Sphingosine Kinase 1 Knockdown Research Articles

SPHK1 potentiates colorectal cancer progression and metastasis via regulating autophagy mediated by TRAF6-induced ULK1 ubiquitination

A sphingolipid metabolite regulator, sphingosine kinase 1 (SPHK1), plays a critical role in the development of colorectal cancer (CRC). Studies have demonstrated that invasion and metastasis of CRC are promoted by SPHK1-driven autophagy. However, the exact mechanism of SPHK1 drives autophagy to promote tumor progression remains unclear. Here, immunohistochemical detection showed the expression of SPHK1 and tumor necrosis factor receptor-associated factor-6 (TRAF6) in human CRC tissues was stronger than in adjacent normal tissues, they were both associated with distance metastasis. It was discovered that knockdown of SPHK1 reduced the expression of TRAF6, inhibited autophagy, and inhibited the growth and metastasis of CRC cells in vitro. Moreover, the effects of SPHK1-downregulating were reversed by overexpression of TRAF6 in CRC cells transfected by double-gene. Overexpression of SPHK1 and TRAF6 promoted the expression of autophagy protein LC3 and Vimentin, while downregulated the expression of autophagy protein P62 and E-cadherin. The expression of autophagy-related ubiquitination protein ULK1 and Ubiquitin protein were significantly upregulated in TRAF6-overexpressed CRC cells. In addition, autophagy inhibitor 3-methyladenine (3MA) significantly inhibited the metastasis-promoting effect of SPHK1 and TRAF6, suppressed the expression of LC3 and Vimentin, and promoted the expression of P62 and E-cadherin, in CRC cells. Immunofluorescence staining showed SPHK1 and TRAF6 were co-localized in HT29 CRC cell membrane and cytoplasm. Immunoprecipitation detection showed SPHK1 was efficiently combined with the endogenous TRAF6, and the interaction was also detected reciprocally. Additionally, proteasome inhibitor MG132 treatment upregulated the expression of TRAF6 and the level of Ubiquitin protein, in SPHK1-downregulating CRC cells. These results reveal that SPHK1 potentiates CRC progression and metastasis via regulating autophagy mediated by TRAF6-induced ULK1 ubiquitination. SPHK1-TRAF6-ULK1 signaling axis is critical to the progression of CRC and provides a new strategy for the therapeutic control of CRC.

Pancreatic Acinar Cells-Derived Sphingosine-1-Phosphate Contributes to Fibrosis of Chronic Pancreatitis via Inducing Autophagy and Activation of Pancreatic Stellate Cells

Studies have demonstrated that activated pancreatic stellate cells (PSCs) play a crucial role in pancreatic fibrogenesis in chronic pancreatitis (CP); however, the precise mechanism for PSCs activation has not been fully elucidated. We analyzed the role of injured pancreatic acinar cells (iPACs) in the activation of PSCs of CP. Sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling was evaluated in experimental CP induced by cerulein injection or pancreatic duct ligation,as well as in PACs injured by cholecystokinin. The activation of PSCs and pancreatic fibrosis in CP samples was evaluated by immunohistochemical and immunofluorescence analyses. Invitro coculture assay of iPACs and PSCs was created to evaluate the effect of the SPHK1/S1P pathway and S1P receptor 2 (SIPR2) on autophagy and activation of PSCs. The pathogenesis of CP was assessed in SPHK1-/- mice or PACs-specific SPHK1-knockdown mice with recombinant adeno-associated virus serotypes 9-SPHK1-knockdown, as well as in mice treated with inhibitor of SPHK1 and S1P receptor 2 (S1PR2). SPHK1/S1P was remarkably increased in iPACs and acinar cells in pancreatic tissues of CP mice. Meanwhile, the pathogenesis, fibrosis, and PSCs activation of CP was significantly prevented in SPHK1-/- mice and recombinant adeno-associated virus serotypes 9-SPHK1-knockdown mice. Meanwhile, iPACs obviously activated PSCs, which was prevented by SPHK1 knockdown in iPACs. Moreover, iPACs-derived S1P specifically combined to S1PR2 of PSCs, by which modulated 5' adenosine monophosphate-activated protein kinase/mechanistic target of rapamycin pathway and consequently induced autophagy and activation of PSCs.Furthermore, hypoxia-inducible factor 1-α and -2α promoted SPHK1 transcription of PACs under hypoxia conditions, which is a distinct characteristic of the CP microenvironment. Coincidently, inhibition of SPHK1 and S1PR2 activity with inhibitor PF-543 and JTE-013 obviously impeded pancreatic fibrogenesis of CP mice. The activated SPHK1/S1P pathway in iPACs induces autophagy and activation of PSCs by regulating the S1PR2/5' adenosine monophosphate-activated protein kinase/mammalian target of rapamycin pathway, which promotes fibrogenesis of CP. The hypoxia microenvironment might contribute to the cross talk between PACs and PSCs in pathogenesis of CP.

