Mechanisms that alter protein phosphatase 2A (PP2A)-dependent lung tumour suppression via the I2PP2A/SET oncoprotein are unknown. We show here that the tumour suppressor ceramide binds I2PP2A/SET selectively in the nucleus and including its K209 and Y122 residues as determined by molecular modelling/simulations and site-directed mutagenesis. Because I2PP2A/SET was found overexpressed, whereas ceramide was downregulated in lung tumours, a sphingolipid analogue drug, FTY720, was identified to mimick ceramide for binding and targeting I2PP2A/SET, leading to PP2A reactivation, lung cancer cell death, and tumour suppression in vivo. Accordingly, while molecular targeting of I2PP2A/SET by stable knockdown prevented further tumour suppression by FTY720, reconstitution of WT-I2PP2A/SET expression restored this process. Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1-dependent programmed necrosis (necroptosis), but not by apoptosis. The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720. Expression of WT- or death-domain-deleted (DDD)-RIPK1, but not the kinase-domain-deleted (KDD)-RIPK1, restored FTY720-mediated necroptosis in RIPK1−/− MEFs. Thus, these data suggest that targeting I2PP2A/SET by FTY720 suppresses lung tumour growth, at least in part, via PP2A activation and necroptosis mediated by the kinase domain of RIPK1.

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Sphingolipids have emerged as bioeffector molecules, controlling various aspects of cell growth and proliferation in cancer, which is becoming the deadliest disease in the world. These lipid molecules have also been implicated in the mechanism of action of cancer chemotherapeutics. Ceramide, the central molecule of sphingolipid metabolism, generally mediates antiproliferative responses, such as cell growth inhibition, apoptosis induction, senescence modulation, endoplasmic reticulum stress responses and/or autophagy. Interestingly, recent studies suggest de novo-generated ceramides may have distinct and opposing roles in the promotion/suppression of tumors, and that these activities are based on their fatty acid chain lengths, subcellular localization and/or direct downstream targets. For example, in head and neck cancer cells, ceramide synthase 6/C 16 -ceramide addiction was revealed, and this was associated with increased tumor growth, whereas downregulation of its synthesis resulted in ER stress-induced apoptosis. By contrast, ceramide synthase 1-generated C 18 -ceramide has been shown to suppress tumor growth in various cancer models, both in situ and in vivo. In addition, ceramide metabolism to generate sphingosine-1-phosphate (S1P) by sphingosine kinases 1 and 2 mediates, with or without the involvement of G-protein-coupled S1P receptor signaling, prosurvival, angiogenesis, metastasis and/or resistance to drug-induced apoptosis. Importantly, recent findings regarding the mechanisms by which sphingolipid metabolism and signaling regulate tumor growth and progression, such as identifying direct intracellular protein targets of sphingolipids, have been key for the development of new chemotherapeutic strategies. Thus, in this article, we will present conclusions of recent studies that describe opposing roles of de novo-generated ceramides by ceramide synthases and/or S1P in the regulation of cancer pathogenesis, as well as the development of sphingolipid-based cancer therapeutics and drug resistance. † Author for correspondence: 86 Jonathan Lucas Street, Room 512A, Charleston, SC, 29425, USA, Tel.: +1 843 792 0941, Fax: +1 843 792 2556, ogretmen@musc.edu. For reprint orders, please contact: reprints@futuremedicine.com Financial & competing interests disclosureThis work was supported by research grants (CA088932, DE016572 and CA097132) from the NIH. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. NIH Public Access Structure & metabolism of ceramideSphingolipids are structural components of biological membranes that were first described by Thudichum, and the chemical structures of various sphingolipid molecules, including sphingosine and ceramide, were later resolved by Carter [1]. The term 'sphingosine' originates from a Greek word, ...

In this study, the inhibitor 2 of protein phosphatase 2A (I2PP2A) was identified in vitro and in situ as a ceramide-binding protein, which exhibits stereoisomer specificity and fatty acid chain length preference. Site- directed mutagenesis coupled with structural details of I2PP2A suggested that VIK 207-209 residues localized on helix 7 are important for ceramide binding and single mutation of K209D altered this interaction. Notably, I2PP2A-ceramide binding decreased the association between PP2A and the inhibitor, preventing the inhibition of PP2A activity in vitro. In addition, studies in A549 human lung cancer cells revealed that ceramide mediates c-Myc degradation via its PP2A-dependent dephosphorylation at S62, and treatment with okadaic acid and expression of c-Myc mutants with S62A or S62D conversions resulted in resistance to ceramide-mediated degradation. Importantly, whereas down-regulation of I2PP2A enhanced PP2A-mediated c-Myc degradation in response to ceramide, ectopic expression of wild-type I2PP2A but not of its K209D mutant protected this degradation in A549 cells. Moreover, expression of wild-type I2PP2A prevented the growth-inhibitory effects of ceramide both against A549 cells and xenograft-driven tumors in situ and in vivo compared with that in controls. Thus, these results suggest that direct interaction of I2PP2A with ceramide plays important biological roles via the regulation of PP2A activity and signaling, which in turn control ceramide-mediated degradation of c-Myc and antiproliferation.

