© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs219204. doi:10.1242/jcs.219204

REVIEW Glycosphingolipid in cell fate specification Domenico Russo1,*, Laura Capolupo1,2, Jaipreet Singh Loomba1,2, Lucia Sticco1 and Giovanni D’Angelo1,2,*

ABSTRACT anchoring. Specifically at the PM, participate in Glycosphingolipids (GSLs) are ubiquitous components of eukaryotic signaling events by recruiting signaling molecules to, or plasma membranes that consist of a backbone linked to a sequestering them at, membrane microdomains for the modulation moiety. Both the ceramide and the glycan parts of GSLs display of their activities and for their processing into the endocytic cycle structural variations that result in a remarkable repertoire of diverse (Holthuis and Menon, 2014; Holthuis et al., 2001; Simons and compounds. This diversity of GSLs is exploited during embryogenesis, Ikonen, 1997). Given these properties, sphingolipids are proposed to when different GSLs are produced at specific developmental stages and function as fundamental membrane organizers and to make up along several differentiation trajectories. Importantly, plasma membrane the fabric of eukaryotic PMs in order to influence the interaction receptors interact with GSLsto modify their activities. Consequently, two with the extracellular environment (Hannun and Obeid, 2018; otherwise identical cells can respond differently to the same stimulus Holthuis et al., 2001). owing to their different GSL composition. The metabolic reprograming of Interestingly, different cell types exhibit a specific GSLs is in fact a necessary part of developmental programs, as its array at their PMs (Hakomori, 2003; Ngamukote et al., 2007) impairment results in developmental failure or tissue-specific defects. (Table S1). Indeed, sphingolipids are subjected to remarkable Moreover, single-cell variability is emerging as a fundamental player in structural variations that lead to the production of hundreds of development: GSL composition displays cell-to-cell variability in different species (Hannun and Obeid, 2008, 2018). A substantial syngeneic cell populations owing to the regulatory gene expression part of this variability derives from the heterogeneous elongation circuits involved in microenvironment adaptation and in differentiation. of glycan chains that are covalently linked to the sphingolipid Here, we discuss how GSLs are synthesized and classified and review backbone in the synthesis of the class of compounds known as the role of GSLs in the establishment and maintenance of cell identity. glycosphingolipids (GSLs). GSL-associated range have We further highlight the existence of the regulatory circuits that between one and more than 20 sugar residues, with 11 different ’ modify GSL pathways and speculate how GSL heterogeneity might monosaccharide types being used in vertebrates (D Angelo et al., contribute to developmental patterning. 2013a). Importantly, the elongation of glycans in GSLs is not driven by a template; instead, it entirely depends on the relative expression KEY WORDS: Differentiation, Glycosphingolipid, Golgi complex and organization of their specific synthetic enzymes (Bieberich et al., 2002; Giraudo and Maccioni, 2003). Still, GSL production Introduction is tightly controlled during differentiation programs; as a result, Cellular membranes serve as both barriers and interfaces between specific GSLs are used as differentiation stage or cell-type-specific topologically distinct biological spaces. The composition of markers (D’Angelo et al., 2013a). In addition, GSL composition can these membranes varies at different cellular locations. For example, substantially vary among single cells in syngeneic cell populations the plasma membrane (PM) is rich in sphingolipids compared to (Majoul et al., 2002; Russo et al., 2018; Snijder et al., 2009). intracellular membranes, which results in the PM having distinct Furthermore, specific GSL glycans appear to organize interactions biophysical properties (Holthuis and Menon, 2014). Sphingolipids with receptors that are located at the PM in order to modulate their contain a hydrophobic ceramide (Cer) backbone that is composed of activity (Bremer and Hakomori, 1982; Bremer et al., 1984; Coskun a saturated fatty acid and sphingoid base. This allows sphingolipids et al., 2011; Farooqui et al., 1999; Liu et al., 2008; Mirkin et al., to establish lateral interactions (both homotypic and with sterols) 2002; Mutoh et al., 1995; Park et al., 2012; Toledo et al., 2004). to yield a tightly packed and thick membrane structure (Hannun This occurs, for instance, in the case of the GM3-dependent and Obeid, 2018; Holthuis et al., 2001). Owing to this lipid inhibition of epidermal growth factor receptor (EGFR) signaling, composition, the PM is less permeable to ions and peptides which maintains EGFR in an inactive state in the absence of its compared to intracellular membranes, which matches with its ligand (Coskun et al., 2011). By contrast, GD1a and GM1 enhance ‘barrier’ function towards the extracellular environment (Holthuis EGFR activation (Li et al., 2001, 2000; Liu et al., 2004). Thus, two and Menon, 2014). Sphingolipids also show incomplete miscibility otherwise identical cells can react differently to the same stimulus with phospholipids, which results in lateral phase partitioning of the owing to their different composition in GSLs. membrane and thus in the formation of membrane microdomains Whereas the role of cell-to-cell variability in GSL composition (Simons and Ikonen, 1997). Such microdomains have different in differentiated cells remains to be understood, non-genetic affinities for proteins depending on the length and composition of heterogeneity has been proposed to contribute to cell-type their transmembrane domains, or on their lipid-based membrane diversification in developmental processes (Huang, 2009). Specifically, non-genetic heterogeneity provides cells with transitory ‘states’ to potentially orient their fates towards diverging directions 1Institute of Protein Biochemistry, National Research Council, Via P. Castellino 111, Napoli, Italy. 2Institute of Bioengineering, Laboratory of Lipid Cell Biology, École (Huang, 2009). Given the role of GSLs in modulating cell responses to polytechnique fédérale de Lausanne (EPFL) CH-1015 Lausanne, Switzerland. environmental cues, along with their extensive structural variation, cell-to-cell heterogeneity in GSL composition might therefore help in *Authors for correspondence ([email protected]; [email protected]) generating identity patterns during tissue morphogenesis. In this