Targeting SPHK1/S1PR3-regulated S-1-P metabolic disorder triggers autophagic cell death in pulmonary lymphangiomyomatosis (LAM)

Lymphangioleiomyomatosis (LAM), a progressive pulmonary disease exclusively affecting females, is caused by defects or mutations in the coding gene tuberous sclerosis complex 1 (TSC1) or TSC2, causing the mammalian target of rapamycin complex 1 (mTORC1) activation and autophagy inhibition. Clinically, rapamycin shows limited cytocidal effects, and LAM recurs after drug withdrawal. In this study, we demonstrated that TSC2 negatively regulated the sphingolipid metabolism pathway and the expressions of sphingosine kinase 1 (SPHK1) and sphingosine-1-phosphate receptor 3 (S1PR3) were significantly elevated in LAM patient-derived TSC2-deficient cells compared to TSC2-addback cells, insensitive to rapamycin treatment and estrogen stimulation. Knockdown of SPHK1 showed reduced viability, migration and invasion in TSC2-deficient cells. Selective SPHK1 antagonist PF543 potently suppressed the viability of TSC2-deficient cells and induced autophagy-mediated cell death. Meanwhile, the cognate receptor S1PR3 was identified to mediating the tumorigenic effects of sphingosine-1-phosphate (S1P). Treatment with TY52156, a selective antagonist for S1PR3, or genetic silencing using S1PR3-siRNA suppressed the viability of TSC2-deficient cells. Both SPHK1 and S1PR3 inhibitors markedly exhibited antitumor effect in a xenograft model of TSC2-null cells, restored autophagy level, and triggered cell death. Together, we identified novel rapamycin-insensitive sphingosine metabolic signatures in TSC2-null LAM cells. Therapeutic targeting of aberrant SPHK1/S1P/S1PR3 signaling may have potent therapeutic benefit for patients with TSC/LAM or other hyperactive mTOR neoplasms with autophagy inhibition.

Targeting SPHK1/PBX1 Axis Induced Cell Cycle Arrest in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) accounts for 85~90% of lung cancer cases, with a poor prognosis and a low 5-year survival rate. Sphingosine kinase-1 (SPHK1), a key enzyme in regulating sphingolipid metabolism, has been reported to be involved in the development of NSCLC, although the underlying mechanism remains unclear. In the present study, we demonstrated the abnormal signature of SPHK1 in NSCLC lesions and cell lines of lung cancers with a potential tumorigenic role in cell cycle regulation. Functionally, ectopic Pre-B cell leukemia homeobox-1 (PBX1) was capable of restoring the arrested G1 phase induced by SPHK1 knockdown. However, exogenous sphingosine-1-phosphate (S1P) supply had little impact on the cell cycle arrest by PBX1 silence. Furthermore, S1P receptor S1PR3 was revealed as a specific switch to transport the extracellular S1P signal into cells, and subsequently activated PBX1 to regulate cell cycle progression. In addition, Akt signaling partially participated in the SPHK1/S1PR3/PBX1 axis to regulate the cell cycle, and the Akt inhibitor significantly decreased PBX1 expression and induced G1 arrest. Targeting SPHK1 with PF-543 significantly inhibited the cell cycle and tumor growth in preclinical xenograft tumor models of NSCLC. Taken together, our findings exhibit the vital role of the SPHK1/S1PR3/PBX1 axis in regulating the cell cycle of NSCLC, and targeting SPHK1 may develop a therapeutic effect in tumor treatment.

Targeting sphingosine kinase 1 (SK1) enhances oncogene-induced senescence through ceramide synthase 2 (CerS2)-mediated generation of very-long-chain ceramides

