Besim Ogretmen

Biologically active sphingolipids in cancer pathogenesis and treatment

Biologically active sphingolipids have key roles in the regulation of several fundamental biological processes that are integral to cancer pathogenesis. Recent significant progress in understanding biologically active sphingolipid synthesis, specifically within ceramide and sphingosine-1-phosphate (S1P)-mediated pathways, has identified crucial roles for these molecules both in cancer development and progression. Ceramide — a central molecule in sphingolipid metabolism — in effect functions as a tumour-suppressor lipid, inducing antiproliferative and apoptotic responses in various cancer cells. Conversely, S1P induces responses that, on aggregate, render S1P a tumour-promoting lipid. These discoveries are paving the way for the advancement of anticancer therapies.

De Novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells.Dependence on protein phosphatase-1

Previous studies have demonstrated that several splice variants are derived from both the caspase 9 andBcl-x genes in which the Bcl-x splice variant, Bcl-x(L) and the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9. In a recent study, we showed that ceramide induces the dephosphorylation of SR proteins, a family of protein factors that regulate alternative splicing. In this study, the regulation of the alternative processing of pre-mRNA of both caspase 9 and Bcl-x(L) was examined in response to ceramide. Treatment of A549 lung adenocarcinoma cells with cell-permeable ceramide, D-e-C6 ceramide, down-regulated the levels of Bcl-x(L) and caspase 9b mRNA and immunoreactive protein with a concomitant increase in the mRNA and immunoreactive protein levels of Bcl-x(s) and caspase 9 in a dose- and time-dependent manner. Pretreatment with calyculin A (5 nm), an inhibitor of protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A) blocked ceramide-induced alternative splicing in contrast to okadaic acid (10 nm), a specific inhibitor of PP2A at this concentrations in cells, demonstrating a PP1-mediated mechanism. A role for endogenous ceramide in regulating the alternative splicing of caspase 9 and Bcl-x was demonstrated using the chemotherapeutic agent, gemcitabine. Treatment of A549 cells with gemcitabine (1 μm) increased ceramide levels 3-fold via the de novo sphingolipid pathway as determined by pulse labeling experiments and inhibition studies with myriocin (50 nm), a specific inhibitor of serine palmitoyltransferase (the first step in de novo synthesis of ceramide). Treatment of A549 cells with gemcitabine down-regulated the levels of Bcl-x(L) and caspase 9b mRNA with a concomitant increase in the mRNA levels of Bcl-x(s) and caspase 9. Again, inhibitors of ceramide synthesis blocked this effect. We also demonstrate that the change in the alternative splicing of caspase 9 and Bcl-x occurred prior to apoptosis following treatment with gemcitabine. Furthermore, doses of D-e-C6 ceramide that induce the alternative splicing of both caspase 9 and Bcl-x-sensitized A549 cells to daunorubicin. These data demonstrate a role for protein phosphatases 1 (PP1) and endogenous ceramide generated via the de novopathway in regulating this mechanism. This is the first report on the dynamic regulation of RNA splicing of members of the Bcl-2 and caspase families in response to regulators of apoptosis.

Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy

Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C18-pyridinium ceramide (C18-Pyr-Cer) treatment, or endogenous C18-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C18-ceramide-induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 beta lipidation (LC3B-II) and selective targeting of mitochondria by LC3B-II-containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite-induced mitophagy, and stable LC3B knockdown protected against CerS1-C18-ceramide-dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a novel receptor function of ceramide for anchoring LC3B-II-autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.

