Prosaposin promotes the proliferation and tumorigenesis of glioma through toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway

Abstract

Background: As a neurotrophic factor, prosaposin (PSAP) can exert neuroprotective and neurotrophic effects. It is involved in the occurrence and development of prostate and breast cancer. However, there is no research about the role of PSAP in glioma.

Methods: The PSAP overexpressed or silenced glioma cells or glioma stem cells were established based on Lentiviral vector transfection. Cell viability assay, Edu assay, neurosphere formation assay and xenograft experiments were used to detect the proliferative ability. Western blot, Elisa and luciferase reporter assays were used to detect the possible mechanism.

Findings: Our study firstly found that PSAP was highly expressed and secreted in clinical glioma specimens, glioma stem cells, and glioma cell lines. It was associated with poor prognosis. We found that PSAP significantly promoted the proliferation of glioma stem cells and cell lines. Moreover, PSAP promoted tumorigenesis in subcutaneous and orthotopic models of this disease. Furthermore, GSEA and KEGG analysis predicted that PSAP acts through the TLR4 and NF-κB signaling pathways, which was confirmed by western blot, immunoprecipitation, immunofluorescence, and use of the TLR4-specific inhibitor TAK-242.

Interpretation: The findings of this study suggest that PSAP can promote glioma cell proliferation via the TLR4/NF-κB signaling pathway and may be an important target for glioma treatment. FUND: This work was funded by National Natural Science Foundation of China (Nos. 81101917, 81270036, 81201802, 81673025), Program for Liaoning Excellent Talents in University (No. LR2014023), and Liaoning Province Natural Science Foundation (Nos. 20170541022, 20172250290). The funders did not play a role in manuscript design, data collection, data analysis, interpretation nor writing of the manuscript.

Keywords: Glioma; Glioma stem cells; Proliferation; Prosaposin; Tumorigenesis.

Fig. 1

PSAP is expressed at higher levels in glioma and is correlated with poor patient survival. a, b: Representative western blots showing higher PSAP expression in glioma patients with different grades compared to normal brain tissue (NBT). β-actin was used as a loading control. Densitometry data are shown as the mean ± S.D. relative to the respective negative control or vector control cells as appropriate (3 experiments). c: Representative immunohistochemistry staining for PSAP in NBT and glioma patients with different grades (grade II, n = 20; grade III, n = 25; grade IV, n = 25; NBT n = 10). Scale bar = 50 μm. (non-tumor vs. grade II, P = 0.0038; non-tumor vs. grade III, P < 0.0001; non-tumor vs. grade IV, P < 0.0001; one-way ANOVA). d: PSAP is expressed at higher levels in glioma patients with different grades of disease compared to NBT as measured by qPCR. e: Kaplan–Meier analysis of the 70 cases of glioma patients with high PSAP expression versus low PSAP expression by IHC. *P, 0.05; **P, 0.01; ***P, 0.001. (t-test).

Supplementary Fig. 1

Representative images of immunostaining with secondary antibody alone (up: control) or PSAP (down, PSAP).

Fig. 2

Endogenous PSAP expression in glioma cells. a: Immunofluorescence staining of CD133 in patient-derived glioma stem cells (GSCs), scale bar = 20 μm. b: After DMEM +10% FBS treatment, the patient-derived GSCs became adherent and differentiated into GFAP- or βIII tubulin-positive cells. Representative micrographs of adherent GSCs are presented, scale bar = 200 μm. Immunofluorescence showed differentiated GSCs expressing GFAP or βIII tubulin (middle and below), scale bar = 50 μm. c: PSAP expression in patient-derived GSCs (left) and different glioma cell lines (right) measured by qPCR. d, e: PSAP protein levels in patient-derived GSCs (d) or different glioma cell lines (e) measured by western blot. f, g: Secreted PSAP levels in the medium of patient-derived GSCs (f) or different glioma cell lines (g) measured by ELISA. All data are shown as the mean ± S.D. (3 experiments). *P, 0.05; **P, 0.01; ***P, 0.001. (t-test).

Fig. 3

PSAP knockdown inhibited glioma proliferation in vitro. a, b, c: Lentiviral-based PSAP-siRNA1, PSAP-siRNA2, or siRNA-control were transfected into U87 or GSC-42, and the knockdown effects were detected by western blot (a), qPCR (b), and ELISA (c). d, e: U87 (d) and GSC-42 (e) cell viability was decreased after PSAP knockdown as measured using the MTS assay. f: PSAP knockdown inhibited the proliferation of U87 and GSC-42 cells as measured by the EdU incorporation assay, scale bar = 100 μm. g The self-renewing capacity of GSC-42 decreased after PSAP knockdown as determined by the neurosphere formation assay, scale bar = 20 μm. All data are shown as the mean ± S.D. (3 experiments). *P, 0.05; **P, 0.01; ***P, 0.001. (t-test).

