Human Metastatic Melanoma Cells Research Articles

CNP mediated selective toxicity on melanoma cells is accompanied by mitochondrial dysfunction.

Cerium (Ce) oxide nanoparticles (CNP; nanoceria) are reported to have cytotoxic effects on certain cancerous cell lines, while at the same concentration they show no cytotoxicity on normal (healthy) cells. Redox-active CNP exhibit both selective prooxidative as well as antioxidative properties. The former is proposed to be responsible for impairment of tumor growth and invasion and the latter for rescuing normal cells from reactive oxygen species (ROS)-induced damage. Here we address possible underlying mechanisms of prooxidative effects of CNP in a metastatic human melanoma cell line. Malignant melanoma is the most aggressive form of skin cancer, and once it becomes metastatic the prognosis is very poor. We have shown earlier that CNP selectively kill A375 melanoma cells by increasing intracellular ROS levels, whose basic amount is significantly higher than in the normal (healthy) counterpart, the melanocytes. Here we show that CNP initiate a mitochondrial increase of ROS levels accompanied by an increase in mitochondrial thiol oxidation. Furthermore, we observed CNP-induced changes in mitochondrial bioenergetics, dynamics, and cristae morphology demonstrating mitochondrial dysfunction which finally led to tumor cell death. CNP-induced cell death is abolished by administration of PEG-conjugated catalase. Overall, we propose that cerium oxide nanoparticles mediate cell death via hydrogen peroxide production linked to mitochondrial dysfunction.

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice.

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Coexpression of Vimentin and Cytokeratins in Human Melanoma Cell Lines

Abstract—Molecular heterogeneity in seven human metastatic melanoma cells lines is revealed using a double immunofluorescent assay by flow cytometry. It is evaluated that quite a small population of cells (up to 10%) expressed only vimentin, the main marker of melanoma. Only epithelial cytokeratins expression is revealed in a significantly greater number of cells (from 25 to 50%). The dominant molecular phenotype (50–80% cells expressed both markers), the coexpression of vimentin and cytokeratins, is identified for the first time. The authors assume that the latter phenotype of melanoma cells may be a molecular marker of the tumor’s aggressiveness and metastatic potential.

Inhibition of glucose metabolism through treatment of BRAF mutated metastatic melanoma with vemurafenib.

e21005 Background: Increased glucose metabolism is a hallmark of neoplastic cells that allows self-promotion of growth and survival. The enzyme 6-phosphofructo-2-kinase (PFKFB3) is an integral controller of glycolysis by promoting the synthesis of fructose 2,6-bisphosphonate (F2,6BP) which activates 6-phoshofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in the glycolytic pathway. Additionally, mitogen-activated protein kinase (MAPK) is a key signaling pathway in a number of cancers with mutations of the BRAF component, described most commonly in melanoma, resulting in constitutive activation of the MAPK pathway. We aim to demonstrate that vemurafenib, a BRAF inhibitor, has antiglycolytic activity in sensitive melanoma cell lines which may help guide development of future therapies with specific attention to PFKFB3 as a potential enzymatic target to decrease glycolytic flux thereby inhibiting tumor growth and survival. Methods: Vemurafenib sensitive and resistant variants of two separate human metastatic melanoma cell lines (451Lu and WM983) were treated with 3 mM Vemurafenib for 24 and 48 hours. Additionally, cells from aforementioned lines were probed for PFKFB3 after 24 hours of treatment with vemurafenib. Glycolysis was measured by incubating cells in complete media containing 1 mCi [5-3H]glucose for 60 minutes. [3H]H2O produced by glycolysis through enolase was measured. Results: A decrease in PFKFB3 protein expression was found in vemurafenib sensitive cells compared to controls but not in resistant cells after 24h treatment with 3 mM vemurafenib in both 451Lu and WM983 metastatic melanoma cell lines (n = 2). Treatment with vemurafenib led to decrease in glycolysis compared to untreated controls in both vemurafenib sensitive metastatic melanoma cell lines but not in resistant cell lines (n = 5). Additionally, there was a significant reduction in glycolysis in vemurafenib resistant WM983 at 48 hours compared to resistant untreated control. Conclusions: BRAF mutated metastatic melanoma cells showed decrease in PFKFB3 protein expression and decreased glycolysis after treatment with BRAF inhibitor vemurafenib. Future studies will focus on assessing metastatic melanoma cell viability and glycolytic activity after treatment with combination BRAF inhibition and PFKFB3 specific inhibition.