Roles of Bioactive Sphingolipids in Cancer Biology and Therapeutics

In this chapter, roles of bioactive sphingolipids in the regulation of cancer pathogenesis and therapy will be reviewed. Sphingolipids have emerged as bioeffector molecules, which control various aspects of cell growth, proliferation, and anti-cancer therapeutics. Ceramide, the central molecule of sphingolipid metabolism, generally mediates anti-proliferative responses such as inhibition of cell growth, induction of apoptosis, and/or modulation of senescence. On the other hand, sphingosine 1-phosphate (S1P) plays opposing roles, and induces transformation, cancer cell growth, or angiogenesis. A network of metabolic enzymes regulates the generation of ceramide and S1P, and these enzymes serve as transducers of sphingolipid-mediated responses that are coupled to various exogenous or endogenous cellular signals. Consistent with their key roles in the regulation of cancer growth and therapy, attenuation of ceramide generation and/or increased S1P levels are implicated in the development of resistance to drug-induced apoptosis, and escape from cell death. These data strongly suggest that advances in the molecular and biochemical understanding of sphingolipid metabolism and function will lead to the development of novel therapeutic strategies against human cancers, which may also help overcome drug resistance.

Sphingolipids and cancer: ceramide and sphingosine-1-phosphate in the regulation of cell death and drug resistance

Sphingolipids have emerged as bioeffector molecules, controlling various aspects of cell growth and proliferation in cancer, which is becoming the deadliest disease in the world. These lipid molecules have also been implicated in the mechanism of action of cancer chemotherapeutics. Ceramide, the central molecule of sphingolipid metabolism, generally mediates antiproliferative responses, such as cell growth inhibition, apoptosis induction, senescence modulation, endoplasmic reticulum stress responses and/or autophagy. Interestingly, recent studies suggest de novo-generated ceramides may have distinct and opposing roles in the promotion/suppression of tumors, and that these activities are based on their fatty acid chain lengths, subcellular localization and/or direct downstream targets. For example, in head and neck cancer cells, ceramide synthase 6/C(16)-ceramide addiction was revealed, and this was associated with increased tumor growth, whereas downregulation of its synthesis resulted in ER stress-induced apoptosis. By contrast, ceramide synthase 1-generated C(18)-ceramide has been shown to suppress tumor growth in various cancer models, both in situ and in vivo. In addition, ceramide metabolism to generate sphingosine-1-phosphate (S1P) by sphingosine kinases 1 and 2 mediates, with or without the involvement of G-protein-coupled S1P receptor signaling, prosurvival, angiogenesis, metastasis and/or resistance to drug-induced apoptosis. Importantly, recent findings regarding the mechanisms by which sphingolipid metabolism and signaling regulate tumor growth and progression, such as identifying direct intracellular protein targets of sphingolipids, have been key for the development of new chemotherapeutic strategies. Thus, in this article, we will present conclusions of recent studies that describe opposing roles of de novo-generated ceramides by ceramide synthases and/or S1P in the regulation of cancer pathogenesis, as well as the development of sphingolipid-based cancer therapeutics and drug resistance.

Alterations of Ceramide/Sphingosine 1-Phosphate Rheostat Involved in the Regulation of Resistance to Imatinib-induced Apoptosis in K562 Human Chronic Myeloid Leukemia Cells

In this study, mechanisms of resistance to imatinib-induced apoptosis in human K562 cells were examined. Continuous exposure to stepwise increasing concentrations of imatinib resulted in the selection of K562/IMA-0.2 and -1 cells, which expressed ∼2.3- and 19-fold resistance, respectively. Measurement of endogenous ceramides by high performance liquid chromatography/mass spectroscopy showed that treatment with imatinib increased the generation of ceramide, mainly C18-ceramide, which is generated by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Inhibition of hLASS1 by small interfering RNA partially prevented imatinib-induced cell death in sensitive cells. In reciprocal experiments, overexpression of hLASS1, and not hLASS6, in drug-resistant cells caused a marked increase in imatinib-induced C18-ceramide generation, and enhanced apoptosis. Interestingly, there were no defects in the levels of mRNA and enzyme activity levels of hLASS1 for ceramide generation in K562/IMA-1 cells. However, expression levels of sphingosine kinase-1 (SK1) and generation of sphingosine 1-phosphate (S1P) were increased significantly in K562/IMA-1 cells, channeling sphingoid bases to the sphingosine kinase pathway. The partial inhibition of SK1 expression by small interference RNA modulated S1P levels and increased sensitivity to imatinib-induced apoptosis in resistant cells. On the other hand, forced expression of SK1 in K562 cells increased the ratio between total S1P/C18-ceramide levels ∼6-fold and prevented apoptosis significantly in response to imatinib. Additional data indicated a role for SK1/S1P signaling in the up-regulation of the Bcr-Abl expression at the post-transcriptional level, which suggested a possible mechanism for resistance to imatinib-mediated apoptosis. In conclusion, these data suggest a role for endogenous C18-ceramide synthesis mainly via hLASS1 in imatinib-induced apoptosis in sensitive cells, whereas in resistant cells, alterations of the balance between the levels of ceramide and S1P by overexpression of SK1 result in resistance to imatinib-induced apoptosis.

