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Abstract Triple-Negative Breast Cancer (TNBC), represents a subtype of breast cancer in which the estrogens receptor (ER) negative, the progesterone receptor (PR) negative and the human epidermal growth factor receptor 2 (HER2) negative, are not expressed. Thusly, TNBC does not respond to hormonal therapies or to those targeting the HER2 protein receptors. To overcome this flawed issue, new alternative therapies based on the use of natural substances, as the (−) - epigallocatechin 3-gallate (EGCG), has been proposed. It is largely documented that EGCG, the principal constituent of green tea, has suppressive effects on different types of cancer, including breast cancer, through the regulation of different signaling pathways. Thus, is reasonable to assume that EGCG could be viewed as a therapeutic option for the prevention and the treatment of TNBC. Here, we summarizing these promising results with the scope of turn a light on the potential roles of EGCG in the treatment of TNBC patients.

Abstract Background Inefficient T-cell access to the tumor microenvironment (TME) is among the causes of tumor immune-resistance. Previous evidence demonstrated that targeting CXCR4 improves anti-PD-1/PD-L1 efficacy reshaping TME. To evaluate the role of newly developed CXCR4 antagonists (PCT/IB2011/000120/ EP2528936B1/US2013/0079292A1) in potentiating anti-PD-1 efficacy two syngeneic murine models, the MC38 colon cancer and the B16 melanoma-human CXCR4-transduced, were employed. Methods Mice were subcutaneously injected with MC38 (1 × 106) or B16-hCXCR4 (5 × 105). After two weeks, tumors bearing mice were intraperitoneally (ip) treated with murine anti-PD-1 [RMP1–14] (5 mg/kg, twice week for 2 weeks), Pep R (2 mg/kg, 5 days per week for 2 weeks), or both agents. The TME was evaluated through immunohistochemistry and flow-cytometry. In addition, the effects of the human-anti-PD-1 nivolumab and/or Peptide-R54 (Pep R54), were evaluated on human melanoma PES43 cells and xenografts treated. Results The combined treatment, Pep R plus anti-PD-1, reduced the MC38 Relative Tumor Volume (RTV) by 2.67 fold (p = 0.038) while nor anti-PD-1, neither Pep R significantly impacted on tumor growth. Significant higher number of Granzyme B (GZMB) positive cells was detected in MC38 tumors from mice treated with the combined treatment (p = 0.016) while anti-PD-1 determined a modest but significant increase of tumor-infiltrating GZMB positive cells (p = 0.035). Also, a lower number of FoxP3 positive cells was detected (p = 0.022). In the B16-hCXCR4 tumors, two weeks of combined treatment reduced tumor volume by 2.27 fold while nor anti-PD-1 neither Pep R significantly impacted on tumor growth. A significant higher number of GRZB positive cells was observed in B16-hCXCR4 tumors treated with combined treatment (p = 0,0015) as compared to anti-PD-1 (p = 0.028). The combined treatment reduced CXCR4, CXCL12 and PD-L1 expression in MC38 tumors. In addition, flow cytometry on fresh B16-hCXCR4 tumors showed significantly higher Tregs number following anti-PD-1 partially reversed by the combined treatment Pep R and anti-PD-1. Combined treatment determined an increase of CD8/Tregs and CD8/MDSC ratio. To dissect the effect of anti-PD-1 and CXCR4 targeting on PD-1 expressed by human cancer cells, PES43 human melanoma xenograft model was employed. In vitro human anti-PD-1 nivolumab or pembrolizumab (10 μM) reduced PES43 cells growth while nivolumab (10 μM) inhibited pERK1/2, P38 MAPK, pAKT and p4EBP. PES43 xenograft mice were treated with Pep R54, a newly developed Pep R derivative (AcHN-Arg-Ala-[DCys-Arg- Nal(2′)-His-Pen]- COOH), plus nivolumab. After 3 weeks of combined treatment a significant reduction in tumor growth was shown (p = 0.038). PES43 lung disseminated tumor cells (DTC) were detected in fresh lung tissues as melanoma positive MCSP-APC+ cells. Although not statistically significant, DTC-PES43 cells were reduced in mice lungs treated with combined treatment while nivolumab or Pep R54 did not affect DTC number. Conclusion Combined treatment with the new developed CXCR4 antagonist, Pep R, plus anti-PD-1, reduced tumor-growth in two syngeneic murine models, anti-PD-1 sensitive and resistant, potentiating Granzyme and reducing Foxp3 cells infiltration. In addition, the human specific CXCR4 antagonist, Pep R54, cooperated with nivolumab in inhibiting the growth of the PD-1 expressing human PES43 melanoma xenograft. This evidence sheds light on PD-1 targeting mechanisms and paves the way for CXCR4/PD-1 targeting combination therapy.

