Regulation of lipid metabolism in breast cancer provides diagnostic and therapeutic opportunities
Cancer cells are characterized by extensive metabolic changes, since the rapid growth of tumors leads to lack of oxygen and hypoxia in the cells. One crucial feature is also the Warburg effect, a process in which glycolysis occurs despite the presence of oxygen in the tumor [1]. Alterations of many other meta-bolic pathways, such as increased nucleotide synthesis, are also well-known hallmarks of rapidly proliferating cancer cells [2]. Another profound phenotypic shift in malignant cells occurs in fatty acid and lipid metabo-lism [3]. The differences in lipid metabolism between normal and malignant tissues make these pathways attractive targets for cancer therapeutics.
The goal of this article is to provide an over-all perspective on the changes in lipid metabo-lism in breast cancer, and more focused reviews on specific topics can be found in the refer-ences. The article focuses on fatty acid and phospholipid metabolism and also presents other cancer-related lipids including lysophos-phatidic acid (LPA), eicosanoids and (glyco)sphingolipids. The changes of these lipids in breast cancer, as well as emerging diagnostic and therapeutic opportunities, are discussed. Steroids are also important cancer-related lipid molecules, but since these compounds have already been utilized for a long time in breast cancer treatment, this field is excluded from the present review.
Fatty acid metabolism in normal & malignant tissuesIn healthy adults, cells obtain the needed fatty acids mainly from nutrition. De novo synthesis of fatty acids occurs only in a very limited num-ber of tissues (i.e., in the liver, adipose tissue and lactating mammary gland). In liver and adipose tissue, high carbohydrate diet together with increased insulin levels stimulate de novo fatty acid synthesis to convert excess sugars into fatty acids and triglycerides for energy storage [4]. In the lactating mammary gland, fatty acid synthase (FASN) produces medium-chain fatty acids, which are easily digested by the offspring [5]. This key enzyme in lipogen-esis, illustrated in Figure 1A, catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA. Another important enzyme in fatty acid synthesis is acetyl-CoA-carboxylase (ACC1, gene name ACACA), whose function is to feed FASN with malonyl-CoA by cata-lyzing the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA, and thus acting as the rate-limiting enzyme in the fatty acid synthesis pathway. One of the key proteins regulating the expression of these enzymes and of membrane phospholipid synthesis is SREBP-1, which is stimulated by insulin, and which has a well-established role in hepato-cytes [6]. In adipocytes, however, PPAR-g is an important regulator of lipogenesis [7], and the role of SREBP-1 still remains unclear, since on
During malignant transformation, the lipid metabolism of cells changes radically and therefore alterations in lipid metabolism are a prominent feature of solid tumors. Lipid metabolism has been investigated at the gene and protein expression levels for several decades, but recent advances in lipidomics technology have also enabled the investigation of pathways at the level of molecular lipids. This review provides an overview of the changes of global lipid metabolism (i.e., fatty acid, phospholipid, eicosanoid and sphingolipid metabolism) in breast cancer, and discusses the diagnostic and therapeutic potential of these pathways.
KEYWORDS: breast cancer n diagnostics n eicosanoids n glycolipids n lipid metabolism n lysophosphatidic acid n phospholipids n sphingolipids n therapeutics
Mika Hilvo* & Matej OrešičBiotechnology for Health & Well-being, VTT Technical Research Centre of Finland, PO Box 1000, FI-02044 VTT, Espoo, Finland *Author for correspondence: Tel.: +358 20 7226131 Fax: +358 20 7227071 [email protected]
the one hand, for example, mice overexpressing SREBP-1 do show a phenotype in adipose tissue [8], but on the other hand, it has been reported that SREBP-1 does not affect the expression of lipogenic genes in adipocytes [9].
In normal tissues the activity of the de novo synthetic pathway is regulated by nutrition, whereas in cancers the pathway is dysregulated and out of nutritional control. The coordinated upregulation of FASN and ACC1 is an early event in tumor progression, as was shown by expression studies in the 1990s [10–12]. In nor-mal breast tissue the expression of these enzymes occurs in scattered cells in the lobules and ter-minal ducts, whereas in carcinomas in situ the expression becomes intense and highest expres-sion is found in high-grade ductal carcinomas in situ [10]. FASN has also been shown to pre-dict poorer disease-free and overall survival in breast cancer patients, and thus be associated with a higher risk of recurrence [11,12]. There are very few studies considering SREBP-1, the key regulator of FASN and ACACA, in breast cancer, and the existing studies are mainly per-formed in vitro. The correlation found between SREBP-1 and FASN expression is weak [13], although it has to be kept in mind that the activ-ity of SREBP-1 is regulated at the post-transla-tional level. Yang et al. have performed in vitro studies indicating that SREBP-1 is regulated by the MAPK and PI3K pathways and that the transcription of FASN is regulated by SREBP-1 [13,14]. However, more recently, Yoon et al. have reported that in breast cancer cells FASN and ACACA are not regulated by SREBP-1, instead,
the key regulator is HER2, and these proteins are regulated at the translational level by the mTOR signaling pathway [15]. Therefore, the exact role of SREBP-1 in regulating FASN and ACACA is still unclear in breast cancer.
