Caractérisation de nouvelles voies régulant l’expression et l’activité des protéines Mcl-1 et PUMA

Cancer is a major public health issue, killing millions of people worldwide each year. The inhibition of apoptosis, a programmed cell death, in its onset and development has been well documented, making it one of the hallmarks of cancer. The regulation of the intrinsic (mitochondrial) pathway of apoptosis is regulated by the Bcl-2 (B cell lymphoma-2) family. Up until now, PUMA, a pro-apoptotic protein, was thought to be mainly expressed at the mitochondria, based on experiments where it had been overexpressed. We showed that endogenous PUMA is mainly expressed in the cytosol of activated or resting B cells. However, upon apoptotic stress, PUMA was able to translocate from the cytosol to the mitochondria, in a caspase-independent but p38-dependent manner, allowing PUMA to bind and inhibit the anti-apoptotic proteins Bcl-2 and Mcl-1, and thereby leading to cell death. The anti-apoptotic proteins, especially Mcl-1, are often overexpressed in tumors. Mcl-1 is a protein with a short half-life, degraded rapidly by the proteasome. This degradation is ubiquitin-dependent, requiring E3 ligases (E3). A handful of E3s and one deubiquitinase (DUB), that hydrolyses the ubiquitin chains, have been reported to regulate Mcl-1 expression. However, they were either very poorly expressed or their inhibition had no impact on Mcl-1 expression in our model. We thus undertook to characterize new E3s and DUBs mediating Mcl-1 ubiquitination. After an immunoprecipitation of Mcl-1 in our cells, followed by a mass spectrometry analysis, we identified the DUB USP14. When knockdown, Mcl-1 expression was selectively increased and its stability enhanced. Our results could help build “double-edge” therapies, removing the breaks on apoptosis on one hand via Mcl-1 downregulation while activating it on the other via PUMA translocation.Le cancer est un problème majeur de santé public, tuant chaque année plusieurs millions de personnes. L’inhibition de la mort cellulaire programmée, l’apoptose, est considérée comme l’un des paramètres principaux impliqués dans son initiation et son développement. La régulation de la voie intrinsèque (mitochondriale) de l’apoptose est régulée par la famille Bcl-2. Jusqu’à maintenant, on pensait que la protéine PUMA, une protéine pro-apoptotique, était principalement exprimée au niveau mitochondrial. Nous avons montré qu’à l’état basal, PUMA était exprimé au niveau du cytosol des lymphocytes B humains. Cependant, suite à un signal apoptotique, PUMA est capable de transloquer du cytosol à la mitochondrie, de façon indépendante des caspases mais dépendante de l’activation de la MAPKinase p38, permettant ainsi son interaction avec les protéines anti-apoptotiques Bcl-2 et Mcl-1 dont l’inhibition conduit à l’apoptose. Les protéines anti-apoptotiques, Mcl-1 notamment, sont souvent surexprimées dans les tumeurs. Mcl-1 est une protéine à courte demi-vie, rapidement dégradée par le protéasome. Cette dégradation dépend de son ubiquitination réalisée par des E3 ligases (E3). Quelques E3 et une déubiquitinase (DUB), hydrolysant les chaînes d’ubiquitine, régulant l’expression de Mcl-1 ont été décrites. Cependant, ces protéines sont soit très peu exprimées, soit leur inhibition n’a pas d’impact sur l’expression de Mcl-1 dans notre modèle. Nous avons donc entrepris de caractériser de nouvelles E3 et DUB régulant l’ubiquitination de Mcl-1. Après une immunoprécipitation de Mcl-1 dans nos cellules, suivie d’une analyse par spectrométrie de masse, nous avons identifié la DUB USP14. Lorsque son expression est diminuée, l’expression et la stabilité de Mcl-1 augmentent de façon sélective. Nos résultats pourraient contribuer à une approche à double-tranchant dans le traitement du cancer, en retirant les freins à l’apoptose via une diminution de l’expression de Mcl-1 d’une part et en l’activant via PUMA de l’autre

Exploiting the potential of autophagy in cisplatin therapy: A new strategy to overcome resistance

Resistance to cisplatin is a major challenge in the current cancer therapy. In order to explore new therapeutic strategies to cisplatin resistance, we evaluated, in a model of lung cancer (H1299 and H460 cell lines), the nature of the pathways leading to cell death. We observed that H1299 displayed a natural resistance to cisplatin due to an inability to trigger an apoptotic response that correlates with the induction of autophagy. However, pharmacological and genetic approaches showed how autophagy was a mechanism associated to cell death rather than to resistance. Indeed, pro-autophagic stimuli such as mTOR or Akt inhibition mediate cell death in both cell lines to a similar extent. We next evaluated the response to a novel platinum compound, monoplatin, able to promote cell death in an exclusive autophagy-dependent manner. In this case, no differences were observed between both cell lines. Furthermore, in response to monoplatin, two molecular hallmarks of cisplatin response (p53 and MAPKs) were not implicated, indicating the ability of this pro-autophagic compound to overcome cisplatin resistance. In summary, our data highlight how induction of autophagy could be used in cisplatin resistant tumours and an alternative treatment for p53 mutated patient in a synthetic lethally approach.This work was supported by grants from Fundación Leticia Castillejo Castillo and Ministerio de Economía y Competitividad (grant SAF2012-30862 to RSP and grant CTQ2011-24434 to FAJ). RSP Research Institute, and the work carried out in his laboratory receive support from the European Community through the regional development funding program (FEDER). JGC received funding from the Regional Ministry of Education and Science of Castilla–La Mancha (FPI-JCCM) and from Fundación Leticia Castillejo Castillo. MCC and RSP have a contract from the INCRECYT progra

