Melanoma Spheroids Grown Under Neural Crest Cell Conditions Are Highly Plastic Migratory/Invasive Tumor Cells Endowed with Immunomodulator Function

International audienceBACKGROUND: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity. CONCLUSION/SIGNIFICANCE: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability

Ubiquitin and plant viruses, let's play together!

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Identification and Molecular Characterization of the Chloroplast Targeting Domain of Turnip yellow mosaic virus Replication Proteins

Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus infecting plants. The TYMV 140K replication protein is a key organizer of viral replication complex (VRC) assembly, being responsible for recruitment of the viral polymerase and for targeting the VRCs to the chloroplast envelope where viral replication takes place. However, the structural requirements determining the subcellular localization and membrane association of this essential viral protein have not yet been defined. In this study, we investigated determinants for the in vivo chloroplast targeting of the TYMV 140K replication protein. Subcellular localization studies of deletion mutants identified a 41-residue internal sequence as the chloroplast targeting domain (CTD) of TYMV 140K; this sequence is sufficient to target GFP to the chloroplast envelope. The CTD appears to be located in the C-terminal extension of the methyltransferase domain—a region shared by 140K and its mature cleavage product 98K, which behaves as an integral membrane protein during infection. We predicted the CTD to fold into two amphipathic α-helices—a folding that was confirmed in vitro by circular dichroism spectroscopy analyses of a synthetic peptide. The importance for subcellular localization of the integrity of these amphipathic helices, and the function of 140K/98K, was demonstrated by performing amino acid substitutions that affected chloroplast targeting, membrane association and viral replication. These results establish a short internal α-helical peptide as an unusual signal for targeting proteins to the chloroplast envelope membrane, and provide new insights into membrane targeting of viral replication proteins—a universal feature of positive-strand RNA viruses

Nanog and Oct4 overexpression increases motility and transmigration of melanoma cells.

International audiencePURPOSE: Melanoma tumors are highly heterogeneous and can undergo phenotypic modifications depending on their plasticity and the microenvironment, with shifts between proliferative and invasive states. We have shown that melanoma cells, grown as spheroids in a neural crest cell medium, polarize toward an invasive and motile phenotype, in agreement with transcriptomic modulations, including the up-regulation of Nanog and Oct4. Overexpression of these genes was shown to be associated with poor prognosis and metastatic forms of some cancers. We thus investigated implication of Nanog and Oct4, two embryonic transcription factors, in melanoma motility. METHODS: Our team used stable transfection of Nanog or Oct4 in A375 melanoma cell line to investigate motility in a wound healing assay and a transendothelial migration assay. Using semiquantitative RT-PCR, expression of two gene panels involved either in mesenchymal motility or in amoeboid migration was studied. RESULTS: Strongly enhanced capacities of motility and extravasation were observed with cells overexpressing Oct4 and Nanog. The A375 cell line has been described as having a mesenchymal migration type. However, in the Oct4 and Nanog transfectants, several amoeboid migration markers are strongly induced. Accordingly, amoeboid migration inhibitors decrease significantly the transmigration of Oct4- and Nanog-expressing cells through endothelial cells. CONCLUSIONS: We propose here that Nanog and Oct4 pluripotency marker expression in melanoma cells increases the transmigration capacity of these cells through the gain of amoeboid motility, leading to higher invasiveness and aggressiveness

Junctional Adhesion Molecules are required for melanoma cell lines transendothelial migration in vitro.

International audienceOne of the main steps of metastasis is extravasation, a phenomenon well described in lymphocytes, but remaining to be fully uncovered for melanoma. Junctional Adhesion Molecules (JAMs) are controlling the transendothelial migration of leukocytes. To date the role of the JAM proteins, notably JAM-A and JAM-C, has not been examined in melanoma. Here, we compared two melanoma tumor cell lines, A375 and SLM8 cells, the A375 cell line being four times more efficient than the SLM8 cells in the crossing of the endothelial monolayer. We evidence the differential expression of JAM-A and JAM-C in these cell lines with JAM-C mainly expressed in the A375 cell line, and JAM-A detected preferentially in the SLM8 cells. To further dissect the respective roles of these proteins, we used both siRNA and blocking antibodies to decrease JAM-A and JAM-C expression

Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis.

International audienceResistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2-related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)-induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system. (Mol Cancer Res 2008;6(1):42-52)

The context of HLA-DR/CD18 complex in the plasma membrane governs HLA-DR-derived signals in activated monocytes.

