Ceramide species are elevated in human breast cancer and are associated with less aggressiveness

Abstract

Sphingolipids have emerged as key regulatory molecules in cancer cell survival and death. Although important roles of sphingolipids in breast cancer progression have been reported in experimental models, their roles in human patients are yet to be revealed. The aim of this study was to investigate the ceramide levels and its biosynthesis pathways in human breast cancer patients. Breast cancer, peri-tumor and normal breast tissue samples were collected from surgical specimens from a series of 44 patients with breast cancer. The amount of sphingolipid metabolites in the tissue were determined by mass spectrometry. The Cancer Genome Atlas was used to analyze gene expression related to the sphingolipid metabolism. Ceramide levels were higher in breast cancer tissue compared to both normal and peri-tumor breast tissue. Substrates and enzymes that generate ceramide were significantly increased in all three ceramide biosynthesis pathways in cancer. Further, higher levels of ceramide in breast cancer were associated with less aggressive cancer biology presented by Ki-67 index and nuclear grade of the cancer. Interestingly, patients with higher gene expressions of enzymes in the three major ceramide synthesis pathways showed significantly worse prognosis. 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 were significantly higher than those in normal tissue, with activation of the three ceramide biosynthesis pathways. We also identified that ceramide levels have a significant association with aggressive phenotype and its enzymes have prognostic impact on breast cancer patients.

Keywords: breast cancer; ceramide; metabolism; prognosis; sphingolipid.

Figure 1. Pathways for ceramide metabolism in breast cancer tissue

Ceramide is generated from the major pathways such as the de novo pathway, the salvage pathway and the sphingomyelin pathway. Ceramide can be phosphorylated to ceramide-1-phosphate (C1P), broken down to sphingosine and phosphorylated to sphingosine-1-phosphate (S1P). The gene expression of ceramide metabolism in The Cancer Genome Atlas (TCGA) cohort (n=112) were analyzed.

Figure 2. Ceramide levels in breast cancer tissue (n = 44), peri-tumor tissue (n = 36), and normal breast tissue (n = 44)

(A, B), Levels of total ceramide (Cer) (A) and each ceramide specie (C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0, C26:1 and C26:0) (B) in cancer tissue (Cancer or C), peri-tumor tissue (Peri or P) and normal tissue (Normal or N) were determined by mass spectrometry. Mean values are shown by the horizontal lines. ^*, P<0.05 for cancer vs. normal tissue; ^**, P<0.05 for cancer vs. peri-tumor tissue. (C, D), Receiver-operating characteristic (ROC) curves and the area under the ROC curve (AUC) were produced to assess the ability of our ceramide assays to distinguish cancer tissue from either normal breast tissue sample (C) or peri-tumor tissue (D). (E, F), Correlation between the ceramide level in breast cancer tissue and that in normal breast tissue (E) or peri-tumor (F) were compared in individual patient. The correlation between two variables is denoted by R². (G) The ceramide levels in interstitial fluid (IF) of cancer tissue and normal breast tissue were determined. (H, I) Correlation between the ceramide level in breast cancer tissue and that in serum (H) or plasma (I) were compared. The correlation between two variables is denoted by R².

Figure 3. The metabolism in the salvage pathway for ceramide synthesis in breast cancer tissue

(A, B) Levels of total monohexocylceramide (HexCer) (A) and each monohexocylceramide specie (C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0, C26:1 and C26:0) (B) in cancer tissue (Cancer or C), peri-tumor tissue (Peri or P) and normal tissue (Normal or N) were determined by mass spectrometry. Mean values are shown by the horizontal lines. ^*, P<0.05 for cancer vs. normal tissue; ^**, P<0.05 for cancer vs. peri-tumor tissue. (C) The expression of glucosylceramidase (GBA) and uridine diphosphoglucose ceramide glucosyltransferase (UGCG) in The Cancer Genome Atlas (TCGA) cohort (n=112) were analyzed. Mean values are shown by the horizontal lines. ^*, P<0.05 for cancer vs. normal tissue.

