Alpha-2-Macroglobulin Is a Novel Anticancer Agent.

INTRODUCTION: Melanoma is the most aggressive skin cancer, with an increasing occurrence. Despite the recent important improvements due to novel immunotherapy approaches, when late diagnosed, melanoma prognosis is poor due to the metastatic progression and drug-resistance onset. Therefore, there is an urgent need to identify additional therapeutic targets. Melanoma invasive behavior is related to the activity of metalloproteases, able to degrade extracellular matrix leading to tumor dissemination. A recent study suggested that the most potent proteases inhibitor alpha-2-macroglobulin (A2MG) from plasma of hibernating fishes exerts potent antiproliferative effects. Our previous studies showed a significant reduction of A2MG in sera from mice/human melanoma models. METHODS: Gene and protein expression studies have been performed by using platforms and databases available online containing expression data from thousands of patients and healthy controls. RESULTS: We carried out an extensive bioinformatics analysis to evaluate the A2MG gene/protein expression on a large cohort of patients affected by many different cancer types, compared to healthy control subjects, and we found a highly significant difference of A2MG expression in 20 out of 31 cancer types (including melanoma) compared to healthy controls. Similar results were also confirmed at the proteomic level using another platform available online. Further, we found that higher A2MG expression is significantly related to overall survival in different cancers including melanoma. CONCLUSION: Our results strongly suggest A2MG as a novel molecular target in melanoma therapy, as well as in other cancer types.

Anti-Melanoma Effects of Miconazole: Investigating the Mitochondria Involvement.

Miconazole is an antimycotic drug showing anti-cancer effects in several cancers. However, little is known on its effects in melanoma. A375 and SK-MEL-28 human melanoma cell lines were exposed to miconazole and clotrimazole (up to 100 mM). Proliferation, viability with MTT assay and vascular mimicry were assayed at 24 h treatment. Molecular effects were measured at 6 h, namely, ATP-, ROS-release and mitochondria-related cytofluorescence. A metabolomic profile was also investigated at 6 h treatment. Carnitine was one of the most affected metabolites; therefore, the expression of 29 genes involved in carnitine metabolism was investigated in the public platform GEPIA2 on 461 melanoma patients and 558 controls. After 24 h treatments, miconazole and clotrimazole strongly and significantly inhibited proliferation in the presence of 10% serum on either melanoma cell lines; they also strongly reduced viability and vascular mimicry. After 6 h treatment, ATP reduction and ROS increase were observed, as well as a significant reduction in mitochondria-related fluorescence. Further, in A375, miconazole strongly and significantly altered expression of several metabolites including carnitines, phosphatidyl-cholines, all amino acids and several other small molecules, mostly metabolized in mitochondria. The expression of 12 genes involved in carnitine metabolism was found significantly modified in melanoma patients, 6 showing a significant impact on patients' survival. Finally, miconazole antiproliferation activity on A375 was found completely abrogated in the presence of carnitine, supporting a specific role of carnitine in melanoma protection toward miconazole effect, and was significantly reversed in the presence of caspases inhibitors such as ZVAD-FMK and Ac-DEVD-CHO, and a clear pro-apoptotic effect was observed in miconazole-treated cells, by FACS analysis of Annexin V-FITC stained cells. Miconazole strongly affects proliferation and other biological features in two human melanoma cell lines, as well as mitochondria-related functions such as ATP- and ROS-release, and the expression of several metabolites is largely dependent on mitochondria function. Miconazole, likely acting via carnitine and mitochondria-dependent apoptosis, is therefore suggested as a candidate for further investigations in melanoma treatments.

Phytochemicals as Immunomodulatory Agents in Melanoma.

