Gaia Barisione

IL-8 induces exocytosis of arginase 1 by neutrophil polymorphonuclears in nonsmall cell lung cancer.

Arginase 1 (ARG1) inhibits T‐cell proliferation by degrading extracellular arginine, which results in decreased responsiveness of T cells to CD3/TCR stimulation. In humans, ARG1 is stored in inactive form within granules of polymorphonuclear neutrophils (PMNs) and gets activated on release. We studied the role of PMNs‐related ARG1 activity in nonsmall cell lung cancer (NSLC), in which tumor‐infiltrating lymphocytes showed reduced proliferation in response to CD3/TCR triggering. Patients with NSCLC had increased ARG1 plasma levels as compared to healthy controls. Furthermore, immunohistochemistry showed that tumor‐infiltrating PMNs display reduced intracellular ARG1, in comparison to intravascular or peritumoral PMNs, suggesting a role of tumor microenvironment in ARG1 release. Indeed, supernatants of NSCLC cell lines induced exocytosis of ARG1 from PMNs. All (4/4) NSCLC cell lines and all (7/7) CD14− cell samples from NSCLC expressed interleukin (IL)‐8 mRNA, whereas TNFα mRNA was expressed by 1 cell line and by 2 tumor specimens. Furthermore, all NSCLC cell lines secreted immunoreactive IL‐8, albeit at different levels. IL‐8 was as effective as TNFα in triggering ARG1 release and the 2 cytokines acted synergistically. Secreted ARG1 was biologically active and catabolized extracellular arginine. The supernatant of IL‐8 gene‐silenced NSCLC cells did not mediate ARG1 release by PMNs. Altogether these findings demonstrate a role of IL‐8 in ARG1 exocytosis by PMNs and indicate that, due at least in part to IL‐8 secreted by NSCLC cells, PMNs infiltrating NSCLC release ARG1. This phenomenon could contribute to local immune suppression.

Exocytosis of azurophil and arginase 1-containing granules by activated polymorphonuclear neutrophils is required to inhibit T lymphocyte proliferation.

ARG1, expressed by human PMNs, inhibits T cell proliferation by depleting extracellular l‐arginine. Here, we report that ARG1, released from gelatinase granules by PMNs, is inactive at physiological pH unless activated by factor(s) stored in azurophil granules. Whereas ARG1 exocytosis was induced by TNF‐α or ionomycin, only the latter mediated the release of both granules, resulting in extracellular ARG enzyme activity at physiological pH. Furthermore, after fractionation of the different classes of granules, only the mixture of gelatinase and azurophil granules resulted in ARG1 activity at physiological pH. The use of protease inhibitors indicated the involvement of a PMSF‐ and leupeptin‐susceptible serine protease in ARG1 processing and activation. Finally, the supernatant of viable PMNs undergoing frustrated phagocytosis, which mediates gelatinase and azurophil granule release, inhibited T cell proliferation through ARG‐dependent mechanisms. In vivo, high ARG1 concentrations and increased ARG enzyme activity, sufficient to inhibit T cell proliferation, were observed in synovial fluids from RA. These findings suggest that PMNs, recruited at sites of immune complex deposition, induce ARG1‐dependent immune suppression through concomitant exocytosis of gelatinase and azurophil granules.

Mda-9/syntenin is expressed in uveal melanoma and correlates with metastatic progression.

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound–healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.

IL-27 induces the expression of IDO and PD-L1 in human cancer cells

IL-27 is a member of the IL-12 family that is produced by macrophages and dendritic cells. IL-27 inhibits the growth and invasiveness of different cancers and therefore represents a potential anti-tumor agent. By contrast, it may exert immune-regulatory properties in different biological systems. We reported that IL-27 induces the expression of the IL-18 inhibitor IL-18BP, in human Epithelial Ovarian Cancer (EOC) cells, thus potentially limiting the immune response. Here, we tested whether IL-27 may modulate other immune-regulatory molecules involved in EOC progression, including Indoleamine 2,3-dioxygenase (IDO) and Programmed Death-Ligand (PD-L)1. IDO and PD-L1 were not constitutively expressed by EOC cells in vitro, but IL-27 increased their expression through STAT1 and STAT3 tyrosine phosphorylation. Differently, cells isolated from EOC ascites showed constitutive activation of STAT1 and STAT3 and IDO expression. These findings, together with the expression of IL-27 in scattered leukocytes in EOC ascites and tissues, suggest a potential role of IL-27 in immune-regulatory networks of EOC. In addition, IL-27 induced IDO or PD-L1 expression in monocytes and in human PC3 prostate and A549 lung cancer cells. A current paradigm in tumor immunology is that tumor cells may escape from immune control due to “adaptive resistance” mediated by T cell-secreted IFN-γ, which induces PD-L1 and IDO expression in tumor cells. Our present data indicate that also IL-27 has similar activities and suggest that the therapeutic use of IL-27 as anti-cancer agent may have dual effects, in some tumors.

