Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumourchemoresistance

International audiencePancreatic ductal adenocarcinoma (PDAC) is extremely stroma-rich. Cancer-associated fibroblasts (CAFs) secrete proteins that activate survival and promote chemoresistance of cancer cells. Our results demonstrate that CAF secretome-triggered chemoresistance is abolished upon inhibition of the protein synthesis mTOR/4E-BP1 regulatory pathway which we found highly activated in primary cultures of -SMA-positive CAFs, isolated from human PDAC resections. CAFs selectively express the sst1 somatostatin receptor. The SOM230 analogue (Pasireotide) activates the sst1 receptor and inhibits the mTOR/4E-BP1 pathway and the resultant synthesis of secreted proteins including IL-6. Consequently, tumour growth and chemoresistance in nude mice xenografted with pancreatic cancer cells and CAFs, or with pieces of resected human PDACs, are reduced when chemotherapy (gemcitabine) is combined with SOM230 treatment. While gemcitabine alone has marginal effects, SOM230 is permissive to gemcitabine-induced cancer cell apoptosis and acts as an antifibrotic agent. We propose that selective inhibition of CAF protein synthesis with sst1-directed pharmacological compounds represents an anti-stromal-targeted therapy with promising chemosensitization potential

Contribution à l'étude des mécanismes moléculaires de chimiorésistance des cellules cancéreuses pancréatiques