In addition, new players have been identified that regulate the activity of ACC1 and hence the whole lipogenic pathway. One of the enzymes regulated by PI3K/AKT together with SREBP-1 is stearoyl-CoA desaturase 1 (SCD1), whose function is to produce monounsaturated fatty acids from saturated ones. The expression of SCD1 is increased in several cancers and it has been reported in lung cancer cells that SCD1 can regulate the activity of ACC1 by regulating the cellular content of palmitic acid, which is a negative regulator of ACC1, and also by con-trolling the phosphorylation status of AMPK, which in turn can regulate ACC1 activity by phosphorylation [16]. Inhibition of expression or activity of SCD1 has been reported to inhibit breast cancer cell growth [17]. SPOT14 (S14, THRSP) is amplified in 15–20% of breast cancers, and its expression correlates with the expression of ACC1 as well as with tumor grade and decreased disease-free survival [18,19]. The expression of SPOT14 is regulated by hormones and SREBP-1 [20] and the link between the lipogenic phenotype (i.e., ACC1 and SPOT14 expression) became more evident when the crys-tal structure of SPOT14 was recently revealed [21]. It was shown that SPOT14 can form het-erodimers with MIG12 protein, which can then induce the polymerization of ACC1 and thereby regulate its activity [21]. The link between SCD1
De novofatty acid synthesis
ACC1
Acetyl-CoA Malonyl-CoA
FASN
Palmitate
Regulation ofredox balance
Membranesynthesis
Proteinpalmitoylation
Formation oflipid rafts
Increased resistance to chemotherapy
PC branch of theKennedy pathway
Choline
ChoK
CT
CDP-choline
CHPT1
PCho
PC
oo–
Figure 1. Production of fatty acids and membrane phospholipids in cancer cells. (A) De novo synthesis of fatty acids may contribute to several different cellular processes in cancer. (B) Illustration of the PC branch of the Kennedy pathway that produces PC. ACC1: Acetyl-CoA carboxylase; ChoK: Choline kinase; CHPT: CDP-choline diacylglycerolphosphocholinetransferase; CT: CTP-choline cytidylyltransferase; FASN: Fatty acid synthase; PC: Phosphatidylcholine; PCho: Phosphocholine.
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and SPOT14 and ACC1 activity may raise the possibility of exploiting these enzymes to reduce lipogenesis in cancer cells.
Upregulation of membrane phospholipid metabolism in breast cancerThe glycerophospholipids, phosphatidyl choline (PC) and phosphatidylethanolamine, account for more than half of the total phospholipid spe-cies in eukaryotic cell membranes, and de novo synthesis of these phospholipids occurs via the so-called Kennedy pathway [22]. The pathway has analogous branches for PC and phospha-tidylethanolamine production, but as the PC branch is investigated more heavily in cancer, this article focuses on the PC branch only (Figure 1B). Choline kinase (ChoK) catalyzes the phosphorylation of free choline using ATP as a phosphate donor, and thus produces phos-phocholine. At least three isoforms of ChoK exist and among these the a-isoform is tumor related [23]. CTP-choline cytidylyltransferase then catalyzes the synthesis of CDP-choline from phosphocholine and CTP. CDP-choline is used by CDP-choline diacyl glycerol phos-phocholinetransferase as a substrate to pro-duce PC. The activity and expression of ChoK is increased in approximately 40% of breast tumors when compared with levels in healthy tissues, and the overexpression is highest in tumors of high grade and estrogen recep-tor (ER)-negative status [24]. It has also been demonstrated in cell line studies that ChoK is both necessary and sufficient for growth factor-induced proliferation and that ChoK inhibi-tors show strong antitumor activity in human breast cancer xenografts [25]. In addition to its recognized therapeutic value, choline metabo-lism also offers diagnostic opportunities. Not only the PC content but also the total choline-containing compounds, consisting of choline, phosphocholine and glycerophosphocholine, are increased in tumors, and these compounds can be detected noninvasively by magnetic reso-nance spectroscopy. Using magnetic resonance spectroscopy combined with MRI, breast can-cer can be separated from benign cases with rea-sonable specificity and sensitivity [26]. Another method for imaging is PET, in which, tradi-tionally, the tumors have been visualized using fluorodeoxyglucose (18F), although labeled [11C]choline has recently become an alternative to this compound. A study in ER-positive breast cancer patients showed that breast tumors can
be visualized using [11C]choline [27], which may also be used to observe patient responses to treatment with trastuzumab [28].
