Toshihiko Kawamori

Role for sphingosine kinase 1 in colon carcinogenesis

Sphingosine kinase 1 (SphKl) phosphory‐lates sphingosine to form sphingosine‐l‐phosphate (S1P) and is a critical regulator of sphingolipid‐mediated functions. Cell‐based studies suggest a tumor‐promoting function for the SphK1/S1P pathway. Also, our previous studies implicated the SphK1/S1P pathway in the induction of the arachidonic acid cascade, a major inflammatory pathway involved in colon carcinogenesis. Therefore, we investigated whether the SphK1/S1P pathway is necessary for mediating carcinogenesis in vivo. Here, we report that 89% (42/47) of human colon cancer samples stained positively for SphK1, whereas normal colon mucosa had negative or weak staining. Adenomas had higher expression of SphK1 vs. normal mucosa, and colon cancers with metastasis had higher expression of SphK1 than those without metastasis. In the azoxymethane (AOM) murine model of colon cancer, SphK1 and S1P were significantly elevated in colon cancer tissues compared to normal mucosa. Moreover, blood levels of S1P were higher in mice with colon cancers than in those without cancers. Notably, SphK1_/_ mice subjected to AOM had significantly less aberrant crypt foci (ACF) formation and significantly reduced colon cancer development. These results are the first in vivo evidence that the SphK1/S1P pathway contributes to colon carcinogenesis and that inhibition of this pathway is a potential target for chemoprevention.—Kawamori, T., Kaneshiro, T., Okumura, M., Maalouf, S., Uflacker, A., Bielawski, J., Hannun, Y. A., Obeid, L. M. Role for sphingosine kinase 1 in colon carcinogenesis. FASEB J. 23, 405‐414 (2009)

Sphingosine kinase 1 is up-regulated in colon carcinogenesis

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to form sphingosine 1‐phosphate (S1P), which has the ability to promote cell proliferation and survival and stimulate angiogenesis. The SK1/S1P pathway also plays a critical role in regulation of cyclooxygenase‐2 (COX‐2), a well‐established pathogenic factor in colon carcinogenesis. Therefore, we examined the expression of SK1 and COX‐2 in rat colon tumors induced by azoxymethane (AOM) and the relationship of these two proteins in normal and malignant intestinal epithelial cells. Strongly positive SK1 staining was found in 21/28 (75%) of rat colon adenocarcinomas induced by AOM, whereas no positive SK1 staining was observed in normal mucosa. The increase in SK1 and COX‐2 expression in AOM‐induced rat colon adenocarcinoma was confirmed at the level of mRNA by real‐time RT‐PCR. In addition, it was found that 1) down‐regulation of SK1 in HT‐29 human colon cancer cells by small interfering RNA (siRNA) decreases COX‐2 expression and PGE2 production; 2) overexpression of SK1 in RIE‐1 rat intestinal epithelial cells induces COX‐2 expression; and 3) S1P stimulates COX‐2 expression and PGE2 production in HT‐29 cells. These results suggest that the SK1/S1P pathway may play an important role in colon carcinogenesis, in part, by regulating COX‐2 expression and PGE2 production.

Communication between host organism and cancer cells is transduced by systemic sphingosine kinase 1/sphingosine 1‐phosphate signalling to regulate tumour metastasis

Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via sphingosine 1‐phosphate (S1P) generated systemically by sphingosine kinase 1 (SK1), rather than via tumour‐derived S1P. Modulation of systemic, but not tumour SK1, prevented S1P elevation, and inhibited TRAMP‐induced prostate cancer growth in TRAMP+/+SK1−/− mice, or lung metastasis of multiple cancer cells in SK1−/− animals. Genetic loss of SK1 activated a master metastasis suppressor, Brms1 (breast carcinoma metastasis suppressor 1), via modulation of S1P receptor 2 (S1PR2) in cancer cells. Alterations of S1PR2 using pharmacologic and genetic tools enhanced Brms1. Moreover, Brms1 in S1PR2−/− MEFs was modulated by serum S1P alterations. Accordingly, ectopic Brms1 in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms1 prevented this process. Importantly, inhibition of systemic S1P signalling using a novel anti‐S1P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms1 knockdown in MB49 cells. Thus, these data suggest that systemic SK1/S1P regulates metastatic potential via regulation of tumour S1PR2/Brms1 axis.

