Ceramide Induced Mitophagy and Tumor Suppression☆

Biochimica et Biophysica Acta 1853 (2015) 2834–2845

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Review Ceramide induced mitophagy and tumor suppression☆

Mohammed Dany, Besim Ogretmen ⁎

Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas Street, Charleston, SC 29425, USA article info abstract

Article history: Sphingolipids are bioactive lipid effectors, which are involved in the regulation of various cellular signaling Received 9 October 2014 pathways. Sphingolipids play essential roles in controlling cell inﬂammation, proliferation, death, migration, Received in revised form 9 December 2014 senescence, metastasis and autophagy. Alterations in sphingolipid metabolism have been also implicated in Accepted 25 December 2014 many human cancers. Macroautophagy (referred to here as autophagy) is a form of nonselective sequestering Available online 26 January 2015 of cytosolic materials by double membrane structures, autophagosomes, which can be either protective or lethal

Keywords: for cells. Ceramide, a central molecule of sphingolipid metabolism is involved in the regulation of autophagy at Ceramide various levels, including the induction of lethal mitophagy, a selective autophagy process to target and eliminate Sphingolipids damaged mitochondria. In this review, we focused on recent studies with regard to the regulation of autophagy, Mitophagy in particular lethal mitophagy, by ceramide, and aimed at providing discussion points for various context- Tumor suppression dependent roles and mechanisms of action of ceramide in controlling mitophagy. This article is part of a Special Issue entitled: Mitophagy. © 2015 Elsevier B.V. All rights reserved.

1. Introduction better understand ceramide's role in inducing selective lethal mitophagy. Several recent studies showed the ability of endogenous or exogenous Sphingolipids are membrane lipids with important functions in ceramides to cause cell death after accumulating in the mitochondria regulating membrane ﬂuidity and subdomain structures. Advances in [11–13]. Our recent study showed that mitochondrial ceramide acts as sphingolipid research suggest that sphingolipids are highly bioactive mol- a receptor for LC3-II [14]. Ceramide binding directly to LC3-II protein ecules and have a great impact on cellular signaling and disease patho- leads to the recruitment of autophagosomes to damaged mitochondria, genesis [1]. Ceramide is one of the central molecules of sphingolipid resulting in lethal mitophagy [14]. This review will focus on the roles metabolism and plays a key role in the regulation of various cellular func- and mechanism of action of ceramide in the regulation of mitophagy tions, including cell proliferation, death, migration, and senescence [2]. and tumor suppression. Ceramide is intimately involved in cancer pathogenesis; alterations in its metabolism are involved in controlling cancer initiation, progression, and/or response of cancer cells to chemotherapeutic agents and radiation. 2. Metabolism and biological roles of ceramide

Endogenous levels of ceramide, especially C18-ceramide, are suppressed in many types of tumor tissues compared to non-cancerous counterparts. 2.1. Structure and metabolism of ceramide Additionally, ceramide levels increase upon exposure of cancer cells to stress-causing agents, such as cytokines, anticancer drugs, and radiation, Ceramide is a bioactive sphingolipid with a peculiar structure. It is leading to cancer cell death and tumor suppression [2,3]. Ceramide- composed of a sphingosine backbone that is esteriﬁed to a fatty acyl mediated tumor suppression is regulated by various mechanisms such chain via an amide linkage at carbon 3 [1,2]. The variety in the length as apoptosis, necroptosis, lethal autophagy, and mitophagy [4–8]. of the fatty acyl chain generates many different ceramides, such as

Mitophagy is a form of autophagy that results in selective degradation C14-toC26-ceramides. A trans-double bond between carbons 4 and 5 in of mitochondria through the autophagic machinery [9]. Ceramide plays a the sphingosine backbone is required for its biological activity, as the key role in the regulation of general autophagy at many levels from initi- loss of the double bond generates dihydro-ceramide [1]. ation to formation of autophagosomes [10]. Recently, we have begun to Ceramide lies at the center of sphingolipid metabolism: acting as a substrate for the generation of more complex sphingolipids, or as a product of the breakdown of complex sphingolipid molecules (Fig. 1). ☆ This article is part of a Special Issue entitled: Mitophagy. As a substrate, ceramide is converted to sphingomyelin, ceramide-1- ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Jonathan Lucas Street, Room 512A, Charleston, SC 29425, phosphate, hexosylceramides, and other complex glycosphingolipids USA. Tel.: +1 843 792 0940; fax: +1 843 792 2556. and gangliosides [1]. As a product, ceramide can be generated by the E-mail address: [email protected] (B. Ogretmen). breakdown of these more complex sphingolipids by specialized enzymes

Fig. 1. Ceramide metabolic pathways. Ceramide lies at the center of sphingolipid metabolism. Ceramide de novo generation starts with a condensation reaction involving serine and palmitoyl CoA by the enzyme serine palmitoyl CoA transferase (SPT), to generate 3-ketosphinganine, which is then converted to sphinganine or dihydrosphingosine. Then, ceramide synthases (CerS1-6) transfer a fatty acyl CoA to the amino group yielding dihydroceramide which gets desaturated to ceramide by dihydro-ceramide desaturase enzyme (DES). Ceramide can be generated back from sphingosine-1-phosphate with the help of S1P phosphatase (S1PP) and ceramide synthase (salvage pathway) or from sphingomyelin by sphingomyelinase (SMase), from glycosphingolipids, or from ceramide-1-phosphate by ceramide kinase (Cer-Kinase). As a metabolic outlet for ceramide, it is converted to sphingosine by the action of ceramidase. Sphingosine kinases 1 and 2 (SK1/2) phosphorylate sphingosine to sphingosine-1-phosphate that can be further degraded by S1P lyase to hexadecanal and ethanolamine phosphate.

