sphingosine-1-phosphate and Breast-Neoplasms

Triple-negative breast cancer (TNBC) is very aggressive with high metastatic and mortality rates and unfortunately, except for chemotherapy, there are few therapeutic options. The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) regulates numerous processes important for cancer progression, metastasis, and neuropathic pain. The pro-drug FTY720 (fingolimod, Gilenya) used to treat multiple sclerosis is phosphorylated in the body to a S1P mimic that binds to S1PRs, except S1PR2, and also acts as a functional antagonist of S1PR1. This review highlights current findings showing that FTY720 has multiple anti-cancer activities and simultaneously prevents formation and actions of S1P. Moreover, in mouse breast cancer models, treatment with FTY720 reduces tumor growth, metastasis, and enhances sensitivity of advanced and hormonal refractory breast cancer and TNBC to conventional therapies. We discuss recent studies demonstrating that neuropathic pain induced by the chemotherapeutic bortezomib is also greatly reduced by administration of clinically relevant doses of FTY720, likely by targeting S1PR1 on astrocytes. FTY720 also shows promising anticancer potential in pre-clinical studies and is FDA approved, thus we suggest in this review that further studies are needed to pave the way for fast-tracking approval of FTY720/fingolimod for enhancing chemotherapy effectiveness and reduction of painful neuropathies.

Topics: Bortezomib; Breast Neoplasms; Chemotherapy, Adjuvant; Female; Fingolimod Hydrochloride; Humans; Lysophospholipids; Neuralgia; Signal Transduction; Sphingosine; Triple Negative Breast Neoplasms

Breast cancer affects 1 out of 8 women in the US and is the second highest cause of death from cancer for women, leading to considerable research examining the causes, progression, and treatment of breast cancer. Over the last two decades, sphingosine-1-phosphate (S1P), a potent sphingolipid metabolite, has been implicated in many processes important for breast cancer including growth, progression, transformation and metastasis, and is the focus of this review. In particular, one of the kinases that produces S1P, sphingosine kinase 1 (SphK1), has come under increasing scrutiny as it is commonly upregulated in breast cancer cells and has been linked with poorer prognosis and progression, possibly leading to resistance to certain anti-cancer therapies. In this review, we will also discuss preclinical studies of both estrogen receptor (ER) positive as well as triple-negative breast cancer mouse models with inhibitors of SphK1 and other compounds that target the S1P axis and have shown good promise in reducing tumor growth and metastasis. It is hoped that in the future this will lead to development of novel combination approaches for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer.

Topics: Breast Neoplasms; Female; Humans; Lysophospholipids; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); Receptors, Estrogen; Sphingosine; United States

Breast cancer metastasizes to lymph nodes or other organs, which determine the prognosis of patients. It is difficult to cure the breast cancer patients with distant metastasis due to resistance to drug therapies. Elucidating the underlying mechanisms of breast cancer metastasis and drug resistance is expected to provide new therapeutic targets. Sphingosine-1-phosphate (S1P) is a pleiotropic, bioactive lipid mediator that regulates many cellular functions, including proliferation, migration, survival, angiogenesis/lymphangiogenesis, and immune responses. S1P is formed in cells by sphingosine kinases and released from them, which acts in an autocrine, paracrine, and/or endocrine manner. S1P in extracellular space, such as interstitial fluid, interacts with components in the tumor microenvironment, which may be important for metastasis. Importantly, recent translational research has demonstrated an association between S1P levels in breast cancer patients and clinical outcomes, highlighting the clinical importance of S1P in breast cancer. We suggest that S1P is one of the key molecules to overcome the resistance to the drug therapies, such as hormonal therapy, anti-HER2 therapy, or chemotherapy, all of which are crucial aspects of a breast cancer treatment.

Topics: Animals; Breast Neoplasms; Humans; Lymphangiogenesis; Lysophospholipids; Models, Biological; Neoplasm Metastasis; Sphingosine

Breast cancer remains the most common malignant disease in women. The estrogen receptor-α (ERα) and its ligand 17β-estradiol (E2) play important roles in breast cancer. E2 elicits cellular effects by binding to ERα in the cytosol followed by receptor dimerization and translocation to the nucleus where it regulates gene expression by binding to ERE response elements. However, it has become apparent that E2 also exerts rapid non-genomic effects through membrane-associated receptors. There is emerging evidence that this induces formation of the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P). S1P in turn has been implicated in many processes important in breast cancer progression. One of the enzymes that produce S1P, sphingosine kinase 1 (SphK1), is upregulated in breast cancer and its expression has been correlated with poor prognosis. This review is focused on the role of the SphK/S1P axis in estrogen signaling and breast cancer progression and will discuss new therapeutic approaches targeting this axis for breast cancer treatment.

Topics: Animals; Breast Neoplasms; Estrogens; Female; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Tamoxifen, a triphenylethylene antiestrogen and one of the first-line endocrine therapies used to treat estrogen receptor-positive breast cancer, has a number of interesting, off-target effects, and among these is the inhibition of sphingolipid metabolism. More specifically, tamoxifen inhibits ceramide glycosylation, and enzymatic step that can adventitiously support the influential tumor-suppressor properties of ceramide, the aliphatic backbone of sphingolipids. Additionally, tamoxifen and metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, have been shown to inhibit ceramide hydrolysis by the enzyme acid ceramidase. This particular intervention slows ceramide destruction and thereby depresses formation of sphingosine 1-phosphate, a mitogenic sphingolipid with cancer growth-promoting properties. As ceramide-centric therapies are becoming appealing clinical interventions in the treatment of cancer, agents like tamoxifen that can retard the generation of mitogenic sphingolipids and buffer ceramide clearance via inhibition of glycosylation, take on new importance. In this review, we present an abridged, lay introduction to sphingolipid metabolism, briefly chronicle tamoxifen's history in the clinic, examine studies that demonstrate the impact of triphenylethylenes on sphingolipid metabolism in cancer cells, and canvass works relevant to the use of tamoxifen as adjuvant to drive ceramide-centric therapies in cancer treatment. The objective is to inform the readership of what could be a novel, off-label indication of tamoxifen and structurally-related triphenylethylenes, an indication divorced from estrogen receptor status and one with application in drug resistance.

Topics: Acid Ceramidase; Antineoplastic Agents, Hormonal; Biotransformation; Breast Neoplasms; Ceramides; Drug Resistance, Neoplasm; Female; Humans; Hydrolysis; Lipid Metabolism; Lysophospholipids; Sphingosine; Tamoxifen

Sphingosine-1-phosphate (S1P) is a potent sphingolipid metabolite that regulates a number of biological processes critical for cancer. S1P produced inside cancer cells is exported and exerts its extracellular functions by binding to its specific receptors in an autocrine, paracrine, and/or endocrine manner, which is known as inside-out signaling. S1P is also known to exert its intracellular functions especially in the inflammatory process, but its relevance to cancer biology remains to be elucidated. Recently, there have been growing interests in the role of S1P in breast cancer progression, including angiogenesis and lymphangiogenesis. Our group demonstrated that activation of sphingosine kinase 1, the enzyme that catalyzes the phosphorylation of sphingosine to S1P, is a key step of this process. In this review, we will cover our current knowledge on the role of S1P signaling pathway in breast cancer progression with an emphasis on its role in tumor-induced lymphangiogenesis.

Topics: Breast Neoplasms; Female; Humans; Lymph Nodes; Lymphangiogenesis; Lymphatic Metastasis; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has been implicated in many biological processes, including cell migration, survival, proliferation, angiogenesis and immune and allergic responses. S1P levels inside cells are regulated tightly by the balance between its synthesis by sphingosine kinases and degradation by S1P lyases and S1P phosphatases. Activation of sphingosine kinase by any of a variety of agonists increases S1P levels, which in turn can function intracellularly as a second messenger or in an autocrine and/or paracrine fashion to activate and signal through S1P receptors present on the surface of the cell. This review summarizes recent findings on the roles of S1P as a mediator of the actions of cytokines, growth factors and hormones.

Topics: Animals; Autocrine Communication; Breast Neoplasms; Gonadal Steroid Hormones; Humans; Inflammation Mediators; Intercellular Signaling Peptides and Proteins; Lysophospholipids; Models, Biological; Neovascularization, Pathologic; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

This review will focus on the role of sphingosine and its phosphorylated derivative sphingosine-1-phosphate (SPP) in cell growth regulation and signal transduction. We will show that many of the effects attributed to sphingosine in quiescent Swiss 3T3 fibroblasts are mediated via its conversion to SPP. We propose that SPP has appropriate properties to function as an intracellular second messenger based on the following: it elicits diverse cellular responses; it is rapidly produced from sphingosine by a specific kinase and rapidly degraded by a specific lyase; its concentration is low in quiescent cells but increases rapidly and transiently in response to the growth factors, fetal calf serum (FCS) and platelet derived growth factor (PDGF); it releases Ca2+ from internal sources in an InsP3-independent manner; and finally, it may link sphingolipid signaling pathways to cellular ras-mediated signaling pathways by elevating phosphatidic acid levels. The effects of this novel second messenger on growth, differentiation and invasion of human breast cancer cells will be discussed.

