Régulation de l'expression de ligands de l'immunité par la voie Rho/ROCK sur les mélanomes

Ma thèse porte sur l'étude des rôles régulateurs des GTPases Rho et de leurs effecteurs ROCK sur l'expression de ligands du système immunitaire, sur des cellules de mélanomes murins et humains ainsi que les conséquences sur le développement tumoral de modulateurs de la voie RhoA/ROCK. A l'heure actuelle, les traitements du mélanome métastatique ont une efficacité limitée, c'est pourquoi les nouvelles stratégies s'orientent vers l'immunothérapie notamment en recherchant de nouvelles molécules pharmacologiques capables d'amplifier les réponses immunes anti mélanome. Mon travail a porté sur l'étude de trois ligands de l'immunité modulés par la voie RhoA/ROCK : - Nous avons étudié la régulation du ligand MICA qui est exprimé sur des mélanomes humains, mais qui est reconnu par les cellules NK humaines et murines du système immun inné. En utilisant des statines, qui sont des inhibiteurs de l'activité des Rho, nous avons induit une surexpression membranaire de MICA sans toxicité cellulaire. Cette surexpression s'accompagne d'une sensibilisation des mélanomes à la lyse par les cellules NK. Elle induit également un ralentissement de leur croissance tumorale sous-cutanée en souris NMRI nu/nu et une diminution de l'implantation des métastases pulmonaires. Nous avons aussi montré que cette régulation de MICA induite par les statines ne dépendait pas de l'inhibition des GTPases Ras ou Rho mais de la voie de PPAR?. - Nous avons ensuite étudié la régulation de la molécule de costimulation CD70 par les GTPases Rho sur des mélanomes humains et son rôle dans ces tumeurs. Nous avons montré que les mélanomes primitifs expriment CD70, que cette expression diminue au cours de la maladie et que la GTPase RhoA et la voie des MAPK contrôlent positivement l'expression de CD70 sur nos lignées de mélanome humain. De façon surprenante, nous avons aussi montré que CD70 possède une fonction non immunologique dans ces tumeurs. En effet, la trimérisation de CD70 favorise l'invasion tumorale et l'apparition de métastases en activant la voie de signalisation BRAF/MEK/ERK/RhoE et en inhibant les fibres de stress d'actine et des points focaux d'adhésion. - Enfin, nous nous sommes intéressés aux conséquences de la modulation de FasL sur le développement tumoral du mélanome murin B16F10. Des travaux précédents de l'équipe ont montré que la protéine RhoA et ses effecteurs ROCK régulent de façon négative l'expression de FasL à la membrane des cellules B16F10. Nous avons étudié le rôle in vivo de la surexpression de FasL induite par l'inhibition de ROCK par le H1152. Nous avons mis en évidence un ralentissement de la croissance tumorale in vivo chez les souris immunocompétentes. Ce contrôle du développement tumoral est dépendant de la voie Fas/FasL et de l'activité des lymphocytes TCD8+ et de l'IFN-?. De plus, l'inhibition de ROCK réduit le nombre de métastases pulmonaires sans intervention de la réponse immune adaptative. L'ensemble de mes travaux montre que le ciblage de la voie des GTPases Rho et de leurs effecteurs ROCK constitue une approche nouvelle pour amplifier les réponses immunes protectrices innées et adaptatives anti mélanome, suggérant que des inhibiteurs de cette voie pourraient être envisagés dans de nouveaux protocoles d'immunothérapie du mélanomeMy thesis focuses on the study of the regulatory roles of Rho GTPases and their effectors ROCK on the expression of immune system ligands in murine and human melanoma cell lines and the impact on tumor development of modulators of the RhoA/ROCK pathway. Current therapies for metastatic melanoma have poor efficiency. It is the reason why new immunotherapeutic strategies are developed for to find new pharmacological molecules that could improve anti-melanoma immune responses. My work is based on the study of three immune ligands: - We studied the regulation by Rho GTPases of MICA ligand expression in human melanoma cell lines and their recognition by NK cells. Using statins, inhibitors of Rho GTPases activity, we have induced MICA over-expression without any cell toxicity. This MICA over-expression enhanced melanoma cells sensitivity to NK cells lysis, then reduced subcutaneous tumor growth in NMRI nu/nu mice and also decreased pulmonary metastases implantation. We also showed that statins-induced MICA over-expression was not linked to Ras or Rho GTPases inhibition but to PPAR? pathway. - Then, we studied the expression and the function of a co-stimulatory molecule, CD70, and its regulation by the Rho pathway in human melanomas. We demonstrated that the RhoA GTPase and MAPK pathway positively regulate CD70 expression in our melanoma cell lines. Surprisingly, we observed a non-immunological function of CD70 in melanoma. Indeed, CD70 trimerization enhanced melanomas invasion and metastatic capacities through an activation of BRAF/MEK/ERK/RhoE pathway, which inhibited stress fibers and focal adhesions. - Finally, we analyzed the consequences of FasL over-expression on B16F10 murine melanoma development in vivo. Our previous studies have showed that RhoA/ROCK pathway negatively regulates membrane FasL expression on B16F10 cells. We studied in vivo the role of this FasL over-expression induced by ROCK inhibitor H1152, on melanoma cells. We showed tumor growth shrinkage in immunocompetent mice, when B16F10 cells were pretreated with H1152. The Fas/FasL pathway and the activity of TCD8+ cells and IFN- ? induced this tumor slowing down. Moreover, ROCK inhibition induced a reduction of pulmonary metastases implantation independently of T lymphocytes response. Altogether, my work showed that targeting Rho GTPases/ROCK pathway could be interesting in order to improve innate and adaptative anti melanoma immune responses, suggesting that inhibitors of this pathway could be envisaged in new melanoma immunotherapy protocol

Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity.

CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors

CD70 expression does not interfere with PLX-4032-induced inhibition of MAPK pathway and tumor cells killing.

<p>LB1319-MEL cells were transfected with control siRNA (siCtrl) or a CD70-specific siRNA (siCD70) for 72 h. At the same time, these cells were treated or not with 1μM of PLX-4032 (PLX) for 72 h. Then cells were analyzed by Western Blot for phospho- and total-ERK expression and phospho- and total-MEK expression. Actin was used as a loading control <b>(A)</b>. Same experiments have been performed in WM-266-4 cells <b>(B)</b>. Illustrations are representative of three independent experiments. Western Blot illustrations are representative of three different experiments. LB1319-MEL (<b>C</b>) and WM-266-4 (<b>D</b>) melanoma cells were transfected with siNeg or siCD70. Then cells were plated at 1x10^<b>4</b> cells and treated with 1 μM of PLX-4032. 72 h after treatment, cells were counted using Coulter Counter. Quantification of three independent experiments is shown as mean values ± SD. Non-significant (ns) <i>p</i>-value using the Tukey ANOVA test (<b>C</b>, <b>D</b>).</p

MEK kinase positively and transcriptionally controls CD70 membrane and global expression.

<p>LB1319-MEL and WM-266-4 cells were treated with 5 μM of U0126 for 72 h then analyzed by Western Blot for CD70, phospho-MEK and MEK expression. Actin was used as a loading control <b>(A)</b>. LB39-MEL CD70+ cells were treated with 5 μM of U0126 for 72 h then fixed and processed for immunofluorescence directed against CD70. Pictures of U0126 treated cells and control (DMSO) conditions are presented (scale bar 50 μm) <b>(B)</b>. LB1319-MEL cells were treated or not with U0126 for 72 h at indicated concentrations then analyzed by flow cytometry for membrane CD70 expression. Fold induction of CD70 membrane expression in triplicate condition is illustrated <b>(C)</b>. Same experiments were performed in LB39-MEL CD70+ cells <b>(D)</b> and WM-226-4 cells <b>(E)</b>. In LB1319-MEL cells treatment with 5 μM of U0126 for 72 h decreases the accumulation of CD70 mRNA, as detected by RT-qPCR <b>(F)</b>. Results are expressed as mean values ± SD (error bars, <i>n</i> = 3 experiments). *P < 0.05; **P < 0.01; ***P < 0.001 versus control siRNA using the Tukey ANOVA test <b>(C, E)</b> or t-test <b>(D, F)</b>.</p

RhoA GTPase positively and transcriptionally controls CD70 membrane and global expression.

<p>LB1319-MEL cells were transfected with control siRNA (siCtrl), two RhoA-specific siRNAs (siRhoA1, siRhoA2), two RhoB-specific siRNAs (siRhoB1, siRhoB2), or two RhoC-specific siRNAs (siRhoC1, siRhoC2). 72 h post transfection, membrane associated CD70 levels were quantified using flow cytometry <b>(A)</b>. Quantification of three different experiments is shown in <b>(B)</b>. Western Blot analyses confirmed RhoA depletion and decreased in CD70 expression in LB1319-MEL cells 72 h post siRNA transfection. Actin was used as a loading control <b>(C).</b> RhoA over-expression was induced in LB1319-MEL cells by infection with an AdenoRhoA (AdRhoA). 36h post infection, levels of membrane associated CD70 were detected by flow cytometry <b>(D)</b>. Results of three different experiments are shown in <b>(E)</b>. siRhoA2 transfection in LB1319-MEL cells decreases the accumulation of CD70 mRNA, as detected by RT-qPCR <b>(F)</b>. Luciferase assay showed that downregulation of RhoA expression by siRhoA2 in LB1319-MEL cells represses CD70 promoter activity <b>(G)</b>. Flow cytometry histograms are illustrated in Fold induction (FI) corresponding to the normalized level of membrane expressed CD70. Results are expressed as mean values ± SD (error bars, <i>n</i> = 3 experiments). *P < 0.05; **P < 0.01; ***P < 0.001 versus control siRNA using the Tukey ANOVA test <b>(B)</b> or <i>t-test</i> <b>(E, G, H).</b></p

