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author:("tretmen, beim")
1.  Ceramide stress in survival versus lethal autophagy paradox 
Autophagy  2013;9(2):258-259.
In a recent study, characterization of novel mechanisms that regulate lethal autophagy revealed that C18-ceramide stress mediates LC3B-II-ceramide binding on mitochondrial membranes to target autophagosomes for mitophagy induction and tumor suppression.
doi:10.4161/auto.22739
PMCID: PMC3552895  PMID: 23182807
sphingolipid; ceramide; mitophagy; mitochondrial fission; lipid-protein interaction; ceramide synthase
2.  PP2A-activating drugs selectively eradicate TKI-resistant chronic myeloid leukemic stem cells 
The Journal of Clinical Investigation  2013;123(10):4144-4157.
The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase–independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression — but not activity — of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/β-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase–independent and PP2A-mediated inhibition of JAK2 and β-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1–positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/β-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.
doi:10.1172/JCI68951
PMCID: PMC3784537  PMID: 23999433
3.  Combining histone deacetylase inhibitors with MDA-7/IL-24 enhances killing of renal carcinoma cells 
Cancer Biology & Therapy  2013;14(11):1039-1049.
In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca2+. Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda-7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.
doi:10.4161/cbt.26110
PMCID: PMC3925659  PMID: 24025359
MDA-7/IL-24; HDACI; ceramide; apoptosis; bystander; cytokine; ROS; caspase; animal study
4.  Off-Target Function of the Sonic-Hedgehog Inhibitor Cyclopamine in Mediating Apoptosis via Nitric Oxide-Dependent Neutral Sphingomyelinase 2/Ceramide Induction 
Molecular cancer therapeutics  2012;11(5):1092-1102.
Sonic hedgehog (SHh) signaling is important in the pathogenesis of various human cancers, such as medulloblastomas, and it has been identified as a valid target for anti-cancer therapeutics. The SHh inhibitor cyclopamine induces apoptosis. The bioactive sphingolipid ceramide mediates cell death in response to various chemotherapeutic agents; however, ceramide’s roles/mechanisms in cyclopamine-induced apoptosis are unknown. Here, we report that cyclopamine mediates ceramide generation selectively via induction of N-SMase2 expression in Daoy human medulloblastoma cells. Importantly, siRNA-mediated knockdown of N-SMase2 prevented cyclopamine-induced ceramide generation, and protected Daoy cells from drug-induced apoptosis. Accordingly, ectopic expression of wild type N-SMase2 caused cell death, compared to controls, expressing the catalytically inactive N-SMase2 mutant. Interestingly, knockdown of smoothened (Smo), a target protein for cyclopamine, or Gli1, a down-stream signaling transcription factor of Smo, did not affect N-SMase2 expression, or apoptosis. Mechanistically, our data showed cyclopamine induced N-SMase2 mRNA and cell death selectively via increased nitric oxide (NO) generation by neuronal-nitric oxide synthase (n-NOS) induction, in Daoy medulloblastoma, and multiple other human cancer cell lines. Accordingly, N-SMase2 activity-deficient skin fibroblasts isolated from homozygous fro/fro (fragilitas ossium) mice exhibited resistance to NO-induced cell death. Thus, our data suggest a novel off-target function of cyclopamine in inducing apoptosis, at least in part, by Nnos/NO-dependent induction of N-SMase2 expression/ceramide axis, independent of Smo/Gli inhibition.
doi:10.1158/1535-7163.MCT-11-0705
PMCID: PMC3709261  PMID: 22452947
sphingolipids; ceramide; sphingomyelin; sphingomyelinase; neutral sphingomyelinase
5.  Sorafenib and pemetrexed toxicity in cancer cells is mediated via SRC-ERK signaling 
Cancer Biology & Therapy  2012;13(9):793-803.
