SUMMARY Inflammatory bowel disease is an important risk factor for colorectal cancer. We show that sphingosine-1-phosphate (S1P) produced by upregulation of sphingosine kinase 1 (SphK1) links chronic intestinal inflammation to colitis-associated cancer (CAC) and both are exacerbated by deletion of Sphk2. S1P is essential for production of the multifunctional NF-κB-regulated cytokine IL-6, persistent activation of the transcription factor STAT3, and consequent upregulation of the S1P receptor, S1PR1. The pro-drug FTY720 decreased SphK1 and S1PR1 expression and eliminated the NF-κB/IL-6/STAT3 amplification cascade and development of CAC even in Sphk2−/− mice and may be useful in treating colon cancer in individuals with ulcerative colitis. Thus, the SphK1/S1P/S1PR1 axis is at the nexus between NF-κB and STAT3 and connects chronic inflammation and CAC.

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that promotes breast cancer progression by diverse mechanisms that remain somewhat unclear. Here we report pharmacological evidence of a critical role for sphingosine kinase 1 (SphK1) in producing S1P and mediating tumor-induced hemangiogenesis and lymphangiogenesis in a murine model of breast cancer metastasis. S1P levels increased both in the tumor and the circulation. In agreement, serum S1P levels were significantly elevated in stage IIIA human breast cancer patients, compared to age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK1 inhibitor SK1-I suppressed S1P levels, reduced metastases to lymph nodes and lungs and decreased overall tumor burden of our murine model. Both S1P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphangiogenesis in vitro whereas SK1-I inhibited each process. We quantified both processes in vivo from the same specimen by combining Directed In Vivo Angiogenesis Assays (DIVAA) with Fluorescence Activated Cell Sorting (DIVAA/FACS), thereby confirming the results obtained in vitro. Notably, SK1-I decreased both processes not only at the primary tumor but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK1-I-treated animals. Taken together, our findings demonstrate that SphK1-produced S1P is a crucial mediator of breast cancer-induced hemangiogenesis and lymphangiogenesis. Our results implicate SphK1 along with S1P as therapeutic targets in breast cancer.

Bile acids have been shown to be important hormones during the feed/fast cycle, allowing the liver to coordinately regulate nutrient metabolism. How they accomplish this has not been fully elucidated. Conjugated bile acids have been shown to activate both the ERK1/2 and AKT signaling pathways via S1PR2 in rodent hepatocytes and in vivo. Here, we report that feeding mice a high fat diet, infusion of taurocholate into the chronic bile fistula rat, or overexpression of the gene encoding S1PR2 in mouse hepatocytes significantly up-regulated hepatic SphK2, but not SphK1. Key genes encoding nuclear receptors/enzymes involved in nutrient metabolism were significantly down-regulated in livers of S1PR2−/− and SphK2−/− mice. In contrast, overexpression of the gene encoding S1PR2 in primary mouse hepatocytes differentially increased SphK2, but not SphK1, and mRNA levels of key genes involved in nutrient metabolism. Nuclear levels of S1P, an endogenous inhibitor of HDAC 1/2, as well as the acetylation of H3K9, H4K5 and H2BK12, were significantly decreased in hepatocytes prepared from S1PR2−/− and SphK2−/− mice. Both S1PR2−/− and SphK2−/− mice rapidly developed fatty livers on a high fat diet suggesting the importance of conjugated bile acids, S1PR2 and SphK2 in regulating hepatic lipid metabolism.

Sphingosine-1-phosphate (S1P), a ligand for 5 specific receptors, is a potent lipid mediator that plays important roles in lymphocyte trafficking and immune responses. S1P is produced inside cells and therefore must be secreted to exert its effects through these receptors. Spinster 2 (Spns2) is one of the cell surface transporters thought to secrete S1P. We have shown that Spns2 can export endogenous S1P from cells and also dihydro-S1P, which is active at all cell surface S1P receptors. Moreover, Spns2 mice have decreased levels of both of these phosphorylated sphingoid bases in blood, accompanied by increases in very long chain ceramide species, and have defective lymphocyte trafficking. Surprisingly, levels of S1P and dihydro-S1P were increased in lymph from Spns2 mice as well as in specific tissues, including lymph nodes, and interstitial fluid. Moreover, lymph nodes from Spns2 mice have aberrant lymphatic sinus that appeared collapsed, with reduced numbers of lymphocytes. Our data suggest that Spns2 is an S1P transporter in vivo that plays a role in regulation not only of blood S1P but also lymph node and lymph S1P levels and consequently influences lymphocyte trafficking and lymphatic vessel network organization.

Although obesity with associated inflammation is now recognized as a risk factor for breast cancer and distant metastases, the functional basis for these connections remain poorly understood. Here, we show that in breast cancer patients and in animal breast cancer models, obesity is a sufficient cause for increased expression of the bioactive sphingolipid mediator sphingosine-1-phosphate (S1P), which mediates cancer pathogenesis. A high-fat diet was sufficient to upregulate expression of sphingosine kinase 1 (SphK1), the enzyme that produces S1P, along with its receptor S1PR1 in syngeneic and spontaneous breast tumors. Targeting the SphK1/S1P/S1PR1 axis with FTY720/fingolimod attenuated key proinflammatory cytokines, macrophage infiltration, and tumor progression induced by obesity. S1P produced in the lung premetastatic niche by tumor-induced SphK1 increased macrophage recruitment into the lung and induced IL6 and signaling pathways important for lung metastatic colonization. Conversely, FTY720 suppressed IL6, macrophage infiltration, and S1P-mediated signaling pathways in the lung induced by a high-fat diet, and it dramatically reduced formation of metastatic foci. In tumor-bearing mice, FTY720 similarly reduced obesity-related inflammation, S1P signaling, and pulmonary metastasis, thereby prolonging survival. Taken together, our results establish a critical role for circulating S1P produced by tumors and the SphK1/S1P/S1PR1 axis in obesity-related inflammation, formation of lung metastatic niches, and breast cancer metastasis, with potential implications for prevention and treatment. These findings offer a preclinical proof of concept that signaling by a sphingolipid may be an effective target to prevent obesity-related breast cancer metastasis. .