Topics: 3T3 Cells; Animals; Blood Physiological Phenomena; Breast Neoplasms; Calcium; Cattle; Cell Division; Cell Movement; Growth Substances; Humans; Lysophospholipids; Mice; Models, Biological; Neoplasm Invasiveness; Phosphatidic Acids; Platelet-Derived Growth Factor; Second Messenger Systems; Signal Transduction; Sphingosine; Tumor Cells, Cultured

Although numerous experiments revealed an essential role of a lipid mediator, sphingosine-1-phosphate (S1P), in breast cancer (BC) progression, the clinical significance of S1P remains unclear due to the difficulty of measuring lipids in patients. The aim of this study was to determine the plasma concentration of S1P in estrogen receptor (ER)-positive BC patients, as well as to investigate its clinical significance. We further explored the possibility of a treatment strategy targeting S1P in ER-positive BC patients by examining the effect of FTY720, a functional antagonist of S1P receptors, on hormone therapy-resistant cells. Plasma S1P levels were significantly higher in patients negative for progesterone receptor (PgR) expression than in those positive for expression (

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Disease Progression; Female; Fingolimod Hydrochloride; Gene Expression; Humans; Lymphatic Metastasis; Lysophospholipids; MCF-7 Cells; Middle Aged; Plasma; Receptors, Estrogen; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Breast cancer MCF-7 cell-line-derived mammospheres were shown to be enriched in cells with a CD44+/CD24- surface profile, consistent with breast cancer stem cells (BCSC). These BCSC were previously reported to express key sphingolipid signaling effectors, including pro-oncogenic sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1P3). In this study, we explored intracellular trafficking and localization of SphK1 and S1P3 in parental MCF-7 cells, and MCF-7 derived BCSC-enriched mammospheres treated with growth- or apoptosis-stimulating agents. Intracellular trafficking and localization were assessed using confocal microscopy and cell fractionation, while CD44+/CD24- marker status was confirmed by flow cytometry. Mammospheres expressed significantly higher levels of S1P3 compared to parental MCF-7 cells (

Topics: Breast Neoplasms; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; MCF-7 Cells; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Tumor Necrosis Factor-alpha

The interleukin-22 (IL-22) signaling pathway is well known to be involved in the progression of various cancer types but its role in bone metastatic breast cancer remains unclear. We demonstrate using human GEO profiling that bone metastatic breast cancer displays elevated interleukin-22 receptor 1 (IL-22R1) and sphingosine-1-phosphate receptor 1 (S1PR1) expression. Importantly, IL-22 stimuli promoted the expression of IL-22R1 and S1PR1 in aggressive MDA-MB-231 breast cancer cells. IL-22 treatment also increased sphingosine-1-phosphate production in mesenchymal stem cells (MSCs) and induced the sphingosine-1-phosphate (S1P)-mediated chemotactic migration of MDA-MB-231 cells. This effect was inhibited by an S1P antagonist. In addition to the S1PR1 axis, IL-22 stimulated the expression of matrix metalloproteinase-9 (MMP-9), thereby promoting breast cancer cell invasion. Moreover, IL-22 induced IL22R1 and S1PR1 expression in macrophages, myeloid cell, and MCP1 expression in MSCs to facilitate macrophage infiltration. Immunohistochemistry indicated that IL-22R1 and S1PR1 are overexpressed in invasive malignant breast cancers and that this correlates with the MMP-9 levels. Collectively, our present results indicate a potential role of IL-22 in driving the metastasis of breast cancers into the bone microenvironment through the IL22R1-S1PR1 axis.

Topics: Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Chemokine CCL2; Chemotaxis; Female; Humans; Interleukin-22; Interleukins; Lysophospholipids; Macrophages; Matrix Metalloproteinase 9; Mesenchymal Stem Cells; Neoplasm Invasiveness; Prognosis; Receptors, Interleukin; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Tumor Microenvironment

Cancer stem cells (CSCs) represent rare tumor cell populations capable of self-renewal, differentiation, and tumor initiation and are highly resistant to chemotherapy and radiotherapy. Thus, therapeutic approaches that can effectively target CSCs and tumor cells could be the key to efficient tumor treatment. In this study, we explored the function of SPHK1 in breast CSCs and non-CSCs. We showed that RNAi-mediated knockdown of SPHK1 inhibited cell proliferation and induced apoptosis in both breast CSCs and non-CSCs, while ectopic expression of SPHK1 enhanced breast CSC survival and mammosphere forming efficiency. We identified STAT1 and IFN signaling as key regulatory targets of SPHK1 and demonstrated that an important mechanism by which SPHK1 promotes cancer cell survival is through the suppression of STAT1. We further demonstrated that SPHK1 inhibitors, FTY720 and PF543, synergized with doxorubicin in targeting both breast CSCs and non-CSCs. In conclusion, we provide important evidence that SPHK1 is a key regulator of cell survival and proliferation in breast CSCs and non-CSCs and is an attractive target for the design of future therapies.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Lineage; Cell Proliferation; Cell Survival; Doxorubicin; Female; HEK293 Cells; Humans; Interferons; Lysophospholipids; Neoplastic Stem Cells; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Proteomics; Signal Transduction; Spheroids, Cellular; Sphingosine; STAT1 Transcription Factor

The metabolic network of sphingolipids plays important roles in cancer biology. Prominent sphingolipids include ceramides and sphingosine-1-phosphate that regulate multiple aspects of growth, apoptosis, and cellular signaling. Although a significant number of enzymatic regulators of the sphingolipid pathway have been described in detail, many remained poorly characterized. Here we applied a patient-derived systemic approach to identify and molecularly define progestin and adipoQ receptor family member IV (PAQR4) as a Golgi-localized ceramidase. PAQR4 was approximately 5-fold upregulated in breast cancer compared with matched control tissue and its overexpression correlated with disease-specific survival rates in breast cancer. Induction of PAQR4 in breast tumors was found to be subtype-independent and correlated with increased ceramidase activity. These findings establish PAQR4 as Golgi-localized ceramidase required for cellular growth in breast cancer. SIGNIFICANCE: Induction of and cellular dependency on

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Ceramidases; Female; Golgi Apparatus; Heterografts; Humans; Lysophospholipids; MCF-7 Cells; Membrane Proteins; Mice, Inbred NOD; Mice, SCID; Sphingosine; Up-Regulation

Although previous experiments have implicated sphingosine-1-phosphate (S1P) as a links between immune reactions and cancer progression, the exact mechanism of this interaction has not comprehensively studied in clinical human samples. This study sought to evaluate the S1P regulation by sphingosine kinase 1 (SPHK1), an S1P-producing enzyme, in the immunity/immuno-reactivity of clinical human breast cancer surgical specimens.. S1P levels were examined in tumor, peritumoral, and normal human breast samples using mass spectrometry. Genomics Data Commons data portal of The Cancer Genome Atlas cohort was used to assess the expression of S1P-related and immune-related genes.. S1P levels were significantly higher in tumor samples compared to peritumoral (P < 0.05) or normal human breast samples (P < 0.001). SPHK1 gene expression was elevated in tumoral samples compared to normal breast samples (P < 0.01). Furthermore, the elevated expression of SPHK1 in breast cancer tissue was associated with an increased expression of the different kinds of immune-related genes, such as CD68, CD163, CD4, and FOXP3 (forkhead box P3), in HER2-negative breast cancer. Network analysis showed the central role of SPHK1 in the interaction of S1P signaling and expression of immune cell-related proteins.. We demonstrated that S1P is mainly produced by tumor tissue, rather than peritumoral tissue, in breast cancer patients. Our data revealed the involvement of S1P signaling in the regulation of immune-related genes, suggesting the links between S1P and complicated immune-cancer interactions in breast cancer patients.

Topics: Breast; Breast Neoplasms; Cell Line, Tumor; Chromatography, High Pressure Liquid; Cohort Studies; Datasets as Topic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Interaction Maps; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Sphingosine; Tandem Mass Spectrometry

Angiogenesis is one of the hallmarks of cancer. We hypothesized that intra-tumoral angiogenesis correlates with inflammation and metastasis in breast cancer patients. To test this hypothesis, we generated an angiogenesis pathway score using gene set variation analysis and analyzed the tumor transcriptome of 3999 breast cancer patients from The Cancer Genome Atlas Breast Cancer (TCGA-BRCA), Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), GSE20194, GSE25066, GSE32646, and GSE2034 cohorts. We found that the score correlated with expression of various angiogenesis-, vascular stability-, and sphingosine-1-phosphate (S1P)-related genes. Surprisingly, the angiogenesis score was not associated with breast cancer subtype, Nottingham pathological grade, clinical stage, response to neoadjuvant chemotherapy, or patient survival. However, a high score was associated with a low fraction of both favorable and unfavorable immune cell infiltrations except for dendritic cell and M2 macrophage, and with Leukocyte Fraction, Tumor Infiltrating Lymphocyte Regional Fraction and Lymphocyte Infiltration Signature scores. High-score tumors had significant enrichment for unfavorable inflammation-related gene sets (interleukin (IL)6, and tumor necrosis factor (TNF)α- and TGFβ-signaling), as well as metastasis-related gene sets (epithelial mesenchymal transition, and Hedgehog-, Notch-, and WNT-signaling). High score was significantly associated with metastatic recurrence particularly to brain and bone. In conclusion, using the angiogenesis pathway score, we found that intra-tumoral angiogenesis is associated with immune reaction, inflammation and metastasis-related pathways, and metastatic recurrence in breast cancer.