Exploiting the potential of autophagy in cisplatin therapy: a new strategy to overcome resistance

Resistance to cisplatin is a major challenge in the current cancer therapy. In order to explore new therapeutic strategies to cisplatin resistance, we evaluated, in a model of lung cancer (H1299 and H460 cell lines), the nature of the pathways leading to cell death. We observed that H1299 displayed a natural resistance to cisplatin due to an inability to trigger an apoptotic response that correlates with the induction of autophagy. However, pharmacological and genetic approaches showed how autophagy was a mechanism associated to cell death rather than to resistance. Indeed, pro-autophagic stimuli such as mTOR or Akt inhibition mediate cell death in both cell lines to a similar extent. We next evaluated the response to a novel platinum compound, monoplatin, able to promote cell death in an exclusive autophagy-dependent manner. In this case, no differences were observed between both cell lines. Furthermore, in response to monoplatin, two molecular hallmarks of cisplatin response (p53 and MAPKs) were not implicated, indicating the ability of this pro-autophagic compound to overcome cisplatin resistance. In summary, our data highlight how induction of autophagy could be used in cisplatin resistant tumours and an alternative treatment for p53 mutated patient in a synthetic lethally approach

Caractérisation de nouvelles voies régulant l’expression et l’activité des protéines Mcl-1 et PUMA

Cancer is a major public health issue, killing millions of people worldwide each year. The inhibition of apoptosis, a programmed cell death, in its onset and development has been well documented, making it one of the hallmarks of cancer. The regulation of the intrinsic (mitochondrial) pathway of apoptosis is regulated by the Bcl-2 (B cell lymphoma-2) family. Up until now, PUMA, a pro-apoptotic protein, was thought to be mainly expressed at the mitochondria, based on experiments where it had been overexpressed. We showed that endogenous PUMA is mainly expressed in the cytosol of activated or resting B cells. However, upon apoptotic stress, PUMA was able to translocate from the cytosol to the mitochondria, in a caspase-independent but p38-dependent manner, allowing PUMA to bind and inhibit the anti-apoptotic proteins Bcl-2 and Mcl-1, and thereby leading to cell death. The anti-apoptotic proteins, especially Mcl-1, are often overexpressed in tumors. Mcl-1 is a protein with a short half-life, degraded rapidly by the proteasome. This degradation is ubiquitin-dependent, requiring E3 ligases (E3). A handful of E3s and one deubiquitinase (DUB), that hydrolyses the ubiquitin chains, have been reported to regulate Mcl-1 expression. However, they were either very poorly expressed or their inhibition had no impact on Mcl-1 expression in our model. We thus undertook to characterize new E3s and DUBs mediating Mcl-1 ubiquitination. After an immunoprecipitation of Mcl-1 in our cells, followed by a mass spectrometry analysis, we identified the DUB USP14. When knockdown, Mcl-1 expression was selectively increased and its stability enhanced. Our results could help build “double-edge” therapies, removing the breaks on apoptosis on one hand via Mcl-1 downregulation while activating it on the other via PUMA translocation.Le cancer est un problème majeur de santé public, tuant chaque année plusieurs millions de personnes. L’inhibition de la mort cellulaire programmée, l’apoptose, est considérée comme l’un des paramètres principaux impliqués dans son initiation et son développement. La régulation de la voie intrinsèque (mitochondriale) de l’apoptose est régulée par la famille Bcl-2. Jusqu’à maintenant, on pensait que la protéine PUMA, une protéine pro-apoptotique, était principalement exprimée au niveau mitochondrial. Nous avons montré qu’à l’état basal, PUMA était exprimé au niveau du cytosol des lymphocytes B humains. Cependant, suite à un signal apoptotique, PUMA est capable de transloquer du cytosol à la mitochondrie, de façon indépendante des caspases mais dépendante de l’activation de la MAPKinase p38, permettant ainsi son interaction avec les protéines anti-apoptotiques Bcl-2 et Mcl-1 dont l’inhibition conduit à l’apoptose. Les protéines anti-apoptotiques, Mcl-1 notamment, sont souvent surexprimées dans les tumeurs. Mcl-1 est une protéine à courte demi-vie, rapidement dégradée par le protéasome. Cette dégradation dépend de son ubiquitination réalisée par des E3 ligases (E3). Quelques E3 et une déubiquitinase (DUB), hydrolysant les chaînes d’ubiquitine, régulant l’expression de Mcl-1 ont été décrites. Cependant, ces protéines sont soit très peu exprimées, soit leur inhibition n’a pas d’impact sur l’expression de Mcl-1 dans notre modèle. Nous avons donc entrepris de caractériser de nouvelles E3 et DUB régulant l’ubiquitination de Mcl-1. Après une immunoprécipitation de Mcl-1 dans nos cellules, suivie d’une analyse par spectrométrie de masse, nous avons identifié la DUB USP14. Lorsque son expression est diminuée, l’expression et la stabilité de Mcl-1 augmentent de façon sélective. Nos résultats pourraient contribuer à une approche à double-tranchant dans le traitement du cancer, en retirant les freins à l’apoptose via une diminution de l’expression de Mcl-1 d’une part et en l’activant via PUMA de l’autre