Pancreatic cancer (PC) is the fourth leading cause of death by cancer in Western countries with a survival rate of less than 5% at five years. Its incidence has been increasing for the last forty years, and, in the absence of improvement in the detection and treatment, PC will become the second leading cause of death by cancer worldwide in 2020. Accordingly, we recently conducted the first gene therapy clinical trial for patients diagnosed with advanced PC, during which patients received using endoscopic ultrasound an intratumoral injection of a non viral vector encoding DCK::UMK and SSTR2 genes. Patients were also treated by gemcitabine. In addition to the feasibility and safety of this protocol, gene therapy results in a clinical benefit in selected patients. We also highlighted two major elements: this beneficial effect (1) is not correlated with the dose administered and (2) reaches a "therapeutic plateau" beyond which the cancer cells are resistant to treatment. These considerations suggest that the intratumoral gene transfer is limiting and that additional mechanisms of drug resistance remain to be identified and defeated to offer new therapeutic perspectives. Therefore, we first investigated the molecular mechanisms that may be involved in the primo resistance of cancer cells to gemcitabine. We highlighted the importance of cytidine deaminase (CDA) in cohort of patients that poorly responded to gemcitabine treatment. This enzyme, which catalyzes the irreversible hydrolytic deamination of CTP in UTP, is also overexpressed in PC as compared to adjacent tissue. In experimental models, the invalidation of CDA results in the profound inhibition of cell proliferation and massive induction of apoptosis, even in the absence of gemcitabine. Using an unbiased proteomic approach, we found that several proteins involved in mitochondrial metabolism are strongly altered in response to CDA targeting. Finally, combining the invalidation of the CDA and gemcitabine treatment results in a rarely achieved inhibition of cancer cell proliferation and tumor progression in vivo. We next investigated new delivery vectors/virus for the gene therapy of pancreatic cancer. We selected oncolytic viruses derived from HSV-1, in which the viral ribonucleotide reductase is mutated, and the expression of the viral protein gamma 34.5 is under the control of the proto-oncogene B-myb promoter (Myb34.5). We first identified a highly favorable molecular context for the replication of Myb34.5 in human pancreatic cancer cells as they over-express not only B-myb, but also cellular ribonucleotide reductase RRM1 and RRM2 whereas expression of ENT1 is decreased. In cancer cell lines, Myb34.5 replicates athigh rate, strongly inhibits cell proliferation and provokes cancer cell death by apoptosis. This effect is increased by the co-treatment with gemcitabine. Finally, Myb34.5 causes the regression of experimental tumors, when combined with gemcitabine. This thesis work opens several interesting prospects: we have identified new therapeutic targets to alleviate the chemoresistance of PC cells and demonstrated the efficacy of oncolytic viruses derived from HSV-1 for the treatment of experimental pancreatic tumors. Ultimately, this work may stem for new combinations of therapeutic genes, delivered or not by oncolytic viruses, to defeat the "therapeutic plateau" observed in PC experimental models and during the Thergap clinical trial and to specifically eradicate pancreatic tumor cells with high efficacy.Le cancer du pancréas (CP) est la quatrième cause de décès par cancer dans les pays occidentaux avec un taux de survie à cinq ans inférieur à 5 %. Son incidence est en augmentation depuis les quarante dernières années ; en l'absence d'amélioration dans le dépistage et le traitement, le CP deviendra la deuxième cause de décès par cancer dans le monde d'ici 2020. Dans ce contexte sombre, nous avons récemment mené le premier essai clinique de thérapie génique pour les formes avancées de ce cancer durant lequel les patients ont reçu une injection intra-tumorale sous écho endoscopie d'un vecteur non viral codant pour le gène de fusion DCK:UMK et le gène SSTR2 combiné au traitement par chimiothérapie. Outre la faisabilité et la sécurité de ce protocole, la thérapie génique se traduit par un bénéfice clinique chez certains patients. Nous avons également mis en évidence deux éléments majeurs: cet effet bénéfique (1) n'est pas corrélé à la dose administrée (2) atteint un "plateau thérapeutique" au-delà duquel les cellules cancéreuses résistent au traitement, suggérant ainsi que le transfert de gène intra-tumoral est limitant et que des mécanismes additionnels de chimiorésistance restent à identifier et à cibler pour proposer de nouvelles stratégies plus efficaces. Ainsi, nous avons recherché les mécanismes moléculaires mis en jeu dans la primo résistance des cellules cancéreuses au traitement par gemcitabine. Dans ce contexte, nous avons ainsi mis en évidence l'importance de la cytidine désaminase (CDA) qui catalyse la désamination hydrolytique irréversible des CTP en UTP. La CDA est surexprimée dans de nombreux cas de patients ne répondant pas à la chimiothérapie et dans les tumeurs pancréatiques par rapport au tissu sain adjacent. Dans des modèles expérimentaux, l'invalidation de la CDA se traduit par une profonde inhibition de la prolifération cellulaire et par l'induction massive d'apoptose, et cela, même en absence de gemcitabine. Une approche protéomique non biaisée nous a permis d'identifier une signature mitochondriale traduisant une reprogrammation métabolique en réponse au ciblage de cette enzyme. Enfin, combiner l'invalidation de la CDA et le traitement par gemcitabine se traduit par une diminution rarement égalée de la prolifération cancéreuse et de la progression tumorale in vivo. Nous avons ensuite recherché de nouveaux vecteurs plus efficaces pour la thérapie génique du cancer du pancréas. Nous avons étudié les virus oncolytiques dérivés d'HSV-1 dans les quels la ribonucléotide réductase virale est mutée, et dont l'expression de la protéine virale gamma 34.5 est sous le contrôle du promoteur du proto-oncogène B-myb (Myb34.5). Nous avons identifié un contexte moléculaire particulièrement favorable à la réplication de ce virus Myb34.5 dans les cellules cancéreuses pancréatiques humaines qui sur-expriment non seulement B-myb, mais aussi les ribonucléotide réductases cellulaires RRM1 et RRM2 alors que l'expression d'ENT1 est diminuée. Dans les lignées cancéreuses, Myb34.5 réplique à fort taux et inhibe de façon drastique la prolifération cellulaire, et induit la mort cellulaire par apoptose. Cet effet est majoré par le co-traitement des cultures par la gemcitabine. Enfin, le virus Myb34.5 en combinaison avec la gemcitabine, provoque la régression de tumeurs expérimentales. Durant ce travail de thèse, nous avons identifié de nouvelles cibles thérapeutiques pour combattre la chimiorésistance des cellules cancéreuses pancréatiques et démontré l'efficacité de virus oncolytiques dérivés d'HSV-1 pour le traitement de tumeurs pancréatiques expérimentales. A terme, ces travaux serviront de base à de nouvelles combinaisons de gènes thérapeutiques, délivrés ou non par des virus oncolytiques, afin de lever le "plateau thérapeutique" observé dans l'essai Thergap et ainsi d'éradiquer efficacement et spécifiquement les cellules tumorales pancréatiques