Topics: Breast Neoplasms; Dendritic Cells; Epithelial-Mesenchymal Transition; Female; Humans; Inflammation; Interferon-gamma; Interleukin-6; Leukocytes; Lymphocytes, Tumor-Infiltrating; Lysophospholipids; Macrophages; Neoplasm Recurrence, Local; Neovascularization, Pathologic; Signal Transduction; Sphingosine; Transforming Growth Factor beta

Breast cancer is one of the most common female malignant cancers. Biorhythm disorder largely increases the risk of breast cancer. We aimed to investigate the biological functions and molecular mechanisms of circadian gene TIMELESS circadian regulator (TIM) in estrogen receptor (ER)-positive breast cancer and provide a new therapeutic target for breast cancer patients. Here, we explored that the expression of TIM was elevated in breast cancer, and high expression of TIM in cancer tissues was associated with poor prognosis, especially in the ER-positive breast cancer patients. In addition, we found that TIM promoted cell proliferation and enhanced mitochondrial respiration. TIM interacted with specificity protein 1 (Sp1) which contributes to upregulate the expression of alkaline ceramidase 2 (ACER2). Moreover, ACER2 is responsible for TIM-mediated promotive effects of cell growth and mitochondrial respiration. Collectively, our research unveiled a novel function of TIM in sphingolipid metabolism through interaction with Sp1. It provides a new theoretical explanation for the pathogenesis of breast cancer, and targeting TIM may serve as a potential therapeutic target for ER-positive breast cancer.

Topics: Alkaline Ceramidase; Animals; Biopsy; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Lysophospholipids; Mice; Mice, Nude; Mitochondria; Sp1 Transcription Factor; Sphingolipids; Sphingosine; Xenograft Model Antitumor Assays

Epithelial-mesenchymal transition (EMT) is a critical process implicated in the initial stage of cancer metastasis, which is the major cause of tumor recurrence and mortality. Although key transcription factors that regulate EMT, such as snail family transcriptional repressor 2 (SNAI2), are well characterized, the upstream signaling pathways controlling these transcriptional mediators are largely unknown, which limits therapeutic strategies. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator, generated by sphingosine kinases (SPHK1 and SPHK2), that mainly exerts its effects by binding to the following 5 GPCRs: S1P

Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lysophospholipids; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); RNA Interference; RNA Stability; RNA, Small Interfering; Snail Family Transcription Factors; Sphingosine; Sphingosine-1-Phosphate Receptors; Trans-Activators; Transcription Factors; YAP-Signaling Proteins

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.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Animals; Breast Neoplasms; Cell Line, Tumor; Culture Media, Conditioned; Cytokines; Diet, High-Fat; Disease Models, Animal; Female; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Inflammation; Interleukin-6; Lung; Lysophospholipids; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Obesity; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid mediator, has been implicated in regulation of many processes important for breast cancer progression. Previously, we observed that S1P is exported out of human breast cancer cells by ATP-binding cassette (ABC) transporter ABCC1, but not by ABCB1, both known multidrug resistance proteins that efflux chemotherapeutic agents. However, the pathologic consequences of these events to breast cancer progression and metastasis have not been elucidated. Here, it is demonstrated that high expression of ABCC1, but not ABCB1, is associated with poor prognosis in breast cancer patients. Overexpression of ABCC1, but not ABCB1, in human MCF7 and murine 4T1 breast cancer cells enhanced S1P secretion, proliferation, and migration of breast cancer cells. Implantation of breast cancer cells overexpressing ABCC1, but not ABCB1, into the mammary fat pad markedly enhanced tumor growth, angiogenesis, and lymphangiogenesis with a concomitant increase in lymph node and lung metastases as well as shorter survival of mice. Interestingly, S1P exported via ABCC1 from breast cancer cells upregulated transcription of sphingosine kinase 1 (SPHK1), thus promoting more S1P formation. Finally, patients with breast cancers that express both activated SPHK1 and ABCC1 have significantly shorter disease-free survival. These findings suggest that export of S1P via ABCC1 functions in a malicious feed-forward manner to amplify the S1P axis involved in breast cancer progression and metastasis, which has important implications for prognosis of breast cancer patients and for potential therapeutic targets.

Topics: Animals; Breast Neoplasms; Female; Humans; Lysophospholipids; Mice; Mice, Inbred BALB C; Mice, Nude; Multidrug Resistance-Associated Proteins; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Survival Analysis

Sphingosine-1-phosphate (S1P) is a bioactive lipid that exerts various pathophysiological functions through binding to its receptor family (S1PRs). Since first report of the breast cancer (BCA) promoting function by S1P production (through the function of sphingosine kinases) and S1P/S1PR signaling, their antagonists have never been successfully progress to clinics after three decades. Taking advantage of bioinformatics linking to gene expression to disease prognosis, we examined the impact of associated genes in BCA patients. We found high gene expressions involved in S1P anabolism suppressed disease progression of patients who are basal cell type BCA or receiving adjuvant therapy. In addition, S1PRs expression also suppressed disease progress of multiple categories of BCA patient progression. This result is contradictory to tumor promoter role of S1P/S1PRs which revealed in the literature. Further examination by directly adding S1P in BCA cells found a cell growth suppression function, which act via the expression of S1PR1. In conclusion, our study is the first evidence claiming a survival benefit function of S1P/S1PR signaling in BCA patients, which might explain the obstacle of relative antagonist apply in clinics.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Female; Humans; Kaplan-Meier Estimate; Lipid Metabolism; Lysophospholipids; Models, Biological; Prognosis; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors

Recently, sphingolipid derivatives, such as ceramide and sphingosine‑1‑phosphate (S1P), have emerged as key modulators in apoptotic cell death and cell proliferation. This study aimed to clarify the underlying signaling pathways of ceramide and S1P involved in breast cancer cell proliferation. Ceramide acyl chain length is determined by six mammalian ceramide synthases (CerS). We overexpressed CerS1 to 6 in MCF‑7 cells to examine whether ceramide signaling propagation varies as a function of acyl chain length. Among the six CerS, only CerS6 overexpression reduced phosphorylation of Akt, S6 kinase (S6K), and extracellular signal‑regulated kinases (ERK) as shown by western blotting. In addition, CerS6 overexpression reduced MCF‑7 cell proliferation. This effect was partially reversed by co‑treatment with MHY1485, an activator of mammalian target of rapamycin (mTOR), demonstrating an important role for the mTOR pathway in the CerS6‑mediated decrease in MCF‑7 cell proliferation. ERK inhibition, but not Akt inhibition, along with mTOR inhibition synergistically reduced MCF‑7 cell proliferation as measured by MTT assay. Notably, the expression of CerS6 and S1P receptor 2 (S1PR2), or CerS6 and sphingosine kinase 1 (SphK1), were negatively correlated according to the invasive breast carcinoma patient cohort in The Cancer Genome Atlas database. In addition, both SphK1 overexpression and S1P addition increased mTOR phosphorylation as shown by ELISA, while S1PR2 inhibition had the inverse effect. These data suggest that CerS6 and SphK1 regulate mTOR signaling in breast cancer cell proliferation. Moreover, mTOR activity can be regulated by the balance between S1P and C16‑ceramide, which is generated by CerS6.

Topics: Breast Neoplasms; Cell Proliferation; Ceramides; Female; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; MAP Kinase Signaling System; MCF-7 Cells; Membrane Proteins; Morpholines; Oncogene Protein v-akt; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine N-Acyltransferase; Sphingosine-1-Phosphate Receptors; TOR Serine-Threonine Kinases; Triazines

Sphingosine-1-phosphate (S1P) is a sphingolipid in plasma that plays a critical role in cardiovascular and immune systems. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. To test the hypothesis that S1P can reduce tumor cell adhesion to microvessel walls by protecting the ESG, we quantified the ESG and MDA-MB-231 tumor cell adhesion in the presence and absence of 1μM S1P, and in the presence of the matrix metalloproteinase (MMP) inhibitor in post-capillary venules of rat mesentery. We also measured the microvessel permeability to albumin as an indicator for the microvessel wall integrity. In the absence of S1P, ESG was ~10% of that in the presence of S1P, whereas adherent tumor cells and the permeability to albumin and were ~3.5-fold (after 30 min adhesion) and ~7.7-fold that in the presence of S1P, respectively. In the presence of the MMP inhibitor, the results are similar to those in the presence of S1P. Our results conform to the hypothesis that protecting ESG by S1P inhibits MDA-MB-231 tumor cell adhesion to the microvessel wall.

Topics: Animals; Blood Vessels; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Endothelial Cells; Female; Glycocalyx; Humans; Lysophospholipids; Microvessels; Rats; Sphingosine; Surface-Active Agents

Doxorubicin is one of the most commonly used chemotherapeutic drugs for breast cancer; however, its use is limited by drug resistance and side effects. We hypothesized that adding FTY720, a sphingosine-1-phosphate (S1P) receptor functional antagonist, to doxorubicin would potentiate its effects by suppression of drug-induced inflammation.. The Cancer Genome Atlas, Gene Expression Omnibus data sets, and National Cancer Institute-60 panel were used for gene expressions and gene set enrichment analysis. E0771 syngeneic mammary tumor cells were used. OB/OB mice fed with western high-fat diet were used as an obesity model.. STAT3 expression was significantly increased after doxorubicin treatment in human breast cancer that implicates that doxorubicin evokes inflammation. Expression of sphingosine kinase 1, the enzyme that produces S1P and links inflammation and cancer, tended to be higher in doxorubicin-resistant human cancer and cell lines. In a murine breast cancer model, sphingosine kinase 1, S1P receptor 1, interleukin 6, and STAT3 were overexpressed in the doxorubicin-treated group, whereas all of them were significantly suppressed with addition of FTY720. Combination therapy synergistically suppressed cancer growth both in vitro and in vivo. Furthermore, combination therapy showed higher efficacy in an obesity breast cancer model, where high body mass index demonstrated trends toward worse disease-free and overall survival, and high-serum S1P levels in human patients and volunteers.. We found that FTY720 enhanced the efficacy of doxorubicin by suppression of drug-induced inflammation, and combination therapy showed stronger effect in obesity-related breast cancer.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Body Mass Index; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; Female; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lysophospholipids; Mammary Neoplasms, Experimental; Mice; Obesity; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Retrospective Studies; Sphingosine; STAT3 Transcription Factor

