Wegner et al. BMC Cancer (2018) 18:153 https://doi.org/10.1186/s12885-018-4084-4

DEBATE Open Access The UDP-glucose (UGCG) and the link to multidrug resistance protein 1 (MDR1) Marthe-Susanna Wegner1* , Lisa Gruber1, Peter Mattjus2, Gerd Geisslinger1 and Sabine Grösch1

Abstract The UDP-glucose ceramide glycosyltransferase (UGCG) is a key in the by generating glucosylceramide (GlcCer), the precursor for all (GSL), which are essential for proper cell function. Interestingly, the UGCG is also overexpressed in several cancer types and correlates with multidrug resistance protein 1 (MDR1) expression. This membrane protein is responsible for efflux of toxic substances and protects cancer cells from cell damage through chemotherapeutic agents. Studies showed a connection between UGCG and MDR1 overexpression and multidrug resistance development, but the precise underlying mechanisms are unknown. Here, we give an overview about the UGCG and its connection to MDR1 in multidrug resistant cells. Furthermore, we focus on UGCG transcriptional regulation, the impact of UGCG on cellular signaling pathways and the effect of UGCG and MDR1 on the lipid composition of membranes and how this could influence multidrug resistance development. To our knowledge, this is the first review presenting an overview about UGCG with focus on the relationship to MDR1 in the process of multidrug resistance development. Keywords: UDP-glucose ceramide glycosyltransferase, UGCG, Glycosphingolipids, MDR1, P-gp, Multidrug resistance, Cancer

Background synthases (CerS), are transported to the Golgi apparatus, Over the last two decades, researchers were able to link where the UGCG transfers UDP-glucose to ceramide the sphingolipid metabolism and the development of resulting in GlcCer. Subsequently, the produced GlcCer multidrug resistance in several cancer types, but the pre- is flipped from the cytosol to the Golgi apparatus lumen cise mechanisms are unknown. There is evidence that for lactosylceramide (LacCer) production by galactosyla- the UDP-glucose ceramide glycosyltransferase (UGCG), tion of GlcCer. LacCer serves as a substrate for a variety which is located in the Golgi apparatus (Fig. 4)is of GSLs producing (reviewed in [2]). From the involved in the process of multidrug resistance develop- Golgi apparatus GSLs are transported to the plasma ment. The UGCG was first cloned by Ichikawa et al. in membrane, where they are integrated (Fig. 4). GSLs the year 1996 [1] and is currently the only enzyme depletion in the plasma membrane is executed by endo- responsible for the de novo production of glucosylcera- cytotic membrane flow of GSLs into the lysosomes, mide (GlcCer). GlcCer is the precursor for all where glycohydrolase activity results in GSLs degrad- glycosphingolipids (GSLs) such as globo-series and ation and subsequently ceramide production [3]. GSL ganglio-series GSLs. Since more than 400 species of are essential for embryonic development and differenti- GSLs are identified, the GSL metabolic pathway is here ation of several tissue types [4] and are involved in lipid described only briefly (reviewed in [2]). , ge- storage disorders called sphingolipidoses, which are nerated in the endoplasmic reticulum by ceramide caused by deficiency of lysosomal enzymes. One of the main sphingolipidoses is the Gaucher disease, which is characterized by deficiency of leading * Correspondence: [email protected] 1pharmazentrum frankfurt/ ZAFES, Institute of Clinical Pharmacology, Johann to GlcCer accumulation in cells and organs. Due to Wolfgang Goethe-University, House 74, Theodor Stern-Kai 7, 60590 Frankfurt different phenotypes the clinical presentation of Gaucher am Main, Germany disease patients ranges from cytopenia to neurological Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wegner et al. BMC Cancer (2018) 18:153 Page 2 of 10

impairment (reviewed in [5]). In patients with Fabry disease an GSL accumulation (in general ) oc- curs also, but in this case due to mutations in the GLA gene encoding α-galactosidase A (reviewed in [6]). The Sandhoff and Tay Sachs disease are characterized by accu- mulation of the GSL GM2 leading to neurodegeneration and early death (reviewed in [7]). Although, the phenotype of the Niemann-Pick disease is based on deficient sphin- gomyelinase activity, GlcCer concentration is found to be the second highest lipid concentration [8]. The different types of sphingolipidoses show how important it is to understand the sphingolipid metabolism and how com- plex it is. To date, only few reviews dealing with the UGCG are available [9–11], therefore we will discuss the impact of the UGCG and its knockout on cell prolifera- tion and cell metabolism shortly. A knockout of the UGCG in mice leads to embryonic lethality, which occurs at the phase of gastrulation. However, Yamashita et al. showed a not discriminable proliferation