Altered sphingolipid metabolism induced by tumor hypoxia - new vistas in glycolipid tumor markers

Abstract

Uncontrolled growth of malignant cells produces hypoxic regions in locally advanced tumors. Recently we showed that tumor hypoxia-induced transcription of multiple genes involved in glycan synthesis, leading to expression of useful glycolipid tumor markers, such as gangliosides having N-glycolyl sialic acid. Our subsequent studies indicated that the ceramide portion of glycolipids, as well as their glycan moiety, was also significantly affected by hypoxia. Tumor hypoxia-induced 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 detecting such distinct molecular species of cancer-associated glycolipids having combined alteration in both glycan- and ceramide moieties.

Fig. 1

Hypoxia-induced expression of ganglioside GM2 having N-glycolyl sialic acid. Panel A, flow-cytometric analysis of a clone of human cultured colon cancer cells Caco-2M using monoclonal antibody specific to NeuGc-GM2 (MK2–34) 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, by hypoxia in Caco-2M cells. Panel C, results of flow-cytometric analysis of Caco-2M cells indicating transfection of Sialin gene confers significant NeuGc-GM2 expression. Panel D, immunohistochemical staining of NeuGc-GM2 in a colon cancer tissue using specific monoclonal antibody, indicating the ganglioside serves as a good marker for advanced stage cancer cells. Advanced cancer cells acquire hypoxia-tolerance, and this accompanies sustained Sialin expression. Upper panel, non-malignant colonic epithelial cells; lower panel, cancer cell nests.

Fig. 2

Hypoxia-induced induction of ganglioside GD3. Panel A, flow-cytometric analysis of a cultured human colon cancer cell line, LS174T, using monoclonal 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 cells.

Fig. 3

Hypoxia-induced changes in ceramide moieties of sphingolipids in cultured human colon cancer cells. Caco-2M cells [8] was 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 hrs (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. *, Significant at 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.

Fig. 4

Schematic representation of cancer-associated changes of glycolipids, 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 the relatively early stages of carcinogenesis. Elevated transcription of the gene forβ GalNAc transferase is also noted in cancers [48], which occurs independent of tumor hypoxia. Cancer cells acquire hypoxia tolerance in the locally advanced stages, and this leads to alterations in both glycan moiety and ceramide moiety of glycolipid. The molecular species of glycolipids exhibiting combination of multiple tumor-associated changes are expected to have higher cancer specificity.