Senescence is an antiproliferative mechanism that can suppress tumor development and can be induced by oncogenes such as genes of the Ras family. Although studies have implicated bioactive sphingolipids (SL) in senescence, the specific mechanisms remain unclear. Here, using MCF10A mammary epithelial cells, we demonstrate that oncogenic K-Ras (Kirsten rat sarcoma viral oncogene homolog) is sufficient to induce cell transformation as well as cell senescence—as revealed by increases in the percentage of cells in the G1 phase of the cell cycle, p21WAF1/Cip1/CDKN1A (p21) expression, and senescence-associated β-galactosidase activity (SA-β-gal). Furthermore, oncogenic K-Ras altered SL metabolism, with an increase of long-chain (LC) C18, C20 ceramides (Cer), and very-long-chain (VLC) C22:1, C24 Cer, and an increase of sphingosine kinase 1 (SK1) expression. Since Cer and sphingosine-1-phosphate have been shown to exert opposite effects on cellular senescence, we hypothesized that targeting SK1 could enhance oncogenic K-Ras-induced senescence. Indeed, SK1 downregulation or inhibition enhanced p21 expression and SA-β-gal in cells expressing oncogenic K-Ras and impeded cell growth. Moreover, SK1 knockdown further increased LC and VLC Cer species (C18, C20, C22:1, C24, C24:1, C26:1), especially the ones increased by oncogenic K-Ras. Fumonisin B1 (FB1), an inhibitor of ceramide synthases (CerS), reduced p21 expression induced by oncogenic K-Ras both with and without SK1 knockdown. Functionally, FB1 reversed the growth defect induced by oncogenic K-Ras, confirming the importance of Cer generation in the senescent phenotype. More specifically, downregulation of CerS2 by siRNA blocked the increase of VLC Cer (C24, C24:1, and C26:1) induced by SK1 knockdown and phenocopied the effects of FB1 on p21 expression. Taken together, these data show that targeting SK1 is a potential therapeutic strategy in cancer, enhancing oncogene-induced senescence through an increase of VLC Cer downstream of CerS2.

Abstract PO-100: Ancestry-related variation in Sphingosine Kinase Type 1-Interacting Protein (SKIP) and Sphingosine Kinase 1 (SPHK1) and response to secondary hormonal therapy in metastatic castration-resistant prostate cancer

Abstract The number of cases and deaths from prostate cancer (PCa) is highest for African American (AA) men compared with men of other racial and ethnic groups, and AA men more frequently have more aggressive disease. However, recent studies have shown that AA PCa patients have a better response to certain therapeutic regimens than white PCa patients. We conducted a DoD Prostate Cancer Clinical Trials Consortium (PCCTC) prospective study of secondary hormonal therapy (HT) in metastatic castration-resistant PCa (mCRPC) patients stratified by race, Abi-Race. This study enrolled 50 AA and 50 white patients with mCRPC and received abiraterone and prednisone daily until disease progression or adverse event. AA men had higher rates of Prostate Specific Antigen (PSA) response and time to PSA progression. Herein we focus on correlative science in the context of Abi-Race to determine novel relationships between ancestry-related genetic variation and response and time to progression on secondary HT in mCRPC. An exploratory genome-wide analysis assessing the role of genotypic and local ancestry variation with respect to time to progression identified a missense variant in Sphingosine Kinase Type 1-Interacting Protein (SKIP) with predicted pathogenicity and potentially high ancestral variation. SKIP plays a role in modulating the conversion of sphingosine to sphingosine-1-phosphate (S1P) by regulating Sphingosine Kinase 1 (SPHK1) activity within the cytosol. S1P is a potent lipid mediator that plays a role in multiple cancer-promoting biofunctions. SKIP directly binds and inhibits SPHK1 activity, resulting in the decreased production of S1P and S1P-associated cell signaling. The relationship between SKIP and SPHK1 and response to secondary HT in mCRPC was investigated. We knocked down SKIP or SPHK1 in LN95 prostate cancer cells and assessed resulting alterations in proliferation with or without abiraterone. Knockdown of SKIP increased proliferation in untreated cells and knockdown cells were more resistant to treatment with abiraterone compared with the control group. Conversely, knockdown of SPHK1 decreased proliferation in untreated cells and knockdown cells were more sensitive to treatment with abiraterone compared with the control group. In addition, we are measuring changes in sphingosine and S1P in serum samples we collected from fasting Abi-Race patients at baseline and cycle 4 of treatment to investigate ancestry-related sphingosine and S1P variations and associated outcomes. Lastly, we are further investigating the potential function of the variant in SKIP associated with time to progression on secondary HT in mCRPC. These findings will further the understanding of ancestry-related biological factors that influence response to secondary HT in mCRPC and could have direct implications for the timing and selection of AA patients for secondary HT and those needing additional therapy. Ultimately, such strategies have the potential to mitigate prostate cancer disparities. Citation Format: Sean A. Piwarski, Tyler A. Allen, Dadong Zhang, Alexander B. Sibley, Patrick Healy, Brendon M. Patierno, Bonnie L. LaCroix, Rick A. Kittles, Kouros Owzar, Terry Hyslop, Steven R. Patierno, Daniel J. George, Jennifer A. Freedman. Ancestry-related variation in Sphingosine Kinase Type 1-Interacting Protein (SKIP) and Sphingosine Kinase 1 (SPHK1) and response to secondary hormonal therapy in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-100.