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that has been shown to serve an important regulatory function in breast cancer progression. This study analyzes plasma S1P levels in breast cancer patients undergoing adjuvant therapy as compared to healthy control volunteers. 452 plasma S1P samples among 158 breast cancer patients, along with 20 healthy control volunteers, were analyzed. Mean S1P levels did not significantly differ between cancer patients and controls. Smoking was associated with higher S1P levels in cancer patients. Baseline S1P levels had weak inverse correlation with levels of the inflammatory mediator interleukin- (IL-) 17 and CCL-2 and positive correlation with tumor necrosis factor alpha (TNF-

Topics: Adult; Body Mass Index; Breast Neoplasms; Chemokine CCL2; Female; Humans; Interleukin-17; Lysophospholipids; Middle Aged; Postoperative Period; Smoking; Sphingosine; Tumor Necrosis Factor-alpha

Sphingolipid metabolism pathway is essential in membrane homeostasis, and its dysfunction has been associated with favorable tumor microenvironment, disease progression, and chemotherapy resistance. Its major components have key functions on survival and proliferation, with opposing effects. We have profiled the components of the sphingolipid pathway on leukocytes of breast cancer (BC) patients undergoing chemotherapy treatment and without, including the five sphingosine 1-phosphate (S1P) receptors, the major functional genes, and cytokines, in order to better understand the S1P signaling in the immune cells of these patients. To the best of our knowledge, this is the first characterization of the sphingolipid pathway in whole blood of BC patients. Skewed gene profiles favoring high

Topics: Breast Neoplasms; Case-Control Studies; Female; Humans; Leukocytes; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tumor Necrosis Factor-alpha

Dynamic interaction between cancer cells and the surrounding microenvironment is critical for cancer progression via changes in cellular behavior including alteration of secreted molecules. However, the molecular mechanisms underlying the influence exerted by the cancer microenvironment on secretion of molecules during cancer progression remain largely unknown. In this study, we report that secretion of spingsine-1-phosphate (S1P) and its regulator, SphK1 expression is dependent of the substrate rigidity, which is critical for the balance between cancer cell invasion and adhesion. Conditioned media (CM) of MDA-MB-231, an aggressive breast cancer cell obtained from soft substrate (~0.5 kPa) induced chemo-attractive invasion, while CM obtained from stiff substrate (~2.5 kPa) increased cell adhesion instead. We found that the expression of SphK1 is upregulated in the stiff substrate, resulting in an increase in S1P levels in the CM. We also found that upregulation of SphK1 expression in the stiff substrate is dominant in metastatic cancer cells but not in primary cancer cells. These results suggest that alterations in the mechanical environment of the ECM surrounding the tumor cells actively regulate cellular properties such as secretion, which in turn, may contribute to cancer progression.

Topics: Biomechanical Phenomena; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Culture Media, Conditioned; Extracellular Matrix; Female; Humans; Lysophospholipids; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tumor Microenvironment

The tumor microenvironment is a determining factor for cancer biology and progression. Sphingosine-1-phosphate (S1P), produced by sphingosine kinases (SphKs), is a bioactive lipid mediator that regulates processes important for cancer progression. Despite its critical roles, the levels of S1P in interstitial fluid (IF), an important component of the tumor microenvironment, have never previously been measured due to a lack of efficient methods for collecting and quantifying IF. The purpose of this study is to clarify the levels of S1P in the IF from murine mammary glands and its tumors utilizing our novel methods. We developed an improved centrifugation method to collect IF. Sphingolipids in IF, blood, and tissue samples were measured by mass spectrometry. In mice with a deletion of SphK1, but not SphK2, levels of S1P in IF from the mammary glands were greatly attenuated. Levels of S1P in IF from mammary tumors were reduced when tumor growth was suppressed by oral administration of FTY720/fingolimod. Importantly, sphingosine, dihydro-sphingosine, and S1P levels, but not dihydro-S1P, were significantly higher in human breast tumor tissue IF than in the normal breast tissue IF. To our knowledge, this is the first reported S1P IF measurement in murine normal mammary glands and mammary tumors, as well as in human patients with breast cancer. S1P tumor IF measurement illuminates new aspects of the role of S1P in the tumor microenvironment.

Topics: Activation, Metabolic; Animals; Antineoplastic Agents; Breast; Breast Neoplasms; Cell Line, Tumor; Extracellular Fluid; Female; Fingolimod Hydrochloride; Humans; Isoenzymes; Lysophospholipids; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice, Inbred BALB C; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Prodrugs; Random Allocation; Sphingosine; Tumor Microenvironment

Tumor cell-derived factors skew macrophages toward a tumor-supporting phenotype associated with the secretion of protumorigenic mediators. Apoptosing tumor cells release sphingosine 1-phosphate (S1P), which stimulates the production of lipocalin 2 (LCN2) in tumor-associated macrophages and is associated with tumor metastasis. We explored the mechanism by which S1P induces LCN2 in macrophages and investigated how this contributed to tumor growth and metastasis. Knockdown of S1P receptor 1 (S1PR1) in primary human macrophages and experiments with bone marrow-derived macrophages from S1PR1-deficient mice showed that S1P signaled through S1PR1 to induce LCN2 expression. The LCN2 promoter contains a consensus sequence for signal transducer and activator of transcription 3 (STAT3), and deletion of the STAT3 recognition sequence reduced expression of an LCN2-controlled reporter gene. Conditioned medium from coculture experiments indicated that the release of LCN2 from macrophages induced tube formation and proliferation in cultures of primary human lymphatic endothelial cells in a manner dependent on the kinase PI3K and subsequent induction of the growth factor VEGFC, which functioned as an autocrine signal stimulating the receptor VEGFR3. Knockout of Lcn2 attenuated tumor-associated lymphangiogenesis and breast tumor metastasis both in the breast cancer model MMTV-PyMT mice and in mice bearing orthotopic wild-type tumors. Our findings indicate that macrophages respond to dying tumor cells by producing signals that promote lymphangiogenesis, which enables metastasis.

Topics: Animals; Breast Neoplasms; Endothelial Cells; Female; Humans; Lipocalin-2; Lymphangiogenesis; Lysophospholipids; Macrophages; Mammary Neoplasms, Experimental; MCF-7 Cells; Mice; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Sphingosine

Sphingolipids, including sphingosine-1-phosphate (S1P) and ceramide, have emerged as key regulatory molecules that control various aspects of cell growth and proliferation in cancer. Although important roles of sphingolipids in breast cancer progression have been reported in experimental models, their roles in human patients have yet to be determined. The aims of this study were to determine the levels of sphingolipids including S1P, ceramides, and other sphingolipids, in breast cancer and normal breast tissue and to compare the difference in levels of each sphingolipid between the two tissues.. Tumor and noncancerous breast tissue were obtained from 12 patients with breast cancer. Sphingolipids including S1P, ceramides, and their metabolites of sphingosine, sphingomyelin, and monohexosylceramide were measured by liquid chromatography-electrospray ionization-tandem mass spectrometry.. The levels of S1P, ceramides, and other sphingolipids in the tumor were significantly higher than those in normal breast tissue. There was a relatively strong correlation in the levels of S1P between the tumor and those of normal breast tissue from the same person. On the other hand, there was no correlation in the levels of most of the ceramide species between the tumor and those of normal breast tissue from the same person.. To our knowledge, this is the first study to reveal that levels of sphingolipids in cancer tissue are generally higher than those of normal breast tissue in patients with breast cancer. The correlation of S1P levels in these tissues implicates the role of S1P in interaction between cancer and the tumor microenvironment.

Topics: Breast Neoplasms; Female; Humans; Lysophospholipids; Sphingolipids; Sphingosine

Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, has been implicated as a key regulatory molecule in cancer through its ability to promote cell proliferation, migration, angiogenesis, and lymphangiogenesis. Previous studies suggested that S1P produced by sphingosine kinase 1 (SphK1) in breast cancer plays important roles in progression of disease and metastasis. However, the associations between S1P and clinical parameters in human breast cancer have not been well investigated to date.. We determined levels of S1P and other sphingolipids in breast cancer tissue by electrospray ionization-tandem mass spectrometry. Associations between S1P levels and clinicopathologic features of the tumors were analyzed. Expression of phospho-SphK1 (pSphK1) in breast cancer tissues was determined by immunohistochemical scoring.. Levels of S1P in breast cancer tissues were significantly higher in patients with high white blood cell count in the blood than those patients without. S1P levels were lower in patients with human epidermal growth factor receptor 2 overexpression and/or amplification than those patients without. Furthermore, cancer tissues with high pSphK1 expression showed significantly higher levels of S1P than cancer tissues without. Finally, patients with lymph node metastasis showed significantly higher levels of S1P in tumor tissues than the patients with negative nodes.. To our knowledge, this is the first study to demonstrate that high expression of pSphK1 is associated with higher levels of S1P, which in turn is associated with lymphatic metastasis in breast cancer.