D.R., 0000-0003-2171-657X; G.D., 0000-0002-0734-4127 Review, we discuss the role of GSLs as cell-fate determinants, focusing Journal of Cell Science

1 REVIEW Journal of Cell Science (2018) 131, jcs219204. doi:10.1242/jcs.219204 on (1) how GSL diversity is generated, (2) what GSL changes occur the PM. For its metabolic conversions, Cer can be galactosylated in when cells differentiate toward alternative fates, and (3) how the the ER to produce (GalCer), extracted from ER GSL metabolism is controlled by differentiation programs. Finally, membranes by the lipid-transfer protein ceramide transfer protein we will speculate on how GSLs can contribute to tissue patterning (CERT) and delivered to the trans-Golgi where SM is synthesized and morphogenesis. (Hanada et al., 2003), or transported in vesicles to the cis-Golgi where it is glucosylated to produce glucosylceramide (GlcCer) GSL synthesis (Funakoshi et al., 2000) (Fig. 1). Whereas SM cannot be further GSL synthesis is initiated at the cytosolic membrane leaflet of the processed in an anabolic direction, GalCer is the precursor of endoplasmic reticulum (ER), where Cer is produced from its GSLs from the gala-series, also known as , which include precursor sphinganine by the consecutive action of enzymes that sulfo-GalCer, (α2-3)-sialylated GalCer (GM4), di-GalCer (i.e. catalyze its acylation and desaturation (Mullen et al., 2012). Cer can Gal-GalCer) and di-sulfo-GalCer, which are produced at the then be converted into several compounds that include , Golgi complex where the enzymes for GalCer processing reside Cer-1-phosphate, acyl-Cers, (SM) and GSLs (Merrill, 2011) (Fig. 1). (Hannun and Obeid, 2018; Holthuis et al., 2001; Merrill et al., Apart from the gala-series GSLs, all other GSLs have GlcCer 2005). SM and GSLs are synthesized at the interface between the as a precursor (D’Angelo et al., 2013a; Merrill, 2011). GlcCer is ER and Golgi complex and constitute the major sphingolipids at converted into (LacCer; Gal-GlcCer) (Kumagai

SSEA-4 Globo ST3GAL2

Gb5 FUT1/2 Globo-H B3GALT5(SSEA-3) fucosyl-Gb5

A4GALT B3GALNT1 GBGT1 Forssman Gb3 Gb4 FAPP2 antigen

− − Ganglio SO3 SO3 P SM TGN sulfo-GalCer ST3GAL5 B4GALNT1 B3GALT4 ST3GAL1 GM3 GM2 GM1a GD1a GT1a () di-sulfo-GalCer Cer-1-P CERT ST8SIA1 GAL3ST1

GAL3ST1 GM4 B4GALNT1 B3GALT4 ST3GAL1 Golgi GD3 GD2 GD1b GT1b di-GalCer B4GALT5 ST8SIA5 GlcCer LacCer GSL precursors GCS B4GALNT1 B3GALT4 GT3 GT2 GT1c GQ1c GalCerS CerS GalCer Cer Sph

ER Asialo GD1c

acyl-Cer ST8SIA5 B4GALNT1 B3GALT4 ST3GAL1 ST6GALNAC4 GA2 GA1 GM1b GD1a

Key

Sia Fuc Glu Gal Cer P Phosphate y Lacto H antigen Lewis − GlcNac GalNac SO3 Sulfate Sph Fatty acid

x LT5 LC4 SSEA-1(Lewis)

B3GA

B3GNT5 B3GALT1 LC3 nLC4 nLC5

Fig. 1. GSL synthesis and classification and schematic representation of the GSL synthetic pathways. Ceramide (Cer) can be acylated (acyl-Cer), phosphorylated (Cer-1-phosphate) or conveyed to the TGN for the synthesis of SM. Alternatively, Cer is glycosylated for the synthesis of the GSL precursors, glucosylceramide (GlcCer) and of galactosylceramide (GalCer) along the secretory pathway (left panel). GalCer is then processed for the production of sulfatides. GlcCer is galactosylated to lactosylceramide (LacCer), which serves as a common precursor for the different GSL series: globo (red), ganglio (green), asialo

(blue) and lacto (purple). Glycosphingolipid-synthetizing enzymes (GSEs) catalyzing the major synthetic reactions are shown in dark orange. Journal of Cell Science

