Développement d'une thérapie anti-angiogène et anti-tumorale utilisant les propriétés de la protéine à doigts de zinc TIS11b

rédigée en 2008In 2002, our team showed that stimulation of primary bovine adrenocortical cells byadrenocorticotropic hormone ACTH leads to a transcription-independent increase in VEGF expression. ACTH-induced VEGF expression is post-transcriptionally regulated through mRNA stabilization/destabilization via AU-rich elements (ARE) located in 3'-untranslated region (3'UTR) of VEGF mRNA. We previously characterized TIS11b as a zinc finger protein that is induced by ACTH concomitantly with VEGF regulation. TIS11b is a member of a protein family known for destabilizing short-lived mRNAs via ARE sequences. TIS11b interaction with VEGF mRNA has been subsequently confirmed and the implicated site has been identified.In this context, the first aim of my thesis was to evaluate the possibility to develop a new anti-angiogenic and anti-tumoral strategy using the mRNA-destabilizing ability of TIS11b in order to decrease VEGF levels in living tumor cells. To this end, TIS11b cDNA has been fused with PTDs (protein transduction domain, Tat derived from HIV, or the polyarginine peptides R7 and R9). PTDs allowed TIS11b to enter living cells and to intracellularly target VEGF mRNA. We report here that 100 nM of Flag-R7- and Flag-R9-TIS11b fusion proteins decrease VEGF mRNA level as well as VEGF protein level, by 40% after 24h in cultured cells. Moreover, we showed that a single injection of Flag-R9-TIS11b fusion protein into mouse adrenal glands decreases VEGF expression levels to 50% of control. Preliminary encouraging results of tumour growth inhibition were obtained with fusion protein injection into pre-established LL2 tumours in nude mice. ACTH is a pituitary hormone that activates cAMP synthesis and the protein kinase A (PKA) pathway. The second aim of my thesis was to characterize PKA-induced TIS11b phosphorylation in response to ACTH and to study its effect on TIS11b-mediated VEGF mRNA decay. For the first time, our team demonstrated that PKA phosphorylates TIS11b on serine 54 in vitro. A second ACTH-induced phosphorylation site, probably PKA-independent, has been identified on TIS11b-serine 334. These observations open a new field of investigation about the regulation of TIS11b mRNA destabilizing activity by specific phosphorylations.En 2002, notre équipe a montré que la stimulation de cellules primaires cortico-surrénaliennes bovines en culture primaire par l'hormone hypophysaire ACTH induisait l'expression du VEGF de manière indépendante de sa transcription. En effet, en réponse à l'ACTH, l'expression du VEGF est régulée au niveau post-transcriptionnel par la stabilisation/déstabilisation de son transcrit via des séquences riches en AU (ARE) situées dans la région 3‘ non traduite (3'UTR) de son ARNm. Le laboratoire a mis en évidence une protéine, TIS11b, exprimée également en réponse à une stimulation des cellules par l'ACTH, mais de manière concomitante avec la phase de déstabilisation de l'ARNm du VEGF. TIS11b appartient à une famille de protéines déstabilisatrices des ARNm via les séquences ARE. L'interaction de TIS11b avec l'ARNm du VEGF a été confirmée par la suite, et le site de liaison de la protéine à l'ARNm a également été identifié.Dans ce contexte, le premier objectif de ma thèse était d'évaluer la possibilité d'utiliser les propriétés déstabilisatrices de TIS11b sur l'ARNm du VEGF pour une thérapie anti-angiogène et anti-tumorale. Pour cela, la protéine a été vectorisée par la fusion de petits peptides ou PTD (protein transduction domain, Tat issu du VIH ou les polyarginines R7 et R9) lui permettant de traverser les membranes et d'atteindre sa cible intracellulaire, l'ARNm du VEGF. Nous avons pu montrer dans cette étude que 100 nM de protéines de fusion Flag-Tat-, Flag-R7- et Flag-R9-TIS11b sont capables d'induire une diminution du taux d'ARNm du VEGF ainsi que de la production de la protéine VEGF, dans des cellules en culture après 24 h d'incubation. De plus, nous avons pu montrer que l'injection de 100 nM de la protéine de fusion Flag-R9-TIS11b dans la glande corticosurrénalienne de souris, induisait une diminution d'environ 50 % de l'expression du VEGF par cette glande et que cette diminution était maintenue à 48 h. Des résultats préliminaires encourageants d'inhibition de croissance tumorale ont été obtenus avec l'injection de cette protéine de fusion dans des tumeurs pré-établies chez la souris nude, et doivent être confirmés.L'ACTH étant une hormone qui active la voie de l'AMPc et de la protéine kinase A (PKA), le deuxième objectif de ma thèse était de caractériser la phosphorylation de TIS11b par la PKA en réponse à une stimulation par l'ACTH et d'étudier les conséquences de cette phosphorylation sur son activité déstabilisatrice des ARNm. Nous avons pu montrer pour la première fois, que la PKA phosphoryle la protéine TIS11b, in vitro, au niveau de la sérine 54. Un deuxième site de phosphorylation en réponse à une stimulation par l'ACTH, via probablement une autre kinase que la PKA, a été mis en évidence au niveau de la sérine 334. Il semblerait que la protéine TIS11b comporte un domaine d'activation et un domaine d'inhibition susceptibles d'être régulés en réponse à une stimulation par l'ACTH. L'attribution de ces domaines aux sérines 54 et 334 nécessite des expériences complémentaires

