Sphingosine metabolism as a therapeutic target in cutaneous melanoma

Accepted Manuscript Sphingosine metabolism as a therapeutic target in cutaneous melanoma Mohammed Dany PII:

S1931-5244(17)30099-3

DOI:

10.1016/j.trsl.2017.04.005

Reference:

TRSL 1148

To appear in:

Translational Research

Received Date: 15 February 2017 Revised Date:

26 March 2017

Accepted Date: 25 April 2017

Please cite this article as: Dany M, Sphingosine metabolism as a therapeutic target in cutaneous melanoma, Translational Research (2017), doi: 10.1016/j.trsl.2017.04.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Sphingosine metabolism as a therapeutic target in cutaneous melanoma Mohammed Dany College of Medicine, Medical University of South Carolina, Charleston, SC

Abbreviated Title: Sphingolipids in melanoma

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Featured New Investigator Submission

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* Corresponding author at: Medical University of South Carolina, Charleston, SC 29425, USA. Tel: +1 843-489-1599; E-mail address: [email protected].

Keywords: Ceramide; Sphingosine-1-phosphate; melanoma; lipid signaling; sphingolipids

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Conflict of interest: none

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Abstract Melanoma is by far the most aggressive type of skin cancer with a poor prognosis in its advanced stages. Understanding the mechanisms involved in melanoma pathogenesis,

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response and resistance to treatment has gained a lot of attention worldwide. Recently, the role of sphingolipid metabolism has been studied in cutaneous melanoma. Sphingolipids are bioactive lipid effector molecules involved in the regulation of various

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cellular signaling pathways such as inflammation, cancer cell proliferation, death, senescence, and metastasis. Recent studies suggest that sphingolipid metabolism

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impacts melanoma pathogenesis and is a potential therapeutic target. This review focuses on defining the role of sphingolipid metabolism in melanoma carcinogenesis, discussing sphingolipid-based therapeutic approaches, and highlighting the areas that

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require more extensive research.

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1. Introduction According to the National Cancer Institute, the incidence of invasive melanoma in the United States was estimated to be about 73,870 cases in 2015, and one American dies

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of melanoma every hour (1). Melanoma treatment depends on the stage of the cancer. Early lesions are often cured by surgical excision alone, while more advanced disease requires systemic therapy (2). The 10-year overall survival rate for advanced melanoma

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is improving, but is still only 10-15% (3,4). One challenge in the field of melanoma is understanding the impact of the different metabolic pathways on melanoma

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pathogenesis. In particular, it is important to understand the role of the metabolism of cancer related bioactive molecules in melanoma pathogenesis. This will help in developing novel therapeutic strategies that target the metabolism of such bioactive molecules.

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Advances in sphingolipid research are continuously proving the impact of sphingolipid metabolism on cancer pathogenesis (5). Sphingolipids are membrane lipids that were discovered for their important functions in regulating membrane fluidity and subdomain

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structures. Indeed, sphingolipids have long been recognized as important structural components of the epidermis, securing the epidermal permeability barrier (6). Recently,

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it was revealed that sphingolipids have signaling roles apart from their structural roles. In particular, several sphingolipid species were shown to act as bioactive signaling lipids in cancer cells. Ceramide is one of the most bioactive sphingolipids that plays a key role in many of the cellular functions including cell proliferation, death, migration, and senescence (7). Ceramides account for 30–40% of stratum corneum lipids (8). Ceramide metabolism is intimately involved in cancer pathogenesis. Ceramide and its

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metabolites are not only involved in cancer initiation and progression, but also in the response of cancer cells to chemotherapeutic agents and radiation induced cell death. Unlike ceramide, Sphingosine 1 Phosphate (S1P) is a tumor promoting bioactive

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sphingolipid. The intricate balance between the levels of ceramide and S1P dictates whether a cancer cell undergoes proliferation or cell death. This is known as the ceramide-S1P rheostat and can be targeted by several approaches to sway the balance

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towards ceramide generation or inhibition of S1P synthesis, to result in cancer cell death. In this review article, we discuss the role of sphingolipid metabolism in the tumor

target sphingolipid metabolism.

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pathogenesis of melanoma and the different potential therapeutic approaches that

2. Metabolism of biologically active sphingolipids: ceramide and S1P Ceramide is composed of a sphingosine backbone esterified to a fatty acyl chain via an

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amide linkage (5,7). The length of the fatty acyl chain conjugated to the sphingosine backbone characterizes the different species of ceramide. For instance, C18-ceramide

ceramides (5).