rate between UGCG deleted embryonic stem cells and wildtype cells in vitro, although GSL syn- thesis was absent in the UGCG disrupted embryonic Fig. 1 High-performance thin-layer chromatography (HPTLC) analysis of diverse sphingolipid species in MCF-7 cells. Representative HPTLC stem cells [4]. Instead, nude mice studies showed that si- plate showing the separation of ceramides, DAG and non-complex lencing the UGCG gene in adriamycin-resistant MCF-7 GSLs like GlcCer, LacCer and complex GLSs like Gb3 and . cells, which overexpress the UGCG, inhibits tumor MCF-7/naiv: untransfected, MCF-7/pTarget: empty vector-transfected, xenograft growth in vivo [12]. This result is in line with MCF-7/NHT: CRISPR-Cas non-human target control, MCF-7/UGCG OE: the studies of the Sabatini Lab (Whitehead Institute, UGCG expression plasmid-transfected, MCF-7/KD: CRISPR-Cas UGCG Knockdown. M = Marker Cambridge, Massachusetts) showing that the UGCG is an essential protein for optimal proliferation of several cancer cell lines [13]. The working group developed a globo-series sphingolipid globotriaosylceramide (Gb3), Clustered Regularly Interspaced Short Palindromic gangliosides and complex GSLs, which are lower in its Repeats (CRISPR) score (CS), which mirrors the loss of concentration in MCF-7 UGCG knockout cells (Fig. 1). fitness evoked by knockout of the respective gene. The Disruption of the UGCG in mice epidermis, causes al- CS is represented „as the average log2 fold-change in the terations in the skin lipid composition leading to the loss abundance of all sgRNAs targeting a given gene” and in- of the barrier function and to death due to dehydration dicates whether the gene knockout leads to an increased [14]. GlcCer is needed for proper lamellar body forma- or decreased cancer cell number. For the UGCG the fol- tion and regular metabolism in the stratum corneum, lowing CS were determined: KBM7 (− 0,243), K562 which is essential for maintaining water permeability (0,163), Jiyoye (− 0,08) and Raji (0,364) cells [13]. These function. Amen et al. showed that an inducible UGCG values show that the UGCG is essential for cell prolifera- knockout in mice keratinocytes leads to a loss of tion, but how severe the impact on the cell metabolism protein-bound ceramides resulting in the same fate, that is seems to be cell-type specific. In summary, contradict- occurs to the constitutive knockout mice [15]. ory results were obtained regarding the essentiality of Beside its essential functions especially in the mainten- the UGCG gene for cell proliferation. Another explan- ance of the epidermal water barrier function, UGCG is ation for the contradictory results could be the existence overexpressed in several cancer types for example in of a second form of this protein, which balances the loss metastatic breast cancer tissue leading to poor patient of the other protein. But our high-performance thin- prognosis [16] (Fig. 4). Gouazé et al. showed that this is layer chromatography (HPTLC) studies show that there not a unique phenomenon for breast cancer cells, be- is no compensation of GlcCer loss in UGCG deficient cause several other cancer cells e.g. human leukemia and MCF-7 cells (lane 5) (Fig. 1). Therefore, UGCG may be colon cancer cells, also exhibit a high UGCG gene a unique protein in cells, solely responsible for the first expression [17]. This overexpression correlates with glycosylation step of ceramides leading to complex increased multidrug resistance protein 1 (MDR1) gene GSLs. Accordingly, knockdown of the UGCG impacts expression, which encodes the protein P-glycoprotein 1 all other glycosylated such as LacCer, the (P-gp) (also ATP-binding cassette sub-family B member Wegner et al. BMC Cancer (2018) 18:153 Page 3 of 10

1, ABCB1), leading to multidrug resistance for example the ceramide for clearing it into less cytotoxic molecules. against vinblastin (human cervix carcinoma KB-3-1 cells; Also in the case of the intrinsic apoptotic pathway, Hela derivatives), vincristine (human leukemia (HL)-60 which recently was shown to be mediated by a cells) and adriamycin (human colon cancer cells mitochondria-specific ceramide pool (reviewed in [24]), SW620). UGCG blockage resensitizes multidrug resist- the residence of the UGCG in the Golgi apparatus seems ant breast cancer cell to anticancer drugs via downregu- to prohibit ceramide clearance. lation of MDR1 [18]. The resistance of tumors to chemotherapeutic Materials and methods agents occurs via highly complex mechanisms and is Cell culture a serious challenge in cancer therapy. The P-gp pro- The human breast adenocarcinoma cell line MCF-7 tein expressed at the plasma and Golgi membrane were purchased from the Health Protection Agency plays an important role in multidrug resistance by (catalogue number: 86,012,803, European Collection of transporting toxic substances out of the cell (reviewed Cell Cultures, ECACC, Salisbury, UK) and was cultured in [19]) (Fig. 4). This efflux pump, which is also in Dulbecco’s Modified Eagle Medium (DMEM) contain- expressed in normal tissue for protection from toxic ing high glucose, no phenol-red and no HEPES. 