2 REVIEW Journal of Cell Science (2018) 131, jcs219204. doi:10.1242/jcs.219204 et al., 2010; Nishie et al., 2010), which is the metabolic branching In the following sections, we will discuss the changes GSLs point for the formation of all remaining GSLs. They are undergo during cellular differentiation in developmental processes, categorized into four classes (i.e. the globo, lacto, ganglio and as well as during oncogenic transformation of tissues. asialo series) and their cumulative number exceeds 400 GSLs (D’Angelo et al., 2013a; Merrill, 2011; van Meer et al., 2008). GSL reprograming in development, cell differentiation and Thus, LacCer is the substrate of (1) GA2 synthase (GA2S) for the cancer synthesis of GA2 (GalNAc-LacCer) and of asialo-series GSLs GSL changes during embryonic development and cellular (Nagata et al., 1992), (2) of GM3 synthase (GM3S) for the differentiation synthesis of GM3 (NeuAc-LacCer) and of ganglio-series GSLs Numerous studies have reported that the composition of GSLs in the (Ishii et al., 1998), (3) Gb3 synthase (Gb3S) for the synthesis of membrane is remodeled during embryonic development (Cochran Gb3 (Gal-LacCer) and of globo-series GSLs (Kojima et al., et al., 1982; Handa and Hakomori, 2017; Kannagi et al., 1983; 2000), (4) Lc3 synthase (Lc3S) for the synthesis of Lc3 (GlcNAc- Ngamukote et al., 2007; Yamashita et al., 1999). These LacCer) and of lacto-series GSLs (Biellmann et al., 2008) compositional changes have been evaluated during the three (Fig. 1). major developmental stages in mice {i.e. preimplantation After being conveyed to one of these four major metabolic [embryonic day (E) 0.5–6.5], gastrulation (E6.5–E10.5) and directions, GSLs are processed in glycosylation pathways. organogenesis (E10.5–E17.5)} (Handa and Hakomori, 2017) There, GSLs are often substrates of multiple possible reactions (Fig. 2). The preimplantation phase is dominated by GSLs of the that lead to further diverging metabolic directions or to the lacto series [i.e. stage-specific embryonic antigen 1 (SSEA-1) and formation of branched glycan structures (Fig. 1) (D’Angelo et al., Ley] and globo series (i.e. Forssman antigen, Gb4, SSEA-3 and 2013a; Merrill, 2011). The elongation of glycan residues in GSLs is SSEA-4) (Handa and Hakomori, 2017; Sato et al., 2007). During indeed the result of the ordered action of glycosyltransferases; their gastrulation, production of the ganglio-series GSLs is induced in relative levels, topological organization within the Golgi stack and both neuronal and glial cell precursors (Goldman et al., 1984), presence in multi-enzymatic complexes are key factors in the whereas SSEA-3, Forssman antigen and Gb4 are determination of the metabolic outcome (Maccioni et al., 2011). restricted to visceral mesoderm cells and to the inner cell mass of Along with these parameters, another factor that influences glycan the growing blastocysts (Handa and Hakomori, 2017). Finally, elongation in GSLs is substrate availability. GlcCer, the common during the organogenesis phase, the GSLs that are most prominently precursor of most GSLs, can be delivered to specific sub-Golgi synthetized are ; their relative amounts change in the regions by different transport mechanisms [i.e. vesicular, or non- nervous system from post gastrulation (E8) to adult ages. Thus, vesicular through the action of the lipid transfer protein 4-phosphate GM3, GD3 and GD2 are expressed at day E8, whereas GM1, GD1a, adapter protein 2 (FAPP2, also known as PLEKHA8); D’Angelo GD1b, GT1b and GQ1b are induced starting from E14 (Ngamukote et al., 2007], where each of these transport routes feeds a distinct et al., 2007) (Fig. 2). glycosylation pathway (D’Angelo et al., 2013b). However, in spite Changes in GSL expression have also been measured during of the non-deterministic nature of the GSL synthetic system, when in vitro differentiation of pluripotent cells into the three germ layers the database of GSL structures was analyzed (Sud et al., 2007), they (i.e. ectoderm, mesoderm and endoderm) (Liang et al., 2010, 2011; were found to be assembled according to regular patterns; Russo et al., 2018) (Table S1). Pluripotent stem cells express GSLs this suggests that structural heterogeneity in GSL structures is of the globo and lacto series (Breimer et al., 2017; Liang et al., 2010, not the result of a random process and points to them having a 2011; Russo et al., 2018), including Gb3, Gb4, Gb5 (SSEA-3), α1-2 biological function. fucosylated-Gb5 (Globo H), sialyl-Gb5 (SSEA-4) and disialyl-Gb5, A major limitation in our understanding of the structural and globo-A, Lc3, Lc4, SSEA-1 and fucosyl-Lc4 (Breimer et al., 2017) functional features of GSLs derives from technical difficulties: (Fig. 3). The levels of globo- and lacto-series GSLs decrease upon determining the GSL composition of a biological sample remains an differentiation of pluripotent stem cells to neuronal progenitors, analytical challenge. GSL composition is specific to the species, which is followed by the increase in the synthesis of GD3, GM3, cell type and condition (Hakomori, 2008). Moreover, GSLs largely GM1 and GD1 (Kwak et al., 2006; Liang et al., 2011; Marconi et al., differ in their abundance, chemical stability and biophysical 2005; Russo et al., 2018). In contrast, when embryonic stem cells properties, which makes their uniform extraction from biological differentiate into definitive endoderm, the major GSL that is samples difficult. In addition, the monosaccharide units in GSL expressed is Gb4 (Liang et al., 2011) (Fig. 3). GSL composition chains have very similar chemical structures, which, together with dynamically changes during the differentiation of mesenchymal heterogeneous positioning and the anomery of the sugar–sugar stem cells (MSCs) from adult bone marrow into multiple cell bonds and glycan chain branching, complicate GSL analysis lineages. Indeed, MSCs express SSEA-4 (Bergante et al., 2014; (Merrill, 2011). However, the accuracy in resolving GSL Gang et al., 2007) along with GD1a and GD2 gangliosides composition has improved as technologies have improved. Thus, (Bergante et al., 2014), whereas in MSC-derived adipocytes, the whereas orcinol-sulfuric acid staining and radioactive labeling with major GSLs are GM3 and GD1a (Kojima et al., 2015), and GM3 3H- or 14C-labeled monosaccharides coupled to chromatographic and GD3 are expressed in MSC-derived chondrocytes (David et al., separation are still valuable procedures for a rapid and inexpensive 1993). Moreover, lacto-series GSLs and GM3 are expressed in pre- assessment of GSL composition (Schnaar and Kinoshita, 2015), B-cells, whereas mature and activated B cells express GM3 and the detection with specific lectins or antibodies and mass spectrometry- globo-series GSLs Gb3 and Gb4 (Taga et al., 1995; Wiels et al., based methods now represent the golden standards for GSL 1991; Wipfler et al., 2011) (Fig. 3). profiling (Wuhrer, 2013). Thanks to these advancements, it is These data indicate that developmental programs are now possible to evaluate GSL changes in biological samples with accompanied by the reprograming of GSL metabolism. good accuracy, although an absolute quantification is often not Importantly, active GSL synthesis is required for embryonic possible owing to lack of complete reference standard samples development: both GlcCer synthase (GLS, encoded by UGCG)and

