Efficacy of the MEK inhibitor trametinib in combination with disulfiram in BRAF-wild type melanoma cells

The MAPK signaling pathway is commonly hyperactivated in melanoma and plays a critical role in the development, proliferation and survival of tumor cells. Thus, in melanoma cells without a BRAF mutations (BRAF-WT), hyperactivation of the MAPK pathway occurs in approximately 40% of cases. The clinical efficacy of MEK inhibitors as monotherapy in NRASmut melanoma has been further investigated, particularly in the phase 3 NEMO clinical trial. The overall response rate of the MEK inhibitor was significantly poor (15%) and progression-free survival improved only marginally (2.8 vs. 1.5 months) over dacarbazine. Therefore, investigations on combinations of MEK inhibitors with additional effective drugs, which are able to improve the response rates of the BRAF-WT melanoma to the MEK inhibitors, are urgently needed. Disulfiram (DSF), a former FDA approved alcohol-aversion drug, is such a drug candidate. DSF is metabolized into bis-diethyldithiocarbamate (ET) by glutathione reductase activity in vivo. ET has a strong chelating capacity and a high affinity for copper II (Cu2+), forming the bis-diethyldithiocarbamate-copper complex (CuET). CuET has a strong anti-tumor effect, which is complete copper-dependent. For these reasons combining CuET with the MEK inhibitor trametinib was tested as novel anticancer therapy in this work. The combination treatment showed enhanced cytotoxicity compared to the monotherapies. It was demonstrated that trametinib/CuET induced a significant nuclear accumulation of copper in BRAF-WT melanoma cells. Copper was transported into the melanoma cells and further translocated to the nucleus by the copper chaperone ATOX1. Interestingly, the copper accumulation in the nucleus was associated with the cellular cytotoxicity mediated by combination therapy. It was further confirmed that melanoma cells with an ATOX1 gene down-regulation was induced were not able to generate nuclear copper accumulation and showed a lower sensitivity to the combination therapy, confirming the major role of ATOX1. Mechanistically trametinib plus CuET induced high levels of reactive oxygen species (ROS) and a significant up-regulation of endoplasmic reticulum (ER) stress-related genes in BRAF-WT melanoma cells. Consistently, this significant up-regulation of ER stress-related genes such as ATF4, CHOP, and NUPR1 leaded to induction of apoptosis. Furthermore, trametinib plus CuET induced high levels of pro-apoptotic proteins of the BCL-2 family such as BIM and BAX, and reduced the anti-apoptotic BCL-2. Additionally, it was demonstrated that the apoptosis mediated by trametinib plus CuET was dependent on c-JUN N-Terminal Kinase (JNK/c-JUN) signaling. Cytotoxicity and apoptosis of the combination were completely mitigated through a selective JNK inhibitor, confirming its crucial role in apoptosis induction. The co-administration of trametinib with CuET affected all melanoma cells tested. Specifically, the growth and survival of melanoma cells with NRASmut, NF1-LoF or Triple-WT were significantly impaired by the treatment and caused significant synergistic cytotoxicity. Trametinib/CuET was very effective in two and three-dimensional model systems. Most importantly, trametinib/CuET suppressed the in vivo growth of BRAF-WT melanoma xenografts. Taken together, these data suggest the combination of trametinib with CuET as a novel targeted therapy for BRAF-WT melanoma

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

Abstract Background During 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. Results Although 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. Conclusion We 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

Marine and coastal biodiversity of Oaxaca, Mexico [with erratum]