1% substances, transports hydrophobic substances like GlutaMAX, 1% sodium pyruvat and 5% charcoaled fetal nonylphenol ethoxylate (component of human urine) bovine serum (FBS) (Sigma-Aldrich, Deisenhofen, (reviewed in [20]). In addition, the P-gp protein Germany) was added. Cells were incubated at 37 °C in transports hydrophobic substances like anthracyclines, an atmosphere containing 5% CO2. For selection of taxanes and kinase inhibitors. P-gp is regulated by a stably transfected cells, G418 (Thermo Fisher Scientific, variety of mechanisms on transcriptional and post- Waltham, Massachusetts, USA) was added. transcriptional level like extracellular-signal regulated kinase (ERK) signaling, Skp1-Cullin1-FBX015 Stable transfection of UDP-glucose ceramide glucosyl- (SCFFBX15) complex and glycogen synthase kinase 3 transferase (UGCG) expression plasmid in MCF-7 cells (GSK-3), which transcriptionally inhibits MDR1 UDP-glucose ceramide glucosyltransferase (UGCG) ex- through wingless (Wnt)/β-catenin signaling (reviewed pression plasmid (pCMV6-ENTRY vector) was purchased in [19]), but also via ubiquitin-proteasomal degrad- from OriGene Technologies Inc. (Rockville, USA). The ation by action of the ubiquitin-activating enzyme E2 pTarget empty vector was purchased from Promega R1 (UBE2R1). For more detailed information of P-gp GmbH (Mannheim, Germany). Stable transfection was please see the following reviews: [21, 22]. performed with Lipofectamine® 2000 Reagent (Invitrogen The UGCG provides a major route for ceramide by Life Technologies) according to the manufacturer’s clearance in the case of an increased activity of CerS6, protocol. MCF-7 cells were transfected with 2 μgofthe which generates the pro-apoptotic C16:0-Cer, accordingly, distinct plasmid and selected over 5 weeks with different ceramide induced apoptosis is not induced. It is likely, G418 concentrations. Under a G418 concentration of that in the case of extrinsic and intrinsic apoptosis 400 μg/ml all untransfected cells died, subsequently, this pathway the UGCG is not capable to clear the ceramide concentration was considered to be sufficient for culturing due to its localization. Through extracellular signals like the stable transfected cells. oxidative stress or Tumor necrosis factor α (TNFα) cellu- lar ceramide concentration increases and induces apop- Sphingolipid concentration determination by high- tosis. Ceramides can either be generated by the performance thin-layer chromatography (HPTLC) activation of the sphingolipid de novo synthesis, which All chemical reagents were of analytical grade or occurs in the endoplasmic reticulum or by induction of higher. Lipid standards where from Avanti Polar the salvage pathway e. g. by hydrolysis of . Lipids (Alabaster, USA) or Matreya LLC (Pleasant Initiation of the extrinsic apoptosis signaling pathway, Gap, USA). Organic solvents were from Rathburn occurs by ligand binding to the Tumor necrosis factor Chemicals Ltd. (Walkerburn Scotland). The high receptor 1 (TNFR1, death receptor). Subsequently, recep- performance thin-layer chromatography (HPTLC) sil- tor trimerization and activation of the protein Factor as- ica plates were from Whatman, UK. 9 × 106 MCF-7 sociated with activation of neutral sphingomyelinase cells were scraped in 1 x PBS, centrifuged and frozen (SMase) (FAN) that binds to the intracellular neutral in liquid nitrogen. Cells were dried under the hood SMase domain (NSD), thereby activating the neutral for three days and stored in a freezer (− 80 °C) until SMase, occurs. The hydrolysis of sphingomyelin in the analyzed. Each dried sample was solubilized in ice plasma membrane leads to C16:0-ceramide release, which cold MQ-water (1 ml) by bath sonication (ice-water activates caspase-dependent apoptosis (reviewed in [23]). bath) for 5 min. The solubilized samples were trans- The UGCG seems not to be capable to logistically reach ferred to clean glass test tubes (with screw-caps). Wegner et al. BMC Cancer (2018) 18:153 Page 4 of 10

