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 ...

Sphingosine kinase 1 (SK1) is an enzyme that catalyses the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that FTY720 (Fingolimod™) and (S)-FTY720 vinylphosphonate are novel inhibitors of SK1 catalytic activity and induce the proteasomal degradation of this enzyme in human pulmonary artery smooth muscle cells, MCF-7 breast cancer cells and androgen-independent LNCaP-AI prostate cancer cells. Proteasomal degradation of SK1 in response to FTY720 and (S)-FTY720 vinylphosphonate is associated with the down-regulation of the androgen receptor in LNCaP-AI cells. (S)-FTY720 vinylphosphonate also induces the apoptosis of these cells. These findings indicate that SK1 is involved in protecting LNCaP-AI from apoptosis. This protection might be mediated by so-called ‘inside-out’ signalling by S1P, as LNCaP-AI cells exhibit increased expression of S1P2/3 receptors and reduced lipid phosphate phosphatase expression (compared with androgen-sensitive LNCaP cells) thereby potentially increasing the bioavailability of S1P at S1P2/3 receptors.

SummaryAutophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.

Various studies in cell lines have previously demonstrated that sphingosine kinase 1 (SK1) and extracellular signal-regulated kinase 1/2 (ERK-1/2) interact in an estrogen receptor (ER)-dependent manner to influence both breast cancer cell growth and migration. A cohort of 304 ER-positive breast cancer patients was used to investigate the prognostic significance of sphingosine 1-phosphate (S1P) receptors 1, 2, and 3 (ie, S1P 1 , S1P 2 , and S1P 3 ), SK1, and ERK-1/2 expression levels. Expression levels of both SK1 and ERK-1/2 were already available for the cohort, and S1P 1 , S1P 2 , and S1P 3 levels were established by immunohistochemical analysis. High membrane S1P 1 expression was associated with shorter time to recurrence (P ‫؍‬ 0.008). High cytoplasmic S1P 1 and S1P 3 expression levels were also associated with shorter disease-specific survival times (P ‫؍‬ 0.036 and P ‫؍‬ 0.019, respectively). Those patients with tumors that expressed high levels of both cytoplasmic SK1 and ERK-1/2 had significantly shorter recurrence times than those that expressed low levels of cytoplasmic SK1 and cytoplasmic ERK-1/2 (P ‫؍‬ 0.00008), with a difference in recurrence time of 10.5 years. Similarly, high cytoplasmic S1P 1 and cytoplasmic ERK-1/2 expression levels (P ‫؍‬ 0.004) and high cytoplasmic S1P 3 expression and cytoplasmic ERK-1/2 expression levels (P ‫؍‬ 0.004) were associated with shorter recurrence times. These results support a model in which the interaction between SK1, S1P 1 , and/or S1P 3 and ERK-1/2 might drive breast cancer progression, and these findings, therefore, warrant further investigation.

No abstract

We demonstrate here a new concept termed "oncogene tolerance" whereby human EGF receptor 2 (HER2) increases sphingosine kinase 1 (SK1) expression in estrogen receptor-positive (ER ؉ ) MCF-7 HER2 cells and SK1, in turn, limits HER2 expression in a negative-feedback manner. The HER2-dependent increase in SK1 expression also limits p21-activated protein kinase 1 (p65 PAK1) and extracellular signal regulated kinase 1/2 (ERK-1/2) signaling. Sphingosine 1-phosphate signaling via S1P 3 is also altered in MCF-7 HER2 cells. In this regard, S1P binding to S1P 3 induces a migratory phenotype via an SK1-dependent mechanism in ER ؉ MCF-7 Neo cells, which lack HER2. This involves the S1P stimulated accumulation of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia and migration. In contrast, S1P failed to promote redistribution of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia or migration of MCF-7 HER2 cells. However, a migratory phenotype in these cells could be induced in response to S1P when SK1 expression had been knocked down with a specific siRNA or when recombinant PAK1 was ectopically overexpressed. Thus, the HER2-dependent increase in SK1 expression functions to desensitize the S1P-induced formation of a migratory phenotype. This is correlated with improved prognosis in patients who have a low HER1-3/SK1 expression ratio in their ER ؉ breast cancer tumors compared to patients that have a high HER1-3/SK1 expression ratio."Oncogene addiction" is a term that has been used to describe the reliance of cancer cells on the continued expression of oncogenes in order to maintain the diseased phenotype, progression, and metastasis of the cancer cell (39). Oncogene addiction is intrinsically susceptible to cross talk and feedback regulation that reflects abnormal signaling wiring in the cancer cell and which potentially makes these cells more susceptible to drug intervention at the level of the oncogene than normal cells. HER2 is a well-established oncogene that has an important role in enhancing breast cancer progression (2). The importance of its functional role as an addictive oncogene is exemplified by the fact that targeting HER2 with antibody mediated therapies, such as herceptin, demonstrates significant clinical efficacy (40). Indeed, this approach is in line with the concept that oncogene addiction is the "Achilles heel" of the cancer cell.There are four members of the human epidermal growth factor (EGF) receptor-related family, termed HER1 to HER4. The HER2/neu (c-erbB-2) gene encodes a 185-kDa transmembrane receptor tyrosine kinase, which is similar in amino acid sequence to other members of the EGF receptor family (30). Moreover, the overexpression of HER2/neu and gene amplification is found in up to 30% of primary breast cancers, and its expression is correlated with increased tumor invasion, poor prognosis, and therapeutic resistance (2). Overexpression of HER2 is also associated with downregulation of the estrogen receptor (ER) but not necessarily the complete elimination of...