The state of Oaxaca in southern Mexico harbors the highest faunistic and floristic biodiversity of the region; however, to date, research effort has focused on terrestrial flora and fauna. Despite this bias, there is a large amount of dispersed information regarding the coastal and marine biodiversity of Oaxaca. The present study aimed to: 1) update and synthesize existing information to improve understanding of coastal and marine biodiversity; and 2) provide a baseline for future biodiversity studies in Oaxaca. The review of 198 references about Oaxaca's flora and fauna produced 2, 157 species records, from 15 taxonomic groups (Division, Phyla or Class). Nevertheless, knowledge about the fauna and flora of Oaxaca remains scarce compared to other regions, such as the Gulf of California. Additionally, floristic and faunistic knowledge is heterogeneous among the taxonomic groups; for instance, 242 (11 .2 °/o) species represent macroalgae, three invertebrate groups (annelids, crustaceans and mollusks) represent about 44.0% (949 species) of all species recorded, while the vertebrates are represented by 762 species (35.3°/o). Similarly, many invertebrate groups recorded in adjacent regions have not yet been recorded on the Oaxaca coast, including some platyhelminthes, rotifers, nematodes, oligochaetes, sipunculids, echiurans, tardigrades, pycnogonids, some crustaceans, brachiopods, chaetognaths, ascidians and cephalochordates. The study of the marine flora and fauna is far from finished; additional effort is required to complete the marine biodiversity inventory of Oaxaca

Therapeutic Efficacy of Pharmacological Ascorbate on Braf Inhibitor Resistant Melanoma Cells In Vitro and In Vivo

High-dose ascorbate paradoxically acts as a pro-oxidant causing the formation of hydrogen peroxide in an oxygen dependent manner. Tumor cells (in particular melanoma cells) show an increased vulnerability to ascorbate induced reactive oxygen species (ROS). Therefore, high-dose ascorbate is a promising pharmacological approach to treating refractory melanomas, e.g., with secondary resistance to targeted BRAF inhibitor therapy. BRAF mutated melanoma cells were treated with ascorbate alone or in combination with the BRAF inhibitor vemurafenib. Viability, cell cycle, ROS production, and the protein levels of phospho-ERK1/2, GLUT-1 and HIF-1α were analyzed. To investigate the treatment in vivo, C57BL/6NCrl mice were subcutaneously injected with D4M.3A (BrafV600E) melanoma cells and treated with intraperitoneal injections of ascorbate with or without vemurafenib. BRAF mutated melanoma cell lines either sensitive or resistant to vemurafenib were susceptible to the induction of cell death by pharmacological ascorbate. Treatment of BrafV600E melanoma bearing mice with ascorbate resulted in plasma levels in the pharmacologically active range and significantly improved the therapeutic effect of vemurafenib. We conclude that intravenous high-dose ascorbate will be beneficial for melanoma patients by interfering with the tumor’s energy metabolism and can be safely combined with standard melanoma therapies such as BRAF inhibitors without pharmacological interference

Therapeutic Efficacy of Pharmacological Ascorbate on Braf Inhibitor Resistant Melanoma Cells In Vitro and In Vivo

High-dose ascorbate paradoxically acts as a pro-oxidant causing the formation of hydrogen peroxide in an oxygen dependent manner. Tumor cells (in particular melanoma cells) show an increased vulnerability to ascorbate induced reactive oxygen species (ROS). Therefore, high-dose ascorbate is a promising pharmacological approach to treating refractory melanomas, e.g., with secondary resistance to targeted BRAF inhibitor therapy. BRAF mutated melanoma cells were treated with ascorbate alone or in combination with the BRAF inhibitor vemurafenib. Viability, cell cycle, ROS production, and the protein levels of phospho-ERK1/2, GLUT-1 and HIF-1α were analyzed. To investigate the treatment in vivo, C57BL/6NCrl mice were subcutaneously injected with D4M.3A (BrafV600E) melanoma cells and treated with intraperitoneal injections of ascorbate with or without vemurafenib. BRAF mutated melanoma cell lines either sensitive or resistant to vemurafenib were susceptible to the induction of cell death by pharmacological ascorbate. Treatment of BrafV600E melanoma bearing mice with ascorbate resulted in plasma levels in the pharmacologically active range and significantly improved the therapeutic effect of vemurafenib. We conclude that intravenous high-dose ascorbate will be beneficial for melanoma patients by interfering with the tumor’s energy metabolism and can be safely combined with standard melanoma therapies such as BRAF inhibitors without pharmacological interference