such as sphingomyelinases (SMAse), which hydrolyze sphingomyelin lipid rafts, which are specialized membrane microdomains that regulate (SM), and cerebrosidases which hydrolyze hexosylceramides [15]. various signaling pathways [24]. De novo generation of ceramide begins with the condensation of serine and palmitoyl CoA to form 3-ketosphinganine, and then 2.2. Biological functions of ceramide synthases 1–6 dihydrosphingosine (sphinganine) [16], involving multiple metabolic enzymes. This is followed by the action of ceramide synthases 1–6 Ceramide synthases 1–6(CerS1–6) were first identified in yeast as (CerS1–6) (also known as dihydro-ceramide synthases) that esterify longevity assurance gene 1 (LAG1). LAG1 regulates longevity in yeast in the dihydrosphingosine to generate dihydroceramide [17]. Ceramide a way that its deletion prolongs the replicative lifespan [17].Themouse is then generated by the action of (dihydroceramide)-desaturase that homologue of LAG1 is LASS1, also known as the upstream of growth irreversibly inserts a double bond between carbons 4 and 5 [18]. and differentiation factor 1 (UOG1), which was discovered to specifically

Ceramide catabolism is regulated mainly by ceramidases, which regulate the synthesis of C18-ceramide [25,26]. There are at least six cleave ceramide to generate sphingosine. Sphingosine then gets phos- mammalian LASS proteins that are currently known as CerS1–6 [17]. phorylated by sphingosine kinases 1 or 2 (SphK1 or SphK2) to produce All of the CerS enzymes share a domain required for their enzymatic sphingosine 1-phosphate (S1P). S1P can be lysed to hexadecanal and activity to generate ceramide called TLC domain (TRAM, LAG1, and ethanolaminephosphate by S1P lyase or dephosphorylated by S1P CLN8 homology), which is made up of ﬁve predicted transmembrane phosphatases [19]. Hence, endogenous levels of ceramide increase helices [27,28].CerS2–6 also contain a HOX domain which CerS1 lacks through de novo synthesis, activation of sphingomyelinases, or de- [29,30]. creased clearance through the inhibition of glucosylceramide syn- CerS1–6 exhibits some speciﬁcity for the generation of endogenous thase (GCS), sphingomyelin synthase (SMS), or ceramidase (CDase) ceramides with different fatty acid chain lengths [31,32]. For example, [20]. CerS1 mainly generates ceramide with an 18-carbon containing fatty

Sphingolipid metabolism is highly compartmentalized in cells, as the acid chain (C18-ceramide), CerS4 generates both C18-ceramide and subcellular localization of different sphingolipid molecules and the C20-ceramide, and CerS5 and CerS6 generate mainly C16-ceramide, and metabolic enzymes play key roles in this process [1].Forexample, to a lesser extent, C12-andC14-ceramides. CerS2 and CerS3 are known CerS1–6 enzymes are localized in the endoplasmic reticulum (ER) to generate very long chain C22–24-andC26-ceramides, respectively where de novo synthesis of ceramide occurs. For the synthesis of SM, [1–3,7,15,33]. ceramide is transported from the ER to the Golgi apparatus by ceramide Different species of ceramide with distinct fatty acyl chain lengths transporter protein (CERT) in a non-vesicular fashion [21,22]. Similarly, have diverse biological functions (Table 1). For example, CerS1 and for the synthesis of glucosylceramide (GlcCer), ceramide is transported CerS6, generating C18-ceramide and C16-ceramide respectively, have to the Golgi by Fabb2 transporter [22]. Recently, a role for a lipid trans- opposing roles in cell death and proliferation in head and neck cancer porter protein GLTPD1 for ceramide 1-phosphate, referred to as CPTP, cells and tumors. CerS1/C18-ceramide axis leads to cancer cell death has been discovered, which plays a critical role in the regulation of in- and decreases head and neck tumor growth [34–36]. Increased levels

ﬂammation [23]. Ceramide is also found in the mitochondria where it of serum C18-ceramide act as a potential biomarker to monitor patients' can be generated by neutral sphingomyelinase (N-SMase) in response response to chemotherapy [37]. On the other hand, C16-ceramide pro- to increased reactive oxygen species (ROS) production [1].Ceramideac- motes head and neck cancer cell proliferation and its increased serum cumulation in the mitochondria can lead to ceramide stress-induced levels associate with a positive lymph node status in breast cancer mitochondrial fragmentation, and decrease in ATP production [14].In patients [38,39]. On the other hand, there are studies showing that lysosomes, ceramide is mainly produced by acid sphingomyelinase C16-ceramide is pro-apoptotic, whereas C24-ceramide protects from (A-SMase), and in the plasma membrane, ceramide is localized within cell death [40,41]. Thus, overall, distinct roles of ceramides appear to 2836 M. Dany, B. Ogretmen / Biochimica et Biophysica Acta 1853 (2015) 2834–2845

Table 1 Diverse biological roles of ceramide synthases 1–6.

