Perspective: Therapeutic Implications for Sphingolipids in Health and Disease.

Long thought to be structural components of cell membranes, sphingolipids (SLs) have emerged as bioactive molecules whose metabolism is tightly regulated. These bioactive lipids and their metabolic enzymes have been implicated in numerous disease states, including lysosomal storage disorders, multiple sclerosis, inflammation, and cancer as well as metabolic syndrome and obesity. In addition, the indications for many of these lipids to potentially serve as biomarkers for disease continue to emerge with increasing metabolomic and lipidomic studies. The implications of these studies have, in turn, led to the examination of SL enzymes and their bioactive lipids as potential therapeutic targets and as markers for therapeutic efficacy. SIGNIFICANCE STATEMENT: Many sphingolipids (SLs) and their metabolizing enzymes have been implicated in disease. This perspective highlights the potential for SLs to serve as therapeutic targets and diagnostic markers and discusses the implications for the studies and reviews highlighted in this Special Section on Therapeutic Implications for Sphingolipids in Health and Disease.

Considerations for defining +80 Da mass shifts in mass spectrometry-based proteomics: phosphorylation and beyond.

Post-translational modifications (PTMs) are ubiquitous and key to regulating protein function. Understanding the dynamics of individual PTMs and their biological roles requires robust characterisation. Mass spectrometry (MS) is the method of choice for the identification and quantification of protein modifications. This article focusses on the MS-based analysis of those covalent modifications that induce a mass shift of +80 Da, notably phosphorylation and sulfation, given the challenges associated with their discrimination and pinpointing the sites of modification on a polypeptide chain. Phosphorylation in particular is highly abundant, dynamic and can occur on numerous residues to invoke specific functions, hence robust characterisation is crucial to understanding biological relevance. Showcasing our work in the context of other developments in the field, we highlight approaches for enrichment and site localisation of phosphorylated (canonical and non-canonical) and sulfated peptides, as well as modification analysis in the context of intact proteins (top down proteomics) to explore combinatorial roles. Finally, we discuss the application of native ion-mobility MS to explore the effect of these PTMs on protein structure and ligand binding.

Upregulation of sphingosine kinase 1 in response to doxorubicin generates an angiogenic response via stabilization of Snail.

Sphingosine kinase 1 (SK1) converts the pro-death lipid sphingosine to the pro-survival sphingosine-1-phosphate (S1P) and is upregulated in several cancers. DNA damaging agents, such as the chemotherapeutic doxorubicin (Dox), have been shown to degrade SK1 protein in cancer cells, a process dependent on wild-type p53. As mutations in p53 are very common across several types of cancer, we evaluated the effects of Dox on SK1 in p53 mutant cancer cells. In the p53 mutant breast cancer cell line MDA-MB-231, we show that Dox treatment significantly increases SK1 protein and S1P. Using MDA-MB-231 cells with CRISPR-mediated knockout of SK1 or the selective SK1 inhibitor PF-543, we implicated SK1 in both Dox-induced migration and in a newly uncovered proangiogenic program induced by Dox. Mechanistically, inhibition of SK1 suppressed the induction of the cytokine BMP4 and of the EMT transcription factor Snail in response to Dox. Interestingly, induction of BMP4 by SK1 increased Snail levels following Dox treatment by stabilizing Snail protein. Furthermore, we found that SK1 was required for Dox-induced p38 MAP kinase phosphorylation and that active p38 MAPK in turn upregulated BMP4 and Snail, positioning p38 downstream of SK1 and upstream of BMP4/Snail. Modulating production of S1P by inhibition of de novo sphingolipid synthesis or knockdown of the S1P-degrading enzyme S1P lyase identified S1P as the sphingolipid activator of p38 in this model. This work establishes a novel angiogenic pathway in response to a commonly utilized chemotherapeutic and highlights the potential of SK1 as a secondary drug target for patients with p53 mutant cancer.

Dihydroceramide desaturase 1 (DES1) promotes anchorage-independent survival downstream of HER2-driven glucose uptake and metabolism.