International audienceHLA-DR-derived signals in activated monocytes mediate both pro-inflammatory cytokine production and caspase-independent death, and have been postulated to play a role in inflammation and in its resolution, respectively. Herein, using the monocytic/macrophagic human cell line THP-1 primed with IFNgamma (IFNgamma-primed THP-1), we investigated how HLA-DR may integrate both signals. Our inhibition studies demonstrated that if cell death is dependent on PKCbeta activation, the induction of TNFalpha gene expression relies on PTK activation, in particular the Src family of kinases, but both cell responses implicate the beta2-integrin CD18. Accordingly, sequential immunoprecipitation experiments demonstrated that following engagement of HLA-DR on IFNgamma-primed THP-1 cells, the HLA-DR/CD18 complex physically associates with PKCbeta and with PTK. Pharmacological disruption of lipid rafts microdomains abolished the assembly of HLA-DR/CD18/PTK signaling complex, HLA-DR-mediated tyrosine activation, and the PTK-dependent TNFalpha expression in IFNgamma-primed THP-1 cells. In contrast, HLA-DR/CD18/PKCbeta complex was still formed and able to mediate cell death after cholesterol depletion of these cells. These results indicate that while the integrity of lipid rafts is necessary for the transduction of cytokine gene expression through the HLA-DR/CD18 complex, it is not necessary for the induction of the HLA-DR/CD18-dependent cell death. Thus, our study provides experimental evidence indicating the compartmentalization of HLA-DR/CD18 complex within or outside lipid rafts as a mechanism through which HLA-DR can integrate both PTK and PKCbeta signals leading to activation and death, respectively, of activated monocytes. This might provide new insights into how MHC class II signaling may regulate inflammatory response

Pathologic expression of MHC class II is driven by mitogen-activated protein kinases.

International audienceThe class II transactivator (CIITA) is the master regulator of MHC class II molecules (MHC II). In melanoma, the MHC II are constitutively expressed due to an abnormal transcription of CIITA from its promoter III (pIII), and requires the presence of a 1-kb enhancer located upstream from this latter. Since mitogen-activated protein kinases (MAPK) have been shown to be activated in most melanomas, we sought to analyze their possible involvement in CIITA expression. Using chemical inhibitors and dominant-negative constructs of MAPK-ERK kinase (Mek1) and MAPK-JNK, we evidenced the inhibition of MHC II and CIITA expression in melanoma cell lines displaying activated MAPK. Transcriptional regulation by MAPK is known to involve the AP-1 transcription factor family. Sequence analysis revealed an AP-1-responsive motif in the enhancer of CIITA pIII at -5954/-5947 from the site of transcription initiation. Its mutagenesis reduced CIITA expression four- to fivefold in melanoma cell lines and alleviated the effect of dominant-negative constructs of the MAPK pathway. Together, our findings demonstrate that MAPK-ERK and MAPK-JNK are regulators of CIITA transcription in melanoma, and pinpoint an AP-1-responsive site in the CIITA gene pIII. This should have considerable impact on our understanding of the physio-pathologic expression of MHC II

Hypoxia-dependent inhibition of tumor cell susceptibility to CTL-mediated lysis involves NANOG induction in target cells.

International audienceHypoxia is a major feature of the solid tumor microenvironment and is known to be associated with tumor progression and poor clinical outcome. Recently, we reported that hypoxia protects human non-small cell lung tumor cells from specific lysis by stabilizing hypoxia-inducible factor-1α and inducing STAT3 phosphorylation. In this study, we show that NANOG, a transcription factor associated with stem cell self renewal, is a new mediator of hypoxia-induced resistance to specific lysis. Our data indicate that under hypoxic conditions, NANOG is induced at both transcriptional and translational levels. Knockdown of the NANOG gene in hypoxic tumor cells is able to significantly attenuate hypoxia-induced tumor resistance to CTL-dependent killing. Such knockdown correlates with an increase of target cell death and an inhibition of hypoxia-induced delay of DNA replication in these cells. Interestingly, NANOG depletion results in inhibition of STAT3 phosphorylation and nuclear translocation. To our knowledge, this study is the first to show that hypoxia-induced NANOG plays a critical role in tumor cell response to hypoxia and promotes tumor cell resistance to Ag-specific lysis

Coexpression of major histocompatibility complex class II with chemokines and nuclear NFkappaB p50 in melanoma: a rational for their association with poor prognosis.

International audienceThe constitutive expression of major histocompatibility complex class II (MHC II) molecules in melanoma is highly unusual and has been associated with unfavorable clinical outcome and higher metastatic dissemination. This association remains poorly understood and therefore, in this study we looked to whether it is caused by intracellular events that promote tumor progression. We previously reported that MHC II expression in melanoma cells requires active mitogen-activated protein kinase/extracellular signal-related kinase. However, our comparative and molecular analyses of a panel of melanoma cell lines herein provide clear evidence that mitogen-activated protein kinase/extracellular signal-related kinase is not sufficient for HLA-DR expression. We found that the expression of HLA-DR in these tumors rather coincides with the expression of CXCL-1 and CXCL-8 chemokines, both known to be expressed in tumors that invade early and are related to invasive stages of melanoma. The expression of HLA-DR also nicely paralleled that of the nuclear NFkappaB p50 subunit, regulating the expression of these chemokines in melanoma and previously correlated with poor prognosis of melanoma patients, although we provide evidence that NFkappaB is not directly regulating MHC II expression level. The molecular basis for class II transactivator and HLA-DR expression in melanoma therefore remains unsolved, but our findings linking together the expression of HLA-DR, of chemokines involved in invasiveness, and of nuclear NFkappaB p50 strongly support the content that MHC II may be a marker of invasive primary melanoma, and could explain the long-standing association of MHC II expression with overall poor prognosis and unfavorable clinical outcome