SCD5-dependent inhibition of SPARC secretion hampers metastatic spreading and favors host immunity in a TNBC murine model.

Dysregulated fatty acid metabolism interacts with oncogenic signals, thereby worsening tumor aggressiveness. The stearoyl-CoA desaturating enzymes, SCD1 and SCD5, convert of saturated fatty acids to monounsaturated fatty acids. While SCD1 is frequently overexpressed in tumor cells and has been widely studied, SCD5 has both limited expression and poor characterization. Here we evaluated, in vitro and in vivo, the effects of SCD5 overexpression in a metastatic clone of 4T1. The results showed SCD5-driven reprogramming of fatty acid metabolism, involving desaturation of stearic acid to oleic acid, which eventually blocked SPARC secretion. The latter event reduced the aggressiveness of the 4T1 subclone by decreasing the ECM deposition and reverting the Epithelial to Mesenchymal Transition (EMT) status. Variation of the fatty acid profile by SCD5-gene transduction or the direct administration oleic acid reduces the immune suppressive activity of myeloid cells and promoting granulocytic myeloid-derived suppressor cell maturation, eventually favoring T-cell activation. The less immunosuppressive microenvironment generated by SCD5 overexpression was enhanced in Sparc-KO mice, indicating that both extracellular and endogenous SPARC additively regulate myeloid cell-suppressive activities. Overall, our data sheds light on exploring the oleic acid-dependent inhibition of SPARC secretion as a possible mechanism to reduce breast cancer malignancy.

Different Susceptibilities of Human Melanoma Cell Lines to G2/M Blockage and Cell Death Activation in Response to the Estrogen Receptor ß agonist LY500307.

Background: Gender differences in melanoma incidence, metastasis formation and disease progression are increasingly evident in epidemiological studies, with women showing significantly better survival than men. Among factors possibly underlying the disparities, sex hormones seem to play a key role. Nonetheless, functional mechanisms are still unclear, except for the antitumor ability of Estrogen Receptor (ER) ß, whose expression determination has often been suggested for melanoma prognosis. In this study, we aimed at evaluating the molecular mechanisms and functional effects associated with ERß signaling by using its agonist LY500307. Methods: We evaluated the antitumor effect of the specific synthetic ERß agonist LY500307 on some human melanoma cell lines, selected for different genetic background, expression levels of ERs and tumor progression. The expression of α and ß estrogen receptors was investigated taking advantage of The Cancer Genome Atlas database and confirmed on some selected melanoma cell lines. The biological effects of LY500307 were determined in vitro looking at melanoma cell proliferation, cell cycle profiles and migration demonstrating by western blot the involvement of some pathway specific markers. The LY500307-dependent induction of cell death was also analyzed by flow cytometry and western blot analysis of caspase 3 and poly adenosine diphosphate-ribose polymerase (PARP). Results: A significant decrease in the expression of both ERs, even more pronounced for ERα, has been found in patients with metastatic NRAS mutation. Treatment with LY500307 significantly reduced the proliferation of melanoma cells showing a cell cycle arrest at the G2/M boundary phase and promoting apoptosis with different sensitivities associated with disease stage and mutation. Indeed, the ERß agonist affects melanoma migration, inducing a reversion of the epithelial-mesenchymal transition, more evident in a low aggressive primary melanoma cell line. Conclusion: These results demonstrate the capability of LY500307 to reduce melanoma malignancy, counteracting cell viability and dissemination, overall suggesting a possible future use of LY500307 in personalized combined therapy.

pH-responsive oleic acid based nanocarriers: Melanoma treatment strategies.

Numerous clinical observations indicate that, despite novel therapeutic approaches, a high percentage of melanoma patients is non-responder or suffers of severe drug-related toxicity. To overcome these problems, we considered the option of designing, preparing and characterizing nanoemulsions and niosomes containing oleic acid, a pH-sensitive monounsaturated fatty acid holding per se an antimetastatic and anti-inflammatory role in melanoma. These new nanostructures will allow in vivo administration of oleic acid, otherwise toxic in its free form. For pulmonary route chitosan, a mucoadhesive agent, was enclosed in these nanocarriers to improve residence time at the lung site. A deep physical and chemical characterization was carried out evaluating size, ζ -potential, microviscosity, polarity as well as stability over time and in culture media. Moreover, their pH-sensitivity was evaluated by fluorometric assay. Cytotoxicity and cellular uptake were assessed in cultured normal fibroblasts and human melanoma cell lines. Interestingly, results obtained confirm nanocarrier stability and pH-sensitivity, associated to absence of cell toxicity, efficient cellular uptake and retention. Therefore, these new pH-sensitive oleic acid-based nanostructures could represent, by combining drug delivery in a pH-dependent manner with the antimetastatic potential of this fatty acid, a powerful strategy for more specific medicine against metastatic melanoma.