RhoA and MAPK pathways are associated to regulate C70 expression.

<p>LB1319-MEL cells were transfected with control siRNA (siCtrl) and two RhoA-specific siRNAs (siRhoA1, siRhoA2). Western Blot analyses were performed 72 h after transfection for phospho- and total-ERK expression. Actin was used as a loading control <b>(A)</b>. Quantification of three independent experiments is shown in <b>(B)</b>. LB1319-MEL cells were treated with 5μM of U0126 for 72 h then analyzed by the TRBD pull-down assay (Rho binding domain of Rhotekin) <b>(C)</b>. Quantification of three independent experiments is shown in <b>(D)</b>. LB1319-MEL cells were transfected with control siRNA (siCtrl) or siRhoA2 for 72 h. At the same time (24 h after transfection), the same cells were treated or not with 5μM of U0126 for 48 h. Finally, cells were analyzed by flow cytometry for CD70 membrane expression. Quantification of three independent experiments by ISF is shown in <b>(E)</b>. Results are expressed as mean values ± SD (error bars, <i>n</i> = 3 experiments). *P < 0.05; ***P < 0.001 versus control (siCtrl or DMSO) using the Tukey ANOVA test <b>(B, E)</b> or t-test <b>(D)</b>.</p

BRAF protein positively controls CD70 membrane and global expression.

<p>WM-266-4 cells were transfected with control siRNA (siCtrl) and BRAF-specific siRNA (siBRAF). Cells were analyzed after 72 h of transfection by flow cytometry for membrane CD70 expression <b>(A-upper)</b> and by Western Blot for BRAF, global CD70, phospho-ERK and total ERK expression. Actin was used as a loading control <b>(A-lower)</b>. Same experiments have been performed in LB1319-MEL cells <b>(C)</b>. WM-266-4 cells were treated with control medium (DMSO) or with PLX-4032 at indicated concentrations for 72 h then analyzed by flow cytometry for CD70 membrane expression <b>(B-upper)</b> or by Western Blot for CD70 global expression. Actin was used as a loading control <b>(B-lower)</b>. Same experiments have been performed in LB1319-MEL cells <b>(C)</b>. Cytometry results are expressed as mean values ± SD (error bars, <i>n</i> = 3 experiments). **P < 0.01; ***P < 0.001 versus control condition (DMSO or siCtrl) using the <i>t-test</i> <b>(A, C)</b> <i>or</i> Tukey ANOVA test (<b>B</b>, <b>D</b>). Western Blot illustrations are representative of three independent experiments.</p

SARS-CoV-2 seroprevalence and associated factors of infection before and after the Delta wave in French Polynesia: a cross-sectional study

Abstract Background French Polynesia (FP) comprises 75 inhabited islands scattered across five archipelagos. Between July and October 2021, the SARS-CoV-2 Delta variant triggered a much stronger second epidemic wave in FP than the original Wuhan strain, which was dominant from August 2020 to March 2021. Although previous seroprevalence surveys made it possible to determine the proportion of the population infected by SARS-CoV-2 on the two most populated islands (Tahiti and Moorea) after the first (20.6% in Tahiti and 9.4% in Moorea) and second (57.7% in Tahiti) epidemic waves, no data are available for more remote islands. We used blood samples and personal data collected before, during, and after the second wave from inhabitants of several islands within the five archipelagos to assess the prevalence of SARS-CoV-2 infections and identify associated factors. Methods Blood samples and personal data were collected between April and December 2021 as part of the MATAEA study, a cross-sectional survey conducted on a random sample of the adult population representative of the five FP archipelagos and stratified by age and gender. IgG antibodies targeting the SARS-CoV-2 nucleocapsid (N) protein were detected using a recombinant antigen-based microsphere immunoassay. Factors associated with anti-SARS-CoV-2-N seropositivity were identified using logistic regression models. Results Of 1,120 participants, 503 (44.9%) tested positive for anti-SARS-CoV-2-N antibodies, corresponding to a weighted prevalence of 56.8% for the FP population aged 18–69 years. The seroprevalence increased from 21.9% to 62.1% before and during/after the Delta wave. Of these infections, only 28.4% had been diagnosed by health professionals. The odds of being seropositive were lower in males, participants recruited before the Delta wave, those who had never been married, those with a diagnosed respiratory allergy, smokers, and those vaccinated against COVID-19. Conclusions Our results confirm the high impact of the Delta wave in FP. By the end of 2021, 56.8% of the FP population aged 18–69 years had been infected by SARS-CoV-2; the majority of these infections went undetected. Individuals with respiratory allergies were found to be less susceptible to SARS-CoV-2 infection

