Les variants d'épissage du VEGF-A : leur rôle dans la progression et la réponse aux thérapies anti-angiogéniques des carcinomes pulmonaires

VEGF-A is one of the most important factors during tumor neoangiogenesis. This role has made it a prime target for the development of therapies. Thus, various drugs targeting VEGF-A (Bevacizumab, Avastin®) or its signaling pathways (VEGFR inhibitors) are currently used in clinical practice, especially in lung adenocarcinomas treatment. However, despite promising initial results, many patients are refractory or escape these therapies, and sometimes, even develop more aggressive tumors. To date, there is no means to identify patients who are likely to respond these treatments. Recently, new splicing variants of VEGF-A resulting from an alternative splicing at the last exon, were identified and called VEGFxxxb. Unlike VEGFxxx, these variants are antiangiogenic. If paracrine effects of VEGF-A isoforms on endothelial cells have been well characterized, few studies have examined their autocrine effects on tumor cells expressing VEGFR1 and VEGFR2. In this context, the main aim of our study was to determine the expression status and biological functions of VEGF165b in primary tumors and derived cell models of non small cell lung carcinoma (NSCLC). Our results identify variable expression profiles of VEGF165b in NSCLC patients, and show that high levels of intratumoral VEGF165b are associated with lymph node metastases. Furthermore, our results identify an autocrine loop through which the VEGFR1/VEGFR2 activation by VEGF165b leads to the appearance a more invasive phenotype on tumor cells. Finally, our results show that treatment of tumor cells by Bevacizumab (BVZ) but also platinum salts, with which it is associated clinics, increases VEGF165b expression and autocrine signaling, which leads to appearance of tumor cells that are more aggressive and resistant to cisplatin-induced apoptosis. These results are the first evidence of the VEGF165b ability to signalize on tumor cells. In a second stage of our work, we investigated the potential role of β1 and β3 integrins in maintaining VEGF165b signaling in NSCLC cells. We show that VEGF165b activates beta1 integrin. This activation leads to a rearrangement of the actin cytoskeleton in stress fibers, which may promote tumor cell migration. Interestingly, BVZ is able to induce this same phenotype by a mechanism requiring the expression of VEGF165b , beta1 integrin and beta3 integrin. The formation of these stress fibers is associated with the activation of downstream signaling pathways involving proteins phosphorylation of FAK and cofilin. In addition, we highlight the existence of neuropilin-2/β1 integrin complexes in NSCLC cells, which are likely to participate in these pathways activation. Thus, VEGF165b and especially BVZ appear able to signal through beata1 and beta3 integrins NSCLC cells, suggesting a role of these integrins in tumor cells response to antiangiogenic therapies. In conclusion, our results are the first evidence of a role of VEGF165b in NSCLC progression and response to antiangiogenic therapies and chemotherapies; they suggest that VEGF165b could be a marker of response to BVZ in NSCLC.Le VEGF-A est l'un des facteurs de croissance les plus importants au cours de la néo-angiogénèse tumorale. Ce rôle en a fait une cible de choix pour le développement de thérapies. Ainsi, différents médicaments le ciblant (Bevacizumab, AvastinR) ou ciblant ses voies de signalisation (inhibiteurs des récepteurs VEGFR) sont actuellement utilisés en clinique, notamment dans les adénocarcinomes pulmonaires. Cependant, malgré des résultats initiaux prometteurs, un grand nombre de patients apparaissent d'emblée résistants ou échappent à ces thérapies, voir même développent des tumeurs plus agressives. A ce jour, il n'existe aucun moyen d'identifier les patients susceptibles de répondre à ces thérapies. Récemment, de nouveaux variants d'épissage du VEGF-A issus d'un épissage alternatif différentiel au niveau du dernier exon ont été identifiés, les isoformes VEGFxxxb. A l'inverse des VEGFxxx, ces variants sont anti-angiogéniques. Si les effets paracrines des isoformes du VEGF-A sur les cellules endothéliales ont été bien caractérisés, bien peu d'études se sont penchées sur leurs effets autocrines sur les cellules tumorales qui expriment à leur surface les récepteurs VEGFR1 et VEGFR2. Dans ce contexte, le but principal de notre étude était de déterminer le statut d'expression et les fonctions biologiques de l'isoforme VEGF165b dans les tumeurs primaires et dans des modèles cellulaires dérivés de Carcinomes Pulmonaires Non à Petites Cellules (CBnPCs). Nos résultats identifient des profils d'expression variables du VEGF165b chez les patients atteints de CBnPCs, et montrent que de hauts niveaux intratumoraux de VEGF165b sont associés à un envahissement ganglionnaire. De plus, nos résultats identifient une boucle autocrine au travers de laquelle l'activation des récepteurs VEGFR1/VEGFR2 par le VEGF165b conduit à l'apparition de cellules tumorales présentant un phénotype plus invasif. Finalement, nous montrons que le traitement des cellules tumorales par le Bevacizumab (BVZ) mais aussi par les sels de platine auquel il est associé en clinique augmente l'expression du VEGF165b, la signalisation autocrine qu'il active et conduit à l'apparition de cellules tumorales plus agressives et résistantes à l'apoptose induite par le cisplatine. Ces résultats sont la première démonstration de la capacité du VEGF165b à signaliser sur les cellules tumorales. Dans une seconde étape de notre travail, nous avons étudié le rôle que pourraient jouer les intégrine β1 et β3 dans le maintien de la signalisation induite par le VEGF165b dans les cellules de CBnPCs. Nous montrons que le VEGF165b active l'intégrine β1. Cette activation aboutit à un réarrangement du cytosquelette d'actine en fibres de stress, ce qui pourrait favoriser la migration de ces cellules. De manière intéressante, le BVZ est capable d'induire ce même phénotype par un mécanisme nécessitant l'expression du VEGF165b, de l'intégrine β1, mais aussi de l'intégrine β3. La formation de ces fibres de stress est associée à l'activation de voies de signalisation d'aval mettant en jeu la phosphorylation des protéines FAK et cofiline. De plus, nous mettons en évidence l'existence de complexes entre la neuropiline 2 et l'intégrine β1 dans les cellules de CBnPCs qui seraient susceptibles de participer à l'activation de ces voies. Ainsi, le VEGF165b et le BVZ apparaissent capable de signaliser à travers les intégrines β1 et β3 dans les cellules de CBnPCs, suggérant un rôle de ces intégrines dans la réponse des cellules tumorales aux thérapies anti-angiogéniques. En conclusion, nos résultats identifient un rôle du variant VEGF165b dans la progression des CBnPCs et la réponse aux thérapies anti-angiogéniques et aux chimiothérapies, ce qui suggère que le VEGF165b pourrait être un marqueur réponse au BVZ dans les CBnPCs