Cutaneous melanoma is an immunogenic highly heterogenic tumor characterized by poor outcomes when it is diagnosed late. Therefore, immunotherapy in combination with other anti-proliferative approaches is among the most effective weapons to control its growth and metastatic dissemination. Recently, a large amount of published reports indicate the interest of researchers and clinicians about plant secondary metabolites as potentially useful therapeutic tools due to their lower presence of side effects coupled with their high potency and efficacy. Published evidence was reported in most cases through in vitro studies but also, with a growing body of evidence, through in vivo investigations. Our aim was, therefore, to review the published studies focused on the most interesting phytochemicals whose immunomodulatory activities and/or mechanisms of actions were demonstrated and applied to melanoma models.

Analysis of gene expression levels and their impact on survival in 31 cancer-types patients identifies novel prognostic markers and suggests unexplored immunotherapy treatment options in a wide range of malignancies.

BACKGROUND: Immunotherapy has dramatically improved cancer treatment by inhibiting or activating specific cell receptors, thus unleashing the host anti-tumor response. However, the engagement of the three main immune checkpoints so far identified, CTLA4, PD-1 and PD-L1, is effective in a fraction of patients, therefore novel targets must be identified and tested. METHODS: We focused our attention on the following nine highly relevant immune checkpoint (ICR) receptors: CTLA4, PD1, PD-L1, LAG3, TIM3, OX40, GITR, 4-1BB and TIGIT. All of them are targets of existing drugs currently under clinical scrutiny in several malignancies. Their expression levels were evaluated in patient tissues of 31 different cancer types vs. proper controls, in a total of 15,038 individuals. This analysis was carried out by interrogating public databases available on GEPIA2 portal and UALCAN portal. By the Principal Component Analysis (PCA) their ability to effectively discriminate patients form controls was then investigated. Expression of the nine ICRs was also related to overall survival in 31 cancer types and expressed as Hazard Ratio, on the GEPIA2 portal and validated, for melanoma patients, in patients-datasets available on PROGgene V2 portal. RESULTS: Significant differential expression was observed for each ICR molecule in many cancer types. A 7-molecules profile was found to specifically discriminate melanoma patients from controls, while two different 6-molecules profiles discriminate pancreatic cancer patients and Testicular Germ Cell Tumors from matched controls. Highly significant survival improvement was found to be related to the expression levels of all nine ICRs in a wide spectrum of malignancies. For melanoma analysis, the relation with survival observed in TCGA datasets was validated in independent GSE melanoma datasets. CONCLUSION: Analysis the nine ICR molecules demonstrates that their expression patterns may be considered as markers of disease and strong survival predictors in a variety of malignancies frequently associated to poor prognosis. Thus, the present findings are strongly advocating that exploratory clinical trials are worth to be performed, using available drugs, targeting these molecules.

Skin Lesion Area Segmentation Using Attention Squeeze U-Net for Embedded Devices.

Melanoma is the deadliest form of skin cancer. Early diagnosis of malignant lesions is crucial for reducing mortality. The use of deep learning techniques on dermoscopic images can help in keeping track of the change over time in the appearance of the lesion, which is an important factor for detecting malignant lesions. In this paper, we present a deep learning architecture called Attention Squeeze U-Net for skin lesion area segmentation specifically designed for embedded devices. The main goal is to increase the patient empowerment through the adoption of deep learning algorithms that can run locally on smartphones or low cost embedded devices. This can be the basis to (1) create a history of the lesion, (2) reduce patient visits to the hospital, and (3) protect the privacy of the users. Quantitative results on publicly available data demonstrate that it is possible to achieve good segmentation results even with a compact model.

Targeting Melanoma-Initiating Cells by Caffeine: In Silico and In Vitro Approaches.

