Increased glucosylceramide production leads to decreased cell energy metabolism and lowered tumor marker expression in non-cancerous liver cells.

Hepatocellular carcinoma (HCC) is one of the most difficult cancer types to treat. Liver cancer is often diagnosed at late stages and therapeutic treatment is frequently accompanied by development of multidrug resistance. This leads to poor outcomes for cancer patients. Understanding the fundamental molecular mechanisms leading to liver cancer development is crucial for developing new therapeutic approaches, which are more efficient in treating cancer. Mice with a liver specific UDP-glucose ceramide glucosyltransferase (UGCG) knockout (KO) show delayed diethylnitrosamine (DEN)-induced liver tumor growth. Accordingly, the rationale for our study was to determine whether UGCG overexpression is sufficient to drive cancer phenotypes in liver cells. We investigated the effect of UGCG overexpression (OE) on normal murine liver (NMuLi) cells. Increased UGCG expression results in decreased mitochondrial respiration and glycolysis, which is reversible by treatment with EtDO-P4, an UGCG inhibitor. Furthermore, tumor markers such as FGF21 and EPCAM are lowered following UGCG OE, which could be related to glucosylceramide (GlcCer) and lactosylceramide (LacCer) accumulation in glycosphingolipid-enriched microdomains (GEMs) and subsequently altered signaling protein phosphorylation. These cellular processes lead to decreased proliferation in NMuLi/UGCG OE cells. Our data show that increased UGCG expression itself does not induce pro-cancerous processes in normal liver cells, which indicates that increased GlcCer expression leads to different outcomes in different cancer types.

UDP-glucose ceramide glucosyltransferase activates AKT, promoted proliferation, and doxorubicin resistance in breast cancer cells.

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.

Influence of glycosphingolipids on cancer cell energy metabolism.

A growing number of studies describe a connection between glycosphingolipids (GSLs) and glutamine metabolism, glucose metabolism and mitochondrial dysfunction in cancer cells. Since deregulated cell energy metabolism is one of cancer cells hallmarks, investigating this connection is an important step in the development of anti-cancer therapies. GSL species are often aberrantly regulated in human cancers. They cluster in signaling platforms in the plasma membrane and organelle membranes in so called glycosphingolipid enriched microdomains (GEMs), thereby regulating cell signaling pathways. The most important glutamine transporter for epithelial cells, alanine-serine-cysteine transporter 2 (ASCT2) locates in GEMs and is regulated by GEM composition. The accumulation of glucosylceramide and lactosylceramide in mitochondria associated ER membranes (MAMs) leads to increased oxidative phosphorylation. This increases mitochondrial reactive oxygen species (ROS) levels and influences mitochondrial dynamics. Here, we review current knowledge about deregulated GSL species in cancer, GSL influence on glutamine and glucose metabolism. In addition, the role of GSLs in MAMs, oxidative phosphorylation (OXPHOS) and mitochondrial dynamics with a special focus on mechanistic target of rapamycin (mTOR) signaling is discussed. mTOR seems to play a pivotal role in the connection between GSLs and glutamine metabolism as well as in mitochondrial signaling.

Ether lipid and sphingolipid expression patterns are estrogen receptor-dependently altered in breast cancer cells.

Identifying co-expression of lipid species is challenging, but indispensable to identify novel therapeutic targets for breast cancer treatment. Lipid metabolism is often dysregulated in cancer cells, and changes in lipid metabolism affect cellular processes such as proliferation, autophagy, and tumor development. In addition to mRNA analysis of sphingolipid metabolizing enzymes, we performed liquid chromatography time-of-flight mass spectrometry analysis in three breast cancer cell lines. These breast cancer cell lines differ in estrogen receptor and G-protein coupled estrogen receptor 1 status. Our data show that sphingolipids and non-sphingolipids are strongly increased in SKBr3 cells. SKBr3 cells are estrogen receptor negative and G-protein coupled estrogen receptor 1 positive. Treatment with G15, a G-protein coupled estrogen receptor 1 antagonist, abolishes the effect of increased sphingolipid and non-sphingolipid levels in SKBr3 cells. In particular, ether lipids are expressed at much higher levels in cancer compared to normal cells and are strongly increased in SKBr3 cells. Our analysis reveals that this is accompanied by increased sphingolipid levels such as ceramide, sphingadiene-ceramide and sphingomyelin. This shows the importance of focusing on more than one lipid class when investigating molecular mechanisms in breast cancer cells. Our analysis allows unbiased screening for different lipid classes leading to identification of co-expression patterns of lipids in the context of breast cancer. Co-expression of different lipid classes could influence tumorigenic potential of breast cancer cells. Identification of co-regulated lipid species is important to achieve improved breast cancer treatment outcome.

UGCG overexpression leads to increased glycolysis and increased oxidative phosphorylation of breast cancer cells.