The regulation of lipid metabolism in breast cancer has been investigated in many stud-ies at the level of genes and proteins. Studies of molecular lipids, the end product of lipid metabolism, have become possible over the past decade with developments in the emerg-ing field of lipid omics. High-throughput lipid-omic methods allow for profiling of hundreds of molecular lipids in a single experiment [29]. For example, the authors’ investigations on the lipidomic changes in breast cancer tis-sue samples revealed that the phospholipids were increased, especially in the grade 3 and ER-negative tumors [30]. Specifically, the PCs containing major or minor products of FASN (i.e., saturated fatty acids C14:0, C16:0 and C18:0) were found at higher levels in these aggressive tumors, and also predicted poorer overall survival for the patients. The finding that these phospho lipids were particularly increased in ER-negative tumors was surpris-ing, as it has been shown that estrogen should upregulate de novo synthesis of fatty acids [31]. However, these results were consistent with the above-mentioned findings that ChoK activity is associated with ER negativity. In addition, in the lipidomic study HER2 was not shown to affect the lipid profiles in the clinical breast cancer samples, although the interconnection between HER2 signaling and FASN activity has been shown, as mentioned earlier [15]. Thus, it is evident that more studies, especially in vivo and clinical studies, of the lipid metabolism at gene, protein and metabolite levels, are needed to solve the discrepancies between the different studies considering the regulation of fatty acid and lipid metabolism in breast cancer.
The role & therapeutic potential of de novo fatty acid synthesis in breast cancerThe most obvious reason for cells to overproduce fatty acids by the de novo pathway is that these fatty acids are used to build membrane phospho-lipids, which are needed in rapidly proliferat-ing cancer cells. In addition, the phospholipids are needed in building lipid rafts, which have an important role in cancer cell signaling, and thereby de novo fatty acid synthesis may also indirectly affect cell signaling processes [32]. Protein palmitoylation affects, for example, the Wnt1/b-catenin pathway in prostate cancer, and
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also in this way the major product of the lipo-genic pathway, palmitate, may contribute to cell signaling [33]. However, the production of fatty acids may not be the only role of the de novo pathway, as this pathway has also been suggested to have relevance in cellular redox balance regu-lation. Cancer cells are usually under glycolytic and hypoxic conditions, in which the cells can-not fully oxidize the reducing equivalents that are formed. However, in lipogenesis NADPH is oxidized and therefore it may consume the exces-sive amounts of reducing compounds present in the cancer cell [3].
Lipogenesis may also have a profound role in the sensitivity of cancers to treatment, as shown by a recent study considering prostate cancer [34]. The study revealed that increased levels of satu-rated fatty acids in cell membranes make cancer cells less susceptible to oxidative stress. This is important because oxidized phospholipids have been shown to be crucial mediators for apopto-sis and unsaturated fatty acids are more easily oxidized [35]. Moreover, increased membrane saturation decreases both the lateral (within the plane of one membrane leaflet) and trans-versal (movement from one membrane leaflet to another) mobility of lipids, which then leads to diminished uptake of chemotherapeutic reagents into cancer cells [34]. The different roles of this pathway are summarized in Figure 1A.
The upregulation of the de novo fatty acid synthesis pathway in cancer, as compared with normal tissue, naturally suggests that target-ing this pathway may be a therapeutic option. Indeed, inhibitors for the FASN enzyme have been generated, including cerulenin, C75 and orlistat. Cerulenin is an antibiotic derived from the fungus Cephalosporum caerulens, which has shown promising antitumoral effects in xeno-graft models, although its chemical instability unfortunately limits its clinical use. C75, a syn-thetic derivative of cerulenin, is more stable and also effective in xenografts [5,36]. The use of C75, however, is limited by the fact that this inhibi-tor has been reported to cause dramatic weight loss, which is explained by the fact that, besides FASN inhibition, C75 also stimulates CPT1 protein, which catalyzes fatty acid oxidation in mitochondria [37,38]. Orlistat is a US FDA-approved drug used for treating obesity, which also inhibits FASN [39]. In vitro breast cancer studies have shown interesting results for this inhibitor but development of its chemical struc-ture would be needed because orlistat possesses poor solubility [40]. Other potential FASN
inhibitors are the antibiotic triclosan and certain polyphenolic compounds [40].