(Farwanah and Kolter, 2012). LacCer synthase (B4GALT5) (Fig. 1) knockout (KO) mice, which are Journal of Cell Science

3 REVIEW Journal of Cell Science (2018) 131, jcs219204. doi:10.1242/jcs.219204

Gastrulation and early Preimplantation development organogenesis Organogenesis and fetal growth

E0.5 E1.5 E2 E3 E3.5 E4.5 E6.5 E7.5 E8.5 E10.5 E14.5 E17.5

Lacto-series LC3, LC4 GSL SSEA-1, Le y

Gb3, Gb4 Globo-series SSEA-3, SSEA-4 GSL Forssman antigen

GM3, GD2, GD3 Ganglio-series GSL GM1, GD1a, GD1b GT1b, GQ1b Sulfatides SM4

Fig. 2. Changes of GSL profile in mouse embryonic development. Stage-specific changes of GSL expression during mouse embryogenesis (Cochran et al., 1982; Handa and Hakomori, 2017; Kannagi et al., 1983; Ngamukote et al., 2007; Yamashita et al., 1999). Globo- and lacto-series GSLs are expressed predominantly during the preimplantation phase and gastrulation (Handa and Hakomori, 2017; Heinrich, 1993). Ganglio-series GSLs, together with sulfatides, start to be synthetized during late gastrulation until the prenatal phase (Goldman et al., 1984; Ngamukote et al., 2007), and concomitantly to embryonic brain development. The three phases of embryonic development are indicated by three different shades of gray. The color-coded rectangles represent that are expressed at the specific stage; colored lines mean that the lipid is not expressed at that specific stage; the absence of a rectangle or line means that the lipid has not been measured at the reported developmental stage. unable to synthesize GSLs through the GlcCer precursor, die by metastasization and the emergence of multi-drug resistance E10.5 (Allende and Proia, 2014; Nishie et al., 2010; Yamashita et al., (Gouaze-Andersson and Cabot, 2006; Hakomori and Zhang, 2002, 1999). In both cases, the embryo is able to progress through pre- 1997; Jacob et al., 2014; Kovbasnjuk et al., 2005). A recent study implantation phase, but not beyond formation of the three germ layers on mammalian target of rapamycin (mTOR)-induced liver cancer (Allende and Proia, 2014; D’Angelo et al., 2013a; Yamashita et al., showed that hyperactive mTOR signaling results in increased GSL 1999). Further analyses of mice that harbor defects in the pathways synthesis (Guri et al., 2017), and that GSL production is strictly of GSL synthesis support the idea that there is a tissue-specific role required for mTOR-dependent cancer development (Guri et al., for the GSL subclasses (Allende and Proia, 2014; D’Angelo et al., 2017), but how exactly do GSLs contribute to the different aspects 2013a). Knockout of B3GNT5 – the gene encoding the first of oncogenesis? enzyme involved in the synthesis of lacto-series GSLs (i.e. Lc3 Signal transducers, adhesion molecules and growth factor synthase) (Fig. 1) – results in either preimplantation lethality or receptors that participate in malignant transformation and multiple postnatal defects (Biellmann et al., 2008). Conversely, development of drug resistance are often GSL targets. For the genetic disruption of globo or ganglio series GSL production instance, in breast cancer, increased GD3 and GD2 synthesis yields a wide range of immune and neurological phenotypes, favors stem cell proliferation by fostering the activation of growth respectively (Allende and Proia, 2014). In addition, loss-of- factor receptors on the PM (Liang et al., 2013) and promoting function mutations in three