| dc.description.abstract |
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. |
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