Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking.

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.

Ceramide synthase 2-C24:1 -ceramide axis limits the metastatic potential of ovarian cancer cells.

Regulation of sphingolipid metabolism plays a role in cellular homeostasis, and dysregulation of these pathways is involved in cancer progression. Previously, our reports identified ceramide as an anti-metastatic lipid. In the present study, we investigated the biochemical alterations in ceramide-centered metabolism of sphingolipids that were associated with metastatic potential. We established metastasis-prone sublines of SKOV3 ovarian cancer cells using an in vivo selection method. These cells showed decreases in ceramide levels and ceramide synthase (CerS) 2 expression. Moreover, CerS2 downregulation in ovarian cancer cells promoted metastasis in vivo and potentiated cell motility and invasiveness. Moreover, CerS2 knock-in suppressed the formation of lamellipodia required for cell motility in this cell line. In order to define specific roles of ceramide species in cell motility controlled by CerS2, the effect of exogenous long- and very long-chain ceramide species on the formation of lamellipodia was evaluated. Treatment with distinct ceramides increased cellular ceramides and had inhibitory effects on the formation of lamellipodia. Interestingly, blocking the recycling pathway of ceramides by a CerS inhibitor was ineffective in the suppression of exogenous C24:1 -ceramide for the formation of lamellipodia. These results suggested that C24:1 -ceramide, a CerS2 metabolite, predominantly suppresses the formation of lamellipodia without the requirement for deacylation/reacylation. Moreover, knockdown of neutral ceramidase suppressed the formation of lamellipodia concomitant with upregulation of C24:1 -ceramide. Collectively, the CerS2-C24:1 -ceramide axis, which may be countered by neutral ceramidase, is suggested to limit cell motility and metastatic potential. These findings may provide insights that lead to further development of ceramide-based therapy and biomarkers for metastatic ovarian cancer.

Targeting defective sphingosine kinase 1 in Niemann-Pick type C disease with an activator mitigates cholesterol accumulation.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder arising from mutations in the cholesterol-trafficking protein NPC1 (95%) or NPC2 (5%). These mutations result in accumulation of low-density lipoprotein-derived cholesterol in late endosomes/lysosomes, disruption of endocytic trafficking, and stalled autophagic flux. Additionally, NPC disease results in sphingolipid accumulation, yet it is unique among the sphingolipidoses because of the absence of mutations in the enzymes responsible for sphingolipid degradation. In this work, we examined the cause for sphingosine and sphingolipid accumulation in multiple cellular models of NPC disease and observed that the activity of sphingosine kinase 1 (SphK1), one of the two isoenzymes that phosphorylate sphingoid bases, was markedly reduced in both NPC1 mutant and NPC1 knockout cells. Conversely, SphK1 inhibition with the isotype-specific inhibitor SK1-I in WT cells induced accumulation of cholesterol and reduced cholesterol esterification. Of note, a novel SphK1 activator (SK1-A) that we have characterized decreased sphingoid base and complex sphingolipid accumulation and ameliorated autophagic defects in both NPC1 mutant and NPC1 knockout cells. Remarkably, in these cells, SK1-A also reduced cholesterol accumulation and increased cholesterol ester formation. Our results indicate that a SphK1 activator rescues aberrant cholesterol and sphingolipid storage and trafficking in NPC1 mutant cells. These observations highlight a previously unknown link between SphK1 activity, NPC1, and cholesterol trafficking and metabolism.

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation.

Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I-type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the "DK switch." Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.

Long-chain acyl-CoA synthetase 1 interacts with key proteins that activate and direct fatty acids into niche hepatic pathways.