Chronic Isolation Stress Affects Central Neuroendocrine Signaling Leading to a Metabolically Active Microenvironment in a Mouse Model of Breast Cancer.

Social isolation is a powerful stressor capable of affecting brain plasticity and function. In the case of breast cancer, previous data indicate that stressful experiences may contribute to a worse prognosis, activating neuroendocrine and metabolism pathways, although the mechanisms underlying these effects are still poorly understood. In this study, we tested the hypothesis that chronic isolation stress (IS) may boost hypothalamic-pituitary-adrenal (HPA) axis activity, leading to changes in the hypothalamic expression of genes modulating both mood and metabolism in an animal model of breast cancer. This centrally activated signaling cascade would, in turn, affect the mammary gland microenvironment specifically targeting fat metabolism, leading to accelerated tumor onset. MMTVNeuTg female mice (a model of breast cancer developing mammary hyperplasia at 5 months of age) were either group-housed (GH) or subjected to IS from weaning until 5 months of age. At this time, half of these subjects underwent acute restraint stress to assess corticosterone (CORT) levels, while the remaining subjects were characterized for their emotional profile in the forced swimming and saccharin preference tests. At the end of the procedures, all the mice were sacrificed to assess hypothalamic expression levels of Brain-derived neurotrophic factor (Bdnf), Neuropeptide Y (NpY), Agouti-Related Peptide (AgRP), and Serum/Glucocorticoid-Regulated Protein Kinase 1 (SgK1). Leptin and adiponectin expression levels, as well as the presence of brown adipose tissue (BAT), were assessed in mammary fat pads. The IS mice showed higher CORT levels following acute stress and decreased expression of NpY, AgRP, and SgK1, associated with greater behavioral despair in the forced swimming test. Furthermore, they were characterized by increased consumption of saccharin in a preference test, suggesting an enhanced hedonic profile. The IS mice also showed an earlier onset of breast lumps (assessed by palpation) accompanied by elevated levels of adipokines (leptin and adiponectin) and BAT in the mammary fat pads. Overall, these data point to IS as a pervasive stressor that is able to specifically target neuronal circuits, mastered by the hypothalamus, modulating mood, stress reactivity and energy homeostasis. The activation of such IS-driven machinery may hold main implications for the onset and maintenance of pro-tumorigenic environments.

Biomarkers for Diagnosis, Prognosis and Response to Immunotherapy in Melanoma.

Cutaneous Melanoma classification is constantly looking for specific and sensitive biomarkers capable of having a positive effect on diagnosis, prognosis and risk assessment, eventually affecting clinical outcome. Classical morphological, immunohistochemical and the well-known BRAF and NRAS genetic biomarkers do not allow the correct categorization of patients, being melanoma conditioned by high genetic heterogeneity. At the same time, classic prognostic methods are unsatisfactory. Therefore, new advances in omics and high-throughput analytical techniques have enabled the identification of numerous possible biomarkers, but their potentiality needs to be validated and standardized in prospective studies. Melanoma is considered an immunogenic tumor, being the first form of cancer to take advantage of the clinical use of the immune-checkpoint blockers. However, as immunotherapy is effective only in a limited number of patients, biomarkers associated with different responses are essential to select the more promising therapeutic approach and maximize clinical benefits. In this review, we summarize the most utilized biomarkers for Cutaneous Melanoma diagnosis, focusing on new prognostic and predictive biomarkers mainly associated with immunotherapy.

Sex and Gender Disparities in Melanoma.