Fig. 4

PSAP overexpression promoted glioma proliferation in vitro. a, b, c: Lentiviral-based PSAP or empty vector were transfected into SNB19 and GSC-21. The effects of overexpression were detected by western blot (a), qPCR (b) and ELISA (c). d: SNB19 (left) and GSC-21 (right) cell viability increased after PSAP overexpression as measured by the MTS assay. e: The proliferation of SNB19 and GSC-21 increased following PSAP overexpression as measured by the EdU incorporation assay, scale bar = 100 μm. f: The self-renewing capacity of GSC-21 increased after PSAP overexpression as determined by the neurosphere formation assay, scale bar = 20 μm. All data are shown as the mean ± S.D. (3 experiments). *P, 0.05; **P, 0.01; ***P, 0.001. (t-test).

Fig. 5

PSAP regulates glioma tumorigenesis. a: Representative photographs show the intracranial tumor size in the coronal position. PSAP knockdown in GSC-42 decreased the intracranial tumor size whereas PSAP overexpression in GSC-21 increased the intracranial tumor size, n scale bar =10 mm. b: Kaplan–Meier survival curves show that PSAP knockdown increased the survival time of GSC-42 tumor-bearing mice (n = 5). c; Kaplan–Meier survival curves show that PSAP overexpression in group reduced the survival time of GSC-21 tumor bearing mice (n = 5). *P, 0.05; **P, 0.01; ***P, 0.001. (t-test).

Fig. 6

PSAP participates in the TLR4 signaling pathway. a: Gene set enrichment analysis (GSEA) indicates that high expression of PSAP is associated with the TLR signaling pathway in both the TCGA and CGGA databases. b: Kyoto Encyclopedia of Genes and Genome (KEGG) analysis suggests that PSAP participates in the TLR signaling pathway in TCGA database. c: TCGA analysis shows the correlations for TLRs/PSAP and Lrp1/PSAP. d: There is a positive correlation between PSAP and TLR4 in the TCGA and CGGA database (r = 0.5847, p < 0.0001 and r = 0.3638, p < 0.0001 respectively, Pearson correlation analysis). e: There is a positive correlation between PSAP and TLR4 in the clinical glioma specimens (r = 0.7342, p < 0.0001, Pearson correlation analysis).

Fig. 7

PSAP regulates NF-κB signaling pathway via TLR4 activation in glioma. a: PSPA and TLR4 levels in GSC-42 cell lysates were measured by western blot following immunoprecipitation (IP) with PSAP (A) or TLR4 (B) antibodies. b: PSAP and TLR4 co-localize in the glioma cell membrane as demonstrated by immunofluorescence, scale bar = 50 μm. c: PSAP modulation affects the expression and nuclear translocation of P65 in glioma cells as measured by immunofluorescence, scale bar = 50 μm. d, e: PSAP overexpression (d) and knockdown (e) regulated the secretion of pro-inflammatory cytokines in glioma cells as measured by ELISA. f, g: PSAP overexpression (f) or knockdown (g) alters the relative P65-mediated luciferase activity after TLR4 knockdown. All data are shown as the mean ± S.D. (3 experiments). *P, 0.05; **P, 0.01; ***P, 0.001. (t-test).

Fig. 8

PSAP regulates NF-κB signaling pathway in glioma. a, c: PSAP knockdown (a) or overexpression (c) alters the levels of NF-κB downstream targets as measured by western blotting. The western blots shown are representative of three independent experiments. b, d: Densitometric analysis (arbitrary units) of the results in a and c. Densitometry for PSAP and the downstream targets are expressed relative to the loading control, β-actin. Densitometry data are shown as the mean ± S.D. relative to the respective negative control or vector control cells as appropriate (3 experiments). *P, 0.05; **P, 0.01; ***P, 0.001. (t-test).

Fig. 9

TLR4 antagonist TAK-242 abrogates the proliferation promoting effects of PSAP. a: TAK-242 decreases the growth of PSAP overexpressing SNB19 and GSC-21 cells as measured by the MTS assay. b, c: The effects of TAK-242 on the growth of PSAP overexpressing and empty control SNB19 and GSC-21 cells measured by the EdU incorporation assay, scale bar = 100 μm. d: TAK-242 decreases the self-renew capacity of PSAP overexpressing GSC-21 cells in the neurosphere formation assay, scale bar = 20 μm. e: TAK-242 alters NF-κB downstream targets in PSAP overexpressing SNB19 and GSC-21 cells as measured by western blotting. All data are shown as the mean ± S.D. (3 experiments). *P, 0.05; **P, 0.01; ***P, 0.001.