Sphingosine Kinase Blockade Leads to Increased Natural Killer T Cell Responses to Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin’s lymphoma. Despite being responsive to combination chemotherapy, median survival remains around 5 years due to high rates of relapse. Sphingolipid metabolism regulates MCL survival and proliferation and we found that sphingosine-1-phosphate (S1P) is upregulated in MCL cells. Therapeutic targeting of the S1P1 receptor or knockdown of sphingosine kinase 1 (SK1), the enzyme responsible for generating S1P, in human MCL cells results in a significant increase in Natural Killer T (NKT) cell activation. NKT cells recognize glycolipid antigens presented on CD1d and can reduce MCL tumor burden in vivo. Lipidomic studies identified cardiolipin, which has been reported to bind to CD1d molecules, as being upregulated in SK1 knockdown cells. We found that the pretreatment of antigen presenting cells with cardiolipin leads to increased cytokine production by NKT cell hybridomas. Furthermore, the ability of cardiolipin to activate NKT cells was dependent on the structure of its acyl chains. Collectively, these studies delineate novel pathways important for immune recognition of malignant cells and could lead to the development of new treatments for lymphoma.

Sphingosine Kinase 1 in Breast Cancer-A New Molecular Marker and a Therapy Target.

It is now well-established that sphingosine kinase 1 (SK1) plays a significant role in breast cancer development, progression, and spread, whereas SK1 knockdown can reverse these processes. In breast cancer cells and tumors, SK1 was shown to interact with various pathways involved in cell survival and chemoresistance, such as nuclear factor-kappa B (NFκB), Notch, Ras/MAPK, PKC, and PI3K. SK1 is upregulated by estrogen signaling, which, in turn, confers cancer cells with resistance to tamoxifen. Sphingosine-1-phosphate (S1P) produced by SK1 has been linked to tumor invasion and metastasis. Both SK1 and S1P are closely linked to inflammation and adipokine signaling in breast cancer. In human tumors, high SK1 expression has been linked with poorer survival and prognosis. SK1 is upregulated in triple negative tumors and basal-like subtypes. It is often associated with high phosphorylation levels of ERK1/2, SFK, LYN, AKT, and NFκB. Higher tumor SK1 mRNA levels were correlated with poor response to chemotherapy. This review summarizes the up-to-date evidence and discusses the therapeutic potential for the SK1 inhibition in breast cancer, with emphasis on the mechanisms of chemoresistance and combination with other therapies such as gefitinib or docetaxel. We have outlined four key areas for future development, including tumor microenvironment, combination therapies, and nanomedicine. We conclude that SK1 may have a potential as a target for precision medicine, its high expression being a negative prognostic marker in ER-negative breast cancer, as well as a target for chemosensitization therapy.

Sphingosine kinase 1 promotes cerebral ischemia-reperfusion injury through inducing ER stress and activating the NF-κB signaling pathway.

Endoplasm reticulum stress and inflammation response have been found to be linked to cerebral ischemia-reperfusion (IR) injury. Sphingosine kinase 1 (SPHK1) has been reported to be a novel endoplasm reticulum regulator. The aim of our study is to figure out the role of SPHK1 in cerebral IR injury and verify whether it has an ability to regulate inflammation and endoplasm reticulum stress. Hydrogen peroxide was used to induce cerebral IR injury. Enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, western blots, and immunofluorescence were used to measure the alterations of cell viability, inflammation response, and endoplasm reticulum stress. The results demonstrated that after exposure to hydrogen peroxide, cell viability was reduced whereas SPHK1 expression was significantly elevated. Knockdown of SPHK1 attenuated hydrogen peroxide-mediated cell death and reversed cell viability. Our data also demonstrated that SPHK1 deletion reduced endoplasm reticulum stress and alleviated inflammation response in hydrogen peroxide-treated cells. In addition, we also found that SHPK1 modulated endoplasm reticulum stress and inflammation response to through the NF-κB signaling pathway. Inhibition of NF-κB signaling pathway has similar results when compared with the cells with SPHK1 deletion. Altogether, our results demonstrated that SPHK1 upregulation, induced by hydrogen peroxide, is responsible for cerebral IR injury through inducing endoplasm reticulum stress and inflammation response in a manner working through the NF-κB signaling pathway. This finding provides new insight into the molecular mechanism to explain the neuron death induced by cerebral IR injury.