Worldwide, the total incidence of cutaneous melanoma is higher in men than in women, with some differences related to ethnicity and age and, above all, sex and gender. Differences exist in respect to the anatomic localization of melanoma, in that it is more frequent on the trunk in men and on the lower limbs in women. A debated issue is if-and to what extent-melanoma development can be attributed to gender-specific behaviors or to biologically intrinsic differences. In the search for factors responsible for the divergences, a pivotal role of sex hormones has been observed, although conflicting results indicate the involvement of other mechanisms. The presence on the X chromosome of numerous miRNAs and coding genes playing immunological roles represents another important factor, whose relevance can be even increased by the incomplete X chromosome random inactivation. Considering the known advantages of the female immune system, a different cancer immune surveillance efficacy was suggested to explain some sex disparities. Indeed, the complexity of this picture emerged when the recently developed immunotherapies unexpectedly showed better improvements in men than in women. Altogether, these data support the necessity of further studies, which consider enrolling a balanced number of men and women in clinical trials to better understand the differences and obtain actual gender-equitable healthcare.

Tumor-derived extracellular vesicles and microRNAs: Functional roles, diagnostic, prognostic and therapeutic options.

In the last few years cancer research more and more highlighted the importance of cell to cell communication in tumor progression. Among many other functional mechanisms, results evidenced the importance of miRNAs loaded into exosomes and their actions as mediators in intercellular communication, either in the tumor microenvironment or at distant sites. Deregulation of miRNA levels is a prerogative of cancer cells and is reflected in the miRNA cargo of tumor derived exosomes. Thus, learning of circulating miRNA activities add the missing piece we need to understand some unclear aspects of cancer biology. Here we summarized the current knowledge on exosome transfer capabilities between cancer cells and all the cells constituting tumor microenvironment with a particular focus on their miRNA cargos and regulatory functions. The clinical relevance of these molecular aspects is emphasized by numerous cell interactions that ultimately result in normal cell function defeat, relevant to increase tumor malignancy. The quantitative and qualitative evaluation of circulating miRNAs offers new perspective for better diagnosis and prognosis of cancer patients, eventually improving their management.

Non-genomic Effects of Estrogen on Cell Homeostasis and Remodeling With Special Focus on Cardiac Ischemia/Reperfusion Injury.

This review takes into consideration the main mechanisms involved in cellular remodeling following an ischemic injury, with special focus on the possible role played by non-genomic estrogen effects. Sex differences have also been considered. In fact, cardiac ischemic events induce damage to different cellular components of the heart, such as cardiomyocytes, vascular cells, endothelial cells, and cardiac fibroblasts. The ability of the cardiovascular system to counteract an ischemic insult is orchestrated by these cell types and is carried out thanks to a number of complex molecular pathways, including genomic (slow) or non-genomic (fast) effects of estrogen. These pathways are probably responsible for differences observed between the two sexes. Literature suggests that male and female hearts, and, more in general, cardiovascular system cells, show significant differences in many parameters under both physiological and pathological conditions. In particular, many experimental studies dealing with sex differences in the cardiovascular system suggest a higher ability of females to respond to environmental insults in comparison with males. For instance, as cells from females are more effective in counteracting the ischemia/reperfusion injury if compared with males, a role for estrogen in this sex disparity has been hypothesized. However, the possible involvement of estrogen-dependent non-genomic effects on the cardiovascular system is still under debate. Further experimental studies, including sex-specific studies, are needed in order to shed further light on this matter.

Autoantibodies Specific to ERα are Involved in Tamoxifen Resistance in Hormone Receptor Positive Breast Cancer.

Tamoxifen resistance is a major hurdle in the treatment of estrogen receptor (ER)-positive breast cancer. The mechanisms of tamoxifen resistance are not fully understood although several underlying molecular events have been suggested. Recently, we identified autoantibodies reacting with membrane-associated ERα (anti-ERα Abs) in sera of breast cancer patients, able to promote tumor growth. Here, we investigated whether anti-ERα Abs purified from sera of ER-positive breast cancer patients could contribute to tamoxifen resistance. Anti-ERα Abs inhibited tamoxifen-mediated effects on cell cycle and proliferation in MCF-7 cells. Moreover, anti-ERα Abs hampered the tamoxifen-mediated reduction of tumor growth in SCID mice xenografted with breast tumor. Notably, simvastatin-mediated disaggregation of lipid rafts, where membrane-associated ERα is embedded, restored tamoxifen sensitivity, preventing anti-ERα Abs effects. In conclusion, detection of serum anti-ERα Abs may help predict tamoxifen resistance and concur to appropriately inform therapeutic decisions concerning hormone therapy in ER-positive breast cancer patients.