The present studies sought to further understand how the anti-folate pemetrexed and the multi-kinase inhibitor sorafenib interact to kill tumor cells. Sorafenib activated SRC, and via SRC the drug combination activated ERK1/2. Expression of dominant negative SRC or dominant negative MEK1 abolished drug-induced ERK1/2 activation, together with drug-induced autophagy, acidic lysosome formation, and tumor cell killing. Protein phosphatase 2A is an important regulator of the ERK1/2 pathway. Fulvestrant resistant MCF7 cells expressed higher levels of the PP2A inhibitor SET/I2PP2A, had lower endogenous PP2A activity, and had elevated basal ERK1/2 activity compared with their estrogen dependent counterparts. Overexpression of I2PP2A blocked drug-induced activation of ERK1/2 and tumor cell killing. PP2A can be directly activated by ceramide and SET/I2PP2A can be inhibited by ceramide. Inhibition of the de novo ceramide synthase pathway blocked drug-induced ceramide generation, PP2A activation and tumor cell killing. Collectively these findings demonstrate that ERK1/2 plays an essential role downstream of SRC in pemetrexed and sorafenib lethality and that PP2A plays an important role in regulating this process.
doi:10.4161/cbt.20562
PMCID: PMC3679099  PMID: 22673740
ERK; I2PP2A; PP2A; SRC; autophagy; ceramide; pemetrexed; sorafenib
6.  Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy 
Nature chemical biology  2012;8(10):831-838.
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.
doi:10.1038/nchembio.1059
PMCID: PMC3689583  PMID: 22922758
7.  OSU-03012 suppresses GRP78/BiP expression that causes PERK-dependent increases in tumor cell killing 
Cancer Biology & Therapy  2012;13(4):224-236.
We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills tumor cells. OSU lethality was suppressed by knock down of PERK and enhanced by knock down of ATF6 and IRE1α. OSU treatment suppressed expression of the chaperone, BiP/GRP78, and did so through reduced stability of the protein. Knock down of BiP/GRP78 further enhanced OSU lethality. Overexpression of BiP/GRP78 abolished OSU toxicity. Pre-treatment of cells with OSU enhanced radiosensitivity to a greater extent than concomitant or sequential drug treatment with radiation exposure. Expression of a mutant active p110 PI3K, or mutant active forms of the EGFR in GBM cells did not differentially suppress OSU killing. In contrast loss of PTEN function reduced OSU lethality, without altering AKT, p70 S6K or mTOR activity, or the drug's ability to radiosensitize GBM cells. Knock down of PTEN protected cells from OSU and radiation treatment whereas re-expression of PTEN facilitated drug lethality and radiosensitization. In a dose-dependent fashion OSU prolonged the survival of mice carrying GBM tumors and interacted with radiotherapy to further prolong survival. Collectively, our data show that reduced BiP/GRP78 levels play a key role in OSU-3012 toxicity in GBM cells, and that this drug has in vivo activity against an invasive primary human GBM isolate.
doi:10.4161/cbt.13.4.18877
PMCID: PMC3336069  PMID: 22354011
OSU-03012; BiP/GRP78; ER stress; PERK; ionizing radiation; ceramide
8.  Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis 
EMBO Molecular Medicine  2012;5(1):105-121.
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.
doi:10.1002/emmm.201201283
PMCID: PMC3569657  PMID: 23180565
ceramide; FTY720; sphingolipids; sphingosine; sphingosine kinase 2
9.  Results of a Phase II Trial of Gemcitabine plus Doxorubicin in Patients with Recurrent Head and Neck Cancers: Serum C18-ceramide as a Novel Biomarker for Monitoring Response 
Purpose
Here we report a phase II clinical trial, which was designed to test a novel hypothesis that treatment with GEM/DOX would be efficacious via reconstitution of C18-ceramide signaling in HNSCC patients for whom first-line platinum-based therapy failed.
Experimental Design
Patients received GEM (1,000 mg/m2) and DOX (25 mg/m2) on days 1 and 8, every 21 days, until disease progression. After completion of 2 treatment cycles, patients were assessed radiographically, and serum samples were taken for sphingolipid measurements.