RED LIGHT IRRADIATION REGULATES ROS SCAVENGING AND ANTI-INflAMMATION THROUGH SPHK1/NF-ΚB PATHWAY IN HACAT CELLS

Objectives Oxidative stress is a well-accepted pathogenesis of several human diseases, which is an increased amount of the oxidants exceeding the capacity of antioxidant defense system. Light-emitting diode irradiation (LEDI) represents an efficient strategy to counteract this condition. The purpose of the present study was to evaluate the ROS scavenging and anti-inflammatory mechanism of LEDI. Findings cDNA microarray, semi quantitative PCR (semi-qPCR), western blotting and small-interfering RNA (siRNA) transfection were processed on PMA induced oxidative stress and inflammation in HaCaT cells. In this study, 625 nm LEDI showed the effect of ROS scavenging and anti-inflammation. One of the most important genes which identified by microarray analysis was sphingosine kinase-1 (SPHK1), which is a key enzyme in sphingolipid metabolism. SPHK1 knockdown drastically reduced the viability of ROS scavenging in the presence of PMA-stimulated HaCaT cells. Furthermore, results with cyclooxygenease-2 (COX-2) and prostaglandin E2(PGE2) further indicated the importance of the SPHK1 in anti-inflammatory process in HaCaT cells. Conclusions The results obtained in this work highlight the possible role of SPHK1 in ROS scavenging and anti-inflammation in PMA-stimulated HaCaT cells, investigating for the first time the possibility its involved molecular mechanisms. And SPHK1 can be used as a therapeutic target in LEDI treatments for treating skin disorders through ROS scavenging and/or anti-inflammation.

Increased SPK1 expression promotes cell growth by activating the ERK1/2 signaling in non-small-cell lung cancer.

Lung cancer remains the leading cause of cancer-associated mortality in China and the world. Increasing numbers of studies have reported that sphingosine kinase 1 (SPK1) is frequently highly expressed in tumors of various origins, including lung cancer, and its high expression contributes toward tumor progression. However, the clinical significance of SPK1 and its role in the growth and metastasis of non-small-cell lung cancer (NSCLC) remain unclear. In the present study, we found that SPK1 expression was expressed highly in NSCLC tissues and cell lines. Knockdown of SPK1 suppressed cell growth, proliferation, migration, and invasion and increased apoptosis. Moreover, knocking down SPK1 expression inhibited the growth of tumors in nude mice. Mechanistically, silencing the expression of SPK1 inhibited the expression of p-extracellular signal-regulated kinase (ERK). Moreover, the ERK-specific inhibitor U0126 suppressed the expression of the epithelial-mesenchymal transition of lung cancer cells. Together, our findings indicated that SPK1 enhanced tumor growth in lung cancer and induced metastasis by activating the ERK1/2 signaling pathway, indicating its potential application in NSCLC diagnosis and therapy.

Links between accelerated replicative cellular senescence and down-regulation of SPHK1 transcription

We have identified a mechanism to diminish the proliferative capacity of cells during cell expansion using human adipose-derived stromal cells (hAD-SCs) as a model of replicative senescence. hAD-SCs of high-passage numbers exhibited a reduced proliferative capacity with accelerated cellular senescence. Levels of key bioactive sphingolipids were significantly increased in these senescent hAD-SCs. Notably, the transcription of sphingosine kinase 1 (SPHK1) was down-regulated in hAD-SCs at high-passage numbers. SPHK1 knockdown as well as inhibition of its enzymatic activity impeded the proliferation of hAD-SCs, with concomitant induction of cellular senescence and accumulation of sphingolipids, as seen in high-passage cells. SPHK1 knockdown-accelerated cellular senescence was attenuated by co-treatment with sphingosine-1-phosphate and an inhibitor of ceramide synthesis, fumonisin B1, but not by treatment with either one alone. Together, these results suggest that transcriptional down-regulation of SPHK1 is a critical inducer of altered sphingolipid profiles and enhances replicative senescence during multiple rounds of cell division.

Up-regulation and tumor-promoting role of SPHK1 were attenuated by miR-330-3p in gastric cancer.

