TP53 is required for BECN1- and ATG5-dependent cell death induced by sphingosine kinase 1 inhibition.

The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) and the enzyme that produces it, SPHK1 (sphingosine kinase 1), regulate many processes important for the etiology of cancer. It has been suggested that SPHK1 levels are regulated by the tumor suppressor protein TP53, a key regulator of cell cycle arrest, apoptosis, and macroautophagy/autophagy. However, little is still known of the relationship between TP53 and SPHK1 activity in the regulation of these processes. To explore this link, we examined the effects of inhibiting SPHK1 in wild-type and TP53 null cancer cell lines. SK1-I, an analog of sphingosine and isozyme-specific SPHK1 inhibitor, suppressed cancer cell growth and clonogenic survival in a TP53-dependent manner. It also more strongly enhanced intrinsic apoptosis in wild-type TP53 cells than in isogenic TP53 null cells. Intriguingly, SK1-I induced phosphorylation of TP53 on Ser15, which increases its transcriptional activity. Consequently, levels of TP53 downstream targets such as pro-apoptotic members of the BCL2 family, including BAX, BAK1, and BID were increased in wild-type but not in TP53 null cells. Inhibition of SPHK1 also increased the formation of autophagic and multivesicular bodies, and increased processing of LC3 and its localization within acidic compartments in a TP53-dependent manner. SK1-I also induced massive accumulation of vacuoles, enhanced autophagy, and increased cell death in an SPHK1-dependent manner that also required TP53 expression. Importantly, downregulation of the key regulators of autophagic flux, BECN1 and ATG5, dramatically decreased the cytotoxicity of SK1-I only in cells with TP53 expression. Hence, our results reveal that TP53 plays an important role in vacuole-associated cell death induced by SPHK1 inhibition in cancer cells.

Identification of SPHK1 as a therapeutic target and marker of poor prognosis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is characterized by a uniquely aggressive behavior and lack of effective targeted therapies. After analyzing the gene expression profiles of seven paired intrahepatic CCA microarrays, a novel sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) pathway and a novel target gene, SPHK1, were identified. We hypothesized that therapeutic targeting of this pathway can be used to kill intrahepatic cholangiocarcinoma (CCA) cells. High levels of SPHK1 protein expression, which was evaluated by immunohistochemical staining of samples from 96 patients with intrahepatic CCA, correlated with poor overall survival. The SPHK1 inhibitor SK1-I demonstrated potent antiproliferative activity in vitro and in vivo. SK1-I modulated the balance of ceramide-sphinogosine-S1P and induced CCA apoptosis. Furthermore, SK1-I combined with JTE013, an antagonist of the predominant S1P receptor S1PR2, inhibited the AKT and ERK signaling pathways in CCA cells. Our preclinical data suggest SPHK1/S1P pathway targeting may be an effective treatment option for patients with CCA.