Results
We enrolled 18 patients in the trial, who were evaluable for toxicity, and 17 for response. The most common toxicity was neutropenia, observed in 9 of 18 patients, and there were no major non-hematological toxicities. Of the 17 patients, 5 patients had progressive disease (PD), 1 had complete response (CR), 3 exhibited partial response (PR), and 8 had stable disease (SD). The median progression-free survival (PFS) was 1.6 months (95% CI, 1.4, 4.2) with a median survival of 5.6 months (95% CI, 3.8, 18.2). Remarkably, serum sphingolipid analysis revealed significant differences in patterns of C18-ceramide elevation in patients with CR/PR/SD in comparison to patients with PD, indicating the reconstitution of tumor suppressor ceramide generation by GEM/DOX treatment.
Conclusions
Our data suggest that the GEM/DOX combination could represent an effective treatment for some patients with recurrent or metastatic HNSCC, and that serum C18-ceramide elevation might be a novel serum biomarker of chemotherapy response.
doi:10.1158/1078-0432.CCR-11-0930
PMCID: PMC3176980  PMID: 21791630
Sphingolipids; ceramide; ceramide synthase; sphingosine 1-phosphate; sphingosine
10.  Communication between host organism and cancer cells is transduced by systemic sphingosine kinase 1/sphingosine 1-phosphate signalling to regulate tumour metastasis 
EMBO Molecular Medicine  2012;4(8):761-775.
Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via sphingosine 1-phosphate (S1P) generated systemically by sphingosine kinase 1 (SK1), rather than via tumour-derived S1P. Modulation of systemic, but not tumour SK1, prevented S1P elevation, and inhibited TRAMP-induced prostate cancer growth in TRAMP+/+SK1−/− mice, or lung metastasis of multiple cancer cells in SK1−/− animals. Genetic loss of SK1 activated a master metastasis suppressor, Brms1 (breast carcinoma metastasis suppressor 1), via modulation of S1P receptor 2 (S1PR2) in cancer cells. Alterations of S1PR2 using pharmacologic and genetic tools enhanced Brms1. Moreover, Brms1 in S1PR2−/− MEFs was modulated by serum S1P alterations. Accordingly, ectopic Brms1 in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms1 prevented this process. Importantly, inhibition of systemic S1P signalling using a novel anti-S1P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms1 knockdown in MB49 cells. Thus, these data suggest that systemic SK1/S1P regulates metastatic potential via regulation of tumour S1PR2/Brms1 axis.
doi:10.1002/emmm.201200244
PMCID: PMC3494075  PMID: 22707406
lung metastasis; sphingolipids; sphingomab; sphingosine kinase 1; sphingosine 1-phosphate
11.  A Role of Sphingosine Kinase 1 in head & neck carcinogenesis 
It is important to identify novel and effective targets for cancer prevention and therapy against head & neck squamous cell carcinoma (HNSCC), one of the most lethal cancers. Accumulating evidence suggests that the bioactive sphingolipids, such as sphingosine-1-phosphate (S1P) and its generating enzyme, sphingosine kinase 1 (SphK1) play pivotal roles in several important biological functions including promoting tumor growth and carcinogenesis. However, roles of SphK1/S1P in HNSCC development and/or progression have not been defined previously. Therefore, in this study, we first analyzed the expression of SphK1 in human HNSCC tumor samples and normal head & neck tissues (n=78 and 17, respectively) using immunohistochemistry. The data showed that SphK1 is overexpressed in all of the HNSCC tumors tested (stages I~IV). We next investigated whether SphK1 is necessary for HNSCC development. To define the role of SphK1/S1P in HNSCC development, we utilized 4-nitroquinoline-1-oxide (4-NQO)-induced HNSCC model in wild type mice compared to SphK1−/− knockout (KO) mice. Remarkably, we found that the genetic loss of SphK1, which reduced S1P generation, significantly prevented 4-NQO-induced HNSCC carcinogenesis, with decreased tumor incidence, multiplicity, and volume when compared to controls. Moreover, our data indicated that prevention of 4-NQO-induced HNSCC development in SphK1−/− KO mice might be associated with decreased cell proliferation, increased levels of cleaved (active) caspase 3, and down-regulation of phospho (active) AKT expression. Thus, these novel data suggest that SphK1/S1P signaling may play important roles in HNSCC carcinogenesis, and that targeting SphK1/S1P might provide a novel strategy for chemoprevention and treatment against HNSCC.
