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Discovery of new actionable targets and functional networks in melanoma is an urgent need as only a fraction of metastatic patients achieves durable clinical benefit by targeted therapy or immunotherapy approaches. Here we show that NFATc2 expression is associated with an EMT-like transcriptional program and with an invasive melanoma phenotype, as shown by analysis of melanoma cell lines at the mRNA and protein levels, interrogation of the TCGA melanoma dataset and characterization of melanoma lesions by immunohistochemistry. Gene silencing or pharmacological inhibition of NFATc2 downregulated EMT-related genes and AXL, and suppressed c-Myc, FOXM1, and EZH2. Targeting of c-Myc suppressed FOXM1 and EZH2, while targeting of FOXM1 suppressed EZH2. Inhibition of c-Myc, or FOXM1, or EZH2 downregulated EMT-related gene expression, upregulated MITF and suppressed migratory and invasive activity of neoplastic cells. Stable silencing of NFATc2 impaired melanoma cell proliferation in vitro and tumor growth in vivo in SCID mice. In NFATc2+EZH2+melanoma cell lines pharmacological co-targeting of NFATc2 and EZH2 exerted strong anti-proliferative and pro-apoptotic activity, irrespective of BRAF or NRAS mutations and of BRAF inhibitor resistance. These results provide preclinical evidence for a role of NFATc2 in shaping the EMT-like melanoma phenotype and reveal a targetable vulnerability associated with NFATc2 and EZH2 expression in melanoma cells belonging to different mutational subsets.

Bortezomib (BTZ) is the first proteasome inhibitor entered in clinical practice. Peripheral neuropathy is likely to be a class side effect of these drugs, although its severity is largely variable, and it deserves to be further investigated, since the mechanisms of BTZ-induced peripheral neurotoxicity (BiPN) are still unknown. In our study, we investigated in vivo and in vitro possible pathogenic events relevant to BiPN using a well-established rat model, with particular reference to the extent of proteasome inhibition and the effects on α-tubulin polymerization in sciatic nerves and dorsal root ganglia specimens obtained from animals treated with chronic regimens at a dose of 0.2 mg/kg intravenously. The same assessments were also performed after a single injection. Moreover, these studies were replicated in vitro using embryonic DRG neurons exposed to 100 nM BTZ and adult DRG neurons exposed to 10–50 nM BTZ for 24 h and 48 h.A significant increase in the polymerized fraction of α-tubulin and prolonged proteasome inhibition were observed after the chronic BTZ treatment in vivo. Recovery to physiological levels was observed after a 4-week follow-up post-treatment period. Proteasome inhibition and increased α-tubulin polymerization were also observed following BTZ treatment of both embryonic and adult DRG neurons in vitro.Our in vivo results suggest that proteasome inhibition and alteration of tubulin dynamics contribute to BiPN. The in vitro systems here described reliably replicate the in vivo results, and might therefore be used for further mechanistic studies on the effects of proteasome inhibitors on neurons.

Abstract: Cancer of head and neck (HN) represents a diffused group of neoplasia; despite advances in clinical treatment its mortality rate has remained high, mainly because it is frequently diagnosed only at an advanced stage. Molecular markers allowing the diagnosis at an early stage would be useful. Mitogen-activated protein kinases (MAPK) are a family of protein kinases, which transduces extracellular stimuli into intracellular responses, controlling proliferation, differentiation and apoptosis. Given their key role in the regulation of cell survival and proliferation, MAPKs have been deeply studied in cancer. Furthermore, they are of particular interest in HN cancer progression, since they are downstream of one of the most frequently found alterations in this cancer: the over-expression of the epidermal growth factor receptor. Although a deregulation of MAPK expression and activation has been reported in several type of cancers, data regarding their role in HN cancer progression are contrasting, thus the aim of this short review is to summarize the data from literature regarding MAPKs activation in this type of cancer.

