SOX1 down-regulates β-catenin and reverses malignant phenotype in nasopharyngeal carcinoma

Abstract

Background: Aberrant activation of the Wnt/β-catenin signaling pathway is an important factor in the development of nasopharyngeal carcinoma (NPC). Previous studies have demonstrated that the developmental gene sex-determining region Y (SRY)-box 1 (SOX1) inhibits cervical and liver tumorigenesis by interfering with the Wnt/β-catenin signaling pathway. However, the role of SOX1 in NPC remains unclear. This study investigates the function of SOX1 in NPC pathogenesis.

Results: Down-regulation of SOX1 was detected in NPC cell lines and tissues. Besides, quantitative methylation-specific polymerase chain reaction revealed that SOX1 promoter was hypermethylated in NPC cell lines. Ectopic expression of SOX1 in NPC cells suppressed colony formation, proliferation and migration in vitro and impaired tumor growth in nude mice. Restoration of SOX1 expression significantly reduced epithelial-mesenchymal transition, enhanced cell differentiation and induced cellular senescence. Conversely, transient knockdown of SOX1 by siRNA in these cells partially restored cell proliferation and colony formation. Notably, SOX1 was found to physically interact with β-catenin and reduce its expression independent of proteasomal activity, leading to inhibition of Wnt/β-catenin signaling and decreased expression of downstream target genes.

Conclusions: SOX1 decreases the expression of β-catenin in a proteasome-independent manner and reverses the malignant phenotype in NPC cells.

Figure 1

Down-regulation of SOX1 in NPC cell lines and tissues is associated with promoter hypermethylation. (A) Endogenous protein level (upper panel) and mRNA level (lower panel) of SOX1 were detected in NPC cell lines via WB and RT-PCR, respectively. (B) SOX1 transcripts of NPC tissues (T) and their corresponding adjacent non-tumor tissues (N) were determined via qRT-PCR and normalized using GAPDH expression. Data were analyzed via the ΔΔCt method and representative results from three samples (numbers 2, 3 and 23) are shown. Bar represents mean ± SD of three independent experiments (***p <0.001, Student’s t test). (C) Methylation status of NPC cell lines was determined by qMS-PCR. M, methylated SOX1; U, unmethylated SOX1. (D) NPC cell lines CNE2 and HONE1 were treated with or without 5 or 25 μM 5-Aza-CdR for 48 h. SOX1 transcripts were analyzed via qRT-PCR and normalized using GAPDH. Data were analyzed using the ΔΔCt method. Bar represents mean ± SD of three independent experiments (**p <0.01, ANOVA followed by the least significant difference test was used to make statistical comparisons).

Figure 2

Ectopic expression of SOX1 represses NPC cells proliferation and migration in vitro . (A) Ectopic expression of SOX1 in NPC cell lines was confirmed by WB (GAPDH as internal control) and IF. (B, C, D) Colony formation assay, cell proliferation assay and Ki67 staining were performed in NPC cells overexpressing SOX1. The colony formation ability reduced from 15.48  ±  3.29% to 4.90  ±  0.09% in CNE2 cells and from 11.14  ±  2.01% to 5.14  ±  0.82% in HONE1 cells. The Ki67 staining rate decreased from 0.54  ±  0.08 to 0.18  ±  0.05 in CNE2 cells and from 0.66  ±  0.11 to 0.29  ±  0.02 in HONE1 cells. Bar represents mean  ±  SD of three independent experiments (*p <0.05, **p <0.01, ***p <0.001, Student’s t test) (E, F) Wound-healing assay and transwell migration assay were performed in NPC cells overexpressing SOX1. The transwell migration cell number for each 20× field decreased from 64.33  ±  9.5 to 21.75  ±  2.99 in CNE2 cells and from 103.0  ±  18.2 to 27.0  ±  5.30 in HONE1 cells. Quantitative data are shown as the mean  ±  SD from three independent experiments. (*p <0.05, **p <0.01, ***p <0.001, Student’s t test). (Scale bars, 50 μm in A, D, 100 μm in E, F).