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contains a fatty acyl chain with n=18 carbons. Ceramide species range from C14- to C26-

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Ceramide lies at the center of sphingolipid metabolism. It can serve as a precursor for more complex sphingolipids, or as a product of the breakdown of other sphingolipids (Figure 1) (5). For example, ceramide acts as a precursor for the generation of glucosylceramides and as a product of the breakdown of sphingomyelin (9). De novo generation of ceramide involves the action of ceramide synthases 1-6 (CerS16). CerS1-6 catalyze the reaction of esterifying a fatty acyl CoA to dihydrosphingosine to

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generate dihydroceramide (10). Ceramide is then generated by the action of desaturase enzyme which inserts a double bond between carbons 4 and 5 (11).

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The catabolism of ceramide starts with the action of the enzyme ceramidase yielding sphingosine. Sphingosine then gets phosphorylated by sphingosine kinases 1 or 2 (SK1 SK2)

to

generate

dephosphorylated

sphingosine

by

S1P

1-phosphate

phosphatases

or

ethanolaminephosphate by S1P lyase (12) (Figure 1).

(S1P). lysed

S1P

to

then

is

hexadecanal

either and

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or

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The subcellular compartmentalization of sphingolipid metabolism is important for the biological function of sphingolipid molecules. The compartmentalization is based on the localization of sphingolipid metabolic enzymes (5,13). For example, de novo ceramide synthesis occurs in the endoplasmic reticulum (ER) because CerS1-6 enzymes are localized to the ER. For the synthesis of SM, ceramide is transported from the ER to the

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Golgi apparatus by ceramide transporter protein (CERT) without the help of vesicles (14,15). Similarly, for the synthesis of glucosylceramide (GlcCer), ceramide is

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transported to the Golgi by Fabb2 transporter (15). Ceramide is also detected in mitochondria due to either de novo synthesis or sphingomyelin degradation (5). In

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addition, Sphingosine kinase enzymes 1 and 2 differ in their localization: SK1 is mainly cytosolic whilst SK2 is mainly nuclear. Thus, it is believed that S1P plays different roles depending on whether it is generated by SK1 or SK2 (16).

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3. Biological roles of ceramide in cancer and melanoma pathogenesis

a. Biological functions of Ceramide Synthases 1-6

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Ceramide Synthases 1-6 have different subcellular localizations and exhibit specificity for the generation of endogenous ceramides with different fatty acid chain lengths (17,18). For example, CerS1 mainly generates C18-ceramide, CerS4 generates both

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C18-ceramide and C20-ceramide, and CerS5 and CerS6 generate mainly C16-ceramide, and to a lesser extent, C12- and C14- ceramides. CerS2 and CerS3 are known to

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generate very long chain C22-24- and C26- ceramides, respectively (5,7,9,19-21). In addition, ceramide synthases are expressed differently in human tissues. For example, CerS1 is mainly expressed in brain and skeletal muscle tissues (22). On the other hand, CerS3 is only expressed in skin and testicular tissues (23,24). CerS4 is also

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expressed in the skin as well as in leukocytes, cardiomyocytes, and hepatocytes (25). Different species of ceramide with distinct fatty acyl chain lengths have diverse biological functions (Table 1). For example, CerS1 and CerS6, generating C18-ceramide

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and C16-ceramide respectively, have opposing roles in cell death and proliferation. CerS1/C18-ceramide axis leads to cancer cell death and decreases tumor growth (26-

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29). Increased levels of serum C18-ceramide act as a potential biomarker to monitor patients’ response to chemotherapy (30). On the other hand, C16-ceramide can promote cancer cell proliferation and its increased serum levels associate with a positive lymph node status (31,32). On the other hand, there are studies showing that C16-ceramide is pro-apoptotic, whereas C24-ceramide protects from cell death (33,34).

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In vivo, Ceramide Synthases play different roles and knocking down their expression result in different phenotypes. Mice expressing a catalytically inactive mutant of CerS1 (toppler mice) exhibit neurological disorders secondary to Purkinjean cells dysfunction.

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Mice with a knockout in CerS2 suffer from liver cirrhosis. Mice with CerS4 knockout have severe alopecia secondary to ceramide content alterations in the sebum. Finally, mice with CerS6 knockout exhibit behavioral alterations (35-37).

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The role of CerSs in melanoma is not extensively studied yet. One study investigated

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the role of CerS6 on the malignant behavior of melanoma (38). The expression of CerS6 was decreased in several melanoma cell lines and the lower expression was correlated with malignant potential. Silencing the expression of CerS6 in three melanoma cell lines resulted in more proliferation and invasion. Silencing of CerS6 induced the expression of GLUT1 (Glucose transporter 1), which in turn downregulated

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the expression of WNT5A, a growth factor that enhances the invasion and proliferation of melanoma cells (38). Thus, in melanoma, CerS6 acts as a tumor suppressor protein

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whose overexpression can provide a therapeutic strategy for melanoma treatment.