Ceramide synthase isoform Ceramide product Biological role

Ceramide synthase 1 C18-ceramide • Induces cancer cell death [34] • Decreases tumor growth [35] • CerS1 mutant mice have neurological disorders [42]

Ceramide synthase 2 C22–24-ceramide • C24-ceramide can protect from cell death [40,41] • CerS2 knockout mice exhibit liver damage [44]

Ceramide Synthase 3 Ultra long chain ceramides N C26 • CerS3 knockout leads to trans-epidermal water loss leading to death after birth [203]

Ceramide synthase 4 C18- and C20-ceramide • CerS4 knockout mice exhibit severe alopecia with alterations in sebaceous glands and sebum contents [43]

Ceramide synthase 5 C12-, C14-, and C16-ceramide • Implicated in induction of autophagy mediated hypertrophy of cardiac myocytes [204]

Ceramide synthase 6 C12-, C14-, and C16-ceramide • Increases cancer cell proliferation [38] • Increased expression associates with positive lymph node status [39] • Induces chemotherapy mediated apoptosis [41] • CerS6 knockout mice exhibit neurological/behavioral alterations [205] be context dependent, especially that knockout mice for multiple CerS generated in lysosomes by A-SMase activates cathepsin D by inducing enzymes exhibit different phenotypes: mice that express a mutant autocatalytic proteolysis, resulting in Bid cleavage and caspase activa- CerS1 (toppler mice) exhibit neurological disorders associated mainly tion [65,66]. with alterations in Purkinjee cells; mice with genetic loss of CerS2 Another mechanism that associates with the tumor suppressor roles result in liver damage; mice with CerS6 knockout exhibit neurological/ of ceramide is its regulation of telomere length by acting as an upstream behavioral alterations; and CerS4 knockout mice exhibit severe alopecia regulator of telomerase. Studies showed that ceramide accumulation in with alterations in sebaceous glands and sebum contents [42,43].The lung cancer cells inhibits telomerase expression by inactivating c-Myc demonstration of the distinct roles of CerS-generated ceramides in the transcription factor, which is an activator of the human telomerase regulation of cell death is also consistent with studies performed in Dro- reverse transcriptase (hTERT) promoter, via increased ubiquitination sophila and Caenorhabditis elegans that showed the distinct roles of and proteasome mediated degradation. The inactivation of telomerase CerS-generated ceramides in these organisms [45–47]. Moreover, re- prevents the cancer cell from elongating the telomeric ends of the chro- cent evidence suggests that changes in the carbon length of the mosomes after each replication cycle, leading eventually to cell death sphingoid base of ceramides with 18 versus 16 carbons play roles in in- [67,68]. ducing survival autophagy versus cell death in cardiomyocytes [48]. Ceramide is also involved in pathways that lead to quiescence and senescence in cancer cells. For example, ceramide inactivates cyclin 2.3. Cancer suppressing role of ceramide dependent kinase 2 (CDK2), and upregulates the expression of CDK inhibitors p21 and p27 in Wi-38 fibroblasts and nasopharyngeal carcino- Ceramide induces a variety of anti-proliferative responses such as ma cells, respectively [69–71]. In addition, exogenous supply of ceramide programmed cell death, cell cycle arrest, senescence, and differentiation. to fibroblasts cultured at low passage induces the biochemical and mor- Indeed, studies show that ceramide is involved in apoptosis, necroptosis, phological features of senescence [1,72]. By inducing senescence, cer- lethal autophagy, and mitophagy, all of which decrease cancer cell via- amide helps in the suppression of key mitogenic pathways, leading to bility [14,49–51]. Exogenously supplied ceramides (C2–C18-ceramides), tumor suppression [73]. also have anti-proliferative activities when added to cells in culture [11, Because of these pro-death characteristics, ceramide analogues or 12,52]. Thus, ceramide metabolism has a significant role in suppressing mimetics have the potential to act as anti-cancer agents. Endogenous cancer progression, and ceramide is emerging as a tumor suppressor accumulation of ceramide might also be beneficial to suppress tumor lipid. growth. One example is the group of ceramide analogues, ceramidoids Ceramide is generated during stress conditions such as hypoxia, or pyridinium ceramides, which preferentially accumulate in the growth factor withdrawal, hyperthermia, and DNA damage which then mitochondria/nuclei, and suppress the tumor growth of lung, breast, mediates cell death [53]. In addition, there is an increase in ceramide and head and neck squamous cell carcinomas [52,74]. The mitochondrial levels during the activation of extrinsic apoptosis via FAS/FAS ligand path- accumulation of pyridinium ceramides results in mitochondrial perme- way or tumor necrosis factor (TNF)-alpha pathway [54]. Ionizing radia- ability transition and either caspase-dependent apoptosis or mitophagy tion causes ceramide formation by activating A-SMase while androgen [14,74]. Another example is the group of glucosylceramide synthase ablation in prostate cancer cells induces de novo generation of ceramide, inhibitors (PPMP and PPPP) that lead to the accumulation of ceramide, leading to cell death [55–57]. which decrease glucosylceramide generation, and suppress solid tumor One mechanism by which ceramide leads to cell death is by activating growth [75,76]. Ceramide can also be delivered exogenously in PEGylated protein phosphatases PP1 and PP2A [58]. PP2A is a tumor suppressor nanoliposomes. Studies using these liposomes show that this delivery protein that acts as a phosphatase to regulate signaling of many targets method of ceramide decreases phosphorylation of AKT, stimulates the including Akt, c-Myc, and Bcr-Abl [59,60]. Ceramide binds to the biologi- activity of caspase 3/7, and prevents the growth of breast cancer cells cal inhibitor of PP2A, I2PP2A or SET oncoprotein, leading to PP2A reacti- in vitro and in vivo [77,78]. vation. PP2A can then dephosphorylate and inactivate several antiapoptotic proteins such as Bcl-2 and AKT, or c-Myc [50,61]. Interestingly, 3. General autophagy and its regulation by ceramide SET/I2PP2A oncoprotein preferentially associates with endogenous

