Abstract
INTRODUCTION: Patients with advanced adrenocortical carcinoma have poor survival and show elevated steroid production. Lipid metabolic profiling of adrenocortical carcinoma has revealed that the upregulation of sphingolipid metabolism, which regulates steroid synthesis, is linked to worse overall survival. Therefore, fingolimod, a Food and Drug Administration-approved drug that targets sphingolipid metabolism, represents a promising option for targeting adrenocortical carcinoma and metastatic spread. METHODS: Adrenocortical carcinoma cell lines were cultured in an appropriate medium. Cell-TiterGlo assessed cell viability. Western-blot and RNA-sequencing examined the targeted pathways. Migration and invasion using decellularized extracellular matrix evaluated metastasis. Seahorse measured metabolic flux. Reverse transcription-polymerase chain reaction determined steroidogenic genes. Mass spectrometry analyzed alterations in sphingolipids. RESULTS: Fingolimod treatment resulted in half-maximal inhibitory concentration (IC(50)) of 7.044 μmol/L, 5.588 μmol/L, and 9.992 μmol/L for the adrenocortical carcinoma 1, adrenocortical carcinoma 2, and NCI-H295R, respectively. Fingolimod showed synergistic effect with the standard of care treatment mitotane. Adrenocortical carcinoma cells treated with fingolimod showed a dose-dependent cleavage of poly (ADP-ribose) polymerase (PARP), an upregulation of LC3-II and p-ERK, and a downregulation of p-Akt and p-P65 with no appreciable change in total proteins. Furthermore, the downregulation of oxygen consumption rate, steroidogenic, and transforming growth factor-β pathway genes was noted after fingolimod treatment. Adrenocortical carcinoma cells in the presence of liver and lung extracellular matrix showed a 2-fold increase in migration and invasion over cells without liver/lung extracellular matrix, which was blocked by >90% after fingolimod treatment. Lipid profiling indicated ceramides, ceramide phospho-ethanolamines and ceramide-1-phosphate, sphingomyelins, triglycerides, phosphatidylcholines as altered lipids. CONCLUSION: Fingolimod induces apoptosis in adrenocortical carcinoma cells by targeting sphingolipid metabolism and prevents liver and lung metastatic invasion in vitro. Further in vivo validation studies alone or in combination with immune checkpoint inhibitor will support its clinical translation as a novel repurposed therapy in adrenocortical carcinoma.