genes that encode enzymes involved in resistance to treatment with Gefitinib, a tyrosine kinase inhibitor the synthesis of ganglio-series GSLs cause neuronal disease in that targets EGFR (Liang et al., 2017). Cisplatin is a humans (Boccuto et al., 2014; Boukhris et al., 2013; Fragaki et al., chemotherapeutic agent that is used for the treatment of a 2013; Harlalka et al., 2013; Simpson et al., 2004). number of cancers, such as non-small cell lung cancer (NSCLC) Altogether, this evidence highlights that (1) GSL cell and malignant pleural mesothelioma (MPM). It induces Cer composition is remodeled when cells differentiate, and (2) that production, leading to cell cycle arrest and apoptosis (Dasari and GSL synthesis has a role in differentiation and development. Along Tchounwou, 2014; Nowak, 2012). Drug-resistant cancer cells these lines, aberrant changes in GSL metabolism are coupled to escape apoptosis by increasing GSL synthesis at the expense of an altered cell differentiation and malignant cell transformation, as accumulation of Cer, which also leads to increased expression of discussed in the following section. the multidrug resistance-associated protein 1 (MRP1), which stimulates drug efflux (Tyler et al., 2015). GSL and cancer GSL reprograming has a role in the epithelial-to-mesenchymal Aberrations in GSL metabolism have also been linked to cancer transition (EMT), which is the process that enables metastatic (Gouaze-Andersson and Cabot, 2006; Morad and Cabot, 2013; cellular invasion in the context of cancer progression. The induction Ogretmen, 2018). In fact, similar to the events during normal of EMT in vitro by transforming growth factor β (TGFβ) treatment is embryonic development and tissue lineage differentiation, cells accompanied by a reduction in the levels of asialo-GSLs GM1 rearrange their GSL composition during oncogenic transformation and GM2, whereas complex gangliosides are, in turn, induced (Hakomori, 1998; Hakomori and Zhang, 1997). This rearrangement during this process (Guan et al., 2009; Mathow et al., 2015). has been suggested to contribute to cellular transformation, Interestingly, a subpopulation of cells that express low levels of Journal of Cell Science

4 REVIEW Journal of Cell Science (2018) 131, jcs219204. doi:10.1242/jcs.219204

Fibroblast GM3 GM1 Gb3 Hepatocyte GM3 GD1a GM1 asialo-GM2 Gb3 Myocyte

Pancreatic cell Adipocyte GM3 GD1a Globo H Endoderm fucosyl-Gb5 (Globo H) Intestine epithelial cells Osteocyte Gb4 LC4 fucosyl-LC4 GM3 GM1a GD3 Le x Gb3 Gb4 MSC Chondrocyte GM3 GD3 SSEA-4 GM1 GD1a GD2 Le a Le b Gb4 Gb3 Proerythroblast Erythrocyte Forssman antigen GD1a

Myelocyte Gb3 Gb4 LC3 nLC4 LC4 Le x sialyl-Le x asialo-GM1 asialo-GM2 Mesoderm HSC Myeloblast GM1 Pluripotent Muscle cell NK cell iGb3 stem cells asialo-GM1 GM1 Gb3 Gb4 SSEA-3 Limphoid progenitor T cell Y

fucosyl-Gb5 (Globo H) Cardiocyte Y Y

Y Y sialyl-Gb5 (SSEA-4) Endothelium B cell Y GM3 Gb3 Gb4

disialyl-Gb5 Globo-A Y GM3 SSEA-1 LC3 LC4 fucosyl-LC4 Neuron GT1b GQ1b Skin and Hair Ceramide GM3 GD3 Axon GalCer Gb3 Gb4 Neural crest stem cell Schwann cell SM4 SM

Ectoderm Neural stem cell Neuronal restricted progenitor Neuron GM3 GM1a GM3 GM1 GM3 GM1 GD3 GD2 GD1a GD1b GD1a GD1b GD2 GD3 GT1b GQ1b GD2 GD3

Sperm Astrocyte GM1 GD1a GT1b Glial restricted progenitor Primordial Oocyte GD3 GalCer germ cell GM1 sialyl-Le x Oligodendrocyte SSEA-1 SM4 GalCer SM SSEA-4

