Identification d'un profil de microARNs plasmatiques comme biomarqueur potentiel dans le mélanome métastasique

Le mélanome métastatique est une pathologie de mauvais pronostic. A ce jour, aucun biomarqueur prédictif de son évolution n'existe, la classification anatomopathologique conventionnelle reste pour le moment le meilleur facteur pronostic. Les microARNs (miARN), petits ARNs non codants, régulent l'expression génique, sont fortement impliqués dans l'oncogénèse et la progression tumorale et sont souvent dérégulés dans les tumeurs, dont le mélanome. Leurs formes circulantes sont depuis peu étudiées comme potentiel biomarqueur diagnostique et pronostique de nombreux cancers. Nous avons identifié, à l'aide de biopuces, des miARNs plasmatiques dérégulés chez des patients atteints de mélanome métastatique. Nous avons dosé ensuite ces miARNs par RT-qPCR sur une cohorte d'apprentissage et de validation. Nous avons également étudié l'expression des miARNs d'intérêt au niveau tissulaire et cellulaire. Cette étude, sur 74 patients, a mis en évidence 8 miARNs plasmatiques discriminant de façon significative les malades et les sujets sains. Nous avons de plus identifié un profil de deux miARNs permettant de discriminer les deux populations avec d'excellentes performances. Quatre miARNs sont apparus associés à la survie. La majorité des miARNs plasmatiques dérégulés ne parait pas avoir une origine tumorale et serait probablement lié au microenvironnement. L'ensemble de nos résultats montre que la détection du profil des deux miARNs dans le plasma pourrait devenir un biomarqueur diagnostique non invasif, sensible, reproductible et prédictif dans le mélanome métastatique, et pourrait être utilisé à terme pour permettre l'identification précoce de patients à haut risque de récurrence.Metastatic melanoma is a disease of very poor prognosis. To date, no predictive biomarker evolution exist, conventional histological classification is currently the best prognostic factor. MicroRNAs (miRNAs), small non-coding RNAs, regulate gene expression, are strongly implicated in oncogenesis and tumor progression and are often deregulated in tumors, including melanoma. Their circulating forms have recently been studied as a potential diagnostic and prognostic biomarker of many cancers. We studied, by microarray, plasma miRNAs deregulated in patients with metastatic melanoma. Then, we assayed these miRNAs by RT-qPCR in a training and validation cohort. We also investigated the expression of miRNAs of interest in tumoral tissue and cell lines. This study of 74 patients showed eight miRNAs plasma significantly discriminating patients and healthy subjects. We have also identified a profile of two miRNAs to discriminate between the two populations with excellent performance. Four appeared miRNAs associated with survival. The majority of plasma miRNAs deregulated does not seem to have a tumor origin and is probably related to the microenvironment. All of our results show that the detection of the profile of the two miRNAs in the plasma may become a sensitive and reproducible non-invasive diagnostic biomarker in metastatic melanoma and could be used eventually to allow early identification of patients high risk of recurrence

Melanoma Cells Treated with GGTI and IFN-γ Allow Murine Vaccination and Enhance Cytotoxic Response against Human Melanoma Cells

International audienceBACKGROUND: Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I) and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298) stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1]. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL), we demonstrated that in vitro treatment with hIFN-gamma and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC) revealed that modifications induced by hIFN-gamma and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-gamma and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i) activation of CD8 T lymphocytes (CD8+/CD69+); ii) proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+); iii) secretion of hIFN-gamma; and iv) anti-melanoma specific cytotoxic cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that pharmacological treatment of melanoma cell lines with IFN-gamma and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells

Etude du rôle régulateur des protéines Rho dans la mise en place de la réponse immune anti-tumorale et sa caractérisation dans des lignées de mélanomes

La lignée murine de mélanome B16F10 présente comme mécanisme d échappement tumoral un défaut d expression des molécules du complexe majeur d histocompatibilité de classe I (CMH-I), lié à un défaut d apprêtement des protéines, réversible par l interféron-g (IFN-g). Nous avions montré que des protéines géranylgéranylées régulent l expression IFN-g dépendante des CMH-I. Nous avons étudié le rôle régulateur des protéines géranylgéranylées Rho dans la mise en place de la réponse immune anti-tumorale, par l utilisation d un inhibiteur (C3 exoenzyme) et d un activateur (CNF-1) des protéines Rho. Nous avons aussi identifié les lymphocytes T activés responsables du ralentissement de la croissance tumorale in vivo. Enfin, nous avons montré l intérêt de nos traitements pour la mise en place de vaccinations de donneurs humains sains.TOULOUSE3-BU Santé-Centrale (315552105) / SudocSudocFranceF

On LB1319-MEL cells, GGTI-298 enhances hIFN-γ-induced TAP1 and TAP2 expressions.

<p>LB1319-MEL cells were cultivated for 4 days in the presence or in the absence of hIFN-γ (50 IU/ml) and/or GGTI-298 (10 µM) as indicated. <b>A</b>) The expression of molecules implicated in the MHC-I Ag processing pathway were tested in these treated tumor cells. β-actin was used as protein loaded control. The expressions were quantified by calculating the ratio between the protein of interest and the β-actin. We defined the ratio of relevant protein over β-actin for untreated cells equal to 1. <b>B</b>) Expressions of TAP1 and TAP2 proteins in these <i>in vitro</i> treated and permeabilized LB1319-MEL were tested by flow cytometry using TAP1 and TAP2 specific mAbs and a PE-conjugated secondary Ab. <b>C</b>) TAP1 membrane expression was also tested by cytofluorometry on LB1319-MEL cells after 4 days treatment with either increasing doses of hIFN-γ (0, 25, 50 and 100 IU/ml) or with the combination of hIFN-γ (50 IU/ml) and GGTI-298 (10 µM). Results are expressed in ISF, related to isotype controls, as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009043#s2" target="_blank">Material and Methods</a>. Data illustrated are representative of 3 independent experiments.</p

GGTI-298 enhances hIFN-γ-induced MHC-I expression on human melanoma cells.