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b. Ceramide as a tumor suppressor lipid

It is well established that ceramide is a pro-cell death molecule in several cancer models (22). Ceramide can trigger several programmed cell death mechanisms such as apoptosis, necroptosis, lethal autophagy, and lethal mitophagy (22,39-44). In fact, ceramide levels increase during stress like hypoxia, growth factor withdrawal, or DNA

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damage (45). Ceramide can then associate with death-associated proteins to trigger programmed cell death.

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There are several mechanisms by which ceramide acts as a tumor suppressor lipid. Ceramide binds and inactivates the inhibitor of protein phosphatase 2A (46). PP2A is itself a tumor suppressor protein (47,48). Ceramide binds to the biological inhibitor of PP2A, I2PP2A or SET oncoprotein, leading to PP2A reactivation. PP2A can then

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0inactivate anti-apoptotic proteins such as Bcl-2 (49,50).

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Another mechanism is that ceramide regulates human telomerase. Ceramide accumulation in cancer cells inhibits inactivates c-myc transcription factor and thus decreases telomerase expression. This results in less elongation of chromosomal telomeric ends after each replication cycle, leading eventually to senescence and cell

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death (51,52).

Recently, a novel mechanism was discovered explaining ceramide’s role in mediating cell death by attaching the mitochondria (53). In response to tyrosine kinase inhibitor

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treatment, ceramide localized to mitochondria of cancer cells and served as a receptor to anchor autophagy related protein LC3B-II and to thus recruit autophagosomes.

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Ceramide localization to mitochondria was mediated by dynamin related protein 1 (Drp1) activation resulting in mitochondrial fission. Inhibition of Drp1 prevented ceramide-dependent lethal mitophagy and reconstitution of WT-Drp1 restored mitochondrial fission and mitophagy (39,54). In addition to tumor suppression, ceramide plays a role in suppressing metastasis by regulating integrin cell surface expression. In particular, treating cancer cells with the

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short chain C6-ceramide resulted in decreased expression of cell surface β1 and β4 integrin subunits and αVβ6 integrin (55). Lower levels of integrin resulted in diminished cellular adhesion, reduced cellular migration, and suppression of tumor

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metastasis (55).

In the context of cutaneous melanoma, few studies have confirmed that upregulation of ceramide generation is required for melanoma induced cell death and better response

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to treatment. Treatment with exogenous ceramide, such as liposomal C6 ceramide, was

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cytotoxic and anti-proliferative against a panel of five human melanoma cell lines. Liposomal C6 ceramide induced caspase-dependent apoptotic cell death by activating protein phosphatase 1 (PP1) (56). In addition, treatment of melanoma cancer cells with nanoliposomal C6-ceramide for only 30 minutes suppressed cell migration and decreased tumor extravasation under shear conditions. This was secondary to reducing

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integrin affinity and activating PI3K and PKCζ tumor-suppressive activities (57). Moreover, ceramide plays an important role in the metastasis of melanoma. Metastatic

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dissemination of melanoma cancer cells is one of the hallmarks of melanoma malignancy and accounts for most of melanoma cancer deaths. Melanoma cells in the

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blood vasculature may aggregate with platelets to form clots and then adhere to endothelial cells in order to finally extravasate to form metastatic niches. Interestingly, the interaction between melanoma cells and platelets results in the activation of acid sphingomyelinase and the release of ceramide. Acid sphingomyelinase activation was shown to be required for trapping of melanoma cells in the lungs of mice. Indeed, preincubation of tumor cells with recombinant acid sphingomyelinase resulted in melanoma cell colony formation in the lung of mice (58).

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4. Acid ceramidase is implicated in melanoma pathogenesis

Acid ceramidase is an enzyme localized to lysosomes and catalyzes the degradation of

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ceramide into sphingosine, thus lowering ceramide levels. One study found that melanoma cells generate lower amounts of ceramides compared with normal

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melanocytes due to suppression of ceramide de novo synthesis pathways (59). In addition, the study found that ceramide hydrolysis is increased in melanoma cancer cells. The expression of acid ceramidase, an enzyme that lowers the levels of ceramides, is significantly elevated in melanoma cell lines compared with non-

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cancerous skin cells. Acid ceramidase expression is also elevated in biopsies from human subjects with Stage II melanoma. Interestingly, treatment with acid ceramidase inhibitor resulted in increased ceramide levels and induction of cell death (59). The

