Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells.

Melissa Mcnaughton,Nigel J Pyne,Susan Pyne,Stuart M Pitson,Melissa Pitman

doi:10.18632/oncotarget.7693

Abstract

Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest.

Highlights

The bioactive lipid, sphingosine 1-phosphate (S1P) is formed by the phosphorylation of sphingosine and this reaction is catalysed by two isoforms of sphingosine kinase (SK1 and SK2), which are encoded by different genes and exhibit distinct subcellular localisations, biochemical properties and functions
We had previously demonstrated that the SK1/2 inhibitor, SKi induced the proteasomal degradation of c-Myc in LNCaP prostate cancer cells [15]
We considered that the ability of ABC294640 to induce proteasomal degradation of c-Myc is not a typical property of SK2 inhibitors but which might be mediated through an indirect effect on the ubiquitin-proteasomal degradation of SK1a in androgen-independent LNCaP-AI cells

Summary

Introduction

The bioactive lipid, sphingosine 1-phosphate (S1P) is formed by the phosphorylation of sphingosine and this reaction is catalysed by two isoforms of sphingosine kinase (SK1 and SK2), which are encoded by different genes and exhibit distinct subcellular localisations, biochemical properties and functions (see [1] for review). A feature of SK1 inhibitors is that they induce the ubiquitin-proteasomal degradation of SK1 in solid cancer cell lines [4,5,6] and proliferating vascular smooth muscle cells [4, 7, 8]. Other inhibitors of SK1 and SK2 such as SKi (2-(p-hydroxyanilino)4-(p-chlorophenyl) thiazole) induce the proteasomal degradation of SK1 via a mechanism that is only partially dependent on direct binding to SK1 [4]. Recent studies have demonstrated that SK2 might have an important role in cancer, For instance, the aryladamantane compound, ABC294640 (3-(4-chlorophenyl)-adamantane1-carboxylic acid (pyridin-4-ylmethyl)amide), which is reported to be a selective inhibitor of SK2 activity, is in phase 2 clinical trials for diffuse B-cell lymphoma. ABC294640 is a competitive (with sphingosine) inhibitor of SK2 activity with a Ki of 9.8 μM, and reduces S1P formation in cancer cells [9]. ABC294640 reduces colorectal cancer growth in vitro and in vivo [12] and decreases tumor incidence in an azoxymethane (AOM)/ dextran sulfate sodium (DSS) mouse model, suggesting that SK2 has a role in inflammation-induced cancer progression [13]

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Conclusion