60P - Investigating the role of nuclear sphingosine kinase 1 (SphK1) in lung cancer

Abstract

Background: Sphingosine kinases (SphKs) are enzymes catalyzing the formation of a bioactive lipid messenger, sphingosine-1-phosphate (S1P) through phosphorylation of sphingosine. So far, two isoforms of SphK have been described: SphK1, which has cytosolic distribution, and SphK2, known to locate predominantly in the nucleus. Overactivation of SphKs is involved in tumor growth and progression through regulation of cell proliferation and apoptosis. Methods: We studied the expression, the intracellular localization and the activity of SphK1 and SphK2 in non-small cell lung cancer (NSCLC) cell lines and in cancer patients. Results: In NSCLC cells, Sphk1 was found in the cytosol, while Sphk2 showed a nuclear localization. Surprisingly, we observed SphK1 expression also in the nuclear compartment. In several of the tested cell lines, the expression of SphK1 was even higher in the nucleus compared to the cytosol. Consistently, in a NSCLC tissue microarray, SphK1 nuclear expression was detected in a wide number of samples. Based on these data, we hypothesize the presence of a nuclear import signal in the SphK1 sequence, probably recognized by Karyopherin beta2. We found, in the aminoacidic sequence of SphK1, a R/K/H-X(5)-P-Y C-terminal motif within a structurally disordered and positively charged regions of about 30 amino acids. In order to dissect the role of nuclear SphK1, we used different tools able to modulate nuclear import/export. Moreover, specific inhibitors of SphKs were tested. Among them, fingolimod (FTY720), an oral drug initially defined as a S1P receptors antagonist but also acting as a SphK1 inhibitor, is effective in NSCLC cell lines: a reduction of cell density of about 50% is obtained with a drug concentration of about 2.5 µM. Interestingly, fingolimod seems to be more effective in cells with a nuclear/cytosolic ratio of SphK1 expression > 1. Conclusions: We demonstrate for the first time that SphK1 has a nuclear localization in NSCLC and we are now investigating its biological role in this context. Moreover, we propose nuclear SphK1 as a novel druggable target in human NSCLC cells. Since fingolimod is a clinically available drug, our results may suggest SphK1 targeting as a therapeutic opportunity to be tested in NSCLC patients. Legal entity responsible for the study: Prof. Roberto Bianco Funding: Has not received any funding Disclosure: All authors have declared no conflicts of interest.