Abstract Background Platelet-rich-plasma (PRP) is largely used, thanks to its properties, as wound therapy after surgical resection. Several studies and clinical findings have demonstrated that the PRP can accelerate the regeneration and the repair of tissues through the action of the platelet-derived growth factors. Material and methods Our study aimed to investigate the effects of PRP-gel on the rate of tumor relapse by using a mouse model of Human Fibrosarcoma (HF). The radical resection of tumors of mice was conducted under fluorescence-guidance (FGR) by using MacroFluo microscope, after a primary tumor removal with bright-light surgery (BLS). Results It was found that the lesion recurrence and the tumor growth were reduced in mice treated with PRP observed in each group of treatment (50%) after 30 days from tumor excision, respect to controls (without statistical significance; p = 0.12). The histopathological and immune-histochemical analysis did not report differences in cellular morphology between the tumors of control and PRP-treated mice. Conclusion Our data suggest that PRP-gel, used as an adjuvant treatment for the stimulation of tissue repair and speed up recovery, can impair tumor growth and slow the tumor.

We have previously shown that secreted BAG3 is a potential target for the treatment of pancreatic ductal adenocarcinoma and that pancreatic tumor growth and metastatic dissemination can be reduced by treatment with an anti‐BAG3 murine antibody. Here, we used complementarity‐determining region (CDR) grafting to generate a humanized version of the anti‐BAG3 antibody that may be further developed for possible clinical use. We show that the humanized anti‐BAG3 antibody, named BAG3‐H2L4, abrogates BAG3 binding to macrophages and subsequent release of IL‐6. Furthermore, it specifically localizes into tumor tissues and significantly inhibits the growth of Mia PaCa‐2 pancreatic cancer cell xenografts. We propose BAG3‐H2L4 antibody as a potential clinical candidate for BAG3‐targeted therapy in pancreatic cancer.

Proper regulation of neurogenesis, the process by which new neurons are generated from neural stem and progenitor cells (NS/PCs), is essential for embryonic brain development and adult brain function. The transcription regulator Patz1 is ubiquitously expressed in early mouse embryos and has a key role in embryonic stem cell maintenance. At later stages, the detection of Patz1 expression mainly in the developing brain suggests a specific involvement of Patz1 in neurogenesis. To address this point, we first got insights in Patz1 expression profile in different brain territories at both embryonic and postnatal stages, evidencing a general decreasing trend with respect to time. Then, we performed in vivo and ex vivo analysis of Patz1-knockout mice, focusing on the ventricular and subventricular zone, where we confirmed Patz1 enrichment through the analysis of public RNA-seq datasets. Both embryos and adults showed a significant reduction in the number of Patz1-null NS/PCs, as well as of their self-renewal capability, compared to controls. Consistently, molecular analysis revealed the downregulation of stemness markers in NS/PCs derived from Patz1-null mice. Overall, these data demonstrate the requirement of Patz1 for NS/PC maintenance and proliferation, suggesting new roles for this key transcription factor specifically in brain development and plasticity, with possible implications for neurodegenerative disorders and glial brain tumors.

Abstract Background A novel prediction algorithm is needed for the identification of effective tumor associated mutated neoantigens. Only those with no homology to self wild type antigens are true predicted neoantigens (TPNAs) and can elicit an antitumor T cell response, not attenuated by central tolerance. To this aim, the mutational landscape was evaluated in HCV-associated hepatocellular carcinoma. Methods Liver tumor biopsies and adjacent non-tumor liver tissues were obtained from 9 HCV-chronically infected subjects and subjected to RNA-Seq analysis. Mutant peptides were derived from single nucleotide variations and TPNAs were predicted using two prediction servers (e.g. NetTepi and NetMHCstabpan) by comparison with corresponding wild-type sequences, non-related self and pathogen-related antigens. Immunological confirmation was obtained in preclinical as well as clinical setting. Results The development of such an improved algorithm resulted in a handful of TPNAs despite the large number of predicted neoantigens. Furthermore, TPNAs may share homology to pathogen’s antigens and be targeted by a pre-existing T cell immunity. Cross-reactivity between such antigens was confirmed in an experimental pre-clinical setting. Finally, TPNAs homologous to pathogen’s antigens were found in the only HCC long-term survival patient, suggesting a correlation between the pre-existing T cell immunity specific for these TPNAs and the favourable clinical outcome. Conclusions The new algorithm allowed the identification of the very few TPNAs in cancer cells, and those targeted by a pre-existing immunity strongly correlated with long-term survival. Only such TPNAs represent the optimal candidates for immunotherapy strategies.