Joint action of miR-126 and MAPK/PI3K inhibitors against metastatic melanoma.

Emerging data support the rationale of combined therapies in advanced melanoma. Specifically, the combined use of drugs with different mechanisms of action can reduce the probability of selecting resistant clones. To identify agents active against melanoma cells, we screened a library of 349 anti-cancer compounds, currently in clinical use or trials, and selected PIK-75, an inhibitor of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway, as the 'top active' drug. PIK-75 was then used alone or in combination with vemurafenib, the first BRAF inhibitor approved for patients with melanoma harboring BRAF mutations. We identified a combined dose of PIK-75 and vemurafenib that inhibited both the PI3K/AKT and mitogen-activated protein kinase pathways, thereby overcoming any compensatory activation. In view of the important tumor suppressor function induced by restoring expression of microRNA (miR)-126 in metastatic melanoma cells, we examined whether miR-126 has a synergistic role when included in a triple combination alongside PIK-75 and vemurafenib. We found that enforced expression of miR-126 (which alone can reduce tumorigenicity) significantly increased PIK-75 activity when used as either a single agent or in combination with vemurafenib. Interestingly, PIK-75 proved to be effective against early passage cell lines derived from patients' biopsies and on melanoma cell lines resistant to either vemurafenib or dabrafenib, thus suggesting that it potentially has the capability to overcome drug resistance. Finally, the synergistic role played by miR-126 in combination with vemurafenib and/or PIK-75 was demonstrated in vivo in mouse xenograft models, in which tumor growth inhibition was associated with increased apoptosis. These results not only show the efficacy of PIK-75 and vemurafenib co-treatment but also indicate that restoration of miR-126 expression in advanced melanoma can enhance their antitumor activity, which may possibly allow dose reduction to decrease adverse events without reducing the therapeutic benefits.

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules.

BACKGROUND: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. METHODS: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. RESULTS: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. CONCLUSIONS: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.

SCD5 restored expression favors differentiation and epithelial-mesenchymal reversion in advanced melanoma.

Our previous data supported a role for the Stearoyl-CoA desaturase (SCD5) in protection against malignancy, whereby it appears to functionally modify tumor stroma impairing tumor spread. SCD5 is significantly expressed in primary melanoma, but becomes barely detectable at tumor advanced stages. Looking for the regulatory mechanisms underlying SCD5 reduced expression during melanoma progression, we demonstrated a significantly lower stability of SCD5 protein as well as the direct targeting of SCD5 mRNA by the oncogenic miR-221&222 in metastatic cell lines. Moreover, our results indicated the existence of a negative feedback loop between SCD5 and miR-221&222, in good agreement with their opposite functions. Also, we showed how SCD5 re-expression and the direct supplementation of its main product oleic acid (OA) can drive advanced melanoma cell lines toward differentiation and reversion of the epithelial-mesenchymal (EMT)-like process, eventually inducing a less malignant phenotype. Indeed, SCD5 re-established the sensitivity to all-trans retinoic acid in A375M metastatic melanoma, associated with increased levels of Tyrosinase, melanin production and reduced proliferation. As evidenced by the correct modulation of some key transcription factors, SCD5 managed by favoring a partial mesenchymal-to-epithelial (MET) transition in in vitro studies. Interestingly, a more complete MET, including E-cadherin re-expression correctly localized at cell membranes, was obtained in in vivo xenograft models, thus indicating the requirement of direct contacts between tumor cells and the surrounding microenvironment as well as the presence of some essential factors for SCD5 complete function.

Cell death-based treatments of melanoma:conventional treatments and new therapeutic strategies.

