Neutralization of NET-associated human ARG1 enhances cancer immunotherapy.

Myeloid cells can restrain antitumor immunity by metabolic pathways, such as the degradation of l-arginine, whose concentrations are regulated by the arginase 1 (ARG1) enzyme. Results from preclinical studies indicate the important role of arginine metabolism in pancreatic ductal adenocarcinoma (PDAC) progression, suggesting a potential for clinical application; however, divergent evolution in ARG1 expression and function in rodents and humans has restricted clinical translation. To overcome this dichotomy, here, we show that neutrophil extracellular traps (NETs), released by spontaneously activated neutrophils isolated from patients with PDAC, create a microdomain where cathepsin S (CTSS) cleaves human (h)ARG1 into different molecular forms endowed with enhanced enzymatic activity at physiological pH. NET-associated hARG1 suppresses T lymphocytes whose proliferation is restored by either adding a hARG1-specific monoclonal antibody (mAb) or preventing CTSS-mediated cleavage, whereas small-molecule inhibitors are not effective. We show that ARG1 blockade, combined with immune checkpoint inhibitors, can restore CD8+ T cell function in ex vivo PDAC tumors. Furthermore, anti-hARG1 mAbs increase the frequency of adoptively transferred tumor-specific CD8+ T cells in tumor and enhance the effectiveness of immune checkpoint therapy in humanized mice. Thus, this study shows that extracellular ARG1, released by activated myeloid cells, localizes in NETs, where it interacts with CTSS that in turn cleaves ARG1, producing major molecular forms endowed with different enzymatic activity at physiological pH. Once exocytosed, ARG1 activity can be targeted by mAbs, which bear potential for clinical application for the treatment of PDAC and require further exploration.

Analysis of A Disintegrin and Metalloprotease 17 (ADAM17) Expression as a Prognostic Marker in Ovarian Cancer Patients Undergoing First-Line Treatment Plus Bevacizumab.

To find prognostic factors for advanced ovarian cancer patients undergoing first-line therapy with carboplatin, paclitaxel and bevacizumab, we investigated the expression of a disintegrin and metalloprotease 17 (ADAM17) in cancer tissues. ADAM17 has been involved in ovarian cancer development, progression and cell resistance to cisplatin. Tissue microarrays from 309 ovarian cancer patients enrolled in the MITO16A/MANGO-OV2 clinical trial were analyzed by immunohistochemistry for ADAM17 protein expression. Intensity and extent of staining were combined into a semi-quantitative visual grading system (H score) which was related to clinicopathological characteristics of cases and the clinical outcome of patients by univariate and multivariate Cox regression models. ADAM17 immunostaining was detected in most samples, mainly localized in the tumor cells, with variable intensity across the cohort. Kaplan-Meier survival curves, generated according to the best cut-off value for the ADAM17 H score, showed that high ADAM17 expression was associated with worse prognosis for PFS and OS. However, after the application of a shrinkage procedure to adjust for overfitting hazard ratio estimates, the ADAM17 value as prognostic factor was lost. As subgroup analysis suggested that ADAM17 expression could be prognostically relevant in cases with no residual disease at baseline, further studies in this patient category may be worth planning.

IL-27 Mediates PD-L1 Expression and Release by Human Mesothelioma Cells.

Malignant mesothelioma (MM) is a rare tumor with an unfavorable prognosis. MM genesis involves asbestos-mediated local inflammation, supported by several cytokines, including IL-6. Recent data showed that targeting PD-1/PD-L1 is an effective therapy in MM. Here, we investigated the effects of IL-6 trans-signaling and the IL-6-related cytokine IL-27 on human MM cells in vitro by Western blot analysis of STAT1/3 phosphorylation. The effects on PD-L1 expression were tested by qRT-PCR and flow-cytometry and the release of soluble (s)PD-L1 by ELISA. We also measured the concentrations of sPD-L1 and, by multiplexed immunoassay, IL-6 and IL-27 in pleural fluids obtained from 77 patients in relation to survival. IL-27 predominantly mediates STAT1 phosphorylation and increases PD-L1 gene and surface protein expression and sPD-L1 release by human MM cells in vitro. IL-6 has limited activity, whereas a sIL-6R/IL-6 chimeric protein mediates trans-signaling predominantly via STAT3 phosphorylation but has no effect on PD-L1 expression and release. IL-6, IL-27, and sPD-L1 are present in pleural fluids and show a negative correlation with overall survival, but only IL-27 shows a moderate albeit significant correlation with sPD-L1 levels. Altogether these data suggest a potential role of IL-27 in PD-L1-driven immune resistance in MM.