Automated in vivo screen in zebrafish identifies Clotrimazole as targeting a metabolic vulnerability in a melanoma model

Therapeutic approaches for cutaneous melanoma are flourishing, but despite promising results, there is an increasing number of reported primary or secondary resistance to the growing sets of drugs approved for therapy in the clinics. Combinatorial approaches may overcome resistance, as they may tackle specific weaknesses of melanoma cells, not sufficient on their own, but effective in combination with other therapies.The transgenic zebrafish line kita:ras develops melanoma with high frequency. At 3 dpf, transgenic kita:ras larvae show a hyperpigmentation phenotype as earliest evidence of abnormal melanocyte growth. Using this model, we performed a chemical screen based on automated detection of a reduction of melanocyte number caused by any of 1280 FDA or EMA approved drugs of the library. The analysis showed that 55 molecules were able to reduce by 60% or more the number of melanocytes per embryo. We further tested two compounds for each of the 5 classes, and a farnesyltransferase inhibitor (Lonafarnib), that inhibits an essential post-translational modification of HRAS and suppresses the hyperpigmentation phenotype. Combinations of Clotrimazole and Lonafarnib showed the most promising results in zebrafish embryos, allowing a dose reduction of both drugs. We performed validation of these observations in the metastatic human melanoma cell line A375M, and in normal human epithelial melanocytes (NHEM) in order to investigate the mechanism of action of Clotrimazole in blocking the proliferation of transformed melanocytes. Viability assay and analysis of energy metabolism in Clotrimazole treated cells show that this drug specifically affects melanoma cells in vitro and transformed melanocytes in vivo, having no effects on NHEM or wild type larvae. Similar effects were observed with another hit of the same class, Miconazole. Furthermore, we show that the effects of Clotrimazole are mediated by the inhibition of hexokinase activity, which is lethal to the abnormal metabolic profile of melanoma cells in vitro and in vivo. Thus, our study shows that the zebrafish can provide a phenotype-rich assay for fully automated screening approaches to identify drugs for synthetic lethal treatment in melanoma and suggest further testing of Clotrimazole in combinatorial treatments.

