Shanmugam Panneer Selvam

Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy

Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C18-pyridinium ceramide (C18-Pyr-Cer) treatment, or endogenous C18-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C18-ceramide-induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 beta lipidation (LC3B-II) and selective targeting of mitochondria by LC3B-II-containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite-induced mitophagy, and stable LC3B knockdown protected against CerS1-C18-ceramide-dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a novel receptor function of ceramide for anchoring LC3B-II-autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.

Sphingolipids and cancer: ceramide and sphingosine-1-phosphate in the regulation of cell death and drug resistance

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.

Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis

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

Communication between host organism and cancer cells is transduced by systemic sphingosine kinase 1/sphingosine 1‐phosphate signalling to regulate tumour metastasis

Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via sphingosine 1‐phosphate (S1P) generated systemically by sphingosine kinase 1 (SK1), rather than via tumour‐derived S1P. Modulation of systemic, but not tumour SK1, prevented S1P elevation, and inhibited TRAMP‐induced prostate cancer growth in TRAMP+/+SK1−/− mice, or lung metastasis of multiple cancer cells in SK1−/− animals. Genetic loss of SK1 activated a master metastasis suppressor, Brms1 (breast carcinoma metastasis suppressor 1), via modulation of S1P receptor 2 (S1PR2) in cancer cells. Alterations of S1PR2 using pharmacologic and genetic tools enhanced Brms1. Moreover, Brms1 in S1PR2−/− MEFs was modulated by serum S1P alterations. Accordingly, ectopic Brms1 in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms1 prevented this process. Importantly, inhibition of systemic S1P signalling using a novel anti‐S1P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms1 knockdown in MB49 cells. Thus, these data suggest that systemic SK1/S1P regulates metastatic potential via regulation of tumour S1PR2/Brms1 axis.

Sphingosine kinase-1 and sphingosine 1-phosphate receptor 2 mediate Bcr-Abl1 stability and drug resistance by modulation of protein phosphatase 2A

The mechanisms by which sphingosine kinase-1 (SK-1)/sphingosine 1-phosphate (S1P) activation contributes to imatinib resistance in chronic myeloid leukemia (CML) are unknown. We show herein that increased SK-1/S1P enhances Bcr-Abl1 protein stability, through inhibition of its proteasomal degradation in imatinib-resistant K562/IMA-3 and LAMA-4/IMA human CML cells. In fact, Bcr-Abl1 stability was enhanced by ectopic SK-1 expression. Conversely, siRNA-mediated SK-1 knockdown in K562/IMA-3 cells, or its genetic loss in SK-1(-/-) MEFs, significantly reduced Bcr-Abl1 stability. Regulation of Bcr-Abl1 by SK-1/S1P was dependent on S1P receptor 2 (S1P2) signaling, which prevented Bcr-Abl1 dephosphorylation, and degradation via inhibition of PP2A. Molecular or pharmacologic interference with SK-1/S1P2 restored PP2A-dependent Bcr-Abl1 dephosphorylation, and enhanced imatinib- or nilotinib-induced growth inhibition in primary CD34(+) mononuclear cells obtained from chronic phase and blast crisis CML patients, K562/IMA-3 or LAMA4/IMA cells, and 32Dcl3 murine progenitor cells, expressing the wild-type or mutant (Y253H or T315I) Bcr-Abl1 in situ. Accordingly, impaired SK-1/S1P2 signaling enhanced the growth-inhibitory effects of nilotinib against 32D/T315I-Bcr-Abl1-derived mouse allografts. Since SK-1/S1P/S1P2 signaling regulates Bcr-Abl1 stability via modulation of PP2A, inhibition of SK-1/S1P2 axis represents a novel approach to target wild-type- or mutant-Bcr-Abl1 thereby overcoming drug resistance.

Binding of the sphingolipid S1P to hTERT stabilizes telomerase at the nuclear periphery by allosterically mimicking protein phosphorylation

Sphingosine 1-phosphate binds and stabilizes telomerase, a process that could be targeted to promote senescence and reduce cancer growth. Telomerase stabilized by a sphingolipid In normal adult cells, the structure at the ends of chromosomes, called the telomere, becomes progressively shorter with each replication cycle. When telomeres get too short or damaged, the cell stops dividing and becomes senescent. The enzyme telomerase maintains the integrity of telomeres, and phosphorylation stabilizes the catalytic subunit (called TERT) of this enzyme. In both normal fibroblasts and lung cancer cells, Panneer Selvam et al. found that binding of the phospholipid S1P to TERT mimicked protein phosphorylation and prevented TERT degration. Disrupting the interaction between S1P and TERT by either depleting S1P or mutating the interaction site in TERT impaired telomere maintenance and promoted senescence in cultured cells and decreased the growth of lung cancer cell xenografts in mice. During DNA replication, the enzyme telomerase maintains the ends of chromosomes, called telomeres. Shortened telomeres trigger cell senescence, and cancer cells often have increased telomerase activity to promote their ability to proliferate indefinitely. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is stabilized by phosphorylation. We found that the lysophospholipid sphingosine 1-phosphate (S1P), generated by sphingosine kinase 2 (SK2), bound hTERT at the nuclear periphery in human and mouse fibroblasts. Docking predictions and mutational analyses revealed that binding occurred between a hydroxyl group (C′3-OH) in S1P and Asp684 in hTERT. Inhibiting or depleting SK2 or mutating the S1P binding site decreased the stability of hTERT in cultured cells and promoted senescence and loss of telomere integrity. S1P binding inhibited the interaction of hTERT with makorin ring finger protein 1 (MKRN1), an E3 ubiquitin ligase that tags hTERT for degradation. Murine Lewis lung carcinoma (LLC) cells formed smaller tumors in mice lacking SK2 than in wild-type mice, and knocking down SK2 in LLC cells before implantation into mice suppressed their growth. Pharmacologically inhibiting SK2 decreased the growth of subcutaneous A549 lung cancer cell–derived xenografts in mice, and expression of wild-type hTERT, but not an S1P-binding mutant, restored tumor growth. Thus, our data suggest that S1P binding to hTERT allosterically mimicks phosphorylation, promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth.

