Eugene Y. Kim

Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2

Tumour-necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key component in NF-κB signalling triggered by TNF-α. Genetic evidence indicates that TRAF2 is necessary for the polyubiquitination of receptor interacting protein 1 (RIP1) that then serves as a platform for recruitment and stimulation of IκB kinase, leading to activation of the transcription factor NF-κB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyses the ubiquitination of RIP1 is lacking. TRAF2 binds to sphingosine kinase 1 (SphK1), one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. Here we show that SphK1 and the production of S1P is necessary for lysine-63-linked polyubiquitination of RIP1, phosphorylation of IκB kinase and IκBα, and IκBα degradation, leading to NF-κB activation. These responses were mediated by intracellular S1P independently of its cell surface G-protein-coupled receptors. S1P specifically binds to TRAF2 at the amino-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, markedly increased recombinant TRAF2-catalysed lysine-63-linked, but not lysine-48-linked, polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data show that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing cofactor for TRAF2 E3 ubiquitin ligase activity, indicating a new paradigm for the regulation of lysine-63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-α signalling and the canonical NF-κB activation pathway important in inflammatory, antiapoptotic and immune processes.

Sphingosine-1-Phosphate Links Persistent STAT3 Activation, Chronic Intestinal Inflammation, and Development of Colitis-Associated Cancer

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 prodrug 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 Produced by Sphingosine Kinase 1 Promotes Breast Cancer Progression by Stimulating Angiogenesis and Lymphangiogenesis

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 pharmacologic 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 with 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 with fluorescence-activated cell sorting, 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 show 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.

Spns2, a transporter of phosphorylated sphingoid bases, regulates their blood and lymph levels and the lymphatic network

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.—Nagahashi, M., Kim, E. Y., Yamada, A., Ramachandran, S., Allegood, J. C., Hait, N. C., Maceyka, M., Milstien, S., Takabe, K., Spiegel, S. Spns2, a transporter of phosphorylated sphingoid bases, regulates their blood and lymph levels and the lymphatic network. FASEB J. 27, 1001–1011 (2013). www.fasebj.org

Role of sphingosine kinase 1 and sphingosine-1-phosphate in CD40 signaling and IgE class switching

The tumor necrosis factor (TNF) receptor family member CD40 plays an essential role in the activation of antigen‐presenting cells, B cell maturation, and immunoglobulin (Ig) class switching critical for adaptive immunity. Although the bioactive sphingolipid metabolite sphingosine‐1‐phosphate (S1P) and the kinase that produces it, sphingosine kinase 1 (SphK1), have long been implicated in the actions of TNF mediated by engagement of TNFR1, nothing is yet known of their role in CD40‐mediated events. We have now found that ligation of CD40 activates and translocates SphK1 to the plasma membrane, leading to generation of S1P. SphK1 inhibition in human tonsil B cells, as well as inhibition or deletion of SphK1 in mouse splenic B cells, significantly reduced CD40‐mediated Ig class switching and plasma cell differentiation ex vivo. Optimal activation of downstream CD40 signaling pathways, including NF‐κB, p38, and JNK, also required SphK1. In mice treated with a SphK1 inhibitor or in SphK1–/– mice, isotype switching to antigen‐specific IgE was decreased in vivo by 70 and 55%, respectively. Our results indicate that SphK1 is important for CD40‐mediated B cell activation and regulation of humoral responses and suggest that targeting SphK1 might be a useful therapeutic approach to control antigen‐specific IgE production.—Kim, E. Y., Sturgill, J. L., Hait, N. C., Avni, D., Valencia, E. C., Maceyka, M., Lima, S., Allegood, J., Huang, W.‐C., Zhang, S., Milstien, S., Conrad, D., Spiegel, S., Role of sphingosine kinase 1 and sphingosine‐1‐phosphate in CD40 signaling and IgE class switching. FASEB J. 28, 4347–4358 (2014). www.fasebj.org

Abstract 5000: Spinster 2 exports S1P, an important player in lymph node metastasis.

Sphingosine-1-phosphate (S1P), a ligand for five specific receptors, is a potent lipid mediator that plays important roles in cancer cell proliferation, migration, and cancer-induced angiogenesis/lymphangiogenesis. S1P is produced inside cells and therefore must be secreted in order to exert its effects through these receptors. We have reported that E2-induced export of S1P mediated by ABCC1 and ABCG2 transporters and consequent activation of S1P receptors may contribute to nongenomic signaling of E2 important for breast cancer pathophysiology. Spinster 2 (Spns2) is one of the cell surface transporters thought to secrete S1P, thus it is important to elucidate its physiological role. Although lymphatic endothelial cells contribute to regulation of S1P levels in lymph, little is known how S1P levels are maintained in the lymphatic system. Spinster 2 knockout mice were used, and the lymphatic system was examined by flow cytometry and immunofluorescent staining. Sphingolipids in the blood, lymph, and organs were determined by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). We found that S1P secreted by breast cancer cells promotes tumor progression in a syngeneic model by stimulating angiogenesis and lymphangiogenesis. Interestingly, suppression of tumor S1P production resulted in less lymph node metastasis. Further, we found that serum S1P levels are elevated in breast cancer patients with lymph node metastasis. We confirmed 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 sinuses, which appear collapsed, with reduced numbers of lymphocytes. Our data suggest that Spns2 is a S1P transporter in vivo that regulate not only of blood S1P but also lymph node and lymph S1P levels, which may implicate a role in lymph node metastasis. M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. S.S. is supported by NIH R37GM043880, and K.T. by NIH R01CA160688 and Susan G. Komen for the Cure Investigator Initiated Research Grant. Citation Format: Masayuki Nagahashi, Eugene Y. Kim, Akimitsu Yamada, Subramaniam Ramachandran, Jeremy C. Allegood, Nitai Hait, Michael Maceyka, Sheldon Milstien, Sarah Spiegel, Kazuaki Takabe. Spinster 2 exports S1P, an important player in lymph node metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5000. doi:10.1158/1538-7445.AM2013-5000