Study of molecular mechanisms involved in pancreatic cancer cell chemoresistance

Le cancer du pancréas (CP) est la quatrième cause de décès par cancer dans les pays occidentaux avec un taux de survie à cinq ans inférieur à 5 %. Son incidence est en augmentation depuis les quarante dernières années ; en l'absence d'amélioration dans le dépistage et le traitement, le CP deviendra la deuxième cause de décès par cancer dans le monde d'ici 2020. Dans ce contexte sombre, nous avons récemment mené le premier essai clinique de thérapie génique pour les formes avancées de ce cancer durant lequel les patients ont reçu une injection intra-tumorale sous écho endoscopie d'un vecteur non viral codant pour le gène de fusion DCK:UMK et le gène SSTR2 combiné au traitement par chimiothérapie. Outre la faisabilité et la sécurité de ce protocole, la thérapie génique se traduit par un bénéfice clinique chez certains patients. Nous avons également mis en évidence deux éléments majeurs: cet effet bénéfique (1) n'est pas corrélé à la dose administrée (2) atteint un "plateau thérapeutique" au-delà duquel les cellules cancéreuses résistent au traitement, suggérant ainsi que le transfert de gène intra-tumoral est limitant et que des mécanismes additionnels de chimiorésistance restent à identifier et à cibler pour proposer de nouvelles stratégies plus efficaces. Ainsi, nous avons recherché les mécanismes moléculaires mis en jeu dans la primo résistance des cellules cancéreuses au traitement par gemcitabine. Dans ce contexte, nous avons ainsi mis en évidence l'importance de la cytidine désaminase (CDA) qui catalyse la désamination hydrolytique irréversible des CTP en UTP. La CDA est surexprimée dans de nombreux cas de patients ne répondant pas à la chimiothérapie et dans les tumeurs pancréatiques par rapport au tissu sain adjacent. Dans des modèles expérimentaux, l'invalidation de la CDA se traduit par une profonde inhibition de la prolifération cellulaire et par l'induction massive d'apoptose, et cela, même en absence de gemcitabine. Une approche protéomique non biaisée nous a permis d'identifier une signature mitochondriale traduisant une reprogrammation métabolique en réponse au ciblage de cette enzyme. Enfin, combiner l'invalidation de la CDA et le traitement par gemcitabine se traduit par une diminution rarement égalée de la prolifération cancéreuse et de la progression tumorale in vivo. Nous avons ensuite recherché de nouveaux vecteurs plus efficaces pour la thérapie génique du cancer du pancréas. Nous avons étudié les virus oncolytiques dérivés d'HSV-1 dans les quels la ribonucléotide réductase virale est mutée, et dont l'expression de la protéine virale gamma 34.5 est sous le contrôle du promoteur du proto-oncogène B-myb (Myb34.5). Nous avons identifié un contexte moléculaire particulièrement favorable à la réplication de ce virus Myb34.5 dans les cellules cancéreuses pancréatiques humaines qui sur-expriment non seulement B-myb, mais aussi les ribonucléotide réductases cellulaires RRM1 et RRM2 alors que l'expression d'ENT1 est diminuée. Dans les lignées cancéreuses, Myb34.5 réplique à fort taux et inhibe de façon drastique la prolifération cellulaire, et induit la mort cellulaire par apoptose. Cet effet est majoré par le co-traitement des cultures par la gemcitabine. Enfin, le virus Myb34.5 en combinaison avec la gemcitabine, provoque la régression de tumeurs expérimentales. Durant ce travail de thèse, nous avons identifié de nouvelles cibles thérapeutiques pour combattre la chimiorésistance des cellules cancéreuses pancréatiques et démontré l'efficacité de virus oncolytiques dérivés d'HSV-1 pour le traitement de tumeurs pancréatiques expérimentales. A terme, ces travaux serviront de base à de nouvelles combinaisons de gènes thérapeutiques, délivrés ou non par des virus oncolytiques, afin de lever le "plateau thérapeutique" observé dans l'essai Thergap et ainsi d'éradiquer efficacement et spécifiquement les cellules tumorales pancréatiques.Pancreatic cancer (PC) is the fourth leading cause of death by cancer in Western countries with a survival rate of less than 5% at five years. Its incidence has been increasing for the last forty years, and, in the absence of improvement in the detection and treatment, PC will become the second leading cause of death by cancer worldwide in 2020. Accordingly, we recently conducted the first gene therapy clinical trial for patients diagnosed with advanced PC, during which patients received using endoscopic ultrasound an intratumoral injection of a non viral vector encoding DCK::UMK and SSTR2 genes. Patients were also treated by gemcitabine. In addition to the feasibility and safety of this protocol, gene therapy results in a clinical benefit in selected patients. We also highlighted two major elements: this beneficial effect (1) is not correlated with the dose administered and (2) reaches a "therapeutic plateau" beyond which the cancer cells are resistant to treatment. These considerations suggest that the intratumoral gene transfer is limiting and that additional mechanisms of drug resistance remain to be identified and defeated to offer new therapeutic perspectives. Therefore, we first investigated the molecular mechanisms that may be involved in the primo resistance of cancer cells to gemcitabine. We highlighted the importance of cytidine deaminase (CDA) in cohort of patients that poorly responded to gemcitabine treatment. This enzyme, which catalyzes the irreversible hydrolytic deamination of CTP in UTP, is also overexpressed in PC as compared to adjacent tissue. In experimental models, the invalidation of CDA results in the profound inhibition of cell proliferation and massive induction of apoptosis, even in the absence of gemcitabine. Using an unbiased proteomic approach, we found that several proteins involved in mitochondrial metabolism are strongly altered in response to CDA targeting. Finally, combining the invalidation of the CDA and gemcitabine treatment results in a rarely achieved inhibition of cancer cell proliferation and tumor progression in vivo. We next investigated new delivery vectors/virus for the gene therapy of pancreatic cancer. We selected oncolytic viruses derived from HSV-1, in which the viral ribonucleotide reductase is mutated, and the expression of the viral protein gamma 34.5 is under the control of the proto-oncogene B-myb promoter (Myb34.5). We first identified a highly favorable molecular context for the replication of Myb34.5 in human pancreatic cancer cells as they over-express not only B-myb, but also cellular ribonucleotide reductase RRM1 and RRM2 whereas expression of ENT1 is decreased. In cancer cell lines, Myb34.5 replicates athigh rate, strongly inhibits cell proliferation and provokes cancer cell death by apoptosis. This effect is increased by the co-treatment with gemcitabine. Finally, Myb34.5 causes the regression of experimental tumors, when combined with gemcitabine. This thesis work opens several interesting prospects: we have identified new therapeutic targets to alleviate the chemoresistance of PC cells and demonstrated the efficacy of oncolytic viruses derived from HSV-1 for the treatment of experimental pancreatic tumors. Ultimately, this work may stem for new combinations of therapeutic genes, delivered or not by oncolytic viruses, to defeat the "therapeutic plateau" observed in PC experimental models and during the Thergap clinical trial and to specifically eradicate pancreatic tumor cells with high efficacy