The incidence of malignant melanoma has continued to rise during the past decades. However, in the last few years, treatment protocols have significantly been improved thanks to a better understanding of the key oncogenes and signaling pathways involved in its pathogenesis and progression. Anticancer therapy would either kill tumor cells by triggering apoptosis or permanently arrest them in the G1 phase of the cell cycle. Unfortunately, melanoma is often refractory to commonly used anticancer drugs. More recently, however, some new anticancer strategies have been developed that are "external" to cancer cells, for example stimulating the immune system's response or inhibiting angiogenesis. In fact, the increasing knowledge of melanoma pathogenetic mechanisms, in particular the discovery of genetic mutations activating specific oncogenes, stimulated the development of molecularly targeted therapies, a form of treatment in which a drug (chemical or biological) is developed with the goal of exclusively destroying cancer cells by interfering with specific molecules that drive growth and spreading of the tumor. Again, after the initial exciting results associated with targeted therapy, tumor resistance and/or relapse of the melanoma lesion have been observed. Hence, very recently, new therapeutic strategies based on the modulation of the immune system function have been developed. Since cancer cells are known to be capable of evading immune-mediated surveillance, i.e., to block the immune system cell activity, a series of molecular strategies, including monoclonal antibodies, have been developed in order to "release the brakes" on the immune system igniting immune reactivation and hindering metastatic melanoma cell growth. In this review we analyze the various biological strategies underlying conventional chemotherapy as well as the most recently developed targeted therapies and immunotherapies, pointing at the molecular mechanisms of cell injury and death engaged by the different classes of therapeutic agents.

Involvement of sperm acetylated histones and the nuclear isoform of Glutathione peroxidase 4 in fertilization.

We previously demonstrated that the nuclear form of Glutathione peroxidase 4 (nGPx4) has a peculiar distribution in sperm head, being localized to nuclear matrix and acrosome and that sperm lacking nGPx4 are more prone to decondensation in vitro. In this study we have hypothesized that sperm retained acetylated histones and nGPx4 are implicated in paternal chromatin decondensation and male pronucleus formation at fertilization. Indeed, significant higher amounts of acetylated histone H4 and acetylated histone H3 were observed by both immunofluorescence and western blotting in nGPx4-KO sperm vs WT ones. In vitro fertilization of zona pellucida-deprived oocytes by WT sperm in the presence of trichostatin (TSA) also demonstrated that paternal histone acetylation was inversely related to the timing of sperm nucleus decondensation at fertilization. In contrast, TSA had no effect on nGPx4-KO sperm, indicating they had a maximal level of histone acetylation. Moreover the paternally imprinted gene Igf2/H19 was hypomethylated in KO sperm compared to WT ones. The lack of nGPx4 negatively affected male fertility, causing a marked decrease in total pups and pregnancies with delivery, a significant reduction in pronuclei (PN) embryos in in vitro fertilization assays and an approximately 2 h delay in egg fertilization in vivo. Because the zona pellucida binding and fusion to oolemma of nGPx4-KO and WT sperm were similar, the subfertility of nGPx4 sperm reflected a decreased sperm progression through egg cumulus/zona pellucida, pinpointing a defective acrosome in line with acrosomal nGPx4 localization. We conclude that paternal acetylated histones and acrosomal nGPx4 are directly involved in fertilization.

Gut Mesenchymal Stromal Cells in Immunity.

Mesenchymal stromal cells (MSCs), first found in bone marrow (BM), are the structural architects of all organs, participating in most biological functions. MSCs possess tissue-specific signatures that allow their discrimination according to their origin and location. Among their multiple functions, MSCs closely interact with immune cells, orchestrating their activity to maintain overall homeostasis. The phenotype of tissue MSCs residing in the bowel overlaps with myofibroblasts, lining the bottom walls of intestinal crypts (pericryptal) or interspersed within intestinal submucosa (intercryptal). In Crohn's disease, intestinal MSCs are tightly stacked in a chronic inflammatory milieu, which causes their enforced expression of Class II major histocompatibility complex (MHC). The absence of Class II MHC is a hallmark for immune-modulator and tolerogenic properties of normal MSCs and, vice versa, the expression of HLA-DR is peculiar to antigen presenting cells, that is, immune-activator cells. Interferon gamma (IFNγ) is responsible for induction of Class II MHC expression on intestinal MSCs. The reversal of myofibroblasts/MSCs from an immune-modulator to an activator phenotype in Crohn's disease results in the formation of a fibrotic tube subverting the intestinal structure. Epithelial metaplastic areas in this context can progress to dysplasia and cancer.

