Altered Sphingolipid Metabolism Induced by Tumor Hypoxia – New Vistas in Glycolipid Tumor Markers

FEBS Letters 584 (2010) 1872–1878

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Review Altered sphingolipid metabolism induced by tumor hypoxia – New vistas in glycolipid tumor markers

Jun Yin a, Keiko Miyazaki a, Rebecca L. Shaner b, Alfred H. Merrill Jr. b, Reiji Kannagi a,* a Department of Molecular Pathology, Aichi Cancer Center, Nagoya 464-8681, Japan b School of Biology, Chemistry and Biochemistry and the Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA article info abstract

Article history: Uncontrolled growth of malignant cells produces hypoxic regions in locally advanced tumors. Received 6 October 2009 Recently we showed that tumor hypoxia-induced transcription of multiple genes involved in glycan Revised 7 November 2009 synthesis, leading to expression of useful glycolipid tumor markers, such as gangliosides having N- Accepted 9 November 2009 glycolyl sialic acid. Our subsequent studies indicated that the ceramide portion of glycolipids, as Available online 11 November 2009 well as their glycan moiety, was also signiﬁcantly affected by hypoxia. Tumor hypoxia-induced Edited by Sandro Sonnino marked accumulation of sphinganine (dihydrosphingosine) long-chain base, and signiﬁcant reduction of unsaturated very long-chain fatty acids in the ceramide moiety. Mass-spectrometry, which yields information on both glycan- and ceramide moieties, is expected to be clinically useful in Keywords: N-Glycolyl sialic acid detecting such distinct molecular species of cancer-associated glycolipids having combined alter- Dihydroceramide synthesis ation in both glycan- and ceramide moieties. Fatty acid 2-hydroxylase Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Dihydroceramide Delta4-desaturase-1/2 Hypoxia-inducible factor Unsaturated very long-chain fatty acid

1. Introduction one of the major mechanisms for induction of cancer-associated glycans in glycolipids and glycoproteins. Here we introduce the no- Cell-surface glycolipids are known to undergo drastic changes tion that tumor hypoxia affects not only the glycan moiety of gly- upon malignant transformation [1,2]. Glycolipids appearing in can- colipids, but also their ceramide moiety. cer cells are shown to serve as good tumor markers, and applied also as therapeutic targets of cancers [3,4]. The mechanisms under- 2. Hypoxia and cancer-associated glycolipids lying tumor-associated changes of glycolipids, however, are com- plicated and remain elusive. Some changes occur at the relatively Recently we showed that tumor hypoxia leads to enhanced early stages of carcinogenesis, while some other changes become expression of some cell-surface glycans, that had been well known apparent only in the advanced stages along with progression of to be associated with cancers, such as sialyl Lewis X and sialyl Le- cancers. In the stages of locally advanced cancers, uncontrolled wis A. This turned out to be due to induction of the transcription of growth of tumor cells produces hypoxic areas in expanded cancer the genes involved in the synthesis of these glycans by hypoxia as cell nests. Tumor hypoxia affects various aspects of intracellular analyzed by DNA microarray [5]. Since then, it is becoming clearer metabolisms of cancer cells, and recently it was disclosed to be year after year that tumor hypoxia affects a wide variety of genes involved in the expression of cell-surface glycoconjugates, and induces profound changes in the expression of glycolipids and glyco- Abbreviations: CerS, ceramide synthase; CMH, ceramide monohexose; DHCer, proteins in cancers. Dihydroceramide; DHCMH, dihydroceramide monohexose; DHSph, dihydrosphing- For instance, it has long been known that gangliosides carrying omyelin; HIF, hypoxia-inducible factor; HPLC–ESI-MS/MS, high-performance liquid N-glycolyl sialic acid increases in human cancers. Such ganglio- chromatography-electrospray ionization-tandem mass spectrometry; HRE, hypoxia-responsive element; LCB, long-chain base; MALDI-MS, matrix-assisted sides are sometimes called Hanganatziu–Deicher antigens [6,7]. laser desorption mass spectrometry; NeuAc, N-acetyl sialic acid; NeuGc, N-glycolyl Recently we have shown that hypoxia-induced up-regulation of a sialic acid; SPT, serine palmitoyl transferase; Sph, sphingomyelin; VLCFA, very long- gene for the sialic acid transporter, Sialin, is closely related to the chain fatty acid enhanced expression of gangliosides carrying N-glycolyl sialic acid * Corresponding author. Address: Department of Molecular Pathology, Research (Fig. 1) [8]. Humans lack the gene for CMP-neuraminic acid Institute, Aichi Cancer Center, 1-1 Kanokoden, Chikusaku, Nagoya 464-8681, Japan. Fax: +81 52 701 1787. hydroxylase, the enzyme required to synthesize N-glycolyl sialic E-mail address: [email protected] (R. Kannagi). acid (NeuGc), and most NeuGc in the human body is thought to

