Tryptophan hydroxylase 1 drives glioma progression by modulating the serotonin/L1CAM/NF-κB signaling pathway

Abstract

Background: Glioma is one of the main causes of cancer-related mortality worldwide and is associated with high heterogeneity. However, the key players determining the fate of glioma remain obscure. In the present study, we shed light on tumor metabolism and aimed to investigate the role of tryptophan hydroxylase 1 (TPH-1) in the advancement of glioma.

Method: Herein, the levels of TPH-1 expression in glioma tissues were evaluated using The Cancer Genome Atlas (TCGA) database. Further, the proliferative characteristics and migration ability of TPH-1 overexpressing LN229/T98G cells were evaluated. Additionally, we performed a cytotoxicity analysis using temozolomide (TMZ) in these cells. We also examined the tumor growth and survival time in a mouse model of glioma treated with chemotherapeutic agents and a TPH-1 inhibitor.

Results: The results of both clinical and experimental data showed that excess TPH-1 expression resulted in sustained glioma progression and a dismal overall survival in these patients. Mechanistically, TPH-1 increased the production of serotonin in glioma cells. The elevated serotonin levels then augmented the NF-κB signaling pathway through the upregulation of the L1-cell adhesion molecule (L1CAM), thereby contributing to cellular proliferation, invasive migration, and drug resistance. In vivo experiments demonstrated potent antitumor effects, which benefited further from the synergistic combination of TMZ and LX-1031.

Conclusion: Taken together, these data suggested that TPH-1 facilitated cellular proliferation, migration, and chemoresistance in glioma through the serotonin/L1CAM/NF-κB pathway. By demonstrating the link of amino acid metabolic enzymes with tumor development, our findings may provide a potentially viable target for therapeutic manipulation aimed at eradicating glioma.

Keywords: Glioma; L1CAM; Serotonin; TPH-1.

Fig. 1

TPH-1 promoted glioma progression. A the transcriptome expression of TPH-1 in 156 glioma tissues and 5 normal tissues. B overall survival of glioma patients divided into high TPH-1 (n = 49) and low TPH-1 (n = 50) groups. C western blotting of TPH-1 in LN229/T98G (VEC) and TPH-1 overexpressing (OE) LN229/T98G cells. D cell proliferation of LN229/T98G (VEC) and TPH-1 overexpressing LN229/T98G cells (OE) determined by CCK8 assay. E relative migrating cells of LN229/T98G (VEC) and TPH-1 overexpressing LN229/T98G cells (OE) determined by transwell assay. F cell apoptosis of LN229/T98G (VEC) and TPH-1 overexpressing LN229/T98G cells (OE) treated with TMZ (48 h)

Fig. 2

Serotonin drove glioma cells proliferation and invasion. A and B 10⁵ LN229/T98G (VEC) or TPH-1 overexpressing LN229/T98G cells (OE) were cultured in DMEM culture medium for 48 h. Trp consumption (A) and serotonin production (B) was determined by Elisa assay. C cell proliferation of LN229/T98G cells treated with PBS or serotonin (10 nM). D relative migrating cells of LN229/T98G cells treated with PBS or serotonin (10 nM). E LN229 or T98G cells were pre-treated with PBS or serotonin (10 nM) for 48 h. Then cell apoptosis of LN229/T98G treated with PBS or TMZ (48 h) were determined. F immunofluorescence staining of TPH-1 in TPH-1^high and TPH-1^low glioma tissues from patients. The scale bar was 25 μm. G serotonin secretion in TPH-1^high and TPH-1^low glioma tissues from patients, detected by Elisa analysis. H the correlation analysis between TPH-1 and serotonin in 20 glioma tissues (R² = 0.466)

Fig. 3

Serotonin upregulated L1CAM signaling to regulate glioma progression. A Heatmap of top 30 overexpressing genes in TPH-1^high glioma patients (n = 49) in comparison with TPH-1^low glioma patients (n = 50). B GO enrichment analysis in TPH-1^high glioma patients (n = 49) in comparison with TPH-1^low glioma patients (n = 50). C western blotting of L1CAM in LN229/T98G cells treated with PBS or serotonin (10 nM), and TPH-1 overexpressing LN229/T98G cells. D relative expression of L1CAM in LN229/T98G cells treated with scrambled and L1CAM siRNA, determined by qPCR. E cell proliferation of PBS cultured LN229/T98G (scrambled siRNA treatment), serotonin (10 nM) cultured LN229/T98G cells treated with scrambled and L1CAM siRNA. F relative migrating cells of PBS cultured LN229/T98G (scrambled siRNA treatment), serotonin (10 nM) cultured LN229/T98G cells treated with scrambled and L1CAM siRNA. G PBS cultured LN229/T98G (scrambled siRNA treatment) was collected, and serotonin (10 nM) cultured LN229/T98G cells were pre-treated with scrambled and L1CAM siRNA. Then cell apoptosis of LN229/T98G treated with PBS or TMZ (1 μg/ml, 48 h) were determined. H overall survival of glioma patients with high L1CAM (n = 49) and low L1CAM (n = 50) expression

Fig. 4

Serotonin upregulated NF-κB expression through LICAM signaling. A Western blotting of NF-κB in LN229/T98G cells, serotonin (10 nM) cultured LN229/T98G cells treated with scrambled and L1CAM siRNA. B Cell proliferation of serotonin (10 nM) cultured LN229/T98G cells treated with PBS and QNZ (10 nM). C Relative migrating cells of serotonin (10 nM) cultured LN229/T98G cells treated with PBS and QNZ (10 nM). D Serotonin (10 nM) cultured LN229/T98G cells were pre-treated with QNZ (10 nM, 48 h). Then cell apoptosis of LN229/T98G treated with PBS or TMZ (1 μg/ml, 48 h) were determined. E Overall survival of glioma patients with high NFKB1 (n = 49) and low NFKB1 (n = 50) expression

Fig. 5

Interrupt of TPH-1 signals attenuated the aggressiveness of glioma in vivo. A LN229 (VEC) and TPH-1 overexpressing LN229 cells (OE) were subcutaneously injected into mice. The tumor volume was recorded. B-C The expression of serotonin (B), L1CAM and NF-κB (C) in tumor tissues from (A) was determined by Elisa assay or immunofluorescence. The scale bar was 25 μm. D Tumor volume of TPH-1 overexpressing LN229 bearing mice treated with PBS, TMZ, LX-1031 and TMZ combining LX-1031. E Survival time of TPH-1 overexpressing LN229 bearing mice treated with PBS, TMZ, LX-1031 and TMZ combining LX-1031