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results of this study suggest that the carcinogenesis of melanoma might involve decreasing intracellular ceramide levels via decreasing its production and increasing its

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degradation by acid ceramidase. Another study found that Dacarbazine, a chemotherapy agent used for the treatment of melanoma, induces cathepsin B-mediated degradation of acid ceramidase in human melanoma cells. Indeed, Dacarbazine elicited a time and dose dependent decrease of acid ceramidase activity and an increase of intracellular ceramide levels (60). The downregulation of acid ceramidase was required for the melanoma cells to respond to

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Dacabazine as inducible overexpression of acid ceramidase reduced ceramide levels and conferred resistance to Dacarbazine. The study suggests that increasing ceramide levels, secondary to degradation of acid ceramidase, is required to induce cell death in

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melanoma cells treated with Dacarbazine (60).

Moreover, a recent study discussed the expression and localization of acid ceramidase in melanoma (59). The findings of this study supported that acid ceramidase is

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overexpressed in melanoma resulting in both: lower levels of ceramide and increasing

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levels of S1P. Interestingly, the localization of acid ceramidase is different in melanocytes vs melanoma cells. In melanocytes, acid ceramidase is localized to both the cytosol and the nucleus, while in melanoma cells it is primarily found in the cytosol (59). This study adds evidence to the role of acid ceramidase in controlling sphingolipid

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metabolism in melanoma and thus melanoma proliferation.

5. Roles of SK in cancer and melanoma pathogenesis

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a. Sphingosine Kinases and S1P in cancer biology and drug resistance

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The ceramide/S1P rheostat impacts the fate of a cancer cell (Figure 2). Stressful stimuli such as radiation or chemotherapy treatment shift the balance towards ceramide to halt cell proliferation and promote cell death (5). However, in some cases, there is increased activity in Sphingosine Kinases, enzymes that will convert ceramide into S1P. The shift in the balance to S1P results in pro-survival, anti-apoptosis, and resistance to radiation or chemotherapy (20).

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Sphingosine kinases (SK1 and SK2) are lipid kinases, encoded by SPHK1 and SPHK2 genes in humans (61). SK1 and SK2 differ structurally by the presence of a transmembrane domain in SK2 that is not found in SK1. In addition, SK1 and SK2

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expression is tissue specific whereby SK1 is more abundant in lung and spleen tissues, whilst SK2 is more expressed in liver and heart tissues. Intracellularly, SK1 and SK2 seem to localize to different organelles, whereby SK1 is found mainly in the cytosol

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whereas SK2 can be detected in both cytoplasm and nucleus (61,62).

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The importance of SK enzymes to human diseases is revealed in the knockout phenotypes; even though SphK1 and SphK2 null mice survive, SphK double knockout is embryonically lethal (63).

When it comes to the context of cancer, SK1 is well established as an onco-protein. Cells overexpressing SK1 demonstrated a transformation potential (64) and formed

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tumors in nude mice (65). SK1 expression is also required for ras mediated transformation. Clinically, the mRNA and protein expression of SK1 is elevated in

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several cancerous tissues such as ovarian, breast, lung, and gastric tumors (66,67). In addition, microarray datasets reveal increased expression of SK1 in melanoma,

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squamous cell carcinomas, cervical cancer, head and neck tumors, and hematological malignancies (68-70).

SK1 acts as an onco-protein by generating S1P onco-lipid. Intracellularly, S1P generated from SK1 can then target several proteins. For example, S1P can bind to TRAF2, an E3 ubiquitin ligase, to modulate TNF-α induced activation of NF-κB mitogenic signaling (71). In addition, extracellularly, serum S1P generated by SK1, can promote lung tumor metastasis by affecting breast cancer metastasis suppressor 1

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(Brms1) expression via S1PR2 signaling. Thus, silencing SK1 expression or treatment with anti-S1P monoclonal antibody (Sphingomab) decreased lung metastasis by

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decreasing serum S1P association with S1PR2 and increasing Brms1 expression (72). SK2 is localized mainly to the nucleus and has a nuclear export signals. Some reports also detected SK2 in cytosol, ER, and mitochondria (73). Inside the nucleus, SK2 can regulate cell cycle and cell proliferation (74,75). S1P generated by SK2 in the nucleus

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can bind to and inhibit HDAC1/2 enzymatic activity thereby preventing deacetylation of

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histone H3. This promoted the expression of p21 and c-Fos genes. Inside the mitochondria, S1P generated by SK2 can bind to prohibitin 2 but not to prohibitin 1, thereby regulating cytochrome c oxidase complex IV (76).