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.11.019 J. Yin et al. / FEBS Letters 584 (2010) 1872–1878 1873

be acquired from the external milieu, mainly of dietary origin [9,10]. Tumor hypoxia induces transcription of Sialin, and enhances incorporation of exogenous sialic acid. This leads to enhanced incorporation of NeuGc as well as N-acetyl sialic acid (NeuAc), and results in signiﬁcant accumulation of unusual gangliosides carrying N-glycolyl sialic acid in cancers. Such gangliosides serve as surrogate markers for the presence of cell masses suffering from chronic hypoxia. De novo synthesis of N-glycolyl sialic acid is based on oxidative hydroxylation of N-acetyl sialic acid catalyzed by CMP-NeuAc hydroxylase, a speciﬁc oxidase, expression of which is phyloge- netically well controlled; it is present in mammals up to higher apes, but absent in humans [11–13]. As the conversion of N-acetyl to N-glycolyl sialic acid is based on oxidative hydroxylation, production of NeuGc through de novo synthesis may be sup- pressed under hypoxic conditions in mammals up to higher apes. In contrast, the expression of NeuGc rather increases under hypoxic conditions in humans, where its level is determined by its salvaging through the transporter Sialin, instead of de novo synthesis. Effects of hypoxia are not limited to changes in sialic acid molecular species, but sometimes extend to the glycan backbone of gangliosides. Marked induction of GD3 expression and moderate induction of that of GM3 were observed in this cell line after hypoxic culture (Fig. 2A). This was accompanied by a prominent induction of ST8Sia-I transcription (Fig. 2B). The ST3GalV gene shows a delayed moderate induction, while genes for other glycosyltrans- ferases show only minimal changes.

