Sphingosine kinase 1 promotes growth of glioblastoma by increasing inflammation mediated by the NF- κ B /IL-6/STAT3 and JNK/PTX3 pathways

Abstract

Glioblastoma (GBM) is the most challenging malignant tumor of the central nervous system because of its high morbidity, mortality, and recurrence rate. Currently, mechanisms of GBM are still unclear and there is no effective drug for GBM in the clinic. Therefore, it is urgent to identify new drug targets and corresponding drugs for GBM. In this study, in silico analyses and experimental data show that sphingosine kinase 1 (SPHK1) is up-regulated in GBM patients, and is strongly correlated with poor prognosis and reduced overall survival. Overexpression of SPHK1 promoted the proliferation, invasion, metastasis, and clonogenicity of GBM cells, while silencing SPHK1 had the opposite effect. SPHK1 promoted inflammation through the NF-κB/IL-6/STAT3 signaling pathway and led to the phosphorylation of JNK, activating the JNK-JUN and JNK-ATF3 pathways and promoting inflammation and proliferation of GBM cells by transcriptional activation of PTX3. SPHK1 interacted with PTX3 and formed a positive feedback loop to reciprocally increase expression, promote inflammation and GBM growth. Inhibition of SPHK1 by the inhibitor, PF543, also decreased tumorigenesis in the U87-MG and U251-MG SPHK1 orthotopic mouse models. In summary, we have characterized the role and molecular mechanisms by which SPHK1 promotes GBM, which may provide opportunities for SPHK1-targeted therapy.

Keywords: ATF3; Drug target; Glioblastoma; Inflammation; NF-κB/IL-6/STAT3 signal pathway; PXT3; SPHK1.

Graphical abstract

Figure 1

SPHK1 is highly expressed in patients with glioma and associated with clinical features. Data from (A) GEPIA (http://gepia.cancer-pku.cn/), (B) Broad Institute (http://gdac.broadinstitute.org/.), (C) Human Protein Atlas (http://www.proteinatlas.org/), (D) Betastasis (https://www.betastasis.com/), and (E) Oncomine website (www.oncomine.org) shows that the expression of SPHK1 is higher in GBM tissues and cells than in normal tissues or other kinds of tumor. (F) Data from Betastasis website (https://www.betastasis.com/), UCSC Xena website (http://xena.ucsc.edu/) and GEPIA website (http://gepia.cancer-pku.cn/) shows that the overall survival rate of patients with high SPHK1 expression is lower than that of patients with low SPHK1 expression. (G) Immunohistochemical analysis shows that the expression of SPHK1 is significantly higher in GBM tissues than in normal brain tissues.

Figure 2

SPHK1 promotes proliferation, migration, invasion, colony formation, and growth of GBM cells. (A) Approach for defining the function of SPHK1 in GBM cells. (B) Western blot and ELISA results show that the protein levels of SPHK1 and S1P were higher in A172 and U87-MG cells and lower in U251-MG and H4 cells. (C) Overexpression of SPHK1 in U251 cells. (D) Overexpression of SPHK1 promotes proliferation, colony formation, and cell growth in 3D Matrigel in U251 cells. (E) Knockdown of SPHK1 in U87-MG and A172 cells. (F) Knockdown of SPHK1 inhibited proliferation, colony formation, and cell growth in 3D Matrigel in U87-MG and A172 cells. (G) Overexpression of SPHK1 promoted migration and invasion of U251-MG cells. (H) Knockdown of SPHK1 had the opposite effects in U87-MG and A172 cells. The data are presented as mean ± SD, and the experiments were performed in triplicate.