VEGF-A splicing variants : their role in lung carcinoma response to antiangiogenic therappies

Le VEGF-A est l'un des facteurs de croissance les plus importants au cours de la néo-angiogénèse tumorale. Ce rôle en a fait une cible de choix pour le développement de thérapies. Ainsi, différents médicaments le ciblant (Bevacizumab, AvastinR) ou ciblant ses voies de signalisation (inhibiteurs des récepteurs VEGFR) sont actuellement utilisés en clinique, notamment dans les adénocarcinomes pulmonaires. Cependant, malgré des résultats initiaux prometteurs, un grand nombre de patients apparaissent d'emblée résistants ou échappent à ces thérapies, voir même développent des tumeurs plus agressives. A ce jour, il n'existe aucun moyen d'identifier les patients susceptibles de répondre à ces thérapies. Récemment, de nouveaux variants d'épissage du VEGF-A issus d'un épissage alternatif différentiel au niveau du dernier exon ont été identifiés, les isoformes VEGFxxxb. A l'inverse des VEGFxxx, ces variants sont anti-angiogéniques. Si les effets paracrines des isoformes du VEGF-A sur les cellules endothéliales ont été bien caractérisés, bien peu d'études se sont penchées sur leurs effets autocrines sur les cellules tumorales qui expriment à leur surface les récepteurs VEGFR1 et VEGFR2. Dans ce contexte, le but principal de notre étude était de déterminer le statut d'expression et les fonctions biologiques de l'isoforme VEGF165b dans les tumeurs primaires et dans des modèles cellulaires dérivés de Carcinomes Pulmonaires Non à Petites Cellules (CBnPCs). Nos résultats identifient des profils d'expression variables du VEGF165b chez les patients atteints de CBnPCs, et montrent que de hauts niveaux intratumoraux de VEGF165b sont associés à un envahissement ganglionnaire. De plus, nos résultats identifient une boucle autocrine au travers de laquelle l'activation des récepteurs VEGFR1/VEGFR2 par le VEGF165b conduit à l'apparition de cellules tumorales présentant un phénotype plus invasif. Finalement, nous montrons que le traitement des cellules tumorales par le Bevacizumab (BVZ) mais aussi par les sels de platine auquel il est associé en clinique augmente l'expression du VEGF165b, la signalisation autocrine qu'il active et conduit à l'apparition de cellules tumorales plus agressives et résistantes à l'apoptose induite par le cisplatine. Ces résultats sont la première démonstration de la capacité du VEGF165b à signaliser sur les cellules tumorales. Dans une seconde étape de notre travail, nous avons étudié le rôle que pourraient jouer les intégrine β1 et β3 dans le maintien de la signalisation induite par le VEGF165b dans les cellules de CBnPCs. Nous montrons que le VEGF165b active l'intégrine β1. Cette activation aboutit à un réarrangement du cytosquelette d'actine en fibres de stress, ce qui pourrait favoriser la migration de ces cellules. De manière intéressante, le BVZ est capable d'induire ce même phénotype par un mécanisme nécessitant l'expression du VEGF165b, de l'intégrine β1, mais aussi de l'intégrine β3. La formation de ces fibres de stress est associée à l'activation de voies de signalisation d'aval mettant en jeu la phosphorylation des protéines FAK et cofiline. De plus, nous mettons en évidence l'existence de complexes entre la neuropiline 2 et l'intégrine β1 dans les cellules de CBnPCs qui seraient susceptibles de participer à l'activation de ces voies. Ainsi, le VEGF165b et le BVZ apparaissent capable de signaliser à travers les intégrines β1 et β3 dans les cellules de CBnPCs, suggérant un rôle de ces intégrines dans la réponse des cellules tumorales aux thérapies anti-angiogéniques. En conclusion, nos résultats identifient un rôle du variant VEGF165b dans la progression des CBnPCs et la réponse aux thérapies anti-angiogéniques et aux chimiothérapies, ce qui suggère que le VEGF165b pourrait être un marqueur réponse au BVZ dans les CBnPCs.VEGF-A is one of the most important factors during tumor neoangiogenesis. This role has made it a prime target for the development of therapies. Thus, various drugs targeting VEGF-A (Bevacizumab, Avastin®) or its signaling pathways (VEGFR inhibitors) are currently used in clinical practice, especially in lung adenocarcinomas treatment. However, despite promising initial results, many patients are refractory or escape these therapies, and sometimes, even develop more aggressive tumors. To date, there is no means to identify patients who are likely to respond these treatments. Recently, new splicing variants of VEGF-A resulting from an alternative splicing at the last exon, were identified and called VEGFxxxb. Unlike VEGFxxx, these variants are antiangiogenic. If paracrine effects of VEGF-A isoforms on endothelial cells have been well characterized, few studies have examined their autocrine effects on tumor cells expressing VEGFR1 and VEGFR2. In this context, the main aim of our study was to determine the expression status and biological functions of VEGF165b in primary tumors and derived cell models of non small cell lung carcinoma (NSCLC). Our results identify variable expression profiles of VEGF165b in NSCLC patients, and show that high levels of intratumoral VEGF165b are associated with lymph node metastases. Furthermore, our results identify an autocrine loop through which the VEGFR1/VEGFR2 activation by VEGF165b leads to the appearance a more invasive phenotype on tumor cells. Finally, our results show that treatment of tumor cells by Bevacizumab (BVZ) but also platinum salts, with which it is associated clinics, increases VEGF165b expression and autocrine signaling, which leads to appearance of tumor cells that are more aggressive and resistant to cisplatin-induced apoptosis. These results are the first evidence of the VEGF165b ability to signalize on tumor cells. In a second stage of our work, we investigated the potential role of β1 and β3 integrins in maintaining VEGF165b signaling in NSCLC cells. We show that VEGF165b activates beta1 integrin. This activation leads to a rearrangement of the actin cytoskeleton in stress fibers, which may promote tumor cell migration. Interestingly, BVZ is able to induce this same phenotype by a mechanism requiring the expression of VEGF165b , beta1 integrin and beta3 integrin. The formation of these stress fibers is associated with the activation of downstream signaling pathways involving proteins phosphorylation of FAK and cofilin. In addition, we highlight the existence of neuropilin-2/β1 integrin complexes in NSCLC cells, which are likely to participate in these pathways activation. Thus, VEGF165b and especially BVZ appear able to signal through beata1 and beta3 integrins NSCLC cells, suggesting a role of these integrins in tumor cells response to antiangiogenic therapies. In conclusion, our results are the first evidence of a role of VEGF165b in NSCLC progression and response to antiangiogenic therapies and chemotherapies; they suggest that VEGF165b could be a marker of response to BVZ in NSCLC