3. Ceramide composition and tumor hypoxia

During the course of study of tumor-associated gangliosides, we have noticed that significant changes occur at the ceramide moiety as well as carbohydrate moiety of the gangliosides during hypoxia. This was initially noticed from changes in the mobilities of ganglioside bands in TLC-immunostaining with specific anti-ganglioside antibodies after hypoxic culture. Broadening of the main band and appearance of additional slow-migrating band were frequently observed, which implied altered ceramide composition in gangliosides by hypoxia. Analyses of sphingolipid molecular species by high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS/MS) indicated a significant accumulation of dihydroceramide (DHCer) under hypoxia (Fig. 3A). A similar accumulation of DHCer moiety was detected also in the glycolipid and sphingomyelin (Sph) fraction (Fig. 3A). This was accompanied with the decrease in ceramide moiety with d18:1 long-chain base (LCB) (Fig. 3B). It is interesting to note that the ratio of sphingosine/sphinganine is reportedly decreased in certain tumors both in ceramide and Sph fractions in the literature [14,15]. Another notable finding was a sharp decrease in the amount of unsaturated very long-chain fatty acids (VLCFAs) in the ceramide moiety by hypoxia. C24:1 was the major unsaturated acyl chain in the given cells, and its amount exhibited a marked de- Fig. 1. Hypoxia-induced expression of ganglioside GM2 having N-glycolyl sialic crease by hypoxia, and tended to recover after reoxygenation. acid. (Panel A) Flow-cytometric analysis of a clone of human cultured colon cancer This was accompanied by an increase of saturated VLCFAs by hy- cells Caco-2M using monoclonal antibody specific to NeuGc-GM2 (MK2-34) poxia, which was most prominent with C22:0 species. Accord- indicating prominent induction of the ganglioside by hypoxia. (Panel B) RT-PCR analysis of transcriptional induction of the gene for a sialic acid transporter, Sialin, ingly, the ratio of unsaturated/saturated VLCFAs showed a by hypoxia in Caco-2M cells. (Panel C) Results of flow-cytometric analysis of Caco- sharp reduction upon hypoxic culture, and tended to recover 2M cells indicating transfection of Sialin gene confers significant NeuGc-GM2 after reoxygenation in all tested sphingolipid fractions (Fig. 3C), expression. (Panel D) Immunohistochemical staining of NeuGc-GM2 in a colon reflecting impaired desaturase reaction for VLCFAs under hypoxic cancer tissue using specific monoclonal antibody, indicating the ganglioside serves condition. as a good marker for advanced stage cancer cells. Advanced cancer cells acquire hypoxia-tolerance, and this accompanies sustained Sialin expression. (Upper panel) Subsequent RT-PCR analysis (Fig. 3D) indicated a significant Non-malignant colonic epithelial cells. (Lower panel) Cancer cell nests. enhancement of transcription of fatty acid 2-hydroxylase (FA2H), 1874 J. Yin et al. / FEBS Letters 584 (2010) 1872–1878

4. Pathophysiological signiﬁcance of hypoxia-induced alteration of glycolipids

Cancer cells must cope with hypoxic environment to survive and expand the size of tumors. Thomlinson–Gray’s equation on tissue oxygen diffusion [16] indicates that cancer cells in an area more than 75 lm distant from a blood vessel suffers from hypoxia, where oxygen tension becomes less than 10 mmHg. The major metabolic change in cancer cells induced by hypoxia is known to be elevated anaerobic glycolysis, accompanied by increased production of angiogenic factors. Enhanced expression of selectin ligand glycans such as sialyl Lewis A and sialyl Lewis X confers a growth advantage to cancer cells under hypoxic environment, because they mediate adhesion of cancer cells to endothelial cells expressing selectins. Induction of genes involved in sialyl Lewis A and sialyl Lewis X by hypoxia is closely involved in tumor angiogenesis [17] and hematogenous metastasis [18–20]. Hypoxia is known to also induce sphingosine kinase-1, and enhances synthesis of sphingosine-1-phosphate, which reportedly has a strong angiogenic activity [21,22]. The advantage for cancer cells of enhanced sialic acid transport through sialin is not clear at this moment. Cancer cells generally have enhanced cell-surface sialylation, and have increased demand for sialic acid donors for use in the synthesis of sialylated glycans. Uptake and reuse of sialic acids through the transporter may econ- omize on energy required for their de novo synthesis. The accumulation of ceramide moieties having a d18:0 long- chain base, and decrease in unsaturated N-acyl chains seems to reflect a passive consequence of oxygen deprivation, rather than an active accommodation of the cells to hypoxic environments. In addition, the difference between cancers and normal cells is usually only quantitative, and not qualitative. Still it is noteworthy that DHCer has been known to have much reduced activity in inhibiting cell growth and inducing apoptosis [23]. This may confer an additional growth advantage to cancer cells accumulating DHCer. Hypoxia-induced alteration in long-chain base and acyl chain composition of ceramide moiety may also influence the physico- chemical properties and affect microdomain formation and signal transduction of cancer cells [24–28]. Accumulation of sphinganine Fig. 2. Hypoxia-induced induction of ganglioside GD3. (Panel A) Flow-cytometric long-chain base and decrease in unsaturated very long-chain fatty analysis of a cultured human colon cancer cell line, LS174T, using monoclonal acids seem to favor more enhanced segregation of sphingolipid-en- antibodies specific to GD3 (GMR19) and GM3 (M2590). (Panel B) RT-PCR analysis of transcriptional induction of genes involved in GD3 synthesis by hypoxia in LS174T riched domains. cells.