Repeated intracerebroventricular (i.c.v.) microinjection of tumor necrosis factor‐α (TNF‐α) into normal rats causes intramyelin and interstitial edema in the white matter of the spinal cord (SC). This response is identical to that observed in the SC white matter of rats made cobalamin (Cbl) deficient by total gastrectomy (TG). Immunoblot analysis showed that: 1) the level of the biologically active form of the TNF‐α protein (17 kDa) is higher in the SC of totally gastrectomized (TGX) rats 2 months after TG, i.e., at the postoperative time when edema is observed; 2) SC levels of TNF‐α protein (17 kDa) in 2‐mo‐TGX‐, Cbl‐treated rats are reduced to control. Repeated i.c.v. microinjections of anti‐TNF‐α antibodies, transforming growth factor‐β1(TGF‐β1) or interleukin‐6 (IL‐6) into TGX rats, begun shortly after TG, substantially reduced both intramyelin and interstitial edema in the SC white matter. This study provides the first evidence that the hallmark myelin damage of Cbl‐deficient central neuropathy, which is a pure myelinolytic disease, is not caused directly by the withdrawal of the vitamin itself, but reflects enhanced production of the biologically active form of TNF‐α by SC cells. This study thus supports the view that TGF‐β1and IL‐6 may act as neuroprotective agents in Cbl deficiency central neuropathy.—Buccellato, F. R., Miloso, M., Braga, M., Nicolini, G., Morabito, A., Pravettoni, G., Tredici, G., Scalabrino, G. Myelinolytic lesions in spinal cord of cobalamin‐deficient rats are TNF‐α‐mediated. FASEB J.13, 297–304 (1999)

We have recently demonstrated that the myelinolytic lesions in the spinal cord (SC) of rats made deficient in vitamin B12(cobalamin) (Cbl) through total gastrectomy (TG) are tumor necrosis factor‐α (TNF‐α)‐mediated. We investigate whether or not permanent Cbl deficiency, induced in the rat either through TG or by chronic feeding of a Cbl‐deficient diet, might modify the levels of three physiological neurotrophic factors—epidermal growth factor (EGF), vasoactive intestinal peptide (VIP), and somatostatin (SS)—in the cerebrospinal fluid (CSF) of these rats. We also investigated the ability of the central nervous system (CNS) in these Cbl‐deficient rats to synthesize EGF mRNA and of the SC to take up labeled Cbl in vivo. Cbl‐deficient rats, however the vitamin deficiency is induced, show a selective decrease in EGF CSF levels and an absence of EGF mRNA in neurons and glia in various CNS areas. In contrast, radiolabeled Cbl is almost exclusively taken up by the SC white matter, but to a much higher degree in totally gastrectomized (TGX) rats. Chronic administration of Cbl to TGX rats restores to normal both the EGF CSF level and EGF mRNA expression in the various CNS areas examined. This in vivostudy presents the first evidence that the neurotrophic action of Cbl in the CNS of TGX rats is mediated by stimulation of the EGF synthesis in the CNS itself. It thus appears that Cbl inversely regulates the expression of EGF and TNF‐α genes in the CNS of TGX rats.—Scalabrino, G., Nicolini, G., Buccellato, F. R., Peracchi, M., Tredici, G., Manfridi, A., Pravettoni, G. Epidermal growth factor as a local mediator of the neurotrophic action of vitamin B12(cobalamin) in the rat central nervous system. FASEB J. 13, 2083–2090 (1999)