C18-ceramide compared with other ceramide species [62].Thisspecific- 3.1. Progression of autophagy ity for binding to a specific species of ceramide is also evident in CERT binding preferentially to C16-andC18-ceramides but not very long Autophagy, or self-eating in Greek, describes the mechanism utilized chain ceramides [63]. Ceramide interacts with another phosphatase, by the cell to self-digest internal organelles and misfolded proteins PP1, which inactivates the pro-apoptotic protein Bid [54]. Moreover, using lysosomal hydrolytic enzymes [79]. Autophagy starts with the ceramide-PP1 is involved in the regulation of retinoblastoma protein formation of cup shaped structures called phagophores, also known as (RB), a tumor suppressor protein that plays an important role in cell isolation membranes, which will elongate to engulf organelles and cycle regulation. Ceramide treatment can dephosphorylate and activate other cytoplasmic components. The maturation of the phagophores RB leading to cell cycle arrest in cancer cells [64]. In addition, ceramide leads to the formation of an autophagosome that fuses with lysosomes M. Dany, B. Ogretmen / Biochimica et Biophysica Acta 1853 (2015) 2834–2845 2837 for the formation of autophagolysosomes [80]. It is at this stage that ly- of autophagy genes and lysosomal flux during the death process [106, sosomal enzymes start the digestion process and the rate of breakdown 108,112–114]. of the cellular components is referred to as lysosomal flux. Autophagy plays a critical role in physiology and cellular homeostasis, allowing 3.3. Autophagy in cancer the cells to recycle nutrients from digested organelles at times of starvation and remove aberrantly folded proteins [81–84]. Dysregulation in There are several lines of evidence supporting that autophagy is a autophagy has been implicated in various human diseases such as neu- tumor-suppressor mechanism: Cancer cells have increased oxidative rodegenerative disorders, cardiovascular diseases, and cancer [84–89]. and metabolic stress that cause chromosomal abnormalities, DNA strand The discovery of autophagy genes (Atg) in yeast had a great impact breaks, and gene mutations. Autophagy helps in scavenging reactive ox- on our understanding of the mechanisms of autophagy process. Most of ygen species by removing the damaged organelles, thus preventing the the Atg genes are conserved in humans and play various roles including genetic abnormalities that might otherwise lead to oncogene activation autophagy initiation, autophagosome formation and maturation or tumor suppressor gene inactivation [115–118]. Some cancer cells [90–92]. During initiation, Atg1 forms a complex with ULK (Unc-51 suppress autophagy as a mechanism to avoid the quality control during like kinase) that integrates inputs from mTOR signaling [87,93–95]. oxidative stress, DNA damage, and genetic instability [119].Oneexample During autophagosome formation, Atg9 allows for membrane addition to illustrate this is the role of autophagy in the turnover of p62 protein. and retrieval to and from sites of autophagosome biogenesis [83,96], When autophagy is suppressed, p62 protein clearance is prevented, while Atg6 (or Beclin 1 in mammals) forms a multimeric complex leading to its accumulation, which in turn activates NRF2 (nuclear factor with Atg14, Vps34/PI3kinase, and Vps15 [94,96–98]. In addition, during erythroid 2 related factor 2). NRF2 can then translocate to the nucleus, autophagosome formation, Atg12 is activated by the enzyme Atg7 (E1- where it activates an anti-oxidant and pro-survival response [117, like ubiquitin ligase) and then transferred to Atg10 (E2-like ubiquitin li- 120–122]. gase) to be conjugated to Atg5 (E3-like ubiquitin ligase), forming an Some of the Atg proteins act as tumor suppressor genes. Beclin 1 autophagosomal precursor. Finally, Atg8 is required during the matura- (Atg6) expression is suppressed in malignant breast epithelial cells, tion phase [93,95,97,100,101]. and it is monoallelically deleted in 40–70% of prostate, ovarian, and LC3, mammalian homologue of Atg8, which plays important roles breast cancers. Overexpression of Beclin 1 in breast cancer cells promoted for the maturation of autophagosomes, has three isoforms: LC3A, LC3B autophagy and inhibited the malignant phenotype [123–125].Invivo, and LC3C. GABARAP and GATE16 are also mammalian homologues of targeted deletion of Beclin 1 in mice led to early embryonic death. Atg8 [102]. LC3 is synthesized in the cell in its cytosolic form, LC3-I, Heterozygous disruption of Beclin 1 resulted in an increased risk of spon- with a carboxy terminal glycine (Gly120) [102]. During autophagy, LC3 taneous tumor development, even though the other allele is intact. This is cleaved by Atg4 protease and then activated by Atg7 to be trans- suggests that the pro-autophagic Beclin 1 is a haplo-insufficient tumor ferred to Atg3 (E2-like ubiquitin ligase) [80,103]. This allows the suppressor protein [126]. Further studies then highlighted that other Gly120 residue on the carboxyl terminal to be conjugated to phosphati- pro-autophagic proteins also act as tumor suppressors, such as Atg5 and dylethanolamine (PE) to form LC3-II. This allows its docking to the Bif1 [83,84,112]. Autophagy can also be suppressed due to its regulation membranes, leading to membrane closure and formation of a mature by signaling pathways that are up- or down-regulated in the cancer autophagosome [104]. This sets LC3-II as a well-established marker of cells [112]. For instance, cancer cells with upregulation of PI3K-AKT- autophagosomes. mTOR signaling cascade, or downregulation of PTEN activity, will have suppressed autophagy, promoting tumor growth/proliferaiton 3.2. Autophagy paradox: cell survival or death regulation [83,127,128]. As cancer progresses to late stages, autophagy can act as a mechanism Autophagy was initially discovered as a mechanism occurring at a to help the cancer cells meet metabolic demands and repair intracellular low rate to remove protein aggregates and damaged organelles that damages inflicted by the aggressive tumor environment [84,112,119]. are otherwise toxic for the cell [81]. In addition, autophagy is considered Pancreatic cancer cell lines demonstrate a high basal rate of autophagy, as a vital process during metabolic stress or nutrient deprivation, in and upon pharmacological inhibition of autophagy by chloroquine, which the degradation of organelles provides macromolecules and pancreatic cancer cell growth was diminished mainly due to increased nutrients that maintain energy production and the basic cellular func- DNA damage and oxidative stress [117,129]. Additionally, cancer cells tions [79,82,99]. Mechanistically, several reports show that upon cell expressing the Ras oncogene have a higher basal rate of autophagy, starvation, LC3-I is modified to LC3-II to promote autophagy [80].In such that Ras expressing Atg5−/− and Atg7−/− cells, have sup- vivo, GFP-LC3 localizes in punctate structures in heart and skeletal pressed autophagy levels and showed reduced tumor growth in vivo muscle tissues in transgenic mice when the animals were under fasting [130]. conditions [102,105]. These functions of autophagy provide pro-survival The implication of autophagy in cancer pathogenesis gives insight and cyto-protective mechanisms. into new pharmacological therapies for cancer. If autophagy is required Recently, autophagy was found to be a pro-death mechanism espe- for the survival of cancer cells in the late stages, then pharmacological cially if it occurs for a sustained period of time with a high intensity. inhibition of autophagy can enhance the anti-cancerous effect of Cells with sustained upregulation of autophagic activity became chemotherapeutic drugs [84,112,112,117,120]. For instance, combining atrophic with loss of vital organelles and cellular functions [106].This vinblastine with C6-ceramide attenuated autophagy and inhibited suggests that over-activated autophagy can lead to cell death when cancer cell growth in a synergistic fashion [131]. In contrast, if autophagy associated with major elimination of essential organelles. Moreover, induction leads to cancer cell death via lethal autophagy, then drugs studies indicate that autophagy can lead to cell death via its ability to de- that induce autophagy will lead to tumor suppression. For example, grade cyto-protective proteins such as catalase, an anti-oxidant enzyme pyridinium ceramide treatment leads to cancer cell death via in part [107]. Autophagy can also result in cell death by upregulating apoptosis activating autophagy [11]. or necroptosis [108–110]. One particular example is the case of Atg5, which during autophagy can translocate to the mitochondria to induce 3.4. Role of ceramide in mediating general autophagy mitochondrial membrane depolarization and caspase dependent cell death [111]. However, autophagic cell death, also known as lethal Ceramide is known to induce both survival and lethal autophagy autophagy, or autosis, can be achieved independent of apoptosis or via several mechanisms that are outlined in Fig. 2 [4,132,133].One necroptosis. This type of cell death is rescued by suppression of autoph- mechanism by which ceramide can induce survival autophagy is by reg- agy by pharmacological or genetic approaches, and involves the action ulating cellular nutrient transporters [134–136]. Transporter proteins 2838 M. Dany, B. Ogretmen / Biochimica et Biophysica Acta 1853 (2015) 2834–2845