Abstract LB-325: Sphingosine-1-phosphate links chronic intestinal inflammation to development of colitis-associated cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Inflammatory bowel disease is an important risk factor for colorectal cancer. The pro-inflammatory cytokines TNF-alpha and IL-6 and their downstream master transcription factors NF-kappaB and Stat3 are critical regulators of chronic inflammation and cancer. There is growing evidence that sphingosine-1-phosphage (S1P), a pleiotropic bioactive sphingolipid metabolite formed inside cells by two closely related sphingosine kinases, SphK1 and SphK2, is involved in inflammation and cancer. In this work, we have shown that S1P produced by upregulation of SphK1 links chronic intestinal inflammation to CAC and both are exacerbated by deletion of SphK2. S1P is essential for production of the multifunctional NF-kappaB-regulated cytokine IL-6, persistent activation of the transcription factor Stat3, and consequent upregulation of the S1P receptor, S1PR1. Adoptive transfer demonstrated that SphK2 deficiency in hematopoietic cells contributed to colitis severity, IL-6 production, and activation of Stat3 and NF-kappaB. The pro-drug FTY720 decreased SphK1 and S1PR1 and eliminated the NF-kappaB-IL-6-Stat3 amplification cascade and development of CAC even in SphK2-/- mice, suggesting that FTY720 may be useful in treating colon cancer in individuals with ulcerative colitis. Thus, the SphK1-S1P-S1PR1 axis is at the nexus between NF-kappaB and Stat3 and connects chronic inflammation and CAC. This work was supported by NIH grants R37GM043880, R01CA61774 (to S.S.) and K12HD055881 and the Susan G. Komen for the Cure Research Foundation grant (to K.T.). M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. E.Y.K. and J.C.A. were supported by NIH training grant T32HL094290. Citation Format: Masayuki Nagahashi, Jie Liang, Eugene Y. Kim, Kuzhuvelil B. Harikumar, Akimitsu Yamada, Wei-Ching Huang, Nitai C. Hait, Jeremy C. Allegood, Megan M. Price, Dorit Avni, Kazuaki Takabe, Tomasz Kordula, Sheldon Milstien, Sarah Spiegel. Sphingosine-1-phosphate links chronic intestinal inflammation to development of colitis-associated cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-325. doi:10.1158/1538-7445.AM2013-LB-325

Abstract 4364: S1P generated by SphK1 is important not only for primary tumor growth but also for tumor-induced hemangiogenesis and lymphangiogenesis

INTRODUCTION: Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, regulates many cellular processes important for breast cancer progression. The aim of this study is to investigate a role for S1P produced by sphingosine kinase 1 (SphK1) in breast cancer progression and tumor-induced hemangiogenesis and lymphangiogenesis. METHODS: An isozyme-specific inhibitor of SphK1 (SK1-I) was used. Tumor burden were quantified using an in vivo imaging system (IVIS 2000). S1P and SK1-I levels were measured by LC-ESI-MS/MS. Hemangiogenesis and lymphangiogenesis are determined by morphological analysis of microvessel density as well as by flow cytometric analysis of endothelial cells. RESULTS: A significant dose dependent effect of SK1-I on inhibition of 4T1-luc2 murine breast cancer cell growth was observed. We confirmed that SK1-I decreased the enzymatic activity of SphK1 and that downregulation of SphK1 with specific siRNA also suppressed growth of these cells. We found that S1P levels increased in the tumor and in the circulation after orthotopic implantation of 4T1-luc2 cells. Similarly, serum S1P levels were significantly elevated in stage IIIA breast cancer patients who have lymph node metastases, compared to age/ethnicity-matched healthy volunteers. SK1-I blocked serum S1P increases in in vivo model and reduced lymph node and lung metastases and overall tumor burden in vivo as well. Importantly, SK1-I also decreased hem- and lymphangiogenesis not only in the primary tumor, but also in lymph nodes, the host tumor microenvironment. Whereas supernatants from 4T1-luc2 cells significantly stimulated tube formation of HUVECs and HLECs, shRNA knockdown of SphK1 markedly reduced them, indicating that S1P produced by SphK1 in 4T1-luc2 breast cancer cells is an important contributor to hem- and lymphangiogenesis. CONCLUSIONS: S1P generated by SphK1 is important for tumor progression, tumor-induced hemangiogenesis and lymphangiogenesis, and lymph node metastais. Therefore targeting SphK1 and its product S1P would be a multi-pronged attack against breast cancer. This work was supported by Sumitomo Life Social Welfare Services Foundation grant to MN, NIH (K12HD055881) and Susan G. Komen for the Cure (KG090510) to KT, and NCI (R01CA61774) to SS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4364. doi:1538-7445.AM2012-4364