D-103 Cancer terminator viruses: A strategy for targeting metastatic cancers

Our laboratory pioneered subtraction hybridization and used this elegant and effective gene identification strategy to clone novel genes of physiological relevance. This methodology defined differentially regulated genes in an unbiased manner that are modified when metastatic human melanoma cells are pharmacologically induced to terminally differentiate and lose irreversibly oncogenic potential following treatment with IFN-band the protein kinase C activator mezerein. This approach, called DISH (differentiation induction subtraction hybridization), identified melanoma differentiation associated (mda) genes relevant to growth control, cancer development, tumor progression, and cancer cell survival. One intriguing gene identified using the DISH scheme was mda-7, which based on chromosomal location, secretory properties and structure has been classified as interleukin-24 (IL-24). mda-7/IL-24 is a novel member of the IL-10 gene family that promotes apoptosis and toxic autophagy in a broad-spectrum of human tumors, exerts potent “bystander” antitumor activity, inhibits tumor angiogenesis, stimulates the immune system and synergizes with conventional therapies (including chemotherapy, radiation, monoclonal antibodies and immunomodulators) to promote selective tumor destruction. mda-7/IL-24 displays profound anti-cancer activity in preclinical animal models (including nude, syngeneic and transgenic mice) and following intratumoral administration using a type 5 adenovirus in a Phase I/II clinical trial in patients with advanced cancers. To enhance the therapeutic efficacy of mda-7/IL-24, we generated a novel class of adenoviruses, called Cancer Terminator Viruses (CTVs), that selectively replicate in cancer cells and simultaneously produce mda-7/IL-24, Ad.5-CTV. A further iteration of this virus employs a chimera between Ad.5 and Ad.3, Ad.5/3, that employs Coxsackie-Adenovirus Receptors (CAR) as well as desmoglien and CD46 to infect cells, thereby increasing its infectivity in cancer cells with reduced CAR. Ad.5/3-CTV displays profound anti-cancer activity in pre-clinical animal models, including GBM, melanoma, and prostate, colorectal and breast cancer. A clinical trial is in preparation to test the effect of the Ad.5/3-CTV in patients with recurrent glioma. To facilitate the delivery of adenoviruses systemically, avoiding immune clearance and liver trapping, we have refined the ultrasound-targeted microbubble-destruction (UTMD) approach. This strategy has allowed us to deliver therapeutic viruses and proteins intravenously to internal target organs, such as the prostate, to evoke pre-clinical antitumor activity. To enhance further the therapeutic efficacy of the UTMD approach, we are employing microbubbles that contain targeting ligands on their surface permitting accumulation in the tumor vasculature, that is, decorated microbubbles. Employing the same cancer-selective promoter used in the original CTV, the rat progression elevated gene-3 promoter (PEG-Prom), we also developed genetic-based imaging approaches for metastases. These approaches use the PEG-Prom (or another cancer-selective promoter) to drive an imaging gene (eg, luciferase or HSV-Tk) linked to a delivery vehicle PEI. These nanoparticle vectors when combined with bioluminescence imaging and SPECT-CT permit non-invasive imaging of tumors and metastases in pre-clinical animal models. As a further iteration of our CTV we have used 2 cancer-selective and a non-selective promoter to generate first in class tripartite Ads that selectively replicate in cancer cells, produce an imaging agent in cancer cells (Lucirerase or HSV-Tk) and simultaneously produce the therapeutic immunogenic cytokine mda-7/IL-24, Ad.Tri-CTV. These “theranostic viruses” permit tumor and metastasis imaging and treatment through application of a single vector. Overall, the novel reagents and methodologies we have developed are providing paradigm shifts in how cancers will be detected and treated in the future.

Mechanism for Regulation of Melanoma Cell Death via Activation of Thermo-TRPV4 and TRPV2.

Background Thermo-TRPs (temperature-sensitive transient receptor potential channels) belong to the TRP (transient receptor potential) channel superfamily. Emerging evidence implied that thermo-TRPs have been involved in regulation of cell fate in certain tumors. However, their distribution profiles and roles in melanoma remain incompletely understood. Methods Western blot and digital PCR approaches were performed to identify the distribution profiles of six thermo-TRPs. MTT assessment was employed to detect cell viability. Flow cytometry was applied to test cell cycle and apoptosis. Calcium imaging was used to determine the function of channels. Five cell lines, including one normal human primary epidermal melanocytes and two human malignant melanoma (A375, G361) and two human metastatic melanoma (A2058, SK-MEL-3) cell lines, were chosen for this research. Results In the present study, six thermo-TRPs including TRPV1/2/3/4, TRPA1, and TRPM8 were examined in human primary melanocytes and melanoma cells. We found that TRPV2/4, TRPA1, and TRPM8 exhibited ectopic distribution both in melanocytes and melanoma cells. Moreover, activation of TRPV2 and TRPV4 could lead to the decline of cell viability for melanoma A2058 and A375 cells. Subsequently, activation of TRPV2 by 2-APB (IC50 = 150 μM) induced cell necrosis in A2058 cells, while activation of TRPV4 by GSK1016790A (IC50 = 10 nM) enhanced apoptosis of A375 cells. Furthermore, TRPV4 mediated cell apoptosis of melanoma via phosphorylation of AKT and was involved in calcium regulation. Conclusion Overall, our studies revealed that TRPV4 and TRPV2 mediated melanoma cell death via channel activation and characterized the mechanism of functional TRPV4 ion channel in regulating AKT pathway driven antitumor process. Thus, they may serve as potential biomarkers for the prognosis and are targeted for the therapeutic use in human melanoma.

Mechanisms Enhancing the Cytotoxic Effects of Bleomycin plus Suicide or Interferon-β Gene Lipofection in Metastatic Human Melanoma Cells.