In bone metastasis miR-34a-5p absence inversely correlates with Met expression, while Met oncogene is unaffected by miR-34a-5p in non-metastatic and metastatic breast carcinomas.

The highlight of the molecular basis and therapeutic targets of the bone-metastatic process requires the identification of biomarkers of metastasis colonization. Here, we studied miR-34a-5p expression, and Met-receptor expression and localization in bone metastases from ductal breast carcinomas, and in ductal carcinomas without history of metastasis (20 cases). miR-34a-5p was elevated in non-metastatic breast carcinoma, intermediate in the adjacent tissue and practically absent in bone metastases, opposite to pair-matched carcinoma. Met-receptor biomarker was highly expressed and inversely correlated with miR-34a-5p using the same set of bone-metastasis tissues. The miR-34a-5p silencing might depend on aberrant-epigenetic mechanisms of plastic-bone metastases, since in 1833 cells under methyltransferase blockade miR-34a-5p augmented. In fact, 1833 cells showed very low endogenous miR-34a-5p, in respect to parental MDA-MB231 breast carcinoma cells, and the restoration of miR-34a-5p with the mimic reduced Met and invasiveness. Notably, hepatocyte growth factor (HGF)-dependent Met stabilization was observed in bone-metastatic 1833 cells, consistent with Met co-distribution with the ligand HGF at plasma membrane and at nuclear levels in bone metastases. Met-protein level was higher in non-metastatic (low grade) than in metastatic (high grade) breast carcinomas, notwithstanding miR-34a-5p-elevated expression in both the specimens. Thus, mostly in non-metastatic carcinomas the elevated miR-34a-5p unaffected Met, important for invasive/mesenchymal phenotype, while possibly targeting some stemness biomarkers related to metastatic phenotype. In personalized therapies against bone metastasis, we suggest miR-34a-5p as a suitable target of epigenetic reprogramming leading to the accumulation of miR-34a-5p and the down-regulation of Met-tyrosine kinase, a key player of the bone-metastatic process.

Combining Type I Interferons and 5-Aza-2'-Deoxycitidine to Improve Anti-Tumor Response against Melanoma.

Resistance to IFN-I-induced antineoplastic effects has been reported in many tumors and arises, in part, from epigenetic silencing of IFN-stimulated genes by DNA methylation. We hypothesized that restoration of IFN-stimulated genes by co-administration of the demethylating drug 5-aza-2'-deoxycitidine (decitabine [DAC]) may enhance the susceptibility to IFN-I-mediated antitumoral effects in melanoma. We show that combined administration of IFN-I and DAC significantly inhibits the growth of murine and human melanoma cells, both in vitro and in vivo. Compared with controls, DAC/IFN-I-treated melanoma cells exhibited reduced cell growth, augmented apoptosis, and diminished migration. Moreover, IFN-I and DAC synergized to suppress the growth of three-dimensional human melanoma spheroids, altering tumor architecture. These direct antitumor effects correlated with induction of the IFN-stimulated gene Mx1. In vivo, DAC/IFN-I significantly reduced melanoma growth via stimulation of adaptive immunity, promoting tumor-infiltrating CD8+ T cells while inhibiting the homing of immunosuppressive CD11b+ myeloid cells and regulatory T cells. Accordingly, exposure of human melanoma cells to DAC/IFN-I induced the recruitment of immune cells toward the tumor in a Matrigel (Corning Life Sciences, Kennebunkport, ME)-based microfluidic device. Our findings underscore a beneficial effect of DAC plus IFN-I combined treatment against melanoma through both direct and immune-mediated anti-tumor effects.

Exosome-mediated transfer of miR-222 is sufficient to increase tumor malignancy in melanoma.