Nouvelles approches thérapeutiques du diabète de type 1

LYON1-BU Santé (693882101) / SudocSudocFranceF

Développement d'une thérapie anti-angiogène et anti-tumorale utilisant les propriétés de la protéine à doigts de zinc TIS11b

En 2002, notre équipe a montré que la stimulation de cellules primaires cortico-surrénaliennes bovines en culture primaire par l'hormone hypophysaire ACTH induisait l'expression du VEGF de manière indépendante de sa transcription. En effet, en réponse à l'ACTH, l'expression du VEGF est régulée au niveau post-transcriptionnel par la stabilisation/déstabilisation de son transcrit via des séquences riches en AU (ARE) situées dans la région 3 non traduite (3'UTR) de son ARNm. Le laboratoire a mis en évidence une protéine, TIS11b, exprimée également en réponse à une stimulation des cellules par l'ACTH, mais de manière concomitante avec la phase de déstabilisation de l'ARNm du VEGF. TIS11b appartient à une famille de protéines déstabilisatrices des ARNm via les séquences ARE. L'interaction de TIS11b avec l'ARNm du VEGF a été confirmée par la suite, et le site de liaison de la protéine à l'ARNm a également été identifié. Dans ce contexte, le premier objectif de ma thèse était d'évaluer la possibilité d'utiliser les propriétés déstabilisatrices de TIS11b sur l'ARNm du VEGF pour une thérapie anti-angiogène et anti-tumorale. Pour cela, la protéine a été vectorisée par la fusion de petits peptides ou PTD (protein transduction domain, Tat issu du VIH ou les polyarginines R7 et R9) lui permettant de traverser les membranes et d'atteindre sa cible intracellulaire, l'ARNm du VEGF. Nous avons pu montrer dans cette étude que 100 nM de protéines de fusion Flag-Tat-, Flag-R7- et Flag-R9-TIS11b sont capables d'induire une diminution du taux d'ARNm du VEGF ainsi que de la production de la protéine VEGF, dans des cellules en culture après 24 h d'incubation. De plus, nous avons pu montrer que l'injection de 100 nM de la protéine de fusion Flag-R9-TIS11b dans la glande corticosurrénalienne de souris, induisait une diminution d'environ 50 % de l'expression du VEGF par cette glande et que cette diminution était maintenue à 48 h. Des résultats préliminaires encourageants d'inhibition de croissance tumorale ont été obtenus avec l'injection de cette protéine de fusion dans des tumeurs pré-établies chez la souris nude, et doivent être confirmés. L'ACTH étant une hormone qui active la voie de l'AMPc et de la protéine kinase A (PKA), le deuxième objectif de ma thèse était de caractériser la phosphorylation de TIS11b par la PKA en réponse à une stimulation par l'ACTH et d'étudier les conséquences de cette phosphorylation sur son activité déstabilisatrice des ARNm. Nous avons pu montrer pour la première fois, que la PKA phosphoryle la protéine TIS11b, in vitro, au niveau de la sérine 54. Un deuxième site de phosphorylation en réponse à une stimulation par l'ACTH, via probablement une autre kinase que la PKA, a été mis en évidence au niveau de la sérine 334. Il semblerait que la protéine TIS11b comporte un domaine d'activation et un domaine d'inhibition susceptibles d'être régulés en réponse à une stimulation par l'ACTH. L'attribution de ces domaines aux sérines 54 et 334 nécessite des expériences complémentaires.In 2002, our team showed that stimulation of primary bovine adrenocortical cells byadrenocorticotropic hormone ACTH leads to a transcription-independent increase in VEGF expression. ACTH-induced VEGF expression is post-transcriptionally regulated through mRNA stabilization/destabilization via AU-rich elements (ARE) located in 3 -untranslated region (3 UTR) of VEGF mRNA. We previously characterized TIS11b as a zinc finger protein that is induced by ACTH concomitantly with VEGF regulation. TIS11b is a member of a protein family known for destabilizing short-lived mRNAs via ARE sequences. TIS11b interaction with VEGF mRNA has been subsequently confirmed and the implicated site has been identified. In this context, the first aim of my thesis was to evaluate the possibility to develop a new anti-angiogenic and anti-tumoral strategy using the mRNA-destabilizing ability of TIS11b in order to decrease VEGF levels in living tumor cells. To this end, TIS11b cDNA has been fused with PTDs (protein transduction domain, Tat derived from HIV, or the polyarginine peptides R7 and R9). PTDs allowed TIS11b to enter living cells and to intracellularly target VEGF mRNA. We report here that 100 nM of Flag-R7- and Flag-R9-TIS11b fusion proteins decrease VEGF mRNA level as well as VEGF protein level, by 40% after 24h in cultured cells. Moreover, we showed that a single injection of Flag-R9-TIS11b fusion protein into mouse adrenal glands decreases VEGF expression levels to 50% of control. Preliminary encouraging results of tumour growth inhibition were obtained with fusion protein injection into pre-established LL2 tumours in nude mice. ACTH is a pituitary hormone that activates cAMP synthesis and the protein kinase A (PKA) pathway. The second aim of my thesis was to characterize PKA-induced TIS11b phosphorylation in response to ACTH and to study its effect on TIS11b-mediated VEGF mRNA decay. For the first time, our team demonstrated that PKA phosphorylates TIS11b on serine 54 in vitro. A second ACTH-induced phosphorylation site, probably PKA-independent, has been identified on TIS11b-serine 334. These observations open a new field of investigation about the regulation of TIS11b mRNA destabilizing activity by specific phosphorylations.GRENOBLE1-BU Sciences (384212103) / SudocPARIS-Académie Médecine (751065201) / SudocSudocFranceF