Fig. 3. Change of GSL expression profile during differentiation of pluripotent stem cells. Schematic representation of the production of GSLs during the differentiation of pluripotent stem cells into definitive endoderm, mesoderm, ectodermal lineages or primordial germ cells (each lineage is represented by a different shade of gray, going from light to dark gray respectively). Detailed information on the changes in the GSL composition during differentiation that are represented in the figure are reported in Table S1 and references therein. epithelial markers has been identified in prostate tumors. This this is the case for a Shigella toxin, which recognizes Gb3 that is subpopulation expresses high levels of SSEA-4 and spontaneously overexpressed in gastric adenocarcinomas (Geyer et al., 2016). escapes from adhesive colonies and forms invadopodia-like Thus, metabolic alterations of GSLs are inherent components migratory structures. This supports the idea that SSEA-4 is a of cancerogenesis as they (1) originate from the malignant marker for metastasizing cells that have acquired a mesenchymal transformation process, (2) contribute to cancer-relevant nature (Sivasubramaniyan et al., 2015). phenotypes and (3) define cancer-specific cell states. Moreover, for a number of tumors, the overproduction of a specific GSL has been reported. These GSLs can be used as The regulatory circuits of GSL expression tumor-associated antigens (TAAs) for the definition of the tumor The aforementioned metabolic changes in GSLs, both in type and stage (Table 1 and references therein). Importantly, cancerogenesis and developmental contexts, are often the GSLs that serve as TAAs have been exploited to develop vaccine consequence of a reprograming in the expression of genes that strategies to elicit a specific cytotoxic and/or humoral immune encode the enzymes that synthesize GSLs. During neural response against tumor cells (Dobrenkov and Cheung, 2014). differentiation, for instance, the expression of genes encoding GD2-targeted immunotherapy of neuroblastoma has become the enzymes for the synthesis of globo- and lacto-series GSLs (i.e. first GSL-targeting immunotherapy to obtain food and drug A4GALT, encoding Gb3 synthase, and B3GNT5, encoding Lc3 administration (FDA) approval for medical care (Dobrenkov and synthase) decreases; at the same time, expression of genes encoding Cheung, 2014). Moreover, innovative strategies to target GSL- enzymes of the ganglio series synthesis pathway (i.e. ST3GAL5, TAAs also imply that toxins that use these GSLs as the natural encoding GM3 synthase, and B4GALNT1, encoding GA2/GM2 receptors in their target cells could be used for cancer treatment; synthase) increases (Liang et al., 2010, 2011; Russo et al., 2018). Journal of Cell Science

5 REVIEW Journal of Cell Science (2018) 131, jcs219204. doi:10.1242/jcs.219204

Table 1. Glycosphingolipids as TAAs Tumor-associated GSL antigens GSL antigen Cancer type Reference Globo-series GSLs Gb3 Burkitt lymphoma Farkas-Himsley et al., 1995 Ovarian cancer Arab et al., 1997 Colon cancer Kovbasnjuk et al., 2005 Gastric adenocarcinoma Geyer et al., 2016 SSEA-3 Breast cancer Chang et al., 2008 Globo H Breast cancer Chang et al., 2008 Ovarian cancer Hakomori, 1989 Thyroid carcinoma Cheng et al., 2016 DSGG Renal cell carcinoma Satoh et al., 1996 Hepatocellular carcinoma Wu et al., 2012 SSEA-4 Breast cancer Aloia et al., 2015 Glioblastoma Lou et al., 2014 Ganglio-series GSLs GM3 Acute myeloid leukemia (AML) Wang et al., 2012 Melanoma Guthmann et al., 2004 Multiple cancer types Zheng et al., 2018 GM1 Lung cancer Fuentes et al., 1997 Fucosyl-GM1 Hepatocellular carcinoma Wu et al., 2012 Small cell lung carcinoma Nilsson et al., 1986 GD3 Melanoma Cheresh et al., 1985 Neuroblastoma Cheresh et al., 1986 Lung cancer Fuentes et al., 1997 GD2 Melanoma Navid et al., 2010 Neuroblastoma Yang and Sondel, 2010 Lacto-series GSLs Lex Breast cancer Hakomori, 1989 Colon cancer Hakomori, 1989 Gastric cancer Hakomori and Zhang, 1997 Lex- Lex Breast cancer Hakomori, 1989 Colon cancer Hakomori, 1989 Gastric cancer Hakomori and Zhang, 1997 Lea- Lea Colon cancer Hakomori, 1989 Gastric cancer Hakomori and Zhang, 1997 Ley- Lex Pancreatic cancer Kim et al., 1988 Multiple cancer types Zheng et al., 2018 SSEA-1 Renal cancer Liebert et al., 1987 Lc3 Acute myeloid leukemia (AML) Wang et al., 2012 Ovarian cancer Alam et al., 2017 nLC4 Acute myeloid leukemia (AML) Wang et al., 2012 Sialylated-Lewisa (sLea) Colon cancer Hakomori, 1989 Pancreatic cancer Hakomori and Zhang, 1997

