Glycosylated sphingolipids (GSLs) are a diverse group of cellular lipids typically reported as being rare in normal mammary tissue. In breast cancer (BCa), GSLs have emerged as noteworthy markers associated with breast cancer stem cells, mediators of phenotypic plasticity, and contributors to cancer cell chemoresistance. GSLs are potential surface markers that can uniquely characterize the heterogeneity of the tumor microenvironment, including cancer cell subpopulations and epithelial-mesenchymal plasticity (EMP). In this study, mass spectrometry analyses of the total sphingolipidome in breast epithelial cells and their mesenchymal counterparts revealed increased levels of Gb3 in epithelial cells and significantly elevated GD2 levels in the mesenchymal phenotype. To elucidate if GSL-related epitopes on BCa cell surfaces reflect EMP and cancer status, we developed and rigorously validated a 12-color spectral flow cytometry panel. This panel enables the simultaneous detection of native GSL epitopes (Gb3, SSEA1, SSEA3, SSEA4, and GD2), epithelial-mesenchymal transition markers (EpCAM, TROP2, and CD9), and lineage markers (CD45, CD31, and CD90) at the single-cell level. Next, the established panel was used for the analysis of BCa primary tumors and revealed surface heterogeneity in SSEA1, SSEA3, SSEA4, GD2, and Gb3, indicative of native epitope presence also on non-tumor cells. These findings further highlighted the phenotype-dependent alterations in GSL surface profiles, with differences between epithelial and stromal cells in the tumor. This study provides novel insights into BCa heterogeneity, shedding light on the potential of native GSL-related epitopes as markers for EMP and cancer status in fresh clinical samples. The developed single-cell approach offers promising avenues for further exploration.

• Keywords
• breast cancer, epithelial cells, glycosphingolipids, phenotypic plasticity, stromal-like cells, surface profiling,
• MeSH
• Single-Cell Analysis * methods   MeSH
• Epithelial-Mesenchymal Transition * MeSH
• Phenotype MeSH
• Glycosphingolipids * metabolism   analysis   MeSH
• Humans MeSH
• Breast Neoplasms * metabolism   pathology   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glycosphingolipids * MeSH

The aryl hydrocarbon receptor (AhR) plays a wide range of physiological roles in cellular processes such as proliferation, migration or control of immune responses. Several studies have also indicated that AhR might contribute to the regulation of energy balance or cellular metabolism. We observed that the AhR is upregulated in tumor epithelial cells derived from colon cancer patients. Using wild-type and the corresponding AhR knockout (AhR KO) variants of human colon cancer cell lines HCT116 and HT-29, we analyzed possible role(s) of the AhR in cell proliferation and metabolism, with a focus on regulation of the synthesis of fatty acids (FAs). We observed a decreased proliferation rate in the AhR KO cells, which was accompanied with altered cell cycle progression, as well as a decreased ATP production. We also found reduced mRNA levels of key enzymes of the FA biosynthetic pathway in AhR KO colon cancer cells, in particular of stearoyl-CoA desaturase 1 (SCD1). The loss of AhR was also associated with reduced expression and/or activity of components of the PI3K/Akt pathway, which controls lipid metabolism, and other lipogenic transcriptional regulators, such as sterol regulatory element binding transcription factor 1 (SREBP1). Together, our data indicate that disruption of AhR activity in colon tumor cells may, likely in a cell-specific manner, limit their proliferation, which could be linked with a suppressive effect on their endogenous FA metabolism. More attention should be paid to potential mechanistic links between overexpressed AhR and colon tumor cell metabolism.

• Keywords
• AhR, Akt pathway, colon cancer cells, fatty acid synthesis, metabolism, proliferation,
• Publication type
• Journal Article MeSH

Sphingolipids (SLs), glycosphingolipids (GSLs), and eicosanoids are bioactive lipids, which play important roles in the etiology of various diseases, including cancer. However, their content and roles in cancer cells, and in particular in the exosomes derived from tumor cells, remain insufficiently characterized. In this study, we evaluated alterations of SL and GSL levels in transformed cells and their exosomes, using comparative HPLC-MS/MS analysis of parental human bronchial epithelial cells HBEC-12KT and their derivative, benzo[a]pyrene-transformed HBEC-12KT-B1 cells with the acquired mesenchymal phenotype. We examined in parallel SL/GSL contents in the exosomes released from both cell lines. We found significant alterations of the SL/GSL profile in the transformed cell line, which corresponded well with alterations of the SL/GSL profile in exosomes derived from these cells. This suggested that a majority of SLs and GSLs were transported by exosomes in the same relative pattern as in the cells of origin. The only exceptions included decreased contents of sphingosin, sphingosin-1-phosphate, and lactosylceramide in exosomes derived from the transformed cells, as compared with the exosomes derived from the parental cell line. Importantly, we found increased levels of ceramide phosphate, globoside Gb3, and ganglioside GD3 in the exosomes derived from the transformed cells. These positive modulators of epithelial-mesenchymal transition and other pro-carcinogenic processes might thus also contribute to cancer progression in recipient cells. In addition, the transformed HBEC-12KT-B1 cells also produced increased amounts of eicosanoids, in particular prostaglandin E2. Taken together, the exosomes derived from the transformed cells with specifically upregulated SL and GSL species, and increased levels of eicosanoids, might contribute to changes within the cancer microenvironment and in recipient cells, which could in turn participate in cancer development. Future studies should address specific roles of individual SL and GSL species identified in the present study.

