We have previously shown that the death receptor ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces an increase of intracellular C₁₆-ceramide in sensitive SW480 but not in resistant SW620 cells. Resistance in SW620 cells was overcome by exogenous ceramide, leading us to propose that defective ceramide signaling contributes to TRAIL resistance. In this study we found that the increase in C₁₆-ceramide in SW480 cells was inhibited by fumonisin B1, an inhibitor of ceramide synthases (CerS). Protein analysis revealed that TRAIL-resistant SW620 cells expressed lower levels of ceramide synthase 6 (CerS6, also known as longevity assurance homologue 6), which prompted us to investigate the effect of CerS6 modulation on TRAIL phenotype. RNAi against CerS6 resulted in a specific and significant decrease of the C₁₆-ceramide species, which was sufficient to inhibit TRAIL-induced apoptosis. In cells with decreased levels of CerS6, caspase-3 was activated but failed to translocate into the nucleus. CerS6 localized primarily to the perinuclear region, suggesting this enzyme may be important in regulation of nuclear permeability. Moderate elevation in CerS6 expression was sufficient to reverse TRAIL resistance in SW620 cells. These results suggest that modulation of CerS6 expression may constitute a new therapeutic strategy to alter apoptotic susceptibility.

Despite recent advances in the development of novel therapies against castration-resistant prostate cancer, the advanced form of the disease remains a major treatment challenge. Aberrant sphingolipid signaling through sphingosine kinases and their product, sphingosine-1-phosphate, can promote proliferation, drug resistance, angiogenesis, and inflammation. The sphingosine kinase 2 inhibitor ABC294640 is undergoing clinical testing in cancer patients, and in this study we investigated the effects this first-in-class inhibitor in castration-resistant prostate cancer. In vitro, ABC294640 decreased prostate cancer cell viability as well as the expression of c-Myc and the androgen receptor, while lysosomal acidification increased. ABC294640 also induced a greater than 3-fold increase in dihydroceramides that inversely correlated with inhibition of dihydroceramide desaturase (DEGS) activity. Expression of sphingosine kinase 2 was dispensable for the ABC294640-mediated increase in dihydroceramides. In vivo, ABC294640 diminished the growth rate of TRAMP-C2 xenografts in syngeneic hosts and elevated dihydroceramides within tumors as visualized by MALDI imaging mass spectroscopy. The plasma of ABC294640-treated mice contained significantly higher levels of C₁₆- and C_24:1-ceramides (but not dihydro-C₁₆-ceramide) compared with vehicle-treated mice. In summary, our results suggest that ABC294640 may reduce the proliferative capacity of castration-resistant prostate cancer cells through inhibition of both sphingosine kinase 2 and dihydroceramide desaturase, thereby providing a foundation for future exploration of this small-molecule inhibitor for the treatment of advanced disease. Mol Cancer Ther; 14(12); 2744–52. ©2015 AACR.

Although adoptive transfer of autologous tumor antigen–specific T-cell immunotherapy can produce remarkable clinical efficacy, most patients do not achieve durable complete responses. We hypothesized that reducing susceptibility of T cells to activation-induced cell death (AICD), which increases during the rapid in vitro expansion of therapeutic T cells before their infusion, might improve the persistence of adoptively transferred cells. Our investigations revealed that repetitive stimulation of the T-cell receptor (TCR) induced AICD, as a result of activating the DNA damage response pathway through ATM-mediated Ser15 phosphorylation of p53. Activation of this DNA damage response pathway also occurred upon antigen-specific restimulation in TCR-transduced TIL1383I T cells prepared for adoptive transfer to patients as part of a clinical trial. Notably, treatment with the antioxidant N-acetyl cysteine (NAC) significantly reduced upregulation of the DNA damage marker γH2AX, subsequent ATM activation, and cell death. In the Pmel mouse model of melanoma, the presence of NAC during ex vivo T-cell expansion improved the persistence of adoptively transferred cells, reduced tumor growth, and increased survival. Taken together, our results offer a preclinical proof of concept for the addition of NAC to current therapeutic T-cell expansion protocols, offering immediate potential to improve the quality and therapeutic efficacy of adoptive T-cell therapeutics infused into patients. Cancer Res; 76(20); 6006–16. ©2016 AACR.