Oncogenic reprogramming of cellular metabolism is a hallmark of many cancers, but our mechanistic understanding of how such dysregulation is linked to tumor behavior remains poor. In this study, we have identified dihydroceramide desaturase (DES1)-which catalyzes the last step in de novo sphingolipid synthesis-as necessary for the acquisition of anchorage-independent survival (AIS), a key cancer enabling biology, and establish DES1 as a downstream effector of HER2-driven glucose uptake and metabolism. We further show that DES1 is sufficient to drive AIS and in vitro tumorigenicity and that increased DES1 levels-found in a third of HER2+ breast cancers-are associated with worse survival outcomes. Taken together, our findings reveal a novel pro-tumor role for DES1 as a transducer of HER2-driven glucose metabolic signals and provide evidence that targeting DES1 is an effective approach for overcoming AIS. Results further suggest that DES1 may have utility as a biomarker of aggressive and metastasis-prone HER2+ breast cancer.

Ca2+-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair.

Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.

Compartmentalization of Sphingolipid metabolism: Implications for signaling and therapy.

Sphingolipids (SLs) are a family of bioactive lipids implicated in a variety of cellular processes, and whose levels are controlled by an interlinked network of enzymes. While the spatial distribution of SL metabolism throughout the cell has been understood for some time, the implications of this for SL signaling and biological outcomes have only recently begun to be fully explored. In this review, we outline the compartmentalization of SL metabolism and describe advances in tools for investigating and probing compartment-specific SL functions. We also briefly discuss the implications of SL compartmentalization for cell signaling and therapeutic approaches to targeting the SL network.

Sublethal doxorubicin promotes migration and invasion of breast cancer cells: role of Src Family non-receptor tyrosine kinases.

BACKGROUND: Doxorubicin (Dox) is a widely used chemotherapy, but its effectiveness is limited by dose-dependent side effects. Although lower Dox doses reduce this risk, studies have reported higher recurrence of local disease with no improvement in survival rate in patients receiving low doses of Dox. To effectively mitigate this, a better understanding of the adverse effects of suboptimal Dox doses is needed. METHODS: Effects of sublethal dose of Dox on phenotypic changes were assessed with light and confocal microscopy. Migratory and invasive behavior were assessed by wound healing and transwell migration assays. MTT and LDH release assays were used to analyze cell growth and cytotoxicity. Flow cytometry was employed to detect cell surface markers of cancer stem cell population. Expression and activity of matrix metalloproteinases were probed with qRT-PCR and zymogen assay. To identify pathways affected by sublethal dose of Dox, exploratory RNAseq was performed and results were verified by qRT-PCR in multiple cell lines (MCF7, ZR75-1 and U-2OS). Regulation of Src Family kinases (SFK) by key players in DNA damage response was assessed by siRNA knockdown along with western blot and qRT-PCR. Dasatinib and siRNA for Fyn and Yes was employed to inhibit SFKs and verify their role in increased migration and invasion in MCF7 cells treated with sublethal doses of Dox. RESULTS: The results show that sublethal Dox treatment leads to increased migration and invasion in otherwise non-invasive MCF7 breast cancer cells. Mechanistically, these effects were independent of the epithelial mesenchymal transition, were not due to increased cancer stem cell population, and were not observed with other chemotherapies. Instead, sublethal Dox induces expression of multiple SFK-including Fyn, Yes, and Src-partly in a p53 and ATR-dependent manner. These effects were validated in multiple cell lines. Functionally, inhibiting SFKs with Dasatinib and specific downregulation of Fyn suppressed Dox-induced migration and invasion of MCF7 cells. CONCLUSIONS: Overall, this study demonstrates that sublethal doses of Dox activate a pro-invasive, pro-migration program in cancer cells. Furthermore, by identifying SFKs as key mediators of these effects, our results define a potential therapeutic strategy to mitigate local invasion through co-treatment with Dasatinib.

Neutral Sphingomyelinase 2 Heightens Anti-Melanoma Immune Responses and Anti-PD-1 Therapy Efficacy.

Dysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by SMPD3, catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8+ tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. In vitro, small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow-derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti-PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing SMPD3 expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8+ T-cell-dependent immune responses and overcome resistance to anti-PD-1.

