Er Stress Induced By 4-Nerolidylcathecol (4-Nc) Promotes Antitumor Effects In Brafi/Meki Sensitive And Resistant Melanoma Models

Melanoma accounts for only 4% of skin malignancies, but it has a high mortality rate. Though the specific inhibitors of MAPK pathway (BRAFi and MEKi) have revolutionized melanoma treatment, most of patients are subject to resistance, justifying the constant search for new therapeutic compounds. Recent data from our laboratory indicated that 4-nerolidylcathecol (4-NC), extracted from Pothomorphe umbellata, a native Brazilian plant, induces apoptosis in melanoma cells by ROS production, DNA damage and p53 expression increase and promoted an antiproliferative effect in reconstructed skin. In this work, we aim to evaluate the 4-NC efficacy in overcoming resistance to BRAF and MEK inhibitors in melanoma cell lines as well in xenograft models. Firstly, the vemurafenib-resistant (R) and trametinib/vemurafenib-resistant (DR) melanoma cell lines were generated and characterized by MTT, fluorescence microscopy and western blotting. The cytotoxicity was evaluated by Trypan Blue Exclusion and Clonogenic Assays. The invasion potential was demonstrated by 2D and 3D models. Besides that, SK-MEL-103 melanoma cells (5x10 5 cells) were injected s.c. in female BALB/c nude mice. Once the tumors appeared, treatments were initiated by i.p. injection for 3 times a week for 3 weeks with 4-NC (10mg/kg), DMSO or no treatment. 4-NC showed cytotoxicity (IC 50 ~30µM), colony formation and invasion decrease in vitro assays. To further investigate that, we dissected the molecular pathways involved with induction of Endoplasmic Reticulum (ER) Stress by Unfolded Protein Response (UPR). As results, we found many altered proteins in naive, R and DR melanoma cells, especially regarding elevated levels of BiP and CHOP. The latter has been implicated in apoptotic events and was confirmed by Flow cytometry. 4-NC, previously reported as a proteasome inhibitor, was able to significantly suppress the xenograft tumor growth by 4-fold compared to controls, with complete tumor remission in one animal. P53, p16 and cleaved PARP expression were increased in the tumors of treated animals indicating apoptosis and cell cycle arrest. MMP2 and MMP14 gene expression were decreased in the same samples, demonstrating a promising role of 4-NC in the inhibition of melanoma invasion also in animal models. Although preliminary, our data suggest that 4-NC compound has an important cytotoxic effect in melanoma models and should be better investigated for future applications in human cutaneous melanoma treatment including resistant patients. Approval of Ethics Committee: Office CEUA/FCF/70-2012 - USP/Brazil. Financial support: FAPESP (2014/06959-0); CNPq (385282/2011-7). Citation Format: Debora Kristina Alves-Fernandes, Erica Aparecida de Oliveira, Fernanda Faiao-Flores, Silvana Sandri, Walter Turato, Silvia Berlanga de Moraes Barros, Silvia Stuchi Maria-Engler. ER stress induced by 4-nerolidylcathecol (4-NC) promotes antitumor effects in BRAFi/MEKi sensitive and resistant melanoma models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A14.