Abstract Meat is a crucial nutrient for human health since it represents a giant supply of proteins, minerals, and vitamins. On the opposite hand, the intake of red and processed meat is taken into account dangerous due to its potential of carcinogenesis and cancer risk improvement, particularly for colorectal cancer (CRC), although it has been reported that also the contaminations of beef infected by oncogenic bovine viruses could increase colorectal cancer’s risk. Regarding the mechanisms underlying the potential carcinogenicity of red and processed meat, different hypotheses have been proposed. A suggested mechanism describes the potential role of the heterocyclic amines (HACs) and polycyclic aromatic hydrocarbons (PHAs) in carcinogenesis induced by DNA mutation. Another hypothesis states that heme, through the lipid peroxidation process and therefore the formation of N-nitroso compounds (NOCs), produces cytotoxic and genotoxic aldehydes, resulting in carcinogenesis. Furthermore, a recent proposed hypothesis, is based on the combined actions between the N-Glycolylneuraminic acid (Neu5Gc) and genotoxic compounds. The purpose of this narrative review is to shed a light on the mechanisms underlying the potential carcinogenicity of red and processed meat, by summarizing the data reported in literature on this topic.

Abstract The development of metastases is a multistep process that requires the activation of physiological and biochemical processes that govern migration, invasion and entry of metastatic cells into blood vessels. The urokinase receptor (uPAR) promotes cell migration by interacting with the Formyl Peptide Receptors (FPRs). Since both uPAR and FPR1 are involved in tumor progression, the uPAR-FPR1 interaction is an attractive therapeutic target. We previously described peptide antagonists of the uPAR-FPR1 interaction that inhibited cell migration and angiogenesis. To develop enzyme-resistant analogues, we applied here the Retro-Inverso (RI) approach, whereby the topology of the side chains is maintained by inverting the sequence of the peptide and the chirality of all residues. Molecular dynamics suggests that peptide RI-3 adopts the turn structure typical of uPAR-FPR1 antagonists. Accordingly, RI-3 is a nanomolar competitor of N-formyl-Met-Leu-Phe for binding to FPR1 and inhibits migration, invasion, trans-endothelial migration of sarcoma cells and VEGF-triggered endothelial tube formation. When sarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density, circulating tumor cells and pulmonary metastases were significantly reduced in animals treated daily with 6 mg/Kg RI-3 as compared to animals treated with vehicle only. Thus, RI-3 represents a promising lead for anti-metastatic drugs.

Abstract C-X-C chemokine receptor 4 (CXCR4) is over-expressed in multiple human cancers and correlates with tumor aggressiveness, poor prognosis and increased risk for distant metastases. Imaging agents for CXCR4 are thus highly desirable. We developed a novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) conjugating the new developed CXCR4 peptidic antagonist Peptide R with the NIR fluorescent dye VivoTag-S750. Specific CXCR4 binding was obtained in cells overexpressing human CXCR4 (B16-hCXCR4 and human melanoma cells PES43), but not in CXCR4 low expressing cells (FB-1). Ex vivo evaluation demonstrated that PepR-NIR750 specifically detects B16-hCXCR4-derived subcutaneous tumors and lung metastases. Fluorescence Molecular Tomography (FMT) in vivo imaging was performed on mice carrying subcutaneous CHO and CHO-CXCR4 tumors. PepR-NIR750 accumulates only in CXCR4-positive expressing subcutaneous tumors. Additionally, an intense NIR fluorescence signal was detected in PES43-derived lung metastases of nude mice injected with PepR-NIR750 versus mice injected with VivoTag-S750. With a therapeutic intent, mice bearing PES43-derived lung metastases were treated with Peptide R. A the dramatic reduction in PES43-derived lung metastases was detected through a decrease of the PepR-NIR750 signal. PepR-NIR750 is a specific probe for non-invasive detection of human high CXCR4-expressing tumors and metastatic lesion and thus a valuable tool for cancer molecular imaging.

