Abstract 2022: Oncogenic protein kinase Cι drives melanoma cell epithelial-mesenchymal transition by activating vimentin through Par6/RhoA signaling

Abstract

Abstract Melanoma is one of the fastest growing cancers in the United States, predicting a 14% increase in 2017 compared to 2016. The five year survival rate drops to 4% when melanoma is metastasized and responsible for >90% melanoma related deaths. Most available drugs target BRAF (V600E) mutation which occurs in ~60% melanoma cases, yet there is a poor prognosis and tumors acquire resistance to BRAF mutation inhibition. PKC-ι (iota) is an oncogene involved in cell cycle progression, tumorigenesis and cell survival in many cancers. We believe PKC-ι is an effective therapeutic target for invasive melanoma. We reported that PKC-ι is overexpressed in melanoma cells [Int. J. Oncol. 51(5), 1370-1382, (2017)]. In the current study, we have investigated the effects of novel PKC-ι inhibitor [4-(5-amino-4-carbamoylimidazol-1-yl)-2, 3-dihydroxycyclopentyl] methyl dihydrogen phosphate (1 µM) along with its nucleoside analog, 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (2.5 μM) on cell migration and invasion of two malignant melanoma cell lines (SK-MEL-2 and MeWo) compared to a normal melanocyte cell line (MEL-NEO-F). Cell viability and WST-1 assays showed that both inhibitors show lesser cytotoxicity to MEL-F-NEO cell line at higher concentrations (>7.5 μM) compare to significant toxicity on melanoma (>1 μM). Both inhibitors decreased the levels of total and phosphorylated levels of PKC-ι. Furthermore, both inhibitors increased the levels of E-cadherin and RhoA while decreasing the levels of Vimentin; a mesenchymal marker associated with EMT. Treatments with inhibitors significantly decreased the phosphorylated Vimentin (S39) and also altered the phosphorylation at S33 and S56, thereby preventing the Vimentin intermediate assembly. Immunoprecipitation and reversed immunoprecipitation showed a strong interaction of PKC-ι and Vimentin and immunofluorescence staining proved the observation. mRNA expression levels of PKC-ι, Vimentin decreased upon PKC-ι and the expression levels transcription factors such as SNAIL1, ZNF703, PRX1 will be tested using qPCR, Western blots and ELISA techniques. PKC-ι inhibition downregulate Par6 thereby stabilize RhoA even upon TGFβ1 stimulation. We have also showed that PKC-ι inhibition limits the translocation of activated NF-κB p65/p52 complex and β-catenin thereby regulates NF-κB and WNT/β-catenin signaling. Overall, results show that PKC-ι is essential for melanoma progression and metastasis through activation of Vimentin via TGFβ/Par6/RhoA pathway. Therefore PKC-ι could be used as effective therapeutic targets for malignant melanoma. Citation Format: Wishrawana S. Ratnayake, André H. Apostolatos, Christopher A. Apostolatos, Avijit Dey, Rekha Patel, Mildred Acevedo-Duncan. Oncogenic protein kinase Cι drives melanoma cell epithelial-mesenchymal transition by activating vimentin through Par6/RhoA signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2022.