Fatty acid channeling into oxidation or storage modes depends on physiological conditions and hormonal signaling. However, the directionality of this channeling may also depend on the association of each of the five acyl-CoA synthetase isoforms with specific protein partners. Long-chain acyl-CoA synthetases (ACSLs) catalyze the conversion of long-chain fatty acids to fatty acyl-CoAs, which are then either oxidized or used in esterification reactions. In highly oxidative tissues, ACSL1 is located on the outer mitochondrial membrane (OMM) and directs fatty acids into mitochondria for ß-oxidation. In the liver, however, about 50% of ACSL1 is located on the endoplasmic reticulum (ER) where its metabolic function is unclear. Because hepatic fatty acid partitioning is likely to require the interaction of ACSL1 with other specific proteins, we used an unbiased protein interaction technique, BioID, to discover ACSL1-binding partners in hepatocytes. We targeted ACSL1 either to the ER or to the OMM of Hepa 1-6 cells as a fusion protein with the Escherichia coli biotin ligase, BirA*. Proteomic analysis identified 98 proteins that specifically interacted with ACSL1 at the ER, 55 at the OMM, and 43 common to both subcellular locations. We found subsets of peroxisomal and lipid droplet proteins, tethering proteins, and vesicle proteins, uncovering a dynamic role for ACSL1 in organelle and lipid droplet interactions. Proteins involved in lipid metabolism were also identified, including acyl-CoA-binding proteins and ceramide synthase isoforms 2 and 5. Our results provide fundamental and detailed insights into protein interaction networks that control fatty acid metabolism.

Decreased ceramide underlies mitochondrial dysfunction in Charcot-Marie-Tooth 2F.

Charcot-Marie-Tooth (CMT) disease is the most commonly inherited neurologic disorder, but its molecular mechanisms remain unclear. One variant of CMT, 2F, is characterized by mutations in heat shock protein 27 (Hsp27). As bioactive sphingolipids have been implicated in neurodegenerative diseases, we sought to determine if their dysregulation is involved in CMT. Here, we show that Hsp27 knockout mice demonstrated decreases in ceramide in peripheral nerve tissue and that the disease-associated Hsp27 S135F mutant demonstrated decreases in mitochondrial ceramide. Given that Hsp27 is a chaperone protein, we examined its role in regulating ceramide synthases (CerSs), an enzyme family responsible for catalyzing generation of the sphingolipid ceramide. We determined that CerSs colocalized with Hsp27, and upon the presence of S135F mutants, CerS1 lost its colocalization with mitochondria suggesting that decreased mitochondrial ceramides result from reduced mitochondrial CerS localization rather than decreased CerS activity. Mitochondria in mutant cells appeared larger with increased interconnectivity. Furthermore, mutant cell lines demonstrated decreased mitochondrial respiratory function and increased autophagic flux. Mitochondrial structural and functional changes were recapitulated by blocking ceramide generation pharmacologically. These results suggest that mutant Hsp27 decreases mitochondrial ceramide levels, producing structural and functional changes in mitochondria leading to neuronal degeneration.-Schwartz, N. U., Linzer, R. W., Truman, J.-P., Gurevich, M., Hannun, Y. A., Senkal, C. E., Obeid, L. M. Decreased ceramide underlies mitochondrial dysfunction in Charcot-Marie-Tooth 2F.

A novel role for ceramide synthase 6 in mouse and human alcoholic steatosis.

Perilipin 2 (PLIN2) is a lipid-droplet protein that is up-regulated in alcoholic steatosis and associated with hepatic accumulation of ceramides, bioactive lipids implicated in alcoholic liver disease pathogenesis. The specific role of ceramide synthetic enzymes in the regulation of PLIN2 and promotion of hepatocellular lipid accumulation is not well understood. We examined the effects of pharmacologic ceramide synthesis inhibition on hepatic PLIN2 expression, steatosis, and glucose and lipid homeostasis in mice with alcoholic steatosis and in ethanol-incubated human hepatoma VL17A cells. In cells, pharmacologic inhibition of ceramide synthase reduced lipid accumulation by reducing PLIN2 RNA stability. The subtype ceramide synthase (CerS)6 was specifically up-regulated in experimental alcoholic steatosis in vivo and in vitro and was up-regulated in zone 3 hepatocytes in human alcoholic steatosis. In vivo ceramide reduction by inhibition of de novo ceramide synthesis reduced PLIN2 and hepatic steatosis in alcohol-fed mice, but only de novo synthesis inhibition, not sphingomyelin hydrolysis, improved glucose tolerance and dyslipidemia. These findings implicate CerS6 as a novel regulator of PLIN2 and suggest that ceramide synthetic enzymes may promote the earliest stage of alcoholic liver disease, alcoholic steatosis.-Williams, B., Correnti, J., Oranu, A., Lin, A., Scott, V., Annoh, M., Beck, J., Furth, E., Mitchell, V., Senkal, C. E., Obeid, L., Carr, R. M. A novel role for ceramide synthase 6 in mouse and human alcoholic steatosis.

