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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may result in a severe pneumonia associated with elevation of blood inflammatory parameters, reminiscent of cytokine storm syndrome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients; however, the mechanisms by which SARS-CoV-2 triggers such an extensive inflammation remain unexplained.To dissect the mechanisms underlying SARS-CoV-2-associated inflammation in patients with severe coronavirus disease 2019 (COVID-19), we studied the role of IL-1β, a pivotal cytokine driving inflammatory phenotypes, whose maturation and secretion are regulated by inflammasomes.We analyzed nod-like receptor protein 3 pathway activation by means of confocal microscopy, plasma cytokine measurement, cytokine secretion following in vitro stimulation of blood circulating monocytes, and whole-blood RNA sequencing. The role of open reading frame 3a SARS-CoV-2 protein was assessed by confocal microscopy analysis following nucleofection of a monocytic cell line.We found that circulating monocytes from patients with COVID-19 display ASC (adaptor molecule apoptotic speck like protein-containing a CARD) specks that colocalize with nod-like receptor protein 3 inflammasome and spontaneously secrete IL-1β in vitro. This spontaneous activation reverts following patient's treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV-2 protein resulted in ASC speck formation.These results provide further evidence that IL-1β targeting could represent an effective strategy in this disease and suggest a mechanistic explanation for the strong inflammatory manifestations associated with COVID-19.

Background and aim of the study: Mutations in the G alpha-genes GNAQ and GNA11 are found in 85-90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes differentially affect tumor characteristics and outcome and if so, to identify potential mechanisms. Methods: We analyzed the association between GNAQ and GNA11 mutations with disease specific survival, gene expression profiles, and cytogenetic alterations in 219 UMs. We used tandem-affinity-purification, mass spectrometry and immunoprecipitation to identify protein interaction partners of the two G-proteins and analyzed their impact on DNA-methylation. Results: GNA11 mutation was associated with: i) an increased frequency of loss of BRCA1- associated protein 1 (BAP1) expression (p = 0.0005), ii) monosomy of chromosome 3 (p < 0.001), iii) amplification of chr8q (p = 0.038), iv) the combination of the latter two (p = 0.0002), and inversely with v) chr6p gain (p = 0.003). Our analysis also showed a shorter disease-specific survival of GNA11-mutated cases as compared to those carrying a GNAQ mutation (HR = 1.97 [95%CI 1.12-3.46], p = 0.02). GNAQ and GNA11 encoded G-proteins have different protein interaction partners. Specifically, the Tet Methylcytosine Dioxygenase 2 (TET2), a protein that is involved in DNA demethylation, physically interacts with the GNAQ protein but not with GNA11, as confirmed by immunoprecipitation analyses. High risk UM cases show a clearly different DNA-methylation pattern, suggesting that a different regulation of DNA methylation by the two G-proteins might convey a different risk of progression. Conclusions: GNA11 mutated uveal melanoma has worse prognosis and is associated with high risk cytogenetic, mutational and molecular tumor characteristics that might be determined at least in part by differential DNA-methylation. (C) 2022 Elsevier Ltd. All rights reserved.

Uveal melanoma (UM) exhibits recurring chromosomal abnormalities and gene driver mutations, which are related to tumor evolution/progression. Almost half of the patients with UM develop distant metastases, predominantly to the liver, and so far there are no effective adjuvant therapies. An accurate UM genetic profile could assess the individual patient's metastatic risk, and provide the basis to determine an individualized targeted therapeutic strategy for each UM patient. To investigate the presence of specific chromosomal and gene alterations, BAP1 protein expression, and their relationship with distant progression free survival (DPFS), we analyzed tumor samples from 63 UM patients (40 men and 23 women, with a median age of 64 years), who underwent eye enucleation by a single cancer ophthalmologist from December 2005 to June 2016. UM samples were screened for the presence of losses/gains in chromosomes 1p, 3, 6p, and 8q, and for mutations in GNAQ, GNA11, BAP1, SF3B1, and EIF1AX. BAP1 protein expression was detected by immunohistochemistry (IHC). Multivariate analysis showed that the presence of monosomy 3, 8q gain, and loss of BAP1 protein were significantly associated to DPFS, while BAP1 gene mutation was not, mainly due to the presence of metastatic UM cases with negative BAP1 IHC and no BAP1 mutation detected by Sanger sequencing. Loss of BAP1 protein expression and monosomy 3 represent the strongest predictors of metastases, and may have important implications for implementation of patient surveillance, properly designed clinical trials enrollment, and adjuvant therapy.

