Carmofur prevents cell cycle progression by reducing E2F8 transcription in temozolomide-resistant glioblastoma cells

Abstract

Sphingolipid metabolism is dysregulated in many cancers, allowing cells to evade apoptosis through increased sphingosine-1-phosphate (S1P) and decreased ceramides. Ceramidases hydrolyze ceramides to sphingosine, which is phosphorylated by sphingosine kinases to generate S1P. The S1P allows cells to evade apoptosis by shifting the equilibrium away from ceramides, which favor cell death. One tumor type that exhibits a shift in the sphingolipid balance towards S1P is glioblastoma (GBM), a highly aggressive brain tumor. GBMs almost always recur despite surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Understanding sphingolipid metabolism in GBM is still limited, and currently, there are no approved treatments to target dysregulation of sphingolipid metabolism in GBM. Carmofur, a derivative of 5-fluorouracil, inhibits acid ceramidase (ASAH1), a key enzyme in the production of S1P, and is in use outside the USA to treat colorectal cancer. We find that the mRNA for ASAH1, but not other ceramidases, is elevated in recurrent GBM. When TMZ-resistant GBM cells were treated with carmofur, decreased cell growth and increased apoptosis were observed along with cell cycle perturbations. RNA-sequencing identified decreases in cell cycle control pathways that were specific to TMZ-resistant cells. Furthermore, the transcription factor and G1 to S phase regulator, E2F8, was upregulated in TMZ-resistant versus parental GBM cells and inhibited by carmofur treatment in TMZ-resistant GBM cells, specifically. These data suggest a possible role for E2F8 as a mediator of carmofur effects in the context of TMZ resistance. These data suggest the potential utility of normalizing the sphingolipid balance in the context of recurrent GBM.