Sphingosine 1-phosphate receptors and sphingosine kinase 1 : novel biomarkers for clinical prognosis in breast, prostate, and hematological cancers

There is substantial evidence for a role in cancer of the bioactive lipid sphingosine 1-phosphate (S1P), the enzyme sphingosine kinase 1 (that catalyses S1P formation) and S1P-specific G protein-coupled receptors. This perspective highlights recent findings demonstrating that sphingosine kinase 1 and S1P receptors are new important biomarkers for detection of early cancer and progression to aggressive cancer. The impact of the sub-cellular distribution of S1P metabolizing enzymes and S1P receptors and their spatial functional interaction with oncogenes is considered with respect to prognostic outcome. These findings suggest that S1P, in addition to being a biomarker of clinical prognosis, might also be a new therapeutic target for intervention in cancer

New FTY720-docetaxel nanoparticle therapy overcomes FTY720-induced lymphopenia and inhibits metastatic breast tumour growth

Purpose: Combining molecular therapies with chemotherapy may offer an improved clinical outcome for chemoresistant tumours. Sphingosine-1-phosphate (S1P) receptor antagonist and sphingosine kinase 1 (SK1) inhibitor FTY720 (FTY) has promising anticancer properties, however, it causes systemic lymphopenia which impairs its use in cancer patients. In this study, we developed a nanoparticle (NP) combining docetaxel (DTX) and FTY for enhanced anticancer effect, targeted tumour delivery and reduced systemic toxicity. Methods: Docetaxel, FTY and glucosamine were covalently conjugated to poly(lactic-co-glycolic acid) (PLGA). NPs were characterised by dynamic light scattering and electron microscopy. The cellular uptake, cytotoxicity and in vivo antitumor efficacy of CNPs were evaluated. Results: We show for the first time that in triple negative breast cancer cells FTY provides chemosensitisation to DTX, allowing a four-fold reduction in the effective dose. We have encapsulated both drugs in PLGA complex NPs (CNPs), with narrow size distribution of ~ 100 nm and excellent cancer cell uptake providing sequential, sustained release of FTY and DTX. In triple negative breast cancer cells and mouse breast cancer models, CNPs had similar efficacy to systemic free therapies, but allowed an effective drug dose reduction. Application of CNPs has significantly reversed chemotherapy side effects such as weight loss, liver toxicity and, most notably, lymphopenia. Conclusions: We show for the first time the DTX chemosensitising effects of FTY in triple negative breast cancer. We further demonstrate that encapsulation of free drugs in CNPs can improve targeting, provide low off-target toxicity and most importantly reduce FTY-induced lymphopenia, offering potential therapeutic use of FTY in clinical cancer treatment

Sphingosine 1-phosphate and cancer

bioactive lipid, sphingosine 1-phosphate (S1P) is produced by phosphorylation of sphingosine and this is catalysed by two sphingosine kinase isoforms (SK1 and SK2). Here we discuss structural functional aspects of SK1 (which is a dimeric quaternary enzyme) that relate to coordinated coupling of membrane association with phosphorylation of Ser225 in the ‘so-called’ R-loop, catalytic activity and protein-protein interactions (e.g. TRAF2, PP2A and Gq). S1P formed by SK1 at the plasma-membrane is released from cells via S1P transporters to act on S1P receptors to promote tumorigenesis. We discuss here an additional novel mechanism that can operate between cancer cells and fibroblasts and which involves the release of the S1P receptor, S1P2 in exosomes from breast cancer cells that regulates ERK-1/2 signalling in fibroblasts. This novel mechanism of signalling might provide an explanation for the role of S1P2 in promoting metastasis of cancer cells and which is dependent on the micro-environmental niche

Aberrant expression of the S1P regulating enzymes, SPHK1 and SGPL1, contributes to a migratory phenotype in OSCC mediated through S1PR2.

