Salih Gencer

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.

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.

Off-Target Function of the Sonic Hedgehog Inhibitor Cyclopamine in Mediating Apoptosis via Nitric Oxide–Dependent Neutral Sphingomyelinase 2/Ceramide Induction

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 anticancer therapeutics. The SHh inhibitor cyclopamine induces apoptosis. The bioactive sphingolipid ceramide mediates cell death in response to various chemotherapeutic agents; however, ceramides roles/mechanisms in cyclopamine-induced apoptosis are unknown. Here, we report that cyclopamine mediates ceramide generation selectively via induction of neutral sphingomyelin phosphodiesterase 3, SMPD3 (nSMase2) in Daoy human medulloblastoma cells. Importantly, short interfering RNA-mediated knockdown of nSMase2 prevented cyclopamine-induced ceramide generation and protected Daoy cells from drug-induced apoptosis. Accordingly, ectopic wild-type N-SMase2 caused cell death, compared with controls, which express the catalytically inactive N-SMase2 mutant. Interestingly, knockdown of smoothened (Smo), a target protein for cyclopamine, or Gli1, a downstream signaling transcription factor of Smo, did not affect nSMase2. Mechanistically, our data showed that cyclopamine induced nSMase2 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. Knockdown of n-NOS prevented nSMase2 induction and cell death in response to cyclopamine. 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 n-NOS/NO-dependent induction of N-SMase2/ceramide axis, independent of Smo/Gli inhibition. Mol Cancer Ther; 11(5); 1092–102. ©2012 AACR.

Concerted functions of HDAC1 and microRNA-574-5p repress alternatively spliced ceramide synthase 1 expression in human cancer cells

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.

Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-Terminal Kinase (JNK)-Dependent Manner

Background: Ceramide is important for cellular signaling. Results: Increasing the expression of ceramide synthase 6 (CerS6) results in transcriptional activation of acid ceramidase independent of catalytic CerS6 activity. Conclusion: Modulation of a single member of the ceramide synthase family impacts on sphingolipid composition and ceramide metabolizing enzymes. Significance: Understanding how CerS impacts gene expression and signaling is important for the development of novel therapeutic approaches. A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14–26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3–5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity.

Ceramide synthesis regulates T cell activity and GVHD development

Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective immunotherapy for a variety of hematologic malignances, yet its efficacy is impeded by the development of graft-versus-host disease (GVHD). GVHD is characterized by activation, expansion, cytokine production, and migration of alloreactive donor T cells. Hence, strategies to limit GVHD are highly desirable. Ceramides are known to contribute to inflammation and autoimmunity. However, their involvement in T-cell responses to alloantigens is undefined. In the current study, we specifically characterized the role of ceramide synthase 6 (CerS6) after allo-HCT using genetic and pharmacologic approaches. We found that CerS6 was required for optimal T cell activation, proliferation, and cytokine production in response to alloantigen and for subsequent induction of GVHD. However, CerS6 was partially dispensable for the T cell-mediated antileukemia effect. At the molecular level, CerS6 was required for efficient TCR signal transduction, including tyrosine phosphorylation, ZAP-70 activation, and PKCθ/TCR colocalization. Impaired generation of C16-ceramide was responsible for diminished allogeneic T cell responses. Furthermore, targeting CerS6 using a specific inhibitor significantly reduced T cell activation in mouse and human T cells in vitro. Our study provides a rationale for targeting CerS6 to control GVHD, which would enhance the efficacy of allo-HCT as an immunotherapy for hematologic malignancies in the clinic.

TGF-β receptor I/II trafficking and signaling at primary cilia are inhibited by ceramide to attenuate cell migration and tumor metastasis