The beneficial effects of coffee on human diseases are well documented, but the molecular mechanisms of its bioactive compounds on cancer are not completely elucidated. This is likely due to the large heterogeneity of coffee preparations and different coffee-based beverages, but also to the choice of experimental models where proliferation, differentiation and immune responses are differently affected. The aim of the present study was to investigate the effects of one of the most interesting bioactive compounds in coffee, i.e., caffeine, using a cellular model of melanoma at a defined differentiation level. A preliminary in silico analysis carried out on public gene-expression databases identified genes potentially involved in caffeine's effects and suggested some specific molecular targets, including tyrosinase. Proliferation was investigated in vitro on human melanoma initiating cells (MICs) and cytokine expression was measured in conditioned media. Tyrosinase was revealed as a key player in caffeine's mechanisms of action, suggesting a crucial role in immunomodulation through the reduction in IL-1ß, IP-10, MIP-1α, MIP-1ß and RANTES secretion onto MICs conditioned media. The potent antiproliferative effects of caffeine on MICs are likely to occur by promoting melanin production and reducing inflammatory signals' secretion. These data suggest tyrosinase as a key player mediating the effects of caffeine on melanoma.

Preliminary results of a counselling programme for fertility preservation in female cancer patients: The experience of the GEMME DORMIENTI network.

OBJECTIVE: To describe a population of patients referred for fertility preservation (FP), how to efficiently provide FP care, and how FP care changed over time. METHODS: This longitudinal observational study enrolled 281 female cancer patients referred between 2013 and 2016 to the non-profit organisation Gemme Dormienti ONLUS (GD) for FP care. All patients underwent the same battery of instrumental and laboratory diagnostic tests. GnRHa therapy was started at least seven days before CTh treatment. RESULTS: From 2013 to 2016, we observed a progressive increase in the number of patients referred for FP care. Out of 251 eligible patients, 135 patients were treated with GnRHa only, and 72 patients underwent GnRHa therapy and cryopreservation. The median time from GD referral to oocyte and ovarian tissue cryopreservation was 11 and 5 days respectively. Tissue cryopreservation requests increased during our study period (from four cases in 2013 to 17 cases in 2016). During follow-up, 17ß-estradiol and FSH levels were significantly increased (p < .0001), and AMH levels were significantly decreased (p < .0001). CONCLUSION: The rapid increase in the number of patients who requested FP care and in the complexity of FP procedures overtime reflects the need to improve quality of life for cancer patients.

Nailfold capillaroscopic parameters and skin telangiectasia patterns in patients with systemic sclerosis.

OBJECTIVE: To correlate nailfold capillaroscopic parameters with the presence of skin telangiectases (TAs) in systemic sclerosis patients (SSc). METHODS: Thirty-three consecutive patients (28 women and 5 men, mean age 59±21years) affected by SSc according to the ACR/EULAR criteria, 30 with limited (lcSSc) and 3 with diffuse (dcSSc) skin disease, displaying the presence of skin TAs on face, hands, forearms, neck, and décolleté were recruited. Nailfold videocapillaroscopy (NVC) was performed to classify the patients into one of the three main patterns of SSc microangiopathy ("early", "active", "late"), and to calculate the microangiopathy evolution score (MES). SSc patients underwent also dermoscopy (DS) for the analysis of the TA score and patterns (spot or reticular). Possible correlations between clinical findings, serum autoantibodies, TA patterns and both NVC patterns and MES were investigated. RESULTS: The "late" NVC pattern was found associated with a highest total number of TAs (p=0.005): in particular both "spot" and "reticular" TA patterns were found equally distributed in SSc patients with the "late" pattern. High MES values were found associated with the highest total number of TAs (p=0.003), with the "reticular" but not with the "spot" DS pattern (p=0.003) and with the "late" pattern of microangiopathy (p=0.001). CONCLUSIONS: The severity of nailfold microangiopathy seems to correlate in SSc patients with both progressive cutaneous microvascular abnormalities and Medsger's severity score, as evaluated by NVC analysis and DS. The assessment of the microvascular damage may be useful not only during the onset of SSc for the early diagnosis, but also to monitor its evolution.

Transglutaminase type 2 affects cell migration through post-translational modification of platelet-derived growth factor-BB.