Sphingolipids in cancer

The bioactive sphingolipids including, ceramide, sphingosine, and sphingosine-1-phosphate (S1P) have important roles in several types of signaling and regulation of many cellular processes including cell proliferation, apoptosis, senescence, angiogenesis, and transformation. Recent accumulating evidence suggests that ceramide- and S1P-mediated pathways have been implicated in cancer development, progression, and chemotherapy. Ceramide mediates numerous cell-stress responses, such as induction of apoptosis and cell senescence, whereas S1P plays pivotal roles in cell survival, migration, and inflammation. These sphingolipids with opposing roles can be interconverted within cells, suggesting that the balance between them is related to cell fate. Importantly, these sphingolipids are metabolically related through actions of enzymes including ceramidases, ceramide synthases, sphingosine kinases, and S1P phosphatases thereby forming a network of metabolically interrelated bioactive lipid mediators whose importance in normal cellular function and diseases is gaining appreciation. In this review, we summarize involvement of sphingolipids and their related enzymes in pathogenesis and therapy of cancer and discuss future directions of sphingolipid field in cancer research.

Acid Ceramidase but Not Acid Sphingomyelinase Is Required for Tumor Necrosis Factor-α-induced PGE2 Production

Sphingolipids are well established effectors of signal transduction downstream of the tumor necrosis factor (TNF) receptor. In a previous study, we showed that the sphingosine kinase/sphingosine 1-phosphate (S1P) pathway couples TNF receptor to induction of the cyclooxygenase 2 gene and prostaglandin synthesis (Pettus, B. J., Bielawski, J., Porcelli, A. M., Reames, D. L., Johnson, K. R., Morrow, J., Chalfant, C. E., Obeid, L. M., and Hannun, Y. A. (2003) FASEB J. 17, 1411-1421). In this study, the requirement for acid sphingomyelinase and sphingomyelin metabolites in the TNFα/prostaglandin E2 (PGE2) pathway was investigated. The amphiphilic compound desipramine, a frequently employed inhibitor of acid sphingomyelinase (ASMase), blocked PGE2 production. However, the action of desipramine was independent of its action on ASMase, since neither genetic loss of ASMase (Niemann-Pick fibroblasts) nor knockdown of ASMase using RNA interference affected TNFα-induced PGE2 synthesis. Further investigations revealed that desipramine down-regulated acid ceramidase (AC), but not sphingosine kinase, at the protein level. This resulted in a time-dependent drop in sphingosine and S1P levels. Moreover, exogenous administration of either sphingosine or S1P rescued PGE2 biosynthesis after desipramine treatment. Interestingly, knockdown of endogenous AC by RNA interference attenuated cyclooxygenase 2 induction by TNFα and subsequent PGE2 biosynthesis. Taken together, these results define a novel role for AC in the TNFα/PGE2 pathway. In addition, the results of this study warrant careful reconsideration of desipramine as a specific inhibitor for ASMase.

A role of sphingosine kinase 1 in head and neck carcinogenesis.