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.

Background Development of resistance to inhibitors of BRAF (BRAFi) and MEK (MEKi) remains a great challenge for targeted therapy in patients with BRAF-mutant melanoma. Here, we explored the role of miRNAs in melanoma acquired resistance to BRAFi. Methods miRNA expression in two BRAF-mutant melanoma cell lines and their dabrafenib-resistant sublines was determined using Affymetrix GeneChip® miRNA 3.1 microarrays and/or qRT-PCR. The effects of miR-126-3p re-expression on proliferation, apoptosis, cell cycle, ERK1/2 and AKT phosphorylation, dabrafenib sensitivity, invasiveness and VEGF-A secretion were evaluated in the dabrafenib-resistant sublines using MTT assays, flow cytometry, immunoblotting, invasion assays in Boyden chambers and ELISA. ADAM9, PIK3R2, MMP7 and CXCR4 expression in the sensitive and dabrafenib-resistant cells was determined by immunoblotting. Small RNA interference was performed to investigate the consequence of VEGFA or ADAM9 silencing on proliferation, invasiveness or dabrafenib sensitivity of the resistant sublines. Long-term proliferation assays were carried out in dabrafenib-sensitive cells to assess the effects of enforced miR-126-3p expression or ADAM9 silencing on resistance development. VEGF-A serum levels in melanoma patients treated with BRAFi or BRAFi+MEKi were evaluated at baseline (T0), after two months of treatment (T2) and at progression (TP) by ELISA. Results miR-126-3p was significantly down-regulated in the dabrafenib-resistant sublines as compared with their parental counterparts. miR-126-3p replacement in the drug-resistant cells inhibited proliferation, cell cycle progression, phosphorylation of ERK1/2 and/or AKT, invasiveness, VEGF-A and ADAM9 expression, and increased dabrafenib sensitivity. VEGFA or ADAM9 silencing impaired proliferation and invasiveness of the drug-resistant sublines. ADAM9 knock-down in the resistant cells increased dabrafenib sensitivity, whereas miR-126-3p enforced expression or ADAM9 silencing in the drug-sensitive cells delayed the development of resistance. At T0 and T2, statistically significant differences were observed in VEGF-A serum levels between patients who responded to therapy and patients who did not. In responder patients, a significant increase of VEGF-A levels was observed at TP versus T2. Conclusions Strategies restoring miR-126-3p expression or targeting VEGF-A or ADAM9 could restrain growth and metastasis of dabrafenib-resistant melanomas and increase their drug sensitivity. Circulating VEGF-A is a promising biomarker for predicting patients’ response to BRAFi or BRAFi+MEKi and for monitoring the onset of resistance. Electronic supplementary material The online version of this article (10.1186/s13046-019-1238-4) contains supplementary material, which is available to authorized users.

Neuroblastoma (NB) tumor substantially contributes to childhood cancer mortality. The design of novel drugs targeted to specific molecular alterations becomes mandatory, especially for high-risk patients burdened by chemoresistant relapse. The dysregulated expression of MYCN, ALK, and LIN28B and the diminished levels of miR-34a and let-7b are oncogenic in NB. Due to the ability of miRNA-mimics to recover the tumor suppression functions of miRNAs underexpressed into cancer cells, safe and efficient nanocarriers selectively targeted to NB cells and tested in clinically relevant mouse models are developed. The technology exploits the nucleic acids negative charges to build coated-cationic liposomes, then functionalized with antibodies against GD2 receptor. The replenishment of miR-34a and let-7b by NB-targeted nanoparticles, individually and more powerfully in combination, significantly reduces cell division, proliferation, neoangiogenesis, tumor growth and burden, and induces apoptosis in orthotopic xenografts and improves mice survival in pseudometastatic models. These functional effects highlight a cooperative down-modulation of MYCN and its down-stream targets, ALK and LIN28B, exerted by miR-34a and let-7b that reactivate regulatory networks leading to a favorable therapeutic response. These findings demonstrate a promising therapeutic efficacy of miR-34a and let-7b combined replacement and support its clinical application as adjuvant therapy for high-risk NB patients.

Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1α, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1α expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells.

