOP) have been engineered. We first determined that VDAC1-âC is involved in the increase of glycolysis and respiration and that VDAC1-âC confers the capability to metabolize more metabolites. We also demonstrated that VDAC1-âC inhibits PC formation, thus participating to the ciliopathic phenotype of tumors. Finally, we demonstrated that the presence of VDAC1-âC can also occur in normoxia upon silencing of the iron-sulfur biosynthetic machinery in mitochondria or with impaired iron homeostasis, attributing a new role to VDAC1 in the context of iron deprivation.Thus, my research demonstrated a new function of VDAC1 and, in particular, of VDAC1-âC in both hypoxic and normoxic contexts
Primary Cilium in Cancer Hallmarks
The primary cilium is a solitary, nonmotile and transitory appendage that is present in virtually all mammalian cells. Our knowledge of its ultrastructure and function is the result of more than fifty years of research that has dramatically changed our perspectives on the primary cilium. The mutual regulation between ciliogenesis and the cell cycle is now well-recognized, as well as the function of the primary cilium as a cellular “antenna” for perceiving external stimuli, such as light, odorants, and fluids. By displaying receptors and signaling molecules, the primary cilium is also a key coordinator of signaling pathways that converts extracellular cues into cellular responses. Given its critical tasks, any defects in primary cilium formation or function lead to a wide spectrum of diseases collectively called “ciliopathies”. An emerging role of primary cilium is in the regulation of cancer development. In this review, we seek to describe the current knowledge about the influence of the primary cilium in cancer progression, with a focus on some of the events that cancers need to face to sustain survival and growth in hypoxic microenvironment: the cancer hallmarks
Dysfunction in the mitochondrial Fe-S assembly machinery leads to formation of the chemoresistant truncated VDAC1 isoform without HIF-1α activation
International audienceBiogenesis of iron-sulfur clusters (ISC) is essential to almost all forms of life and involves complex protein machineries. This process is initiated within the mitochondrial matrix by the ISC assembly machinery. Cohort and case report studies have linked mutations in ISC assembly machinery to severe mitochondrial diseases. The voltage-dependent anion channel (VDAC) located within the mitochondrial outer membrane regulates both cell metabolism and apoptosis. Recently, the C-terminal truncation of the VDAC1 isoform, termed VDAC1-ÎC, has been observed in chemoresistant late-stage tumor cells grown under hypoxic conditions with activation of the hypoxia-response nuclear factor HIF-1α. These cells harbored atypical enlarged mitochondria. Here, we show for the first time that depletion of several proteins of the mitochondrial ISC machinery in normoxia leads to a similar enlarged mitochon-dria phenotype associated with accumulation of VDAC1-ÎC. This truncated form of VDAC1 accumulates in the absence of HIF-1α and HIF-2α activations and confers cell resistance to drug-induced apoptosis. Furthermore, we show that when hypoxia and siRNA knock-down of the ISC machinery core components are coupled, the cell phenotype is further accentuated , with greater accumulation of VDAC1-ÎC. Interestingly, we show that hypoxia promotes the downregulation of several proteins (ISCU, NFS1, FXN) involved in the early steps of mitochondrial Fe-S cluster biogenesis. Finally, we have identified the mitochondria-associated membrane (MAM) localized Fe-S protein CISD2 as a link between ISC machinery downregulation and accumulation of anti-apoptotic VDAC1-ÎC. Our results are the first to associate dysfunction in Fe-S cluster biogenesis with cleavage of VDAC1, a form which has previously been shown to promote tumor resistance to chemotherapy, and raise new perspectives for targets in cancer therapy
Knock-down of CISD2 induces the accumulation of the truncated VDAC1 form.
<p>(A) Total protein extracts of either non-transfected (-) or transfected HeLa cells for 3 days with scramble (NC)-, <i>cisd2</i>-, or <i>nfs1</i>-siRNA were analyzed by immunoblotting using antibodies against VDACs poly, CISD2, and α-tubulin, which was used as loading control. Eight independent experiments were performed and a representative western blot is shown. (B) Total mRNA was extracted from HeLa cells transfected with scramble (NC)-, <i>cisd2</i>-, or <i>nfs1</i>-siRNA. Twenty-four hours after transfection, the mRNA levels of <i>cisd2</i> and <i>nfs1</i> were determined by quantitative RT-qPCR. Data were normalized to <i>gapdh</i> mRNA levels. Means ± standard deviation of n = 4 independent experiments are presented (*** p < 0.001).</p