Sphingolipids in mitochondria.

Sphingolipids are bioactive lipids found in cell membranes that exert a critical role in signal transduction. In recent years, it has become apparent that sphingolipids participate in growth, senescence, differentiation and apoptosis. The anabolism and catabolism of sphingolipids occur in discrete subcellular locations and consist of a strictly regulated and interconnected network, with ceramide as the central hub. Altered sphingolipid metabolism is linked to several human diseases. Hence, an advanced knowledge of how and where sphingolipids are metabolized is of paramount importance in order to understand the role of sphingolipids in cellular functions. In this review, we provide an overview of sphingolipid metabolism. We focus on the distinct pathways of ceramide synthesis, highlighting the mitochondrial ceramide generation, transport of ceramide to mitochondria and its role in the regulation of mitochondrial-mediated apoptosis, mitophagy and implications to disease. We will discuss unanswered questions and exciting future directions. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

Tumor Necrosis Factor-α (TNFα)-induced Ceramide Generation via Ceramide Synthases Regulates Loss of Focal Adhesion Kinase (FAK) and Programmed Cell Death.

Ceramide synthases (CerS1-CerS6), which catalyze the N-acylation of the (dihydro)sphingosine backbone to produce (dihydro)ceramide in both the de novo and the salvage or recycling pathway of ceramide generation, have been implicated in the control of programmed cell death. However, the regulation of the de novo pathway compared with the salvage pathway is not fully understood. In the current study, we have found that late accumulation of multiple ceramide and dihydroceramide species in MCF-7 cells treated with TNFα occurred by up-regulation of both pathways of ceramide synthesis. Nevertheless, fumonisin B1 but not myriocin was able to protect from TNFα-induced cell death, suggesting that ceramide synthase activity is crucial for the progression of cell death and that the pool of ceramide involved derives from the salvage pathway rather than de novo biosynthesis. Furthermore, compared with control cells, TNFα-treated cells exhibited reduced focal adhesion kinase and subsequent plasma membrane permeabilization, which was blocked exclusively by fumonisin B1. In addition, exogenously added C6-ceramide mimicked the effects of TNFα that lead to cell death, which were inhibited by fumonisin B1. Knockdown of individual ceramide synthases identified CerS6 and its product C16-ceramide as the ceramide synthase isoform essential for the regulation of cell death. In summary, our data suggest a novel role for CerS6/C16-ceramide as an upstream effector of the loss of focal adhesion protein and plasma membrane permeabilization, via the activation of caspase-7, and identify the salvage pathway as the critical mechanism of ceramide generation that controls cell death.

Sustained PKCßII activity confers oncogenic properties in a phospholipase D- and mTOR-dependent manner.