Assessment of interferon-γ in pleural fluid as a prognostic factor of survival in malignant pleural mesothelioma.

BACKGOUND: Literature reports suggest that the host immune system may control Malignant Pleural Mesothelioma (MPM) growth, although its activity is limited by regulatory mechanisms. In this retrospective study, we analyzed the levels of pro-inflammatory (IL-1, IL-6, TNF), immune-regulatory (IL-10) and Th1/CTL-related cytokines (IL-12p70, IFN-γ) in the pleural exudate and their relationship with overall survival (OS) in MPM. METHODS: Cytokines were quantified by multiplexed immunoassay. Concentrations were dichotomized with respect to the median value. Correlation between cytokine level and OS was assessed using univariate (Kaplan-Meier curves) and multivariate (Cox regression) analyses. RESULTS: Regarding outcome, tumor histology, therapies undergone and IFN-γ were independent prognostic factors of OS in a 72 MPM training cohort. Notably, high concentrations of IFN-γ halved death probability (HR of high vs low IFN-γ concentration = 0.491, 95%CI 0.3-0.8, p = 0.007). Also in patients with epithelioid histology and those receiving at least one line of therapy, high IFN-γ level was an independent factor predictive of OS (HR of high vs low IFN-γ concentration were 0.497, p = 0.007 and 0.324, p = 0.006, respectively). However, these data were not confirmed in a 77 MPM validation cohort, possibly due to the low IFN-γ levels encountered in this population, and the heterogeneous distribution of disease stages between the training and the validation cohorts. None of the other cytokines showed any effect on survival. CONCLUSIONS: High level of IFN-γ in pleural effusion may be associated with better survival in MPM patients and potentially serve as a prognostic biomarker. Larger prospective studies are needed to ascertain this hypothesis.

Potential Onco-Suppressive Role of miR122 and miR144 in Uveal Melanoma through ADAM10 and C-Met Inhibition.

Uveal melanoma (UM) is a rare tumor of the eye that leads to deadly metastases in about half of the patients. ADAM10 correlates with c-Met expression in UM and high levels of both molecules are related to the development of metastases. MiR122 and miR144 modulate ADAM10 and c-Met expression in different settings. We hypothesized a potential onco-suppressive role for miR122 and miR144 through modulation of ADAM10 and c-Met in UM. We analyzed the UM Cancer Genome Atlas data portal (TCGA) dataset, two other cohorts of primary tumors and five human UM cell lines for miR122 and miR144 expression by miR microarray, RT-qPCR, Western blotting, miR transfection and luciferase reporter assay. Our results indicate that miR122 and miR144 are expressed at low levels in the UM cell lines and in the TCGA UM dataset and were down-modulated in a cohort of seven UM samples, compared to normal choroid. Both miR122 and miR144 directly targeted ADAM10 and c-Met. Overexpression of miR122 and miR144 led to reduced expression of ADAM10 and c-Met in the UM cell lines and impaired cell proliferation, migration, cell cycle and shedding of c-Met ecto-domain. Our results show that miR122 and miR144 display an onco-suppressive role in UM through ADAM10 and c-Met modulation.

Cytokine-Induced Guanylate Binding Protein 1 (GBP1) Release from Human Ovarian Cancer Cells.

We showed that IL-27 shares several effects with IFN-γ in human cancer cells. To identify novel extracellular mediators, potentially involved in epithelial ovarian cancer (EOC) biology, we analyzed the effect of IL-27 or IFN-γ on the secretome of cultured EOC cells by mass-spectrometry (nano-UHPLC-MS/MS). IL-27 and IFN-γ modulate the release of a limited fraction of proteins among those induced in the whole cell. We focused our attention on GBP1, a guanylate-binding protein and GTPase, which mediates several biological activities of IFNs. Cytokine treatment induced GBP1, 2, and 5 expressions in EOC cells, but only GBP1 was secreted. ELISA and immunoblotting showed that cytokine-stimulated EOC cells release full-length GBP1 in vitro, through non-classical pathways, not involving microvesicles. Importantly, full-length GBP1 accumulates in the ascites of most EOC patients and ex-vivo EOC cells show constitutive tyrosine-phosphorylated STAT1/3 proteins and GBP1 expression, supporting a role for Signal Transducer And Activator Of Transcription (STAT)-activating cytokines in vivo. High GBP1 gene expression correlates with better overall survival in the TCGA (The Cancer Genome Atlas) dataset of EOC. In addition, GBP1 transfection partially reduced EOC cell viability in an MTT assay. Our data show for the first time that cytokine-stimulated tumor cells release soluble GBP1 in vitro and in vivo and suggest that GBP1 may have anti-tumor effects in EOC.