Targeting sphingosine kinase 1 (SK1) enhances oncogene-induced senescence through ceramide synthase 2 (CerS2)-mediated generation of very-long-chain ceramides.

Senescence is an antiproliferative mechanism that can suppress tumor development and can be induced by oncogenes such as genes of the Ras family. Although studies have implicated bioactive sphingolipids (SL) in senescence, the specific mechanisms remain unclear. Here, using MCF10A mammary epithelial cells, we demonstrate that oncogenic K-Ras (Kirsten rat sarcoma viral oncogene homolog) is sufficient to induce cell transformation as well as cell senescence-as revealed by increases in the percentage of cells in the G1 phase of the cell cycle, p21WAF1/Cip1/CDKN1A (p21) expression, and senescence-associated ß-galactosidase activity (SA-ß-gal). Furthermore, oncogenic K-Ras altered SL metabolism, with an increase of long-chain (LC) C18, C20 ceramides (Cer), and very-long-chain (VLC) C22:1, C24 Cer, and an increase of sphingosine kinase 1 (SK1) expression. Since Cer and sphingosine-1-phosphate have been shown to exert opposite effects on cellular senescence, we hypothesized that targeting SK1 could enhance oncogenic K-Ras-induced senescence. Indeed, SK1 downregulation or inhibition enhanced p21 expression and SA-ß-gal in cells expressing oncogenic K-Ras and impeded cell growth. Moreover, SK1 knockdown further increased LC and VLC Cer species (C18, C20, C22:1, C24, C24:1, C26:1), especially the ones increased by oncogenic K-Ras. Fumonisin B1 (FB1), an inhibitor of ceramide synthases (CerS), reduced p21 expression induced by oncogenic K-Ras both with and without SK1 knockdown. Functionally, FB1 reversed the growth defect induced by oncogenic K-Ras, confirming the importance of Cer generation in the senescent phenotype. More specifically, downregulation of CerS2 by siRNA blocked the increase of VLC Cer (C24, C24:1, and C26:1) induced by SK1 knockdown and phenocopied the effects of FB1 on p21 expression. Taken together, these data show that targeting SK1 is a potential therapeutic strategy in cancer, enhancing oncogene-induced senescence through an increase of VLC Cer downstream of CerS2.

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics.

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely 'non-canonical' substrates. Surprisingly, sequence interrogation of ∼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling.

A role for ceramide glycosylation in resistance to oxaliplatin in colorectal cancer.

There is growing evidence to support a role for the ceramide-metabolizing enzyme, glucosylceramide synthase (GCS), in resistance to a variety of chemotherapeutic agents. Whether GCS contributes to oxaliplatin resistance in colorectal cancer (CRC) has not yet been determined. We have addressed this potentially important clinical issue by examining GCS function in two panels of oxaliplatin-resistant, isogenic CRC cell lines. Compared to parental cell lines, oxaliplatin-resistant cells have increased expression of GCS protein associated with increased levels of the pro-survival ceramide metabolite, glucosylceramide (GlcCer). Inhibition of GCS expression by RNAi-mediated gene knockdown resulted in a reduction in cellular GlcCer levels, with restored sensitivity to oxaliplatin. Furthermore, oxaliplatin-resistant CRC cells displayed lower ceramide levels both basally and after treatment with oxaliplatin, compared to parental cells. GlcCer, formed by GCS-mediated ceramide glycosylation, is the precursor to a complex array of glycosphingolipids. Differences in cellular levels and species of gangliosides, a family of glycosphingolipids, were also seen between parental and oxaliplatin-resistant CRC cells. Increased Akt activation was also observed in oxaliplatin-resistant CRC cell lines, together with increased expression of the anti-apoptotic protein survivin. Finally, this study shows that GCS protein levels are greatly increased in human CRC specimens, compared to matched, normal colonic mucosa, and that high levels of UGCG gene expression are significantly associated with decreased disease-free survival in colorectal cancer patients. These findings uncover an important cellular role for GCS in oxaliplatin chemosensitivity and may provide a novel cellular target for augmenting chemotherapeutic drug effectiveness in CRC.