Three metastatic human melanoma cell lines generated from patient removed lymph nodes and spleen metastasis were established in our laboratory. To investigate the mechanisms enhancing the cytotoxic effects of Bleomycin (BLM), herpes simplex virus thymidine kinase/ganciclovir Suicide Gene (SG) and human interferon-β gene (hIFNβ) lipofection in early passages of these melanoma cell lines. In these cell lines, we determined: cytotoxicity, bystander effect, lipofection efficiencies, apoptosis, necrosis, senescence, colony forming capacity and mitochondrial membrane depolarization after treatments. The three assayed cell lines displayed sensitivity to single and combined BLM/gene treatments. BLM improved the antitumor and anti-clonogenic effects of SG and hIFNβ genes. Considering the low lipofection efficiencies (<10%), one of the main causes of the SG and hIFNβ gene effectiveness was their bystander effect. In one of these cell lines, this effect eradicated up to 60% of the cells although <1% expressed the transgene. In the three cell lines, BLM alone or combined with SG or hIFNβ gene significantly increased the percentage of cells exhibiting membrane compromise, DNA damage, and senescence. Interestingly, the strong BLM/hIFNβ gene combination was able to generate from 73% to 98% of non-viable cells. The high proportion of senescent cells induced by BLM alone or combined with genes strongly decreased the clonogenic capacity of surviving cells. The presented results indicate that BLM improves the antitumor effects of SG and hIFNβ transgene expression. Altogether, these findings strongly support the clinical potential of these combined approaches.

Synthesis, Molecular Modelling and Anticancer Activities of New Molecular Hybrids Containing 1,4-Naphthoquinone, 7-Chloroquinoline, 1,3,5-Triazine and Morpholine Cores as PI3K and AMPK Inhibitors in the Metastatic Melanoma Cells

Three molecular hybrids containing 1,4-naphthoquinones, 1,3,5-triazines, morpholine and 7-chloroquinoline, which have recognized contributions to the biological activity of many drugs, were synthesized in yields ranging from 43-84%. All hybrids were obtained in three steps starting from readily available reactants: lawsone, cyanuric chloride, morpholine and 4,7-dichloroquinoline. A previous docking study was carried out to identify the binding energy and pharmacophore conformation of the promising anticancer compounds with PI3Kγ (phosphoinositide 3-kinase) and AMPK (5’ AMP-activated protein kinase). The cancer activity in human metastatic melanoma cells (SKMEL-103) were performed, and the synthetized compounds presented half maximal inhibitory concentration (IC50) values around 25 µM. The expressions of PI3K and AMPK were also determined using western blotting technique, and all molecular hybrids negatively modulated both targets.

Metformin increases antitumor activity of MEK inhibitor binimetinib in 2D and 3D models of human metastatic melanoma cells

Melanoma is one of the most aggressive and treatment-resistant tumors that responsible for majority of skin-cancer related deaths. Here we propose a combination of MEK inhibitor binimetinib with metformin as a promising therapy against human melanoma cells in vitro, including BRAF -mutated A375, Mel Z, and Mel IL cells, and NRAS-mutated Mel MTP and Mel Me cells. Additionally, we obtained two close to clinical practice models of melanoma progression. The first one was vemurafenib-resistant Mel IL/R melanoma cells with acquired resistance to BRAF inhibition-targeted therapy, and the second one was tumor spheroids, which are 3D in vitro model of small-size solid tumors in vivo. The cytotoxicity of binimetinib and metformin was synergistic in both 2D and 3D melanoma culture and mediated through apoptotic pathway. The combination reduced the number of melanoma-formed colonies, inhibited cell invasion and migration, and led to G0/G1 cell cycle arrest through cyclin D/CDK4/CDK6 pathway. The mechanism of metformin and binimetinib synergy in melanoma cells was associated with increased activation of p-AMPKα and decreased p-ERK, but not with alterations in p-mTOR. In summary, the combination of metformin and binimetinib resulted in stronger anti-proliferative effects on melanoma cells compared to binimetinib alone, and therefore could be promising for clinical applications.