Oral squamous cell carcinoma (OSCC) is a lethal disease with a 5-year mortality rate of around 50%. Molecular targeted therapies are not in routine use and novel therapeutic targets are required. Our previous microarray data indicated sphingosine 1-phosphate (S1P) metabolism and signalling was deregulated in OSCC. In this study, we have investigated the contribution of S1P signalling to the pathogenesis of OSCC. We show that the expression of the two major enzymes that regulate S1P levels were altered in OSCC: SPHK1 was significantly upregulated in OSCC tissues compared to normal oral mucosa and low levels of SGPL1 mRNA correlated with a worse overall survival. In in vitro studies, S1P enhanced the migration/invasion of OSCC cells and attenuated cisplatin-induced death. We also demonstrate that S1P receptor expression is deregulated in primary OSCCs and that S1PR2 is over-expressed in a subset of tumours, which in part mediates S1P-induced migration of OSCC cells. Lastly, we demonstrate that FTY720 induced significantly more apoptosis in OSCC cells compared to non-malignant cells and that FTY720 acted synergistically with cisplatin to induce cell death. Taken together, our data show that S1P signalling promotes tumour aggressiveness in OSCC and identify S1P signalling as a potential therapeutic target.This article is freely available via Open Access. Click on the 'Additional Link' above to access the full-text via the publisher's site.Published (Open Access