There are several mechanisms by which FASN inhibitors can reduce cancer cell growth. One hypothesis is that the inhibition of FASN reduces de novo fatty acid synthesis, and that the cancer cells are therefore lacking an adequate pool of fatty acids for building membranes for new cells. This hypothesis is supported by stud-ies showing that the addition of palmitate or oleate to the cell culture medium restores cell viability despite fatty acid inhibition, whereas this effect is not seen with non-FASN-related fatty acids [41]. Another hypothesis is that accu-mulation of malonyl-CoA, the product of ACC1 and the reactant of FASN, is cytotoxic to cancer cells. This hypothesis is supported by a study in which ACC1 inhibition was not cytotoxic to breast cancer cells, and inhibition of both ACC1 and FASN showed reduced cytotoxicity as com-pared with the inhibition of FASN alone [36]. However, another study using RNAi showed that knock-down of both ACACA and FASN reduces proliferation and induces apoptosis in cancer cells, supporting the idea that malonyl-CoA is not a prerequisite for cytotoxicity but that fatty acid synthesis is the crucial event [42]. There is also evidence for other cytotoxic mechanisms. It has been shown that inhibition of FASN can increase chemotherapy-induced cytotoxicity by activating proapoptotic and inactivating antiapoptotic signaling cascades in breast cancer cells [43]. p53 protein plays an important role in cellular responses to nongeno-toxic metabolic stress, and it also has connection to FASN, as in breast cancer cells FASN inhibi-tion together with p53 silencing leads to apopto-sis, whereas FASN inhibition with functioning p53 leads only to growth arrest [44]. FASN gene silencing causes wide changes in the expression of genes related to metabolism, cell survival and proliferation as well as DNA replication processes [45]. Finally, inhibition of FASN may also decrease HER2 activity, because silencing of FASN leads to downregulation of HER2 mRNA [46], although other data show that actually HER2 regulates FASN [15]. Data from colon cancer studies indicate that inhibition of FASN causes stress in the endoplasmic reticu-lum, which may also explain the antitumoral properties of FASN inhibitors [47]. It is likely that several nonexclusive mechanisms exist, and therefore more studies are still needed to elabo-rate their relative importance in the antitumoral properties of FASN inhibition.
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LPA regulates several cellular processesThe synthesized phospholipids contain fatty acyl chains coming from both de novo synthesis and nutrition. The phospholipids are not only static molecules residing in the cell membrane, as, for example, cleavage of their fatty acyl chains has important consequences in cell signaling. This section discusses LPA and the next section dis-cusses eicosanoids, both of which can be formed by cleavage of membrane phospholipids and have important roles in cancer cell signaling.
The role of LPA in the initiation and progres-sion of several cancers has been investigated, and thorough reviews of this lipid and cancer have been published [48,49]. LPA consists of a glycerol backbone, a fatty acid bonded at the sn1 or sn2 position, and a phosphate group bonded at the sn3 position. LPA can be produced in two ways; either by hydrolysis of one fatty acyl chain from phosphatidic acid or by hydrolysis of the choline group from lysophosphatidylcholine [48]. The latter reaction is catalyzed by the lysophospholi-pase D activity of the extracellular enzyme auto-taxin (ATX). Even before the lysophospholipase activity of ATX was known, ATX was reported to be associated with cancer progression. LPA exerts its effect by binding to G protein-coupled receptors, and at the moment six of these have been reported (LPA
1–6) [50]. The LPA receptors
affect many cancer signaling pathways, such as PI3K, MAPK and Wnt/b-catenin, and thereby affect processes related to, for example, cell pro-liferation and survival, angiogenesis and inflam-mation [49], as shown in Figure 2. Both ATX and the LPA receptors are overexpressed in breast cancer as compared with normal mammary epi-thelial cells, and the overexpression of ATX and LPA
1–3 receptors has been reported to induce
mammary carcinomas in mice [51]. LPA1 is also
associated with cytokine production and bone metastasis in breast cancer [52]. In vitro studies have revealed that ATX protects breast cancer cells against Taxol-induced apoptosis [53].