Biochemical Mechanisms of the Generation of Endogenous Long Chain Ceramide in Response to Exogenous Short Chain Ceramide in the A549 Human Lung Adenocarcinoma Cell Line ROLE FOR ENDOGENOUS CERAMIDE IN MEDIATING THE ACTION OF EXOGENOUS CERAMIDE

Treatment of A549 cells with C6-ceramide resulted in a significant increase in the endogenous long chain ceramide levels, which was inhibited by fumonisin B1 (FB1), and not by myriocin (MYR). The biochemical mechanisms of generation of endogenous ceramide were investigated using A549 cells treated with selectively labeled C6-ceramides, [sphingosine-3-3H]d-erythro-, andN-[N-hexanoyl-1-14C]d-erythro-C6-ceramide. The results demonstrated that 3H label was incorporated into newly synthesized long chain ceramides, which was inhibited by FB1 and not by MYR. Interestingly, the 14C label was not incorporated into long chain ceramides. Taken together, these results show that generation of endogenous ceramide in response to C6-ceramide is due to recycling of the sphingosine backbone of C6-ceramide via deacylation/reacylation and not due to the elongation of its fatty acid moiety. Moreover, the generation of endogenous long chain ceramide in response to C6-ceramide was completely blocked by brefeldin A, which causes Golgi disassembly, suggesting a role for the Golgi in the metabolism of ceramide. In addition, the generation of endogenous ceramide in response to short chain exogenous ceramide was induced byd-erythro- but notl-erythro-C6-ceramide, demonstrating the stereospecificity of this process. Interestingly, several key downstream biological activities of ceramide, such as growth inhibition, cell cycle arrest, and modulation of telomerase activity were induced byd-erythro-C6-ceramide, and notl-erythro-C6-ceramide (and inhibited by FB1) in A549 cells, suggesting a role for endogenous long chain ceramide in the regulation of these responses.

Antiapoptotic roles of ceramide-synthase-6-generated C16-ceramide via selective regulation of the ATF6/CHOP arm of ER-stress-response pathways

Emerging results suggest that ceramides with different fatty acid chain lengths might play distinct functions in the regulation of tumor growth and therapy. Here we report that de novo‐generated C18‐ and C16‐ceramides by ceramide synthases 1 and 6 (CerS1 and CerS6) play opposing proapoptotic and prosurvival roles, respectively, in human head and neck squamous cell carcinomas (HNSCCs). Unexpectedly, knockdown of CerS6/C16‐ceramide using small interfering RNA induced endoplasmic reticulum (ER)‐stress‐mediated apoptosis. Reconstitution of C16‐ceramide generation by induced expression of wild‐type CerS6, but not its catalytically inactive mutant, protected cells from cell death induced by knockdown of CerS6. Moreover, using molecular tools coupled with analysis of sphingo‐lipid metabolism showed that generation of C16‐cer‐amide, and not dihydro‐C16‐ceramide, by induced expression of CerS6 rescued cells from ER stress and apoptosis. Mechanistically, regulation of ER‐stress‐in‐duced apoptosis by CerS6/C16‐ceramide was linked to the activation of a specific arm, ATF6/CHOP, of the unfolded protein response pathway. Notably, while expression of CerS1/C18‐ceramide inhibited HNSCC xenograft growth, CerS6/C16‐ceramide significantly protected ER stress, leading to enhanced tumor development and growth in vivo, consistent with their pro‐ and antiapoptotic roles, respectively. Thus, these data reveal an unexpected and novel prosur‐vival role of CerS6/C16‐ceramide involved in the protection against ER‐stress‐induced apoptosis and induction of HNSCC tumor growth.—Senkal, C. E., Ponnusamy, S., Bielawski, J., Hannun, Y. A., Ogret‐men, B. Antiapoptotic roles of ceramide‐synthase‐6‐generated C16‐ceramide via selective regulation of the ATF6/CHOP arm of ER‐stress‐response pathways. FASEB J. 24, 296–308 (2010). www.fasebj.org