Characterization of New Regulatory Pathways for Mcl-1 and PUMA Expression and Activity

Le cancer est un problème majeur de santé public, tuant chaque année plusieurs millions de personnes. L’inhibition de la mort cellulaire programmée, l’apoptose, est considérée comme l’un des paramètres principaux impliqués dans son initiation et son développement. La régulation de la voie intrinsèque (mitochondriale) de l’apoptose est régulée par la famille Bcl-2. Jusqu’à maintenant, on pensait que la protéine PUMA, une protéine pro-apoptotique, était principalement exprimée au niveau mitochondrial. Nous avons montré qu’à l’état basal, PUMA était exprimé au niveau du cytosol des lymphocytes B humains. Cependant, suite à un signal apoptotique, PUMA est capable de transloquer du cytosol à la mitochondrie, de façon indépendante des caspases mais dépendante de l’activation de la MAPKinase p38, permettant ainsi son interaction avec les protéines anti-apoptotiques Bcl-2 et Mcl-1 dont l’inhibition conduit à l’apoptose. Les protéines anti-apoptotiques, Mcl-1 notamment, sont souvent surexprimées dans les tumeurs. Mcl-1 est une protéine à courte demi-vie, rapidement dégradée par le protéasome. Cette dégradation dépend de son ubiquitination réalisée par des E3 ligases (E3). Quelques E3 et une déubiquitinase (DUB), hydrolysant les chaînes d’ubiquitine, régulant l’expression de Mcl-1 ont été décrites. Cependant, ces protéines sont soit très peu exprimées, soit leur inhibition n’a pas d’impact sur l’expression de Mcl-1 dans notre modèle. Nous avons donc entrepris de caractériser de nouvelles E3 et DUB régulant l’ubiquitination de Mcl-1. Après une immunoprécipitation de Mcl-1 dans nos cellules, suivie d’une analyse par spectrométrie de masse, nous avons identifié la DUB USP14. Lorsque son expression est diminuée, l’expression et la stabilité de Mcl-1 augmentent de façon sélective. Nos résultats pourraient contribuer à une approche à double-tranchant dans le traitement du cancer, en retirant les freins à l’apoptose via une diminution de l’expression de Mcl-1 d’une part et en l’activant via PUMA de l’autre.Cancer is a major public health issue, killing millions of people worldwide each year. The inhibition of apoptosis, a programmed cell death, in its onset and development has been well documented, making it one of the hallmarks of cancer. The regulation of the intrinsic (mitochondrial) pathway of apoptosis is regulated by the Bcl-2 (B cell lymphoma-2) family. Up until now, PUMA, a pro-apoptotic protein, was thought to be mainly expressed at the mitochondria, based on experiments where it had been overexpressed. We showed that endogenous PUMA is mainly expressed in the cytosol of activated or resting B cells. However, upon apoptotic stress, PUMA was able to translocate from the cytosol to the mitochondria, in a caspase-independent but p38-dependent manner, allowing PUMA to bind and inhibit the anti-apoptotic proteins Bcl-2 and Mcl-1, and thereby leading to cell death. The anti-apoptotic proteins, especially Mcl-1, are often overexpressed in tumors. Mcl-1 is a protein with a short half-life, degraded rapidly by the proteasome. This degradation is ubiquitin-dependent, requiring E3 ligases (E3). A handful of E3s and one deubiquitinase (DUB), that hydrolyses the ubiquitin chains, have been reported to regulate Mcl-1 expression. However, they were either very poorly expressed or their inhibition had no impact on Mcl-1 expression in our model. We thus undertook to characterize new E3s and DUBs mediating Mcl-1 ubiquitination. After an immunoprecipitation of Mcl-1 in our cells, followed by a mass spectrometry analysis, we identified the DUB USP14. When knockdown, Mcl-1 expression was selectively increased and its stability enhanced. Our results could help build “double-edge” therapies, removing the breaks on apoptosis on one hand via Mcl-1 downregulation while activating it on the other via PUMA translocation

SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production

B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt’s lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK–Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response

image_2_SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production.tiff

<p>B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt’s lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK–Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response.</p

image_3_SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production.tiff

<p>B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt’s lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK–Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response.</p

image_1_SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production.tiff

<p>B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt’s lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK–Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response.</p