The Sphingosine Kinase 1 Inhibitor 2-(p-Hydroxyanilino)-4-(p-chlorophenyl)thiazole Induces Proteasomal Degradation of Sphingosine Kinase 1 in Mammalian Cells

Abstract

Sphingosine kinase 1 (SK1) is an enzyme that catalyzes the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) induces the proteasomal degradation of SK1 in human pulmonary artery smooth muscle cells, androgen-sensitive LNCaP prostate cancer cells, MCF-7 and MCF-7 HER2 breast cancer cells and that this is likely mediated by ceramide as a consequence of catalytic inhibition of SK1 by SKi. Moreover, SK1 is polyubiquitinated under basal conditions, and SKi appears to increase the degradation of SK1 by activating the proteasome. In addition, the proteasomal degradation of SK1a and SK1b in androgen-sensitive LNCaP cells is associated with the induction of apoptosis. However, SK1b in LNCaP-AI cells (androgen-independent) is less sensitive to SKi-induced proteasomal degradation and these cells are resistant to SKi-induced apoptosis, thereby implicating the ubiquitin-proteasomal degradation of SK1 as an important mechanism controlling cell survival.Sphingosine 1-phosphate (S1P) 5 is a bioactive lipid that has an important role in regulating the growth, survival, and migration of mammalian cells. S1P binds to a family of five GPCR termed S1P n (where n ϭ 1-5) that regulate various effectors, such as MAP kinase (1). S1P is produced by the enzyme sphingosine kinase (SK1 and SK2 isoforms), which catalyzes the phosphorylation of sphingosine to produce S1P (2, 3). There are three N-terminal variants of SK1. SK1a (GenBank TM number: NM_001142601) is a 42.5 kDa protein, while SK1b (GenBank TM number: NM_182965) is a 51 kDa protein identical to SK1a, but with an 86 amino acid N-terminal extension. The third form has a molecular mass of 43.9 kDa and is identical to SK1a except for a 14 amino acid Nterminal extension (GenBank TM number: NM_021972) and migrates with similar mobility as SK1a on SDS-PAGE. The SK1a annotation used here therefore includes SK1a and possibly SK1aϩ14.SK1 has been demonstrated to have an important role in cancer (4). For instance, enforced overexpression of SK1 increases V12-Ras-dependent transformation of cancer cells (5), S1P levels, estrogen-dependent tumorigenesis, and blocks apoptosis of MCF-7 cells induced by anti-cancer drugs (6). SK1/S1P is also required for EGF-induced MCF-7 cell migration, proliferation and survival (7) and breast cancer cell growth (8). High SK1 expression is also correlated with poor prognosis in ER ϩ breast cancer, and SK1 induces a migratory phenotype in response to S1P in MCF-7 cells, via SK1-dependent changes in S1P 3 expression and PAK1/ERK-1/2 regulation (9). There is no evidence that mutations occur in the SK1 gene linked to cancer and therefore, the term non-oncogene addiction has been used to describe its role in cancer progression (10). The S1P signaling pathway has also been implicated in promoting the proliferation of androgen-independent prostate cancer PC-3 cells (11). Moreover, irradiation of a radiation-sensitive cancer cell ...

Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II

“…In contrast, SK1 protein levels were substantially reduced in cells exposed to SKI II (10 M) ( Fig. 1E ), which agrees with published reports in other cell models ( 65 ). Figure 2 summarizes the results of UPLC/TOFMS analyses of sphingolipids extracted from HGC 27 cells harvested at different time points after incubation with 10 M SKI II.…”

Autophagosomal Membrane Serves as Platform for Intracellular Death-inducing Signaling Complex (iDISC)-mediated Caspase-8 Activation and Apoptosis

“…Importantly, we also found that SKI-I promotes the degradation of p62 through proteasome in AML cells. Consistently, activation of the proteasome machinery in response to SK inhibition has been reported (47). As the addition of bortezomib stabilized p62 and enhanced the autophagydependent activation of caspase-8 and apoptosis upon SKI-I treatment, inhibition of proteasome may be a key factor to increase the efficacy of SKI-I-induced cell death.…”

Role of sphingosine 1-phosphate receptors, sphingosine kinases and sphingosine in cancer and inflammation

“…We have demonstrated that SKi induces the proteasomal degradation of SK1a and SK1b (which has an 86 amino-acid N-terminal extension compared to SK1a) in androgen-sensitive LNCaP prostate cancer cells and this results in a reduction in S1P levels and an increase in sphingosine and C22:0 and C24:0 ceramide levels and the induction of apoptosis (Loveridge et al, 2010). SKi also induces proteasomal degradation of SK1a in androgen-independent LNCaP-AI cells, but fails to reduce SK1b levels (Loveridge et al, 2010), which is possibly a consequence of a compensatory increase in SK1b mRNA expression in these cells. This is associated with a failure to increase C22:0 and C24:0 ceramide levels and the absence of apoptosis (Loveridge et al, 2010;Lim et al, 2012).…”

“…A common feature of these inhibitors is that they induce the ubiquitinproteasomal degradation of SK1 in solid cancer cell lines (Loveridge et al, 2010;Tonelli et al, 2010;Lim et al, 2011b) and proliferating vascular smooth muscle cells (Loveridge et al, 2010;Baek et al, 2013;Byun et al, 2013). This leads to inhibition of DNA synthesis (Byun et al, 2013) and is recapitulated by siRNA knockdown of SK1 expression.…”

“…However, other very poor inhibitors of SK1 and SK2 such as SKi (2-(p-hydroxyanilino)-4-(pchlorophenyl) thiazole)) also induce the proteasomal degradation of SK1 via a mechanism that is only partially dependent on direct binding to SK1 (Loveridge et al, 2010). We have demonstrated that SKi induces the proteasomal degradation of SK1a and SK1b (which has an 86 amino-acid N-terminal extension compared to SK1a) in androgen-sensitive LNCaP prostate cancer cells and this results in a reduction in S1P levels and an increase in sphingosine and C22:0 and C24:0 ceramide levels and the induction of apoptosis (Loveridge et al, 2010).…”