Arginase 2 is expressed by human lung cancer, but it neither induces immune suppression, nor affects disease progression

In human prostate cancer, Arginase 2 (ARG2) and nitric oxide synthase (NOS) are concomitantly expressed by tumor cells, and induce tumor immune escape via peroxynitrite‐dependent Tyrosine nitrosylation. Since there were no data regarding this immune suppressive mechanism in other tumor types, and an evaluation of its clinical relevance in human tumors had still to be provided, we have investigated presence and clinical relevance of ARG2 and NOS expression in lung cancer. No evidence of NOS expression was found, no significant NOS enzymatic activity was detected. Instead, ARG2 protein was expressed by tumor cells. In a cohort of 120 patients, the amount of ARG2‐positive tumor cells was significantly higher in small cell lung cancers (SCLC) than in non‐small cell lung cancers (NSCLC). Large cell undifferentiated carcinomas had twice ARG2 than the other NSCLC subtypes. ARG2 expression was increased in Grade 3 tumors, as compared to Grades 1 and 2. However, no relationship was found with tumor size and stage, and with patient survival. Indeed, the enzyme was active, since the Arginine catabolite Ornithine was produced, but Arginine depletion was not attained. In addition, nitrotyrosine was not found in tumor tissue. Accordingly, when tumor cells isolated from lung cancer were incubated with activated autologous T cells, no inhibition of proliferation was detected. Our results indicate that ARG2 is expressed in lung cancer, but it does not induce tumor immune escape and does not affect disease progression, most probably due to the lack of concomitant NOS expression.

Different Subcellular Localization of ALCAM Molecules in Neuroblastoma: Association with Relapse

Background: The Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD), involved in nervous system development, has been linked to tumor progression and metastasis in several tumors. No information is available on ALCAM expression in neuroblastoma, a childhood neoplasia originating from the sympathetic nervous system. Methods: ALCAM expression was analysed by immunofluorescence and immunohistochemistry on differentiated neuroblastoma cell lines and on archival specimens of stroma-poor, not MYCN amplified, resectable neuroblastoma tumors, respectively. Results: ALCAM is variously expressed in neuroblastoma cell lines, is shed by metalloproteases and is cleaved by ADAM17/TACE in vitro. ALCAM is expressed in neuroblastoma primary tumors with diverse patterns of subcellular localization and is highly expressed in the neuropil area in a subgroup of cases. Tumor specimens showing high expression of ALCAM at the membrane of the neuroblast body or low levels in the neuropil area are associated with relapse (P = 0.044 and P < 0.0001, respectively). In vitro differentiated neuroblastoma cells show strong ALCAM expression on neurites, suggesting that ALCAM expression in the neuropil is related to a differentiated phenotype. Conclusions: Assessment of ALCAM localization by immunohistochemistry may help to identify patients who, in the absence of negative prognostic factors, are at risk of relapse and require a more careful follow-up.

Potential Role of Soluble c-Met as a New Candidate Biomarker of Metastatic Uveal Melanoma