Sphingosine Kinase/Sphingosine 1-Phosphate Signaling in Cancer Therapeutics and Drug Resistance

In this chapter, roles of bioactive sphingolipids, specifically sphingosine kinase 1 (SK1) and 2 (SK2) and their product-sphingosine 1-phosphate (S1P)-will be reviewed with respect to regulation of cancer growth, metastasis, chemotherapeutics, and drug resistance. Sphingolipids are known to be key bioeffector molecules that regulate cancer proliferation, angiogenesis, and cell death. Sphingolipid molecules such as ceramide and S1P have been shown to control cancer cell death and proliferation, respectively. Roles of S1P have been described with respect to their intracellular and extracellular pro-survival and drug resistance functions mostly through S1P receptor (S1PR1-5) engagement. Identification of novel intracellular SK/S1P targets has broadened the existing complex regulatory roles of bioactive sphingolipids in cancer pathogenesis and therapeutics. Thus, deciphering the biochemical and molecular regulation of SK/S1P/S1PR signaling could permit development of novel therapeutic interventions to improve cancer therapy and/or overcome drug resistance.

HPV/E7 induces chemotherapy‐mediated tumor suppression by ceramide‐dependent mitophagy

Human papillomavirus (HPV) infection is linked to improved survival in response to chemo‐radiotherapy for patients with oropharynx head and neck squamous cell carcinoma (HNSCC). However, mechanisms involved in increased HNSCC cell death by HPV signaling in response to therapy are largely unknown. Here, using molecular, pharmacologic and genetic tools, we show that HPV early protein 7 (E7) enhances ceramide‐mediated lethal mitophagy in response to chemotherapy‐induced cellular stress in HPV‐positive HNSCC cells by selectively targeting retinoblastoma protein (RB). Inhibition of RB by HPV‐E7 relieves E2F5, which then associates with DRP1, providing a scaffolding platform for Drp1 activation and mitochondrial translocation, leading to mitochondrial fission and increased lethal mitophagy. Ectopic expression of a constitutively active mutant RB, which is not inhibited by HPV‐E7, attenuated ceramide‐dependent mitophagy and cell death in HPV(+) HNSCC cells. Moreover, mutation of E2F5 to prevent Drp1 activation inhibited mitophagy in HPV(+) cells. Activation of Drp1 with E2F5‐mimetic peptide for inducing Drp1 mitochondrial localization enhanced ceramide‐mediated mitophagy and led to tumor suppression in HPV‐negative HNSCC‐derived xenograft tumors in response to cisplatin in SCID mice.

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

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

Balance between senescence and apoptosis is regulated by telomere damage–induced association between p16 and caspase-3

Telomerase activation protects cells from telomere damage by delaying senescence and inducing cell immortalization, whereas telomerase inhibition mediates rapid senescence or apoptosis. However, the cellular mechanisms that determine telomere damage–dependent senescence versus apoptosis induction are largely unknown. Here, we demonstrate that telomerase instability mediated by silencing of sphingosine kinase 2 (SPHK2) and sphingosine 1-phosphate (S1P), which binds and stabilizes telomerase, induces telomere damage–dependent caspase-3 activation and apoptosis, but not senescence, in p16-deficient lung cancer cells or tumors. These outcomes were prevented by knockdown of a tumor-suppressor protein, transcription factor 21 (TCF21), or by ectopic expression of WT human telomerase reverse transcriptase (hTERT) but not mutant hTERT with altered S1P binding. Interestingly, SphK2-deficient mice exhibited accelerated aging and telomerase instability that increased telomere damage and senescence via p16 activation especially in testes tissues, but not in apoptosis. Moreover, p16 silencing in SphK2−/− mouse embryonic fibroblasts activated caspase-3 and apoptosis without inducing senescence. Furthermore, ectopic WT p16 expression in p16-deficient A549 lung cancer cells prevented TCF21 and caspase-3 activation and resulted in senescence in response to SphK2/S1P inhibition and telomere damage. Mechanistically, a p16 mutant with impaired caspase-3 association did not prevent telomere damage–induced apoptosis, indicating that an association between p16 and caspase-3 proteins forces senescence induction by inhibiting caspase-3 activation and apoptosis. These results suggest that p16 plays a direct role in telomere damage–dependent senescence by limiting apoptosis via binding to caspase-3, revealing a direct link between telomere damage–dependent senescence and apoptosis with regards to aging and cancer.