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

Statins Stimulate In Vitro Membrane FasL Expression and Lymphocyte Apoptosis through RhoA/ROCK Pathway in Murine Melanoma Cells12

The capacity of FasL molecules expressed on melanoma cells to induce lymphocyte apoptosis contributes to either antitumor immune response or escape depending on their expression level. Little is known, however, about the mechanisms regulating FasL protein expression. Using the murine B16F10 melanoma model weakly positive for FasL, we demonstrated that in vitro treatment with statins, inhibitors of 3-hydroxy-3-methylgutaryl CoA reductase, enhances membrane FasL expression. C3 exotoxin and the geranylgeranyl transferase I inhibitor GGTI-298, but not the farnesyl transferase inhibitor FTI-277, mimic this effect. The capacity of GGTI-298 and C3 exotoxin to inhibit RhoA activity prompted us to investigate the implication of RhoA in FasL expression. Inhibition of RhoA expression by small interfering RNA (siRNA) increased membrane FasL expression, whereas overexpression of constitutively active RhoA following transfection of RhoAV14 plasmid decreased it. Moreover, the inhibition of a RhoA downstream effector p160ROCK also induced this FasL overexpression. We conclude that the RhoA/ROCK pathway negatively regulates membrane FasL expression in these melanoma cells. Furthermore, we have shown that B16F10 cells, through the RhoA/ROCK pathway, promote in vitro apoptosis of Fas-sensitive A20 lymphoma cells. Our results suggest that RhoA/ROCK inhibition could be an interesting target to control FasL expression and lymphocyte apoptosis induced by melanoma cells

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

Colon-specific immune microenvironment regulates cancer progression versus rejection

International audienceImmunotherapies have achieved clinical benefit in many types of cancer but remain limited to a subset ofpatients in colorectal cancer (CRC). Resistance to immunotherapy can be attributed in part to tissuespecificfactors constraining antitumor immunity. Thus, a better understanding of how the colon microenvironmentshapes the immune response to CRC is needed to identify mechanisms of resistance toimmunotherapies and guide the development of novel therapeutics.In an orthotopic mouse model of MC38-CRC, tumor progression was monitored by bioluminescenceimaging and the immune signatures were assessed at a transcriptional level using NanoString and ata cellular level by flow cytometry. Despite initial tumor growth in all mice, only 25% to 35% of micedeveloped a progressive lethal CRC while the remaining animals spontaneously rejected their solid tumor.No tumor rejection was observed in the absence of adaptive immunity, nor when MC38 cells were injectedin non-orthotopic locations, subcutaneously or into the liver. We observed that progressive CRC tumorsexhibited a protumor immune response, characterized by a regulatory T-lymphocyte pattern, discernibleshortly post-tumor implantation, as well as suppressive myeloid cells. In contrast, tumor-rejecting micepresented an early inflammatory response and an antitumor microenvironment enriched in CD8+ T cells.Taken together, our data demonstrate the role of the colon microenvironment in regulating thebalance between anti or protumor immune responses. While emphasizing the relevance of the CRCorthotopic model, they set the basis for exploring the impact of the identified signatures in colon cancerresponse to immunotherapy

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

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