TotallipidswereextractedbyamodifiedBlightand Retrospective analysis Dyer protocol: 1 ml of MQ-water was added to each UGCG and MDR1 sample (2 ml of total water volume), 3 ml of chloro- Upstream effectors form:isopropanol (2:1, vol:vol) was added to each sample, samples were vortexed thoroughly for 10 s, UGCG promoter methylation Recently, Liu et al. [25] and rotated end-over-end for 20 min at RT. The sam- showed that the CpG island methylation at the UGCG ples were centrifuged at 4.000 rpm for 20 min at RT promoter (Fig. 2) correlates inversely with its gene to separate the phases. The organic phase (bottom) expression and multidrug resistance development in in- was carefully extracted with a glass Pasteur pipette vasive ductal breast cancer tissue. UGCG CpG island and placed in a clean glass tube, without disturbing methylation was negatively associated with estrogen the protein precipitate at the interphase. The chloro- receptor (ER) positive status (ERα expressing) and posi- form:isopropanol (2:1, vol:vol) extractions were re- tively associated with HER-2 positive status [25]. In peated once, as described above, and combined with addition, in vitro experiments showed that 5-Aza-2`- the previous extracts. 3 ml of hexane was then added deoxycytidine (decitabine; DNA methylation inhibitor) to each sample, samples were vortexed, rotated and could reverse the DNA methylation status of the UGCG centrifuged as described above. The hexane phases promoter, which restores UGCG and (top) were extracted and combined with the previous correlates with multidrug resistance development. The chloroform:isopropanol extracts. The organic solvent study also showed that the methylation status of the containing the extracted lipids was dried under a UGCG promoter is related to the expression of DNA stream of nitrogen. The dry samples were stored at − (cytosine-5)-methyltransferase 3a (DNMT3a) (Fig. 2), 20 °C. Each of the dried lipid sample was re- which catalyzes the transfer of methyl groups to CpG solubilized in an appropriate volume of chloroform:i- structures in the DNA [25]. However, previous data sopropanol (2:1, vol:vol). The lipid samples were nor- from the same group showed a correlation of UGCG ex- malized according to their corresponding protein pression with ER positive and HER-2 positive status in concentrations and applied on an HPTLC plate using breast cancer cells [26]. Since the DNMT3a encoding an autosampler. The HPTLC plate was developed gene is frequently mutated in several cancer types [27], using the solvent system chloroform:methanol:aceto- more studies are needed to shed light on the DNMT3a- ne:acetic acid:water (10:2:4:2:1). The sugar residues of UGCG-P-gp axes. Studies from Raghavan et al. show the glycolipids were visualized by spraying the plate that beside UGCG, also other enzymes involved in the with an orcinol solution (0.3% orcinol in 20% are methylation-dependently regulated sulphuric acid) and by heating (< 5 min, 120 °C). and thus leading to altered sensitivity against several Lipids were identified with the help of commercial drugs [28]. The cell membrane composition of doxo- standards that were run in parallel with the samples rubicin resistant MCF-7 cells differs from membrane on the HPTLC plate. composition of sensitive MCF-7 cells (Fig. 4). E. g. cell The precipitated protein was isolated as follows: 1 ml membranes of resistant cells exhibit an increased of chloroform and 1.5 ml of methanol was added to the sphingomyelin concentration [28]. By decitabine treat- remaining aqueous phases of the samples. Samples were ment the changes in resistant MCF-7 cell membranes briefly vortexed and centrifuged at 4.000 rpm for could partially be reversed. The question remains 20 min. The aqueous upper phase was carefully removed whether the altered membrane composition leads to with a glass Pasteur pipette and discarded, leaving the multidrug resistance induction by altering gene expres- interphase (with the precipitated proteins) untouched. sion or the overexpression of the UGCG induces altered 2 ml of methanol was added to each of the samples. membrane composition and thus leading to multidrug Samples were again briefly vortexed and centrifuged at resistance. 4.000 rpm for 20 min, to pellet the precipitated proteins. The chloroform:methanol solution was carefully re- P-gp and others on UGCG promoter In 2012, studies moved and discarded with a glass Pasteur pipette, showed that doxorubicin treatment leads to an increase without disturbing the pelleted proteins. The pellets of UGCG mRNA and protein concentration in ER posi- were carefully dried to completion, under a stream of tive MCF-7 cells, but only to a slight increase in ER nitrogen. Each protein sample was re-solubilized in negative MDA-MB-231 cells [29]. This is accompanied 500 μlofan8Mureasolution(8MureainPBS, by development of resistance to doxorubicin treatment. pH 6.8, 0.5% SDS) by vigorous vortexing at RT. The When the transcription factor Sp1, which has a binding protein samples were stored at − 20 °C. The concen- site at the UGCG promoter, is inhibited by Sp1 decoy trations of the solubilized proteins were determined ODNs transfection, doxorubicin does not lead to an in- by the method of Lowry. creased UGCG mRNA and protein expression in MCF-7 Wegner et al. BMC Cancer (2018) 18:153 Page 5 of 10

Fig. 2 Upstream effectors – Schematic display of UGCG promoter activity regulating processes in the context of multidrug resistance.