Migration is a key cellular function with important implications in cell physiology. Impairment of such function is observed in angiogenesis, cancer, central nervous system development, and many other physiological and pathological events. Serum is considered among the most potent physiological chemotactic stimuli. Transglutaminase 2 (TG2) is involved in most of the mentioned processes, suggesting the hypothesis that TG2 may modulate cell movement and chemotaxis by acting on serum factors. Cell biology and biochemistry studies confirmed this hypothesis, showing that human serum contains potent chemotactic signals significantly impaired by activated TG2. Bioinformatics studies indicated that one potent serum factor potential substrate of TG2-dependent transamidation is platelet-derived growth factor-BB (PDGF-BB). Cell biology and immunometric experiments carried out with U87MG human glioma cell line showed that human recombinant PDGF-BB pre-incubated with calcium-activated TG2 lost about 70 % of its chemotactic activity and antigenicity. These data indicate that PDGF-BB is a substrate of TG2-transamidating activity, and such modification may play a key role in the modulation of PDGF's chemotactic features. Further, these findings suggest a novel point of view to study the extracellular functions of TG2 and to understand how protein signals, such as growth factors and cytokines, act in the extracellular space to reach their specific targets.

Letter to the Editor: "Ion Channels in Brain Metastasis"-Ion Channels in Cancer Set up and Metastatic Progression.

The review by Klumpp, L. et al. entitled Ion Channels in Brain Metastasis [1] discusses the role of ion channels in breast cancer, lung cancer and melanoma in metastatic tropism to the brain [...].

Lipid Storage and Autophagy in Melanoma Cancer Cells.

Cancer stem cells (CSC) represent a key cellular subpopulation controlling biological features such as cancer progression in all cancer types. By using melanospheres established from human melanoma patients, we compared less differentiated melanosphere-derived CSC to differentiating melanosphere-derived cells. Increased lipid uptake was found in melanosphere-derived CSC vs. differentiating melanosphere-derived cells, paralleled by strong expression of lipogenic factors Sterol Regulatory Element-Binding Protein-1 (SREBP-1) and Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ). An inverse relation between lipid-storing phenotype and autophagy was also found, since microtubule-associated protein 1A/1B-Light Chain 3 (LC3) lipidation is reduced in melanosphere-derived CSC. To investigate upstream autophagy regulators, Phospho-AMP activated Protein Kinase (P-AMPK) and Phospho-mammalian Target of Rapamycin (P-mTOR) were analyzed; lower P-AMPK and higher P-mTOR expression in melanosphere-derived CSC were found, thus explaining, at least in part, their lower autophagic activity. In addition, co-localization of LC3-stained autophagosome spots and perilipin-stained lipid droplets was demonstrated mainly in differentiating melanosphere-derived cells, further supporting the role of autophagy in lipid droplets clearance. The present manuscript demonstrates an inverse relationship between lipid-storing phenotype and melanoma stem cells differentiation, providing novel indications involving autophagy in melanoma stem cells biology.

c-Flip KO fibroblasts display lipid accumulation associated with endoplasmic reticulum stress.

c-Flip proteins are well-known apoptosis modulators. They generally contribute to tissue homeostasis maintenance by inhibiting death-receptor-mediated cell death. In the present manuscript, we show that c-Flip knock-out (KO) mouse embryonic fibroblasts (MEFs) kept in culture under starvation conditions gradually modify their phenotype and accumulate vacuoles, becoming progressively larger according to the duration of starvation. Large vacuoles are present in KO MEFs though not in WT MEFs, and are Oil Red-O positive, which indicates that they represent lipid droplets. Western blot experiments reveal that, unlike WT MEFs, KO MEFs express high levels of the lipogenic transcription factor PPAR-γ. Lipid droplet accumulation was found to be associated with endoplasmic reticulum (ER) stress activation and autophagic modulation valuated by means of BIP increase, LC3 lipidation and AMP-activated protein kinase (AMPK) phosphorylation, and p62 accumulation. Interestingly, XBP-1, an ER stress-induced lipogenic transcription factor, was found to preferentially localize in the nucleus rather than in the cytoplasm of KO MEFs. These data demonstrate that, upon starvation, c-Flip affects lipid accumulation, ER stress and autophagy, thereby pointing to an important role of c-Flip in the adaptive response and ER stress response programs under both normal and pathological conditions.

