Russell W. Jenkins

Remodeling of cellular cytoskeleton by the acid sphingomyelinase/ceramide pathway

The chemotherapeutic agent cisplatin is widely used in treatment of solid tumors. In breast cancer cells, cisplatin produces early and marked changes in cell morphology and the actin cytoskeleton. These changes manifest as loss of lamellipodia/filopodia and appearance of membrane ruffles. Furthermore, cisplatin induces dephosphorylation of the actin-binding protein ezrin, and its relocation from membrane protrusions to the cytosol. Because cisplatin activates acid sphingomyelinase (ASMase), we investigate here the role of the ASMase/ceramide (Cer) pathway in mediating these morphological changes. We find that cisplatin induces a transient elevation in ASMase activity and its redistribution to the plasma membrane. This translocation is blocked upon overexpression of a dominant-negative (DN) ASMaseS508A mutant and by a DN PKCδ. Importantly; knockdown of ASMase protects MCF-7 cells from cisplatin-induced cytoskeletal changes including ezrin dephosphorylation. Reciprocally, exogenous delivery of D-e-C16-Cer, but not dihydro-C16-Cer, recapitulates the morphotropic effects of cisplatin. Collectively, these results highlight a novel tumor suppressor property for Cer and a function for ASMase in cisplatin-induced cytoskeletal remodeling.

Drug targeting of sphingolipid metabolism: sphingomyelinases and ceramidases

Sphingolipids represent a class of diverse bioactive lipid molecules that are increasingly appreciated as key modulators of diverse physiologic and pathophysiologic processes that include cell growth, cell death, autophagy, angiogenesis, and stress and inflammatory responses. Sphingomyelinases and ceramidases are key enzymes of sphingolipid metabolism that regulate the formation and degradation of ceramide, one of the most intensely studied classes of sphingolipids. Improved understanding of these enzymes that control not only the levels of ceramide but also the complex interconversion of sphingolipid metabolites has provided the foundation for the functional analysis of the roles of sphingolipids. Our current understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from loss‐of‐function/gain‐of‐function studies utilizing genetic deletion/downregulation/overexpression of enzymes of sphingolipid metabolism (e.g. knockout animals, RNA interference) and from the use of pharmacologic inhibitors of these same enzymes. While genetic approaches to evaluate the functional roles of sphingolipid enzymes have been instrumental in advancing the field, the use of pharmacologic inhibitors has been equally important in identifying new roles for sphingolipids in important cellular processes.The latter also promises the development of novel therapeutic targets with implications for cancer therapy, inflammation, diabetes, and neurodegeneration. In this review, we focus on the status and use of pharmacologic compounds that inhibit sphingomyelinases and ceramidases, and we will review the history, current uses and future directions for various small molecule inhibitors, and will highlight studies in which inhibitors of sphingolipid metabolizing enzymes have been used to effectively treat models of human disease.

Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism

Mammalian ceramide synthases 1 to 6 (CerS1–6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced downregulation of each CerS resulted in counter-regulation of nontargeted CerS. Additionally, each CerS knockdown produced unique effects on the levels of multiple sphingolipid species. For example, downregulation of CerS2 decreased very long-chain Cer but increased levels of CerS4, CerS5, and CerS6 expression and upregulated long-chain and medium-long-chain sphingolipids. Conversely, CerS6 knockdown decreased C16:0-Cer but increased CerS5 expression and caused non-C16:0 sphingolipids to be upregulated. Knockdown of individual CerS failed to decrease total sphingolipids or upregulate sphingoid bases. Treatment with siRNAs targeting combined CerS, CerS2, CerS5, and CerS6, did not change overall Cer or sphingomyelin mass but caused upregulation of dihydroceramide and hexosylceramide and promoted endoplasmic reticulum stress. These data suggest that sphingolipid metabolism is robustly regulated by both redundancy in CerS-mediated Cer synthesis and counter-regulation of CerS expression.

Regulated Secretion of Acid Sphingomyelinase: IMPLICATIONS FOR SELECTIVITY OF CERAMIDE FORMATION*

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMaseWT were treated with inflammatory cytokines. Interleukin-1β and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C16-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMaseWT exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMaseS508A MCF7. Secretion of the S508A mutant was also defective in response to IL-1β, as was the regulated generation of C16-ceramide. Taken together, these data support a crucial role for Ser508 in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.