<p>LB1319-MEL cells were grown for 4 days in culture medium (filled profiles) or in the presence of hIFN-γ alone (50 IU/ml) (thin lines) or GGTI-298 alone (10 µM) (dotted lines) or IFN-γ and GGTI-298 (thick lines). <b>A</b>) HLA-A,B,C ; HLA-A0201; HLA-DP,DQ,DR and isotypic controls expressions were measured by flow cytometry after staining with PE-conjugated mAbs. <b>B</b>) HLA-A0201 (White Column) HLA-A,B,C (Grey Column) and HLA-DP,DQ,DR (Black Column) membrane expressions were tested by cytofluorometry on LB1319-MEL cells after 4 days treatment with either increasing doses of hIFN-γ (0, 25, 50 and 100 IU/ml) or with the combination of hIFN-γ (50 IU/ml) and GGTI-298 (10 µM). Results are expressed in ISF, related to isotype controls, as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009043#s2" target="_blank">Material and Methods</a>. Data are representative of 3 independent experiments.</p

Vaccination of syngeneic mice with mIFN-γ+GGTI-298 pretreated B16F10 cells induces resistance to untreated tumor growth.

<p>C57BL/6 mice were vaccinated with untreated B16F10 cells ; mIFN-γ (50 IU/mL); GGTI-298 (10 µM); or mIFN-γ (50 IU/mL) and GGTI-298 (10 µM) pretreated B16F10 cells. (<b>A</b>) One month after vaccination tumor growth was measured after s.c. injection of 1×10⁵ live B16F10 cells, by Vernier caliper every 3 days. Groups of 6 or 7 C57Bl/6 mice were tested. Data are representative of three independent experiments. (<b>B</b>) The number of tumor free mice were calculated for each kind of vaccination. Results are pooled for the three independent experiments.</p

Syngeneic mice vaccination with mIFN-γ+GGTI-298 pretreated B16F10 cells induces resistance to pulmonary metastases implantation.

<p>Four groups of C57Bl/6 mice were vaccinated with untreated B16F10 cells (NT); mIFN-γ (50 IU/mL) (IFN); GGTI-298 (10 µM) (GGTI); or mIFN-γ (50 IU/mL) and GGTI-298 (10 µM) (IFN+GGTI). One month later, they were challenged intravenously with untreated B16F10 cells. (<b>A</b>) Metastases were screened macroscopically and microscopically as illustrated. (<b>B</b>) Lungs from the 4 groups of mice were slidded vertically and micro-metastases were counted and reported to the lung surfaces (mm²).</p

Enhanced IFN-γ secretion by PBMC co-cultivated with LB1319-MEL cells pre-treated <i>in vitro</i> with hIFN-γ+GGTI-298.

<p>PBMC from 3 HLA-A0201 HD were stimulated twice <i>in vitro</i> with LB1319-MEL cells untreated (NT); or pretreated with 50 UI/mL hIFN-γ (IFN); 10 µM GGTI-298 (GGTI); or the combination of 50 UI/mL hIFN-γ and 10 µM GGTI-298 (IFN+GGTI) as indicated. 24h after the second <i>in vitro</i> stimulation, cytokine secretions in the culture supernatants were measured by Multi-Plex human cytokine assays. Results are illustrated in pg/mL for secretions of IFN-γ, IL-2, IL-4 and IL-10. Data are shown as mean (+/− standard deviation) of duplicate values and were obtained from one donor representative of the two others giving comparable results.</p

On LB1319-MEL cells, GGTI-298 induces enhancement of CD86 and reduction of IFN-γ-induced PD-1L expression.

<p>Membrane expression of CD86 (<b>A</b>) and its isotype control (<b>B</b>) was determined by flow cytometry with PE-conjugated specific antibodies on LB1319-MEL cells after 4 days <i>in vitro</i> treatment with medium alone (filled profiles); hIFN-γ (50 IU/mL) alone (thin lines); GGTI-298 (10 µM) alone (dotted lines); or the combination of both (thick lines). Data are representative of three independent experiments. <b>C</b>) CD86 membrane expression was also tested by cytofluorometry on LB1319-MEL cells after 4 days treatment with either increasing doses of GGTI-298 (0, 5,10, 15 and 20 µM) or with the combination of hIFN-γ (50 IU/ml) and GGTI-298 (10 µM). Results are expressed in percentage of CD86 positive cells. D) Membrane expression of inhibitory molecule PD-1L was determined by flow cytometry with PE-conjugated specific Ab on LB1319-MEL cells after 4 days <i>in vitro</i> treatment with or without hIFN-γ (50 IU/ml) and/or GGTI-298 (10 µM). Results are expressed in ISF, related to isotype controls, as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009043#s2" target="_blank">Material and Methods</a>. Data are representative of 3 independent experiments.</p

Enhanced activation of CD8+ T lymphocytes in PBMC co-cultivated with LB1319-MEL cells pre-treated with hIFN-γ+GGTI-298.

<p>PBMC from HD HLA-A0201 were stimulated twice with LB1319-MEL cells either untreated; or pretreated with hIFN-γ (50 IU/mL); GGTI-298 (10 µM); or hIFN-γ (50 IU/mL) plus GGTI-298 (10 µM). CD8+ activation was evaluated by CD69 membrane expression. Experiments were performed in triplicate with PBMC from one healthy donor representative of one other giving comparable results.</p