Topics: Breast Neoplasms; Female; Genes, erbB-2; Humans; Lymphatic Metastasis; Lysophospholipids; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Receptors, Estrogen; Receptors, Progesterone; Sphingosine; Triple Negative Breast Neoplasms

A crucial role of the inflammatory lipid sphingosine-1-phosphate (S1P) in breast cancer aggressiveness has been reported. Recent clinical studies have suggested that C-reactive protein (CRP) has a role in breast cancer development. However, limited information is available on the molecular basis for the expression of CRP and its functional significance in breast cell invasion. The present study aimed to elucidate the molecular link between S1P and CRP during the invasive process of breast epithelial cells. This is the first report showing that transcription of CRP was markedly activated by S1P in breast cells. Our data suggest that not only S1P treatment but also the endogenously produced S1P may upregulate CRP in breast carcinoma cells. Transcription factors CCAAT/enhancer-binding protein beta and c-fos were required for S1P-induced CRP expression. Coupling of S1P3 to heterotrimeric Gαq triggered the expression of CRP, utilizing signaling pathways involving reactive oxygen species (ROS), Ca(2+) and extracellular signal-related kinases (ERKs). S1P-induced CRP expression was crucial for the transcriptional activation of matrix metalloproteinase-9 through ERKs, ROS and c-fos, leading to breast cell invasion. Using a xenograft mice tumor model, we demonstrated that S1P induced CRP expression both in vitro and in vivo. Taken together, our findings have revealed a molecular basis for S1P-induced transcriptional activation of CRP and its functional significance in the acquisition of the invasive phenotype of human breast epithelial cells under inflammatory conditions. Our findings may provide useful information on the identification of useful therapeutic targets for inflammatory breast cancer.

Topics: Animals; Breast Neoplasms; C-Reactive Protein; Calcium; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Disease Progression; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Inflammation; Lysophospholipids; Matrix Metalloproteinase 9; Mice; Neoplasm Invasiveness; Proto-Oncogene Proteins c-fos; Reactive Oxygen Species; Sphingosine; Transcriptional Activation; Up-Regulation; Xenograft Model Antitumor Assays

The potential antitumor activity of cannabinoid receptor agonists, such as the aminoalklylindole WIN55,212-2 (WIN2), has been studied extensively, but their potential interaction with conventional cancer therapies, such as radiation, remains unknown. In the present work, the influence of WIN2 on the antiproliferative activity of radiation in human (MCF-7 and MDA-MB231) and murine (4T1) breast cancer cells was investigated. The antiproliferative effects produced by combination of WIN2 and radiation were more effective than either agent alone. The stereoisomer of WIN2, WIN55,212-3 (WIN3), failed to inhibit growth or potentiate the growth-inhibitory effects of radiation, indicative of stereospecificity. Two other aminoalkylindoles, pravadoline and JWH-015 [(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenyl-methanone], also enhanced the antiproliferative effects of radiation, but other synthetic cannabinoids (i.e., nabilone, CP55,940 [(+)-rel-5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-phenol], and methanandamide) or phytocannabinoids [i.e., Δ⁹-tetrahydrocannabinol (THC) and cannabidiol] did not. The combination treatment of WIN2 + radiation promoted both autophagy and senescence but not apoptosis or necrosis. WIN2 also failed to alter radiation-induced DNA damage or the apparent rate of DNA repair. Although the antiproliferative actions of WIN2 were mediated through noncannabinoid receptor-mediated pathways, the observation that WIN2 interfered with growth stimulation by sphingosine-1-phosphate (S1P) implicates the potential involvement of S1P/ceramide signaling pathways. In addition to demonstrating that aminoalkylindole compounds could potentially augment the effectiveness of radiation treatment in breast cancer, the present study suggests that THC and nabilone are unlikely to interfere with the effectiveness of radiation therapy, which is of particular relevance to patients using cannabinoid-based drugs to ameliorate the toxicity of cancer therapies.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Autophagy; Benzoxazines; Breast Neoplasms; Cannabinoid Receptor Agonists; Cannabinoids; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Female; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; Mice; Morpholines; Naphthalenes; Neoplasm Proteins; Phytochemicals; Receptors, Cannabinoid; Sphingosine; Stereoisomerism

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.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; CSK Tyrosine-Protein Kinase; Female; Fingolimod Hydrochloride; Humans; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Estrogen; Receptors, Lysosphingolipid; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; src-Family Kinases; Thiazoles; Tyrosine

Sphingosine kinase 1 (SK1) is a signaling enzyme that catalyzes the formation of sphingosine-1-phosphate. Overexpression of SK1 is causally associated with breast cancer progression and resistance to therapy. SK1 inhibitors are currently being investigated as promising breast cancer therapies. Two major transcriptional isoforms, SK143 kDa and SK151 kDa, have been identified; however, the 51 kDa variant is predominant in breast cancer cells. No studies have investigated the protein-protein interactions of the 51 kDa isoform and whether the two SK1 isoforms differ significantly in their interactions. Seeking an understanding of the regulation and role of SK1, we used a triple-labeling stable isotope labeling by amino acids in cell culture-based approach to identify SK1-interacting proteins common and unique to both isoforms. Of approximately 850 quantified proteins in SK1 immunoprecipitates, a high-confidence list of 30 protein interactions with each SK1 isoform was generated via a meta-analysis of multiple experimental replicates. Many of the novel identified SK1 interaction partners such as supervillin, drebrin, and the myristoylated alanine-rich C-kinase substrate-related protein supported and highlighted previously implicated roles of SK1 in breast cancer cell migration, adhesion, and cytoskeletal remodeling. Of these interactions, several were found to be exclusive to the 43 kDa isoform of SK1, including the protein phosphatase 2A, a previously identified SK1-interacting protein. Other proteins such as allograft inflammatory factor 1-like protein, the latent-transforming growth factor β-binding protein, and dipeptidyl peptidase 2 were found to associate exclusively with the 51 kDa isoform of SK1. In this report, we have identified common and isoform-specific SK1-interacting partners that provide insight into the molecular mechanisms that drive SK1-mediated oncogenicity.

Topics: Breast Neoplasms; Calcium-Binding Proteins; Cell Adhesion; Cell Line, Tumor; Cell Movement; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; DNA-Binding Proteins; Female; Humans; Latent TGF-beta Binding Proteins; Lysophospholipids; MCF-7 Cells; Microfilament Proteins; Phosphotransferases (Alcohol Group Acceptor); Protein Isoforms; Signal Transduction; Sphingosine

Many tumours originate from cancer stem cells (CSCs), which is a small population of cells that display stem cell properties. However, the molecular mechanisms that regulate CSC frequency remain poorly understood. Here, using microarray screening in aldehyde dehydrogenase (ALDH)-positive CSC model, we identify a fundamental role for a lipid mediator sphingosine-1-phosphate (S1P) in CSC expansion. Stimulation with S1P enhances ALDH-positive CSCs via S1P receptor 3 (S1PR3) and subsequent Notch activation. CSCs overexpressing sphingosine kinase 1 (SphK1), an S1P-producing enzyme, show increased ability to develop tumours in nude mice, compared with parent cells or CSCs. Tumorigenicity of CSCs overexpressing SphK1 is inhibited by S1PR3 knockdown or S1PR3 antagonist. Breast cancer patient-derived mammospheres contain SphK1(+)/ALDH1(+) cells or S1PR3(+)/ALDH1(+) cells. Our findings provide new insights into the lipid-mediated regulation of CSCs via Notch signalling, and rationale for targeting S1PR3 in cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; Lysophospholipids; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Receptors, Notch; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Sphingosine kinase is an enzyme that catalyses the phosphorylation of sphingosine to form sphingosine 1-phosphate. Sphingosine 1-phosphate is a bioactive lipid, which has been shown to have an important role in promoting the survival, growth and invasiveness of cancer cells. Sphingosine 1-phosphate binds to five different plasma membrane sphingosine 1-phosphate receptors (S1P(1-5) ) and can regulate intracellular target proteins. We have used immunohistochemical analysis to determine the concurrent expression levels of sphingosine kinase 1 or S1P receptors and other signaling proteins in estrogen receptor-positive breast cancer tumors and have then assessed the impact of these combinations on clinical outcome. This approach has enabled identification of (i) novel biomarkers and (ii) several spatially controlled associations between either sphingosine kinase 1 or S1P(1-3) and other signaling proteins whose combination affect prognosis. For instance, the translocation of sphingosine kinase 1 to the plasma membrane has been shown to be a critical determinant in cancer progression. However, our findings identify an additional novel role for the nuclear localization of sphingosine kinase 1 combined with either ERK-1/2 or SFK or LYN or AKT or NF-κB, which significantly shortens disease-specific survival and/or recurrence. We also demonstrate that nuclear S1P(2) receptor and c-SRC are associated with improved prognosis and this is linked with a reduction in the nuclear localization of sphingosine kinase 1. These findings identify potential novel biomarker associations, which might serve as new targets for drug intervention designed to improve treatment of estrogen receptor-positive breast cancer.

Topics: Aged; Biomarkers, Tumor; Breast Neoplasms; Cell Membrane; Cell Nucleus; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Lysophospholipids; Middle Aged; NF-kappa B; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; src-Family Kinases; Tamoxifen

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.

Topics: Amino Alcohols; Animals; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cells, Cultured; Disease Progression; Enzyme Inhibitors; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Lymphangiogenesis; Lymphatic Metastasis; Lysophospholipids; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Staging; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sphingosine; Tumor Burden; Vascular Endothelial Growth Factor A

The ceramide-sphingosine-1-phosphate rheostat is a promising therapeutic target. Here, the novel ceramide analog (S)-2-(benzylideneamino)-3-hydroxy-N-tetrade-cylpropanamide is shown to block proliferation and enhance the efficacy of the clinical chemotherapeutics, etoposide and doxorubicin. These results demonstrate the therapeutic potential of this compound in treating both endocrine resistant and chemoresistant breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Ceramides; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Etoposide; Female; Humans; Ki-67 Antigen; Lysophospholipids; Sphingosine; Stereoisomerism; Structure-Activity Relationship

Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Proliferation; Chromatography, Liquid; Cytokinesis; Enzyme Inhibitors; Female; Fluorescent Antibody Technique; Humans; Indoles; Lysophospholipids; Mitosis; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingosine; Tumor Cells, Cultured

Sphingosine-1-phosphate (S1P) is an important regulator of cancer development and progression. Its cellular concentration is controlled predominantly by sphingosine kinase (SK) and sphingosine-1-phosphate lyase (SPL). In the current study we showed that mRNA expressions for both SK and SPL were up-regulated throughout all four disease stages in human breast cancer patients. Exogenous administration of S1P produced a bell-shaped dose response for apoptosis in normal mammary gland MCF12A cells but a sigmoid-shaped apoptotic response in breast cancer MCF7 cells. Co-administration of S1P enhanced the cytotoxicity of anticancer drug docetaxel against MCF7 cells.