Ceramide Synthase-dependent Ceramide Generation and Programmed Cell Death INVOLVEMENT OF SALVAGE PATHWAY IN REGULATING POSTMITOCHONDRIAL EVENTS

The sphingolipid ceramide has been widely implicated in the regulation of programmed cell death or apoptosis. The accumulation of ceramide has been demonstrated in a wide variety of experimental models of apoptosis and in response to a myriad of stimuli and cellular stresses. However, the detailed mechanisms of its generation and regulatory role during apoptosis are poorly understood. We sought to determine the regulation and roles of ceramide production in a model of ultraviolet light-C (UV-C)-induced programmed cell death. We found that UV-C irradiation induces the accumulation of multiple sphingolipid species including ceramide, dihydroceramide, sphingomyelin, and hexosylceramide. Late ceramide generation was also found to be regulated by Bcl-xL, Bak, and caspases. Surprisingly, inhibition of de novo synthesis using myriocin or fumonisin B1 resulted in decreased overall cellular ceramide levels basally and in response to UV-C, but only fumonisin B1 inhibited cell death, suggesting the presence of a ceramide synthase (CerS)-dependent, sphingosine-derived pool of ceramide in regulating programmed cell death. We found that this pool did not regulate the mitochondrial pathway, but it did partially regulate activation of caspase-7 and, more importantly, was necessary for late plasma membrane permeabilization. Attempting to identify the CerS responsible for this effect, we found that combined knockdown of CerS5 and CerS6 was able to decrease long-chain ceramide accumulation and plasma membrane permeabilization. These data identify a novel role for CerS and the sphingosine salvage pathway in regulating membrane permeability in the execution phase of programmed cell death.

A novel role for protein kinase Cδ-mediated phosphorylation of acid sphingomyelinase in UV light-induced mitochondrial injury

Multiple studies have addressed the mechanisms by which ultraviolet (UV) light induces cell death, and a few have focused on stress mediators such as acid sphingomyelinase (ASMase) or protein kinase Cδ (PKCδ). Based on a recent study that identified a novel mechanism of activation of ASMase through phosphorylation (1), the current study was undertaken to determine the upstream mechanisms regulating ASMase in response to UV and to investigate the role of ASMase and its phosphorylation at S508 as an integral event during UV light‐induced cell death. Exposure of MCF‐7 breast cancer cells to UV light type C (UVC) transiently activated ASMase with maximal activity detected at 10 min postirradiation. A significant increase in C16‐ceramide was detected concomitant with a decrease in C16‐sphingomyelin. In marked contrast, cells overexpressing the ASMaseS508A mutant, which could not be phosphorylated, had no change in either ASMase activity or ceramide levels post‐UV radiation. Loss of PKCδ by RNA interference or its inhibition by rottlerin blocked ASMase phosphorylation and membrane targeting, thus implicating PKCδ upstream of ASMase activation by UV light. Further investigations revealed that UV radiation altered mitochondrial morphology from elongated tubules to fragmented perinuclear organelles, consistent with the onset of the apoptotic cascade. Importantly, cells overexpressing ASMaseS508A were protected (>50%) from UV light‐induced mito‐chondrial fragmentation. Mechanistically, the results showed that ASMaseS508A cells had 50% less active Bax than ASMaseWT cells. These molecular differences culminated in resistance of ASMaseS508 cells to UVC‐induced cell death (25%) as compared to ASMaseWT cells (46%). Taken together, this study provides key molecular insights into activation of ASMase in response to UV light, the role of PKCδ in this activation, and the role of ASMase in mediating apoptotic re‐sponses.— Zeidan, Y. H., Wu, B. X., Jenkins, R. W., Obeid, L. M., Hannun, Y. A. A novel role for protein kinase cδ‐mediated phosphorylation of acid sphingo‐myelinase in UV light‐induced mitochondrial injury. FASEB J. 22, 183–193 (2008)

Molecular Targeting of Acid Ceramidase: Implications to Cancer Therapy

Increasingly recognized as bioactive molecules, sphingolipids have been studied in a variety of disease models. The impact of sphingolipids on cancer research facilitated the entry of sphingolipid analogues and enzyme modulators into clinical trials. Owing to its ability to regulate two bioactive sphingolipids, ceramide and sphingosine-1-phosphate, acid ceramidase (AC) emerges as an attractive target for drug development within the sphingolipid metabolic pathway. Indeed, there is extensive evidence supporting a pivotal role for AC in lipid metabolism and cancer biology. In this article, we review the current knowledge of the biochemical properties of AC, its relevance to tumor promotion, and its molecular targeting approaches.

Protein Kinase C-induced Activation of a Ceramide/Protein Phosphatase 1 Pathway Leading to Dephosphorylation of p38 MAPK

