Abstract B22: Inhibition of c-MYC expression in oral squamous carcinoma cells contributes to the antiproliferative activity of the alkylphosphocholine erufosine

Abstract

Abstract Erufosine is a third generation alkylphosphocholine, which has been shown to inhibit the proliferation of oral squamous cell carcinoma (OSCC) cells. It simultaneously induces apoptosis and autophagy by modulating the Akt–mTOR signaling pathway and is known to down regulate the expression of cell cycle related proteins, but the exact mechanism of action of this membrane seeking compound is still unknown. For investigating the mechanism of action in a systematic way, we studied the effects of erufosine in a panel of OSCC cell lines (Cal-27, FaDu and HN-5). The concentration effecting the 50% inhibition in cell proliferation (IC50) in response to erufosine was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using concentrations ranging from 0.6µM – 100µM at various time points (24h, 48h and 72h) after exposure. Based on these results, FaDu cells were exposed to erufosine at concentrations effecting IC25, IC50 and IC75, and then the mRNA of respectively treated cells was isolated and subjected to microarray analysis by Illumina Chip array. Selected genes with highly modulated mRNA expression were further investigated by quantitative RT-PCR and Western Blot. IC50 values in response to erufosine, following exposure for 24h, 48h and 72h exposure ranged from 20µM to 9.7µM for Cal-27 -, 27µM to 14µM for FaDu - , and 43µM to 37µM for HN-5 cells. Analysis of microarray data by the Ingenuity Pathways Analysis (IPA; Ingenuity® Systems, www.ingenuity.com) software revealed that 725 genes were modulated at 16h, averaged for all concentrations, with 47 genes being modulated more than 3 fold. The respective 24h gene expression analysis revealed that 1215 genes were significantly modulated, with 77 genes deregulated over 3 fold, whereas a total of 1305 genes were altered at the end of 48h, with 93 genes being deregulated over 3 fold. In a first approach, we concentrated on genes related to the c-MYC signaling pathway. The cyclin-dependent kinase inhibitor 1A (p21, Cip1) was found to be upregulated 9.2, 7.9 and 9.9 fold, averaged for all concentrations, at 16h, 24h and 48h, respectively. The Max dimerization protein 1 (MDX1) was upregulated 3.7 fold and 3.3 fold over the untreated control at 24h and 48h post treatment. The combined modulation of the two mRNA species hints at the involvement of c-MYC as a target of erufosine treatment. These results were further validated by qRT-PCR, which also confirmed the modulation of these genes across the OSCC cell panel. In line with this, the Western blots also showed a clear decrease in c-MYC protein levels and a corresponding increase in p21 in a concentration- and time-dependent manner. In summary, the known effect of erufosine on cell cycle related proteins as well as the increased mRNA (p21, MDX-1) and protein (p21) expression reported here is in accordance with the decreased expression of c-MYC at protein level. These findings support the hypothesis that c-MYC is an indirect target of erufosine in OSCC cells. Citation Format: Shariq S. Ansari, Martin R. Berger. Inhibition of c-MYC expression in oral squamous carcinoma cells contributes to the antiproliferative activity of the alkylphosphocholine erufosine. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B22.