Because of their crucial role in cancer, LPA, ATX and LPA receptors provide attractive tar-gets for cancer therapeutics. Zhang et al. have generated LPA analogs that perform a dual role; they inhibit the lysophospholipase D activity of ATX as well as abrogate signaling through the LPA receptor. These inhibitors were found to reduce cell migration and invasion by breast tumors in vivo [54]. Lpath Inc. (CA, USA) has generated monoclonal antibodies recognizing LPA. These antibodies are in preclinical trials;
the concept is that the monoclonal antibody neutralizes the action of LPA, thereby prevent-ing its function in cancer [201]. LPA is also a potential diagnostic biomarker, as most patients with ovarian or other gynecologic cancer show increased concentrations of LPA circulating in the plasma [55]. However, in breast cancer patients no elevated levels have been observed [55], and therefore, based on the available evi-dence, LPA is more likely to be beneficial as a therapeutic target rather than a diagnostic tool in this cancer type.
Role of eicosanoids in breast cancerThe term eicosanoid is used to refer to a group of oxygenated, 20-carbon fatty acids, and these molecules have an important role in cellular signaling. Eicosanoids differ from the lipids in previous sections in that they are essential (i.e., the precursors come solely from the diet). Even today, reliable measurements of eicosanoids from in vivo and clinical samples are very chal-lenging – despite the availability of sensitive instruments and analytical techniques – due to the sensitivity of many eicosanoids to sample collection and pretreatment. Most of the infor-mation regarding these molecules in cancer, therefore, still comes from studies investigat-ing the enzymes and receptors related to these signaling molecules, rather than the molecules themselves.
Eicosanoids have been investigated extensively in cancer, and a thorough review considering these molecules has been published by Wang and DuBois [56]. The precursors of eicosanoids are stored as fatty acyl chains in membrane phospholipids, from which they are cleaved for eicosanoid synthesis. Arachidonic acid, an w-6 fatty acid with four double bonds, is the major precursor for various eicosanoids and it can be converted by the COX pathway to prostanoids (prostaglandins, thromboxanes and prostacy-clins), by the lipoxygenase (LOX) pathway to leukotrienes and hydroxyeicosatetraenoic acids and by the P450 pathway to epoxyeicosatrienoic acids and hydroxyperoxyeicosatetraenoic acids.
Under normal conditions in healthy cells COX-2 is mainly unexpressed but during inflammation its levels are rapidly elevated lead-ing to increased production of prostaglandins. The significance of COX-2 in breast cancer is still highly controversial, although expression studies were carried out in the 1990s [57,58]. Some studies have reported high expression of COX-2 in most breast cancer samples with no expression
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in normal breast tissue [57], whereas other studies have shown that COX-2 is upregulated only in some breast cancer samples [58] or downregulated in breast cancer as compared with healthy tis-sue [59]. The use of NSAIDs inhibiting COX in reducing breast cancer risk is also under debate, although there is evidence that NSAIDs may be beneficial in reducing cancer risk [60].
Regardless of the specific role of COX-2, it is nevertheless clear that one of the prostaglan-dins, prostaglandin E2 (PGE2), has many roles in breast cancer. It is known that PGE2 can increase the breast cancer cell proliferation rate by inducing aromatase enzyme expression, and thus local synthesis of estrogens [61], as well as promote invasion of breast cancer cells to lymph nodes by upregulating the CC chemokine recep-tor CCR7 [62]. Myeloid-derived suppressor cells, which are found in cancers and shown to inhibit T-cell activity, thus leading to immunosuppres-sion, are induced by PGE2 in a breast cancer mouse model [63]. Finally, PGE2 can promote angiogenesis in breast cancer by stimulating angiogenic regulatory genes [64] and affecting endothelial cell motility [65]. Based on this evi-dence, which is far from comprehensive, it can be concluded that PGE2 affects the processes of cell proliferation, cell invasion, immunology and angiogenesis in breast cancer (Figure 2). However, the role of PGE2 may be even more complex. Recently the authors have shown that
the key enzyme degrading PGE2, 15-hydroxy-prostaglandin dehydrogenase, has a dual role in breast cancer. On the one hand previous stud-ies have indicated HPGD as a tumor-suppressor gene and downregulated in cancer as compared with normal tissues [66], but on the other hand it is highly upregulated in those breast cancer patients with poor outcome [67].