It is important to identify novel and effective targets for cancer prevention and therapy against head and neck squamous cell carcinoma (HNSCC), one of the most lethal cancers. Accumulating evidence suggests that the bioactive sphingolipids, such as sphingosine-1-phosphate (S1P) and its generating enzyme, sphingosine kinase 1 (SphK1) play pivotal roles in several important biological functions including promoting tumor growth and carcinogenesis. However, roles of SphK1/S1P in HNSCC development and/or progression have not been defined previously. Therefore, in this study, we first analyzed the expression of SphK1 in human HNSCC tumor samples and normal head & neck tissues (n = 78 and 17, respectively) using immunohistochemistry. The data showed that SphK1 is overexpressed in all of the HNSCC tumors tested (stages I–IV). We next investigated whether SphK1 is necessary for HNSCC development. To define the role of SphK1/S1P in HNSCC development, we utilized 4-nitroquinoline-1-oxide (4-NQO)-induced HNSCC model in wild-type mice compared with SphK1−/− knockout (KO) mice. Remarkably, we found that the genetic loss of SphK1, which reduced S1P generation, significantly prevented 4-NQO–induced HNSCC carcinogenesis, with decreased tumor incidence, multiplicity, and volume when compared with controls. Moreover, our data indicated that prevention of 4-NQO–induced HNSCC development in SphK1−/− KO mice might be associated with decreased cell proliferation, increased levels of cleaved (active) caspase 3, and downregulation of phospho (active) AKT expression. Thus, these novel data suggest that SphK1/S1P signaling may play important roles in HNSCC carcinogenesis, and that targeting SphK1/S1P might provide a novel strategy for chemoprevention and treatment against HNSCC. Cancer Prev Res; 4(3); 454–62. ©2011 AACR.

Loss of neutral ceramidase increases inflammation in a mouse model of inflammatory bowel disease.

Sphingolipids are emerging as important mediators of immune and inflammatory responses. We have previously demonstrated that sphingosine-1-phosphate (S1P) and its synthetic enzyme sphingosine kinase-1 (SK1) play an important role in inflammatory bowel disease. S1P generation is dependent on SK phosphorylation of sphingosine. Generation of sphingosine results only from the breakdown of ceramide by ceramidases (CDase). In this study, we set out to determine the role of neutral CDase (nCDase) in S1P generation and inflammatory bowel disease. To this end, we established nCDase expression is increased in patients with ulcerative colitis. Using the dextran sulfate sodium (DSS)-induced colitis model, we determined nCDase activity increased in colon epithelium, but not submucosa, in wild-type (WT) mice. Following DSS, ceramide levels were elevated in colon epithelium from WT and nCDase(-/-) mice, while S1P levels were significantly elevated only in the epithelium of nCDase(-/-) mice. Similarly, cyclooxygenase-2 (Cox-2) levels were significantly elevated only in the epithelium of nCDase(-/-) mice. Neutral CDase(-/-) mice also exhibited higher endotoxin levels in circulation, as well as higher circulating levels of S1P. This increase in S1P in nCDase(-/-) mice was accompanied by a marked leukocytosis, most notably circulating neutrophils and lymphocytes. Taken together these data demonstrate that loss of nCDase results in an unexpected increase in S1P generation in inflammation, and suggests that nCDase may actually protect against inflammation.

Distinct roles for hematopoietic and extra-hematopoietic sphingosine kinase-1 in inflammatory bowel disease.

Sphingosine kinase 1 (SK1), one of two SK enzymes, is highly regulated and has been shown to act as a focal point for the action of many growth factors and cytokines. SK1 leads to generation of sphingosine-1-phosphate (S1P) and potentially the activation of S1P receptors to mediate biologic effects. Our previous studies implicated SK1/S1P in the regulation of inflammatory processes, specifically in inflammatory bowel disease (IBD). These studies were conducted using a total body knockout mouse for SK1 and were unable to determine the source of SK1/S1P (hematopoietic or extra-hematopoietic) involved in the inflammatory responses. Therefore, bone marrow transplants were performed with wild-type (WT) and SK1-/- mice and colitis induced with dextran sulfate sodium (DSS). Irrespective of the source of SK1/S1P, bone marrow or tissue, DSS induced colitis in all mice; however, mice lacking SK1 in both hematopoietic and extra-hematopoietic compartments exhibited decreased crypt damage. Systemic inflammation was assessed, and mice with WT bone marrow demonstrated significant neutrophilia in response to DSS. In the local inflammatory response, mice lacking SK1/S1P in either bone marrow or tissue exhibited decreased induction of cytokines and less activation of STAT3 (signal transducer and activator of transcription 3). Interestingly, we determined that extra-hematopoietic SK1 is necessary for the induction of cyclooxygenase 2 (COX2) in colon epithelium in response to DSS-induced colitis. Taken together our data suggest that hematopoietic-derived SK1/S1P regulates specific aspects of the systemic inflammatory response, while extra-hematopoietic SK1 in the colon epithelium is necessary for the autocrine induction of COX2 in DSS-induced colitis.