The ErbB2 blocker trastuzumab improves survival in oncologic patients, but can cause cardiotoxicity. The late Na+ current inhibitor ranolazine has been shown to counter experimental HF, including doxorubicin cardiotoxicity (a condition characterized by derangements in redox balance), by lowering the levels of reactive oxygen species (ROS). Since ErbB2 can modulate ROS signaling, we tested whether trastuzumab cardiotoxicity could be blunted by ranolazine via redox-mediated mechanisms. Trastuzumab decreased fractional shortening and ejection fraction in mice, but ranolazine prevented heart dysfunction when co-administered with trastuzumab. Trastuzumab cardiotoxicity was accompanied by elevations in natriuretic peptides and matrix metalloproteinase 2 (MMP2) mRNAs, which were not elevated with co-treatment with ranolazine. Trastuzumab also increased cleavage of caspase-3, indicating activation of the proapoptotic machinery. Again, ranolazine prevented this activation. Interestingly, Neonatal Rat Ventricular Myocytes (NRVMs), labeled with MitoTracker Red and treated with trastuzumab, showed only a small increase in ROS compared to baseline conditions. We then stressed trastuzumab-treated cells with the beta-agonist isoproterenol to increase workload, and we observed a significant increase of probe fluorescence, compared with cells treated with isoproterenol alone, reflecting induction of oxidative stress. These effects were blunted by ranolazine, supporting a role for INa inhibition in the regulation of redox balance also in trastuzumab cardiotoxicity.

Several nutraceuticals have been investigated for preventing or retarding the progression of different neurodegenerative diseases, including Alzheimer's disease (AD). Because Nigella sativa (NS) and its isolated compound thymoquinone (TQ) have significant anti-oxidant and anti-inflammatory proprieties, they could represent effective neuroprotective agents. The purpose of this manuscript is to analyze and to recapitulate the results of in vitro and in vivo studies on the potential role of NS/TQ in AD's prevention and treatment. The level of evidence for each included animal study has been assessed by using a modified CAMARADES (Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies) 10-item checklist. We used MEDLINE and EMBASE databases to screen relevant articles published up to July 2017. A manual search was also performed. The database search yielded 38 studies, of which 18 were included in this manuscript. Results from these approaches suggest that NS or TQ could represent an effective strategy against AD due to the balancing of oxidative processes and the binding to specific intracellular targets. The overall effects mainly regard the prevention of hippocampal pyramidal cell loss and the increased cognitive functions.

We have recently generated knockout mice for the Cbx7 gene, coding for a polycomb group protein that is downregulated in human malignant neoplasias. These mice develop liver and lung adenomas and carcinomas, which confirms a tumour suppressor role for CBX7. The CBX7 ability to downregulate CCNE1 expression likely accounts for the phenotype of the Cbx7-null mice. Unexpectedly, Cbx7-knockout mice had a higher fat tissue mass than wild-type, suggesting a role of CBX7 in adipogenesis. Consistently, we demonstrate that Cbx7-null mouse embryonic fibroblasts go towards adipocyte differentiation more efficiently than their wild-type counterparts, and this effect is Cbx7 dose-dependent. Similar results were obtained when Cbx7-null embryonic stem cells were induced to differentiate into adipocytes. Conversely, mouse embryonic fibroblasts and human adipose-derived stem cells overexpressing CBX7 show an opposite behaviour. These findings support a negative role of CBX7 in the control of adipocyte cell growth and differentiation.

The HMGA1 and HMGA2 genes code for proteins belonging to the High Mobility Group A family. Several genes are negatively or positively regulated by both these proteins, but a number of genes are specifically regulated by only one of them. Indeed, knock-out of the Hmga1 and Hmga2 genes leads to different phenotypes: cardiac hypertrophy and type 2 diabetes in the former case, and a large reduction in body size and amount of fat tissue in the latter case. Therefore, to better elucidate the functions of the Hmga genes, we crossed Hmga1-null mice with mice null for Hmga2. The Hmga1−/−/Hmga2−/− mice showed reduced vitality and a very small size (75% smaller than the wild-type mice); they were even smaller than pygmy Hmga2-null mice. The drastic reduction in E2F1 activity, and consequently in the expression of the E2F-dependent genes involved in cell cycle regulation, likely accounts for some phenotypic features of the Hmga1−/−/Hmga2−/− mice.