5. Mechanisms for gene transcription induced by tumor hypoxia while only marginal changes were observed with dihydroceramide Delta4-desaturase-1/2 (DES1/2) transcription. Lack of DES1 induc- A transcription factor called hypoxia-inducible factor (HIF) fig- tion indicated that the impaired DHCer desaturation due to paucity ures heavily in gene transcription induced by hypoxia. Although of oxygen is not going to be compensated by the induction of gene several genes are known to be induced by hypoxia independent transcription. The increase of DHCer species in sphingolipids by of HIF, HIF is the major player in the gene transcription in hypoxia. hypoxia seems to simply reflect the uncompensated result of oxy- The a-subunit of HIF usually resides in cytoplasm, and is rapidly gen deprivation. At this moment the relationship between changes ubiquitinated and degraded in proteasomes under normoxic condi- in LASS (CerS) and SPTLC (SPT) transcription and alteration of cera- tions. Ubiquitination stops under hypoxic conditions due to oxygen mide acyl chain composition is not clear. The increase in FA2H may sensing by proline hydroxylases and several other molecules, and be related to the appearance of slow-migrating band in TLC- this leads to nuclear translocation of a-subunit, where it forms immunostaining. an active heterodimer with the b-subunit present in the nucleus, The FA2H and DES1/2 products require oxygen in their catalytic and induces transcription of genes having a hypoxia-responsive action. It is highly conceivable that such enzymes requiring oxygen element (HRE). Transcription of many cellular genes is thus in- would increase under hypoxic conditions to cope and compensate duced, which are beneficial for cells to adapt to, or to cope with, for the paucity of oxygen. It, however, seems to apply to only a part hypoxic environments. Some genes, especially those with multiple of such enzymes, in this case, FA2H, and does not apply to others HREs in their regulatory region, are readily induced by HIF alone, such as DES1/2. In the latter case, the deficiency of oxygen would while other genes require cooperation of transcription factors be straightforwardly reflected in changes in the ceramide compo- other than HIF for maximal induction of transcription; thus, the sition of glycolipids. set of genes induced by hypoxia is not the same in every cell and J. Yin et al. / FEBS Letters 584 (2010) 1872–1878 1875

Fig. 3. Hypoxia-induced changes in ceramide moieties of sphingolipids in cultured human colon cancer cells. Caco-2M cells [8] were cultured under normoxic conditions