Similarly, during EMT, the production of GSLs is switched from the differentiation of stem cells into neural cells, the decrease in asialo to ganglio series (Fig. 4A) owing to the induction of globo-series GSLs (owing to A4GALT/Gb3 synthase repression) ST3GAL5 and ST8SIA1 (encoding GD3 synthase) and to the triggers the expression of the chromatin-remodeling factor autism repression of B3GALT4 (encoding GA1/GM1 synthase) (Mathow susceptibility gene 2 protein (AUTS2), which, in turn, binds to the et al., 2015). These data suggest that dedicated regulatory circuits ST3GAL5 promoter where it stimulates local histone acetylation and exist to reorient the GSL pathways. transcriptional activation of ganglio-series GSLs (Russo et al., During TGFβ-induced EMT, the mothers against 2018). Similar to what is seen during EMT, the addition of decapentaplegic homolog 3 and 4 (Smad3–Smad4) complex GSLs that are repressed in differentiated cells (globo-series GSLs) represses B3GALT4 by binding to its promoter (Guo et al., 2015), counteracts both the differentiation process and metabolic whereas zinc finger E-box-binding homeobox 1 (Zeb1) (a reprograming (Russo et al., 2018) (Fig. 4B). Interestingly, transcriptional target of Smad3–Smad4) binds to and activates the ganglio-series GSLs (i.e. GM1) have been found to stimulate promoters of both ST3GAL5 (Mathow et al., 2015) and ST8SIA1 neuronal differentiation and to sustain the expression of enzymes (Dae et al., 2009). Importantly, exogenous provision of GA1 (one of that synthesize ganglio-series GSLs (i.e. GM2S) by promoting the products of B3GALT4/GA1 synthase) inhibits TGFβ-induced histone acetylation at their promoters (Tsai et al., 2016; Tsai and Yu, EMT, which suggests that GSLs are both targets and regulators of 2014), which ultimately leads to maturation of the neuronal the same signaling pathway (Guan et al., 2009; Guo et al., 2015) population (Fig. 4C). An increase in histone acetylation at the

(Fig. 4A). Moreover, we recently demonstrated that, during GM2S (B4GALNT1) promoter was indeed observed in developing Journal of Cell Science

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A EMT Epithelial phenotype Mesenchymal phenotype

Asialo-series GM2S GA1S GM2S GA1S Asialo-series GSLs GSLs LacCer GA2 GA1 LacCer GA2 GA1 TGFb1

ZEB1 Ganglio-series GM3S Ganglio-series GM3S GM3S GM3S GSLs mRNA enzyme GSLs mRNA enzyme GM3S promoter GM3 GM3 GM3S promoter

B Neuronal differentiation Day 0 Day 13

Globo-series GSLs Gb3 Gb3 AUTS2 Ganglio-series GM3S GM3S GSLs AUTS2 mRNA enzyme AUTS2 promoter GM3S promoter AUTS2 promoter GM3S promoter GM3 Repressive histone modifications Active histone modifications

C Neuronal maturation Immature neurons Mature neurons Simple Complex ganglio-series ganglio-series GM1S GM1S GSLs GM2S GSLs GM2S GM2 GM1a GM3 enzyme GM3 enzyme GM2 GM1a

GM2S GM2S mRNA mRNA GM2S promoter GM2S promoter Active histone modifications

Key Sia Glu Gal GlcNac GalNac Cer

Fig. 4. Regulatory circuits for GSL expression and metabolism. (A) The asialo- to ganglio-GSL switch during EMT. During the acquisition of the mesenchymal phenotype, which is induced by TGFβ1, the transcription factor Zeb1 binds the GM3S (ST3GAL5) promoter and activates its transcription. Increased expression of GM3S and the concomitant suppression of GA1S (B3GALT4) tip GSL metabolism toward the synthesis of ganglio-series GSLs. (B) The globo- to ganglio-GSL switch during neuronal differentiation. In parallel with the decrease in the globo-series GSL, the epigenetic regulator AUTS2 is induced. AUTS2, in turn, binds and activates the promoters of neuronal genes – and of GM3S – inducing GM3S gene expression and favoring neuronal differentiation. (C) Establishment of complex ganglio-series GSL synthesis during neuronal maturation. GM1 fosters the synthesis of complex gangliosides and neuronal maturation by promoting the expression of the upstream ganglio-series GSL-synthesizing enzymes (GM2S). mouse brains, where it correlates with GM2S mRNA expression (2) their accessibility to ligands, (3) their oligomerisation state (Suzuki et al., 2011). This evidence reveals the existence of a two- and/or (4) their partitioning into membrane microdomains (Russo way relationship between GSL metabolism and transcriptional et al., 2016). programs that affect cell fate determination. Thus, GSLs can modify cell signaling in response to specific How exactly do GSLs influence gene expression? One stimuli. Examples for this are the interactions of GSLs with fundamental feature of GSLs is to organize protein– EGFR (Hofman et al., 2008; Park et al., 2017; Coskun et al., 2011) or carbohydrate–carbohydrate interactions with structural proteins and the notch ligand delta-like 1 (Dll1) (Heuss et al., 2013). Here, and receptors at the PM (Chakrabandhu et al., 2008; Coskun et al., their signaling – and as a consequence, the downstream 2011; Kawashima et al., 2009; Liang et al., 2017; Liu et al., 2004; transcriptional responses – are influenced by their interaction with Mutoh et al., 1995; Park et al., 2012; Russo et al., 2016). The GSL- GSLs at the PM. GSLs also affect signal transduction and gene glycan moiety can, indeed, interact directly with a specific amino expression by regulating endocytosis (Lakshminarayan et al., 2014). acid residue (Coskun et al., 2011) within a protein domain, or with GSLs interact with the secreted carbohydrate-binding protein a glycan portion (Heuss et al., 2013) of PM proteins. In doing so, galectin-3, which in turn triggers GSL-dependent biogenesis of