The term ‘personalized medicine’ refers to a medical procedure that consists in the grouping of patients based on their predicted individual response to therapy or risk of disease. In oncologic patients, a ‘tailored’ therapeutic approach may potentially improve their survival and well-being by not only reducing the tumour, but also enhancing therapeutic response and minimizing the adverse effects. Diagnostic tests are often used to select appropriate and optimal therapies that rely both on patient genome and other molecular/cellular analysis. Several studies have shown that lifestyle and environmental factors can influence the epigenome and that epigenetic events may be involved in carcinogenesis. Thus, in addition to traditional biomarkers, epigenetic factors are raising considerable interest, because they could potentially be used as an excellent tool for cancer diagnosis and prognosis. In this review, we summarize the role of conventional cancer genetic biomarkers and their association with epigenomics. Furthermore, we will focus on the so-called ‘homeostatic biomarkers’ that result from the physiological response to cancer, emphasizing the concept that an altered ‘new’ homeostasis influence not only tumour environment, but also the whole organism.

Cl-IB-MECA, synthetic A3 adenosine receptor agonist, is a potential anticancer agent. In this study, we have examined the effect of Cl-IB-MECA in a mouse melanoma model. Cl-IB-MECA significantly inhibited tumor growth in immune-competent mice. Notably, the number of tumor-infiltrating NK1.1+ cells and CD8+ T cells was significantly increased in Cl-IB-MECA-treated mice. This effect was correlated with high levels of tumor necrosis factor α (TNF-α) and interferon γ in melanoma tissue. Depletion of either CD8+ T cells or NK1.1+ cells completely abrogated the antitumor effect of Cl-IB-MECA. Accordingly, Cl-IB-MECA did not affect tumor growth in nude mice. In addition, we also found that the number of mature and active conventional dendritic cells at the tumor site was increased after Cl-IB-MECA administration. Moreover, Cl-IB-MECA significantly increased TNF-α and IL-12p40 release from splenic CD11c+ cells. In conclusion, our study provides novel insights into the mechanism by which Cl-IB-MECA leads to an effective antitumor response that involves the activation of natural killer cells and CD8+ T cells and further highlights its therapeutic potential.

The cross-talk at the prefronto-striatal interface involves excitatory amino acids, different receptors, transducers and modulators. We investigated long-term effects of a prepuberal, subchronic 5-HT7-R agonist (LP-211) on adult behaviour, amino acids and synaptic markers in a model for Attention-Deficit/Hyperactivity Disorder (ADHD). Naples High Excitability rats (NHE) and their Random Bred controls (NRB) were daily treated with LP-211 in the 5th and 6th postnatal week. One month after treatment, these rats were tested for indices of activity, non selective (NSA), selective spatial attention (SSA) and emotionality. The quantity of L-Glutamate (L-Glu), L-Aspartate (L-Asp) and L-Leucine (L-Leu), dopamine transporter (DAT), NMDAR1 subunit and CAMKIIα, were assessed in prefrontal cortex (PFC), dorsal (DS) and ventral striatum (VS), for their role in synaptic transmission, neural plasticity and information processing. Prepuberal LP-211 (at lower dose) reduced horizontal activity and (at higher dose) increased SSA, only for NHE but not in NRB rats. Prepuberal LP-211 increased, in NHE rats, L-Glu in the PFC and L-Asp in the VS (at 0.250 mg/kg dose), whereas (at 0.125 mg/kg dose) it decreased L-Glu and L-Asp in the DS. The L-Glu was decreased, at 0.125 mg/kg, only in the VS of NRB rats. The DAT levels were decreased with the 0.125 mg/kg dose (in the PFC), and increased with the 0.250 mg/kg dose (in the VS), significantly for NHE rats. The basal NMDAR1 level was higher in the PFC of NHE than NRB rats; LP-211 treatment (at 0.125 mg/kg dose) decreased NMDAR1 in the VS of NRB rats. This study represents a starting point about the impact of developmental 5-HT7-R activation on neuro-physiology of attentive processes, executive functions and their neural substrates.