Identification of novel biomarkers for clinical prognosis in breast cancer

Sphingosine kinase/sphingosine 1-phosphate (SK/S1P) signalling interacts with major cellular pathways controlling cell proliferation, migration, survival, and resistance to chemotherapeutics. Moreover, extensive research has shown that the SK/S1P signalling is up-regulated in numerous human cancers (e.g. stomach cancer, colon, rectal, glioblastoma, ovarian, renal, lung and breast) making S1P signalling an important candidate as a biomarker and a key player in promoting cancer progression. Several inhibitors of SK/S1P signalling pathway have been identified and have shown to inhibit cancer cell survival and resistance to chemo- and radio-therapies. In this study, human breast cancer tissue microarrays at various tissue histological grades of ERα negative breast tumours were analysed for the expression of S1P signalling proteins (e.g. SKs and S1P₁₋₅ receptors) to identify the impact of expression level of these proteins on clinical outcomes. High SK1 and S1P₄ receptor tumour expression is associated with poor cancer prognosis in ERα negative breast cancer patients. Moreover, high SK1 and S1P₄ receptor expression in these tumours was also associated with cancer recurrence and this was dependent on the HER2 receptor expression. Indeed, the SK1/2 dual inhibitor SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) abrogated the S1Pstimulated ERK-1/2 activation in ERα-/HER2+ MDA-MB-453 cells suggesting that SK1 activity is required for the S1P₄/HER2-mediated ERK-1/2 activation that is known to promote cancer progression. In addition, a diverse array of kinases and transcription factors e.g. c-RAF-1, ERK-1/2, AKT, LYN, SRC family kinases (SFKs) and NFκB (p50 RelA) were analysed in combination with SK1 and S1P receptors to discover novel prognostic interactions that drive cancer progression in ERα positive breast cancer patients. High tumour SK1 expression in combination with high expression of either S1P₁ receptor or S1P₃ receptor or phosphorylated ERK-1/2 or phosphorylated AKT or phosphorylated NFκB or phosphorylated RAF-1 or Y416 phosphorylated SFK or LYN is associated with shorter disease-specific patient survival and disease-free cancer recurrence. Similarly, high S1P₁ receptor tumour expression in combination with high expression of either Y216 phosphorylated SRC or c-RAF-1 or ERK-1/2 or AKT kinase is associated with shorter disease-specific patient survival and disease-free cancer recurrence. High S1P₂ receptor tumour expression is associated with prolonged patient survival and this is enhanced in combination with high expression of c-SRC and Y416 phosphorylated SFK in ERα positive breast cancer tumours. Finally, high tumour S1P3 receptor expression in combination with high expression of LYN or c-RAF-1 kinases is associated with shorter disease-specific patient survival and disease-free cancer recurrence. Lastly, a new signalling pathway involving SK2, Y416 phosphorylated c-SRC, S1P₄ receptor and S1P₂ receptor was identified using pharmacological agents/gene silencing in ERα negative MDA-MB-231 breast cancer cells. In this pathway, SK2 possibly through 'inside out' S1P signalling activates the S1P₄ receptor that promotes cellular growth by preventing the nuclear accumulation of S1P₂ receptor. Moreover, SK2 activity also prevents the accumulation of Y416 phosphorylated c-SRC into the nucleus that might be crucial for tumour growth. Thus, this study shows that the high tumour expression of S1P signalling proteins is associated with poor disease prognosis in both ERα positive and ERα negative breast cancer patients. However, cancer progression is mediated by distinct set of S1P signalling proteins in different types of breast cancer. Hence, different treatment regiments including SK inhibitors and S1P receptor antagonist must be employed in treatment of ERα positive and ERα negative breast cancer patients.Sphingosine kinase/sphingosine 1-phosphate (SK/S1P) signalling interacts with major cellular pathways controlling cell proliferation, migration, survival, and resistance to chemotherapeutics. Moreover, extensive research has shown that the SK/S1P signalling is up-regulated in numerous human cancers (e.g. stomach cancer, colon, rectal, glioblastoma, ovarian, renal, lung and breast) making S1P signalling an important candidate as a biomarker and a key player in promoting cancer progression. Several inhibitors of SK/S1P signalling pathway have been identified and have shown to inhibit cancer cell survival and resistance to chemo- and radio-therapies. In this study, human breast cancer tissue microarrays at various tissue histological grades of ERα negative breast tumours were analysed for the expression of S1P signalling proteins (e.g. SKs and S1P₁₋₅ receptors) to identify the impact of expression level of these proteins on clinical outcomes. High SK1 and S1P₄ receptor tumour expression is associated with poor cancer prognosis in ERα negative breast cancer patients. Moreover, high SK1 and S1P₄ receptor expression in these tumours was also associated with cancer recurrence and this was dependent on the HER2 receptor expression. Indeed, the SK1/2 dual inhibitor SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) abrogated the S1Pstimulated ERK-1/2 activation in ERα-/HER2+ MDA-MB-453 cells suggesting that SK1 activity is required for the S1P₄/HER2-mediated ERK-1/2 activation that is known to promote cancer progression. In addition, a diverse array of kinases and transcription factors e.g. c-RAF-1, ERK-1/2, AKT, LYN, SRC family kinases (SFKs) and NFκB (p50 RelA) were analysed in combination with SK1 and S1P receptors to discover novel prognostic interactions that drive cancer progression in ERα positive breast cancer patients. High tumour SK1 expression in combination with high expression of either S1P₁ receptor or S1P₃ receptor or phosphorylated ERK-1/2 or phosphorylated AKT or phosphorylated NFκB or phosphorylated RAF-1 or Y416 phosphorylated SFK or LYN is associated with shorter disease-specific patient survival and disease-free cancer recurrence. Similarly, high S1P₁ receptor tumour expression in combination with high expression of either Y216 phosphorylated SRC or c-RAF-1 or ERK-1/2 or AKT kinase is associated with shorter disease-specific patient survival and disease-free cancer recurrence. High S1P₂ receptor tumour expression is associated with prolonged patient survival and this is enhanced in combination with high expression of c-SRC and Y416 phosphorylated SFK in ERα positive breast cancer tumours. Finally, high tumour S1P3 receptor expression in combination with high expression of LYN or c-RAF-1 kinases is associated with shorter disease-specific patient survival and disease-free cancer recurrence. Lastly, a new signalling pathway involving SK2, Y416 phosphorylated c-SRC, S1P₄ receptor and S1P₂ receptor was identified using pharmacological agents/gene silencing in ERα negative MDA-MB-231 breast cancer cells. In this pathway, SK2 possibly through 'inside out' S1P signalling activates the S1P₄ receptor that promotes cellular growth by preventing the nuclear accumulation of S1P₂ receptor. Moreover, SK2 activity also prevents the accumulation of Y416 phosphorylated c-SRC into the nucleus that might be crucial for tumour growth. Thus, this study shows that the high tumour expression of S1P signalling proteins is associated with poor disease prognosis in both ERα positive and ERα negative breast cancer patients. However, cancer progression is mediated by distinct set of S1P signalling proteins in different types of breast cancer. Hence, different treatment regiments including SK inhibitors and S1P receptor antagonist must be employed in treatment of ERα positive and ERα negative breast cancer patients