Fig. 2. Ceramide's role in survival and lethal autophagy. Ceramide regulates several signaling pathways in autophagy, some of which lead to cytoprotective autophagy and survival while some lead to cell death through lethal autophagy. In the context of survival autophagy, ceramide can induce ER stress that activates the pro-survival IRE1 (inositol-requiring element 1), downregulate nutrient transporters and improves catabolic metabolism, and can result in activation of Atg5 via CD95 and PERK. In the context of lethal autophagy, ceramide can induce ER stress that inhibits mTOR through TRB3, activate ceramide associated phosphatases (CAPPs) that also inhibit mTOR by inactivating Akt, increase the expression of Beclin-1 by activating JNK-c-Jun axis or by inactivating Bax, leading to loss of mitochondrial membrane potential and activation of BNIP3, and bind to LC3-II to recruit autophagosomes to engulf mitochondria.

are required by cells to move nutrients across the plasma membrane. downregulation enhanced ER stress and induced autophagy in an Since these transporters control the cellular fuel supply, alteration of mTOR independent fashion [146]. the expression of the nutrient transporters is one way to affect survival and cell growth. Ceramide was shown to down-regulate the expression of amino acid and nutrient transporters leading to 4. Ceramide-mediated mitophagy starvation, a state that induces survival autophagy by reducing mTOR signaling or activating AMPK [135,137]. Survival autophagy 4.1. Mitophagy: selective autophagy of the mitochondria was also induced in the context of CerS2 downregulation that dys- regulated the normal trafficking of ceramide in the ER, leading to Autophagy was considered to be a general process whereby the long chain ceramide accumulation, and activation of pro-survival autophagosomes engulf many cytoplasmic elements, including mito- IRE-1 (inositol requiring element 1) to prevent induction of cell death chondria, endoplasmic reticulum, and peroxisomes [147]. However, [133]. recent findings suggest that autophagy can be selective to a specificor- Moreover, ceramide is shown to induce lethal autophagy by affecting ganelle. The findings are based on the discovery of specific proteins of the expression of pro-autophagic protein Beclin 1 whereby exogenous the organelles that are required to instigate the autophagy process. treatment of cells with C2-ceramide increased Beclin 1 expression and These include peroxin 14 of peroxisomes in yeast for pexophagy, the induced lethal autophagy [138]. This is due to the conversion of autophagic degradation of peroxisomes; and Uth1p, an outer mitochon-