S1P can be secreted to the extracellular matrix and bind to S1P Receptors (S1PR1-5) in an autocrine or paracrine fashion. The major downstream responses are related to

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increased inflammation, cell migration, and angiogenesis (77). In particular, S1P-S1PR1 interaction helps in activating receptor tyrosine kinases such as EGF and PDGF. A

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recent study that identified the X-ray structure of ligand bound S1PR1, suggested that S1PR activation does not necessarily require the export of S1P to the extracellular

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surroundings. However, S1P can interact with its receptor within the plasma membrane via lateral movements of S1P in the membrane (78). It is well established in several studies that S1P protects cancer cells from cell death in response to various cytotoxic agents. For instance, SK1 upregulation resulted in camptothecin resistance in prostate cancer cells, which was reversed upon inhibiting SK1 pharmacologically (76). In another study, upregulation of SK1 resulted in resistance of prostate cancer cells to radiation therapy, but were re-sensitized if pre-

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treated with SK inhibitor N,N-dimethyl-sphingosine (79). Other examples include SK1 mediated resistance in FAS mediated cell death in melanoma and tamoxifen induced

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cell death in breast cancer cells (80). In the context of AML-M6, known as erythro-leukemia, SK1 overexpression in proerythroblasts transformed them into tumor cells resistant to cell death. In addition,

which was required for leukemic progression (81).

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microarray datasets revealed upregulation of SK1 in tumorigenic pro-erythroblasts

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In Chronic Myeloid Leukemia (CML), ceramide/S1P rheostat plays an important role in inducing drug resistance to bcr-abl targeted therapy. CML cells resistant to imatinib treatment had higher levels of SK1. Silencing SK1 expression sensitized the cells to imatinib induced cell death (81). S1P generated by SK1 binds and inhibits PP2A to prevent

bcr-abl

dephosphorylation

and

subsequent

degradation.

Inhibition

of

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SK1/S1P/S1PR2 signaling either by pharmacological or molecular approaches restored PP2A mediated dephosphorylation of Bcr-Abl1 and enhanced imatinib mediated

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apoptosis (82).

Most of the studies highlight the role of S1P generated by SK1 in drug resistance.

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However, recently S1P generated by SK2 also had a role in conferring resistance in several types of cancer. For example, in estrogen receptor positive breast cancer cells, SK2 increased transcription of ER-regulated genes like SDF1 and progesterone receptor. Pharmacological inhibition of SK2 using ABC294640 decreased the expression of these genes and sensitized the cells to treatment (83). Another described mechanism is the ability of ABC294640 (SK2 inhibitor) to decrease NF-κB pro survival

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signaling by decreasing activation p65 (84). In vivo, oral administration of ABC294640 inhibited the growth of xenograft-derived tumors mice (83).

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In the context of lung cancer, hypoxia can increase SK2 generation of S1P, which can then be secreted to activate S1PR1/S1PR3 signaling and activate MAP kinase signaling. This resulted in resistance to etoposide chemotherapeutic agent (85). In colon

outside the nucleus to confer the resistance (86).

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cancer cells resistant to sodium butyrate, SK2 was phosphorylated and exported

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In some cases, S1P can paradoxically induce apoptosis in cancer cells. This paradoxical effect was observed when S1P was generated by SK2. Indeed, some studies demonstrated that overexpression of SK2, but not SK1, suppresses growth and enhances apoptosis (87). These studies imply that the cellular location of S1P generation determines the impact of S1P on cell death. SK1 is mostly cytosolic and thus

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S1P generated from SK1 impacts cytosolic signaling pathways and signals via S1PRs, while SK2 is mostly nuclear and thus S1P generated from SK2 impacts nuclear

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signaling pathways (41).

Similarly, sphingosine can also induce cell death in response to DNA damage. One

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recent study showed that sphingosine generated from alkaline ceramidase 2 (ACER2) induces programmed cell death (88). Upon treating cancer cells with DNA damaging agents such as doxorubicin, ACER2 expression was upregulated significantly and sphingosine levels increased resulting in apoptosis and necroptosis (88).

b. S1P in the skin

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As mentioned earlier, ceramide inhibits keratinocyte growth and induces apoptosis as in most other systems (89). Unexpectedly, S1P, normally a ceramide antagonist, also inhibits keratinocyte proliferation. However, S1P does not drive keratinocytes into

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apoptosis, but rather induces their differentiation (90,91). Treating keratinocytes with S1P results in cell cycle arrest, inhibition of cell division, and an increase in intracellular calcium (92). Calcium is an important signaling molecule in keratinocytes for their

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differentiation to into corneocytes (93). S1P induces these effects in keratinocytes by

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binding to S1PR2 receptor (92).