The LOX enzymes metabolize arachi-donic acid to hydroxyperoxyeicosatetraenoic acids that can be further reduced to hydroxy-eicosatetraenoic acids. In the case of 5-LOX, hydroxyperoxyeicosatetraenoic acid is further metabolized to leukotriene A4, which can be further transformed to other leukotrienes [68]. 15-LOX-1 and 15-LOX-2 are suggested to have tumor-suppressor roles in breast cancer, as their expression is reduced in malignant breast cells and tissues compared with healthy ones [69,70], whereas the expression of 5-LOXAP is increased in breast cancer and together with 12-LOX also associated with poor survival of the patient [71]. Indeed, inhibition of both 5-LOX and 12-LOX reduces cell proliferation and induces apoptosis in breast cancer cells in vitro [72]. By contrast, the activity of 15-LOX-2 has been implicated in the increased adhesion of breast cancer cells to the extracellular matrix [73]. Thus, in summary, 5-LOX and 12-LOX possess protumorigenic activity while 15-LOX-1 and 15-LOX-2 pos-sess antitumorigenic activity in breast cancer (Figure 3) [68].w-6 fatty acids form the majority of the poly-
unsaturated fatty acids in the western diet. In addition to the pathways derived from arachi-donic acid, there can also be parallel competing pathways derived from eicosapentaenoic acid, which is an w-3 fatty acid. In general, arachi-donic acid-derived eicosanoids promote inflam-mation, whereas eicosapentaenoic acid-derived eicosanoids are less inflammatory or even anti-inflammatory, and eicosapentaenoic acid com-petes with arachidonic acid for prostaglandin and leukotriene synthesis [74]. w-3 fatty acids have also shown promising results in breast cancer. It was shown two decades ago, in mice, that diets rich in w-3 fatty acids can reduce breast cancer cell growth and metastasis [75]. It has been shown that dietary w-3 fatty acids can be found in breast tis-sue [76], and that w-3 fatty acids have a protective effect in breast adipose tissue [77]. This suggests that lipid metabolism can be seen not only as a promising source of targets for drug development but also as a source of novel disease prevention strategies via nutritional interventions.
Signaling
Proliferation
Arachidonic acid PGE2
COX-2
AngiogenesisInvasion
Inflammation andimmunologicalprocesses
LPA receptor
LPALPCATX/LPC
Figure 2. Effects of lysophosphatidic acid and prostaglandin E2 in breast cancer. LPA: Lysophosphatidic acid; LPC: Lysophosphatidylcholine.
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Sphingolipids & glycosphingolipids are either pro- or antitumorigenicSphingolipids form a class of lipids of which the common structural feature is the sphingosine backbone. It has been suggested that the balance between different sphingolipids affects the char-acteristics of a cancer cell (Figure 3). For exam-ple, ceramides and sphingosines are associated with cell growth arrest and cell death, whereas glycosylated sphingolipids together with sphin-gosine-1-phosphate (S1P) are involved in cellular proliferation, survival and drug resistance [78]. Actually, the so-called ceramide–sphingosine–S1P rheostat was proposed some time ago [79]. In this model, ceramide and sphingosine are apoptotic molecules while S1P promotes cell survival [79].
In cells, ceramides can be generated by de novo synthesis, either by condensation of serine and palmitoyl-CoA, by catabolism of sphingomyelin or glycosphingolipids or by desphosphorylation of phosphorylated ceramides [78]. In many can-cers chemotherapeutic agents increase ceramide levels, thereby leading to cell death. Ceramide levels can also be increased exogenously, and exogenous ceramide analogs induce apopto-sis in several cancers, including breast cancer, and also show selectivity towards cancer cells versus normal breast epithelial cells [80,81]. The hydrophobicity and physicochemical properties of short-chain ceramides limit their use in vivo, but delivery of these ceramides using liposomal carriers was reported to be effective in a breast cancer mouse model [82]. The downregulation of ceramides, however, is not straightforward, as it has also been reported that ceramide synthase, and thereby some long-chain ceramides, are upregulated in breast cancer tissue as compared with benign breast tissue [83].
Ceramide can be converted to sphingosine by the enzyme ceramidase and then sphingo-sine kinase (SK) converts the sphingosine to S1P. SK exists as two isozymes, SK1 and SK2, and of these two isozymes the role of SK1 in cancer is clearer. In breast cancer, SK1 is highly expressed, especially in ER-negative tumors, but also among ER-positive patients high expression of SK1 is associated with poor outcome [84]. High expression of SK2 in breast cancer has not been reported in the literature, and there are also controversial results on its effect on cancer cells. On the one hand SK2 can induce apoptosis in cells [85] and may also have an opposing role in the regulation of ceramide biosynthesis com-pared with SK1 [86], but on the other hand the
activity of SK2 has been implicated in enhanc-ing breast cancer tumorigenesis, for instance by affecting tumor-associated macrophages [87] or by participating in the EGF-catalyzed migration of cells [88].