doi:10.1158/1940-6207.CAPR-10-0299
PMCID: PMC3342666  PMID: 21209394
Sphingolipids; Sphingosine kinase 1; 4-NQO-induced animal model; head & neck squamous cell carcinoma; SphK1 KO mice
12.  Concerted functions of HDAC1 and microRNA-574-5p repress alternatively spliced ceramide synthase 1 expression in human cancer cells 
EMBO Molecular Medicine  2012;4(2):78-92.
Histone deacetylases (HDACs) and microRNAs (miRs) have pro-survival roles, but the mechanism behind this is unclear. Repression of ceramide synthase 1 (CerS1), altering C18-ceramide generation, was linked to drug resistance and metastasis. Here we report that the CerS1 promoter was repressed by HDAC1-dependent inhibition of Sp1 recruitment to two specific GC-boxes spanning the −177 and −139 region. Moreover, an alternatively spliced variant CerS1 mRNA (CerS1-2) was detected mainly in cancer cells or primary tumour tissues compared to controls, which was targeted by miR-574-5p for degradation. A specific 3′UTR-targeting site, localized within the retained intron between exons 6 and 7, was identified, and its mutation, or miR-574-5p knockdown prevented the degradation of CerS1-2 mRNA. Interference with HDAC1 and miR-574-5p reconstituted CerS1-2 expression and C18-ceramide generation in multiple human cancer cell lines, which subsequently inhibited proliferation and anchorage-independent growth. Accordingly, knockdown of CerS1 partially protected cancer cells from MS-275/miR-574-5p siRNA-mediated growth inhibition. Thus, these data suggest that the HDAC1/miR-574-5p axis might provide a novel therapeutic target to reconstitute tumour suppressor CerS1/ceramide signalling.
doi:10.1002/emmm.201100189
PMCID: PMC3376837  PMID: 22180294
ceramide synthase 1; lipid signalling; sphingolipids; sphingosine; sphingosine 1-phosphate
13.  Vorinostat and sorafenib increase CD95 activation in gastrointestinal tumor cells through a Ca2+ - de novo ceramide - PP2A - ROS dependent signaling pathway 
Cancer research  2010;70(15):6313-6324.
The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat but not the individual drugs rapidly increased ROS, Ca2+ and ceramide levels in GI tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation and cell killing was also blocked by quenching of induced Ca2+ levels or by inhibition of PP2A. Inhibition of acidic sphingomyelinase or de novo ceramide generation blocked the induction of ROS however combined inhibition of both acidic sphingomyelinase and de novo ceramide generation was required to block the induction of Ca2+. Quenching of ROS did not impact on drug-induced ceramide/dihydro-ceramide levels whereas quenching of Ca2+ reduced the ceramide increase. Sorafenib and vorinostat treatment radiosensitized liver and pancreatic cancer cells, an effect that was suppressed by quenching ROS or knock down of LASS6. Further, sorafenib and vorinostat treatment suppressed the growth of pancreatic tumors in vivo. Our findings demonstrate that induction of cytosolic Ca2+ by sorafenib and vorinostat is a primary event that elevates dihydroceramide levels, each essential steps in ROS generation that promotes CD95 activation.
doi:10.1158/0008-5472.CAN-10-0999
PMCID: PMC2918282  PMID: 20631069
14.  Sorafenib activates CD95 and promotes autophagy and cell death via Src family kinases in GI tumor cells 
Molecular cancer therapeutics  2010;9(8):2220-2231.
Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and the present studies have determined individually how sorafenib and vorinostat contribute to CD95 activation. Sorafenib (3-6 μM) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib (3 μM) –induced CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 μM), or sorafenib (3 μM) and vorinostat (500 nM) treatment promoted higher levels of CD95 phosphorylation that correlated with DISC formation, receptor surface localization and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to plasma membrane localize or induce autophagy. Knock down / knock out of Src family kinases abolished sorafenib –induced: CD95 tyrosine phosphorylation; DISC formation; and the induction of cell death and autophagy. Knock down of PDGFRβ enhanced Src Y416 and CD95 tyrosine phosphorylation that correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased ROS levels; and in a delayed NFκB-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95 however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by ~50%. Thus sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation whereas vorinostat contributes to CD95 activation via initial facilitation of ROS generation and subsequently of FAS-L expression.
doi:10.1158/1535-7163.MCT-10-0274
PMCID: PMC2933415  PMID: 20682655
Vorinostat; Sorafenib; CD95; c-FLIP-s; FAS-L; cell death; autophagy
15.  Sphingolipids and cancer: ceramide and sphingosine-1-phosphate in the regulation of cell death and drug resistance 
Future oncology (London, England)  2010;6(10):1603-1624.
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/C16-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 C18-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.
doi:10.2217/fon.10.116
PMCID: PMC3071292  PMID: 21062159
apoptosis; autophagy; chemoresistance; endoplasmic reticulum stress; sphingolipid; sphingolipid; protein binding
16.  PERK–Dependent Regulation of Ceramide Synthase 6 and Thioredoxin Play a Key Role in mda-7/IL-24–Induced Killing of Primary Human Glioblastoma Multiforme Cells 
Cancer research  2010;70(3):1120-1129.
Melanoma differentiation associated gene-7(mda-7) encodes IL-24, a cytokine that can selectively trigger apoptosis in transformed cells. Recombinant mda-7 adenovirus (Ad.mda-7) effectively kills glioma cells, offering a novel gene therapy strategy to address deadly brain tumors. In this study, we defined the proximal mechanisms by which Ad-mda-7 kills glioma cells. Key factors implicated included activation of the endoplasmic reticulum stress kinase protein kinase R–like endoplasmic reticulum kinase (PERK), Ca++ elevation, ceramide generation and reactive oxygen species (ROS) production. PERK inhibition blocked ceramide or dihydroceramide generation, which were critical for Ca++ induction and subsequent ROS formation. Activation of autophagy and cell death relied upon ROS formation, the inhibition of which ablated Ad.mda-7–killing activity. In contrast, inhibiting TRX induced by Ad.MDA-7 enhanced tumor cytotoxicity and improved animal survival in an orthotopic tumor model. Our findings indicate that mda-7/IL-24 induces an endoplasmic reticulum stress response that triggers production of ceramide, Ca2+, and ROS, which in turn promote glioma cell autophagy and cell death.
doi:10.1158/0008-5472.CAN-09-4043
PMCID: PMC2890071  PMID: 20103619
17.  DIRECT BINDING OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE TO TELOMERIC DNA PROTECTS TELOMERES AGAINST CHEMOTHERAPY-INDUCED RAPID DEGRADATION 
Journal of molecular biology  2009;394(4):789-803.
GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel shift assays, we show that recombinant GAPDH binds directly with high affinity (Kd = 45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats and that nucleotides T1, G5 and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA: GAPDH), and GAPDH appears to form a high-molecular weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition, and this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells.
doi:10.1016/j.jmb.2009.09.062
PMCID: PMC2789664  PMID: 19800890
protection of telomeres; glycolytic enzyme; A549 lung carcinoma cells; chemotherapeutics & protein-DNA interactions
18.  A New Mixed-Backbone Oligonucleotide against Glucosylceramide Synthase Sensitizes Multidrug-Resistant Tumors to Apoptosis 
PLoS ONE  2009;4(9):e6938.
Enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) limits therapeutic efficiencies of antineoplastic agents including doxorubicin in drug-resistant cancer cells. Aimed to determine the role of GCS in tumor response to chemotherapy, a new mixed-backbone oligonucleotide (MBO-asGCS) with higher stability and efficiency has been generated to silence human GCS gene. MBO-asGCS was taken up efficiently in both drug-sensitive and drug-resistant cells, but it selectively suppressed GCS overexpression, and sensitized drug-resistant cells. MBO-asGCS increased doxorubicin sensitivity by 83-fold in human NCI/ADR-RES, and 43-fold in murine EMT6/AR1 breast cancer cells, respectively. In tumor-bearing mice, MBO-asGCS treatment dramatically inhibited the growth of multidrug-resistant NCI/ADR-RE tumors, decreasing tumor volume to 37%, as compared with scrambled control. Furthermore, MBO-asGCS sensitized multidrug-resistant tumors to chemotherapy, increasing doxorubicin efficiency greater than 2-fold. The sensitization effects of MBO-asGCS relied on the decreases of gene expression and enzyme activity of GCS, and on the increases of C18-ceramide and of caspase-executed apoptosis. MBO-asGCS was accumulation in tumor xenografts was greater in other tissues, excepting liver and kidneys; but MBO-asGCS did not exert significant toxic effects on liver and kidneys. This study, for the first time in vivo, has demonstrated that GCS is a promising therapeutic target for cancer drug resistance, and MBO-asGCS has the potential to be developed as an antineoplastic agent.
doi:10.1371/journal.pone.0006938
PMCID: PMC2735002  PMID: 19742320
19.  Neutral Sphingomyelinase-3 Is a DNA Damage and Nongenotoxic Stress-Regulated Gene That Is Deregulated in Human Malignancies 
Molecular cancer research : MCR  2008;6(5):795-807.
In this study, we report the characterization of a novel genotoxic and nongenotoxic stress-regulated gene that we had previously named as SKNY. Our results indicate that SKNY encodes the recently identified neutral sphingomyelinase-3 (nSMase3; hereafter SKNY is referred to as nSMase3). Examination of nSMase3 subcellular distribution reveals nSMase3 to localize to the endoplasmic reticulum (ER), and deletion of a COOH-terminal region containing its putative transmembrane domain and ER targeting signal partly alters its compartmentalization to the ER. Treatment with genotoxic Adriamycin and nongenotoxic tumor necrosis factor-α up-regulates endogenous nSMase3 expression, albeit with different kinetics. Tumor necrosis factor-α up-regulates nSMase3 expression within 2 h that lasts beyond 24 h and declines to control levels by 36 h. Adriamycin up-regulation of nSMase3 is transient, occurs within 30 min, and declines to control levels by 120 min. Prolonged treatment with Adriamycin by 24 h and beyond, however, causes a down-regulation in nSMase3 expression. Activation of wild-type p53 also down-regulates nSMase3 expression, suggesting that DNA damage-mediated nSMase3 down-regulation seems to occur partly through the tumor suppressor p53. Overexpression of exogenous nSMase3 sensitizes cells to Adriamycin-induced cell killing, a finding consistent with the proposed proapoptotic role of nSMase enzymes and nSMase-generated ceramide. We further investigated nSMase3 expression in various human malignancies and found its expression to be deregulated in several types of primary tumors when compared with their matching normal tissues. Collectively, our results have identified nSMase3 to be an important molecule that is linked to tumorigenesis and cellular stress response.
doi:10.1158/1541-7786.MCR-07-2097
PMCID: PMC2642592  PMID: 18505924
20.  Roles of Bioactive Sphingolipids in Cancer Biology and Therapeutics 
Sub-cellular biochemistry  2008;49:413-440.
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.
doi:10.1007/978-1-4020-8831-5_16
PMCID: PMC2636716  PMID: 18751921
Apoptosis; ceramide; drug resistanc; cancer therapeutic; sphingolipids
21.  Clinical Relevance of Ceramide Metabolism in the Pathogenesis of Human Head and Neck Squamous Cell Carcinoma (HNSCC): Attenuation of C18-ceramide in HNSCC Tumors Correlates with Lymphovascular Invasion and Nodal Metastasis 
Cancer letters  2007;256(1):101-111.