Background Prostate cancer (PCa), the second most common cancer affecting men worldwide, shows a broad spectrum of biological and clinical behaviour representing the epiphenomenon of an extreme heterogeneity. Androgen deprivation therapy is the mainstay of treatment for advanced forms but after few years the majority of patients progress to castration-resistant prostate cancer (CRPC), a lethal form that poses considerable therapeutic challenges. Methods Western blotting, immunocytochemistry, invasion and reporter assays, and in vivo studies were performed to characterize androgen resistant sublines phenotype in comparison to the parental cell line LNCaP. RNA microarray, mass spectrometry, integrative transcriptomic and proteomic differential analysis coupled with GeneOntology and multivariate analyses were applied to identify deregulated genes and proteins involved in CRPC evolution. Results Treating the androgen-responsive LNCaP cell line for over a year with 10 μM bicalutamide both in the presence and absence of 0.1 nM 5-α-dihydrotestosterone (DHT) we obtained two cell sublines, designated PDB and MDB respectively, presenting several analogies with CRPC. Molecular and functional analyses of PDB and MDB, compared to the parental cell line, showed that both resistant cell lines were PSA low/negative with comparable levels of nuclear androgen receptor devoid of activity due to altered phosphorylation; cell growth and survival were dependent on AKT and p38MAPK activation and PARP-1 overexpression; their malignant phenotype increased both in vitro and in vivo. Performing bioinformatic analyses we highlighted biological processes related to environmental and stress adaptation supporting cell survival and growth. We identified 15 proteins that could direct androgen-resistance acquisition. Eleven out of these 15 proteins were closely related to biological processes involved in PCa progression. Conclusions Our models suggest that environmental factors and epigenetic modulation can activate processes of phenotypic adaptation driving drug-resistance. The identified key proteins of these adaptive phenotypes could be eligible targets for innovative therapies as well as molecules of prognostic and predictive value. Electronic supplementary material The online version of this article (10.1186/s12964-017-0206-x) contains supplementary material, which is available to authorized users.

Despite advances in surgery and radiotherapy of uveal melanoma (UM), many patients develop distant metastases that poorly respond to therapy. Improved therapies for the metastatic disease are therefore urgently needed. Expression of the epidermal growth factor receptor (EGFR), a target of kinase inhibitors and humanised antibodies in use for several cancers, had been reported. Forty-eight human UMs were analysed by expression profiling. Signalling was tested in three EGFR expressing UM cell lines by Western blotting using phosphorylation specific antibodies for EGFR and the downstream mediators AKT (v-akt murine thymoma viral oncogene homolog) and extracellular signal-regulated kinase (ERK). Evidence for signalling in tumours was obtained through the application of a UM-specific EGF-signature. The EGFR specific kinase inhibitor, Gefitinib and the humanised monoclonal antibody, Cetuximab, were tested for their effect on EGFR signalling. Natural killer cell mediated antibody-dependent cellular cytotoxicity (ADCC) and tumour necrosis factor α (TNF-α) release was analysed for Cetuximab. Fourteen of 48 UMs and three of 14 cell lines (over-)express EGFR, at least in part due to trisomy of the EGFR locus on chromosome 7p12. EGFR and the downstream mediator, AKT, are phosphorylated upon stimulation with EGF in EGFR expressing cell lines. EGFR over-expressing tumours but not EGFR negative tumours show an activated EGF-signature. Gefitinib inhibits EGFR and AKT phosphorylation and Cetuximab induces EGFR phosphorylation but inhibits signalling to AKT induced with EGF. Cetuximab triggers natural killer (NK) cells to lyse EGFR+ cell lines and to release TNF-α. EGFR appears suited as a novel molecular drug target for therapy of uveal melanoma.