Cibler la dégradation des ARNms médiée par les éléments riches en A et U à l'aide d'une forme active tronquée de la protéine à doigts de zincs TIS11b/BRF1 affecte les principales propriétés de la tumorigenèse mammaire.

International audienceAltered expression of regulatory RNA-binding proteins (RBPs) in cancer leads to abnormal expression of mRNAs encoding many factors involved in cancer hallmarks. While conventional anticancer therapies usually target one pathway at a time, targeting key RBP would affect multiple genes and thus overcome drug resistance. Among the Tristetraprolin family of RBP, TIS11b/BRF1/ZFP36L1 mediates mRNA decay through binding to Adenylate/Uridylate (AU-rich elements) in mRNA 3'-untranslated region and recruitment of mRNA degradation enzymes. Here, we show that TIS11b is markedly underexpressed in three breast cancer cell lines, as well as in breast tumor samples. We hypothesized that restoring intracellular TIS11b levels could impair cancer cell phenotypic traits. We thus generated a derivative of TIS11b called R9-ZnCS334D, by combining N-terminal domain deletion, serine-to-aspartate substitution at position 334 to enhance the function of the protein and fusion to the cell-penetrating peptide polyarginine R9. R9-ZnCS334D not only blunted secretion of vascular endothelial growth factor (VEGF) but also inhibited proliferation, migration, invasion, and anchorage-independent growth of murine 4T1 or human MDA-MB-231 breast cancer cells. Moreover, R9-ZnCS334D prevented endothelial cell organization into vessel-like structures, suggesting that it could potentially target various cell types within the tumor microenvironment. In vivo, injection of R9-ZnCS334D in 4T1 tumors impaired tumor growth, decreased tumor hypoxia, and expression of the epithelial-to-mesenchymal transition (EMT) markers Snail, Vimentin, and N-cadherin. R9-ZnCS334D also hindered the expression of chemokines and proteins involved in cancer-related inflammation and invasion including Fractalkine (CX3CL1), SDF-1 (CXCL12), MCP-1 (CCL2), NOV (CCN3), and Pentraxin-3 (PTX3). Collectively, our data indicate that R9-ZnCS334D counteracts multiple traits of breast cancer cell aggressiveness and suggest that this novel protein could serve as the basis for innovative multi-target therapies in cancer

S100A8/A9 mRNA induction in an ex vivo model of endotoxin tolerance: roles of IL-10 and IFNγ.