Relationship between deletion and point mutations of p53 and drug resistance to aranoza in human melanoma cell lines

Aranosa, nitrozourea derivative is a DNA-methylating agent that has been approved for treatment of patients with disseminated melanoma. Aranoza effect is based on DNA damage and then as a result apoptosis mechanisms start launching. The important role in this process must be played by p53 protein, and its different dysfunctions can result in drug resistance. Objective. The purpose is to study p53 mutational status in cell lines of human skin metastatic melanoma and to estimate its connection with сell lines resistance to aranoza. Materials and methods. The research was conducted on 14 cell lines of human skin metastatic melanoma. Aranoza IC50 for cell lines was determined by MTT-test. The 17р chromosome’s condition was estimated by fluorescence in situ hybridization. The presence of point mutations in DNA-binding domain of human p53 was researched by Sanger sequencing. Results. Skin metastatic melanoma cell lines had different sensitivity to aranoza. Almost all cell lines were heterogeneous in the condition of 17 th chromosome. P53 point mutations were found in 2 cell lines. But one part of resistant cell lines almost didn’t have any mutational disorders of p53, another part of resistant lines on the contrary had plenty of p53 mutational disorders. Conclusion. The correlation of resistance and p53 mutations can be established for one part of human skin metastatic melanoma cell lines. But for another part of human skin metastatic melanoma cell lines resistance most likely are driven by other mechanisms.

Celecoxib inhibits mitochondrial O2 consumption, promoting ROS dependent death of murine and human metastatic cancer cells via the apoptotic signalling pathway

Capecitabine induced toxicities such as hand-foot syndrome (HFS) and progression of metastatic cancer are both treatable with concurrent celecoxib as shown in the ADAPT (Activating Cancer Stem Cells from Dormancy And Potentiate for Targeting) trial. In the present study, five commonly used NSAIDs, including celecoxib were compared for their pro-oxidative capacities as cytotoxic drugs against human and mouse metastatic melanoma or breast cancer cells in vitroand the source of cellular ROS production induced by celecoxib was examined in greater detail. ResultsCelecoxib was unique among the NSAIDs in that it showed particular potency as a cytotoxic drug against the metastatic cancer cells with IC50 values in the low micromolar range. Celecoxib rapidly enhanced mitochondrial superoxide production in situ from cancer cells within minutes, leading to a decrease in cellular respiration and dissipation of the mitochondrial transmembrane potential (Δψm), followed by extensive ROS-dependent apoptosis of the metastatic cancer cells. Celecoxib also showed rapid and direct effects on isolated mitochondria, inducing extensive ROS production in a dose-dependent manner, whilst it inhibited respiration via Complex I or Complex II when tested in whole cells. Mitochondrial ROS production was necessary for the celecoxib induced cell death. Innovation and ConclusionThese novel findings for direct effects of celecoxib on mitochondria to induce metastatic cancer cell death via a ROS-dependent pro-oxidative mechanism provide supportive evidence for its combinatorial use as a chemosensitizing agent complementing chemotherapies to improve response rates in patients with advanced metastatic cancers.

P-105 - Effect of a novel biscumarin derivative on human melanoma cells: Regulation of cell survival, proliferation, migration and kinases

We have investigated the effects of a newly synthesized biscumarin derivative on cell survival, proliferation, migration and signal transduction pathways in the human metastatic melanoma cells A375. Among the modulated pathways of cell survival and tumor suppression we found highly significant effects of this novel synthetic derivative on TOR (Target of Rapamycin), Checkpoint Kinase 2 (Chk- 2), Tumor Suppressor Protein Kinase p53 ( P53), Additionally the compound was found to modulate a number of pathways of cell proliferation with varying degree of efficacy. We have shown that biscumarin derivative induced activation of Epidermal Growth Factor Receptor (EGFR), Focal Adhesion Kinase (FAK), cAMP response Element-Binding Protein (CREB), Glycogen Synthase Kinase (GSK3 α/f3) and f3 –catenin: this effect may be at thebases of observed motility inhibition of melanoma cell caused by the compound. We have shown that the biscumarin derivative diminish phosphorylation of CHK-2 protein, TOR and P53 an event that is likely be the basis of observed inhibition of melanoma cell survival and tumor growth. Inhibition of cell survival and growth possibly due to the deminish phosphorylation of the chk-2 kinase on Threonine: T68, of TOR on Serine: S2448 of P53 on Serine: S392.