Targeted Therapy of Uveal Melanoma: Recent Failures and New Perspectives.

Among Uveal Melanoma (UM) driver mutations, those involving GNAQ or GNA11 genes are the most frequent, while a minor fraction of tumors bears mutations in the PLCB4 or CYSLTR2 genes. Direct inhibition of constitutively active oncoproteins deriving from these mutations is still in its infancy in UM, whereas BRAFV600E-targeted therapy has obtained relevant results in cutaneous melanoma. However, UM driver mutations converge on common downstream signaling pathways such as PKC/MAPK, PI3K/AKT, and YAP/TAZ, which are presently considered as actionable targets. In addition, BAP1 loss, which characterizes UM metastatic progression, affects chromatin structure via histone H2A deubiquitylation that may be counteracted by histone deacetylase inhibitors. Encouraging results of preclinical studies targeting signaling molecules such as MAPK and PKC were unfortunately not confirmed in early clinical studies. Indeed, a general survey of all clinical trials applying new targeted and immune therapy to UM displayed disappointing results. This paper summarizes the most recent studies of UM-targeted therapies, analyzing the possible origins of failures. We also focus on hyperexpressed molecules involved in UM aggressiveness as potential new targets for therapy.

ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions.

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is a cell adhesion molecule of the immunoglobulin superfamily and has been implicated in diverse pathophysiological processes including T cell activation, leukocyte trafficking, and (lymph)angiogenesis. However, exploring the therapeutic potential of ALCAM blockade in immune-mediated inflammatory disorders has been difficult due to the lack of antibodies with blocking activity toward murine ALCAM. In this study, we identified and characterized a monoclonal antibody with high affinity and specificity for murine ALCAM. This antibody reduced in vitro T cell activation induced by antigen-presenting dendritic cells (DCs) as well as (trans)migration of murine DCs across lymphatic endothelial monolayers. Moreover, it reduced emigration of DCs from in vitro-cultured human skin biopsies. Similarly, antibody-based blockade of ALCAM reduced (lymph)angiogenic processes in vitro and decreased developmental lymphangiogenesis in vivo to levels observed in ALCAM-deficient mice. Since corneal allograft rejection is an important medical condition that also involves (lymph)angiogenesis, DC migration and T cell activation, we investigated the therapeutic potential of ALCAM blockade in murine corneal disease. Blocking ALCAM lead to DC retention in corneas and effectively prevented corneal allograft rejection. Considering that we also detected ALCAM expression in human corneal DCs and lymphatics, our findings identify ALCAM as a potential novel therapeutic target in human corneal allograft rejection.

A High-Content Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy.

Novel druggable targets have been discovered in neuroblastoma (NB), paving the way for more effective treatments. However, children with high-risk NB still show high mortality rates prompting for a search of novel therapeutic options. Here, we aimed at repurposing FDA-approved drugs for NB treatment by performing a high-content screening of a 349 anticancer compounds library. In the primary screening, we employed three NB cell lines, grown as three-dimensional (3D) multicellular spheroids, which were treated with 10 µmol/L of the library compounds for 72 hours. The viability of 3D spheroids was evaluated using a high-content imaging approach, resulting in a primary hit list of 193 compounds. We selected 60 FDA-approved molecules and prioritized drugs with multi-target activity, discarding those already in use for NB treatment or enrolled in NB clinical trials. Hence, 20 drugs were further tested for their efficacy in inhibiting NB cell viability, both in two-dimensional and 3D models. Dose-response curves were then supplemented with the data on side effects, therapeutic index, and molecular targets, suggesting two multiple tyrosine kinase inhibitors, ponatinib and axitinib, as promising candidates for repositioning in NB. Indeed, both drugs showed induction of cell-cycle block and apoptosis, as well as inhibition of colony formation. However, only ponatinib consistently affected migration and inhibited invasion of NB cells. Finally, ponatinib also proved effective inhibition of tumor growth in orthotopic NB mice, providing the rationale for its repurposing in NB therapy. Mol Cancer Ther; 17(7); 1405-15. ©2018 AACR.