DNMT3a = DNA (cytosine-5)-methyltransferase 3a,CH3 = methyl group, MDR1 = multidrug resistance protein 1,ERα = estrogen receptor α, UGCG = UDP-glucose ceramide glycosyltransferase,RTN-1C=Reticulon-1C cells (Fig. 2). The doxorubicin resistance is reversed and effect on the UGCG promoter is exactly mediated since apoptotic events occur. Additionally, blocking the ER this chemotherapeutic agent leads to cell stress by subtype α (ERα) also prevents UGCG upregulation after induction of reactive oxygen species (ROS) or p53 accu- doxorubicin treatment (Fig. 2)[29]. In ER positive MCF- mulation and subsequently induces apoptotic signaling 7 cells blocking of ERα has a severe impact on the cell pathways. However, since doxorubicin is a known in- metabolism, since ERα promotes cell proliferation by ducer of P-gp it has to be clearified whether or not this direct transcriptional upregulation of several prolifera- induction is mediated via a doxorubicin-dependent tion promoting like Myc, B-cell lymphoma 2 activation of UGCG gene transcription resulting in an (BCL2) and E2F transcription factor 1 (E2F1) and UGCG-regulated MDR1 gene expression (Fig. 4). indirectly like FOS (reviewed in [30, 31]). This led us to Whether or not this mechanism is possible for other P- suggest, that the decrease of UGCG mRNA after ERα in- gp inducer or inhibitors such as verapamil or ciclosporin hibition and induction of apoptosis after doxorubicin should be investigated in the future. treatment may be based on an entire block of the cell Most studies show an UGCG-dependent regulation of metabolism. In addition, although in the study from MDR1 in the context of multidrug resistance induction Zhang et al. Sp1 decoy ODNs were used, which contain (Fig. 3). However, studies by Zhang et al. revealed that the sequence of the UGCG putative Sp1/GC-rich this dependency occurs possibly vice versa, meaning that binding site, it cannot be excluded that another Sp1- P-gp can directly influence UGCG expression in breast regulated gene is blocked in its expression, which could cancer cells [32] (Fig. 2). Zhang et al. showed that treat- be responsible for the UGCG promoter activity increase. ment of adriamycin resistant MCF-7 cells, which exhibit If this is the case, the altered UGCG promoter activity is an increased MDR1 and UGCG expression as compared only a downstream effect and not directly mediated by to control cells, with siRNA against MDR1 results in Sp1. The same might be true for the ERα effect. It is also decreased MDR1 and UGCG mRNA expression and possible that additional transcription factors to Sp1 are protein concentration indicating that MDR1 regulates involved in UGCG promoter activity regulation. UGCG expression. How exactly this UGCG upregulation Therefore, it would be helpful to perform reporter gene is accomplished by MDR1 has not been investigated. studies with deleted transcription factor binding sites of the UGCG promoter to verify this result. In addition, Downstream effectors DNA-protein interactions of the UGCG promotor se- Induction of apoptosis and potential heterodimerization of quence and e.g. Sp1 should be monitored by chromatin UGCG immunoprecipitation (ChIP) assays or by electrophoretic Most studies published in the last two decades, which mobility shift assay (EMSA) to proof the interaction. investigated the involvement of the UGCG and MDR1 Furthermore, it has to be clarified how the doxorubicin in multidrug resistance, have focused on the association Wegner et al. BMC Cancer (2018) 18:153 Page 6 of 10

ab

Fig. 3 Downstream effectors – Schematic display of UGCG regulated processes in the context of multidrug resistance. a UGCG mRNA inhibition leads to induction of apoptotic processes and prevents multidrug resistance development. b UGCG activity derived GSLs such as gangliosides regulate MDR1 promoter activity. UGCG = UDP-glucose ceramide glycosyltransferase,ERK=extracellular-signal regulated kinase,Bcl-2=B-cell lymphoma 2,Bcl-xL=B-cell lymphoma-extra large,NFκB=nuclear factor (NF) κB,cSrc=tyrosine kinase,Gb3=Globotriasoylceramide,MDR1= multidrug resistance protein 1 of UGCG and its downstream effectors. Progress was the sphingolipid signaling pathway is the main factor in made regarding the elucidation of processes following this process, because the UGCG overexpressing cells UGCG inhibition, especially processes leading to an in- showed only a slight increase in GlcCer amount (see also duction of apoptotic signaling pathways. In 2010 already, Fig. 1). This may be due to a low substrate supply or low Sun et al. showed that inhibition of the UGCG leads to UGCG activity, a fast processing of GlcCer to complex high ceramide levels in breast cancer cells and results in GSLs or degradation of GlcCer. Furthermore, the UGCG caspase-3 activation and subsequently cell death (Figs. 3a can interact with other unknown proteins and induces and 4). This study also demonstrated that in a mice thereby the drug resistance. It was already shown that xenograft model treatment with shRNA against the UGCG protein from rat is organized as a heterodimer