Exploring ceramide metabolism as a potential target in dedifferentiated liposarcoma.

Abstract

e22558 Background: Dedifferentiated liposarcomas (DDLPS) are highly aggressive mesenchymal tumors characterized by MDM2 amplification. Elevated MDM2 amplification levels are associated with tumor growth, chemoresistance, and changes in cellular metabolism. Previous work from our laboratory has demonstrated that lipid metabolism, particularly increased glycosylated ceramides (Gly-Cer), is profoundly altered as a function of MDM2 amplification. Importantly, ceramides have been identified to play a key role in proliferation arrest and apoptosis or autophagy by stabilizing p53 through the binding and disrupting the MDM2-p53 interaction. Gly-Cer clears out ceramides, thereby preventing the ceramides from performing their anti-proliferative activity. We hypothesized that restoring Gly-Cer levels would induce anti-proliferative effects in DDLPS. Methods: 6 DDLPS cell lines (MDM2-high: Lipo224, Lipo141, Lipo224B, Lipo246; MDM2-low: Lipo815, Lipo863) were selected for in vitro assessment. MDM2 levels were ascertained via Western blot and RNA-sequencing. Metabolomic (Metabolon) and lipidomic (SCIEX 5600 TripleTOF-MS) profiling were conducted on all cell lines. Inhibitory concentration 50% (IC50) of the C6-ceramide (C6C) and nanoliposomal C6-Ceramides (C6CNL) were tested in vitro by XTT . Adjusted t-tests were used to determine metabolite relevance. Results: Metabolomic/lipidomic analyses identified a correlation between MDM2 amplification status and elevated levels of glycosylated ceramides (nom: Pval < 0.001), including GlcCer_NS(d18:1/24:1), GlcCer_NS(d19:1/24:1), GlcCer_NS(d18:1/25:0), glycosyl-N-palmitoyl-sphingosine, and glycosyl-N-stearoyl-sphingosine. Further, the ratio of glycosylated to un-glycosylated ceramides in DDLPS cell was also associated with MDM2 status for Cer(d19:1/24:1) (Pval < 0.01) and borderline associated for Cer(d18:1/16:0) and Cer(d18:1/25:0) (Pval < 0.2). In vitro treatment of DDLPS cell lines with C6C resulted in a reduction of cellular viability most prominent in MDM2-high cell lines (IC50: 17 vs. 55 µM). When treated with C6CNL, all DDLPS cell lines exhibited equal sensitivity (IC50: ~9 µM). Western blots demonstrate C6CNL treatment resulted in a reduction in MDM2 expression and restoration of p53 function. Conclusions: Exogenous ceramide treatment reduces MDM2 expression, restores TP53 status, and manifests anti-proliferative activity in DDLPS. C6CNL exhibited lower effective concentrations relative to free C6 ceramide. Prospective in vivo studies are underway to confirm these findings.