Protein kinase C (PKC) is a family of serine/threonine kinases implicated in a variety of physiological processes. We have shown previously that sustained activation of the classical PKCα and PKCßII induces their phospholipase D (PLD)-dependent internalization and translocation to a subset of the recycling endosomes defined by the presence of PKC and PLD (the pericentrion), which results in significant differences in phosphorylation of PKC substrates. Here, we have investigated the biological consequences of sustained PKC activity and the involvement of PLD in this process. We find that sustained activation of PKC results in activation of the mammalian target of rapamycin (mTOR)/S6 kinase pathway in a PLD- and endocytosis-dependent manner, with both pharmacologic inhibitors and siRNA implicating the PLD2 isoform. Notably, dysregulated overexpression of PKCßII in A549 lung cancer cells was necessary for the enhanced proliferation and migration of these cancer cells. Inhibition of PKCßII with enzastaurin reduced A549 cell proliferation by >60% (48 h) and migration by >50%. These biological effects also required both PLD activity and mTOR function, with both the PLD inhibitor FIPI and rapamycin reducing cell growth by >50%. Reciprocally, forced overexpression of wild-type PKCßII, but not an F666D mutant that cannot interact with PLD, was sufficient to enhance cell growth and increase migration of noncancerous HEK cells; indeed, both properties were almost doubled when compared to vector control and PKC-F666D-overexpressing cells. Notably, this condition was also dependent on both PLD and mTOR activity. In summary, these data define a PKC-driven oncogenic signaling pathway that requires both PLD and mTOR, and suggest that inhibitors of PLD or mTOR would be beneficial in cancers where PKC overexpression is a contributing or driving factor.

Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy.

Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C(18)-pyridinium ceramide treatment or endogenous C(18)-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C(18)-ceramide-induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 ß-lipidation, forming 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- and C(18)-ceramide-dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a new receptor function of ceramide for anchoring LC3B-II autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.

Sphingosine kinase-1 and sphingosine 1-phosphate receptor 2 mediate Bcr-Abl1 stability and drug resistance by modulation of protein phosphatase 2A.

The mechanisms by which sphingosine kinase-1 (SK-1)/sphingosine 1-phosphate (S1P) activation contributes to imatinib resistance in chronic myeloid leukemia (CML) are unknown. We show herein that increased SK-1/S1P enhances Bcr-Abl1 protein stability, through inhibition of its proteasomal degradation in imatinib-resistant K562/IMA-3 and LAMA-4/IMA human CML cells. In fact, Bcr-Abl1 stability was enhanced by ectopic SK-1 expression. Conversely, siRNA-mediated SK-1 knockdown in K562/IMA-3 cells, or its genetic loss in SK-1(-/-) MEFs, significantly reduced Bcr-Abl1 stability. Regulation of Bcr-Abl1 by SK-1/S1P was dependent on S1P receptor 2 (S1P2) signaling, which prevented Bcr-Abl1 dephosphorylation, and degradation via inhibition of PP2A. Molecular or pharmacologic interference with SK-1/S1P2 restored PP2A-dependent Bcr-Abl1 dephosphorylation, and enhanced imatinib- or nilotinib-induced growth inhibition in primary CD34(+) mononuclear cells obtained from chronic phase and blast crisis CML patients, K562/IMA-3 or LAMA4/IMA cells, and 32Dcl3 murine progenitor cells, expressing the wild-type or mutant (Y253H or T315I) Bcr-Abl1 in situ. Accordingly, impaired SK-1/S1P2 signaling enhanced the growth-inhibitory effects of nilotinib against 32D/T315I-Bcr-Abl1-derived mouse allografts. Since SK-1/S1P/S1P2 signaling regulates Bcr-Abl1 stability via modulation of PP2A, inhibition of SK-1/S1P2 axis represents a novel approach to target wild-type- or mutant-Bcr-Abl1 thereby overcoming drug resistance.

Cholesterol-dependent homeostatic regulation of very long chain sphingolipid synthesis.

Sphingomyelin plays a key role in cellular cholesterol homeostasis by binding to and sequestering cholesterol in the plasma membrane. We discovered that synthesis of very long chain (VLC) sphingomyelins is inversely regulated by cellular cholesterol levels; acute cholesterol depletion elicited a rapid induction of VLC-sphingolipid synthesis, increased trafficking to the Golgi apparatus and plasma membrane, while cholesterol loading reduced VLC-sphingolipid synthesis. This sphingolipid-cholesterol metabolic axis is distinct from the sterol responsive element binding protein pathway as it requires ceramide synthase 2 (CerS2) activity, epidermal growth factor receptor signaling, and was unaffected by inhibition of protein translation. Depletion of VLC-ceramides reduced plasma membrane cholesterol content, reduced plasma membrane lipid packing, and unexpectedly resulted in the accumulation of cholesterol in the cytoplasmic leaflet of the lysosome membrane. This study establishes the existence of a cholesterol-sphingolipid regulatory axis that maintains plasma membrane lipid homeostasis via regulation of sphingomyelin synthesis and trafficking.