or UGCG leads to decreased tumor growth and that beside heterooligomer [36]. Approved binding partners for the the UGCG also the MDR1 gene expression and subse- UGCG are c-Fos [37] and the neuroendocrine-specific quently P-gp protein is inhibited in vivo [12]. Years later protein Reticulon-1C [38], whereby the UGCG activity Wang et al. showed that UGCG knockdown decreases does not depend on complex formation with these pro- the mRNA and protein levels of Bcl-2 (anti-apoptotic) teins (Figs. 2 and 4). Future studies should investigate via reduction of ERK phosphorylation, which was in- the influence of these complexing partners in the duced by ceramide accumulation in K562/A02 cells [33] process of multidrug resistance. The idea of UGCG pro- (Fig. 3a). Beside Bcl-2, also B-cell lymphoma-extra large tein interaction with another protein could be supported (Bcl-xL), which has an anti-apoptotic effect by prevent- by a study showing that curcumin inhibits UGCG ing e. g. cytochrome c release from mitochondria, is de- activity in drug-resistant cells only. This means that the creased in its expression when the UGCG is inhibited multidrug resistant cells express proteins, which mediate [34] (Fig. 3a). However, Liu et al. published that MCF-7 the curcumin-induced UGCG inhibition [39]. In Hela cells, which overexpress the UGCG are resistant against cells only the combination of mifepristone (progesterone adriamycin and ceramide toxicity, but P-gp was not de- receptor antagonist) and mitomycin c (DNA synthesis tectable [35]. In addition, the phosphorylation status of block) inhibited UGCG [40], meaning that after mitomy- the anti-apoptotic Bcl-2 protein was not altered indicat- cin c treatment one or more specific genes are not tran- ing that the multidrug resistance in these cells is inde- scribed, which are essential for induction of multidrug pendent from both, MDR1 and Bcl-2. It is possible that resistance. It is possible that also after curcumin Wegner et al. BMC Cancer (2018) 18:153 Page 7 of 10

Fig. 4 Summary of the molecular mechanisms regarding the connection between UGCG and MDR1 in the process of multidrug resistance development. In sensitive cells (green) cytotoxic agents lead to apoptosis by, amongst other mechanisms, UGCG gene expression inhibition and following ceramide accumulation. During the process of multidrug resistance development (red) cytotoxic agents may lead to an increased gene expression of UGCG and MDR1 resulting in increased UGCG protein concentration and P-gp concentration. P-gp functions as an efflux transporter for the cytotoxic agents and works also as a GlcCer flippase in the Golgi apparatus supporting ceramide and GlcCer clearance. The increased GSL production leads to alteration of raft structures in the plasma membrane resulting in increased P-gp and kinase activity. The kinases induce signal- ing pathways, which lead to increased MDR1 gene transcription. Besides, the altered membrane composition blocks the diffusion of extracellular cytotoxic agents. MDR1 and UGCG are able to influence each other on transcriptional level. The influence of dimerization partners of UGCG in the context of multidrug resistance development is unknown. UGCG = UDP-glucose ceramide glycosyltransferase, MDR1 = multidrug resistance protein 1, P-gp = P-glycoprotein, GSL = , Gb3 = Globotriasoylceramide treatment a potential dimer partner of UGCG is not target protein for UGCG interaction would be the P-gp expressed anymore. However, curcumin does also induce protein itself. Furthermore, it has to be clarified in what necroptosis and is thereby able to revert multidrug kind of way the UGCG-derived GSL are involved in resistance [41]. A possible partner of UGCG protein apoptotic processes since ceramides induce apoptosis via interaction could be the cellular tumor antigen p53. membrane bound receptors. Inhibition of the UGCG by thre-1-Phenyl-2-decanoyl- amino-3-morpholino-1-propanol hydrochloride (PDMP) Glycosphingolipids on MDR1 or siRNA treatment in p53 deficient human osteosar- A few studies describe the direct influence of UGCG ac- coma cells (U20S) leads to increased sensitivity to mito- tivity on MDR1 expression, indicating that also ganglio- mycin c resulting in ceramide accumulation, decreased sides resulting from UGCG activity may have an effect cell viability and apoptosis induction shown by increased on MDR1. These studies are described in this chapter. It caspase 3 and 7 activity [42]. p53 deficiency in U2OS could be shown that inhibition of the transcription fac- cells was accomplished by stable transfection with the tor nuclear factor (NF) ΚB results in decreased MDR1 E6 oncoprotein of Human Papillomavirus 16 (HPV16), gene and P-gp protein expression [43]. Thereby, NFΚB which mediates accelerated degradation of p53. The data seems to bind directly to the MDR1 promoter through of Haynes et al. may indicate a p53-dependent UGCG an intronic site (Fig. 3b). In this respect, it is interesting regulation (Fig. 2), but studies with p53 proficient cells that a -dependent activation of NFΚB via should be performed in the future. Another interesting PKC and NDAPH oxidase could be shown in microglia Wegner et al. BMC Cancer (2018) 18:153 Page 8 of 10

