SOX2 promotes tumor growth of esophageal squamous cell carcinoma through the AKT/mammalian target of rapamycin complex 1 signaling pathway

Abstract

The transcription factor SOX2 is essential for the maintenance of embryonic stem cells and normal development of the esophagus. Our previous study revealed that the SOX2 gene is an amplification target of 3q26.3 in esophageal squamous cell carcinoma (ESCC), and that SOX2 promotes ESCC cell proliferation in vitro. In the present study, we aimed to identify the mechanisms by which SOX2 promotes proliferation of ESCC cells. Using a phosphoprotein array, we assayed multiple signaling pathways activated by SOX2 and determined that SOX2 activated the AKT/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. LY294002, an inhibitor of phosphatidylinositol 3-kinase, and rapamycin, an inhibitor of mTORC1, suppressed the ability of SOX2 to enhance proliferation of ESCC cells in vitro. Effects of SOX2 knockdown, including reduced levels of phosphorylated AKT and decreased ESCC cell proliferation, were reversed with constitutive activation of AKT with knockdown of phosphatase and tensin homolog. In mouse xenografts, SOX2 promoted in vivo tumor growth of ESCC, which was dependent on AKT/mTORC1 activation. LY294002 suppressed the ability of SOX2 to enhance tumor growth of ESCC by reducing cell proliferation, but not by enhancing apoptosis. Furthermore, tissue microarray analysis of 61 primary ESCC tumors showed a positive correlation between expression levels of SOX2 and phosphorylated AKT. Our findings suggest that SOX2 promotes in vivo tumor growth of ESCC through activation of the AKT/mTORC1 signaling pathway, which enhances cell proliferation.

Figure 1

Activation of AKT/mammalian target of rapamycin complex 1 and ERK1/2 by SOX2. (a,b) Phosphoprotein array. (a) Chemiluminescent images of phosphoproteins in KYSE70 and KYSE140 esophageal squamous cell carcinoma cells transfected with SOX2 siRNA (si) or control siRNA. (b) Fold change in the expression levels of each phosphoprotein in SOX2 siRNA‐transfected KYSE70 or KYSE140 cells relative to control siRNA‐transfected cells. (c) Immunoblot analysis of the indicated proteins in KYSE70 and KYSE140 cells transfected with SOX2 siRNA or control siRNA. (d,e) Immunofluorescence. KYSE70 cells treated with SOX2 siRNA or control siRNA were triple‐labeled with anti‐SOX2 (green), anti‐phosphorylated AKT (p‐AKT) (red in d) or anti‐p‐ERK1/2 (red in e), and DAPI (blue; nuclei). Arrows indicate SOX2 knocked‐down cells. (f) Expression levels of SOX2 and p‐AKT in the indicated cell lines. 4E‐BP1, 4E‐binding protein 1; EGFR, epidermal growth factor receptor; p70‐S6K, ribosomal S6 kinase; S6, S6 ribosomal protein.

Figure 2

Effects of LY294002 and U0126 on in vitro cell viability. (a,b) KYSE70 and KYSE140 esophageal squamous cell carcinoma cells were treated with LY294002 (a) or U0126 (b) at the indicated concentrations for 48 h then subjected to cell viability experiments (MTT assay) and immunoblot analysis for the indicated proteins. (c) KYSE70 and KYSE140 cells were transfected with siRNA targeting SOX2 or negative control siRNA. After a 24‐h incubation, cells were treated with U0126 at the indicated concentrations for 48 h then subjected to cell viability experiments and immunoblot analysis for the indicated proteins. Relative cell viability was normalized to DMSO treated controls for each cell line. (d) KYSE70 cells were transfected with negative control siRNA, siRNA targeting SOX2, or siRNAs targeting SOX2 and phosphatase and tensin homologue (PTEN). After a 72‐h incubation, cell viability was assessed. (E) KYSE70 cells were treated with rapamycin at the indicated concentrations for 48 h then subjected to cell viability experiments and immunoblot analysis for the indicated proteins. Bars represent the means ± SD for three independent experiments carried out in triplicate (*P < 0.05 vs control). p‐, phosphorylated; S6, S6 ribosomal protein.

Figure 3

Promotion of in vivo tumor growth of esophageal squamous cell carcinoma and activation of the AKT/mammalian target of rapamycin complex 1 signaling pathway by SOX2. (a) Representative macroscopic appearance of xenograft tumors (arrows) on day 24 after KYSE30‐SOX2 (clone stably expressing SOX2) or KYSE30‐EV (clone expressing empty vector) cells were injected s.c. into nude mice. (b) Tumor growth curves in the xenograft mouse model (n = 5 each). Tumor size was assessed every 3 days. *P < 0.05. (c) Immunoblot analysis of the indicated proteins in the xenograft tumors. Representative images of two samples are shown. (d) Immunohistochemical analysis of SOX2 and phosphorylated AKT (p‐AKT) in the xenograft tumors of mice inoculated with KYSE30‐SOX2 or KYSE30‐EV. 4E‐BP1, 4E‐binding protein 1; p70‐S6K, ribosomal S6 kinase; S6, S6 ribosomal protein.

Figure 4

Effects of LY294002 on in vivo tumor growth. (a) Macroscopic appearance of representative xenograft tumors (arrows) of nude mice that were inoculated with KYSE30‐SOX2 esophageal squamous cell carcinoma cells then treated with LY294002 (right) or vehicle (left). (b) Tumor growth curves of the xenograft mouse models that were treated with LY294002 or vehicle (n = 4 each). (c) Immunoblot analyses of phosphorylated AKT (p‐AKT), total AKT, and p‐S6 in the xenograft tumors of mice that were treated with LY294002 or vehicle (n = 4 each). (d) Immunohistochemical staining for Ki‐67 and TUNEL in sections from the xenograft tumors that were treated with LY294002 or vehicle. Arrows indicate TUNEL‐positive cells. (e) Proliferation index (percentage of Ki‐67‐positive cells) or apoptosis index (the percentage of TUNEL‐positive cells) in xenograft tumors treated with LY294002 or vehicle. *P < 0.05. S6, S6 ribosomal protein.

Figure 5

Representative immunostaining of SOX2 and phosphorylated AKT (p‐AKT) in two primary esophageal squamous cell carcinoma tumors. Expression levels of SOX2 and p‐AKT are low in Tumor #1 and high in Tumor #2. Original magnification, ×200.