Microenvironmental regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence.

Although the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12Rß1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rß1 chain when cocultured with activated T cells or CD40L+ cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies.

Combined immunotherapy with anti-PDL-1/PD-1 and anti-CD4 antibodies cures syngeneic disseminated neuroblastoma.

Anti-PD-1 or anti-PD-L1 blocking monoclonal antibodies (mAbs) have shown potent anti-tumor effects in adult cancer patients and clinical studies have recently been started in pediatric cancers, including high-risk/relapsing neuroblastoma (NB). Therefore, we studied the effects of anti-PD-1/PD-L1 mAbs in two syngeneic models of disseminated NB generated by the injection of either Neuro2a or NXS2 cells, which express PD-L1. In addition, we tested the combination of these agents with the immune-enhancing cytokine IL-21, the Ecto-NTPDase inhibitor POM-1, an anti-CD25 mAb targeting Treg cells, or an anti-CD4 mAb. We previously showed that CD4-transient depletion removes CD4+CD25+ Treg cells and other CD4+CD25- regulatory subsets. Here we show that mono-therapy with anti-PD-1/PD-L1 mAbs had no effect on systemic NB progression in vivo, and also their combination with IL-21, POM-1 or anti-CD25 mAb was ineffective. The combined use of anti-PD-1 with an anti-CD4 mAb mediated a very potent, CD8-dependent, synergistic effect leading to significant elongation of tumor-free survival of mice, complete tumor regression and durable anti-NB immunity. Similar results were obtained by combining the anti-PD-L1 and anti-CD4 mAbs. These findings indicate that both PD-1/PD-L1 and CD4+ T cell-related immune-regulatory mechanisms must be simultaneously blocked to mediate therapeutic effects in these models.

IL-27 mediates HLA class I up-regulation, which can be inhibited by the IL-6 pathway, in HLA-deficient Small Cell Lung Cancer cells.

BACKGROUND: Recently, immunotherapy with anti-PD-1 antibodies has shown clinical benefit in recurrent Small Cell Lung Cancer (SCLC). Since anti-PD-1 re-activates anti-tumor Cytotoxic T Lymphocyte (CTL) responses, it is crucial to understand the mechanisms regulating HLA class I, and PD-L1 expression in HLA-negative SCLC. Here we addressed the role of IL-27, a cytokine related to both IL-6 and IL-12 families. METHODS: The human SCLC cell lines NCI-N592, -H69, -H146, -H446 and -H82 were treated in vitro with different cytokines (IL-27, IFN-γ, IL-6 or a soluble IL-6R/IL-6 chimera [sIL-6R/IL-6]) at different time points and analyzed for tyrosine-phosphorylated STAT proteins by Western blot, for surface molecule expression by immunofluorescence and FACS analyses or for specific mRNA expression by QRT-PCR. Relative quantification of mRNAs was calculated by the ΔΔCT method. The Student's T test was used for the statistical analysis of experimental replicates. RESULTS: IL-27 triggered STAT1/3 phosphorylation and up-regulated the expression of surface HLA class I antigen and of TAP1 and TAP2 mRNA in four out of five SCLC cell lines tested. The IL-27-resistant NCI-H146 cells showed up-regulation of HLA class I by IFN-γ. IFN-γ also induced expression of PD-L1 in SCLC cells, while IL-27 was less potent in this respect. IL-27 failed to activate STAT1/3 phosphorylation in NCI-H146 cells, which display a low expression of the IL-27RA and GP130 receptor chains. As GP130 is shared in IL-27R and IL-6R complexes, we assessed its functionality in response to sIL-6R/IL-6. sIL-6R/IL-6 failed to trigger STAT1/3 signaling in NCI-H146 cells, suggesting low GP130 expression or uncoupling from signal transduction. Although both sIL-6R/IL-6 and IL-27 triggered STAT1/3 phosphorylation, sIL-6R/IL-6 failed to up-regulate HLA class I expression, in relationship to the weak activation of STAT1. Finally sIL-6R/IL-6 limited IL-27-effects, particularly in NCI-H69 cells, in a SOCS3-independent manner, but did not modify IFN-γ induced HLA class I up-regulation. CONCLUSIONS: In conclusion, IL-27 is a potentially interesting cytokine for restoring HLA class I expression for SCLC combined immunotherapy purposes. However, the concomitant activation of the IL-6 pathway may limit the IL-27 effect on HLA class I induction but did not significantly alter the responsiveness to IFN-γ.

