K. Moro1,4, T. Kawaguchi2, J. Tsuchida1, E. Gabriel2, Q. Yan3, L. Yan3, N. Sato4, T. Wakai1, K. Takabe2,5, M. Nagahashi1 1Niigata University Graduate School Of Medical And Dental Sciences,Digestive And General Surgery,Niigata, NIIGATA, Japan 2Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NEW YORK, USA 3Roswell Park Cancer Institute,Department Of Biostatistics And Bioinformatics,Buffalo, NEW YORK, USA 4Niigata Cancer Center Hospital,Surgery,Niigata, NIIGATA, Japan 5University At Buffalo Jacobs School Of Medicine And Biomedical Sciences,Surgery,Buffalo, NEW YORK, USA
Introduction: Sphingolipids have emerged as key regulatory molecules that control various aspects of cell biology. Among them, sphingosine-1-phosphate (S1P) and ceramide are known to form a “rheostat” where former promote cell growth and survival, and the latter apoptosis in cancer. There are three biosynthesis pathways that generate ceramide; De novo pathway; Sphingomyelin pathway; and Salvage pathway, and it is metabolized to S1P through Catabolic pathway. Despite their critical roles, the levels of sphingolipids have never been measured in patients due to lack of methods to precisely quantify them until recently. We have recently published high levels of S1P not only in breast tumor, but also in tumor microenvironment, such as tumor interstitial fluid, and reported that S1P plays pivotal roles in breast cancer progression. On the other hand, the levels of ceramide, a bioactive metabolite of S1P, in breast cancer patients have not yet well investigated to date. The aim of this study is to clarify the ceramide levels and its biosynthesis pathways in breast cancer patients.
Methods: Breast cancer, peri-tumor normal breast defined as tissue within 1 cm from the gross edge of cancer and normal breast tissue samples were collected from surgical specimens from a series of 44 patients with breast cancer. Sphingolipids, including ceramides (C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0, C26:0) and their metabolites of monohexosylceramides, dihydroceramide and sphingomyelin in the tissue samples were determined by mass spectrometry. Results were analyzed for statistical significance with the Kruskal-Wallis test. The Cancer Genome Atlas (TCGA) was used to analyze gene expressions related to the sphingolipid metabolism.
Results: Ceramide levels were higher in breast cancer compared from both normal and peri-tumor breast tissue. Substrates and enzymes that generate ceramide were significantly increased in all three ceramide biosynthesis pathways in cancer; Monohexocylceramide and glucosylceramides beta in Salvage pathway; Sphingomyelin and sphingomyelin phosphodiesterase 2 and 4 in Sphingomyelin pathway; Dihydroceramide and dihydroceramide desaturase 1 in De novo pathway. Sphingosine and ceramide synthases 2, 4, 5 and 6 in Catabolic pathway were also significantly elevated in cancer. On the contrary, gene expression of enzymes that catalyze ceramide, sphingomyelin synthase 2 and ceramide kinase were significantly suppressed, all contribute to ceramide increase in cancer.
Conclusion: This is the first study to reveal the clinical relevance of ceramide metabolism in breast cancer patients. We demonstrated that ceramide levels in breast cancer tissue are significantly higher than those in normal tissue, with activation of the three ceramide biosynthesis pathways. Our finding is in agreement with the classic notion that apoptotic cell destruction signal is activated in cancer, however, it is not enough to overcome its proliferative drive.