Topics: Aldehyde-Lyases; Apoptosis; Breast Neoplasms; Cell Proliferation; Docetaxel; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Sphingosine; Taxoids

Sphingosine kinase 1 (SK1) catalyzes the conversion of sphingosine to the bioactive lipid sphingosine 1-phosphate. We have previously demonstrated that FTY720 and (S)-FTY720 vinylphosphonate are novel inhibitors of SK1 activity. Here, we show that (S)-FTY720 vinylphosphonate binds to a putative allosteric site in SK1 contingent on formation of the enzyme-sphingosine complex. We report that SK1 is an oligomeric protein (minimally a dimer) containing noncooperative catalytic sites and that the allosteric site exerts an autoinhibition of the catalytic site. A model is proposed in which (S)-FTY720 vinylphosphonate binding to and stabilization of the allosteric site might enhance the autoinhibitory effect on SK1 activity. Further evidence for the existence of allosteric site(s) in SK1 was demonstrated by data showing that two new FTY720 analogues (a conjugate of sphingosine with a fluorophore and (S)-FTY720 regioisomer) increased SK1 activity, suggesting relief of autoinhibition of SK1 activity. Comparisons with the SK1 inhibitor, SKi or siRNA knockdown of SK1 indicated that (S)-FTY720 vinylphosphonate and FTY720 behave as typical SK1 inhibitors in preventing sphingosine 1-phosphate-stimulated rearrangement of actin in MCF-7 cells. These findings are discussed in relation to the anticancer properties of SK1 inhibitors.

Topics: Actins; Allosteric Regulation; Breast Neoplasms; Catalytic Domain; Cell Line, Tumor; Enzyme Inhibitors; Female; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lysophospholipids; Models, Chemical; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Proteasome Endopeptidase Complex; Sphingosine

Recent evidence suggests that inflammation is involved in malignant progression of breast cancer. Sphingosine 1-phosphate (S1P), acting on the G-protein-coupled receptors, is known as a potent inflammatory mediator. In this study, the effect of the inflammatory lipid S1P on the regulation of invasive/migratory phenotypes of MCF10A human breast epithelial cells was investigated to elucidate a causal relationship between inflammation and the control of invasiveness of breast cells. We show that S1P causes induction of matrix metalloproteinase-9 (MMP-9) in vitro and in vivo, and thus enhances invasion and migration. We also show that fos plays a crucial role in the transcriptional activation of MMP-9 by S1P. In addition, activation of extracellular-signal-regulated kinases 1 and 2 (ERK1/2), p38 and alpha serine/threonine-protein kinase (Akt) are involved in the process of S1P-mediated induction of MMP-9 expression and invasion. Activation of the S1P receptor S1P₃ and G(αq) are required for S1P-induced invasive/migratory responses, suggesting that the enhancement of S1P-mediated invasiveness is triggered by the specific coupling of S1P₃ to the heterotrimeric G(αq) subunit. Activation of phospholipase C-β₄ and intracellular Ca²⁺ release are required for S1P-induced MMP-9 upregulation. Taken together, this study demonstrated that S1P regulates MMP-9 induction and invasiveness through coupling of S1P₃ and G(αq) in MCF10A cells, thus providing a molecular basis for the crucial role of S1P in promoting breast cell invasion.

Topics: Animals; Breast; Breast Neoplasms; Cell Line; Cell Movement; Female; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Lysophospholipids; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Oncogene Proteins v-fos; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

Sphingosine 1-phosphate (S1P), a potent sphingolipid mediator produced by sphingosine kinase isoenzymes (SphK1 and SphK2), regulates diverse cellular processes important for breast cancer progression acting in an autocrine and/or paracrine manner. Here we show that SphK1, but not SphK2, increased S1P export from MCF-7 cells. Whereas for both estradiol (E(2)) and epidermal growth factor-activated SphK1 and production of S1P, only E(2) stimulated rapid release of S1P and dihydro-S1P from MCF-7 cells. E(2)-induced S1P and dihydro-S1P export required estrogen receptor-alpha, not GPR30, and was suppressed either by pharmacological inhibitors or gene silencing of ABCC1 (multidrug resistant protein 1) or ABCG2 (breast cancer resistance protein). Inhibiting these transporters also blocked E(2)-induced activation of ERK1/2, indicating that E(2) activates ERK via downstream signaling of S1P. Taken together, our findings suggest that E(2)-induced export of S1P mediated by ABCC1 and ABCG2 transporters and consequent activation of S1P receptors may contribute to nongenomic signaling of E(2) important for breast cancer pathophysiology.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Estradiol; Estrogens; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrometry, Mass, Electrospray Ionization; Sphingosine

The lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are small lipid molecules with a variety of physiological roles. Additionally, their involvement in the initiation and progression of malignant tumors has been increasingly recognized in recent years. However, the data on the expression of S1P and LPA receptors on different cancer cells are very few. Real-time polymerase chain reaction was used for the analysis of mRNA expression of five S1P((1-5)) and three LPA((1-3)) receptors on a large panel of 13 colon, breast, melanoma, and lung cancer cell lines. Furthermore, the modulation of S1P and LPA receptor mRNA expression was studied upon xenotransplantation of tumor cells into severe combined immunodeficient (SCID) mice. The S1P and LPA receptors were expressed to a variable degree on all tumor cell lines tested (with exception of colon cancer SW480). Most notably, tumor cell lines in vitro expressed S1P(2) mRNA that was down-regulated upon xenotransplantation, whereas LPA(2) receptor mRNA was strongly expressed both in vitro and in vivo (except by breast cancer cells). The latter was especially distinctive for small cell lung tumor cells. The S1P and LPA receptors are differentially expressed on tumor cell lines in vitro. Their expression is modulated upon xenografting into SCID mice in vivo.

Topics: Animals; Breast Neoplasms; Colonic Neoplasms; Endothelium, Vascular; Female; Humans; Immunoenzyme Techniques; Lung Neoplasms; Lysophospholipids; Melanoma; Mice; Mice, SCID; Neoplasm Transplantation; Receptors, Lysophosphatidic Acid; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine; Transplantation, Heterologous; Tumor Cells, Cultured; Umbilical Cord

We demonstrate here a new concept termed "oncogene tolerance" whereby human EGF receptor 2 (HER2) increases sphingosine kinase 1 (SK1) expression in estrogen receptor-positive (ER(+)) MCF-7 HER2 cells and SK1, in turn, limits HER2 expression in a negative-feedback manner. The HER2-dependent increase in SK1 expression also limits p21-activated protein kinase 1 (p65 PAK1) and extracellular signal regulated kinase 1/2 (ERK-1/2) signaling. Sphingosine 1-phosphate signaling via S1P(3) is also altered in MCF-7 HER2 cells. In this regard, S1P binding to S1P(3) induces a migratory phenotype via an SK1-dependent mechanism in ER(+) MCF-7 Neo cells, which lack HER2. This involves the S1P stimulated accumulation of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia and migration. In contrast, S1P failed to promote redistribution of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia or migration of MCF-7 HER2 cells. However, a migratory phenotype in these cells could be induced in response to S1P when SK1 expression had been knocked down with a specific siRNA or when recombinant PAK1 was ectopically overexpressed. Thus, the HER2-dependent increase in SK1 expression functions to desensitize the S1P-induced formation of a migratory phenotype. This is correlated with improved prognosis in patients who have a low HER1-3/SK1 expression ratio in their ER(+) breast cancer tumors compared to patients that have a high HER1-3/SK1 expression ratio.

Topics: Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Estrogen Receptor alpha; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; p21-Activated Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Receptors, Estrogen; RNA, Small Interfering; Signal Transduction; Sphingosine

Weight gain in women receiving chemotherapy for breast cancer is associated with a higher risk of recurrence. Using metabonomic profiling, we recently reported that plasma lactate and alanine were prognostic for weight gain in individuals with breast cancer receiving chemotherapy. The role of lipid second messengers has not been studied. We assessed serum levels of sphingosine-1-phosphate (S1P), a known secreted lipid second messenger with a role in cell growth, in sequential samples from post-menopausal women receiving standard chemotherapy for early breast cancer and correlated these with body mass measurements and metabonomic profiling. While serum S1P levels prior to treatment did not correlate with body weight changes or circulating alanine and lactate, S1P levels measured during therapy were inversely correlated with weight gain (P = 0.04), but not weight loss (P = 0.74) or no change in weight (P = 0.5), suggesting a role of dynamic circulating S1P in adipocyte growth. These data provide evidence for an association between serum S1P and weight gain during chemotherapy cycles in women with breast cancer. Lipid second messengers have a role in chemotherapy-induced weight gain in breast cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Body Mass Index; Breast Neoplasms; Chemotherapy, Adjuvant; Down-Regulation; Female; Humans; Lysophospholipids; Metabolomics; Neoadjuvant Therapy; Risk Assessment; Risk Factors; Sphingosine; Time Factors; Treatment Outcome; Weight Gain