[44] (Fig. 3b). The precursor of all gangliosides is GlcCer clearance (reviewed in [48]. Subsequently, ceramides are generated by the UGCG and accordingly an increased not able to induce apoptotic signaling pathways any- UGCG activity leads to increased gangliosides and/or ce- more. However, De Rosa et al. showed that P-gp activity rebrosides levels [45] (Figs. 1 and 4). The influence of as a flippase is required for neutral GSL production, but NFΚB on multidrug resistance should be also investi- not for acidic GSL (e. g. gangliosides) generation [49]. gated in other cell types to determine whether or not The hypothesis of the flippase task of P-gp is also sup- this is a unique mechanism. Liu et al. showed for the ported by the fact that beside ceramides, GlcCer seem to first time that globo series GSL produced by UGCG induce toxic effects in the cell [50]. Cer are anabolized activity mediate MDR1 expression through cSrc and β- to GlcCer, which undergo clearance by flipping and catenin signaling [46]. Figure 3b shows that UGCG usage for non-toxic complex GSL synthesis (Fig. 4). It is overexpression combined with chemotherapeutic agent an ongoing discussion whether or not the Cer and treatment leads to increased concentrations of e. g. Gb3 GlcCer clearance or the production of complex GSL are and Gb5 in GSL-enriched microdomains (GEM) result- the mechanisms to overcome drug sensitivity. The in- ing in tyrosine kinase cSrc activation and decreased β- creased GSL production and subsequent transport to catenin phosphorylation and increased nuclear β-catenin cell membranes alters P-gp activity. There are a variety (Fig. 4). cSrc-induced phosphorylation of GSK-3β, which of parameters, which influence the P-gp activity is inactive when phosphorylated and therefore leads no (reviewed in [20]). E. g. the presence of cholesterol, more to phosphorylation of β-catenin, results in nuclear which alters membrane packing and fluidity affects cata- β-catenin. The nuclear β-catenin may bind in a complex lytic activity of P-gp and its stimulation by substrates with the T-cell factor 4 (Tcf4) to the Tcf4/lymphoid and modulators (Fig. 4). It is to add, that possibly the enhancer factor (LEF) binding motif at the MDR1 UGCG structure may allow the GlcCer to be released to promoter and thus enhancing promoter activity leading the inner as well as to the outer leaflet of the Golgi ap- to increased efflux of anticancer drugs of the cell [46]. paratus. The structure of the ceramide determined by its Another possibility to increase GSK-3β phosphorylation chain length could determine a preference for the inner and subsequently reduce GSK-3β activity is to inhibit or outer leaflet of the cell organell. In this case the cell protein phosphatase 2 (PP2A) activity, which dephos- could discriminate between GlcCer intended for com- phorylates the GSK-3β protein leading to activation of plex GSL synthesis and GlcCer taking the sphingolipid the protein and degradation of β-catenin. However, transport protein FAPP2 route to the trans-Golgi appar- currently, a correlation of β-arrestin 2 and MDR1 atus and a flippase would be unnecessary. expression could be shown in breast cancer [47]. The protein β-arrestin 2 is highly expressed in multidrug re- Impact of UGCG activity induced alterations of membrane sistant cells and a knockout of β-arrestin 2 decreased composition by GSL MDR1 gene expression and P-gp protein expression dra- Beside the UGCG-P-gp connection, it could be shown matically leading to increased doxorubicin sensitvity. that the composition of the cell membrane plays an im- Since β-arrestin 2 is able to stabilize β-catenin by inter- portant role in the induction of multidrug resistance. acting with the intracellular proteins Dishevelled and The question is how UGCG activity can influence P-gp Axin, an increased expression leads to translocation of activity not directly, but via GSL synthesis, which lead to β-catenin to the nucleus, where the previous described alteration of the membrane composition. Tyler et al. mechanisms occur (Fig. 3b). In the future it has also to showed that the globo-series GSL Gb3 concentration is be investigated whether or not β-arrestin 2 is able to increased in non-small cell lung cancer (NSCLC) and bind directly to β-catenin and thereby execute the multi- malignant pleural mesothelioma (MPM) cells, which are drug resistance inducing effect. cisplatin resistant [51] (Fig. 4). Treatment with the UGCG inhibitor 1-phenyl-2-palmitoylamino-3-morpho- P-gp as a flippase lino-1-propanol (PPMP) decreased the induced cell sur- The UGCG protein modifies ceramide on the cytosolic face Gb3, but not P-gp protein expression (MDR1 gene surface of the Golgi apparatus (Fig. 4). The conversion expression pattern similar). Nevertheless, Gb3 silencing of GlcCer to LacCer by adding a galactose molecule is restores cisplatin sensitivity, indicating that the UGCG accomplished in the luminal leaflet of the Golgi appa- protein does not have a direct effect on P-gp. This leads ratus. For this reaction GlcCer has to be flipped from to the assumption that Gb3 alters membrane compo- the cytosol to the Golgi apparatus lumen. Morad and sition leading to a changed P-gp activity status. Another Cabot postulate that P-gp contributes to multidrug study already showed that membrane lipids of drug re- resistance by adopting GlcCer transfer from the Golgi sistant MCF-7 cells exhibit an increased lipid packing apparatus cytosol to the Golgi lumen in form of a and membrane fluidity as compared to membrane lipids GlcCer flippase and thereby promoting ceramide of sensitive cells. Following decitabine treatment these Wegner et al. BMC Cancer (2018) 18:153 Page 9 of 10