IMPORTANCE Conventional melanoma serum biomarkers (S100 and lactate dehydrogenase [LDH]) perform poorly in patients with uveal melanoma, and the search for new biomarkers is needed. A high expression of the oncoprotein c-Met in primary uveal melanoma is associated with metastatic progression, and c-Met is released as a soluble ectodomain through ADAM10- and ADAM17-mediated cleavage, suggesting a possible role as biomarker. OBJECTIVE To determine the potential role of soluble c-Met (sc-Met) as a biomarker of uveal melanoma progression in comparison with S100 and LDH. DESIGN, SETTING, AND PARTICIPANTS Soluble c-Met was studied in the conditioned medium of 9 uveal melanoma cell lines and in the blood serum samples of 24 mice with uveal melanoma xenografts, 57 patients with uveal melanoma (17 patients whose tumors metastasized and 40 patients whose tumors did not metastasize), and 37 healthy donors. We collected blood samples for as long as 5 years after treatment of the primary tumor. The concentration of sc-Met was measured using enzyme-linked immunosorbent assays, and the receiver operating characteristic curve was used to evaluate sensitivity and specificity in the identification of metastatic uveal melanoma. The study began on May 2, 2011, and the last samples were collected in January 2015. MAIN OUTCOMES AND MEASURES Levels of sc-Met in uveal melanoma cell cultures and in the blood serum samples of xenotransplanted mice, of healthy donors, and of patients with uveal melanoma during follow-up. RESULTS The conditioned medium of uveal melanoma cell lines and the blood serum samples of mice with uveal melanoma xenografts contained significant levels of sc-Met. Patients with metastatic disease had significantly higher serum levels of sc-Met (median level, 590 ng/mL [range, 246-12,856 ng/mL]) than did patients without metastatic disease (median level, 296 ng/mL [range, 201-469 ng/mL]) (P < .001) and healthy donors (median level, 285 ng/mL [range, 65-463 ng/mL]) (P < .001). Analysis of receiver operating characteristic curves for sc-Met levels in patients with nonmetastatic uveal melanoma vs patients with metastatic uveal melanoma yielded an area under the curve of 0.82 (95% CI, 0.68-0.95) (P < .001), which was superior to the areas under the curve achieved with S100 or LDH markers. Patients with progressive metastatic disease showed further increases in sc-Met level, whereas stable patients did not. CONCLUSIONS AND RELEVANCE The present pilot study suggests that sc-Met should be further exploited as a biomarker for monitoring of uveal melanoma.

The biology of uveal melanoma

Uveal melanoma (UM), a rare cancer of the eye, is distinct from cutaneous melanoma by its etiology, the mutation frequency and profile, and its clinical behavior including resistance to targeted therapy and immune checkpoint blockers. Primary disease is efficiently controlled by surgery or radiation therapy, but about half of UMs develop distant metastasis mostly to the liver. Survival of patients with metastasis is below 1 year and has not improved in decades. Recent years have brought a deep understanding of UM biology characterized by initiating mutations in the G proteins GNAQ and GNA11. Cytogenetic alterations, in particular monosomy of chromosome 3 and amplification of the long arm of chromosome 8, and mutation of the BRCA1-associated protein 1, BAP1, a tumor suppressor gene, or the splicing factor SF3B1 determine UM metastasis. Cytogenetic and molecular profiling allow for a very precise prognostication that is still not matched by efficacious adjuvant therapies. G protein signaling has been shown to activate the YAP/TAZ pathway independent of HIPPO, and conventional signaling via the mitogen-activated kinase pathway probably also contributes to UM development and progression. Several lines of evidence indicate that inflammation and macrophages play a pro-tumor role in UM and in its hepatic metastases. UM cells benefit from the immune privilege in the eye and may adopt several mechanisms involved in this privilege for tumor escape that act even after leaving the niche. Here, we review the current knowledge of the biology of UM and discuss recent approaches to UM treatment.

ADAM10 correlates with uveal melanoma metastasis and promotes in vitro invasion

Uveal melanoma (UM) is a rare ocular tumor that may lead to deadly metastases in 50% of patients. A disintegrin and metalloproteinase (ADAM)10, ADAM17, and the HGF‐receptor c‐Met support invasiveness in different tumors. Here, we report that high ADAM10, MET, and, to a lesser extent, ADAM17 gene expression correlates with poor progression‐free survival in UM patients (hazard ratio 2.7, 2.6, and 1.9, respectively). About 60% of primary UM expresses c‐Met and/or ADAM10 proteins. Four UM cell lines display high levels of ADAM10 and ADAM17, which constitutively cleave c‐Met, inducing the release of soluble c‐Met. ADAM10/17 pharmacological inhibition or gene silencing reduces c‐Met shedding, but has limited impact on surface c‐Met, which is overexpressed. Importantly, ADAM10 silencing inhibits UM cell invasion driven by FCS or HGF, while ADAM17 silencing has a limited effect. Altogether our data indicate that ADAM10 has a pro‐invasive role and may contribute to UM progression.