It has been documented previously that defects in the generation of C18-ceramide, a product of ceramide synthase 1 (CerS1), also known as longevity assurance gene 1 (hLASS1), play important roles in the pathogenesis and/or progression of HNSCC. However, whether altered levels of ceramide generation in HNSCC tumors have any clinical relevance remains unknown. In this study, the levels of endogenous ceramides were measured in tumor tissues of 45 HNSCC patients as compared to their normal tissues using high-pressure liquid chromatography/mass spectrometry (LC/MS), and then possible link between ceramide levels and the clinical parameters of HNSCC were examined. The data showed that the levels of C16-, C24-, C24:1-ceramide were significantly elevated in the majority of tumor tissues compared to their normal tissues, while the levels of only C18-ceramide were significantly decreased in HNSCC tumors, especially in tumor tissues of male patients. Importantly, it was also shown here that decreased C18-ceramide levels in HNSCC tumor tissues were significantly associated with the higher incidences of lymphovascular invasion, and pathologic nodal metastasis. Importantly, attenuation of C18-ceramide was also positively linked to the higher overall stages of the primary HNSCC tumors. Therefore, these data suggest, for the first time, that the defects in the generation/accumulation of C18-ceramide might have important clinical roles in HNSCC, especially in lymphovascular invasion and nodal disease.
doi:10.1016/j.canlet.2007.06.003
PMCID: PMC2084356  PMID: 17619081
Ceramide; Ceramide synthase; Longevity assurance gene (LASS); Head and neck cancer; Lymphovascular spread; Nodal metastasis
22.  Involvement Of The Dihydroceramide Desaturase In Cell Cycle Progression In Human Neuroblastoma Cells 
The Journal of biological chemistry  2007;282(23):16718-16728.
The role of dihydroceramide desaturase as a key enzyme in the de-novo pathway of ceramide generation was investigated in human neuroblastoma cells (SMS-KCNR). A novel assay using water soluble analogs of dihydroceramide, dihydroceramidoids (D-e-dhCCPS analogs) was used to measure desaturase activity in-situ. Conversion of D-e-C12-dhCCPS (C12-dhCCPS) to its 4,5-desaturated counterpart: D-e-C12-CCPS (C12-CCPS) was determined by LC/MS analysis. The validity of the assay was confirmed by C8-cyclopropenylceramide, a competitive inhibitor of dihydroceramide desaturase. A human homologue (DEGS-1) of the Drosophila melanogaster degenerative-spermatocyte-gene-1 (des-1) was recently identified, and reported to have desaturase activity. Transfection of SMS-KCNR cells with siRNA to DEGS-1 significantly blocked the conversion of C12-dhCCPS to C12-CCPS. The associated accumulation of endogenous dihydroceramides confirmed DEGS-1 as the main active dihydroceramide desaturase in these cells. The partial loss of DEGS-1 inhibited cell growth with cell cycle arrest at G0/G1. This was accompanied by significant decrease in the amount of phosphorylated retinoblastoma protein (pRb). This hypophosphorylation was inhibited by tautomycin and not by okadaic acid, suggesting the involvement of protein phosphatase 1. Additionally, we found that treatment of SMS-KCNR cells with fenretinide inhibited desaturase activity in a dose dependent manner. Increase of dihydroceramides, but not ceramides, paralled this process as measured by LC/MS. There were no effects on the mRNA or protein levels of DEGS-1, suggesting that fenretinide acts at the post-translational level as an inhibitor of this enzyme. Tautomycin was also able to block the hypophosphorylation of Rb observed with fenretinide treatment. These findings suggest a novel biologic function for dihydroceramides.
doi:10.1074/jbc.M700647200
PMCID: PMC2084375  PMID: 17283068

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