Figure 3

SPHK1 is associated with inflammatory signaling pathways. (A) Strategy for investigating the mechanisms of SPHK1. (B) Volcano plot of differentially expressed genes in U251 cells after overexpression of SPHK1 (up-regulated genes are in red; down-regulated genes are in blue; fold change ≥2). (C) Gene ontology analysis of DEGs after overexpression of SPHK1. (D) KEGG pathway enrichment of DEGs (fold change ≥2 and P < 0.05) (E) RT-qPCR results confirmed that the mRNA levels of key genes involved in inflammatory signaling were higher after overexpression of SPHK1 in U251-MG cells. (F) The concentration of IL-6 and IL-1β was higher in U251 cells after overexpression of SPHK1. (G) The concentration of IL-6 was lower in U87-MG and A172 cells after silencing of SPHK1. (H) The protein levels of NF-κB and STAT3 were increased in U251 cells after overexpression of SPHK1. (I) The protein levels of NF-κB and STAT3 were decreased in U87-MG and A172 cells after silencing of SPHK1. The data are presented as mean ± SD, and the experiments were performed in triplicate.

Figure 4

PTX3 is regulated by SPHK1 and promotes inflammation and proliferation of GBM cells. (A) Heatmap of top-ranked DEGs after SPHK1 overexpression in U251 cells. (B) The mRNA and protein expression of PTX3 was increased in U251 cells after overexpression of SPHK1. (C) The mRNA and protein expression of PTX3 was decreased in A172 and U87-MG cells after knockdown of SPHK1. (D) The concentration of PTX3 was increased in U251 cells after overexpression of SPHK1. (E) Confocal immunofluorescence staining of SPHK1 and PTX3 showed that protein expression of PTX3 was increased after overexpression of SPHK1. (F) Data from GEPIA website (http://gepia.cancer-pku.cn/) shows that expression of PTX3 is higher in GBM tissues than in healthy tissues. (G) Data from the Broad Institute website (http://gdac.broadinstitute.org/.) shows that the expression of PTX3 is higher in GBM tissues than in healthy tissues. Data from (H, I) Oncomine (www.oncomine.org) and (J) UALCAN (http://ualcan.path.uab.edu/index.html) website shows that the expression of PTX3 is higher in GBM tissues than in normal tissues. (K) Data from Betastasis (https://www.betastasis.com/) and GEPIA (http://gepia.cancer-pku.cn/) website shows that the overall survival rate in patients with high expression of PTX3 is lower than in those with low expression of PTX3. (L) Immunohistochemical results indicate that the expression of PTX3 was higher in GBM tissues than in normal tissues. Representative images showing the expression of PTX3 in GBM and adjacent normal tissues. The data are presented as mean ± SD, and the experiments were performed in triplicate.

Figure 5

PTX3 mediates the function of SPHK1 in GBM cells. (A) Protein expression of PTX3 in normal vs. GBM cells. (B) Overexpression of PTX3 increases the ratio of p-STAT3/STAT3 and the release of IL-6, and promotes colony formation of U251-MG cells. (C) Knockdown of PTX3 decreases the ratio of p-STAT3/STAT3 and the release of IL-6, and inhibits colony formation of U138-MG cells. (D) Strategy for investigating PTX3 function after overexpression of SPHK1. (E) Knockdown of PTX3 in U251 cells with overexpressed SPHK1. (F) ELISA results show that silencing PTX3 impairs release of IL-6 caused by overexpression of SPHK1 in U251-MG cells. (G) Silencing PTX3 decreases colony formation caused by overexpression of SPHK1 in U251-MG cells. (H) Silencing PTX3 inhibits growth of U251 cells in 3D Matrigel caused by overexpression of SPHK1. EdU analysis (I) and CCK-8 analysis (J) show that silencing PTX3 inhibits proliferation of U251 cells caused by overexpression of SPHK1. (K) Results from confocal immunofluorescence show that SPHK1 and PTX3 were co-localized in the cytoplasm and nuclei in U251-MG and U87-MG cells. The data are presented as mean ± SD, and the experiments were performed in triplicate.