C2-ceramide to long chain ceramide as the effect was inhibited when drial membrane protein required for selective mitochondrial autophagy, de novo ceramide synthesis was blocked using myriocin, the pharmaco- also known as mitophagy [148–150]. logical inhibitor of SPT [138]. Further evidence indicated that ceramide Aged and dysfunctional mitochondria are removed from cells to increases Beclin 1 expression by activating JNK kinase, which in turn ac- prevent the harm of unhealthy mitochondria, which generate reactive tivates c-Jun, a known transcription factor for Beclin 1 expression [139]. oxygen species, and release pro-apoptotic proteins [151]. This turnover In addition, JNK activation by ceramide leads to Bcl2 phosphorylation process involves the action of autophagosomes and lysosomal hydrolytic allowing it to dissociate from Beclin 1 [140]. In addition, chemothera- enzymes. The term mitophagy has been suggested to refer to such process peutic drugs and arsenic trioxide lead to ceramide production that in- that selectively removes the mitochondria by autophagy [151–153]. crease Beclin1 expression and promote lethal autophagy [141]. Other It has been shown that mitochondria in hepatocytes that had un- drugs such as cannabinoids also lead to ceramide accumulation to in- dergone a mitochondrial permeability transition or a depolarization duce ER stress, mTOR inhibition via TRB3 (tribbles homologue 3), and of the mitochondrial membrane potential are selectively removed by lethal autophagy [142]. autophagosomes [153]. These studies showed that upon loss of mito- Ceramide can be hydrolyzed for the generation of S1P, which plays chondrial membrane potential or during mitochondrial permeability important roles in the regulation of autophagy [19,143]. When cells transition, mitochondria are engulfed by GFP-LC3 positive auto- were subjected to starvation, the activity of sphingosine kinase 1 phagosomes [9,154]. Photo-damaged mitochondria also recruited (SphK1) increased, leading to increased accumulation of S1P [144]. GFP-LC3 positive structures to the damaged areas [155].Itissuggested SphK1 overexpression was able to induce autophagy by inhibiting the that reactive oxygen species (ROS) act as a signal in damaged mitochon- mTOR pathway, but unlike the case of ceramide, this mechanism was dria to recruit the LC3 positive autophagosomes. ROS can activate Atg4B, independent of AKT dephosphorylation [143–145]. In addition, SphK1 theproteasethatisrequiredforLC3-ItobeconvertedtoLC3-II[156,157]. M. Dany, B. Ogretmen / Biochimica et Biophysica Acta 1853 (2015) 2834–2845 2839

