Can E. Senkal

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.

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.

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

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)

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)/C18-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 C18-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 C18-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/C18-ceramide in gemcitabine/doxorubicin-induced cell death via the activation of caspase-9/3 in HNSCC. [Mol Cancer Ther 2007;6(2):712–22]

Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis

Mechanisms that alter protein phosphatase 2A (PP2A)‐dependent lung tumour suppression via the I2PP2A/SET oncoprotein are unknown. We show here that the tumour suppressor ceramide binds I2PP2A/SET selectively in the nucleus and including its K209 and Y122 residues as determined by molecular modelling/simulations and site‐directed mutagenesis. Because I2PP2A/SET was found overexpressed, whereas ceramide was downregulated in lung tumours, a sphingolipid analogue drug, FTY720, was identified to mimick ceramide for binding and targeting I2PP2A/SET, leading to PP2A reactivation, lung cancer cell death, and tumour suppression in vivo. Accordingly, while molecular targeting of I2PP2A/SET by stable knockdown prevented further tumour suppression by FTY720, reconstitution of WT‐I2PP2A/SET expression restored this process. Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1‐dependent programmed necrosis (necroptosis), but not by apoptosis. The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720. Expression of WT‐ or death‐domain‐deleted (DDD)‐RIPK1, but not the kinase‐domain‐deleted (KDD)‐RIPK1, restored FTY720‐mediated necroptosis in RIPK1−/− MEFs. Thus, these data suggest that targeting I2PP2A/SET by FTY720 suppresses lung tumour growth, at least in part, via PP2A activation and necroptosis mediated by the kinase domain of RIPK1.

Ceramide synthase 6 modulates TRAIL sensitivity and nuclear translocation of active caspase 3 in colon cancer cells

We have previously shown that the death receptor ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces an increase of intracellular C16-ceramide in sensitive SW480 but not in resistant SW620 cells. Resistance in SW620 cells was overcome by exogenous ceramide, leading us to propose that defective ceramide signaling contributes to TRAIL resistance. In this study we found that the increase in C16-ceramide in SW480 cells was inhibited by fumonisin B1, an inhibitor of ceramide synthases (CerS). Protein analysis revealed that TRAIL-resistant SW620 cells expressed lower levels of ceramide synthase 6 (CerS6, also known as longevity assurance homologue 6), which prompted us to investigate the effect of CerS6 modulation on TRAIL phenotype. RNAi against CerS6 resulted in a specific and significant decrease of the C16-ceramide species, which was sufficient to inhibit TRAIL-induced apoptosis. In cells with decreased levels of CerS6, caspase-3 was activated but failed to translocate into the nucleus. CerS6 localized primarily to the perinuclear region, suggesting this enzyme may be important in regulation of nuclear permeability. Moderate elevation in CerS6 expression was sufficient to reverse TRAIL resistance in SW620 cells. These results suggest that modulation of CerS6 expression may constitute a new therapeutic strategy to alter apoptotic susceptibility.

Potent Antitumor Activity of a Novel Cationic Pyridinium-Ceramide Alone or in Combination with Gemcitabine against Human Head and Neck Squamous Cell Carcinomas in Vitro and in Vivo

In this study, a cationic water-soluble ceramide analog l-threo-C6-pyridinium-ceramide-bromide (l-t-C6-Pyr-Cer), which exhibits high solubility and bioavailability, inhibited the growth of various human head and neck squamous cell carcinoma (HNSCC) cell lines at low IC50 concentrations, independent of their p53 status. Consistent with its design to target negatively charged intracellular compartments, l-t-C6-Pyr-Cer accumulated mainly in mitochondria-, and nuclei-enriched fractions upon treatment of human UM-SCC-22A cells [human squamous cell carcinoma (SCC) of the hypopharynx] at 1 to 6 h. In addition to its growth-inhibitory function as a single agent, the supra-additive interaction of l-t-C6-Pyr-Cer with gemcitabine (GMZ), a chemotherapeutic agent used in HNSCC, was determined using isobologram studies. Then, the effects of this ceramide, alone or in combination with GMZ, on the growth of UM-SCC-22A xenografts in SCID mice was assessed following the determination of preclinical parameters, such as maximum tolerated dose, clearance from the blood, and bioaccumulation. Results demonstrated that treatment with l-t-C6-Pyr-Cer in combination with GMZ significantly prevented the growth of HNSCC tumors in vivo. The therapeutic efficacy of l-t-C6-Pyr-Cer/GMZ combination against HNSCC tumors was approximately 2.5-fold better than that of the combination of 5-fluorouracil/cis-platin. In addition, liquid chromatography/mass spectroscopy analysis showed that the levels of l-t-C6-Pyr-Cer in HNSCC tumors weresignificantly higher than its levels in the liver and intestines; interestingly, the combination with GMZ increased the sustained accumulation of this ceramide by approximately 40%. Moreover, treatment with l-t-C6-Pyr-Cer/GMZ combination resulted in a significant inhibition of telomerase activity and decrease in telomere length in vivo, which are among downstream targets of ceramide.

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.