Abstract 5204: ICAM-1 and IL-17E upregulate FOXO1 to suppress the expression of oncogenic PKC-ι in human melanoma

Abstract

Abstract Throughout the span of the last 15 years, the number of cases of melanoma has been on a steady increase. Approximately, 92,000 new melanoma cases will be diagnosed in the United States in 2018. Our previous studies demonstrated that protein kinase C-iota (PKC-ι) is crusial for melanoma progression, which promotes the activation of nuclear factor (NF)-κB through a PI3K/AKT manner, thereby supporting survival and progression. In addition, PKC-ι induced the metastasis of melanoma cells by stimulating ephithilial-mesenchmal transition. Results also showed that PKC- specific inhibitors diminished the expression of both PKC-ι and phospo-PKC-ι, suggesting that PKC-ι plays a role in regulating its own expression in melanoma cells in-vitro {Ratnayake, W.S., et al. Cell Adhes. Migr. (2018) https://doi.org/10.1080/19336918.2018.1471323}. In this study, we report the transcriptional regulation mechanisms of PRKCI (PKC-ι gene) expression in melanoma. PROMO and Genomatix Matinspector methods were used to identify c-Jun, interferon-stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI and they were analyzed for their roles in PKC-ι expression. siRNAs were used to silence selected transcription factors, and the results revealed that, silencing of FOXO1 and c-Jun significantly altered the expression of PRKCI. In addtion, we knocked down NF-κB in order to demonstrate changes on activities of c-Jun and FOXO1. Both phosphorylated and total PKC-ι levels increased upon FOXO1 silencing and decreased upon c-Jun silencing, suggesting that c-Jun turns is an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. Multiplex ELISA assays were used to analyze multiple pathways and the expression of cytokines with respect to crosstalk between signaling pathways affected by specific inhibiton PKC-ι. Western blot data for all siRNA treatments of PKC-ι, NF-κB, FOXO1 and c-Jun were supported by real-time qPCR. Taken together, results suggested that the regulation of PKC-ι expression was strongly associated with FOXO1 and c-Jun through the trascriptional deactivation/activation. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)-6 and IL-8, with a significant increase in the levels of IL-17E and intercellular adhesion molecule 1 (ICAM-1) upon the knockdown of expression of PKC-ι in both cell lines. This suggested that PKC-ι expression was affected by said cytokines in an autocrine modus. Overall, the findings suggest that PKC-ι inhibition suppresses its own expression, diminishing oncogenic signaling such as NF-κB, PI3K/AKT and JNK/c-Jun while upregulating anti-tumor signaling such as ICAM-1/FOXO1, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma metastasis. Citation Format: Wishrawana Sarathi Ratnayake, Christopher Apostolatos, Sloan Breedy, André Apostolatos, Mildred Acevedo-Duncan. ICAM-1 and IL-17E upregulate FOXO1 to suppress the expression of oncogenic PKC-ι in human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5204.