BACKGROUND:Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR). EXPERIMENTAL: FINDINGS:We report that the pure anti-androgen Casodex inhibits the growth of HT1080 cell xenografts in immune-depressed mice, revealing a novel role of AR in fibrosarcoma progression. In HT1080 cultured cells EGF, but not androgens, robustly increases DNA synthesis. Casodex abolishes the EGF mitogenic effect, implying a crosstalk between EGFR and AR. The mechanism underlying this crosstalk has been analyzed using an AR-derived small peptide, S1, which prevents AR/Src tyrosine kinase association and androgen-dependent Src activation. Present findings show that in HT1080 cells EGF induces AR/Src Association, and the S1 peptide abolishes both the assembly of this complex and Src activation. The S1 peptide inhibits EGF-stimulated DNA synthesis, cell matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of HT1080 cells. Both Casodex and S1 peptide also prevent DNA synthesis and migration triggered by EGF in various human cancer-derived cells (prostate, breast, colon and pancreas) that express AR. CONCLUSION:This study shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells. The EGF-elicited processes inhibited by the peptide (DNA synthesis, MMP-9 secretion and invasiveness) cooperate in increasing the aggressive phenotype of HT1080 cells. Therefore, AR represents a new potential therapeutic target in human fibrosarcoma, as supported by Casodex inhibition of HT1080 cell xenografts. The extension of these findings in various human cancer-derived cell lines highlights the conservation of this process across divergent cancer cells and identifies new potential targets in the therapeutic approach to human cancers.

The CXCR4/CXCL12 axis plays a role in cancer metastases, stem cell mobilization and chemosensitization. Proof of concept for efficient CXCR4 inhibition has been demonstrated in stem cell mobilization prior to autologous transplantation in hematological malignancies. Nevertheless CXCR4 inhibitors suitable for prolonged use as required for anticancer therapy are not available. To develop new CXCR4 antagonists a rational, ligand-based approach was taken, distinct from the more commonly used development strategy. A three amino acid motif (Ar-Ar-X) in CXCL12, also found in the reverse orientation (X-Ar-Ar) in the vMIP-II inhibitory chemokine formed the core of nineteen cyclic peptides evaluated for inhibition of CXCR4-dependent migration, binding, P-ERK1/2-induction and calcium efflux. Peptides R, S and I were chosen for evaluation in in vivo models of lung metastases (B16-CXCR4 and KTM2 murine osteosarcoma cells) and growth of a renal cells xenograft. Peptides R, S, and T significantly reduced the association of the 12G5-CXCR4 antibody to the receptor and inhibited CXCL12-induced calcium efflux. The four peptides efficiently inhibited CXCL12-dependent migration at concentrations as low as 10 nM and delayed CXCL12-mediated wound healing in PES43 human melanoma cells. Intraperitoneal treatment with peptides R, I or S drastically reduced the number of B16-CXCR4-derived lung metastases in C57/BL mice. KTM2 osteosarcoma lung metastases were also reduced in Balb/C mice following CXCR4 inhibition. All three peptides significantly inhibited subcutaneous growth of SN12C-EGFP renal cancer cells. A novel class of CXCR4 inhibitory peptides was discovered. Three peptides, R, I and S inhibited lung metastases and primary tumor growth and will be evaluated as anticancer agents.

The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR(88-92) chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPAR(S90P) exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPAR(S90P) cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPAR(S90E) cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPAR(S90P). In conclusion, our findings indicate that Ser(90) is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPAR(S90E) and uPAR(S90P) are forced into inactive and active forms, respectively, suggesting important implications for the development of novel drugs targeting uPAR function.

Cl-IB-MECA is a selective A3 adenosine receptor agonist, which plays a crucial role in limiting tumor progression. In mice, Cl-IB-MECA administration enhances the anti-tumor T cell-mediated response. However, little is known about the activity of Cl-IB-MECA on CD8+ T cells. The aim of this study was to investigate the effect of ex vivo Cl-IB-MECA treatment of CD8+ T cells, adoptively transferred in melanoma-bearing mice. Adoptive transfer of Cl-IB-MECA-treated CD8+ T cells or a single administration of Cl-IB-MECA (20 ng/mouse) inhibited tumor growth compared with the control group and significantly improved mouse survival. This was associated with the release of Th1-type cytokines and a greater influx of mature Langerin+ dendritic cells (LCs) into the tumor microenvironment. CD8+ T cells treated with Cl-IB-MECA released TNF-α which plays a critical role in the therapeutic efficacy of these cells when injected to mice. Indeed, neutralization of TNF-α by a specific monoclonal Ab significantly blocked the anti-tumor activity of Cl-IB-MECA-treated T cells. This was due to the reduction in levels of cytotoxic cytokines and the presence of fewer LCs. In conclusion, these studies reveal that ex vivo treatment with Cl-IB-MECA improves CD8+ T cell adoptive immunotherapy for melanoma in a TNF-α-dependent manner.