Pistacia lentiscus L. vegetable oil: Physicochemical quality, composition and antibacterial capacity

Recently, there has been a growing interest in natural products of plant origin as herbal remedies. In this study, the profile of bioactive volatile organic compounds and odorous compounds in a sample of Pistacia lentiscus L. vegetable oil has been determined. The study indicated that the oil extraction yield is influenced by the method, the extraction conditions, the stage of maturity of plant and the sampling area. Analysis by HS‐SPME‐GC–MS and GC–MS showed that monoterpenes and sesquiterpenes represent the most numerous groups among the 57 compounds identified. The most abundant compounds are beta‐myrcene, d‐limonene and linoleic acid ethyl ester. Among other compounds humulene, caryophyllene, a‐muurolene and copaene have been detected known for facilitating wound healing. Moreover, β‐caryophyllene and α‐humulene with anti‐inflammatory effects have been determined. 38 different odorous compounds with herbal, citrus and vegetable aromas were detected by panellists using HS‐SPME‐GC‐O‐MS. Research confirmed that the analysed samples have only pleasant aromas that make it a perfect active agent for medicinal use and may gain acceptance of consumers

Abnormal expression of the pre-mRNA splicing regulators SRSF1, SRSF2, SRPK1 and SRPK2 in non small cell lung carcinoma.

Splicing abnormalities frequently occur in cancer. A key role as splice site choice regulator is played by the members of the SR (Ser/Arg-rich) family of proteins. We recently demonstrated that SRSF2 is involved in cisplatin-mediated apoptosis of human lung carcinoma cell lines. In this study, by using immunohistochemistry, we demonstrate that the SR proteins SRSF1 and SRSF2 are overexpressed in 63% and 65% of lung adenocarcinoma (ADC) as well as in 68% and 91% of squamous cell lung carcinoma (SCC), respectively, compared to normal lung epithelial cells. In addition, we show that SRSF2 overexpression correlates with high level of phosphorylated SRSF2 in both ADC (p<0.0001) and SCC (p = 0.02), indicating that SRSF2 mostly accumulates under a phosphorylated form in lung tumors. Consistently, we further show that the SR-phosphorylating kinases SRPK1 and SRPK2 are upregulated in 92% and 94% of ADC as well as in 72% and 68% of SCC, respectively. P-SRSF2 and SRPK2 scores are correlated in ADC (p = 0.01). Using lung adenocarcinoma cell lines, we demonstrate that SRSF1 overexpression leads to a more invasive phenotype, evidenced by activation of PI3K/AKT and p42/44MAPK signaling pathways, increased growth capacity in soft agar, acquisition of mesenchymal markers such as E cadherin loss, vimentin and fibronectin gain, and increased resistance to chemotherapies. Finally, we provide evidence that high levels of SRSF1 and P-SRSF2 proteins are associated with extensive stage (III-IV) in ADC. Taken together, these results indicate that a global deregulation of pre-mRNA splicing regulators occurs during lung tumorigenesis and does not predict same outcome in both Non Small Cell Lung Carcinoma histological sub-types, likely contributing to a more aggressive phenotype in adenocarcinoma