Regulation of CEACAM1 Protein Expression by the Transcription Factor ETS-1 in BRAF-Mutant Human Metastatic Melanoma Cells

BRAF becomes constitutively activated in 50% to 70% of melanoma cases. CEACAM1 has a dual role in melanoma, including facilitation of cell proliferation and suppression of infiltrating lymphocytes, which are consistent with its value as a marker for poor prognosis in melanoma patients. Here we show that BRAFV600E melanoma cells treated with BRAF and MEK inhibitors (MAPKi) downregulate CEACAM1 mRNA and protein expression in a dose- and exposure time–dependent manners. Indeed, there is a significant correlation between the presence of BRAFV600E and CEACAM1 expression in melanoma specimens obtained from 45 patients. Vemurafenib-resistant cell systems reactivate the MAPK pathway and restore basal CEACAM1 mRNA and protein levels. These combined results suggest transcriptional regulation. Indeed, luciferase reporting assays show that CEACAM1 promoter (CEACAM1p) activity is significantly reduced by MAPKi. Importantly, we show that the MAPK-driven CEACAM1p activity is mediated by ETS1, a major transcription factor and downstream effector of the MAPK pathway. Phosphorylation mutant ETS1T38A shows a dominant negative effect over CEACAM1 expression. The data are consistent with independent RNAseq data from serial biopsies of melanoma patients treated with BRAF inhibitors, which demonstrate similar CEACAM1 downregulation. Finally, we show that CEACAM1 downregulation by MAPKi renders the cells more sensitive to T-cell activation. These results provide a new view on a potential immunological mechanism of action of MAPKi in melanoma, as well as on the aggressive phenotype observed in drug-resistant cells.

Wnt-signaling enhances neural crest migration of melanoma cells and induces an invasive phenotype

BackgroundDuring embryonic development Wnt family members and bone morphogenetic proteins (BMPs) cooperatively induce epithelial-mesenchymal transition (EMT) in the neural crest. Wnt and BMPs are reactivated during malignant transformation in melanoma. We previously demonstrated that the BMP-antagonist noggin blocked the EMT phenotype of melanoma cells in the neural crest and malignant invasion of melanoma cells in the chick embryo; vice-versa, malignant invasion was induced in human melanocytes in vivo by pre-treatment with BMP-2.ResultsAlthough there are conflicting results in the literature about the role of β-catenin for invasion of melanoma cells, we found Wnt/β-catenin signaling to be analogously important for the EMT-like phenotype of human metastatic melanoma cells in the neural crest and during invasion: β-catenin was frequently expressed at the invasive front of human primary melanomas and Wnt3a expression was inversely correlated with survival of melanoma patients. Accordingly, cytoplasmic β-catenin levels were increased during invasion of melanoma cells in the rhombencephalon of the chick embryo. Fibroblast derived Wnt3a reduced melanoma cell adhesion and enhanced migration, while the β-catenin inhibitor PKF115–584 increased adhesion and reduced migration in vitro and in the chick embryonic neural crest environment in vivo. Similarly, knockdown of β-catenin impaired intradermal melanoma cell invasion and PKF115–584 efficiently reduced liver metastasis in a chick chorioallantoic membrane model. Our observations were accompanied by specific alterations in gene expression which are linked to overall survival of melanoma patients.ConclusionWe present a novel role for Wnt-signaling in neural crest like melanoma cell invasion and metastasis, stressing the crucial role of embryonic EMT-inducing neural crest signaling for the spreading of malignant melanoma.

Vitamin C Sensitizes Melanoma to BET Inhibitors.