biophysical changes in membrane lipids are altered Acknowledgements meaning that the packing density is decreased and the The knockdown of the UGCG by CRISPR-Cas was kindly performed by Martin Wegner from the working group Manuel Kaulich (Institute for Biochemistry II, membrane fluidity is increased even more [28]. The University Clinic Frankfurt am Main). Matti Kjellberg is acknowledged for the authors hypothesized that the resistant cells exhibit HPTLC work. increased cholesterol-sphingomyelin raft structures, Funding which may influence drug transport and therefore induce This work was funded by the Deutsche Forschungsgemeinschaft (DFG) (WE multidrug resistance. Ghetie et al. could already show that 5825/1–1; MSW) and the SFB 1039, the August Scheidel-Stiftung and the P-gp is more active in cholesterol and sphingomyelin rich Heinrich und Fritz Riese-Stiftung and the Sigrid Jusélius Foundation. raft structures as compared to a localization outside these Availability of data and materials membrane structures [52](Fig.4). The changes regarding The publications quoted are publically available from the publishers. All data the biophysical properties in the study of Raghavan et al. supporting the fact that the UGCG is a unique protein in cells are presented are blocked by decitabine treatment [28]indicatingan within the manuscript. Raw data as required may be requested from the corresponding author. epigenetic mechanism meaning methylation-dependent regulation of enzymes of the lipid metabolism in the cell Authors’ contributions as it has already been shown for the UGCG. Since decita- All authors have read and approved the final version of this manuscript. MSW wrote the main parts of all sections. LG added the section Materials bine inhibits the methylation of all genes in the cell, no and Methods and illustrated Figs. 2 and 3. PM analyzed and interpreted the detailed information about the molecular mechanisms are HPTLC data and added valuable information regarding the flippase function. available. Peetla et al. reviewed in 2013 that in drug resis- SG and GG edited the manuscript. tant cell membranes the sphingolipid and/or cholesterol Ethics approval and consent to participate content is increased leading to a higher membrane order Not applicable. and less permeability resulting in reduced lipophilic drug diffusion and less effective cytotoxic substances [53] inde- Consent for publication Not applicable. pendent from P-gp (Fig. 4). This shows, not only the impact on membrane proteins is altered by changed lipid Competing interests composition, but also the behavior of the toxic substance The authors declare that they have no competing interests. in the lipid bilayer. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Conclusions published maps and institutional affiliations. The influence of the UGCG on multidrug resistance was investigated in depth for the last two decades. But still Author details 1pharmazentrum frankfurt/ ZAFES, Institute of Clinical Pharmacology, Johann many pieces of the puzzle need to be analyzed. The link Wolfgang Goethe-University, House 74, Theodor Stern-Kai 7, 60590 Frankfurt between UGCG overexpression and multidrug resistance am Main, Germany. 2Biochemistry, Faculty of Science and Engineering, Åbo induction is more complex than it was anticipated. Par- Akademi University, Artillerigatan 6A, III, BioCity, FI-20520 Turku, Finland. ticularly, the influence of membrane properties should Received: 2 August 2017 Accepted: 31 January 2018 be investigated in future studies since during malignant transformation of cells the biophysical parameters of the References membranes changes. 1. Ichikawa S, Sakiyama H, Suzuki G, Hidari KI, Hirabayashi Y. Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first Abbreviations glycosylation step of glycosphingolipid synthesis. Proc Natl Acad Sci U S A. ABCB1: ATP-binding cassette sub-family B member 1; Bcl-2: B-cell lymphoma 1996;93:12654. 2; Bcl-xL: B-cell lymphoma-extra large; Cer: Ceramide; CerS6: Ceramide 2. D'Angelo G, Capasso S, Sticco L, Russo D. Glycosphingolipids: synthesis and synthase 6; ChIP: Chromatin immunoprecipitation; CRISPR: Clustered functions. FEBS J. 2013;280:6338–53. Regularly Interspaced Short Palindromic Repeats; CS: CRISPR score; 3. 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