Septic syndromes are the leading cause of death in intensive care units. They are characterized by the development of immune dysfunctions such as endotoxin tolerance (ET), whose intensity and duration are associated with increased risk of nosocomial infections and mortality. Alarmins S100A8 and S100A9 have been shown to be increased after septic shock. Importantly, a delayed S100A9 mRNA increase predicts hospital-acquired infection in patients. The aim of this study was to investigate the regulation of S100A8 and S100A9 mRNA expression in an ex vivo model of ET.ET was reproduced ex vivo by priming healthy peripheral blood mononuclear cells (number of donors  = 9 to 10) with low-dose endotoxin (2 ng/ml) before stimulation with high dose endotoxin (100 ng/ml). S100A8 and S100A9 mRNA levels were measured by quantitative real-time polymerase chain reactions.ET was established by observing decreased TNFα and increased IL-10 transcriptomic responses to two subsequent endotoxin challenges. Interestingly, ET was associated with increased S100A8 and S100A9 mRNA expression ex vivo. We showed that IL-10 played a role in this process, since S100A8 and S100A9 mRNA increases were significantly abrogated by IL-10 blockade in the model. Conversely, treatment with rIFN-γ, a pro-inflammatory and immunostimulating molecule known to block ET induction, was able to restore normal S100A8 and S100A9 mRNA in this model.In this ex vivo model, we observed that S100A8 and S100A9 mRNA expression was significantly increased during ET. This reproduced ex vivo the observations we had previously made in septic shock patients. Interestingly, IL-10 blockade and rIFN-γ treatment partially abrogated S100A8/A9 mRNA increases in this model. Pending confirmation in larger, independent clinical studies, these preliminary results suggest that S100A8 and S100A9 mRNA levels might be used as surrogate markers of ET and as stratification tools for personalized immunotherapy in septic shock patients

The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

International audienceTPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3'-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein-protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function

S100A8 and S100A9 mRNA level increases were significantly abrogated by rIFN-γ in endotoxin tolerance model.

<p>Messenger RNA (mRNA) level of TNFα, IL-10, S100A8 and S100A9 in an <i>ex vivo</i> model of endotoxin tolerance. The mRNA level was normalized to that of the reference gene peptidylpropylisomerase B and then compared to the control group. Black columns represent controls (cells without any lipopolysaccharide (LPS)), white columns represent LPS-unprimed cells (only stimulated once with 100 ng/ml LPS) and grey columns represent LPS-primed cells (stimulated three times: 2 ng/ml LPS followed by vehicle or IFN-γ followed by 100 ng/ml LPS). †p<0.01, ‡p<0.05, Wilcoxon paired test. Median (+/− interquartile range) data from 10 independent experiments are given.</p

Quantitative PCR and primers.

a<p>Top sequence is forward primer, bottom is reverse.</p

Endotoxin tolerance is associated with decreased TNFα and increased IL-10, S100A8 and S100A9 mRNA expressions.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100909#pone-0100909-g002" target="_blank">Figures 2A, B, C and D</a>. Messenger RNA (mRNA) level of tolerizable gene (TNFα) and non-tolerizable genes (IL-10, S100A8 and S100A9) in an <i>ex vivo</i> model of endotoxin tolerance. The mRNA level was normalized to that of the reference gene peptidylpropylisomerase B (PPIB) and then compared to the control group. Black columns represent controls (cells without any lipopolysaccharide (LPS)), white columns represent LPS-unprimed cells (only stimulated once with 100 ng/ml LPS), light grey columns represent LPS-primed cells (stimulated twice: 2 ng/ml followed by 100 ng/ml) and dark grey columns represent cells stimulated once with 2 ng/ml LPS without any second stimulation. **<0.01, *<0.05, Wilcoxon signed-rank test. Median (+/− interquartile range) data from 10 independent experiments are given. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100909#pone-0100909-g002" target="_blank">Figure 2E</a>. Correlation between S100A8 mRNA expression (x-axis) and S100A9 mRNA expression (y-axis) in the endotoxin tolerance model. S100A8 and S100A9 mRNA levels were normalized to that of PPIB. Data were obtained from the 10 independent experiments described above.</p