4.2. Types of mitophagy the translocation of DRP-1 (dynamin related protein 1) to the mitochondria, where it oligomerizes to bind to Fission 1 (Fis1) in the It is proposed that there are at least three types of mitophagy de- outer mitochondrial membrane [170]. Fusion, a process that fuses pending on the cellular mechanism of sequestration of mitochondria two mitochondria together, involves mitofusin 1 and mitofusin 2, lo- into autophagosomes [152]. cated in the outer mitochondrial membrane, and OPA1 (optic atro- Type 1 mitophagy refers to the mitophagy process that occurs during phy protein 1) located in the inner mitochondrial membrane [171]. nutrient deprivation. The process occurs in coordination with mitochon- OPA1 is processed by mitochondrial peptidase OMA1 and i-AAA pro- drial fission, which starts with the formation of phagophores that enlarge tease YME1L and is regulated by mitofusin 1 during inner mitochondri- to surround mitochondria to form structures called mitophagosomes. al membrane fusion [200,201]. Mitophagosomes are then acidified to activate the hydrolytic enzymes The role of fission and fusion during the mitophagy process is illus- of lysosomes [152,155,158]. trated in several studies. Cells with a knockdown of DRP-1 had sup- Type 2 mitophagy refers to the degradation of mitochondria during pressed rates of mitophagy, whereas cells with overexpression of DRP-1 photo damage. In this case, depolarized mitochondria recruit LC3 positive had excessive mitochondrial disappearance [172–174]. In addition, mito- structures to aggregate onto their surface. These structures then fuse tochondria going through a round of fusion followed by fission generate gether to sequester the mitochondria into a mitophagosome [159–161]. two populations of mitochondria: those that re-fuse and are healthy, This type of mitophagy is not coordinated with mitochondrial fission, un- and those that never re-fuse, have a depolarized membrane potential, like Type 1. Another difference is that Beclin-1 protein is required for and get degraded by mitophagy [173,175]. The loss of the pro-fusion Type 1 mitophagy but not for Type 2. This came from studies showing OPA-1 is a key process for mitophagy, such that OPA-1 overexpression that Type 1 mitophagy can be prevented by pharmacological inhibition wasabletodecreasemitophagy[173,175]. of PI3K using 3-methyladenine or wortmannin. On the other hand, The pro-fission function of DRP-1 makes it an important player during Type 2 mitophagy was shown to be independent of Beclin-1 and PI3K mitophagy. DRP-1 is a cytosolic GTPase with three domains: GTP binding [152]. domain, bundle signaling element (BSE), and a stalk that allows for stable Type 3 mitophagy, also known as micromitophagy, involves the dimerization and oligomerization [176]. Upon its activation, DRP-1 trans- formation of mitochondria-derived vesicles, which translocate to the locates to the mitochondria to form dimers and oligomers that are neces- lysosomes [152,162,163]. The release of mitochondrial derived vesicles sary for fission. It is believed that DRP-1 translocation requires adapter depends on oxidative stress in the mitochondria and involves pink 1 proteins, such as mitochondrial fission factor (MFF), mitochondrial and parkin proteins. Micromitophagy does not involve LC3 or Atg5, elongation factor 1/mitochondrial dynamics proteins of 49 and and it is independent of mitochondrial depolarization or mitochondrial 51 kDa (MIEF1/MiD49/MiD51), and mitochondrial fission protein fission [164]. This process allows the cell to selectively remove damaged Fis1 [177–181]. DRP-1 is regulated by several post-transcriptional modi- or oxidized components of the mitochondria without total degradation. fications such as phosphorylation. There are two sites of phosphorylation for DRP-1: Ser637 and Ser616. DRP-1 is activated when it is phosphory- 4.3. Progression of mitophagy and the involvement of mitochondrial lated by cyclin B1-CDK1 at Ser616 and dephosphorylated by calcineurin fission/fusion at Ser637 [170,182]. DRP-1 is inactivated when it is phosphorylated by protein kinase A at Ser637 [170,182]. Another form of regulation of The signaling pathways involved in the progression of mitophagy DRP-1 is nitrosylation by nitric oxide in the mitochondria, and share many similarities with general autophagy. At baseline, LC3 is dis- deSUMOylation by SENP5 protease, both of which promote DRP-1 acti- persed throughout the cytosol, and some LC3 is found in pre-autophagic vation and dimer formation [182]. structures close to the mitochondrial membrane [80]. During mitophagy, Two other proteins, which are associated with Parkinson's disease, LC3 is conjugated to phosphatidylethanolamine (PE), forming LC3-II. are involved in DRP-1 mediated mitophagy: pink1 and parkin. Pink1 is During type 1 mitophagy, already existing preautophagic structures a serine/threonine protein kinase located inside the mitochondria, enlarge in size to envelope and sequester the mitochondria. This event while parkin is an E3 ubiquitin protein ligase located mainly in the cytosol forms the mitophagosome, which then fuses with a lysosome or a late en- [183–186]. It is believed that Parkin and Pink1 promote mitochondrial dosome to form a mitophagolysosome that digests the mitochondrial fission such that silencing their expression leads to mitochondrial defects content [152,155]. due to lack of fission [184,187]. Pink 1 and Parkin also regulate other mi- The molecular events contributing to mitophagy initiation were first tochondrial functions, including mitochondrial biogenesis, mitochondrial identified in yeast. Studies showed that there are three yeast proteins transport, and calcium homeostasis [188–192]. In healthy mitochondria, participating in mitophagy initiation: outer mitochondrial protein Uth1, Pink1 is cleaved and exported to the cytosol where it is rapidly degraded intermembrane space protein phosphatase Aup1, and inner membrane by proteasomes [200–202]. Upon uncoupling or depolarization of the mi- protein required for K+/H+ exchange Mdm38p [9].Atg32wasidentified tochondria, Pink1 is stabilized, and Parkin is translocated from the cytosol as the main signal to direct autophagosomes to mitochondria after to the mitochondria in a Pink 1-dependent fashion. This leads to ubiquitin interacting with Atg8 and Atg11 [165,166,202]. There are no mammalian mediated proteasomal degradation of outer mitochondrial proteins such homologues for Atg32; however, studies showed that there are some as mitofusin 1 and mitofusin 2, leading to mitochondrial fragmentation receptors on the mitochondrial outer membrane that signal for the and initiation of mitophagy [177,184,193]. DRP-1 is recruited to mito- mitophagy process. For instance, optineurin acts as an autophagy recep- chondrial sites in close proximity of Pink1 and Parkin highlighting their tor in parkin-mediated mitophagy, and FUNDC1 mediates hypoxia importance in DRP-1-dependent mitophagy. In addition, Parkin can in- induced mitophagy [196,197]. Autophagy receptors can also be lipids duce mitochondrial fission independent of Pink1 by affecting DRP-1 in the mitochondrial membrane such as cardiolipin and ceramide phosphorylation [184]. [14,198,199]. Moreover, ROS serve as candidates to initiate mitophagy. Cells Moreover, proteins involved in mitochondria fission/fusion are key supplied exogenously with hydrogen peroxide or superoxide showed regulators for the selective elimination of mitochondria in mammalian evidence of autophagosomal structure formation [166]. It has been cells [167]. Mitochondria are dynamic mobile organelles continuously shown that upon ROS generation in the mitochondria, the mitochondrial dividing or fusing [147].Theprocessesoffusionandfission are intrinsic membrane was depolarized, leading to Parkin translocation and initiation for mitochondrial viability, and they are important in the regulation of of mitophagy [195]. Interestingly, overexpression of the anti-oxidant calcium homeostasis and the generation of ATP and ROS [168,169]. enzyme superoxide dismutase 2 or pre-treatment with antioxidants In addition, fission and fusion of the mitochondria are important prevented ROS-induced mitophagy [194,195].Thissuggeststhatoxida- during mitophagy [9]. Fission, or mitochondrial division, involves tive stress may be an important signal to initiate mitophagy. 2840 M. Dany, B. Ogretmen / Biochimica et Biophysica Acta 1853 (2015) 2834–2845