Immunohistochemical Distribution of Sphingosine Kinase 1 in Normal and Tumor Lung Tissue

Sphingosine kinase 1 (SK1) is a key enzyme critical to the sphingolipid metabolic pathway responsible for catalyzing the formation of the bioactive lipid sphingosine-1-phosphate. SK1-mediated production of sphingosine-1-phosphate has been shown to stimulate such biological processes as cell growth, differentiation, migration, angiogenesis, and inhibition of apoptosis. In this study, cell type–specific immunolocalization of SK1 was examined in the bronchus/terminal bronchiole of the lung. Strong immunopositive staining was evident at the apical surface of pseudostratified epithelial cells of the bronchus and underlying smooth muscle cells, submucosal serous glands, immature chondrocytes, type II alveolar cells, foamy macrophages, endothelial cells of blood vessels, and neural bundles. Immunohistochemical screening for SK1 expression was performed in 25 samples of normal/tumor patient matched non–small-cell lung cancer tissue and found that 25 of 25 tumor samples (carcinoid [5 samples], squamous [10 samples], and adenocarcinoma tumors [10 samples]), exhibited overwhelmingly positive immunostaining for SK1 as compared with patient-matched normal tissue. In addition, an approximately 2-fold elevation of SK1 mRNA expression was observed in lung cancer tissue versus normal tissue, as well as in several other solid tumors. Taken together, these findings define the localization of SK1 in lung and provide clues as to how SK1 may play a role in normal lung physiology and the pathophysiology of lung cancer.

Direct interaction between the inhibitor 2 and ceramide via sphingolipid-protein binding is involved in the regulation of protein phosphatase 2A activity and signaling

In this study, the inhibitor 2 of protein phosphatase 2A (I2PP2A) was identified in vitro and in situ as a ceramide‐binding protein, which exhibits stereoisomer specificity and fatty acid chain length preference. Site‐directed mutagenesis coupled with structural details of I2PP2A suggested that VIK 207‐209 residues localized on helix 7 are important for ceramide binding and single mutation of K209D altered this interaction. Notably, I2PP2A‐ceramide binding decreased the association between PP2A and the inhibitor, preventing the inhibition of PP2A activity in vitro. In addition, studies in A549 human lung cancer cells revealed that ceramide mediates c‐Myc degradation via its PP2A‐dependent dephosphorylation at S62, and treatment with okadaic acid and expression of c‐Myc mutants with S62A or S62D conversions resulted in resistance to ceramidemediated degradation. Importantly, whereas down‐regulation of I2PP2A enhanced PP2A‐mediated c‐Myc degradation in response to ceramide, ectopic expression of wild‐type I2PP2A but not of its K209D mutant protected this degradation in A549 cells. Moreover, expression of wild‐type I2PP2A prevented the growth‐inhibitory effects of ceramide both against A549 cells and xenograft‐driven tumors in situ and in vivo compared with that in controls. Thus, these results suggest that direct interaction of I2PP2A with ceramide plays important biological roles via the regulation of PP2A activity and signaling, which in turn control ceramide‐mediated degradation of c‐Myc and antiproliferation.— Mukhopadhyay, A., Saddoughi, S. A., Song, P., Sultan, I., Ponnusamy, S., Senkal, C. E., Snook, C. F., Arnold, H. K., Sears, R. C., Hannun, Y. A., Ogretmen, B. Direct interaction between the inhibitor 2 and ceramide via sphingolipid‐protein binding is involved in the regulation of protein phosphatase 2A activity and signaling. FASEB J. 23, 751–763 (2009)