MHC class II engagement by its ligand LAG-3 (CD223) contributes to melanoma resistance to apoptosis.

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.

Autocrine effect of EGFR ligands on the pro-inflammatory response induced by PM(2.5) exposure in human bronchial epithelial cells.

Human exposure to PM(2.5) (particulate matter with an aerodynamic diameter below 2.5 µm) is known to be responsible for airway inflammation and may also induce airway remodelling. In respiratory epithelial cells exposed to PM(2.5), releases of pro-inflammatory cytokines such as granulocyte macrophage-colony stimulating factor (GM-CSF) and growth factor ligands of the epidermal growth factor receptor (EGFR) are increased. The present study aimed at determining the involvement of EGFR ligands by autocrine effects in PM(2.5)-induced GM-CSF release. PM(2.5) exposure triggers GM-CSF release by human bronchial epithelial (HBE) cells. This release is dependent on EGFR activation by ligand binding as it is inhibited by AG1478, an inhibitor of EGFR tyrosine kinase activity as well as by a neutralizing anti-EGFR antibody. The use of conditioned medium from cells previously exposed to PM(2.5) demonstrates that PM(2.5)-exposed cells release soluble EGFR ligands able to induce GM-CSF release by an autocrine manner. It was further demonstrated by inhibiting tumour-necrosis factor-alpha converting enzyme (TACE) that is involved in some EGFR ligand shedding. TAPI-2 and GM-6001, two TACE inhibitors, prevented the PM(2.5)-induced GM-CSF release as well as the silencing of TACE by siRNA. We provide evidence that the pro-inflammatory response induced by PM(2.5) exposure on HBE cells, results from an autocrine effect of EGFR ligands released by TACE activity. This autocrine loop by eliciting and sustaining inflammation could contribute to exacerbation of airway remodelling in respiratory-compromised individuals.

Size-partitioning of an urban aerosol to identify particle determinants involved in the proinflammatory response induced in airway epithelial cells.

BACKGROUND: The contribution of air particles in human cardio-respiratory diseases has been enlightened by several epidemiological studies. However the respective involvement of coarse, fine and ultrafine particles in health effects is still unclear. The aim of the present study is to determine which size fraction from a chemically characterized background aerosol has the most important short term biological effect and to decipher the determinants of such a behaviour. RESULTS: Ambient aerosols were collected at an urban background site in Paris using four 13-stage low pressure cascade impactors running in parallel (winter and summer 2005) in order to separate four size-classes (PM0.03-0.17 (defined here as ultrafine particles), PM0.17-1 (fine), PM1-2.5(intermediate) and PM2.5-10 (coarse)). Accordingly, their chemical composition and their pro-inflammatory potential on human airway epithelial cells were investigated. Considering isomass exposures (same particle concentrations for each size fractions) the pro-inflammatory response characterized by Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) release was found to decrease with aerosol size with no seasonal dependency. When cells were exposed to isovolume of particle suspensions in order to respect the particle proportions observed in ambient air, the GM-CSF release was maximal with the fine fraction. In presence of a recombinant endotoxin neutralizing protein, the GM-CSF release induced by particles is reduced for all size-fractions, with exception of the ultra-fine fraction which response is not modified. The different aerosol size-fractions were found to display important chemical differences related to the various contributing primary and secondary sources and aerosol age. The GM-CSF release was correlated to the organic component of the aerosols and especially its water soluble fraction. Finally, Cytochrome P450 1A1 activity that reflects PAH bioavailability varied as a function of the season: it was maximal for the fine fraction in winter and for the ultrafine fraction in summer. CONCLUSION: 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.

Effects of PM2.5 components in the release of amphiregulin by human airway epithelial cells.

Exposure to ambient particulate matter (PM) is responsible for airway inflammation and tissue remodeling. Urban PM(2.5) (aerodynamic diameter <2.5microm) is a complex mixture rich in soots and containing hydrosoluble and organic components. We previously showed that the exposure of airway epithelial cells to PM(2.5) triggers the release of amphiregulin (AR), ligand of the epidermal growth factor receptor (EGFR) involved in proinflammatory and repair responses. The effect of Paris PM(2.5) organic and aqueous fractions in AR expression and secretion was investigated on the bronchial epithelial cell line 16HBE and normal human nasal epithelial (NHNE) cells. Both a macroarray specific for inflammation pathways and RT-PCR showed an AR upregulation in organic extract-treated 16HBE cells. AR release is induced in 16HBE and NHNE cells grown on plastic and exposed to native PM(2.5), organic extract and to a lesser extent washed PM(2.5) (deprived of its hydrosoluble content) and aqueous extract. Furthermore, as assessed by using NHNE cells grown on Transwell inserts, this secretion is polarized toward the basolateral side where the EGFR is expressed. To conclude, both PM(2.5) organic and hydrosoluble components are involved in the expression and secretion of AR; organic compounds exhibiting a strong effect when they are easily bioavailable.

TGF-ß-induced (TGFBI) protein in melanoma: a signature of high metastatic 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.

Melanoma spheroids grown under neural crest cell conditions are highly plastic migratory/invasive tumor cells endowed with immunomodulator function.

BACKGROUND: 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.

Fine particulate matter induces amphiregulin secretion by bronchial epithelial cells.

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.