Abstract 3110: CBF regulates cell senescence and autophagy through sphingolipid metabolism.

Core Binding Factor (CBF) is a heterodimeric transcription factor comprised of one of three RUNX DNA binding proteins (Runx1, Runx2, or Runx3) and a non-DNA binding component termed CBFβ. CBFβ is a dedicated and required component of CBF. CBF is commonly altered by chromosomal translocations leading to acute myeloid and lymphocytic leukemias. More recently, altered CBF activity was associated with the malignant phenotype of various solid tumors. Recent studies placed RUNX factors in direct transcriptional control of several sphingolipid pathway enzymes as well as autophagy in murine fibroblast and myofibrils. However, CBF regulation of sphingolipid metabolism in cancer cells has not been investigated. Purpose: To study CBF function in solid tumors. Hypothesis: Previous work in the laboratory lead to a hypothesis that suggested a requirement for CBF in the malignant phenotype of prostate and ovarian cancers. Method and Results: To determine if CBF contributed to the malignant phenotype of cancer cells, we used lentiviral delivery of CBFβ-specific shRNA to decrease CBFβ expression. Cells knocked down for CBFβ expression exhibited a growth defect that was not associated with large perturbances in the cell cycle or with increased apoptosis. Since both sphingolipid metabolism and autophagy are central to survival pathways, these processes were examined in the CBFβ knockdown cell lines. Prostate (PPC1 and PC-3) and ovarian (SKOV-3) cancer cell lines that were infected with the CBFβ-specific shRNA but not those cells infected with a non-target-shRNA displayed a significant increase in autophagic markers, including LC3B-II, suggesting that autophagy was upregulated in CBFβ knocked-down cells. Treatment of CBFβ knockdown cells with the autophagy inhibitors chloroquine and 3-methyladenosine modestly increased cell survival, suggesting that cell death was associated with increased autophagy. Real time PCR analysis revealed that Ugcg, an enzyme critical for sphingolipid metabolism, was decreased in SKOV-3 cells after CBFβ knockdown. These data lead to a hypothesis that loss of CBFβ increases cell death and autophagy by, in part, perturbing sphinglolipid metabolism and, thus, increasing ceramide pools. Since sphingolipid metabolism is linked to senescence the process was examined using senescence-associated B-galactosidase activity, which was found to be increased in CBFβ knockdown cells. Treatment of cells knocked down for CBFβ with Fumonsin B1 (FB1), which decreases ceramide production, improved cell survival and decreased autophagy. Further, direct lipodomic analysis of the CBFβ knockdown cells identified significant increases in the levels of ceramide. Conclusion: Together, these data suggest that CBF regulates senescence and autophagy via control of the sphingolipid pathway. These studies further suggest that CBF is a putative therapeutic target in prostate and ovarian cancers. Citation Format: Adam H. Greer, Thomas Yong, Katie Fennell, Shari Meyers, Nathan Davis. CBF regulates cell senescence and autophagy through sphingolipid metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3110. doi:10.1158/1538-7445.AM2013-3110