Ion channels expression and function are strongly modified in solid tumors and vascular malformations.

BACKGROUND: Several cellular functions relate to ion-channels activity. Physiologically relevant chains of events leading to angiogenesis, cell cycle and different forms of cell death, require transmembrane voltage control. We hypothesized that the unordered angiogenesis occurring in solid cancers and vascular malformations might associate, at least in part, to ion-transport alteration. METHODS: The expression level of several ion-channels was analyzed in human solid tumor biopsies. Expression of 90 genes coding for ion-channels related proteins was investigated within the Oncomine database, in 25 independent patients-datasets referring to five histologically-different solid tumors (namely, bladder cancer, glioblastoma, melanoma, breast invasive-ductal cancer, lung carcinoma), in a total of 3673 patients (674 control-samples and 2999 cancer-samples). Furthermore, the ion-channel activity was directly assessed by measuring in vivo the electrical sympathetic skin responses (SSR) on the skin of 14 patients affected by the flat port-wine stains vascular malformation, i.e., a non-tumor vascular malformation clinical model. RESULTS: Several ion-channels showed significantly increased expression in tumors (p < 0.0005); nine genes (namely, CACNA1D, FXYD3, FXYD5, HTR3A, KCNE3, KCNE4, KCNN4, CLIC1, TRPM3) showed such significant modification in at least half of datasets investigated for each cancer type. Moreover, in vivo analyses in flat port-wine stains patients showed a significantly reduced SSR in the affected skin as compared to the contralateral healthy skin (p < 0.05), in both latency and amplitude measurements. CONCLUSIONS: All together these data identify ion-channel genes showing significantly modified expression in different tumors and cancer-vessels, and indicate a relevant electrophysiological alteration in human vascular malformations. Such data suggest a possible role and a potential diagnostic application of the ion-electron transport in vascular disorders underlying tumor neo-angiogenesis and vascular malformations.

Comprehensive analysis of PTEN status in Sezary syndrome.

Sézary syndrome (SS) is an incurable leukemic variant of cutaneous T-cell lymphoma characterized by recurrent chromosomal alterations, among which, chromosome 10q deletion is very frequent. In this study, we investigated the PTEN status, on locus 10q23, in 44 SS patients; our findings show that PTEN is deleted in 36% of SS cases, whereas PTEN downregulation is observed in almost all of the samples evaluated by quantitative reverse-transcriptase polymerase chain reaction and Western blotting analysis. Neither DNA sequence mutation nor promoter hypermethylation were found at the PTEN locus, but we demonstrate that PTEN level can be also reduced by a group of miRs previously found upregulated and of prognostic relevance in SS; particularly, miR-21, miR-106b, and miR-486 were able to control PTEN abundance either in vitro or in vivo. Finally, because reduced PTEN activates the PI3/AKT-mediated pathway of cell growth and survival, we demonstrate that PTEN deficiency is associated with activated AKT in skin resident but not circulating SS cells, suggesting that the cutaneous milieu may strongly contribute to the SS cell growth. To our knowledge, this is the first study fully exploring the PTEN status in a large cohort of SS patients, unveiling potential elements of clinical utility in this malignancy.

Cancer Microenvironment and Endoplasmic Reticulum Stress Response.

Different stressful conditions such as hypoxia, nutrient deprivation, pH changes, or reduced vascularization, potentially able to act as growth-limiting factors for tumor cells, activate the unfolded protein response (UPR). UPR is therefore involved in tumor growth and adaptation to severe environments and is generally cytoprotective in cancer. The present review describes the molecular mechanisms underlying UPR and able to promote survival and proliferation in cancer. The critical role of UPR activation in tumor growth promotion is discussed in detail for a few paradigmatic tumors such as prostate cancer and melanoma.