S1P exerts its effects by binding to five G pro-tein-coupled receptors, named S1PR
1–5. S1P has
numerous roles in the cell; it is associated with cancer cell survival, growth, motility, trans-formation, neoangiogenesis and inflammation [89]. There seems to be an interesting interac-tion between S1P and hypoxia. The expression of SK1 is activated by hypoxia-inducible factors [90], but SK1 also regulates the levels of HIF1-a, as shown in breast cancer cells [91]. Studies in glioma cells have revealed that HIF2-a has an important role in the regulation of SK1 expres-sion and, interestingly, S1P released to the extra-cellular environment induces neoangiogenesis [92]. In fact, S1P has been shown to display a synergistic effect with VEGF in neoangiogenesis [93] and, indeed, monoclonal antibodies against S1P have been shown to inhibit angiogenesis in tumor xenograft models [94]. In breast cancer S1P is associated with hormonal regulation, since it is involved in a pathway in which estrogen stimu-lates the activity of the EGF receptor [95]. In addition, recent reports indicate a role of S1P in the generation of tamoxifen resistance in breast cancer. Studies with cell lines showed that over-expression of SK1 results in enhanced cell prolif-eration and resistance to tamoxifen-induced cell growth arrest and apoptosis, and moreover that tamoxifen-resistant cells exhibit higher expres-sion of SK1 [96]. Tamoxifen responsiveness can be rescued by silencing SK1 expression with RNAi [96]. These results are supported by clini-cal findings showing that high expression of SK1 and the S1P
1 and S1P
3 receptors predict a poorer
Prot
umor
igen
ic Proapoptotic
Multidrug resistanceGlycosylated ceramides
S1P
5-LOX
12-LOX
Ceramides
Sphingosine
15-LOX-1
15-LOX-2
Figure 3. The balance of sphingolipids and the activity of lipoxygenase enzymes affect the malignancy state of the cell. Glycosylated ceramides are associated with multidrug resistance of breast cancer cells.
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prognosis in ER-positive breast cancer patients [97]. Interestingly, S1P has also been implicated in epigenetic regulation, as SK2 associates with histone H3 and histone deacetylases to produce S1P, which regulates histone acetylation and can thereby affect cell cycle progression [98]. The role of S1P in several cellular processes has made it an interesting target for cancer therapeutics. There are three major strategies for drug development: S1P-specific antibodies that neutralize the extra-cellular action of S1P; SK inhibitors that inhibit the production of S1P; and inhibitors that block the activity of S1P receptors. These approaches have yielded promising results in vitro and in vivo, but so far it seems that, in the context of cancer, the only one that has proceeded to a Phase I clinical trial is the antibody approach, which has also shown promising results in mouse breast cancer models [201].
Glycosphingolipids include cerebrosides, gan-gliosides and globosides. Cerebrosides consist of a ceramide with a hexose at the 1-hydroxyl moiety. The sugar residue can be either glu-cose or galactose, and therefore cerebrosides can be classified as either glucocerebrosides or galacto cerebrosides [99]. Glucosylceramide syn-thase (GCS) is the enzyme producing glucosyl-ceramide (glucocerebroside) by catalyzing the transfer of glucose to ceramide [78]. The activity of GCS, and thereby the increased concentration of glycosylated ceramides, is thought to poten-tiate the development of multidrug resistance in cancer cells [100,101]. Interestingly, however, in clinical breast cancer samples the expression of GCS is associated with lower histological grading and ER-positive status [102]. This is also supported by the authors’ own findings show-ing dramatically decreased concentrations of cer-tain glycosylated ceramides in tumors of higher grade and ER-negative status [30]. Nevertheless, inhibition of the activity of GCS downregulates P-glycoprotein and resensitizes breast cancer cells to, for example, vinblastine [103].