• Keywords
• eicosanoids, exosomes, glycosphingolipid, in vitro cell transformation, sphingolipid,
• MeSH
• Benzo(a)pyrene toxicity   MeSH
• Bronchi cytology   MeSH
• Cell Line MeSH
• Exosomes metabolism   MeSH
• Carcinogens toxicity   MeSH
• Humans MeSH
• Cell Transformation, Neoplastic * MeSH
• Respiratory Mucosa drug effects   metabolism   MeSH
• Sphingolipids metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Benzo(a)pyrene MeSH
• Carcinogens MeSH
• Sphingolipids MeSH

The development of colon cancer, one of the most common malignancies, is accompanied with numerous lipid alterations. However, analyses of whole tumor samples may not always provide an accurate description of specific changes occurring directly in tumor epithelial cells. Here, we analyzed in detail the phospholipid (PL), lysophospholipid (lysoPL), and fatty acid (FA) profiles of purified EpCAM+ cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients. We found that a number of FAs increased significantly in isolated tumor cells, which also included a number of long polyunsaturated FAs. Higher levels of FAs were associated with increased expression of FA synthesis genes, as well as with altered expression of enzymes involved in FA elongation and desaturation, including particularly fatty acid synthase, stearoyl-CoA desaturase, fatty acid desaturase 2 and ELOVL5 fatty acid elongase 5 We identified significant changes in ratios of specific lysoPLs and corresponding PLs. A number of lysophosphatidylcholine and lysophosphatidylethanolamine species, containing long-chain and very-long chain FAs, often with high numbers of double bonds, were significantly upregulated in tumor cells. Increased de novo synthesis of very long-chain FAs, or, altered uptake or incorporation of these FAs into specific lysoPLs in tumor cells, may thus contribute to reprogramming of cellular phospholipidome and membrane alterations observed in colon cancer.

• Keywords
• EpCAM, colorectal carcinoma, desaturation, epithelial cells, fatty acid synthesis, lipidomics, lysophospholipids, phospholipids,
• MeSH
• Adenocarcinoma enzymology   genetics   metabolism   MeSH
• Fatty Acid Desaturases genetics   metabolism   MeSH
• Fatty Acid Elongases genetics   metabolism   MeSH
• Epithelial Cells enzymology   metabolism   MeSH
• Phospholipids metabolism   MeSH
• Humans MeSH
• Lipidomics MeSH
• Lipogenesis MeSH
• Fatty Acids metabolism   MeSH
• Lipid Metabolism * MeSH
• Colonic Neoplasms enzymology   genetics   metabolism   MeSH
• Gene Expression Regulation, Neoplastic * MeSH
• Aged MeSH
• Stearoyl-CoA Desaturase genetics   metabolism   MeSH
• Fatty Acid Synthases genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fatty Acid Desaturases MeSH
• Fatty Acid Elongases MeSH
• ELOVL5 protein, human MeSH Browser
• FADS2 protein, human MeSH Browser
• Phospholipids MeSH
• Fatty Acids MeSH
• Stearoyl-CoA Desaturase MeSH
• Fatty Acid Synthases MeSH

The development and progression of colorectal cancer (CRC), a major cause of cancer-related death in the western world, is accompanied with alterations of sphingolipid (SL) composition in colon tumors. A number of enzymes involved in the SL metabolism have been found to be deregulated in human colon tumors, in experimental rodent studies, and in human colon cancer cells in vitro. Therefore, the enzymatic pathways that modulate SL levels have received a significant attention, due to their possible contribution to CRC development, or as potential therapeutic targets. Many of these enzymes are associated with an increased sphingosine-1-phosphate/ceramide ratio, which is in turn linked with increased colon cancer cell survival, proliferation and cancer progression. Nevertheless, more attention should also be paid to the more complex SLs, including specific glycosphingolipids, such as lactosylceramides, which can be also deregulated during CRC development. In this review, we focus on the potential roles of individual SLs/SL metabolism enzymes in colon cancer, as well as on the pros and cons of employing the current in vitro models of colon cancer cells for lipidomic studies investigating the SL metabolism in CRC.

• Keywords
• colon cancer (CRC) sphingolipidomics, colon cancer cells, colorectal cancer, glycosphingolipid, lactosylceramide, sphingolipid, sphingosine-1-phosphate,
• MeSH
• Alkaline Ceramidase genetics   metabolism   MeSH
• Ceramides metabolism   MeSH
• Phosphotransferases (Alcohol Group Acceptor) genetics   metabolism   MeSH
• Acid Ceramidase genetics   metabolism   MeSH
• Lactosylceramides metabolism   MeSH
• Humans MeSH
• Lysophospholipids metabolism   MeSH
• Lipid Metabolism genetics   MeSH
• Disease Models, Animal MeSH
• Tumor Cells, Cultured MeSH
• Colonic Neoplasms enzymology   genetics   pathology   MeSH
• Neutral Ceramidase genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-akt genetics   metabolism   MeSH
• Gene Expression Regulation, Neoplastic * MeSH
• Sphingolipids metabolism   MeSH
• Sphingosine N-Acyltransferase genetics   metabolism   MeSH
• Sphingosine analogs & derivatives   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• ACER2 protein, human MeSH Browser
• Alkaline Ceramidase MeSH
• ASAH1 protein, human MeSH Browser
• ASAH2 protein, human MeSH Browser
• ceramide 1-phosphate MeSH Browser
• Ceramides MeSH
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• Acid Ceramidase MeSH
• Lactosylceramides MeSH
• Lysophospholipids MeSH
• Neutral Ceramidase MeSH
• Proto-Oncogene Proteins c-akt MeSH
• Sphingolipids MeSH
• Sphingosine N-Acyltransferase MeSH
• Sphingosine MeSH
• sphingosine 1-phosphate MeSH Browser
• sphingosine kinase MeSH Browser