(normoxia), hypoxic conditions (1% O2) for 7 days (hypoxia), or hypoxic conditions for 7 days followed by normoxic conditions for 24 h (hypoxia + reoxygenation). Lipids were extracted and analyzed by HPLC–ESI-MS/MS according to the protocol shown in [35]. (Panel A) Effects of hypoxia on sphingolipids having dihydrosphingosine LCB (d18:0, sphinganine). (Panel B) Effects of hypoxia on sphingolipids having sphingosine LCB (d18:1) with ceramides. (Panel C) Effects of hypoxia on the ratio of monounsaturated/ saturated VLCFAs (sum of monounsaturated C20:1–C26:1/sum of saturated C20–26) in the ceramide moiety of sphingolipids. *P < 0.05; **P < 0.01; ***P < 0.001. Panel D indicates the results of RT-PCR analyses of gene transcription involved in sphingolipid synthesis indicating significant transcriptional induction of LASS1 (CerS1) and FA2H by hypoxia. Transcription of LASS3 (CerS3) was markedly reduced. Only modest changes were observed in transcription of other genes. tissue. This results in the enhanced expression of various glycolip- of the genes for enzymes requiring oxygen are induced under hyp- ids and gangliosides upon hypoxic culture depending on the cells oxic conditions in normal cells, but enzymatic products are not used in experiments. effectively generated because of paucity of oxygen. On the other hand, when genes for such enzymes are irreversibly induced in 6. Paradoxical effects of hypoxia-induced genes in cancers cancer cells, this sometimes leads to an abnormal overproduction of primary enzymatic metabolites, because of sustained gene tran- Hypoxia-induced gene transcription is usually reversible in nor- scription even under normoxic conditions. There are not many en- mal cells, since re-oxygenation rapidly restores ubiquitination of zymes requiring oxygen for glycan synthesis, while many such the a-subunit of HIF in normal cells. On the other hand, cancer cells enzymes are involved in lipid synthesis, such as in biosynthesis acquire hypoxia resistance through sustained HIF expression, in of sphingolipids and plasmalogens, or in hydroxylation and unsat- most cases by irreversibly interfering with HIF ubiquitination. For uration of fatty acids, where the paradoxical effects may be ob- instance, loss of anti-oncogenes such as p53 or VHL inhibits ubiqui- served in cancers. tination of HIF and leads to its sustained nuclear translocation. Tu- mor hypoxia promotes malignant progression by facilitating 7. Increase of cancer specificity by combination of multiple selection and clonal expansion of the more aggressive cancer cells, tumor-associated changes which can overcome oxygen deprivation or escape the hypoxic environments. Such cancer cells have constitutively elevated tran- Malignant transformation affects cell-surface glycan expression scription of hypoxia-inducible genes even if under the normoxic through various mechanisms [29]. One of the major mechanisms is condition. Once HIF is irreversibly activated, cancer cells tend to so-called incomplete synthesis of glycans. For instance, mature exhibit enhanced expression of sialyl Lewis A/X or Hanganatziu– nerve cells express higher gangliosides such as GD1a and GM1, Deicher gangliosides, irrespective of oxygen tension in their but malignant neuroblastoma cells frequently express shorter gan- environments. gliosides such as GM2 or GD3. Cancer cells tend to express shorter This sometimes leads to paradoxical consequences, especially chain glycolipids compared to the corresponding non-malignant when the enzymes require oxygen for their catalytic activity. Some cells of the same tissue origin, and this is mainly due to epigenetic 1876 J. Yin et al. / FEBS Letters 584 (2010) 1872–1878 silencing of a part of the genes required for the synthesis of normal colyl sialic acid. Modification of cancer-associated glycans by N- complex gangliosides (Fig. 4). Similarly, normal epithelial cells ex- glycolyl sialic acid would further improve their cancer specificity. press sialyl 6-sulfo Lewis X and disialyl Lewis A, the glycans having In contrast, if normal epithelial cells were subjected to hypoxic more complex structures than cancer-associated glycans, sialyl Le- condition, normal glycans in epithelial cells such as sialyl 6-sulfo wis X and sialyl Lewis A. Epigenetic silencing of genes involved in Lewis X and disialyl Lewis A would be mainly modified by N-gly- GlcNAc 6-sulfation or a2-6 sialylation upon carcinogenesis results colyl sialic acid. in the appearance of sialyl Lewis X and sialyl Lewis A in cancer cells [30–32]. This mechanism affects cancer cell glycans at the relatively early stages of carcinogenesis. This mechanism, however, is 8. Cancer-associated changes affect both glycan and ceramide not strictly cancer-specific, since a similar set of genes is some- moieties of glycolipids times silenced epigenetically also under non-malignant conditions such as in inflammatory diseases. Our results as described here indicated that tumor hypoxia af- Later in the locally advanced tumor stages, cancer cells cope and fects not only glycan moiety, but also the ceramide moiety of tu- overcome hypoxic environments by acquiring hypoxia-resistance. mor-associated glycolipids. For