(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the errorfree DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.autophagy | DNA repair | cell death | synthetic lethality T he preservation of genome integrity is critical for the prevention of human disease. In addition, the maintenance of proteome integrity is also considered central to healthy cellular homeostasis. Macroautophagy, hereafter referred to as autophagy, is a process that is paramount in counteracting damage to cytoplasmic constituents (1). Upon initiation of autophagy, double-membraned vesicles termed "autophagosomes" form to encapsulate cargoes including damaged or misfolded proteins and organelles. These vesicles ultimately fuse with lysosomes and the acidic hydrolases provided by the lysosome degrade cargoes into constituent parts, which can be recycled into biosynthetic pathways or in some situations, further catabolized to produce energy for the cell (1). Autophagy functions at basal levels in virtually all cells and is a major mechanism for protein turnover and the only known mechanism for degradation of organelles (1). Due to its crucial role in maintaining cytoplasmic and therefore cellular homeostasis, perturbations in autophagy have been reported to be an important contributing factor in a spectrum of diseases, including Crohn's disease, lysosomal storage disorders, neurodegenerative diseases, and cancer (2-6).Autophagy operates in the cytoplasm and yet studies have shown that autophagy-deficient cells accumulate DNA damage (5). The reasons behind this observation, however, are not completely clear. Because the cellular environment of autophagy-deficient cells will cause accrual of damaged proteins with abnormal function and as a result accumulation of reactive oxygen species, it is easily conceivable that this will ultimately lead to a higher incidence of genetic lesions. However, even when autophagy is competent, our cells are already subject to an extremely high frequency of spontaneous DNA damage. The fact that this damage does not persist is ...

There is an increasing body of evidence demonstrating a critical role for the bioactive lipid S1P (sphingosine 1-phosphate) in cancer. S1P is synthesized and metabolized by a number of enzymes, including sphingosine kinase, S1P lyase and S1P phosphatases. S1P binds to cell-surface G-protein-coupled receptors (S1P1-S1P5) to elicit cell responses and can also regulate, by direct binding, a number of intracellular targets such as HDAC (histone deacetylase) 1/2 to induce epigenetic regulation. S1P is involved in cancer progression including cell transformation/oncogenesis, cell survival/apoptosis, cell migration/metastasis and tumour microenvironment neovascularization. In the present paper, we describe our research findings regarding the correlation of sphingosine kinase 1 and S1P receptor expression in tumours with clinical outcome and we define some of the molecular mechanisms underlying the involvement of sphingosine kinase 1 and S1P receptors in the formation of a cancer cell migratory phenotype. The role of sphingosine kinase 1 in the acquisition of chemotherapeutic resistance and the interaction of S1P receptors with oncogenes such as HER2 is also reviewed. We also discuss novel aspects of the use of small-molecule inhibitors of sphingosine kinase 1 in terms of allosterism, ubiquitin-proteasomal degradation of sphingosine kinase 1 and anticancer activity. Finally, we describe how S1P receptor-modulating agents abrogate S1P receptor-receptor tyrosine kinase interactions, with potential to inhibit growth-factor-dependent cancer progression.