We demonstrate here that the bioactive lipid sphingosine 1-phosphate (S1P) uses sphingosine 1-phosphate receptor 4 (S1P(4)) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 cells. This was based on several lines of evidence. First, the S1P stimulation of ERK-1/2 was abolished by JTE013, which we show here is an S1P(2/4) antagonist and reduced by siRNA knockdown of S1P(4). Second, the S1P-stimulated activation of ERK-1/2 was almost completely abolished by a HER2 inhibitor (ErbB2 inhibitor II) and reduced by siRNA knockdown of HER2 expression. Third, phyto-S1P, which is an S1P(4) agonist, stimulated ERK-1/2 activation in an S1P(4)- and HER2-dependent manner. Fourth, FTY720 phosphate, which is an agonist at S1P(1,3,4,5) but not S1P(2) stimulated activation of ERK-1/2. Fifth, S1P stimulated the tyrosine phosphorylation of HER2, which was reduced by JTE013. HER2 which is an orphan receptor tyrosine kinase is the preferred dimerization partner of the EGF receptor. However, EGF-stimulated activation of ERK-1/2 was not affected by siRNA knockdown of HER2 or by ErbB2 (epidermal growth factor receptor 2 (or HER2)) inhibitor II in MDA-MB-453 cells. Moreover, S1P-stimulated activation of ERK-1/2 does not require an EGF receptor. Thus, S1P and EGF function in a mutually exclusive manner. In conclusion, the magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by HER2 in MDA-MB-453 cells. The linkage of S1P with an oncogene suggests that S1P and specifically S1P(4) may have an important role in breast cancer progression.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; Epidermal Growth Factor; Female; Fingolimod Hydrochloride; HEK293 Cells; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Propylene Glycols; Pyrazoles; Pyridines; Receptor, ErbB-2; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Sphingosine

Various studies in cell lines have previously demonstrated that sphingosine kinase 1 (SK1) and extracellular signal-regulated kinase 1/2 (ERK-1/2) interact in an estrogen receptor (ER)-dependent manner to influence both breast cancer cell growth and migration. A cohort of 304 ER-positive breast cancer patients was used to investigate the prognostic significance of sphingosine 1-phosphate (S1P) receptors 1, 2, and 3 (ie, S1P1, S1P2, and S1P3), SK1, and ERK-1/2 expression levels. Expression levels of both SK1 and ERK-1/2 were already available for the cohort, and S1P1, S1P2, and S1P3 levels were established by immunohistochemical analysis. High membrane S1P1 expression was associated with shorter time to recurrence (P=0.008). High cytoplasmic S1P1 and S1P3 expression levels were also associated with shorter disease-specific survival times (P=0.036 and P=0.019, respectively). Those patients with tumors that expressed high levels of both cytoplasmic SK1 and ERK-1/2 had significantly shorter recurrence times than those that expressed low levels of cytoplasmic SK1 and cytoplasmic ERK-1/2 (P=0.00008), with a difference in recurrence time of 10.5 years. Similarly, high cytoplasmic S1P1 and cytoplasmic ERK-1/2 expression levels (P=0.004) and high cytoplasmic S1P3 expression and cytoplasmic ERK-1/2 expression levels (P=0.004) were associated with shorter recurrence times. These results support a model in which the interaction between SK1, S1P1, and/or S1P3 and ERK-1/2 might drive breast cancer progression, and these findings, therefore, warrant further investigation.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; HEK293 Cells; Humans; Lysophospholipids; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphotransferases (Alcohol Group Acceptor); Protein Isoforms; Receptors, Estrogen; Receptors, Lysosphingolipid; RNA, Small Interfering; Sphingosine; Survival Rate; Tamoxifen

Resistance to chemotherapy and endocrine therapy is a major cause of breast cancer treatment failure. We have synthesized six novel analogues using C8-ceramide as the lead analogue and studied their effect on hormone therapy resistant (MDA-MB-231) and chemoresistant (MCF-7TN-R) breast cancer cells. Pharmacologic intervention using these ceramide analogues inhibited clonogenic survival and induced apoptosis, with one analogue being more effective than C8-ceramide. Our results show ceramide-based therapy has therapeutic potential in treating drug resistant breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Ceramides; Drug Design; Drug Resistance, Neoplasm; Hormones; Humans; Inhibitory Concentration 50; Lysophospholipids; Sphingosine

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are structurally related bioactive lipids with growth factor-like activities. LPA and S1P are naturally produced in vivo by blood platelets upon platelet aggregation and at least in vitro by fibroblasts, adipocytes, and multiple types of tumor cells. Breast cancer cells respond to LPA and S1P. However, their specific actions on breast cancer cell biological functions remain unclear. We therefore conducted an in vitro side-by-side study of these two lipids on breast cancer cells. LPA mediates human breast cancer MDA-BO2 cell proliferation, migration, and invasion through activation of a G(alpha i)/ERK1/2-dependent signaling pathway, whereas activation of G(alpha i)/PI3K predominates upon S1P stimulation. In MDA-BO2 cells, LPA but not S1P activities were dependent on active type 1 insulin-like growth factor and epithelial growth factor receptors. LPA and S1P act directly on endothelial cells to induce angiogenesis. We demonstrate that LPA and S1P have indirect angiogenic properties as judged by induced secretion of angiogenic factors by breast cancer cells primed with these lysophospholipids. S1P, but not LPA, controlled the expression of VEGF-A by breast cancer cells, while LPA, but not S1P, controlled the expression of GM-CSF, Gro-alpha, MCP-1, and IL-6. According to the secretion of these paracrine osteoclastic factors, LPA, but not S1P, stimulates breast cancer cell-induced osteoclastogenesis. These findings suggest that, in vivo, LPA and S1P can coordinate their action on tumor and surrounding cells to induce breast cancer progression both at primary and bone metastatic sites.

Topics: Bone Marrow Cells; Breast Neoplasms; Cell Adhesion; Cell Movement; Cell Proliferation; Cells, Cultured; Chemokine CCL2; Chemokine CXCL1; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-6; Interleukin-8; Lysophospholipids; Neovascularization, Physiologic; Osteoclasts; Phosphatidylinositol 3-Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine; Umbilical Veins; Vascular Endothelial Growth Factor A

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lysophospholipid mediators of diverse cellular processes important for cancer progression. S1P is produced by two sphingosine kinases, SphK1 and SphK2. Expression of SphK1 is elevated in many cancers. Here, we report that LPA markedly enhanced SphK1 mRNA and protein in gastric cancer MKN1 cells but had no effect on SphK2. LPA also up-regulated SphK1 expression in other human cancer cells that endogenously express the LPA(1) receptor, such as DLD1 colon cancer cells and MDA-MB-231 breast cancer cells, but not in HT29 colon cancer cells or MDA-MB-453 breast cancer cells, which do not express the LPA(1) receptor. An LPA(1) receptor antagonist or down-regulation of its expression prevented SphK1 and S1P(3) receptor up-regulation by LPA. LPA transactivated the epidermal growth factor receptor (EGFR) in these cells, and the EGFR inhibitor AG1478 attenuated the increased SphK1 and S1P(3) expression induced by LPA. Moreover, down-regulation of SphK1 attenuated LPA-stimulated migration and invasion of MNK1 cells yet had no effect on expression of neovascularizing factors, such as interleukin (IL)-8, IL-6, urokinase-type plasminogen activator (uPA), or uPA receptor induced by LPA. Finally, down-regulation of S1P(3), but not S1P(1), also reduced LPA-stimulated migration and invasion of MKN1 cells. Collectively, our results suggest that SphK1 is a convergence point of multiple cell surface receptors for three different ligands, LPA, EGF, and S1P, which have all been implicated in regulation of motility and invasiveness of cancer cells.

Topics: Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Chemotaxis; Colonic Neoplasms; ErbB Receptors; Humans; Interleukin-6; Interleukin-8; Lysophospholipids; Neoplasm Invasiveness; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysophosphatidic Acid; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine; Stomach Neoplasms; Transcriptional Activation; Tumor Cells, Cultured; Up-Regulation; Urokinase-Type Plasminogen Activator

Beside their structural role for the cell membrane the family of sphingolipids act as effector molecules in signal transduction with links to various aspects of cancer initiation, progression and treatment response. The "sphingolipid rheostat" balances between apoptosis inducing ceramid and growth promoting sphingosine-1-phosphate. We analyzed gene expression of 43 proteins from this pathway in different subtypes of breast cancer using microarray data of 1,269 tumor samples (test set n=171; validation sets n=1098) and observed significant differences for several genes. Sphingosine kinase 1 (SPHK1), ceramide galactosyltransferase (UGT8), and Ganglioside GD3-Synthase (ST8SIA1) displayed higher expression among ER negative tumors. In contrast, glucosylceramidsynthase (GCS), dihydroceramidsynthases (LASS4, LASS 6) and acid ceramidase (ASAH1) were higher expressed in ER positive samples. Survival analysis revealed a worse outcome of patients with high SPHK1 expression. To avoid a confounding effect of the ER status we also restricted the analysis to 750 patients with ER positive tumors. Again a worse outcome was observed for tumors displaying high SPHK1 expression. While 75.8+/-1.9% of the patients with tumors low in SPHK1 expression were free of metastasis at 5 years, this was the case for only 64.9+/-3.6% of patients with tumors displaying high SPHK1 expression (P=0.008). Immunohistochemistry identified the carcinoma cells as the major source of SPHK1 expression in the tumor. The correlation of SPHK1 with a poor prognosis as well as its high expression among ER negative tumors are in line with the antiapoptotic and proliferative properties of its product sphingosine-1-phosphate. Targeting of the sphingolipid rheostat may thus open new treatment options.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Female; Gene Expression Profiling; Humans; Immunoenzyme Techniques; Lysophospholipids; Middle Aged; Oligonucleotide Array Sequence Analysis; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Sphingosine; Survival Rate