GSLs regulate PM proteins through (1) their conformation, specific cargo-laden endocytic carriers (Lakshminarayan et al., Journal of Cell Science

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Pluripotent daughter stem cells Symmetrical cell division Pluripotent stem cells

Pluripotent stem cells Pluripotent daughter Asymmetrical cell division stem cells Stable equilibrium Symmetry breaking event

Multiple attractor states Committed cell progenitors Differentiated cells

Key GSL profile of GSL profile of Progenitors Pluripotent stem cells and differentiated cells

Fig. 5. Symmetry breaking in cell differentiation. During stem cell differentiation, symmetrical or asymmetrical cell divisions occur. Symmetrical cell divisions sustain the maintenance of pluripotent stem state whereby a stable transcriptional and epigenetic profile is inherited by pluripotent daughter stem cells in a ‘stable equilibrium’. Asymmetrical cell divisions lead to the generation of cell progenitors diverging toward multiple differentiation trajectories due to uneven partitioning of key differentiation factors between the two daughter cells. Here, destabilized daughter cells transition towards new stable transcriptional and epigenetic arrangements (i.e. attractor states). GSLs influence differentiation programs, and their asymmetrical segregation might sustain bifurcation events in cell fate decisions.

2014). Through this mechanism, GSLs regulate the exposure Cell-to-cell variability in gene expression (either stochastic or of specific receptors and proteins at the PM. Another level of action dependent on the cellular microenvironment) has been proposed to for GSLs on gene expression is directly within the nucleus, where drive these early morphogenetic events by changing cell GSLs have been found at the nuclear lamina where they directly differentiation potential (Huang, 2009). This provides otherwise contact chromatin and influence the activity of promoters (Tsai identical cells with the capability to break symmetry within the et al., 2016). population (Huang, 2009) (Fig. 5). According to this concept, Thus, the impact of GSLs on signal transduction pathways and on progenitor cells can follow alternative differentiation trajectories to transcriptional programs upon their activation (Regina Todeschini achieve one of multiple stable states; this eventually leads to cell and Hakomori, 2008). As outlined above, the transcriptional fate decisions that depend on the oscillating expression of a key responses to changes in cellular GSL content often involve factor (Huang, 2009). Besides proteins and nucleic acids, small enzymes of the GSL synthetic pathway itself, thus resulting in molecules, which include lipids and, specifically, GSLs, are able self-contained regulatory loops that lead to metabolic switches. The to influence these cell differentiation programs. Moreover, GSL final output of these transcriptional metabolic interplays influences composition varies among cells in a syngeneic cell population cell fate decisions and differentiation programs. owing to cell cycle phase (Majoul et al., 2002), the local microenvironment (Snijder et al., 2009) or to metabolic circuits Conclusions and future directions (Russo et al., 2018). Whether this variability is involved in Glycans of GSLs protrude out of the PM towards the extracellular symmetry-breaking events in morphogenesis remains to be space. This peculiar position makes GSLs specifically suited to addressed (Fig. 5). interact with glycans and proteins that are present either on the same Research devoted to the dissection of the role for GSLs PM or on the PM of adjacent cells. Through these interactions, in regulatory gene expression circuits at single-cell resolution in GSLs influence signaling, receptor trafficking, cell–cell contacts advanced models of development and morphogenesis (i.e. in and adhesion, and, thus, ultimately gene expression and cell fate organoids) is probably the missing and required step to attain a determination. The structural diversity in GSLs and their tissue- sufficient body of knowledge on the role of GSLs in development, specific production suggest that distinct GSLs influence cell fate morphogenesis and tissue patterning. decisions towards differentiation trajectories. During development and tissue pattering, individual progenitor Competing interests cells are subjected to specific differentiation programs in order to The authors declare no competing or financial interests. achieve the formation of functional anatomical structures through morphogenesis (Basson, 2012). Whereas specific hormone Funding gradients sustain morphogenetic processes, the events that initiate G.D.A. acknowledges the financial support of Associazione Italiana per la morphogenesis usually happen in a uniform context, that is, among Ricerca sul Cancro (AIRC) (MFAG 10585), of the Italian Ministry of Health (GR-2011-02352256) and of Ministero dell’Istruzione, dell’Universitàe della Ricerca undifferentiated and genetically identical cells that are exposed to a (MIUR) (PON_00862). J.S.L. is supported by the H2020 Marie Skłodowska-Curie homogeneous environment (Tabata and Takei, 2004). Actions INCIPIT PhD program. Journal of Cell Science

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