In this chapter, roles of bioactive sphingolipids in the regulation of cancer pathogenesis and therapy will be reviewed. Sphingolipids have emerged as bioeffector molecules, which control various aspects of cell growth, proliferation, and anti-cancer therapeutics. Ceramide, the central molecule of sphingolipid metabolism, generally mediates anti-proliferative responses such as inhibition of cell growth, induction of apoptosis, and/or modulation of senescence. On the other hand, sphingosine 1-phosphate (S1P) plays opposing roles, and induces transformation, cancer cell growth, or angiogenesis. A network of metabolic enzymes regulates the generation of ceramide and S1P, and these enzymes serve as transducers of sphingolipid-mediated responses that are coupled to various exogenous or endogenous cellular signals. Consistent with their key roles in the regulation of cancer growth and therapy, attenuation of ceramide generation and/or increased S1P levels are implicated in the development of resistance to drug-induced apoptosis, and escape from cell death. These data strongly suggest that advances in the molecular and biochemical understanding of sphingolipid metabolism and function will lead to the development of novel therapeutic strategies against human cancers, which may also help overcome drug resistance.

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Key Points• The tumor suppressor PP2A is repressed in Jak2 V617F -driven myleoproliferative neoplasms by a Jak2/PI3K/ PKC/SET signaling pathway.• PP2A-activating (eg, FTY720, OSU-2S) but not sphingosine-1-phosphate agonistic (eg, FTY720-P) drugs selectively kill Jak2 V617F1 cells.FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2 V617F oncogene. PP2A inactivation occurs in a Jak2 V617F dose/kinase-dependent manner through the PI-3Kg-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PADmediated PP2A reactivation induces Jak2 V617F inactivation/downregulation and impairs clonogenic potential of Jak2 V617F cell lines and PV but not normal CD34 1 progenitors.Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2 V617F leukemic mice without adverse effects. Mechanistically, we show that in Jak2 V617F cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2 V617F also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphatereceptor-1 agonist, elicits signals leading to the Jak2-PI-3Kg-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2

Introduction CML is a clonal disorder of pluripotent hematopoietic stem cells characterized by the Philadelphia (Ph) chromosome, which results from the reciprocal translocation between the long arms of chromosomes 9 and 22. 1-4 This hybrid B-cell receptor (BCR)-ABL1 gene encodes for a fusion protein Bcr-Abl1 with a constitutive tyrosine kinase activity. 3,4 Despite high rates of clinical responses in early chronic phase CML (CML-CP) to the Bcr-Abl1 kinase inhibitor imatinib, 5-8 development of resistance is a major problem in late CML-CP and in the treatment of blast crisis CML (CML-BC). 9-12 Although Bcr-Abl1-independent mechanisms also exist, 13-15 resistance in CML-CP is usually associated with the expression of mutant Bcr-Abl1 proteins, including T315I and Y253F/H mutations against which the second generation ABL tyrosine kinase inhibitors (TKI) such as nilotinib and/or dasatinib show limited effect. 15-17 Nonetheless , BCR-ABL1 mutations may not account for all cases of drug resistance in CML (CP and BC); indeed, alternative Bcr-Abl1-dependent mechanisms including alterations of sphingolipid metabolism and signaling, 18 might account for TKI resistance. Sphingolipids, ceramide and sphingosine 1-phosphate (S1P) included, are a family of membrane lipids with important roles in the regulation of the fluidity and subdomain structure of membranes. 19-21 Ceramide can be hydrolyzed by ceramidases to release sphingosine, which is phosphorylated by sphingosine kinases-1 or-2 (SK-1 or SK-2) to generate S1P. 20 Ceramide plays proapoptotic roles 21 whereas S1P mediates proliferation and/or resistance to apoptosis 22,23 generally via G-protein-coupled S1P1-5 receptor signaling. 24 However, receptor-independent intracellular functions of S1P were also reported. 25 Recently, alteration of the balance between the proapoptotic ceramide and antiapoptotic S1P via up-regulation of SK-1 was shown to mediate imatinib resistance in K562 CML-BC patient-derived cells by an unknown mechanism. 18 Here, we report the identification of a novel mechanism by which SK-1/S1P mediates imatinib resistance by regulation of the PP2A-dependent and SHP-1-mediated Bcr-Abl1 dephosphoryla-tion and stability selectively via receptor 2 (S1P2) signaling in CML (CP and BC). In addition, our data suggest that targeting the SK-1/S1P2 signaling axis provides a novel strategy to modulate wild-type (wt) or mutant (T315I or Y253H) Bcr-Abl1 stability by restoring PP2A function, and attenuate drug resistance both in cell culture and in mice bearing 32D/T315I-Bcr-Abl1 allografts. Human CML cell lines K562, LAMA4, and their imatinib-resistant derivatives K562/IMA-0.1,-1,-3, or LAMA4/IMA, were maintained as described. 18 The Bcr-Abl-expressing 32Dcl3 cells, 32D-p210 Bcr-Abl (wt), 32D-p210 Bcr-Abl (Y253H) and (T315I) were maintained in RPMI containing 15% FBS, 2mM L-glutamine, and penicillin and streptomycin (P/S; 100 ng/mL each). MEFs (wt and SK-1 /) were maintained in DMEM with 10% FBS and P/S. Human CD34 primary cells from CML patients and normal donor were obt...