Bromodomain and extraterminal inhibitors (BETi) are promising cancer therapies, yet prominent side effects of BETi at effective doses have been reported in phase I clinical trials. Here, we screened a panel of small molecules targeting epigenetic modulators against human metastatic melanoma cells. Cells were pretreated with or without ascorbate (vitamin C), which promotes DNA demethylation and subsequently changes the sensitivity to drugs. Top hits were structurally unrelated BETi, including JQ1, I-BET151, CPI-203, and BI-2536. Ascorbate enhanced the efficacy of BETi by decreasing acetylation of histone H4, but not H3, while exerting no effect on the expression of BRD proteins. Histone acetyltransferase 1 (HAT1), which catalyzes H4K5ac and H4K12ac, was downregulated by ascorbate mainly via the TET-mediated DNA hydroxymethylation pathway. Loss of H4ac, especially H4K5ac and H4K12ac, disrupted the interaction between BRD4 and H4 by which ascorbate and BETi blocked the binding of BRD4 to acetylated histones. Cotreatment with ascorbate and JQ1 induced apoptosis and inhibited proliferation of cultured melanoma cells. Ascorbate deficiency as modeled in Gulo-/- mice diminished the treatment outcome of JQ1 for melanoma tumorgraft. In contrast, ascorbate supplementation lowered the effective dose of JQ1 needed to successfully inhibit melanoma tumors in mice. On the basis of our findings, future clinical trials with BETi should consider ascorbate levels in patients. Furthermore, ascorbate supplementation might help reduce the severe side effects that arise from BETi therapy by reducing the dosage necessary for treatment.Significance: This study shows that ascorbate can enhance the efficacy of BET inhibitors, providing a possible clinical solution to challenges arising in phase I trials from the dose-dependent side effects of this class of epigenetic therapy. Cancer Res; 78(2); 572-83. ©2017 AACR.

6- and 8-Prenylnaringenin, Novel Natural Histone Deacetylase Inhibitors Found in Hops, Exert Antitumor Activity on Melanoma Cells

Background/Aims: Prenylnaringenins are natural prenylflavonoids with anticancer properties. However, the underlying mechanisms have not been elucidated yet. Here we report a novel mode of action of 6- and 8-prenylnaringenin (PN) on human melanoma cells: Inhibition of cellular histone deacetylases (HDACs). Methods: We performed in silico and in vitro analyses using 6-PN or 8-PN to study a possible interaction of 6-PN or 8-PN with HDAC as well as Western blot and FACS analyses, real-time cell proliferation and cell viability assays to assess the impact of 6-PN and 8-PN on human metastatic melanoma cells. Results: In silico, 6-PN and 8-PN fit into the binding pocket of HDAC2, 4, 7 and 8, binding to the zinc ion of their catalytic center that is essential for enzymatic activity. In vitro, 100 µmol/L of 6-PN or 8-PN inhibited all 11 conserved human HDAC of class I, II and IV. In clinical oncology HDAC inhibitors are currently investigated as new anticancer compounds. In line, treatment of SK-MEL-28 cells with 6-PN or 8-PN induced a hyperacetylation of histone complex H3 within 2 h. Further, 6-PN or 8-PN mediated a prominent, dose-dependent reduction of cellular proliferation and viability of SK-MEL-28 and BLM melanoma cells. This effect was apoptosis-independent and accompanied by down-regulation of mTOR-specific pS6 protein via pERK/pP90 in SK-MEL-28 cells. Conclusion: The identification of a broad inhibitory capacity of 6-PN and 8-PN for HDAC enzymes with antiproliferative effects on melanoma cells opens the perspective for clinical application as novel anti-melanoma drugs and the usage as innovative lead structures for chemical modification to enhance pharmacology or inhibitory activities.

Abstract A56: Effect of long-chain omega-3 polyunsaturated fatty acids on cisplatin chemoresistance in murine and human melanoma cells