The Spatiotemporal Deposition of Lysophosphatidylcholine Within Starch Granules of Maize Endosperm and its Relationships to the Expression of Genes Involved in Endoplasmic Reticulum-Amyloplast Lipid Trafficking and Galactolipid Synthesis

International audienceThe presence of lipids within starch granules is specific to cereal endosperm starches. These starch lipids are composed of lysophospholipids, especially lysophosphatidylcholine (LysoPC) and free fatty acids that strongly impact the assembly and properties of cereal starches. However, the molecular mechanisms associated with this specific lipid routing have never been investigated. In this study, matrix-assisted laser desorption ionization mass spectrometry imaging revealed decreasing gradients in starch LysoPC concentrations from the periphery to the center of developing maize endosperms. This spatiotemporal deposition of starch LysoPC was similar to that previously observed for endoplasmic reticulum (ER)-synthesized storage proteins, i.e. zeins, suggesting that LysoPC might originate in the ER, as already reported for chloroplasts. Furthermore, a decrease of the palmitate concentration of amyloplast galactolipids was observed during endosperm development, correlated with the preferential trapping of palmitoyl-LysoPC by starch carbohydrates, suggesting a link between LysoPC and galactolipid synthesis. Using microarray, the homologous genes of the Arabidopsis ER-chloroplast lipid trafficking and galactolipid synthesis pathways were also expressed in maize endosperm. These strong similarities suggest that the encoded enzymes and transporters are adapted to managing the differences between chloroplast and amyloplast lipid homeostasis. Altogether, our results led us to propose a model where ER-amyloplast lipid trafficking directs the LysoPC towards one of two routes, the first towards the stroma and starch granules and the other towards galactolipid synthesis