G protein-coupled receptors (GPCRs) for lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) transduce signals to many functions of normal cells. Most human cancer cells upregulate S1P and LPA GPCRs, in patterns distinctive for each type of tumor. The findings that 1-alpha, 25-dihydroxy-vitamin D(3) (VD3) and all-trans retinoic acid (RA) differentially alter expression of the predominant S1P(3) (Edg-3) R and S1P(2) (Edg-5) R in human breast cancer cells (BCCs) permitted analyses of their individual activities, despite a lack of selective pharmacological probes. S1P-evoked increases in [Ca(2+)](i) in S1P(3) R-predominant BCCs were suppressed by concentrations of VD3 and RA which decreased expression of S1P(3) Rs, despite RA-induced increases in S1P(2) Rs. S1P-elicited chemokinetic migration of S1P(3) R-predominant BCCs across a type IV collagen-coated micropore filter also was inhibited by concentrations of VD3 and RA which decreased expression of S1P(3) Rs. The RA-induced increase in expression of S1P(2) Rs did not prevent suppression by RA of S1P-elicited chemokinesis, which appears to be mediated by S1P(3) Rs, but instead exposed S1P(2) R-mediated inhibition of epidermal growth factor-stimulated chemotaxis of BCCs. In contrast, expression of the predominant LPA(2) Rs, LPA-evoked increase in [Ca(2+)](i) and LPA-stimulated chemokinetic migration were suppressed concomitantly by RA but not VD3. Thus two structurally-homologous S1P Rs of BCCs differ in coupling to [Ca(2+)](i) signaling and have opposite effects on protein growth factor-stimulated chemotaxis.

Topics: Breast Neoplasms; Calcitriol; Calcium; Cell Movement; Cells, Cultured; Chemotaxis; Epidermal Growth Factor; Female; Gene Expression Regulation; Humans; Lysophospholipids; Male; Membrane Proteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Receptors, Lysophospholipid; Signal Transduction; Sphingosine; Tretinoin

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), formed by phosphorylation of sphingosine, has been implicated in cell growth, suppression of apoptosis, and angiogenesis. In this study, we have examined the contribution of intracellular S1P to tumorigenesis of breast adenocarcinoma MCF-7 cells. Enforced expression of sphingosine kinase type 1 (SPHK1) increased S1P levels and blocked MCF-7 cell death induced by anti-cancer drugs, sphingosine, and TNF-alpha. SPHK1 also conferred a growth advantage, as determined by proliferation and growth in soft agar, which was estrogen dependent. While both ERK and Akt have been implicated in MCF-7 cell growth, SPHK1 stimulated ERK1/2 but had no effect on Akt. Surprisingly, parental growth of MCF-7 cells was only weakly stimulated by S1P or dihydro-S1P, ligands for the S1P receptors which usually mediate growth effects. When injected into mammary fat pads of ovariectomized nude mice implanted with estrogen pellets, MCF-7/SPHK1 cells formed more and larger tumors than vector transfectants with higher microvessel density in their periphery. Collectively, our results suggest that SPHK1 may play an important role in breast cancer progression by regulating tumor cell growth and survival.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Breast Neoplasms; Estrogens; Humans; Lysophospholipids; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasms, Hormone-Dependent; Phosphotransferases (Alcohol Group Acceptor); Receptors, Cell Surface; Receptors, Estrogen; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Sphingosine; Tumor Cells, Cultured

Matriptase is an epithelial-derived, cell surface serine protease. This protease activates hepatocyte growth factor (HGF) and urokinase plasminogen activator (uPA), two proteins thought to be involved in the growth and motility of cancer cells, particularly carcinomas, and in the vascularization of tumors. Thus, matriptase may play an important role in the progression of carcinomas, such as breast cancer. We examined the regulation of activation of matriptase in human breast cancer cells, in comparison to non-transformed mammary epithelial cells 184A1N4 and MCF-10A. Results clearly indicated that unlike non-transformed mammary epithelial cells, breast cancer cells do not respond to the known activators of matriptase, serum and sphingosine 1-phosphate (S1P). Similar levels of activated matriptase were detected in breast cancer cells, grown in the presence or absence of S1P. However, up to five-fold higher levels of activated matriptase were detected in the conditioned media from the cancer cells grown in the absence of serum and S1P, when compared to non-transformed mammary epithelial cells. S1P also induces formation of cortical actin structures in non-transformed cells, but not in breast cancer cells. These results show that in non-transformed cells, S1P induces a rearrangement of the actin cytoskeleton and stimulates proteolytic activity on cell surfaces. In contrast, S1P treatment of breast cancer cells does not activate matriptase, and instead these cells constitutively activate the protease. In addition, breast cancer cells respond differently to S1P in terms of the regulation of actin cytoskeletal structures. Matriptase and its cognate inhibitor, HGF activator inhibitor 1 (HAI-1) colocalize on the cell periphery of breast cancer cells and form stable complexes in the extracellular milieu, suggesting that the inhibitor serves to prevent undesired proteolysis in these cells. Finally, we demonstrate that treatment of T-47D cells with epidermal growth factor (EGF), which promotes cell ruffling, stimulates increased accumulation of activated matriptase at the sites of membrane ruffling, suggesting a possible functional role at these sites.

Topics: Actins; Breast; Breast Neoplasms; Cell Line, Transformed; Cell Membrane; Cytoskeleton; Enzyme Activation; Epidermal Growth Factor; Epithelial Cells; Female; Humans; Lysophospholipids; Membrane Glycoproteins; Proteinase Inhibitory Proteins, Secretory; Serine Endopeptidases; Sphingosine; Trypsin; Tumor Cells, Cultured

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397-transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.

Topics: Breast Neoplasms; Cell Adhesion; Cell Adhesion Molecules; Cell Division; Cell Movement; Chemotaxis; Collagen; Cytoskeletal Proteins; Drug Combinations; Enzyme Activation; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gelatinases; Humans; Laminin; Lysophospholipids; Matrix Metalloproteinase 2; Metalloendopeptidases; Paxillin; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Proteoglycans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-crk; Sphingosine; Tumor Cells, Cultured; Tyrosine

Endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) Edg-1, Edg-3, and Edg-5 bind sphingosine 1-phosphate (S1P), and Edg-2 and Edg-4 Rs bind lysophosphatidic acid (LPA). LPA and S1P initiate ras- and rho-dependent signaling of cellular growth. Cultured lines of human breast cancer cells (BCCs) express Edg-3 > Edg-4 > Edg-5 > or = Edg-2, without detectable Edg-1, by both assessment of mRNA and Western blots with rabbit and monoclonal mouse anti-Edg R antibodies. BCC proliferation was stimulated significantly by 10(-9) M to 10(-6) M LPA and S1P. Luciferase constructs containing the serum response element (SRE) of growth-related gene promoters reported mean activation of BCCs by LPA and S1P of up to 85-fold. LPA and S1P stimulated BCC secretion of type II insulin-like growth factor (IGF-II) by 2-7-fold, to levels at which exogenous IGF-II stimulated increased proliferation and SRE activation of BCCs. All BCC responses to LPA and S1P were suppressed similarly by pertussis toxin, mitogen-activated protein kinase kinase inhibitors, and C3 exoenzyme inactivation of rho, suggesting mediation by Edg Rs. Monoclonal anti-IGF-II and anti-IGFR1 antibodies suppressed proliferation and SRE reports of BCCs to LPA and S1P by means of up to 65%. Edg Rs thus transduce LPA and S1P enhancement of BCC growth, both directly through SRE and indirectly by enhancing the contribution of IGF-II.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Breast Neoplasms; Cell Division; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Proteins; Immediate-Early Proteins; Lysophospholipids; Mice; NF-KappaB Inhibitor alpha; Nuclear Proteins; Rabbits; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Receptors, Lysophospholipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Exogenous sphingosine-1-phosphate (SPP) inhibits chemotactic motility of several transformed cell lines. We have found that SPP at high micromolar concentrations decreased chemotaxis of estrogen-independent (MDA-MB-231 and BT 549) and estrogen-dependent (MCF-7 and ZR-75-1) human breast cancer cells. Because SPP has been implicated as a lipid-signaling molecule with novel dual intra- and intercellular actions, it was of interest to determine whether the effect of SPP on chemotactic motility of human breast cancer cells is mediated intracellularly or through the recently identified endothelial differentiation gene (EDG) family of G protein-coupled SPP receptors. There was no detectable specific binding of [32P]SPP to MDA-MB-231 or MCF-7 cells; however, reverse transcription-PCR analysis revealed that both MDA-MB-231 and MCF-7 cells expressed moderate levels of EDG-3, neither expressed EDG-1, and EDG-5 mRNA was expressed in MCF-7 but not in MDA-MB-231 cells. In contrast to SPP, sphinganine-1-phosphate, which binds to and signals through SPP receptors EDG-1, EDG-3, and EDG-5, had no effect on chemotactic motility of MDA-MB-231 or MCF-7 cells. To further discriminate between intracellular and receptor-mediated actions of SPP, we used caged SPP, a photolyzable derivative of SPP that elevates intracellular levels of SPP after illumination. Caged SPP inhibited chemotactic motility of MDA-MB-231 cells only upon UV irradiation. In addition, in MCF-7 cells, overexpression of sphingosine kinase, the enzyme that produces SPP, inhibited chemotactic motility compared with vector-transfected cells and markedly increased cellular SPP levels in the absence of detectable secretion. Our results suggest that the inhibitory effect of SPP on chemotactic motility of human breast cancer cells is likely mediated through intracellular actions of SPP rather than through cell surface receptors.

Topics: Breast Neoplasms; Chemotaxis; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Photolysis; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Sphingosine; Tumor Cells, Cultured