sphingosine-1-phosphate and Skin-Neoplasms

Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine 1-phosphate. In the present study, we evaluated the role of AC-regulated sphingolipid signaling in melanoma. We found that AC expression is markedly elevated in normal human melanocytes and proliferative melanoma cell lines, compared with other skin cells (keratinocytes and fibroblasts) and non-melanoma cancer cells. High AC expression was also observed in biopsies from human subjects with Stage II melanoma. Immunofluorescence studies revealed that the subcellular localization of AC differs between melanocytes (where it is found in both cytosol and nucleus) and melanoma cells (where it is primarily localized to cytosol). In addition to having high AC levels, melanoma cells generate lower amounts of ceramides than normal melanocytes do. This down-regulation in ceramide production appears to result from suppression of the de novo biosynthesis pathway. To test whether AC might contribute to melanoma cell proliferation, we blocked AC activity using a new potent (IC50 = 12 nM) and stable inhibitor. AC inhibition increased cellular ceramide levels, decreased sphingosine 1-phosphate levels, and acted synergistically with several, albeit not all, antitumoral agents. The results suggest that AC-controlled sphingolipid metabolism may play an important role in the control of melanoma proliferation.

Topics: Acid Ceramidase; Cell Line, Tumor; Cell Proliferation; Cell Survival; Ceramides; Down-Regulation; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation, Neoplastic; HCT116 Cells; Hep G2 Cells; Humans; Inhibitory Concentration 50; Keratinocytes; Lipids; Lysophospholipids; MCF-7 Cells; Melanocytes; Melanoma; Microscopy, Confocal; Microscopy, Fluorescence; Oxidoreductases; RNA, Small Interfering; Serine C-Palmitoyltransferase; Signal Transduction; Skin Neoplasms; Sphingolipids; Sphingosine; Uracil

Despite progress in the understanding of the biology and genetics of melanoma, no effective treatment against this cancer is available. The adjacent microenvironment has an important role in melanoma progression. Defining the molecular signals that control the bidirectional dialog between malignant cells and the surrounding stroma is crucial for efficient targeted therapy. Our study aimed at defining the role of sphingosine-1-phosphate (S1P) in melanoma-stroma interactions. Transcriptomic analysis of human melanoma cell lines showed increased expression of sphingosine kinase-1 (SPHK1), the enzyme that produces S1P, as compared with normal melanocytes. Such an increase was also observed by immunohistochemistry in melanoma specimens as compared with nevi, and occurred downstream of ERK activation because of BRAF or NRAS mutations. Importantly, migration of melanoma cells was not affected by changes in SPHK1 activity in tumor cells, but was stimulated by comparable modifications of S1P-metabolizing enzymes in cocultured dermal fibroblasts. Reciprocally, incubation of fibroblasts with the conditioned medium from SPHK1-expressing melanoma cells resulted in their differentiation to myofibroblasts, increased production of matrix metalloproteinases and enhanced SPHK1 expression and activity. In vivo tumorigenesis experiments showed that the lack of S1P in the microenvironment prevented the development of orthotopically injected melanoma cells. Finally, local tumor growth and dissemination were enhanced more efficiently by coinjection of wild-type skin fibroblasts than by fibroblasts from Sphk1(-/-) mice. This report is the first to document that SPHK1/S1P modulates the communication between melanoma cells and dermal fibroblasts. Altogether, our findings highlight SPHK1 as a potential therapeutic target in melanoma progression.

Topics: Aldehyde-Lyases; Animals; Cell Communication; Cell Differentiation; Cell Movement; Cell Transformation, Neoplastic; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Gene Expression Profiling; GTP Phosphohydrolases; HEK293 Cells; Humans; Lysophospholipids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanoma; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myofibroblasts; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins B-raf; RNA Interference; RNA, Messenger; RNA, Small Interfering; Skin Neoplasms; Sphingosine

Resistance to therapies develops rapidly for melanoma leading to more aggressive disease. Therefore, agents are needed that specifically inhibit proteins or pathways controlling the development of this disease, which can be combined, dependent on genes deregulated in a particular patient's tumors. This study shows that elevated sphingosine-1-phosphate (S-1-P) levels resulting from increased activity of sphingosine kinase-1 (SPHK1) occur in advanced melanomas. Targeting SPHK1 using siRNA decreased anchorage-dependent and -independent growth as well as sensitized melanoma cells to apoptosis-inducing agents. Pharmacological SPHK1 inhibitors SKI-I but not SKI-II decreased S-1-P content, elevated ceramide levels, caused a G2-M block and induced apoptotic cell death in melanomas. Targeting SPHK1 using siRNA or the pharmacological agent called SKI-I decreased the levels of pAKT. Furthermore, SKI-I inhibited the expression of CYCLIN D1 protein and increased the activity of caspase-3/7, which in turn led to the degradation of PARP. In animals, SKI-I but not SKI-II retarded melanoma growth by 25-40%. Thus, targeting SPHK1 using siRNAs or SKI-I has therapeutic potential for melanoma treatment either alone or in combination with other targeted agents.

Topics: Animals; Apoptosis; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Down-Regulation; Fibroblasts; G1 Phase Cell Cycle Checkpoints; Humans; Lysophospholipids; Melanocytes; Melanoma; Mice; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Resting Phase, Cell Cycle; RNA, Small Interfering; Skin Neoplasms; Sphingosine; Staurosporine; Thiazoles; Up-Regulation; Xenograft Model Antitumor Assays

Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti-cancer treatment have been sought from natural resources. Here, we have investigated anti-proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the de novo sphingolipid pathway, and its mechanism in B16F10 melanoma cells.. We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine-1-phosphate levels were analysed by HPLC.. Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G(2) /M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21(waf1/cip1) was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine-1-phosphate in myriocin-treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells.. Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21(waf1/cip1) , followed by inhibition of cyclin B1 and cdc2, resulting in G(2) /M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism-based therapy for this type of skin cancer.

Topics: Animals; Antineoplastic Agents; CDC2 Protein Kinase; cdc25 Phosphatases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Ceramides; Cyclin B1; Fatty Acids, Monounsaturated; Gene Expression Regulation, Neoplastic; Lysophospholipids; Melanoma, Experimental; Mice; Proto-Oncogene Proteins p21(ras); Serine C-Palmitoyltransferase; Skin Neoplasms; Sphingomyelins; Sphingosine; Tumor Suppressor Protein p53