Circulating interleukin-8 and osteopontin are promising biomarkers of clinical outcomes in advanced melanoma patients treated with targeted therapy.

BACKGROUND: Circulating cytokines can represent non-invasive biomarkers to improve prediction of clinical outcomes of cancer patients. Here, plasma levels of IL-8, CCL4, osteopontin, LIF and BDNF were determined at baseline (T0), after 2 months of therapy (T2) and, when feasible, at progression (TP), in 70 melanoma patients treated with BRAF and MEK inhibitors. The association of baseline cytokine levels with clinical response, progression-free survival (PFS) and overall survival (OS) was evaluated. METHODS: Cytokine concentrations were measured using the xMAP technology. Their ability to discriminate between responding (Rs) and non-responding (NRs) patients was assessed by Receiver Operating Characteristics analysis. PFS and OS were estimated with the Kaplan-Meier method. The Cox proportional hazard model was used in the univariate and multivariate analyses to estimate crude and adjusted hazard ratios with 95% confidence intervals. RESULTS: CCL4 and LIF were undetectable in the majority of samples. The median osteopontin concentration at T0 and T2 was significantly higher in NRs than in Rs. The median T0 and T2 values of IL-8 were also higher in NRs than in Rs, although the statistical significance was not reached. No differences were detected for BDNF. In 39 Rs with matched T0, T2, and TP samples, osteopontin and IL-8 significantly decreased from T0 to T2 and rose again at TP, while BDNF levels remained unchanged. In NRs, none of the cytokines showed a significant decrease at T2. Only osteopontin demonstrated a good ability to discriminate between Rs and NRs. A high IL-8 T0 level was associated with significantly shorter PFS and OS and higher risk of progression and mortality, and remained an independent negative prognostic factor for OS in multivariate analysis. An elevated osteopontin T0 concentration was also significantly associated with worse OS and increased risk of death. Patients with high IL-8 and high osteopontin showed the lowest PFS and OS, and in multivariate analysis this cytokine combination remained independently associated with a three- to six-fold increased risk of mortality. CONCLUSION: Circulating IL-8 and osteopontin appear useful biomarkers to refine prognosis evaluation of patients undergoing targeted therapy, and deserve attention as potential targets to improve its clinical efficacy.

Cholangiocarcinoma Malignant Traits Are Promoted by Schwann Cells through TGFß Signaling in a Model of Perineural Invasion.

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial-mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFß receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFß signaling in regulating this process.

Tissue transglutaminase activity protects from cutaneous melanoma metastatic dissemination: an in vivo study.

The role of tissue transglutaminase (TG-2, TGase-2) in cancer development is still a fascinating field of research. The available reports do not elucidate fully its mechanism of action, due to the limitations of in vitro approaches. Therefore, to understand TG-2 role in cancer, we carried out an in vivo study with a more direct approach. TG-2 was in vivo overexpressed in a murine model of melanoma (intravenous injection of B16 melanoma cells in C57BL/6N mice) by means of a plasmid carrying the TG-2 cDNA. The evaluation of the frequency and size of the metastases indicated that the number of melanoma lung foci was more markedly reduced by TG-2 overexpression than the metastatic size. Then, TG-2 overexpressing mice showed a prolonged survival with respect to control mice. Further analyses were carried by means of proteomic analysis of melanoma cell lysates and meta-analysis of published transcriptomic datasets. Proteomic analysis of cell lysates from a human melanoma cell line compared to human keratinocytes showed significant differences in the expression of TG-2 substrates known to be involved in proliferation/differentiation and cancer progression. Taken together, these findings indicate a protective role of TG-2 enzymatic activity in melanoma progression in vivo.