In addition, S1P modulates some of the functions of dendritic cells such as uptake of antigens and interaction with T cells. S1P decreases the endocytic ability of dendritic cells, thus limiting their antigen uptake capacity (94). In fact, S1P topical application in a mouse model reduced foreign antigen uptake by dendritic cells down to 40%. This is

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again secondary to S1P mediated inhibition of Akt (94). In addition, S1P modulates the migration of dendritic cells. Studies show that dendritic cells migrate towards lymph nodes, to areas rich with S1P, following the S1P gradient. Disrupting the gradient by

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topical application of S1P prevented the migration of dendritic cells from the epidermis

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to the lymph nodes (95). Finally, S1P exerts anti-inflammatory roles in the skin by decreasing the levels of IL-12 release from activated dendritic cells and by decreasing the activation of T helper cells (96). c. Sphingosine kinases and S1P in melanoma pathogenesis and drug resistance Even though S1P inhibits the proliferation of keratinocytes, this is not the case in human melanoma cells. Treatment of melanoma cells with S1P inhibited drug induced cell death. This was prevented by the pre-treatment with short-chain C6-ceramide (97). In

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addition, elevated S1P levels secondary to increased activity of SK1 occur in advanced melanomas. Targeting SK1 using siRNA decreased growth and induced apoptosis in melanoma cells. Pharmacological inhibition of SK1 in melanoma using the inhibitor SKI-

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I decreased S1P content, elevated ceramide levels, caused a G2-M block, and induced apoptotic cell death (98). This suggests that targeting SK1 using siRNAs or SKI-I inhibitor has therapeutic potential for melanoma treatment.

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Indeed, several studies demonstrate that human melanoma cell death is induced upon

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the inhibition of sphingosine kinases or the blockade of S1P receptors. In one study, the sphingosine analogue FTY720, which functionally antagonizes S1P receptors, induced cell death in human melanoma cells through a mechanism involving the vacuolar ATPase activity. Interestingly, cell death was characterized by features of necrosis and autophagy (99). In another study, FTY720 significantly decreased the viability and

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expression of apoptosis markers in cisplatin resistant human melanoma cell lines (100). These finding suggest that FTY720 or other sphingosine receptor inhibitors are promising candidates for further development as new therapeutic agents in the

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treatment of melanoma.

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Sphingosine kinases play several roles in melanoma pathogenesis. For instance, downregulation of sphingosine kinase-1 induces protective tumor immunity in melanoma. The infiltration of melanoma tumors by M2 macrophages is often correlated with poor prognosis. Downregulation of SK1 in melanoma cells reduced the percentage of M2 macrophages in favor of increased M1 macrophages into the tumor. Infiltration by M1 macrophages recruited T lymphocytes and hence tumor rejection. This suggest that

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SK1 plays a key role in the recruitment and phenotypic shift of the tumor macrophages that promote melanoma growth (101).

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Moreover, S1P plays an important role in the metastasis of melanoma. SK1 facilitates the communication between melanoma cells and fibroblasts in metastasis. SK1 in myofibroblasts promotes S1P-dependent dissemination of melanoma cells via a S1P receptor 3 (102). These findings clarify the role of S1P in melanoma’s

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microenvironment.

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One paradox; however, is that one study show that S1P can inhibit cell migration in B16F10 mouse melanoma cells (103). Inhibition of migration was dependent on the endogenous expression of S1P receptor 2 (S1PR2). Overexpression of S1PR2 in melanoma cells potentiated S1P’s effect in inhibiting tumor cell migration. These observations led to the suggestion that sphingosine agonists specific for S1PR2 might

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be therapeutically beneficial in the treatment of melanoma. In addition, some nonphosphorylated sphingosine analogs, N-methyl sphingosine and N-acetyl sphingosine,

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have been shown to protect against induction of skin carcinomas in a mouse skin carcinogenesis model (104). It seems that S1P’s effect on melanoma depends on the

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receptor it is binding to. S1P signaling through S1PR2 can inhibit melanoma migration while S1P signaling through other S1PRs can result in metastasis and viability of melanoma cells. Clearly, the role of S1P and the different S1PRs need to be studies more extensively in melanoma cells, fibroblasts, myofibroblasts, and platelet. In addition, the role of SK2, which is usually located in the nucleus, needs to be studied in melanoma.