Chémotypage de l’albumen de maïs : relation avec la construction de la vitrosité du grain

Chémotypage de l’albumen de maïs : relation avec la construction de la vitrosité du grain. 4. Colloque National du Réseau Français de Biologie des Graine

Comparative Analysis of Transcriptomes from Secondary Reproductives of Three Reticulitermes Termite Species

International audienceTermites are eusocial insects related to cockroaches that feed on lignocellulose. These insects are key species in ecosystems since they recycle a large amount of nutrients but also are pests, exerting major economic impacts. Knowledge on the molecular pathways underlying reproduction, caste differentiation or lignocellulose digestion would largely benefit from additional transcriptomic data. This study focused on transcriptomes of secondary reproductive females (nymphoid neotenics). Thirteen transcriptomes were used: 10 of Reti-culitermes flavipes and R. grassei sequenced from a previous study, and two transcrip-tomes of R. lucifugus sequenced for the present study. After transcriptome assembly and read mapping, we examined interspecific variations of genes expressed by termites or gut microorganisms. A total of 18,323 orthologous gene clusters were detected. Functional annotation and taxonomic assignment were performed on a total of 41,287 predicted con-tigs in the three termite species. Between the termite species studied, functional categories of genes were comparable. Gene ontology (GO) terms analysis allowed the discovery of 9 cellulases and a total of 79 contigs potentially involved in 11 enzymatic activities used in wood metabolism. Altogether, results of this study illustrate the strong potential for the use of comparative interspecific transcriptomes, representing a complete resource for future studies including differentially expressed genes between castes or SNP analysis for population genetics

Alignments of O. edulis in silico sequences

For the in silico SNPs, we investigated O. edulis transcriptome sequence data from eight individuals from the natural range (Cahais et al. 2012, Gayral et al. 2011). For the present study, the 454 and Illumina reads were assembled using a multi-kmer strategy (kmers: 37, 41, 45, 49, 53, 57 and 61, assembled with Velvet version 1.1.03). Contigs longer than 100 bp from every assembly were then meta-assembled with TGICL (http://compbio.dfci.harvard.edu/tgi/software/). The Illumina reads were remapped on the contigs using BWA (0.5.9-r16) and a compressed alignment file was produced using SAMtools view (version 0.1.11). The alignment file was then used to call the SNPs with SAMtools pileup and varFilter (version 0.1.11). In this database, we looked for SNPs that represented different contigs, with a depth ranging from 20 to 500 at the position and no other SNPs in the surrounding 120 bp. The SNP quality score was initially set at 20 but finally, due to the high number of SNPs available, we only used SNPs with the highest score of 227

Lipid Partitioning in Maize ([i]Zea mays[/i] L.) Endosperm Highlights Relationships among Starch Lipids, Amylose, and Vitreousness

Content and composition of maize endosperm lipids and their partition in the floury and vitreous regions were determined for a set of inbred lines. Neutral lipids, i.e., triglycerides and free fatty acids, accounted for more than 80% of endosperm lipids and are almost 2 times higher in the floury than in the vitreous regions. The composition of endosperm lipids, including their fatty acid unsaturation levels, as well as their distribution may be related to metabolic specificities of the floury and vitreous regions in carbon and nitrogen storage and to the management of stress responses during endosperm cell development. Remarkably, the highest contents of starch lipids were observed systematically within the vitreous endosperm. These high amounts of starch lipids were mainly due to lysophosphatidylcholine and were tightly linked to the highest amylose content. Consequently, the formation of amyloselysophosphatidylcholine complexes has to be considered as an outstanding mechanism affecting endosperm vitreousness