Abstract The incidence of melanoma is continuously growing worldwide, and an effective therapy for the metastatic disease still does not exist. Moreover, an intrinsic/extrinsic resistance to conventional chemotherapeutics, such as cisplatin (CPt), often arises in this tumor. The increased expression of a number of DNA repair genes, including the Excision Repair Cross-Complementation group 1 (ERCC1), has been related to the melanoma resistance to CPt. ERCC1 is a component of an important chemoresistance pathway [tyrosine-phosphatase dual-specificity phosphatase-6 (DUSP6)/extracellular signal-regulated kinase phosphorylation (p-ERK)/ERCC1]. New therapeutic strategies are under study, and one possibility is the administration of natural products combined with CPt therapy, in order to revert the CPt resistance. Many experimental in vitro and in vivo studies have contributed to support the antineoplastic properties of long-chain omega-3 polyunsaturated fatty acids (LC-omega-3 PUFA), especially docosahexaenoic acid (DHA), in tumor cells of different origin, including melanoma. In the present work the effects of CPt treatment combined with LC-omega-3 PUFA have been evaluated in highly metastatic murine (B16F10) and human (WM266-4) melanoma cell lines. Melanoma cells were exposed to increasing concentrations of CPt (1, 2.5 and 5 μM), alone or in combination with DHA (10 μM). Cell viability was analysed by the Trypan blue dye exclusion method 24, 48, and 72 h after treatment. The in vitro invasive ability of melanoma cells was assessed by the Wound Healing assay. The effects of increasing DHA concentrations combined or not with CPt on the expression of several proteins (ERCC1, pERK and DUSP6) related to CPt chemoresistance in melanoma cells were also analyzed by Western Bbotting. The data obtained demonstrated that the CPt/DHA combined treatment, performed by adding suboptimal concentrations of both the compounds, was significantly more efficient in inhibiting cell growth than the treatments with each of the two compounds administered alone. We confirmed that CPt was able to increase ERCC1 and decrease DUSP- 6 in a dose-dependent manner in both WM266-4 and B16F10 cell lines. Moreover, in the present work it was observed that DHA was able to revert the effects of CPt on ERCC1, DUSP6, and p-ERK expression. Similarly to what was observed with DHA, the LC-omega-3 PUFA eicosapentaenoic acid (EPA) exerted similar effects, even though at lower extent, on ERCC1, p-ERK, and DUSP6 expression in the same experimental conditions. Overall, the findings indicate that the combination of CPt with a dietary supplementation with LC-omega-3 PUFA could potentially represent a new, promising, complementary therapeutic strategy in order to overcome CPt resistance in melanoma. Moreover, the results suggest that the CPt/LC-omega-3 PUFA combination may allow the use of lower and suboptimal doses of CPt, thus probably reducing the undesirable side effects often accompanying the treatment with this antineoplastic drug. Citation Format: Renata O. Vasconcelos, Simona Serini, Ana Paula S. Votto, Gilma S. Trindade, Gabriella Calviello. Effect of long-chain omega-3 polyunsaturated fatty acids on cisplatin chemoresistance in murine and human melanoma cells [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A56.

Oncolytic Properties of a Mumps Virus Vaccine Strain in Human Melanoma Cell Lines

The oncolytic potential of the attenuated mumps virus (MV) vaccine strain Leningrad-3 (L-3) was evaluated in a panel of four human metastatic melanoma cell lines. The lines were shown to be susceptible and permissive to MV infection. Efficient MV replication led to death of melanoma cells, but the effect differed among the cell lines. Possible mechanisms mediating the selectivity of MV L-3 towards the cell lines were explored. Replicative and oncolytic activity of MV was found to depend on the expression pattern of type I interferon genes. None of the melanoma cell lines showed induction of expression of the total spectrum of genes required to inhibit virus replication. Based on the results, MV L-3 was assumed to be a promising oncolytic agent for human melanoma cells.

A Potent, Metabolically Stable Tubulin Inhibitor Targets the Colchicine Binding Site and Overcomes Taxane Resistance.

Antimitotics that target tubulin are among the most useful chemotherapeutic drugs, but their clinical activity is often limited by the development of multidrug resistance. We recently discovered the novel small-molecule DJ101 as a potent and metabolically stable tubulin inhibitor that can circumvent the drug efflux pumps responsible for multidrug resistance of existing tubulin inhibitors. In this study, we determined the mechanism of action of this drug. The basis for its activity was illuminated by solving the crystal structure of DJ101 in complex with tubulin at a resolution of 2.8Å. Investigations of the potency of DJ101 in a panel of human metastatic melanoma cell lines harboring major clinically relevant mutations defined IC50 values of 7-10 nmol/L. In cells, DJ101 disrupted microtubule networks, suppressed anchorage-dependent melanoma colony formation, and impaired cancer cell migration. In melanoma-bearing mice, DJ101 administration inhibited tumor growth and reduced lung metastasis in the absence of observable toxicity. DJ101 also completely inhibited tumor growth in a paclitaxel-resistant xenograft mouse model of human prostate cancer (PC-3/TxR), where paclitaxel was minimally effective. Our findings offer preclinical proof of concept for the continued development of DJ101 as a next-generation tubulin inhibitor for cancer therapy.Significance: These findings offer preclinical proof of concept for the continued development of DJ101 as a next-generation antitubulin drug for cancer therapy. Cancer Res; 78(1); 265-77. ©2017 AACR.