A genome-wide microRNA profiling indicates miR-424-5p and miR-503-5p as regulators of ALK expression in neuroblastoma.

The discovery of missense mutations of ALK gene identified this receptor tyrosine kinase as a therapeutic target in neuroblastoma (NB). Moreover, a high level of ALK protein has been associated with metastatic NB cases and with a worse prognosis, suggesting that also ALK overexpression is involved in NB tumorigenesis. Since miRNAs play key roles in the regulation of gene expression we aimed at identifying those miRNAs that can regulate ALK in NB. We therefore analyzed the genome-wide expression profile of miRNAs in two sample sets of 16 NB cell lines and 22 NB samples by using miRNA microarrays. Both sample sets were then divided into two subgroups showing high (ALK+) or low/absent (ALK-) expression of ALK. Results showed a down-regulation of 30 and 23 miRNAs (p-value <0.05) in the ALK+ group in NB cell lines and samples, respectively. Validation analysis indicated that miR-424-5p and miR-503-5p, belonging to the same cluster, were differentially expressed in both NB cell lines and tumor samples. Although only miR-424-5p showed a direct binding to ALK 3'-UTR, both miRNAs led to a remarkable decreasing of ALK protein as well as to the inhibition of cell viability in ALK+ NB cell lines. In conclusion, our data indicate that both miR-424-5p and miR-503-5p are involved in regulating ALK expression in NB, either by directly targeting ALK receptor or indirectly, and may thus serve as potential therapeutic tools in ALK dependent NBs.

Altered erythropoiesis and decreased number of erythrocytes in children with neuroblastoma.

Neuroblastoma (NB) is a pediatric tumor presenting at diagnosis either as localized or metastatic disease, which mainly involves the bone marrow (BM). The physical occupancy of BM space by metastatic NB cells has been held responsible for impairment of BM function. Here, we investigated whether localized or metastatic NB may alter hematopoietic lineages' maturation and release of mature cells in the periphery, through gene expression profiling, analysis of BM smears, cell blood count and flow cytometry analysis. Gene ontology and disease-associated analysis of the genes significantly under-expressed in BM resident cells from children with localized and metastatic NB, as compared to healthy children, indicated anemia, blood group antigens, and heme and porphyrin biosynthesis as major functional annotation clusters. Accordingly, in children with NB there was a selective impairment of erythrocyte maturation at the ortho-chromic stage that resulted in reduced erythrocyte count in the periphery, regardless of the presence of metastatic cells in the BM. By considering all NB patients, low erythrocyte count at diagnosis associated with worse survival. Moreover, in the subset of metastatic patients, low erythrocyte count, hemoglobin and hematocrit and high red cell distribution width at follow-up also associated with worse outcome. These observations provide an alternative model to the tenet that infiltrating cells inhibit BM functions due to physical occupancy of space and may open a new area of research in NB to understand the mechanism(s) responsible for such selective impairment.

Dual Roles of IL-27 in Cancer Biology and Immunotherapy.

IL-27 is a pleiotropic two-chain cytokine, composed of EBI3 and IL-27p28 subunits, which is structurally related to both IL-12 and IL-6 cytokine families. IL-27 acts through a heterodimer receptor consisting of IL-27Rα (WSX1) and gp130 chains, which mediate signaling predominantly through STAT1 and STAT3. IL-27 was initially reported as an immune-enhancing cytokine that supports CD4+ T cell proliferation, T helper (Th)1 cell differentiation, and IFN-γ production, acting in concert with IL-12. However, subsequent studies demonstrated that IL-27 displays complex immune-regulatory functions, which may result in either proinflammatory or anti-inflammatory effects in relationship to the biological context and experimental models considered. Several pieces of evidence, obtained in preclinical tumor models, indicated that IL-27 has a potent antitumor activity, related not only to the induction of tumor-specific Th1 and cytotoxic T lymphocyte (CTL) responses but also to direct inhibitory effects on tumor cell proliferation, survival, invasiveness, and angiogenic potential. Nonetheless, given its immune-regulatory functions, the effects of IL-27 on cancer may be dual and protumor effects may also occur. Here, we will summarize IL-27 biological activities and its functional overlaps with the IFNs and discuss its dual role in tumors in the light of potential applications to cancer immunotherapy.