// Adriana Amaro 1, * , Giovanna Angelini 1, * , Valentina Mirisola 1 , Alessia Isabella Esposito 1 , Daniele Reverberi 1 , Serena Matis 1 , Massimo Maffei 1 , Walter Giaretti 1 , Maurizio Viale 2 , Rosaria Gangemi 2 , Laura Emionite 3 , Simonetta Astigiano 4 , Michele Cilli 3 , Beatrice E. Bachmeier 5 , Peter H. Killian 5 , Adriana Albini 6 , Ulrich Pfeffer 1 1 Molecular Pathology, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 2 Biotherapy, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 3 Animal Facility, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 4 Immunology, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 5 Institute of Laboratory Medicine, Ludwig-Maximilians-University, Munich, Germany 6 Scientific and Technology Park, IRCCS MultiMedica, Milan, Italy * These authors have contributed equally to the work Correspondence to: Adriana Albini, email: adriana.albini@multimedica.it Ulrich Pfeffer, email: patologia.molecolare.integrata@gmail.com Keywords: breast cancer, invasion, apocrine breast cancer, metastasis, aneuploidy Received: May 16, 2016 Accepted: August 13, 2016 Published: September 10, 2016 ABSTRACT The acquisition of an invasive phenotype is a prerequisite for metastasization, yet it is not clear whether or to which extent the invasive phenotype is linked to other features characteristic of metastatic cells. We selected an invasive subpopulation from the triple negative breast cancer cell line MDA-MB-231, performing repeated cycles of preparative assays of invasion through Matrigel covered membranes. The invasive sub-population of MDA-MB-231 cells exhibits stronger migratory capacity as compared to parental cells confirming the highly invasive potential of the selected cell line. Prolonged cultivation of these cells did not abolish the invasive phenotype. ArrayCGH, DNA index quantification and karyotype analyses confirmed a common genetic origin of the parental and invasive subpopulations and revealed discrete structural differences of the invasive subpopulation including increased ploidy and the absence of a characteristic amplification of chromosome 5p14.1-15.33. Gene expression analyses showed a drastically altered expression profile including features of apocrine breast cancers and of invasion related matrix-metalloproteases and cytokines. The invasive cells showed accelerated proliferation, increased apoptosis, and an altered pattern of chemo-sensitivity with lower IC50 values for drugs affecting the mitotic apparatus. However, the invasive cell population is significantly less tumorigenic in orthotopic mouse xenografts suggesting that the acquisition of the invasive capacity and the achievement of metastatic growth potential are distinct events.

Colorectal cancer is characterized by exquisite genomic instability either in the form of microsatellite instability or chromosomal instability. Microsatellite instability is the result of mutation of mismatch repair genes or their silencing through promoter methylation as a consequence of the CpG island methylator phenotype. The molecular causes of chromosomal instability are less well characterized. Genomic instability and field cancerization lead to a high degree of intratumoral heterogeneity and determine the formation of cancer stem cells and epithelial-mesenchymal transition mediated by the TGF-β and APC pathways. Recent analyses using integrated genomics reveal different phases of colorectal cancer evolution. An initial phase of genomic instability that yields many clones with different mutations (big bang) is followed by an important, previously not detected phase of cancer evolution that consists in the stabilization of several clones and a relatively flat outgrowth. The big bang model can best explain the coexistence of several stable clones and is compatible with the fact that the analysis of the bulk of the primary tumor yields prognostic information.

Importance Chromosome 6p amplification is associated with more benign behavior for uveal melanomas (UMs) with an otherwise high risk of metastasis conferred by chromosome 3 monosomy. Chromosome 6p contains several members of the B7 family of immune regulator genes, including butyrophilin-like 2 ( BTNL2 ; OMIM,606000), which is associated with prostate cancer risk and autoimmune diseases. Objective To investigate the expression and variant allele frequencies of BTNL2 , a candidate gene for chromosome 6 amplification, in patients with UM. Design, Setting, and Participants In this case-control study, we analyzed the expression of BTNL2 in UM cell lines and human macrophages in patients with UM. Variants of BTNL2 were analyzed using probes for polymerase chain reaction and high-resolution melting. The association of missense variantsrs28362679andrs41441651with tumor risk was analyzed in 209 patients with UM and 116 matched control patients as well as 12 UM and 64 other tumor cell lines. Genes that were differentially expressed in M1- and M2-polarized macrophages were identified by microarray analysis of 111 patients with UM, and the association of the expression of these genes with disease-free survival was analyzed by Cox regression analysis. Data were collected from September 2013 to November 2015. Main Outcomes and Measures Butyrophilin-like 2 single-nucleotide variants were associated with UM risk; M1 and M2 macrophage-specific gene expression was associated with disease-free survival. Results We genotyped a total of 325 patients. Of the 209 patients with UM, 124 (59.3%) were male, 114 (54.5%) were Italian, and 95 (45.5%) were German; the mean (range) age was 65 (27-94) years. Of the 116 Italian control patients, 67 (57.8%) were female, and the mean (range) age was 39 (21-88) years. Butyrophilin-like 2 is expressed in patients with UM and macrophages. The frequency of thers28362679variant was higher in patients with UM (16 of 209 [7.7%]; 95% CI, 4.7-12.2) than frequencies from European Variation Archive and Exome Aggregation Consortium data (2134 of 118 564 [1.8%]; 95% CI, 1.7-1.9) and Exome Sequencing Project data (100 of 4540 [2.2%]; 95% CI, 1.8-2.7) but were not higher compared with Italian control patients (10 of 116 [8.6%]; 95% CI, 4.6-15.4). Thers41441651variant was present in 5 patients with UM (2.4%; 95% CI, 0.9-5.7), 2 Italian control patients (1.7%; 95% CI, 0.1-6.5), 2846 patients from European Variation Archive and Exome Aggregation Consortium data (2.4%; 95% CI, 2.3-2.5), and 23 patients from Exome Sequencing Project data (0.5%; 95% CI, 0.3-0.8). Human UM cells express M1 and M2 macrophage-specific genes, whose expression is associated with disease-free survival. Conclusions and Relevance Butyrophilin-like 2, expressed at various levels by UM cells and macrophages, might interfere with the immune control of the tumor. Butyrophilin-like 2 variants showed highly variable frequencies among ethnically related cohorts. There was no enrichment of BTNL2 variants in patients with UM compared with control patients.