Phenolic compounds profile of macerates of different edible parts of carob tree (Ceratonia siliqua L.) using UPLC-ESI-Q-TOF-MSE: Phytochemical screening and biological activities

Locust bean pulp and gum extracts were prepared, and phytochemical tests based on color reactions and chromatographic analyzes were performed. A profile of seventy-six phenolic compounds was obtained by the ultra-high performance liquid chromatography with electrospray ionization and quadrupole time-of-flight mass spectrometry. The main groups of phenolic compounds identified in the both extracts of Ceratonia siliqua L., were flavonoids, tannins and phenolic acids. Moreover, carob pulp and gum extracts were tested for their antimicrobial activity using disk diffusion tests which showed sensitivity of the different strains to the analyzed extracts at a concentration of 100 mg/mL. Additionally, the antioxidant activity of Ceratonia siliqua L. extracts was assessed by the 2,2-diphenyl-1-picrylhydrazyl acid test, which confirmed stronger antioxidant properties in the case of the pulp extract. To sum up, carob pulp and gum extracts present promising alternatives to synthetic additives within the medicinal industry, serving as potential antioxidant agents and preservatives that combat bacterial contamination, thereby offering a more natural approach to enhancing product safety and efficacy

Expression of SRSF1, SRSF2 and phosphorylated SRSF2 proteins in NSCLCs.

<p>A, Representative immunostaining from frozen section of normal lung parenchyma and lung cancer tissue with anti SRSF1 (a, b, c), anti SRSF2 (d,e,f) and anti phospho-SRSF2 (g, h, i) antibodies [(a, d, g) normal lung; (b, e, h) ADC; (c, f, i) SCC; immunoperoxidase and haematoxylin counterstaining]. B, Representative western blots demonstrating overexpression of SRSF1 and SRSF2 proteins in lung tumors compared with their matched normal lung tissues. (NL, normal lung; ADC, adenocarcinoma; SCC, squamous cell carcinoma).</p

Demographics and clinical characteristics of the patients.

<p>Abbreviations: ADC, adenocarcinoma; SCC, squamous cell carcinoma; SD, standard deviation.</p

Immunohistochemical analysis of SRPK1 and SRPK2 proteins expression in non-small cell lung cancer according to histological subtype.

<p>Abbreviations: ADC, adenocarcinoma; SCC, squamous cell carcinoma; NSCLC, non-small cell lung carcinoma. Immunostaining scores were calculated by multiplying the number of labeled cells (0–100%) by the level of intensity (1–3). According to this, tumor samples were grouped into three classes (see ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046539#s2" target="_blank">Materials and Methods</a>’ section): class 0 (normal expression, as compared to normal lung), class 1 (moderate overexpression) and class 2 (high overexpression). Statistical analysis was done using Fisher’s exact test.</p

Relationship between SRPK2 protein expression and phospho-SRSF2 status in NSCLC subtypes.

<p>A, Distribution of phospho-SRSF2 scores in tumors displaying normal (class 0), moderate (class +) or overexpression (class ++) of SRPK2, in all the tumors (left panels, NSCLC) and in histological subtypes (right panels, ADC and SCC). B, Distribution of SRPK2 scores according to the tumor stage, in all the tumors (left panels, NSCLC) and in histological subtypes (right panels, ADC and SCC). Statistical analysis was done using Mann-Whitney’s U test.</p