The role of sphingosine 1-phosphate in inflammation and cancer

The enzymes that catalyze formation of the bioactive sphingolipid, sphingosine 1-phosphate, sphingosine kinase 1 and 2, are predictive markers in inflammatory diseases and cancer as evidenced by data from patients, knockout mice and the use of available molecular and chemical inhibitors. Thus, there is a compelling case for therapeutic targeting of sphingosine kinase. In addition, there are several examples of functional interaction between sphingosine 1-phosphate receptors and sphingosine kinase 1 that can drive malicious amplification loops that promote cancer cell growth. These novel aspects of sphingosine 1-phosphate pathobiology are reviewed herein

Sphingosine kinase 2 prevents the nuclear translocation of sphingosine 1-phosphate receptor-2 and tyrosine 416 phosphorylated c-Src and increases estrogen receptor negative MDA-MB-231 breast cancer cell growth : the role of sphingosine 1-phosphate receptor-4

We demonstrate that pre-treatment of estrogen receptor negative MDA-MB-231 breast cancer cells containing ectopically expressed HA-tagged sphingosine 1-phosphate receptor-2 (S1P2) with the sphingosine kinase 1/2 inhibitor SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) or the sphingosine kinase 2 selective inhibitor (R)-FTY720 methyl ether (ROMe) or sphingosine kinase 2 siRNA induced the translocation of HA-tagged S1P2 and Y416 phosphorylated c-Src to the nucleus of these cells. This is associated with reduced growth of HA-tagged S1P2 over-expressing MDA-MB-231 cells. Treatment of HA-S1P2 over-expressing MDA-MB-231 cells with the sphingosine 1-phosphate receptor-4 (S1P4) antagonist CYM50367 or with S1P4 siRNA also promoted nuclear translocation of HA-tagged S1P2. These findings identify for the first time a signaling pathway in which sphingosine 1-phosphate formed by sphingosine kinase 2 binds to S1P4 to prevent nuclear translocation of S1P2 and thereby promote the growth of estrogen receptor negative breast cancer cells

Expression of sphingosine 1-phosphate receptor 4 and sphingosine kinase 1 is associated with outcome in oestrogen receptor-negative breast cancer

BACKGROUND: We previously reported that sphingosine 1-phosphate receptor 4 (S1P(4)) is expressed and stimulates the ERK-1/2 pathway via a human epidermal growth factor receptor 2 (HER2)-dependent mechanism in oestrogen receptor-negative (ER(-)) MDA-MB-453 breast cancer cells. METHODS: Clinical relevance of S1P(4) and sphingosine kinase 1 (SK1, which catalyses the formation of S1P) was assessed in a cohort of 140 ER(-) breast tumours by immunohistochemistry (IHC) and the weighted histoscore method. Additional evidence for a functional interaction between S1P(4) and SK1 and between HER2 and SK1 was obtained using MDA-MB-453 cells. RESULTS: High S1P(4) expression is associated with shorter disease-free (P=0.014) and disease-specific survival (P=0.004), and was independent on multivariate analysis. In addition, patients with tumours that contain high and low levels of SK1 and S1P(4), respectively, have a significantly shorter disease-free survival (P=0.043) and disease-specific survival (P=0.033) compared with patients whose tumours contain both low S1P(4) and SK1 levels. In addition, high tumour expression of SK1 was significantly associated with shorter disease-specific survival (P=0.0001) in patients with HER2-positive tumours. Treatment of MDA-MB-453 cells with the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) reduced the basal and S1P/S1P(4)-induced activation of ERK-1/2 and altered HER2 trafficking in these cells. CONCLUSION: These findings highlight an important role for S1P(4) and SK1 in ER(-) breast cancer progression