instance, it could be suggested This process further promotes changes in cell-surface glycans. that the tumor-associated glycolipids having DHCer moiety with These hypoxia-induced changes are also not strictly cancer-spe- less unsaturated very long-chain fatty acids would have higher cific, since a similar set of genes is also induced by hypoxia in cancer specificity. non-malignant diseases such as ischemic disorders. It means that To date detection of cancer-associated glycans largely relied on the Hanganatziu–Deicher gangliosides are not strictly specific to the use of specific monoclonal antibodies. A good example is an cancers, but can appear in non-malignant cells under chronic hyp- antibody specific to the GM2 ganglioside having N-glycolyl sialic oxic conditions. Normal gangliosides would acquire N-glycolyl sia- acid. Such a ganglioside having DHCer would be more specific to lic acid under hypoxic conditions, and such gangliosides may not hypoxia-resistant clones of cancer cells. Although the ceramide serve as specific markers for cancers. moiety of glycolipid is known to significantly affect its antigenicity When, however, well-known cancer-associated gangliosides, [33], it is difficult to expect anti-glycolipid antibodies to specifi- such as GM2 or GD3, are modified by N-glycolyl sialic acid, such cally detect glycolipids having definite molecular species of cera- gangliosides exhibit higher cancer specificity, and are competent mide. In contrast, mass-spectrometry is theoretically best fit for to serve as excellent markers of cancer cells. If normal neural tis- this purpose, because it directly detects molecular species of cera- sues were attacked by hypoxia, normal gangliosides such as GM1 mide moiety of given glycolipids [34,35]. and GD1a would be mainly modified by N-glycolyl sialic acid. It is noteworthy that the glycan molecules reflecting the combined 9. Clinical application of glycolipids as future cancer markers effects of both epigenetic silencing and tumor hypoxia have rela- detected by mass spectrometry tively higher cancer specificity. The GM2 ganglioside having N-glycolyl sialic acid could be one of such examples. An increasing number of papers has reported examples of suc- The same principle can be applied to other cancer-associated cessful large-scale routine determination of glycolipids from small glycans such as sialyl Lewis X and sialyl Lewis A carrying N-gly- amounts of clinical samples using mass-spectrometry. A significant increase in plasma ceramide levels was detected in type 2 diabetic patients accompanied with C18:1 and C18:0 subspecies [36].An increased plasma level of ceramide was shown to be a risk factor also for early atherosclerosis using LC–MS/MS [37]. Determination of urinary globotriaosylceramide levels by LC–MS/MS is used for screening of Fabry disease [38]. Matrix-assisted laser desorption mass-spectrometry (MALDI-MS) is successfully applied for detection of ceramides or non-sialylated glycolipids such as monohexo- syl ceramide and sulfatide [39–41]. Although these methods utilizing mass spectrometry supply precious information on the ceramide composition, the pathophysiological significance and clinical utility of such information do not seem to be widely appre- ciated. Recent methods applying mass spectrometry for detection of disease-marker glycolipids in patients sera or tissue samples sometimes include procedures to isolate the glycan portion from glycolipids through chemical or enzymatic cleavage [42–44]. Infor- mation on ceramide moiety becomes unavailable by such procedures. Quality of tissue imaging by matrix-assisted laser desorption mass-spectrometry (MALDI-MS) is significantly improved recently [45–47]. At present the sensitivity and resolution are not enough for detection of minor molecular species, but ultimately, the imaging MALDI-MS should be able to demonstrate hypoxic lesions in tissue sections, which are occupied by cancer cells expressing the Fig. 4. Schematic representation of cancer-associated changes of glycolipids, cancer-associated glycolipids exhibiting a distinct long-chain base exemplified by GM2. Epigenetic silencing of a part of genes required for normal complex glycolipids results in accumulation of shorter glycolipids such as GM2 at and amide-linked fatty acid composition. Differential display of the the relatively early stages of carcinogenesis. Elevated transcription of the gene for ratio of hypoxia-related molecular species of ceramide moiety of a bGalNAc transferase is also noted in cancers [48], which occurs independent of given cancer-associated glycolipid to those non-related normoxic tumor hypoxia. Cancer cells acquire hypoxia tolerance in the locally advanced constituents using appropriate software will help to obtain more stages, and this leads to alterations in both glycan moiety and ceramide moiety of clear imaging of tumor cell nests composed of hypoxia-resistant glycolipid. The molecular species of glycolipids exhibiting combination of multiple tumor-associated changes are expected to have higher cancer specificity. cancer cells. J. Yin et al. / FEBS Letters 584 (2010) 1872–1878 1877

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