Recently we showed that, in human breast cancer cells, activation of protein kinase C by 4β-phorbol 12-myristate 13-acetate (PMA) produced ceramide formed from the salvage pathway (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). In this study, we investigated intracellular signaling events mediated by this novel activated pathway of ceramide generation. PMA treatment resulted in transient activation of mitogen-activated protein kinases (ERK1/2, JNK1/2, and p38) followed by dephosphorylation/inactivation. Interestingly, fumonisin B1 (FB1), an inhibitor of the salvage pathway, attenuated loss of phosphorylation of p38, suggesting a role for ceramide in p38 dephosphorylation. This was confirmed by knock-down of longevity-assurance homologue 5, which partially suppressed the formation of C16-ceramide induced by PMA and increased the phosphorylation of p38. These results demonstrate a role for the salvage pathway in feedback inhibition of p38. To determine which protein phosphatases act in this pathway, specific knock-down of serine/threonine protein phosphatases was performed, and it was observed that knock-down of protein phosphatase 1 (PP1) catalytic subunits significantly increased p38 phosphorylation, suggesting activation of PP1 results in an inhibitory effect on p38. Moreover, PMA recruited PP1 catalytic subunits to mitochondria, and this was significantly suppressed by FB1. In addition, phospho-p38 resided in PMA-stimulated mitochondria. Upon PMA treatment, a mitochondria-enriched/purified fraction exhibited significant increases in C16-ceramide, a major ceramide specie, which was suppressed by FB1. Taken together, these data suggest that accumulation of C16-ceramide in mitochondria formed from the protein kinase C-dependent salvage pathway results at least in part from the action of longevity-assurance homologue 5, and the generated ceramide modulates the p38 cascade via PP1.

Differential Effects of Ceramide and Sphingosine 1-Phosphate on ERM Phosphorylation PROBING SPHINGOLIPID SIGNALING AT THE OUTER PLASMA MEMBRANE

ERM proteins are regulated by phosphorylation of the most C-terminal threonine residue, switching them from an activated to an inactivated form. However, little is known about the control of this regulation. Previous work in our group demonstrated that secretion of acid sphingomyelinase acts upstream of ERM dephosphorylation, suggesting the involvement of sphingomyelin (SM) hydrolysis in ERM regulation. To define the role of specific lipids, we employed recombinant bacterial sphingomyelinase (bSMase) as a direct probe of SM metabolism at the plasma membrane. bSMase induced a rapid dose- and time-dependent decrease in ERM dephosphorylation. ERM dephosphorylation was driven by ceramide generation and not by sphingomyelin depletion, as shown using recombinant sphingomyelinase D. The generation of ceramide at the plasma membrane was sufficient for ERM regulation, and no intracellular SM hydrolysis was required, as was visualized using Venus-tagged lysenin probe, which specifically binds SM. Interestingly, hydrolysis of plasma membrane bSMase-induced ceramide using bacterial ceramidase caused ERM hyperphosphorylation and formation of cell surface protrusions. The effects of plasma membrane ceramide hydrolysis were due to sphingosine 1-phosphate formation, as ERM phosphorylation was blocked by an inhibitor of sphingosine kinase and induced by sphingosine 1-phosphate. Taken together, these results demonstrate a new regulatory mechanism of ERM phosphorylation by sphingolipids with opposing actions of ceramide and sphingosine 1-phosphate. The approach also defines a tool kit to probe sphingolipid signaling at the plasma membrane.

Response to Crizotinib in a Patient With Lung Adenocarcinoma Harboring a MET Splice Site Mutation

MET (c-MET; mesenchymal-epithelial transition factor) is a receptor tyrosine kinase that was first characterized as a proto-oncogene in 1984, in a chemically transformed osteosarcoma cell line [1]. Located on chromosome 7q21-31, the MET gene encodes a cell surface receptor tyrosine kinase comprised of a 50-kDa α-chain and a 140-kDa transmembrane β-chain linked by a disulfide bond. The natural ligand for cMet is hepatocyte growth factor (HGF), also known as scatter factor [2]. Following binding of HGF the c-MET receptor undergoes dimerization and phosphorylation, which in turn promotes recruitment of downstream effector proteins, leading to activation of multiple signaling cascades, including the MAPK, PI3K/Akt, STAT and NF-κB pathways [3]. Physiologic roles for HGF/MET signaling include embryogenesis, development, and wound healing [3–5]. Aberrant activation of the c-Met receptor tyrosine kinase promotes oncogenicity in a subset of lung adenocarcinomas. A variety of mechanisms can result in constitutive c-Met signaling, including MET gene amplification, protein overexpression, activating point mutations, and induction of its ligand HGF [3, 6, 7]. Crizotinib, a multitargeted tyrosine kinase inhibitor (TKI) that is FDA approved for the therapy of lung adenocarcinomas harboring ALK or ROS1 fusions [8–10], was also recently found to be clinically active in tumors with high level MET amplification [11]. These findings have prompted the clinical development of more selective c-MET inhibitors for evaluation in this particular patient population. MET splice site mutation has also been demonstrated to induce constitutive activity and confer sensitivity to c-Met inhibition in vitro [12, 13]. Indeed, frequent activating MET splice site mutations were recently described in whole exome sequencing (WES) discovery efforts in lung adenocarcinoma [14]. However, the clinical activity of c-Met inhibition in this context remains unknown. We identified one such mutation using targeted next generation sequencing (NGS) in a patient with lung adenocarcinoma and treated him with crizotinib.