We intended to clarify the role of sphingosine kinase 1 (SPHK1) in gastric cancer (GC) using both in vitro and in vivo assays. The study was designed to identify novel therapeutic targets for GC treatment. Differential analysis was utilized to dissect two gene expression omnibus series (GSE49515 and GSE79973) microarray data form Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) dataset. MRNA and protein expressions were determined by quantitative polymerase chain reaction and Western blot, respectively. GC cell growth was measured by MTT assays and verified by in vivo analysis. Cell cycle and cell apoptosis were detected via flow cytometer observation. Cell migration and invasion were assessed by wound healing assays and Transwell assays. The targeting relationship between miRNA and SPHK1/S1PR1 was identified via dual-luciferase assay. Twenty-four common differentially expressed genes were screened out from two gene expression omnibus series (GSE49515 and GSE79973), among which SPHK1 was chosen for its higher fold change. We found elevated SPHK1 expression in GC tissues and cells, along with an increased concentration of SPHK1-generated sphingosine-1-phosphate (S1P) in both GC serum and tissue. SPHK1 knockdown significantly suppressed cell proliferation, migration, and invasion of MKN1 and KATO3 cells. It also blocked cell cycle and induced apoptosis in MKN1 and KATO3 cells. Silencing of SPHK1 also refrained tumor growth and inhibited S1P level. MiR-330-3p directly targeted SPHK1 and S1PR1. Overexpressed miR-330-3p in MKN1 cells repressed SPHK1 and S1PR1 expressions like their chemical inhibitors-SPHK1 inhibitors (FTY720) and S1PR1 inhibitors (VPC23019), and acted anti-tumor both in vitro and in vivo. Our study provides evidence that SPHK1 was promotive for GC tumor growth and cell biological behaviors, and that miR-330-3p targeted 3'-UTR of SPHK1 and inhibited its expression. SPHK1 was expected to become a new molecular marker and miR-330-3p a novel therapeutic target for GC. © 2018 IUBMB Life, 70(11):1164-1176, 2018.

Lentivirus-mediated siRNA knockdown of SPHK1 inhibits proliferation and tumorigenesis of neuroblastoma.

BackgroundThe overexpression of sphingosine kinase 1 (SPHK1) is responsible for the progress of many cancers. However, the role of SPHK1 in the development and progression of neuroblastoma (NB) remain largely unknown. Here in this study, we explored whether silencing SPHK1 by lentivirus-mediated siRNA could be employed as a potential therapeutic target for NB.Materials and methodsLentivirus was adopted to load SPHK1 siRNA. The results were obtained using RT-qPCR, Western blot, cell proliferation assay, transwell cell migration/invasion assays as well as in vivo xenograft tumor models in nude mice.ResultsOur results demonstrated that SPHK1 mRNA was upregulated in SH-SY5Y and SK-N-SH cells as well as in human NB tissues. SPHK1 knockdown by siRNA resulted in impaired proliferation, increased apoptosis, as well as impaired migration and invasion of SH-SY5Y and SK-N-SH cells. In addition, the in vivo study suggested that SPHK1 knockdown significantly reduced the tumorigenesis of SH-SY5Y xenograft model. Furthermore, intratumorally administered lentivirus-SPHK1 siRNA could significantly inhibit tumor growth in an SH-SY5Y xenograft mice model. Intensive investigations on mechanism revealed that these effects were achieved through the deactivation of STAT3 pathways.ConclusionThese data suggest that SPHK1 inhibition via downregulation of STAT3 pathways by lentivirus-mediated siRNA knockdown can significantly suppress NB progression, which could be a promising target for future gene therapy of NB.

Red light-emitting diode irradiation regulates oxidative stress and inflammation through SPHK1/NF-κB activation in human keratinocytes

Oxidative stress, in which the amount of oxidants exceeds the capacity of antioxidant defense system, is a well-accepted pathogenesis of several human diseases. Light-emitting diode irradiation (LEDI) is an efficient strategy to counteract this condition. The biological effect of phototherapy, using visible light, has attracted recent attention especially in dermatological practice. However, little is known about the molecular mechanism of the anti-oxidant and anti-inflammatory effects of red light irradiation. We evaluated these effects of LEDI in HaCaT human keratinocyte cells under phorbol-12-myristate-13-acetate (PMA) induced reactive oxygen species (ROS). Microarray analysis revealed changes in 309 genes after LEDI. LEDI at 625 nm produced ROS scavenging and anti-inflammatory effects. One of the most important genes identified by microarray analysis was sphingosine kinase-1 (SPHK1), which is a key molecule in sphingolipid metabolism. SPHK1 knock-down drastically reduced ROS scavenging efficiency as well as expression levels of inflammation-related proteins in PMA-treated HaCaT cells. These results not only indicate the potential for the clinical application of 625-nm LEDI in treating skin disorders via ROS and/or inflammation, but also suggest SPHK1 as a potential therapeutic target in phototherapy.

Oncogenic S1P signalling in EBV-associated nasopharyngeal carcinoma activates AKT and promotes cell migration through S1P receptor 3.