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.

Proteomic analysis uncovers common effects of IFN-γ and IL-27 on the HLA class I antigen presentation machinery in human cancer cells.

IL-27, a member of the IL-12-family of cytokines, has shown anti-tumor activity in several pre-clinical models due to anti-proliferative, anti-angiogenic and immune-enhancing effects. On the other hand, IL-27 demonstrated immune regulatory activities and inhibition of auto-immunity in mouse models. Also, we reported that IL-27, similar to IFN-γ, induces the expression of IL-18BP, IDO and PD-L1 immune regulatory molecules in human cancer cells. Here, a proteomic analysis reveals that IL-27 and IFN-γ display a broad overlap of functions on human ovarian cancer cells. Indeed, among 990 proteins modulated by either cytokine treatment in SKOV3 cells, 814 showed a concordant modulation by both cytokines, while a smaller number (176) were differentially modulated. The most up-regulated proteins were common to both IFN-γ and IL-27. In addition, functional analysis of IL-27-regulated protein networks highlighted pathways of interferon signaling and regulation, antigen presentation, protection from natural killer cell-mediated cytotoxicity, regulation of protein polyubiquitination and proteasome, aminoacid catabolism and regulation of viral protein levels.Importantly, we found that IL-27 induced HLA class I molecule expression in human cancer cells of different histotypes, including tumor cells showing very low expression. IL-27 failed only in a cancer cell line bearing a homozygous deletion in the B2M gene. Altogether, these data point out to a broad set of activities shared by IL-27 and IFN-γ, which are dependent on the common activation of the STAT1 pathway. These data add further explanation to the anti-tumor activity of IL-27 and also to its dual role in immune regulation.

Sugar-Based Arylsulfonamide Carboxylates as Selective and Water-Soluble Matrix Metalloproteinase-12 Inhibitors.

Matrix metalloproteinase-12 (MMP-12) can be considered an attractive target to study selective inhibitors useful in the development of new therapies for lung and cardiovascular diseases. In this study, a new series of arylsulfonamide carboxylates, with increased hydrophilicity resulting from conjugation with a ß-N-acetyl-d-glucosamine moiety, were designed and synthesized as MMP-12 selective inhibitors. Their inhibitory activity was evaluated on human MMPs by using the fluorimetric assay, and a crystallographic analysis was performed to characterize their binding mode. Among these glycoconjugates, a nanomolar MMP-12 inhibitor with improved water solubility, compound 3 [(R)-2-(N-(2-(3-(2-acetamido-2-deoxy-ß-d-glucopyranosyl)thioureido)ethyl)biphenyl-4-ylsulfonamido)-3-methylbutanoic acid], was identified.

Targeting ADAM17 Sheddase Activity in Cancer.

A disintegrin and metalloprotease (ADAM)17 is a sheddase, capable of releasing the ectodomains of membrane proteins such as growth factors (e.g. Epidermal Growth Factor Receptor ligands), cytokines and their receptors, adhesion and signaling molecules. These activities regulate several physiological and pathological processes including inflammation, tumor growth and metastatic progression. In this review, we will summarize ADAM17 biology and focus on its role in cancer and the possible usage of ADAM17 inhibitors in cancer therapy. Recent achievements in this area include the development of small molecule metalloprotease inhibitors with enhanced specificity for ADAM17, monoclonal antibodies, and synthetic short RNA molecules for gene silencing. These approaches successfully inhibited cancer cell growth and invasiveness or sensitized them to cytotoxic drugs, ionizing radiations or targeted therapies, in preclinical studies. These findings suggest the repositioning of ADAM17 inhibitors, which have yet proven unsuccessful as anti-inflammatory agents, for the development of new anti-cancer therapies, particularly in EGFR ligand-dependent cancers. Future studies should address ADAM17 inhibitors as short-term treatments in combination with different anti-cancer therapies.

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.