In this study we evaluated the effect of recombinant human nerve growth factor (rhNGF) on cisplatin (CDDP)-induced sensory neuronopathy in an experimental paradigm in the rat. Young adult female Wistar rats were treated with CDDP (2 mg/kg ip twice weekly for nine times) alone or in combination with rhNGF (1 mg/kg sc on alternate days). The effect of CDDP ± NGF treatment was evaluated with behavioral (tail-flick test) and neurophysiological (nerve conduction velocity in the tail) methods immediately after treatment and after a follow-up period of 6 weeks. Pathological and morphometrical examinations of the dorsal root ganglia (DRG) and sciatic and saphenous nerves were also performed. rhNGF treatment induced a significant reduction in the CDDP-induced decrease in nerve conduction velocity (P<0.05), and this was associated with a significant protection against the decrease in somatic (P<0.05), nuclear (P<0.05), and nucleolar size (P<0.01) caused by CDDP treatment. However, for each of the parameters examined the neuroprotection obtained with rhNGF treatment was not complete. At the follow-up examination no differences between the three groups were observed in tail-flick test and nerve conduction velocity. We conclude that rhNGF, administered according to the schedule used in this experiment, exerts a biologically significant neuroprotective effect against CDDP peripheral neurotoxicity.

We report here the identification of a new shared human melanoma antigen recognized by a human leukocyte antigen (HLA)-A*68011–restricted cytotoxic T lymphocyte clone (CTL 128). The cDNA encoding this antigen is composed of a partially spliced form of the melanocyte differentiation antigen tyrosinase-related protein (TRP)-2, containing exons 1–4 with retention of intron 2 and part of intron 4 (TRP-2–INT2). The sequence coding for the antigenic epitope is located at the 5′ end of intron 2 and is available for translation in the same open reading frame of the fully spliced TRP-2 mRNA. This peptide is also recognized by CTL 128 when presented by the HLA-A*3301, a member of the HLA-A3–like supertype that includes the HLA-A*68011. Quantitative reverse transcription PCR analysis carried out on total and/or cytoplasmic mRNA demonstrated that, in contrast to the fully spliced TRP-2 mRNA expressed in melanomas, normal skin melanocytes, and retina, the TRP-2–INT2 mRNA could be detected at significant levels in melanomas but not in normal cells of the melanocytic lineage. Instead, in these normal samples, both the spliced and the unspliced transcript of gp100 were expressed at high levels. Absence of endogenous TRP-2–INT2 expression in melanocytes was also confirmed by lack of recognition of HLA-A*68011–transduced, TRP-2+ melanocyte lines by CTL 128. These results indicate that a partially spliced form of a differentiation antigen mRNA, present in the cytoplasmic compartment of neoplastic but not normal cells of the melanocytic lineage, can be the source of a melanoma-restricted T cell epitope.

Expression of epidermal growth factor receptor (EGF-R) antisense RNA results in a drastic reduction of EGF-R levels in the human carcinoma KB cell line and induces a reversion of their transformed phenotype (Moroni, M. C., Willingham, M. C., and Beguinot, L. (1992) J. Biol. Chem.267, 2714-2722). We used parental and EGF-R antisense KB clones as a genetic system to study, in the same cell line, the role of transforming growth factor α (TGF-α) in the establishment and maintenance of the transformed phenotype. KB cells produce TGF-α mRNA, and their conditioned medium is able to sustain growth of antisense cells, mimicking the effect of exogenous EGF or TGF-α. In antisense cells there is a marked reduction of TGF-α mRNA steady-state levels. In addition, the decrease in TGF-α parallels the levels of residual EGF-R in the various antisense clones, indicating a direct correlation between receptors and growth factor levels. The addition of exogenous TGF-α (10 ng/ml) to antisense clones induces TGF-α levels. The half-life of TGF-α mRNA is 40-60 min in antisense cells and more than 8 h in parental KB cells, as determined by actinomycin D decay curves. This result indicates a predominant regulation of TGF-α mRNA at the post-transcriptional level. Nuclear run-on experiments show that there is only a marginal effect at the transcriptional level. We conclude that the autocrine loop responsible for the transformed phenotype of the human carcinoma KB cell line is dependent on both elevated levels of EGF-R and the presence of TGF-α. In addition, TGF-α is able to induce its own mRNA via a signal due to activation of the EGF-R acting predominantly at the post-transcriptional level.