Figure 3

SOX1 suppresses tumor formation in nude mice model. (A) CNE2 cells (1 × 10⁶) virally transformed with vector-alone or SOX1 plasmid were injected into the left (vector-only) and right (SOX1) flank of nude mice, respectively (upper panel). Tumors were taken from mice of both the control group and SOX1 overexpression group after 20 days (lower panel). (B) Tumor growth curve of SOX1-overexpressing cells compared with that of vector-only cells. Points represent the mean tumor volumes of six independent experiments; bars represent the SD. (**p <0.01, ***p <0.001, Student’s t test) (C) Tumor weight from the vector-only and SOX1 groups decreased from 1.15 ± 0.20 g to 0.15 ±  0.04 g. The results were obtained from six independent experiments; bars represent the SD. (***p <0.001, Student’s t test).

Figure 4

SOX1 reduces EMT, stimulates cell differentiation and triggers cellular senescence, whereas transient knockdown of SOX1 partially reverses the malignant phenotype. (A) Presence of the EMT-related proteins E-cadherin and Vimentin was detected via IF and WB. IF (left panel) Blue, DAPI; Red, E-cadherin; Green, Vimentin. EMT-related markers were detected by WB (right panel) in HONE1 cells with forced SOX1 expression. GAPDH served as an internal control. (B) Morphology of differentiated HONE1 cells induced by SOX1 was observed under microscopy. WB was used to detect the cell surface markers related to cell differentiation in HONE1 cells with or without SOX1 overexpression. (C) HONE1 cells with or without SOX1 overexpression were fixed and stained for SA-β-gal activity. The number of senescent colonies within each 20× field of HONE1 cells increased from 8.33 ± 1.53 to 50.33 ± 9.87. Bar represents mean ± SD of three independent experiments. (*p <0.05, Student’s t test) (D) SOX1 expression level in SOX1-overexpressing-NPC cells following transfection of SOX1-specific siRNA was determined using WB. (E, F) Ki67 staining rate and colony formation ability following knockdown of SOX1 using siRNA in SOX1-overexpressing NPC cells. Ki67 staining rate increased from 0.17 ± 0.02 to 0.49 ± 0.10 in CNE2 cells and from 0.14 ± 0.02 to 0.28 ± 0.06 in HONE1 cells. Colony formation ability increased from 6.82 ± 1.77% to 15.47 ± 5.28% in HONE1 cells. Bar represents mean ± SD of three independent experiments (*p <0.05, **p <0.01, Student’s t test). (Scale bars, 50 μm in A, 25 μm in B, C, 50 μm in E).

Figure 5

SOX1 suppresses Wnt/β-catenin signaling by interacting with β-catenin and stimulating its down-regulation in a proteasome-independent manner. (A) IP was performed on whole-cell lysate from HONE1cells expressing SOX1-myc using an anti-myc-tag antibody. The same amount of mouse immunoglobulin G and blank (empty) were used as negative controls. Immunoprecipitated protein complexes were analyzed by WB to detect β-catenin and SOX1 with myc-tag. (B) Merged images depicting co-localization of SOX1 and β-catenin by laser confocal microscopy. Blue, DAPI; Red, SOX1; Green, β-catenin. (Scale bar, 20 μm in B) (C, D) The SOX1 and β-catenin protein and mRNA levels of HONE1 cells with or without SOX1 overexpression were detected via WB (C) and qRT-PCR (D), (**p <0.01, ***p <0.001, Student’s t test). (E) SOX1-overexpressing HONE1 cells were transfected with mock and SOX1 siRNA. SOX1 and β-catenin expression were determined by WB. (F) Expression of SOX1 and β-catenin were determined by WB in HONE1 cells transfected with 0, 0.5, 1, or 2 μg of the SOX1 plasmid either with or without the addition of 20 μg/ml MG132 for 12 h. GAPDH was used as an internal control.

Figure 6

SOX1 inhibits Wnt/β-catenin downstream genes and modulates the expression of molecules involved in the cell cycle. (A) WB for expression of c-Myc and Cyclin D1 in SOX1-overexpressing HONE1 cells. (B) Flow cytometry cell cycle analysis of HONE1 cells overexpressing SOX1. The percentage of cells in G1 phase increased from 67.77 ± 0.9% to 71.82 ± 1.05% while the percentage of cells in G2/M phase decreased from 12.7 ± 0.10% to 9.3 ± 0.10% in HONE1 cells overexpressing SOX1. Bar represents mean ± SD of three independent experiments. (*p <0.05, Student’s t test). (C) Proteins involved in the cell cycle after SOX1 expression was forced in HONE1 cells were identified via WB. GAPDH was used as an internal control.