4.4. Ceramide mediated mitophagy as a tumor suppressor mechanism

There is evidence to support a role for mitophagy in cell survival or death, which appears to be context dependent. Mitophagy promotes cell survival under circumstances where it degrades the mitochondria that are about to activate caspase dependent apoptosis. In this case, disrupting the autophagic and lysosomal processes will prevent survival and lead to apoptosis [152,153]. On the other hand, when mitophagy occurs excessively or for a sustained period of time, enzymes from the lysosomal ﬂux such as cathepsins can leak to the cytosol where they initiate caspase dependent cell death [79,83,152]. Therefore, the functional outcome of mitophagy inducing cell death or survival depends on the intensity and duration of the stress as well as cellular contact [152]. Moreover, mitophagy can serve as a programmed cell death mechanism independent of apoptosis. This mechanism of cell death depends on ceramide synthase 1 (CerS1) and its metabolic product C18-ceramide. Sentelle et al. showed that CerS1 and C18-ceramide selectively induce non-apoptotic lethal mitophagy independent of Bax, Bak, or caspase activity, in head and neck squamous cell carcinoma cells and tumors.

Ectopic expression of CerS1 or treatment with C18-pyridinium-ceramide resulted in LC3-II formation, and promoted its direct binding to ceramide on the mitochondrial membranes. This lipid–protein binding then allowed the mitochondria to be targeted by the LC3-II containing autophagosomes. This report was the ﬁrst to describe the role of ceramide signaling in mediating lethal mitophagy through ceramide-LC3-II binding

(Fig. 3). Interestingly, endogenous C16-ceramide generated by CerS6 did not show any mitophagy promoting function in these cells. However, treatment with C16-pyridinium ceramide, which accumulates in the mitochondria, induced mitophagy. This suggested that the subcellular localization of endogenous ceramides, and not their fatty acid chain length per se, is of great importance to determine their distinct biological actions during mitophagy [14]. The binding of ceramide to LC3-II indicates that ceramide acts as a tumor suppressor lipid that can directly bind proteins. Ceramide is shown to have a higher afﬁnity to the PE-conjugated LC3-II than LC3-I. This interaction was proposed to involve the central hydrophobic domain of LC3 that has structural similarities to the domain of CERT

(ceramide transporter protein) that binds C16-andC18-ceramides. Within this hydrophobic domain, the Ile35 and Phe52 residues of LC3-II were required for ceramide binding. Computational docking simulations and molecular modeling suggested that conjugation of LC3-I to phosphatidylethanolamine hides a low-affinity ceramide-binding sites allowing ceramide to bind selectively to the opposite end of the protein [14,49]. More importantly, although point mutations at the Ile35 and/or Phe52 for conversion to Ala did not prevent ceramide-mediated LC3 lipidation, and inhibition of ceramide binding modulated mitophagy and resulted Fig. 3. Regulation of mitophagy by ceramide. Endogenous generation of C18-ceramide via CerS1 or exogenous treatment by C18-pyridinium-ceramide is followed by two processes: in resistance to ceramide-mediated tumor suppression. Thus, these data A. conjugation of LC3-I to phosphatidylethanolamine on the carboxy terminal to form LC3-II support that ceramide plays a novel receptor role at the mitochondrial and B. accumulation of ceramide in the mitochondrial outer membrane. Ceramide in the mi- membranes to recruit LC3-II-containing autophagosomes to the mito- tochondrial membrane acts as a receptor to LC3-II by binding to its amino terminal, opposite chondria, which have been subjected to DRP-1-mediated fission. These to where PE is conjugated. This results in C. recruiting the autophagosome to engulf the mitochondria. Lysosomes then fuse with the autophagosomes (D) for hydrolytic degradation of data also suggest that targeting LC3 containing autophagosomes to mito- the contents. chondria by ceramide at the mitochondrial membranes results in cancer cell death and tumor suppression, which seems to be regulated down- stream of DRP1-mediated mitochondrial fission. Interestingly, in the regulates lethal autophagic signaling pathways is its activation of c-Jun absence of CerS1 overexpression, tumor cells with knockdown of LC3 through JNK signaling, causing upregulated Beclin 1 expression and had a reduced growth in vivo suggesting that at baseline, LC3 mediated autophagic cell death [138]. Exogenous supply of C18-pyridinium cer- autophagy is required for tumor growth in head and neck squamous amide or overexpression of CerS1 resulted in caspase independent cell carcinoma tumors [14,49]. mitophagy where ceramide acts as a mitochondrial receptor for LC3- II-containing autophagosomes by interacting directly with LC3-II, 5. Conclusions and future perspectives recruiting autophagolysosomes to damaged mitochondria [14]. Exoge- nous ceramide mediated autophagic cell death is believed to involve Ceramide, as a bioactive sphingolipid, plays key roles in the regula- BNIP3 activation after a reduction in mitochondrial membrane potential tion of general and selective autophagy and/or mitophagy. This role of [140]. Chemotherapeutic drugs and arsenic trioxide lead to ceramide pro- ceramide is of great importance in tumor biology as in most cases duction that increases Beclin1 expression and promotes lethal autophagy ceramide mediated autophagy leads to cell death and is thus called [141]. Other drugs such as cannabinoids also lead to ceramide accumula- lethal autophagy or autosis [108,114]. One example by which ceramide tion to induce ER stress, mTOR inhibition via TRB3 (tribbles homologue M. Dany, B. Ogretmen / Biochimica et Biophysica Acta 1853 (2015) 2834–2845 2841

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