Correction: PKCα is required for Akt-mTORC1 activation in non-small cell lung carcinoma (NSCLC) with EGFR mutation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Meeting Report - The 2019 FEBS special meeting on sphingolipid biology: sphingolipids in physiology and pathology.

Sphingolipids are a fundamental class of molecules that are involved in structural, organizational and signaling properties of eukaryotic membranes. Defects in their production or disposal lead to acquired and inherited human diseases. A growing community of scientists has embraced the challenge to dissect different aspects of sphingolipid biology using a variety of approaches, and a substantial part of this community met last May in the beautiful town of Cascais in Portugal. Over 200 scientists from 26 countries animated the conference, held in a 15th century citadel, sharing their data and opinions on the current understanding and future challenges in sphingolipid research. Here, we report some of their contributions to provide the readers with a bird's-eye view of the themes discussed at the meeting.

Strong anion exchange-mediated phosphoproteomics reveals extensive human non-canonical phosphorylation.

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site-specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other "non-canonical" amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non-canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry-based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non-canonical phosphosites is approximately one-third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high-throughput exploration of non-canonical phosphorylation in all organisms.

PKCα is required for Akt-mTORC1 activation in non-small cell lung carcinoma (NSCLC) with EGFR mutation.

Mutational activation of the epidermal growth factor receptor (EGFR) is a major player in the pathogenesis of non-small cell lung cancer (NSCLC). NSCLC patients with constitutively active EGFR mutations eventually develop drug resistance against EGFR tyrosine-kinase inhibitors; therefore, better understandings of key components of mutant EGFR (mtEGFR) signaling are required. Here, we initially observed aberrantly high expression of protein kinase Cα (PKCα) in lung adenocarcinomas, especially those with EGFR mutations, and proceeded to examine the role of PKCα in the regulation of the signaling pathways downstream of mtEGFR. The results showed that NSCLC cell lines with constitutively active EGFR mutations tend to have very or moderately high PKCα levels. Furthermore, PKCα was constitutively activated in HCC827 and H4006 cells which have an EGFR deletion mutation in exon 19. Interestingly, mtEGFR was not required for the induction of PKCα at protein and message levels suggesting that the increased levels of PKCα are due to independent selection. On the other hand, mtEGFR activity was required for robust activation of PKCα. Loss of functions studies revealed that the NSCLC cells rely heavily on PKCα for the activation of the mTORC1 signaling pathway. Unexpectedly, the results demonstrated that PKCα was required for activation of Akt upstream of mTOR but only in cells with the mtEGFR and with the increased expression of PKCα. Functionally, inhibition of PKCα in HCC827 led to caspase-3-dependent apoptosis and a significant decrease in cell survival in response to cellular stress induced by serum starvation. In summary, the results identified important roles of PKCα in regulating mTORC1 activity in lung cancer cells, whereby a primary switching occurs from PKCα-independent to PKCα-dependent signaling in the presence of EGFR mutations. The results present PKCα as a potential synergistic target of personalized treatment for NSCLC with constitutively active mutant forms of EGFR and constitutively active PKCα.

Probing compartment-specific sphingolipids with targeted bacterial sphingomyelinases and ceramidases.

Sphingolipids contribute to the regulation of cell and tissue homeostasis, and disorders of sphingolipid metabolism lead to diseases such as inflammation, stroke, diabetes, and cancer. Sphingolipid metabolic pathways involve an array of enzymes that reside in specific subcellular organelles, resulting in the formation of many diverse sphingolipids with distinct molecular species based on the diversity of the ceramide (Cer) structure. In order to probe compartment-specific metabolism of sphingolipids in this study, we analyzed the Cer and SM species preferentially produced in the inner plasma membrane (PM), Golgi apparatus, ER, mitochondria, nucleus, and cytoplasm by using compartmentally targeted bacterial SMases and ceramidases. The results showed that the length of the acyl chain of Cer becomes longer according to the progress of Cer from synthesis in the ER to the Golgi apparatus, then to the PM. These findings suggest that each organelle shows different properties of SM-derived Cers consistent with its emerging distinct functions in vitro and in vivo.