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.

Observations on the role of ovarian hormones in breast cancer growth, as well as interest in contraception, stimulated research into the biology of estrogens. The identification of the classical receptors ERα and ERβ and the transmembrane receptor GPER and the resolution of the structure of the ligand bound to its receptor established the principal molecular mechanisms of estrogen action. The presence of estrogen-like compounds in many plants used in traditional medicine or ingested as food ingredients, phytoestrogens, as well as the estrogenic activities of many industrial pollutants and pesticides, xenoestrogens, have prompted investigations into their role in human health. Phyto- and xenoestrogens bind to the estrogen receptors with a lower affinity than the endogenous estrogens and can compete or substitute the hormone. Xenoestrogens, which accumulate in the body throughout life, are believed to increase breast cancer risk, especially in cases of prenatal and prepuberal exposure whereas the role of phytoestrogens is still a matter of debate. At present, the application of phytoestrogens appears to be limited to the treatment of post-menopausal symptoms in women where the production of endogenous estrogens has ceased. In this review we discuss chemistry, structure and classification, estrogen signaling and the consequences of the interactions of estrogens, phytoestrogens and xenoestrogens with their receptors, the complex interactions of endogenous and exogenous ligands, the evaluation of the health risks related to xenoestrogens, and the perspectives toward the synthesis of potent third generation selective estrogen receptor modulators (SERMs).

Several substances widely dispersed in the environment including hormones, industrial by-products and pollutants exert hormone like activity affecting steroid-responsive physiological systems. These compounds, named endocrine disruptors, are suspected to affect the mammalian reproductive system. However it is still unclear whether these substances are able to elicit estrogen like activity at the low concentrations encountered in the environment. Here we compare the effects of the endocrine disruptor nonylphenol with the effects elicited by 17-β -estradiol on gene transcription in the human breast cancer cell line MCF7. The correlation of the nonylphenol induced gene expression alterations with a reference profile of estradiol treated cells shows that nonylphenol at a concentration of 100 nM exerts a significant effect on estrogen responsive gene transcription in MCF7 cells. Most of the genes regulated by 17-β -estradiol respond to the nonylphenol in the same direction though to a much lesser extent. Molecular modeling of the potential interaction of nonylphenol with the estrogen receptor α shows that nonylphenol is likely to bind to the estrogen receptor α.