Figure 6

SPHK1 promotes the expression of PTX3 through ATF3. (A) Strategy for identifying ATF3 as the key transcription factor in regulating the expression of PTX3. (B) In silico analysis of TCGA data shows that SPHK1 is highly correlated with PTX3. (C) Results from confocal immunofluorescence show that SPHK1 and ATF3 or ATF3 and PTX3 are co-localized in the nucleus in U251-MG and U87-MG cells. (D) Prediction of the binding site of ATF3 in the promoter region of PTX3 using PROMO website (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3.). (E) ChIP-qPCR analysis shows that ATF3 binds to PTX3 promoter in U251 cells. (F) Gel electrophoresis of ChIP-qPCR products. (G) Confocal immunofluorescence assay revealing that overexpression of ATF3 increases PTX3 expression in U251-MG cells. (H) Western blot results show that silencing ATF3 impairs the effects of SPHK1 on the expression of ATF3, PTX3, and the ratio of p-STAT3/STAT3 in U251-MG cells. Silencing ATF3 impairs the effect of SPHK1 on the expression of PTX3 (I), colony formation (J), 3D growth (K) in U251-MG cells. The data are presented as mean ± SD, and the experiments were performed in triplicate.

Figure 7

SPHK1 activates ATF3 through the JNK pathway and directly interacts with PTX3. (A) Strategy for identifying which pathway mediates the effects of SPHK1 on the regulation of PTX3. (B) Western blot results show that overexpression of SPHK1 increases the ratio of p-ERK/ERK and p-JNK/JNK in U251 cells. (C) Western blot results show that knockdown of SPHK1 and A172 cells reduces the ratio of P-ERK/ERK, p-JNK/JNK, and p-JUN/JUN in A172 and U87 cells. (D) The JNK inhibitor, SP600125, but not the ERK inhibitor, PD98059, suppresses expression of p-STAT3/STAT3, ATF3, and PTX3 after overexpression of SPHK1 in U251-MG cells. The JNK inhibitor, SP600125, but not the ERK inhibitor, PD98059, suppresses the release of IL-6 (E), colony formation (F) and growth in 3D Matrigel (G) in U251-MG cells after overexpression of SPHK1. (H) Co-IP analysis shows that SPHK1 and PTX3 co-immunoprecipitated in U251-MG cells and GBM tissues. (I) Confocal immunofluorescence results show that SPHK1 and PTX3 co-localize in the cytoplasm and nuclei in U251-MG cells. (J) Overexpression of PTX3 increases the expression of ATF3, p-JUN/JUN, and p-JNK/JNK in U251 cells. (K) Overexpression of SPHK1 or PTX3 increases the expression of ATF3, p-JUN/JUN, and p-STAT3/STAT3 in the nuclei of U251 cells. The data are presented as mean ± SD, and the experiments were performed in triplicate.

Figure 8

SPHK1 promotes tumor growth and SPHK1 inhibitor, PF543, suppresses tumorigenesis in vivo. (A) Strategy for studying the tumor promotion effects of SPHK1 in vivo. (B) Overexpression of SPHK1 in U251-MG cells increases tumor volume and weight in nude mice. The data are presented as mean ± SD, n = 6 in each group. (C) Overexpression of SPHK1 in U251-MG cells increases the expression of PTX3 and S1P levels in nude mice. The data are presented as mean ± SD, n = 3 in each group. (D) Knockdown of SPHK1 in U87-MG cells suppresses tumor growth in nude mice. The data are presented as mean ± SD, n = 6 in each group. (E) Knockdown of SPHK1 in U87-MG cells decreases the expression of PTX3 and S1P levels in nude mice. The data are presented as mean ± SD, n = 3 in each group. (F) PF543 significantly reduces tumor volume in mice transplanted with U87-MG cells in the orthotopic model. The data are presented as mean ± SD, n = 3 for MRI and tumor volume and n = 6 for body weight. (G) PF543 significantly reduces the expression of PTX3 and S1P levels in mice transplanted with U87-MG cells in the orthotopic model. The data are presented as mean ± SD, n = 3 in each group. (H) PF543 significantly inhibits tumor growth in mice transplanted with U251-MG SPHK1 cells in the orthotopic model. The data are presented as mean ± SD, n = 3 in each group. (I) PF543 significantly reduces the expression of PTX3 and S1P levels in mice transplanted with U251-MG SPHK1 cells in the orthotopic model. The data are presented as mean ± SD, n = 3 for MRI and tumor volume and n = 6 for body weight.

Figure 9

Diagram showing the proposed mechanism of SPHK1 action in GBM cells. SPHK1 promotes growth of GBM by increasing inflammation mediated by the NF-κB/IL-6/STAT3 and JNK/PTX3 pathways.