The heat shock protein Hsp27 controls mitochondrial function by modulating ceramide generation.

Sphingolipids have key functions in membrane structure and cellular signaling. Ceramide is the central molecule of the sphingolipid metabolism and is generated by ceramide synthases (CerS) in the de novo pathway. Despite their critical function, mechanisms regulating CerS remain largely unknown. Using an unbiased proteomics approach, we find that the small heat shock protein 27 (Hsp27) interacts specifically with CerS1 but not other CerS. Functionally, our data show that Hsp27 acts as an endogenous inhibitor of CerS1. Wild-type Hsp27, but not a mutant deficient in CerS1 binding, inhibits CerS1 activity. Additionally, silencing of Hsp27 enhances CerS1-generated ceramide accumulation in cells. Moreover, phosphorylation of Hsp27 modulates Hsp27-CerS1 interaction and CerS1 activity in acute stress-response conditions. Biologically, we show that Hsp27 knockdown impedes mitochondrial function and induces lethal mitophagy in a CerS1-dependent manner. Overall, we identify an important mode of CerS1 regulation and CerS1-mediated mitophagy through protein-protein interaction with Hsp27.

Alteration of ceramide synthase 6/C16-ceramide induces activating transcription factor 6-mediated endoplasmic reticulum (ER) stress and apoptosis via perturbation of cellular Ca2+ and ER/Golgi membrane network.

Mechanisms that regulate endoplasmic reticulum (ER) stress-induced apoptosis in cancer cells remain enigmatic. Recent data suggest that ceramide synthase1-6 (CerS1-6)-generated ceramides, containing different fatty acid chain lengths, might exhibit distinct and opposing functions, such as apoptosis versus survival in a context-dependent manner. Here, we investigated the mechanisms involved in the activation of one of the major ER stress response proteins, ATF-6, and subsequent apoptosis by alterations of CerS6/C(16)-ceramide. Induction of wild type (WT), but not the catalytically inactive mutant CerS6, increased tumor growth in SCID mice, whereas siRNA-mediated knockdown of CerS6 induced ATF-6 activation and apoptosis in multiple human cancer cells. Down-regulation of CerS6/C(16)-ceramide, and not its further metabolism to glucosylceramide or sphingomyelin, activated ATF-6 upon treatment with ER stress inducers tunicamycin or SAHA (suberoylanilide hydroxamic acid). Induction of WT-CerS6 expression, but not its mutant, or ectopic expression of the dominant-negative mutant form of ATF-6 protected cells from apoptosis in response to CerS6 knockdown and tunicamycin or SAHA treatment. Mechanistically, ATF-6 activation was regulated by a concerted two-step process involving the release of Ca(2+) from the ER stores ([Ca(2+)](ER)), which resulted in the fragmentation of Golgi membranes in response to CerS6/C(16)-ceramide alteration. This resulted in the accumulation of pro-ATF-6 in the disrupted ER/Golgi membrane network, where pro-ATF6 is activated. Accordingly, ectopic expression of a Ca(2+) chelator calbindin prevented the Golgi fragmentation, ATF-6 activation, and apoptosis in response to CerS6/C(16)-ceramide down-regulation. Overall, these data suggest a novel mechanism of how CerS6/C(16)-ceramide alteration activates ATF6 and induces ER-stress-mediated apoptosis in squamous cell carcinomas.