Uveal melanoma is the most common primary tumour of the eye with an annual incidence of approximately two cases per million in southern European countries to eight cases in northern European countries (Virgili et al, 2007). Incidence increases with latitude in a highly significant manner (Virgili et al, 2007). Whether this association can be attributed to the exposure to sunlight of variable intensity or not, remains a matter of discussion (Gallagher et al, 1985; Pane and Hirst, 2000; Guenel et al, 2001; Singh et al, 2004). Uveal melanoma shows a mutation pattern that is clearly distinct from cutaneous (for recent reviews, see Coupland et al, 2013; Harbour, 2013; Zeschnigk and Lohmann, 2013), mucosal (Furney et al, 2013) and conjunctival melanomas (Griewank et al, 2013c). The mutations typically encountered in cutaneous and conjunctival melanomas, BRAF and NRAS, are rare in uveal melanomas that are characterised by mutations of the G-proteins GNAQ and GNA11 occurring in mutual exclusive manner in ∼85% of the cases (Zeschnigk and Lohmann, 2013). The mutation pattern observed by exome sequencing in cutaneous melanoma is clearly consistent with an aetiological role of sunlight exposure (Hodis et al, 2012). Cytidine-to-thymidine transition frequency has not been addressed by the two exome sequencing studies of uveal melanoma (Harbour et al, 2013; Martin et al, 2013) but the complete lack of overlap among the driver mutations in the two pathologies is consistent with a different aetiology. Recently, three mutations in the promoter of the telomerase reverse transcriptase (TERT) needed for telomere maintenance in cancer cells, close to the transcriptional start site, have been described for sporadic (Huang et al, 2013) and familiar (Horn et al, 2013) forms of cutaneous melanoma. The familiar cases all derived from one family and showed a mutation different from those detected in sporadic melanomas and melanoma cell lines (Huang et al, 2013). Two mutations were identified in sporadic cases where they occurred in a mutually exclusive manner in over 70% of cases. The same mutations were also present, though less frequently, in other tumours and tumour-derived cell lines. The mutations consisted in cytidine-to-thymidine transitions at a dipyrimidine motif consistent with ultraviolet (UV) light-induced damage. All three mutations created novel binding sites for the transcription factor E-twenty-six (ETS) in the TERT promoter within 100 bp upstream of the transcription start site (TSS) in sporadic cases and closer to the TTS (−57 bp) in the familiar cases. Reporter assays showed the functionality of the new binding sites observed in sporadic cases (Huang et al, 2013). The mutation is highly penetrant in the melanoma family analysed and it is linked to early onset (mean 34 years) and short survival (mean 3.5 years in 8 patients who died from the disease of a total of 11 cases). It is therefore probable that the mutation strongly contributes to the development of aggressively growing cutaneous melanoma. Griewank et al (2013a) have addressed whether the same mutations are present also in ocular melanomas reporting an incidence of 32% in conjunctival melanomas and the absence from uveal (ciliary body or choroid) melanomas (n=47). In the present study, we describe the analysis of TERT promoter mutations in a series of 50 uveal melanomas. A mutation was detected in a single case that also carried mutations in GNA11 and EIF1AX, typical for this disease and absent from conjunctival and cutaneous melanomas. The tumour showed disomy of chromosome 3 and had wild-type sequences of GNAQ, BAP1 and SF3B1 which frequently show mutations in uveal melanoma.

Biomarkers are important for early detection of cancer, prognosis, response prediction, and detection of residual or relapsing disease. Special attention has been given to diagnostic markers for prostate cancer since it is thought that early detection and surgery might reduce prostate cancer-specific mortality. The use of prostate-specific antigen, PSA (KLK3), has been debated on the base of cohort studies that show that its use in preventive screenings only marginally influences mortality from prostate cancer. Many groups have identified alternative or additional markers, among which PCA3, in order to detect early prostate cancer through screening, to distinguish potentially lethal from indolent prostate cancers, and to guide the treatment decision. The large number of markers proposed has led us to the present study in which we analyze these indicators for their diagnostic and prognostic potential using publicly available genomic data. We identified 380 markers from literature analysis on 20,000 articles on prostate cancer markers. The most interesting ones appeared to be claudin 3 (CLDN3) and alpha-methysacyl-CoA racemase highly expressed in prostate cancer and filamin C (FLNC) and keratin 5 with highest expression in normal prostate tissue. None of the markers proposed can compete with PSA for tissue specificity. The indicators proposed generally show a great variability of expression in normal and tumor tissue or are expressed at similar levels in other tissues. Those proposed as prognostic markers distinguish cases with marginally different risk of progression and appear to have a clinically limited use. We used data sets sampling 152 prostate tissues, data sets with 281 prostate cancers analyzed by microarray analysis and a study of integrated genomics on 218 cases to develop a multigene score. A multivariate model that combines several indicators increases the discrimination power but does not add impressively to the information obtained from Gleason scoring. This analysis of 10 years of marker research suggests that diagnostic and prognostic testing is more difficult in prostate cancer than in other neoplasms and that we must continue to search for better candidates. Electronic supplementary material The online version of this article (doi:10.1007/s10555-013-9470-4) contains supplementary material, which is available to authorized users.