BACKGROUND: Growing evidence is showing that metastatic cell populations are able to transfer their characteristics to less malignant cells. Exosomes (EXOs) are membrane vesicles of endocytic origin able to convey their cargo of mRNAs, microRNAs (miRs), proteins and lipids from donors to proximal as well as distant acceptor cells. Our previous results indicated that miR-221&222 are key factors for melanoma development and dissemination. The aim of this study was to verify whether the tumorigenic properties associated with miR-222 overexpression can be also propagated by miR-222-containing EXOs. METHODS: EXOs were isolated by UltraCentrifugation or Exoquick-TC(®) methods. Preparations of melanoma-derived vesicles were characterized by using the Nanosight™ technology and the expression of exosome markers analyzed by western blot. The expression levels of endogenous and exosomal miRNAs were examined by real time PCR. Confocal microscopy was used to evaluate transfer and uptake of microvesicles from donor to recipient cells. The functional significance of exosomal miR-222 was estimated by analyzing the vessel-like process formation, as well as cell cycle rates, invasive and chemotactic capabilities. RESULTS: Besides microvesicle marker characterization, we evidenced that miR-222 exosomal expression mostly reflected its abundance in the cells of origin, correctly paralleled by repression of its target genes, such as p27Kip1, and induction of the PI3K/AKT pathway, thus confirming its functional implication in cancer. The possible differential significance of PI3K/AKT blockade was assessed by using the BKM120 inhibitor in miR-222-transduced cell lines. In addition, in vitro cultures showed that vesicles released by miR-222-overexpressing cells were able to transfer miR-222-dependent malignancy when taken-up by recipient primary melanomas. Results were confirmed by antagomiR-221&222 treatments and by functional observations after internalization of EXOs devoid of these miRs. CONCLUSION: All together these data, besides generally confirming the role of miR-222 in melanoma tumorigenesis, supported its responsibility in the exosome-associated melanoma properties, thus further indicating this miR as potential diagnostic and prognostic biomarker and its abrogation as a future therapeutic option.

SCD5-induced oleic acid production reduces melanoma malignancy by intracellular retention of SPARC and cathepsin B.

A proper balance between saturated and unsaturated fatty acids (FAs) is required for maintaining cell homeostasis. The increased demand of FAs to assemble the plasma membranes of continuously dividing cancer cells might unbalance this ratio and critically affect tumour outgrowth. We unveiled the role of the stearoyl-CoA desaturase SCD5 in converting saturated FAs into mono-unsaturated FAs during melanoma progression. SCD5 is down-regulated in advanced melanoma and its restored expression significantly reduced melanoma malignancy, both in vitro and in vivo, through a mechanism governing the secretion of extracellular matrix proteins, such as secreted protein acidic and rich in cysteine (SPARC) and collagen IV and of their proteases, such as cathepsin B. Enforced expression of SCD5 or supplementation of its enzymatic product, oleic acid, reduced the intracellular pH (pHe > pHi) and, in turn, vesicular trafficking across plasma membranes as well as melanoma dissemination. This intracellular acidification appears also to depend on SCD5-induced reduction of the C2 subunit of the vacuolar H(+) -ATPase, a proton pump whose inhibition changes the secretion profile of cancer cells. Our data support a role for SCD5 and its enzymatic product, oleic acid, in protection against malignancy, offering an explanation for the beneficial Mediterranean diet. Furthermore, SCD5 appears to functionally connect tumour cells and the surrounding stroma toward modification of the tumour microenvironment, with consequences on tumour spread and resistance to treatment.

The nuclear form of glutathione peroxidase 4 colocalizes and directly interacts with protamines in the nuclear matrix during mouse sperm chromatin assembly.

The testis-specific nuclear form of Phospholipid Hydroperoxide Glutathione Peroxidase (nGPx4) is associated with the nuclear matrix during spermiogenesis and is implicated in sperm chromatin condensation. In this study, we have addressed the question whether nGPx4 directly interacts with protamines by transiently sharing a nuclear matrix localization. We first expressed tagged protamine 1-myc and protamine 2-V5 in HeLa and COS-1 cells and showed by both confocal microscopy and immunoblotting analyses that protamines were produced in vitro and colocalized correctly to the nucleus. Co-transfection experiments demonstrated that protamine 1 was physically associated with flag-nGPx4 specifically at the level of nuclear matrix. The peculiar presence of protamines together with nGPx4 in this subnuclear compartment was also confirmed in mouse elongated spermatids by immunofluorescence, suggesting that nGPx4 is a physiological component of a novel protein complex relevant to chromatin assembly in condensing haploid cells. Also, in epididymal sperm, nGPx4 and protamine 1 co-immunoprecipitated, indicating that nGPx4, although localized to a subnuclear compartment different from that of protamines, represents a constant link between nuclear matrix and chromatin in mammalian male gamete.