Effect of sphingosine kinase 1 inhibition on blood pressure

Accumulating evidence suggests that sphingosine kinase 1 (SphK1) plays a key role in carcinogenesis by regulating cyclooxygenase‐2 (COX‐2) expression. Recent clinical studies have revealed that COX‐2 inhibitors cause adverse cardiovascular side effects, likely due to inhibition of prostacyclin (PGI2). In this work, we investigated the roles of SphK1 inhibition on blood pressure (BP). The results show that lack of SphK1 expression did not exacerbate angiotensin II (Ang II)‐induced acute hypertension, whereas celecoxib, a COX‐2 inhibitor, augmented and sustained higher BP in mice. Interestingly, SphK1‐knockout mice inhibited prostaglandin E2 (PGE2) but not PGI2 production in response to Ang II, whereas celecoxib blocked both PGE2 and PGI2 production. Mechanistically, SphK1 down‐regulation by siRNA in human umbilical vein endothelial cells decreased cytokine‐induced PGE2 production primarily through inhibition of microsomal PGE synthase‐1 (mPGES‐1), not COX‐2. SphK1 down‐regulation also decreased MKK6 expression, which phosphorylates and activates P38 MAPK, which, in turn, regulates early growth response‐1 (Egr‐1), a transcription factor of mPGES‐1. Together, these data indicate that SphK1 regulates PGE2 production by mPGES‐1 expression via the p38 MAPK pathway, independent of COX‐2 signaling, in endothelial cells, suggesting that SphK1 inhibition may be a promising strategy for cancer chemoprevention with lack of the adverse cardiovascular side effects associated with coxibs.—Furuya, H., Wada, M., Shimizu, Y., Yamada, P. M., Hannun, Y. A., Obeid, L. M., Kawamori, T. Effect of sphingosine kinase 1 inhibition on blood pressure. FASEB J. 27, 656–664 (2013). www.fasebj.org

Inhibition of prostaglandin E2 signaling through the EP1 receptor does not affect prostacyclin production in human endothelial cells

Accumulating evidence suggests that cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) may play an important role in colon carcinogenesis. Thus, blockage of this pathway may be a suitable strategy for colon cancer chemoprevention. Recent clinical studies suggest that COX-2 inhibitors cause adverse cardiovascular effects due to prostacyclin (PGI(2)) inhibition. To test our hypothesis that inhibition of PGE(2) signaling through E-prostanoid (EP) receptors may offer a safer cardiovascular profile than COX-2 inhibition, we analyzed expression of 6-keto PGF(1alpha), a hydrated form of PGI(2) and PGI(2) synthase, which was stimulated with cytokines in human umbilical vein endothelial cells (HUVECs) treated with the EP(1) receptor antagonist ONO-8711 or the COX-2 inhibitor celecoxib. ONO-8711 did not inhibit both 6-keto PGF(1alpha) production and PGIS expression, whereas celecoxib did in HUVECs. ONO-8711 also inhibited cytokine-induced tissue factor expression in HUVECs. These results suggest that ONO-8711 may be a safer chemopreventive agent with respect to cardiovascular events.