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7. Sphingolipid based anti-cancer therapeutics

a. Anti-cancerous ceramide analogues and mimetics

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Exogenous ceramide analogues or mimetics have been used in several studies as a strategy to induce programmed cell death in cancer cells. Several modifications in the structure and delivery methods of ceramide were adopted in order to maximize the

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efficiency of exogenous ceramide (Table 1). Most notably, modifying ceramide by adding a pyridinium ring in the sphingosine backbone is an ingenious method to

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increase water and cell membrane solubility and allows targeting and accumulation of ceramide analogues into the negatively charged mitochondria, and to a lesser extent, to the nucleus of cancer cells (39). In addition, this offers a selective delivery of the ceramide analogues to cancer cells versus normal cells since it is suggested that

pyridinium-ceramide

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cancer cells acquire more negative charge in their mitochondria (105). Indeed, the analogues

L-threo-C6-Pyr-Cer

and

D-erythro-C16-Pyr-Cer

accumulate in mitochondria and to a lesser extent in nuclei fractions in vitro (106-110).

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In addition, ceramide analogues accumulated in vivo in the tumor site more so than in other intact organs (39,109). Therefore, Pyr-Cer can preferentially target cancer cells

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with minimum toxicity to normal cells. Other structural analogs of ceramide include C16-serinol and 4,6-diene-ceramide, 5ROH-3E-C8-ceramide, adamantyl-ceramide, and benzene-C4-ceramide. These analogues were also able to either induce apoptosis in various human cancer cells (111,112) or inhibit the growth of drug-resistant human cancer cell lines (113). It should also be noted that treatment of cells with exogenous ceramides may result in the generation of

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endogenous long chain ceramides via the sphingosine-recycling pathway and this might add to the anti-cancerous effect.

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The delivery method of ceramide was also modified so that ceramide can be packaged in PEGylated liposomes, which are known to be more effective for crossing the cell membrane. This formulation increased the accumulation of ceramide and thus the procell death effect in cancer cells (114). Mechanistically, ceramide delivered in liposomes

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prevented the phosphorylation of Akt and stimulated the activity of caspase-3/7 more

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effectively than non-liposomal ceramide (114). The liposomal formulation was also effective in vivo, resulting in slower tumor growth in murine models of breast cancer (115).

Another approach is to encapsulated vincristine in sphingomyelin-liposomes, also called

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sphingosomes. This method had a higher effect compared to the conventional vincristine injections in animal models of adult acute lymphocytic leukemia (ALL). Sphingosomal vincristine is now in phase II clinical trials for recurrent and refractory ALL

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(116). Another method is polymeric nano-particle delivery system to improve the delivery and efficacy of ceramides to overcome resistance (117).

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b. SK/S1P anticancer therapeutics Given the importance of SK1-SK2/S1P signaling in promoting tumor progression and developing resistance, several approaches were developed to target SK enzymes and S1P for therapeutic purposes (Table 2). Non-selective sphingosine kinase inhibitors: N, N-dimethyl sphingosine (DMS) exhibited anti-tumor growth properties in cancer cell lines and nude mice. Side effects of DMS

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include liver toxicity and hemolysis (118). L-threo dihyrosphingosine (Safingol) is currently in phase I clinical trials (French, Schwartz). Another SK inhibitor is Phenoxodiol with anti-cancerous and anti-angiogenic effects, and is currently in clinical

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trials for ovarian and prostate cancers (119,120). Another inhibitor is SKI-II, which has excellent oral bioavailability and anti-tumor effects in vivo (121).

SK1 selective inhibitors: SK1-I inhibitor is specific for SK1 with established in vivo

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orthotopic anti-tumor effects, sub-cutaneous glioblastoma multiforme, and AML (122). In

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breast cancer mouse models, SK1-I decreased serum S1P levels and decreased tumor burden (121).

SK2 selective inhibitors: ABC294640 is the SK2 specific inhibitor. It has a very high oral bioavailability and a low toxicology profile. ABC294640 has anti-proliferative effects in a panel of cancer cell lines and in vivo mouse models (83,84,123). ABC294640 is

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currently in phase II clinical trials for diffuse large B cell lymphoma. Antibody based therapeutics: A monoclonal antibody against S1P (Sphingomab) was

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developed and was shown to be effective in neutralizing S1P and exert anti-cancerous effects in breast, ovarian, lung, and melanoma cancer models (72). In addition,

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Sphingomab (LT1009) can neutralize VEGF-induced angiogenesis (visentin). LT1009 is currently in Phase I/ II clinical trials for neovascular Age Related Macular Degeneration and some types of cancer (124).