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine-1-phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV-encoded latent genes (EBNA1, LMP1, and LMP2A) can up-regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is overexpressed in EBV-positive NPC patient-derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P-induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A novel role of sphingosine kinase-1 in the invasion and angiogenesis of VHL mutant clear cell renal cell carcinoma.

Sphingosine kinase 1 (SK1), the enzyme responsible for sphingosine 1-phosphate (S1P) production, is overexpressed in many human solid tumors. However, its role in clear cell renal cell carcinoma (ccRCC) has not been described previously. ccRCC cases are usually associated with mutations in von Hippel-Lindau (VHL) and subsequent normoxic stabilization of hypoxia-inducible factor (HIF). We previously showed that HIF-2α up-regulates SK1 expression during hypoxia in glioma cells. Therefore, we hypothesized that the stabilized HIF in ccRCC cells will be associated with increased SK1 expression. Here, we demonstrate that SK1 is overexpressed in 786-0 renal carcinoma cells lacking functional VHL, with concomitant high S1P levels that appear to be HIF-2α mediated. Moreover, examining the TCGA RNA seq database shows that SK1 expression was ∼2.7-fold higher in solid tumor tissue from ccRCC patients, and this was associated with less survival. Knockdown of SK1 in 786-0 ccRCC cells had no effect on cell proliferation. On the other hand, this knockdown resulted in an ∼3.5-fold decrease in invasion, less phosphorylation of focal adhesion kinase (FAK), and an ∼2-fold decrease in angiogenesis. Moreover, S1P treatment of SK1 knockdown cells resulted in phosphorylation of FAK and invasion, and this was mediated by S1P receptor 2. These results suggest that higher SK1 and S1P levels in VHL-defective ccRCC could induce invasion in an autocrine manner and angiogenesis in a paracrine manner. Accordingly, targeting SK1 could reduce both the invasion and angiogenesis of ccRCC and therefore improve the survival rate of patients.

Abstract B09: Sphingosine kinase 1 (SPHK1) is a novel mediator of tumor-stroma interaction in ovarian cancer

Abstract Sphingosine kinase-1 (SPHK1) is an enzyme that catalyzes the formation of the prosurvival second messenger sphingosine-1-phosphate (S1P) from the proapoptotic lipid sphingosine. The balance between sphingosine and S1P forms a sphingolipid rheostat that is primed for cell death when the balance shifts towards sphingosine/ceramide or to cell survival when S1P levels are increased. Elevated levels of S1P have been observed in several cancers, including ovarian cancer where it is elevated in both the ascites and serum of patients. Correspondingly, high expression of SPHK1 has also been described in multiple cancer types and has been linked to disease progression. Here we report that SPHK1 is overexpressed in a subset of epithelial ovarian cancers (EOC) and correlates with poor progression-free survival. Overexpression and knockdown of SPHK1 in multiple human EOC cell lines modulates in vitro cell proliferation, anchorage-independent growth, and chemosensitivity. In mouse xenograft studies, intraperitoneal administration of a SPHK1-specific inhibitor (SKI-5c) decreases tumor size, indicating that SPHK1 may be a potential therapeutic target in EOC. S1P is a secreted factor that has been shown to influence the tumor microenvironment of other cancers; however its role in EOC has not been fully established. Using a co-culture model, we show that overexpression of SPHK1 in EOC cells stimulates the transition of normal ovarian stromal fibroblasts to myofibroblasts, and enhances stromal SPHK1 expression. In a reciprocal manner, overexpression of SPHK1 in stromal fibroblast results in increased expression of MMP2 and MMP9 in EOC cells, which may contribute to a more invasive phenotype. Interestingly, stromal SPHK1 expression also enhanced expression of SPHK1 and the S1P receptor (S1PR1) in the EOC cells. Biostatistical analysis suggests a significant correlation between SPHK1 expression in EOC cells and the expression of several extracellular matrix genes (FN1, POSTN, VCAN), which have been shown to have role in the EOC tumor microenvironment. Overall, these results suggest that SPHK1 is a critical regulator of ovarian tumor cell proliferation and survival, and a mediator of tumor-stroma interaction. Citation Format: Jessica A. Beach, Paul-Joseph Aspuria, Dong-Joo Cheon, Maricel C. Gozo, Beth Y. Karlan, Sandra Orsulic. Sphingosine kinase 1 (SPHK1) is a novel mediator of tumor-stroma interaction in ovarian cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B09. doi:10.1158/1538-7445.CHTME14-B09

Sphingosine kinase 1 contributes to leptin-induced STAT3 phosphorylation through IL-6/gp130 transactivation in oestrogen receptor-negative breast cancer.