Globosides are glycosphingolipids with two or more sugars, usually glucose, galactose or N-acetylgalactosamine, but they have not been investigated extensively in the context of breast cancer. Acidic glycosphingolipids containing one or more sialic acid (e.g., N-acetylneuraminic acid) residue(s) in their carbohydrate moiety are called gangliosides [104]. Gangliosides are the most complex sphingolipids, and those with one sialic acid residue are referred to as the GM series, those with two as the GD series, those with three, the GT series, and those with four,
the GQ series. Gangliosides have diverse roles in cancer, as they have been implicated in immuno-logical processes, angiogenesis, cell adhesion and motility as well as cell signaling [105]. However, gangliosides have not been investigated heavily in breast cancer. It has been reported that the overall ganglioside levels are higher as compared with normal breast tissue, and GM3-, GD3- and GT3-containing gangliosides, not present in nor-mal breast tissue, are found in malignant tissues [106]. Indeed, ganglioside-based vaccines have been developed for cancer treatment, and two of these are racotumomab and NGcGM3/VSSP vaccine [107]. Racotumomab is an anti-idiotype monoclonal antibody, which means that it is a mirror image of the original antibody, and can act as an antigen, inducing a response against the original antigen. The NGcGM3/VSSP vaccine is a conjugation of the ganglioside with bacte-rial proteoliposomes. These therapies, developed by the RECOMBIO company (Madrid, Spain), have not caused severe side effects in patients and have progressed into Phase III clinical trials [107].
ConclusionIt is apparent that the regulation of lipid metabo-lism in malignant tissues is very different from that occurring in normal tissues. The observed changes in lipids, such as membrane lipids, may also provide an explanation for the comorbidity of obesity and cancer. In fact, during the last few years, interesting studies have emerged sug-gesting a link between the metabolic syndrome and cancer. Hirsch et al. investigated, in cell lines, the changes in gene expression that occur during the malignant transformation [108]. This study, which also included transformed mam-mary epithelial cells, revealed that many of the genes differentially regulated during transforma-tion are also related to lipid metabolism, inflam-mation, obesity and the metabolic syndrome. Moreover, several drugs designed for the treat-ment of metabolic diseases showed either com-plete suppression or delay in tumor growth in mouse models [108].
Therefore, lipid metabolism provides thera-peutic opportunities not only for drug develop-ment but also for lifestyle changes, including nutrition, which may be considered in cancer prevention.
Future perspectiveThe importance of lipid metabolism in the cancer field has been largely neglected until recently, possibly because the other metabolic
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changes in cancer are highly prominent and due to technological limitations. However, recent developments in the lipidomics field have made lipid metabolism investigations more tempt-ing, and therefore the authors believe that in the future lipid metabolism will become a more important field in cancer drug develop-ment and diagnostics. The authors believe that in the future:
nThe development of analytical methods will lead to increased knowledge of lipid metabo-lism in cancer and also provide diagnostic opportunities;
nThe integration of data from gene, protein and lipid studies will enable a more comprehensive understanding of the metabolism in malig-nant cells and also reveal new signaling pathways contributing to carcinogenesis;
nThe importance of nutrition on lipid metabo-lism and cancer pathogenesis will become more clear, which may lead to new nutritional recommendations;
nThe first clinical trials of drugs targeting lipid metabolism will be finished and prove that lipid metabolism is an attractive target for future drug development.
Financial & competing interests disclosureM Hilvo was financially supported by the Academy of Finland postdoctoral researcher’s fellowship 138201. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Executive summary
Fatty acid metabolism in normal & malignant tissues � In normal tissues, de novo synthesis of fatty acids occurs in a limited number of tissues and is under nutritional control.
� In tumors, upregulation of the de novo fatty acid synthesis pathway is a universal feature and out of nutritional control.Upregulation of membrane phospholipid metabolism in breast cancer � The activity and expression of choline kinase and the Kennedy pathway is increased in breast tumors and leads to increased membrane phospholipid synthesis.
� Phospholipids containing saturated de novo synthesized fatty acids are especially increased in grade 3 and estrogen receptor-negative breast cancers.
The role & therapeutic potential of de novo fatty acid synthesis in breast cancer � Inhibitors of fatty acid synthase may exert their antitumoral activities by affecting membrane phospholipid synthesis, malonyl-CoA accumulation, apoptotic pathways, HER2 activity and stress in the endoplasmic reticulum.
Lysophosphatidic acid regulates several cellular processes � Lysophosphatidic acid exerts its function on cells via G protein-coupled receptors, and affects cell proliferation and survival, angiogenesis and inflammation.
Role of eicosanoids in breast cancer � The role of COX-2 in breast cancer is controversial.
� Prostaglandin E2 promotes carcinogenesis by multiple mechanisms.
� Lipoxygenase isozymes are either pro- or antitumorigenic.Sphingolipids & glycosphingolipids are either pro- or antitumorigenic � The balance between different sphingolipids determines the malignancy of cells.
� According to the so-called ceramide–sphingosine–sphingosine-1-phosphate rheostat model, ceramide and sphingosine are apoptotic molecules while sphingosine-1-phosphate promotes cell survival.
� Glycosphingolipids are associated with the multidrug resistant phenotype of cancer cells.
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