Melanoma is the most aggressive skin cancer in humans that often expresses MHC class II (MHC II) molecules, which could make these tumors eliminable by the immune system. However, this MHC II expression has been associated with poor prognosis, and there is a lack of immune-mediated eradication. The lymphocyte activation gene-3 (LAG-3) is a natural ligand for MHC II that is substantially expressed on melanoma-infiltrating T cells including those endowed with potent immune-suppressive activity. Based on our previous data showing the signaling capacity of MHC II in melanoma cells, we hypothesized that LAG-3 could contribute to melanoma survival through its MHC II signaling capacity in melanoma cells. In this study, we demonstrate that both soluble LAG-3 and LAG-3–transfected cells can protect MHC II-positive melanoma cells, but not MHC II-negative cells, from FAS-mediated and drug-induced apoptosis. Interaction of LAG-3 with MHC II expressed on melanoma cells upregulates both MAPK/Erk and PI3K/Akt pathways, albeit with different kinetics. Inhibition studies using specific inhibitors of both pathways provided evidence of their involvement in the LAG-3–induced protection from apoptosis. Altogether, our data suggest that the LAG-3–MHC II interaction could be viewed as a bidirectional immune escape pathway in melanoma, with direct consequences shared by both melanoma and immune cells. In the future, compounds that efficiently hinder LAG-3–MHC II interaction might be used as an adjuvant to current therapy for MHC II-positive melanoma.

Particulate matter (PM) is thought to be responsible for respiratory health problems. Epithelial cells exposed to particles release pro-inflammatory cytokines leading to inflammation of airways. However, the signaling cascades triggered by particles are poorly understood. We demonstrate that PM with an aerodynamic diameter < 2.5 microm (PM2.5) or diesel exhaust particles upregulate the expression of amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), in human bronchial epithelial cells. AR secretion was blocked by an inhibitor of the EGFR tyrosine kinase (AG1478), or a selective mitogen-activated protein (MAP) kinase/extracellular regulated kinase (Erk) inhibitor (PD98059), but not by the p38 MAP kinase inhibitor (SB203580). Thus, AR secretion is mediated through the activation of the EGFR and Erk MAP kinase pathway. In addition, AR secretion was inhibited by the antioxidant N-acetyl cysteine, but not by a neutralizing anti-EGFR, suggesting an EGFR transactivation via oxidative stress. AR may be involved in cytokine secretion, as AR can induce granulocyte macrophage-colony-stimulating factor (GM-CSF) release and a neutralizing anti-EGFR reduces the particle-induced GM-CSF release. This study indicates that PM2.5 induces the expression and secretion of AR, an EGFR ligand contributing to GM-CSF release, which may reflect an important mechanism for sustaining the proinflammatory response.

BackgroundThe 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 FindingsWe 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/SignificanceThe 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.

In the frame of future regulations, a particular attention should thus be paid to the ultrafine/fine (here referred to as PM1) fraction due to their overwhelming anthropogenic origin and predominance in the urban aerosol and their pro-inflammatory potential.

Tumor-produced extracellular matrix (ECM) proteins can be key elements in tumor growth and metastasis. Transforming growth factor beta-inducible (TGFBI) protein is a secreted ECM component that can have dual function in cancer, acting as tumor suppressor or promoter. Although TGFBI is expressed in human melanoma cells, the exact role it might have in melanoma metastasis remains elusive. Assessing the expression and secretion of TGFBI, we show that human metastatic melanomas express and secrete significantly higher amounts of TGFBI, compared with nevus lesions and primary melanoma tumors. Intravenous injection of highly metastatic human melanoma cells expressing shRNA that targets TGFBI assigns a critical role for TGFBI in the formation of melanoma distal metastases in nude mice. In vivo assays demonstrate that TGFBI silencing does not interfere with melanoma cells' dissemination to distal sites but rather with their proliferation and outgrowth within new microenvironment. In line, TGFBI silencing increases melanoma cells motility/invasion/extravasation in vitro but interferes with their progression through the cell cycle, drastically reducing their proliferation. Furthermore, we show that TGFBI is a regulator of cyclins and cyclin-dependent kinases in melanoma. Collectively, our data describe a mechanism of melanoma metastatic outgrowth via promotion of growth/survival by the ECM protein TGFBI.

The aim of the current study was to establish the epidermal growth factor receptor (EGFR) ligand expression profile in human airway epithelial cells exposed to either particulate matter (PM) with an aerodynamic diameter ,2.5 mm (PM2.5) or its components and the involvement of EGFR ligands in PM2.5-provoked airway inflammation.EGFR ligand mRNA and protein expression were studied in a human bronchial epithelial cell line and normal nasal cells exposed to noncytotoxic concentrations of PM2.5 or its components. The autocrine role of EGFR ligands in airway epithelial cell pro-inflammation was determined by adding conditioned media from PM2.5-treated cells to fresh cells and measuring the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF), a pro-inflammatory biomarker.PM2.5 increased amphiregulin, transforming growth factor-a and heparin-binding EGF-like growth factor mRNA expression and protein secretion, with a slight contribution of aqueous metallic compounds and a strong participation of organic components putatively attributed to PM polyaromatic hydrocarbon content. PM2.5-induced EGFR ligands were involved in cellular GM-CSF release.The current study revealed upregulation of several epidermal growth factor receptor ligands by airway epithelial cells exposed to particulate matter with an aerodynamic diameter ,2.5 mm and their contribution to bronchial epithelial cell granulocyte-macrophage colony-stimulating factor secretion by an autocrine action, suggesting that these ligands could elicit and sustain the particulate matter-induced airway pro-inflammatory response and contribute to bronchial remodelling.

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