The anti-hyperglycaemic drug metformin has important anticancer properties as shown by the direct inhibition of cancer cells proliferation. Tumor cells avidly use glucose as a source for energy production and cell building blocks. Critical to this phenotype is the production of glucose-6-phosphate (G6P), catalysed by hexokinases (HK) I and II, whose role in glucose retention and metabolism is highly advantageous for cell survival and proliferation. Here we show that metformin impairs the enzymatic function of HKI and II in Calu-1 cells. This inhibition virtually abolishes cell glucose uptake and phosphorylation as documented by the reduced entrapment of (18)F-fluorodeoxyglucose. In-silico models indicate that this action is due to metformin capability to mimic G6P features by steadily binding its pocket in HKII. The impairment of this energy source results in mitochondrial depolarization and subsequent cell death. These results could represent a starting point to open effective strategies in cancer prevention and treatment.

Survival after diagnosis of laryngeal cancer has not improved over the last 20 years. Selection of patients for radio- and chemotherapy or surgery or follow-up strategies based on a prognostic classifier could improve survival without unduly extending radical surgery. We performed microarray gene expression analysis and developed a four-gene classifier for laryngeal cancer using Prediction Analysis of Microarray and leave-one-out cross validation. A four-gene classifier containing the non-coding gene H19, the histone HIST1H3F and the two small nucleolar RNAs, SNORA16A and SNORD14C was developed that assigns cases to low and high risk classes. The high risk class has a relative risk of 6.5 (CI = 1.817–23.258, Fisher exact test p

// Giovanna Bianchi 1,* , Roberto Martella 1,* , Silvia Ravera 2 , Cecilia Marini 3 , Selene Capitanio 4 , Annamaria Orengo 4 , Laura Emionite 5 , Chiara Lavarello 6 , Adriana Amaro 7 , Andrea Petretto 6 , Ulrich Pfeffer 7 , Gianmario Sambuceti 4 , Vito Pistoia 1 , Lizzia Raffaghello 1,** and Valter D. Longo 8,9,10,** 1 Laboratorio di Oncologia Istituto G. Gaslini, Genoa, Italy 2 Department of Pharmacy, University of Genoa, Genova, Italy 3 CNR Institute of Bioimages and Molecular Physiology, Milan, Section of Genoa, Genoa, Italy 4 Nuclear Medicine Unit, Department of Health Sciences, University of Genoa and IRCCS AOU San Martino - IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 5 Animal facility, IRCCS AOU San Martino - IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 6 Core Facility, Istituto G. Gaslini, Genoa, Italy 7 Functional Genomics, IRCCS AOU San Martino - IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 8 Longevity Institute, School of Gerontology, Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA 9 Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 10 IFOM, FIRC Institute of Molecular Oncology, Milan, Italy * These authors have contributed equally as first authors ** These authors have contributed equally as last authors Correspondence to: Valter D. Longo, email: // Keywords : fasting, Warburg effect, colon cancer, oxidative phosphorylation, glucose uptake Received : March 04, 2015 Accepted : March 11, 2015 Published : March 30, 2015 Abstract Tumor chemoresistance is associated with high aerobic glycolysis rates and reduced oxidative phosphorylation, a phenomenon called “Warburg effect” whose reversal could impair the ability of a wide range of cancer cells to survive in the presence or absence of chemotherapy. In previous studies, Short-term-starvation (STS) was shown to protect normal cells and organs but to sensitize different cancer cell types to chemotherapy but the mechanisms responsible for these effects are poorly understood. We tested the cytotoxicity of Oxaliplatin (OXP) combined with a 48hour STS on the progression of CT26 colorectal tumors. STS potentiated the effects of OXP on the suppression of colon carcinoma growth and glucose uptake in both in vitro and in vivo models. In CT26 cells, STS down-regulated aerobic glycolysis, and glutaminolysis, while increasing oxidative phosphorylation. The STS-dependent increase in both Complex I and Complex II-dependent O 2 consumption was associated with increased oxidative stress and reduced ATP synthesis. Chemotherapy caused additional toxicity, which was associated with increased succinate/Complex II-dependent O 2 consumption, elevated oxidative stress and apoptosis . These findings indicate that the glucose and amino acid deficiency conditions imposed by STS promote an anti-Warburg effect characterized by increased oxygen consumption but failure to generate ATP, resulting in oxidative damage and apoptosis.