Figure 4. The metabolism in the sphingomyelin pathway for ceramide synthesis in breast cancer patients

(A, B) Levels of total sphingomyelin (SM) (A) and each sphingomyelin species (C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0, C26:1 and C26:0) (B) in cancer tissue (Cancer or C), peri-tumor tissue (Peri or P) and normal tissue (Normal or N) were determined by mass spectrometry. Mean values are shown by the horizontal lines. ^*, P<0.05 for cancer vs. normal tissue; ^**, P<0.05 for cancer vs. peri-tumor tissue. (C) The expression of sphingomyelin phosphodiesterase 2 (SMPD2), sphingomyelin phosphodiesterase 4 (SMPD4), sphingomyelin phosphodiesterase 5 (SMPD5) and sphingomyelin synthase 2 (SGMS2) in The Cancer Genome Atlas (TCGA) cohort (n=112) were analyzed. Mean values are shown by the horizontal lines. ^*, P<0.05 for cancer vs. normal tissue.

Figure 5. The metabolism in the de novo pathway for ceramide synthesis in breast cancer patients

(A, B) Levels of total dihydroceramide (DH-Cer) (A) and each dihydroceramide species (C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0, C26:1 and C26:0) (B) in cancer tissue (Cancer or C), peri-tumor tissue (Peri or P) and normal tissue (Normal or N) were determined by mass spectrometry. Mean values are shown by the horizontal lines. ^*, P<0.05 for cancer vs. normal tissue; ^**, P<0.05 for cancer vs. peri-tumor tissue. (C) The expression of Dihydroceramide desaturase 1 (DES1) in The Cancer Genome Atlas (TCGA) cohort (n=112) were analyzed. Mean values are shown by the horizontal lines. ^*, P<0.05 for cancer vs. normal tissue.

Figure 6. The other sphingolipid metabolic pathways and correlation between ceramide levels in breast cancer tissue and cell proliferation potency

(A-J) The expression of genes related to the other sphingolipid metabolic pathways in The Cancer Genome Atlas (TCGA) cohort (n=112) were analyzed. Mean values are shown by the horizontal lines. ^*, P<0.05 for cancer vs. normal tissue.

Figure 7. The correlation between ratio of ceramide, the ratio of ceramide to S1P or sphingosine and cell proliferation potency

(A) The comparison of ceramide levels in breast cancer tissue between patients with Ki-67 index. Mean values are shown by the horizontal lines. ^*, P<0.05 for high Ki-67 index vs. low Ki-67 index. (B) The comparison of ceramide levels in breast cancer tissue between patients with high nuclear grade (Nuclear Grade 3) and patients with low nuclear grade (Nuclear Grade 1 and 2). Mean values are shown by the horizontal lines. ^*, P<0.05 for high nuclear grade vs. low nuclear grade. (C) The correlation between ceramide levels in breast cancer tissue and Ki-67 index. The correlation between two variables is denoted by R². (D) The correlation between ceramide levels in breast cancer tissue and nuclear grade. The correlation between two variables is denoted by R². (E) The comparison of the ratio of S1P to ceramide in breast cancer tissue between patients with high Ki-67 index and patients with low Ki-67 index. Mean values are shown by the horizontal lines. ^*, P<0.05 for high Ki-67 index vs. low Ki-67 index. (F) The comparison of the ratio of S1P to ceramide in breast cancer tissue between patients with high nuclear grade (Nuclear Grade 3) and patients with low nuclear grade (Nuclear Grade 1 and 2). Mean values are shown by the horizontal lines. ^*, P<0.05 for high nuclear grade vs. low nuclear grade. (G) The correlation between the ratio of S1P to ceramide in breast cancer tissue and Ki-67 index. The correlation between two variables is denoted by R². (H) The correlation between the ratio of S1P to ceramide in breast cancer tissue and nuclear grade. The correlation between two variables is denoted by R².

Figure 8. Overall survival of breast cancer patients in The Cancer Genome Atlas (TCGA) cohort

The gene expression levels of enzymes related to ceramide metabolism in breast cancer tissue were examined from TCGA cohort (A-V). High, patients with high gene expression. Low, patients with low gene expression. Genes are listed at the top of each figure.