Real-time surveillance of international SARS-CoV-2 prevalence using systematic traveller arrival screening: An observational study.

BackgroundEffective Coronavirus Disease 2019 (COVID-19) response relies on good knowledge of population infection dynamics, but owing to under-ascertainment and delays in symptom-based reporting, obtaining reliable infection data has typically required large dedicated local population studies. Although many countries implemented Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) testing among travellers, it remains unclear how accurately arrival testing data can capture international patterns of infection, because those arrival testing data were rarely reported systematically, and predeparture testing was often in place as well, leading to nonrepresentative infection status among arrivals.Methods and findingsIn French Polynesia, testing data were reported systematically with enforced predeparture testing type and timing, making it possible to adjust for nonrepresentative infection status among arrivals. Combining statistical models of polymerase chain reaction (PCR) positivity with data on international travel protocols, we reconstructed estimates of prevalence at departure using only testing data from arrivals. We then applied this estimation approach to the United States of America and France, using data from over 220,000 tests from travellers arriving into French Polynesia between July 2020 and March 2022. We estimated a peak infection prevalence at departure of 2.1% (95% credible interval: 1.7, 2.6%) in France and 1% (95% CrI: 0.63, 1.4%) in the USA in late 2020/early 2021, with prevalence of 4.6% (95% CrI: 3.9, 5.2%) and 4.3% (95% CrI: 3.6, 5%), respectively, estimated for the Omicron BA.1 waves in early 2022. We found that our infection estimates were a leading indicator of later reported case dynamics, as well as being consistent with subsequent observed changes in seroprevalence over time. We did not have linked data on traveller demography or unbiased domestic infection estimates (e.g., from random community infection surveys) in the USA and France. However, our methodology would allow for the incorporation of prior data from additional sources if available in future.ConclusionsAs well as elucidating previously unmeasured infection dynamics in these countries, our analysis provides a proof-of-concept for scalable and accurate leading indicator of global infections during future pandemics

Towards elimination of chronic viral hepatitis in French Polynesia: results from a national population-based survey

International audienceBackground: In French Polynesia, hepatitis B virus (HBV) infection appears as a major risk factor for hepatocellular carcinoma (HCC), which detection rate in the Austral archipelago is among the highest in the world. Through a nationally representative cross-sectional survey of the adult population, this study aimed at assessing the prevalence of HBV, but also hepatitis C virus (HCV), and hepatitis delta virus (HDV).Methods: A total of 1942 blood samples from participants aged 18-69 years were tested for anti-HBc, anti-HBs, HBsAg, anti-HCV IgG, and HDV RNA. Complete genome sequencing of detected HBV strains was performed.Findings: Among participants, 315/1834, 582/1834, 33/1834, 0/1857, and 0/33 tested positive for anti-HBc, anti-HBs, HBsAg, anti-HCV IgG, and HDV RNA, respectively. The population prevalence of HBsAg was estimated at 1.0% (95% CI: 0.6-1.7). All HBsAg carriers were born in French Polynesia before vaccination at birth became mandatory. In multivariate analyses, identified factors associated with HBsAg carriage included: the archipelago of residence (p &lt; 0.0001), age (p &lt; 0.0001), and education level (p = 0.0077). HBV genotypes B, C, and F were detected.Interpretation: French Polynesia has a low endemicity level of HBV and its population may be considered at low risk for HCV and HDV infection. However, prevalence of HBsAg was found concerning in Austral (3.8%; 95% CI: 1.9-7.5) and Marquesas (6.5%; 95% CI: 3.8-11) archipelagoes. In the Austral archipelago, the presence of genotype C may account for the elevated rate of HCC. Our findings warrant more efforts to improve access to detection, prevention and care to people born before the systematic vaccination policy application, and residing in higher-risk areas, to achieve HBV elimination in French Polynesia