8. Conclusion

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The last decade has witnessed a dramatic increase in researching the mechanisms involved in melanoma pathogenesis, response to treatment, and resistance. Several studies

show

convincingly

that

sphingolipid

signaling

affects

melanoma’s

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carcinogenesis, regulate the fate of treated melanoma cells, and impact migration and metastasis. Specifically, the accumulation of intracellular ceramide lipid seems to drive melanoma cell death and response to treatment, while SK generated S1P seem to

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oppose cell death and promote resistance. This suggests that ceramide analogues, SK inhibitors, and S1P antibodies, can potentially be used in the treatment of melanoma. In

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addition, acid ceramidase is emerging as another target in melanoma cells. By overexpressing acid ceramidase, melanoma cells hydrolyze ceramide ending up with lower levels of ceramide. There are several acid ceramidase inhibitors that can be tested in vivo or in phase 1 clinical trials.

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The role of S1P in the skin should be studied more extensively. It is interesting to understand the reason behind why keratinocytes and melanoma cells respond differently to S1P: S1P promotes keratinocyte cell death but melanoma cell survival.

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Other questions to answer are whether the ceramide levels are the same in these two

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cell types and whether the S1P downstream signaling molecules, such as Akt, are altered in melanoma.

Finally, the role of S1P receptors S1PR1-5 in melanoma should be further characterized. Even though S1P binding to S1PR3 has been reported to promote metastasis, the underlying mechanism is not fully understood. S1PR inhibitors, such as FTY720, have been shown to be very effective in inducing cell death and decreasing

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melanoma tumor burden in vivo. More studies need to be performed to characterize the

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role of each S1PR in melanoma and their role as therapeutic targets.

Acknowledgements •

There is no conflict of interest to be declared.

•

All authors have read the journal's policy on disclosure of potential conflicts of

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interest. •

No editorial sources have been used for preparation of the manuscript.

•

All authors have read the journal's authorship agreement and that the manuscript

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has been reviewed by and approved by all named authors.

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Figure Legends

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Figure 1. Metabolism of ceramide and S1P. Ceramide lies at the center of sphingolipid metabolism. de novo generation of ceramide requires the condensation of Serine and Palmitoyl CoA by the enzyme Serine Palmitoyl CoA Transferase (SPT), to generate 3-ketosphinganine, which is then converted to dihydrosphingosine. Ceramide

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Synthases (CerS1-6) generate dihydroceramide which gets desaturated to ceramide by Desaturase enzyme (DES). Ceramide can be converted to S1P via ceramidase followed by the action of SK1 or SK2. Ceramide can be generated back from S1P via

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sphingosine-1-phosphate-phosphatase (S1PP). Ceramide can be generated from sphingomyelin via sphingomyelinase (SMase) and from Glycosphingolipids via

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hexosylceramidase. Ceramide also serves as the precursor of sphingomyelin via Sphingomyelin Synthase (SMS) and gangliosides using several enzymes such as hexosylceramide synthase and GM synthase.

Figure 2. Ceramide/S1P rheostat. The balance between ceramide and S1P signaling can determine whether a cancer cell would survive or proceed to cell death.

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Accumulation of ceramide promotes susceptibility to anti-cancer drugs and cell death;

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however, accumulation of S1P is implicated with drug resistance and survival.

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Table 1. Ceramide based anticancer therapeutics Mechanism of action

Cancer Type

Pyridinium-Ceramides

Mitochondrial accumulation

Lung, breast, melanoma

C16-serinol

Ceramide analogue

Breast

4,6-diene-ceramide

Ceramide analogue

Neuroblastoma

Ceramide nanoparticles

Liposomes to improve delivery

D-MAPP

Ceramidase inhibitor

B13

Acid Ceramidase inhibitor

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Compound

Breast, leukemia

Squamous Cell Carcinomas

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Prostate, Colon, Head and Neck

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Table 2. Sphingosine kinase/S1P/S1PR based anti-cancer therapeutics Target

Status

Cancer Type

Dimethyl sphingosine

SK1,SK2

in vitro, in vivo

Leukemia, colon, breast, melanoma

Safingol

SK1,SK2

Phase I

Solid tumors

SKI-II

SK1,SK2

in vitro, in vivo

Breast

Phenoxodiol

SK1,SK2

in vitro, in vivo

Ovarian and prostate

SKI-I

SK1

in vitro, in vivo

Glioblastoma, breast, AML, melanoma

ABC294640

SK2

Phase II

Breast, prostate and renal

FTY-720

S1PR

in vitro, in vivo

Bladder, prostate, breast, lymphoma, melanoma

Anti-S1P-mAb

S1P

Phase I/II

Lung, ovarian, breast and melanoma

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Compound

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