The only enzyme in the glycosphingolipid (GSL) metabolic pathway, which produces glucosylceramide (GlcCer) de novo is UDP-glucose ceramide glucosyltransferase (UGCG). UGCG is linked to pro-cancerous processes such as multidrug resistance development and increased proliferation in several cancer types. Previously, we showed an UGCG-dependent glutamine metabolism adaption to nutrient-poor environment of breast cancer cells. This adaption includes reinforced oxidative stress response and fueling the tricarboxylic acid (TCA) cycle by increased glutamine oxidation. In the current study, we investigated glycolytic and oxidative metabolic phenotypes following UGCG overexpression (OE). UGCG overexpressing MCF-7 cells underwent a metabolic shift from quiescent/aerobic to energetic metabolism by increasing both glycolysis and oxidative glucose metabolism. The energetic metabolic phenotype was not associated with increased mitochondrial mass, however, markers of mitochondrial turnover were increased. UGCG OE altered sphingolipid composition of the endoplasmic reticulum (ER)/mitochondria fractions that may contribute to increased mitochondrial turnover and increased cell metabolism. Our data indicate that GSL are closely connected to cell energy metabolism and this finding might contribute to development of novel therapeutic strategies for cancer treatment.

UGCG influences glutamine metabolism of breast cancer cells.

UDP-glucose ceramide glucosyltransferase (UGCG) is the key enzyme in glycosphingolipid (GSL) metabolism by being the only enzyme that generates glucosylceramide (GlcCer) de novo. Increased UGCG synthesis is associated with pro-cancerous processes such as increased proliferation and multidrug resistance in several cancer types. We investigated the influence of UGCG overexpression on glutamine metabolism in breast cancer cells. We observed adapted glucose and glutamine uptake in a limited energy supply environment following UGCG overexpression. Glutamine is used for reinforced oxidative stress response shown by increased mRNA expression of glutamine metabolizing proteins such as glutathione-disulfide reductase (GSR) resulting in increased reduced glutathione (GSH) level. Augmented glutamine uptake is also used for fueling the tricarboxylic acid (TCA) cycle to maintain the proliferative advantage of UGCG overexpressing cells. Our data reveal a link between GSL and glutamine metabolism in breast cancer cells, which is to our knowledge a novel correlation in the field of sphingolipid research.

GPER1 influences cellular homeostasis and cytostatic drug resistance via influencing long chain ceramide synthesis in breast cancer cells.

The G protein-coupled estrogen receptor 1 (GPER1) is involved in the regulation of physiological processes such as cellular growth and proliferation, but also in pathophysiological processes such as tumor development. The role of GPER1 in breast cancer is contradictory. Therefore, we investigated the influence of GPER1 overexpression on cellular processes in MCF-7 breast cancer cells. GPER1 overexpression leads to a cell cycle arrest in the G1 phase, induction of autophagy and reduced proliferation. Reduced proliferation was accompanied by a reduced basal respiration and reduced glycolysis rate in GPER1 overexpressing cells. This is presumably ascribable to mitophagy induction following GPER1 overexpression. However, GPER1 overexpressing cells were less sensitive against doxorubicin as compared to control cells. In previous work we showed the effect of transient GPER1 overexpression on the synthesis of several ceramide synthases (CerS) thereby influencing the sphingolipid pathway. Therefore, we investigated CerS expression and sphingolipid level in stable GPER1 overexpressing and control cells. Stable GPER1 overexpression strongly reduced CerS4, CerS5 and CerS6 promoter activity and CerS5 and CerS6 mRNA expression, whereas CerS2 mRNA expression was upregulated. The GPER1 effect on CerS5 promoter is mediated by GSK-3ß signaling. In addition, other enzymes of the sphingolipid pathway were upregulated. Our study provides new insights into the role of GPER1 and the activated sphingolipid pathways and how GPER1 may influence cellular processes such as cancer cell survival following chemotherapy. Further studies are needed to investigate the molecular mechanisms leading to these cellular effects. Finding new therapeutic targets for modulating specifically GPER1 in breast tumors may improve endocrine breast cancer therapy.

Characterization of cholesterol homeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts reveals a Niemann-Pick disease type C-like phenotype with enhanced lysosomal Ca2+ storage.

Sphingosine-1-phosphate (S1P) lyase irreversibly cleaves S1P, thereby catalysing the ultimate step of sphingolipid degradation. We show here that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1-/--MEFs), in which S1P and sphingosine accumulate, have features of Niemann-Pick disease type C (NPC) cells. In the presence of serum, overall cholesterol content was elevated in Sgpl1-/--MEFs, due to upregulation of the LDL receptor and enhanced cholesterol uptake. Despite this, activation of sterol regulatory element-binding protein-2 was increased in Sgpl1-/--MEFs, indicating a local lack of cholesterol at the ER. Indeed, free cholesterol was retained in NPC1-containing vesicles, which is a hallmark of NPC. Furthermore, upregulation of amyloid precursor protein in Sgpl1-/--MEFs was mimicked by an NPC1 inhibitor in Sgpl1+/+-MEFs and reduced by overexpression of NPC1. Lysosomal pH was not altered by S1P lyase deficiency, similar to NPC. Interestingly, lysosomal Ca2+ content and bafilomycin A1-induced [Ca2+]i increases were enhanced in Sgpl1-/--MEFs, contrary to NPC. These results show that both a primary defect in cholesterol trafficking and S1P lyase deficiency cause overlapping phenotypic alterations, and challenge the present view on the role of sphingosine in lysosomal Ca2+ homeostasis.