Obesity is a known risk factor for breast cancer. We have recently identified that adipokine leptin regulates the expression of a proto-oncogenic enzyme sphingosine kinase 1 (SK1). Signal transducer and activator of transcription 3 (STAT3) has been linked to breast cancer progression and here we investigate the mechanism of leptin-induced STAT3 activation in ER-negative breast cancer. Gene and protein expression in human primary and secondary breast cancer tissues was analysed using quantitative real-time polymerase chain reaction (qRT-PCR) assay and immunofluorescence. Leptin-induced signalling was analysed in human ER-negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays. Gene expression and receptor signalling was modified using small interfering RNA and neutralising antibodies. In human ER-negative breast tumours and lymph node metastases, the expression of leptin receptor significantly correlated with SK1. In ER-negative breast cancer cells, SK1 knockdown led to a significant reduction in leptin-induced STAT3 phosphorylation. Knockdown of another known activator of STAT3 signalling, gp130 also resulted in a significant decrease in leptin-induced STAT3 phosphorylation. ELISA assay showed that leptin produces a significant amount of IL-6 in an SK1-dependent manner. IL-6 neutralising antibodies significantly reduced p-STAT3. Immunofluorescent staining of human primary and secondary breast tumours showed significant correlation between SK1 and IL-6 (P < 0.001), SK1 and p-STAT3 (P < 0.01) and IL-6 and p-STAT3 (P < 0.01). Our findings demonstrate that leptin-induced STAT3 is partially cross activated through SK1-mediated IL6 secretion and gp130 activation. Positive correlations in human tissues suggest the potential significance of this pathway in ER-negative breast cancer.

Abstract 2010: SPHK1 promotes lung metastasis of breast cancer

Abstract Breast cancer is the second most commonly diagnosed cancer and second leading cause of cancer death in American women. The majority of cancer-related fatalities are a result of metastases to distant organs, rather than due to the primary tumors themselves. The role of the lipid metabolic pathways in breast cancer progression and metastasis has become a focal point of interest in recent years and our work is mainly focused on sphingolipids. Sphingosine-1-phosphate (S1P) is a lipid signaling mediator that functions both intracellularly, as a second messenger signaling molecule, and extracellularly, as a ligand for G-protein coupled S1P receptors (S1PRs). S1P plays important roles in modulating diverse cellular functions involved in cancer, i.e. cell growth, survival, and motility. Sphingosine Kinase 1 (SPHK1) catalyzes the phosphorylation of sphingosine, an amino alcohol, to generate S1P. Recently, studies have shown that SPHK1 is overexpressed in breast cancer and correlates with poor prognosis; thus, its role in breast cancer has received substantial attention. It is still unclear if SPHK1 plays any role in spontaneous metastasis of breast cancer to the lungs, and if so, the underlying mechanism behind it. To study this, we used the highly metastatic human triple negative breast cancer cell line MDA-MB-435, as it expressed high levels of endogenous SPHK1. We generated SPHK1 knockdown (435 shSPHK1) and control (435 shScramble) cells using SPHK1-targeting shRNA and scrambled shRNA, respectively. Knocking down SPHK1 decreased the invasive and migration potential of MDA-MB-435 cells in vitro, but had no significant effect on in vitro cell proliferation. Then, the control and knockdown cells were orthotopically injected into mammary fat pads (MFP) of nude mice to form MFP tumors. MFP tumors were excised from mice with survival surgery at day 30. Consistent with our in vitro proliferation data, there was no significant difference in MFP tumor proliferation, indicated by Ki-67 staining, between control and SPHK1 knockdown cells by day 30. However, TUNEL staining of MFP tumors showed that tumors formed by 435 shSPHK1 cells have increased apoptosis compared to tumors formed by 435 shScramble cells. After tumor excision at day 30, mice were monitored for another 4 weeks for spontaneous metastases. Remarkably, although ∼100% of mice injected with 435 shScramble developed spontaneous lung metastasis, only ∼20% of mice injected with 435 shSPHK1 had spontaneous lung metastasis. No metastasis was detected in the liver and the brain in this time frame. These data suggest that SPHK1 might play an important role in promoting spontaneous lung metastasis of breast cancer. Currently, we are investigating the specific step(s) of the metastatic cascade regulated by SPHK1. An understanding of the molecular mechanism of SPHK1-mediated breast cancer lung metastasis may enable SPHK1 to be developed as a molecular marker and a therapeutic target of metastatic breast cancer. Citation Format: Sunil Acharya, Chenyu Zhang, Frank L. Lowery, Qingling Zhang, Dihua Yu. SPHK1 promotes lung metastasis of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2010. doi:10.1158/1538-7445.AM2014-2010