Ceramide forms complexes with Smad7 to inhibit cell migration–associated TGF-β–sonic hedgehog signaling in primary cilia. The good side of ceramides Ceramides are lipids that contribute to various cellular structures and functions. In the context of cancer, some ceramides, and the enzymes that produce them, contribute to tumor growth because they provide a critical component of the plasma membrane, enabling cells to divide. However, Gencer et al. found that certain long-chain ceramides synthesized by the enzyme CerS4 play a critical tumor suppressor role. C18- to C20-ceramides mediated the interaction between an inhibitory Smad protein and a TGF-β receptor complex, thus blocking subsequent cross-talk activation of the sonic hedgehog (Shh) pathway in tumor cells’ primary cilia, a region of the cell that coordinates motility. Depleting CerS4 in tumor cells increased the incidence of distant metastases from mammary tumors in mice. The disruption of TGF-β–Shh cross-talk by CerS4 may also prevent the development of the hair loss disorder alopecia. Both the TGF-β and Shh pathways are challenging to target pharmacologically; these findings suggest that some ceramides may have therapeutic potential against these pathways in various disorders. Signaling by the transforming growth factor–β (TGF-β) receptors I and II (TβRI/II) and the primary cilia-localized sonic hedgehog (Shh) pathway promote cell migration and, consequently, tumor metastasis. In contrast, the sphingolipid ceramide inhibits cell proliferation and tumor metastasis. We investigated whether ceramide metabolism inhibited TβRI/II trafficking to primary cilia to attenuate cross-talk between TβRI/II and the Shh pathway. We found that ceramide synthase 4 (CerS4)–generated ceramide stabilized the association between TβRI and the inhibitory factor Smad7, which limited the trafficking of TβRI/II to primary cilia. Expression of a mutant TβRI that signals but does not interact with Smad7 prevented the CerS4-mediated inhibition of migration in various cancer cells. Genetic deletion or knockdown of CerS4 prevented the formation of the Smad7-TβRI inhibitory complex and increased the association between TβRI and the transporter Arl6 through a previously unknown cilia-targeting signal (Ala31Thr32Ala33Leu34Gln35) in TβRI. Mutating the cilia-targeting signal abolished the trafficking of TβRI to the primary cilia. Localization of TβRI to primary cilia activated a key mediator of Shh signaling, Smoothened (Smo), which stimulated cellular migration and invasion. TβRI-Smo cross-talk at the cilia in CerS4-deficient 4T1 mammary cancer cells induced liver metastasis from orthotopic allografts in both wild-type and CerS4-deficient mice, which was prevented by overexpression of Smad7 or knockdown of intraflagellar transport protein 88 (IFT88). Overall, these data reveal a ceramide-dependent mechanism that suppresses cell migration and invasion by restricting TβRI/II-Shh signaling selectively at the plasma membrane of the primary cilium.

Abstract 4122: Ceramide is a key factor that regulates the crosstalk between TGF-ß and sonic hedgehog signaling at the basal cilia to control cell migration and tumor metastasis

Recent studies indicate that ceramide species with different fatty-acid chain lengths play diverse biological functions in various cellular processes, highlighting the importance of ceramide synthases (CerS) in these processes. Migration and cell mobility, a part of these processes, also are effected by ceramide metabolism. However, the molecular mechanism of CerS/and ceramide involved is unknown. Here, we investigated the effect of CerS/and ceramide on migration and its related signal pathways in situ and in vivo model. Interestingly, our data show that among CerS only CerS4/ceramide is involved to cell migration and tumor metastasis. Here, we also have generated CerS4-/- mice for in vivo studies. Interestingly, we observed that genetically loss of CerS4 resulted in severe irreversible alopecia, which was associated with hyper-proliferation and migration of keratinocytes. Mechanistically, we show here that genetic loss or shRNA-mediated knockdown of CerS4 enhances cell migration by which ligand-independent signaling of TGF-beta receptors I and II in various cell types, including keratinocytes, mouse embryonic fibroblasts and cancer cells. Moreover, we found that ceramide directly interact with Smad7 and this interaction was decreased by shRNA-mediated knockdown of CerS4. Thus, ceramide-Smad7 binding modulates plasma membrane association of TGF-sR1 at primary basal cilia, and inhibits its signaling through Sonic-Hedgehog (Shh) for migration. Furthermore, Ceramide accumulation at the primary basal cilia was decreased by knockdown of CerS4. Thus, these data revealed that CerS4/ceramide signaling plays key roles in the regulation of cell migration and metastasis via controlling the TGF-sR and Shh axis at primary basal cilia. Citation Format: Salih Gencer, Natalia Oleinik, Mohammed Dany, Besim Ogretmen. Ceramide is a key factor that regulates the crosstalk between TGF-s and sonic hedgehog signaling at the basal cilia to control cell migration and tumor metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4122.