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 C(18)- and C(16)-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/C(16)-ceramide using small interfering RNA induced endoplasmic reticulum (ER)-stress-mediated apoptosis. Reconstitution of C(16)-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 sphingolipid metabolism showed that generation of C(16)-ceramide, and not dihydro-C(16)-ceramide, by induced expression of CerS6 rescued cells from ER stress and apoptosis. Mechanistically, regulation of ER-stress-induced apoptosis by CerS6/C(16)-ceramide was linked to the activation of a specific arm, ATF6/CHOP, of the unfolded protein response pathway. Notably, while expression of CerS1/C(18)-ceramide inhibited HNSCC xenograft growth, CerS6/C(16)-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 prosurvival role of CerS6/C(16)-ceramide involved in the protection against ER-stress-induced apoptosis and induction of HNSCC tumor growth.

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 ceramide-mediated 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.

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.

Mechanisms of ceramide-mediated repression of the human telomerase reverse transcriptase promoter via deacetylation of Sp3 by histone deacetylase 1.

In this study, distinct roles of de novo-generated endogenous ceramides and mechanisms by which deacetylated Sp3 regulates the hTERT promoter activity in response to ceramide signaling were explored. The generation of C18-ceramide via the expression of ceramide synthase 1 (CerS1), and not C16-ceramide by CerS5 or CerS6 expression, resulted in repression of the hTERT promoter via deacetylation of Sp3 by histone deacetylase 1 (HDAC1) in A549 human lung adenocarcinoma cells. Then roles and mechanisms of action of ceramide-mediated deacetylation of Sp3 in inhibiting the hTERT promoter were determined using constitutively deacetylated or acetylated Sp3 mutants at lysine (K) 551. Expression of the deacetylated Sp3 mutant resulted in repression, whereas its acetylated mutant induced basal hTERT promoter activity in Drosophila S2 cells, which do not express any endogenous Sp3, and in A549 cells. Remarkably, chromatin immunoprecipitation data revealed that acetylated Sp3 mutant (K551Q-Sp3) did not bind whereas deacetylated Sp3 (K551R-Sp3) mutant bound strongly to the promoter DNA, resulting in the recruitment of histone deacetylase 1 (HDAC1) and inhibition of the association of RNA polymerase II with the promoter. Mechanistically, increased generation of C18-ceramide by hCerS1 expression, but not by its catalytically inactive mutant, mediated the association and recruitment of the deacetylated Sp3/HDAC1 complex to the hTERT promoter DNA, resulting in the local histone H3 deacetylation and repression of the promoter.

Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas.

In this study, quantitative isobologram studies showed that treatment with gemcitabine and doxorubicin, known inducers of ceramide generation, in combination, supra-additively inhibited the growth of human UM-SCC-22A cells in situ. Then, possible involvement of the human homologue of yeast longevity assurance gene 1 (LASS1)/C(18)-ceramide in chemotherapy-induced cell death in these cells was examined. Gemcitabine/doxorubicin combination treatment resulted in the elevation of mRNA and protein levels of LASS1 and not LASS2-6, which was consistent with a 3.5-fold increase in the endogenous (dihydro)ceramide synthase activity of LASS1 for the generation of C(18)-ceramide. Importantly, the overexpression of LASS1 (both human and mouse homologues) enhanced the growth-inhibitory effects of gemcitabine/doxorubicin with a concomitant induction of caspase-3 activation. In reciprocal experiments, partial inhibition of human LASS1 expression using small interfering RNA (siRNA) prevented cell death by about 50% in response to gemcitabine/doxorubicin. In addition, LASS1, and not LASS5, siRNA modulated the activation of caspase-3 and caspase-9, but not caspase-8, in response to this combination. Treatment with gemcitabine/doxorubicin in combination also resulted in a significant suppression of the head and neck squamous cell carcinoma (HNSCC) tumor growth in severe combined immunodeficiency mice bearing the UM-SCC-22A xenografts. More interestingly, analysis of endogenous ceramide levels in these tumors by liquid chromatography/mass spectroscopy showed that only the levels of C(18)-ceramide, the main product of LASS1, were elevated significantly (about 7-fold) in response to gemcitabine/doxorubicin when compared with controls. In conclusion, these data suggest an important role for LASS1/C(18)-ceramide in gemcitabine/doxorubicin-induced cell death via the activation of caspase-9/3 in HNSCC.