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. 2013 Apr 16;104(7):810–816. doi: 10.1111/cas.12155

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

Yasuyuki Gen 1, Kohichiroh Yasui 1,✉, Taichiro Nishikawa 1, Toshikazu Yoshikawa 1
PMCID: PMC7657236  PMID: 23510069

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.

SOX2 is a member of the SOX family of transcription factors.1, 2, 3 SOX2 is critical for the maintenance of pluripotency and self‐renewal of embryonic stem cells4, 5 and generation of induced pluripotent stem cells.6, 7, 8 In the esophagus, SOX2 plays an important role in differentiation and morphogenesis.9 In the developing foregut endoderm, the highest levels of SOX2 expression occur in the future esophagus and the anterior stomach.10, 11 Mutations in the SOX2 gene cause anophthalmia‐esophageal‐genital syndrome, a condition that involves esophageal atresia and tracheoesophageal fistula.12

We previously showed that SOX2 is the amplification target at chromosome 3q26.3 in esophageal squamous cell carcinoma (ESCC), and that SOX2 promotes ESCC cell proliferation in vitro.13 Furthermore, we showed that the expression of SOX2 is elevated in most primary ESCCs (70%).13 These findings are consistent with several publications from other groups.14, 15 However, the mechanisms by which SOX2 promotes ESCC remain to be elucidated.

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. Here we show that SOX2 promotes in vivo tumor growth of ESCC through activation of the AKT/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, which promotes cell proliferation. The serine/threonine kinase, AKT, regulates many biological processes, such as proliferation, apoptosis, and growth.16, 17 AKT is activated by extracellular signals through phosphatidylinositol 3‐kinase (PI3K) activation. Conversely, AKT activity is negatively regulated by phosphatase and tensin homologue (PTEN). Deregulated AKT activity is common in various tumors, including ESCC.16, 17, 18, 19 Although inactivating mutations or deletions of PTEN occur frequently in malignant tumors,20 they are very rare in ESCC.21, 22 AKT promotes cell proliferation mainly through mTORC1 activation.23, 24 mTORC1 is a major regulator of ribosomal biogenesis and protein synthesis. mTORC1 regulates these processes largely by the phosphorylation and inactivation of the repressors of mRNA translation 4E‐binding proteins (4E‐BPs) and the phosphorylation and activation of ribosomal S6 kinase (p70‐S6K). The phosphorylation status of 4E‐BP1 and p70‐S6K is used to evaluate mTORC1 activity in vivo.24, 25

Materials and Methods

Reagents and antibodies

LY294002, an inhibitor of PI3K, U0126, an inhibitor of MEK1/2, and rapamycin, an inhibitor of mTORC1, were purchased from Cayman Chemical Company (Ann Arbor, MI, USA), Sigma‐Aldrich (Tokyo, Japan), and Selleck Chemicals (Houston, TX, USA), respectively. Antibodies against SOX2, AKT, phosphorylated AKT (p‐AKT) (Ser473), phosphorylated p70‐S6 kinase (p‐p70‐S6K) (Thr389), phosphorylated S6 ribosomal protein (p‐S6) (Ser235/236), phosphorylated 4E‐BP1 (p‐4E‐BP1) (Thr37/Ser46), ERK1/2, p‐ERK1/2 (Thr202/Tyr204), and PTEN were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody against SOX2 for immunohistochemistry was purchased from R&D Systems (Minneapolis, MN, USA). Alexa Fluor 488‐conjugated anti‐mouse IgG and Alexa Fluor 555‐conjugated anti‐rabbit IgG for immunofluorescence were also obtained from Cell Signaling Technology. The antibody against β‐actin was purchased from Sigma‐Aldrich, and the anti‐mouse Ki‐67 antibody (clone MIB‐1) was obtained from Dako (Glostrup, Denmark).

Cell culture

Three ESCC cell lines (KYSE30, KYSE70, and KYSE140) were obtained and maintained as described previously.13, 26

RNA interference

Small interfering RNA and control (non‐silencing) siRNA were delivered into KYSE70 or KYSE140 cells, as described previously.13 Small interfering RNA duplex oligoribonucleotides targeting PTEN were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The cells were harvested for analysis 48 h after transfection.

Phosphoprotein array

KYSE70 and KYSE140 cells were transfected with siRNA targeting SOX2 or negative control siRNA. After 48 h of incubation, the cell lysates were analyzed using the PathScan RTK Signaling Antibody Array Kit (Cell Signaling Technology), according to the manufacturer's instructions. This array can simultaneously detect 28 receptor tyrosine kinases that are phosphorylated at tyrosine residues and 11 downstream signaling kinases that are phosphorylated at tyrosine/serine/threonine residues (Table S1).

Immunoblotting

Immunoblotting was carried out as described previously.13 All antibodies were used at dilutions of 1:1000, with the exception of anti‐β‐actin antibody (1:5000). For secondary immunodetection, anti‐rabbit IgG or anti‐mouse IgG (Amersham, Piscataway, NJ, USA) were used at a dilution of 1:5000.

Immunofluorescence

Immunofluorescence was carried out as described previously.27 Briefly, KYSE70 cells were transfected with siRNA targeting SOX2 or control siRNA. After a 48‐h incubation, cells were fixed then incubated with a combination of anti‐SOX2 (1:50) and anti‐p‐AKT (1:200) or anti‐SOX2 (1:50) and anti‐p‐ERK1/2 (1:200) antibodies. Alexa Fluor 488‐conjugated anti‐mouse IgG and Alexa Fluor 555‐conjugated anti‐rabbit IgG were used to detect the primary antibodies. Nuclei were counterstained with DAPI (Sigma‐Aldrich).

Epidermal growth factor receptor pathway mutation PCR array

The somatic mutation status for the epidermal growth factor receptor gene (EGFR) and eight additional key genes in the EGFR pathway (AKT, BRAF, KRAS, HRAS, NRAS, MEK1, PIK3CA, and PTEN) was analyzed using the EGFR Pathway Mutation PCR Array (Qiagen, Tokyo, Japan), according to the manufacturer's instructions.

Cell viability experiments

Cells were seeded at a density of 8 × 104 cells/mL in 96‐well plates and maintained for 24 h in normal culture conditions. The medium was then replaced with medium containing LY294002, U0126, rapamycin, or DMSO, and cells were cultured for 48 h. Cell viability was assessed by measuring MTT (Nacalai Tesque, Kyoto, Japan) dye absorbance, according to the manufacturer's instructions.

Plasmid construction and transfection

Full‐length human SOX2 cDNA (clone ID, IOH13706) was obtained from Invitrogen and subcloned into pcDNA3.2/V5‐DEST (Invitrogen) to generate a mammalian expression vector. The SOX2 expression vector or the empty vector was transfected into KYSE30 cells using the Effectene Transfection Reagent kit (Qiagen), according to the manufacturer's instructions. After selection with 0.5 mg/mL geneticin (G418; Invitrogen), individual clones were isolated. A clone stably expressing SOX2, which was named KYSE30‐SOX2, was established. A clone expressing an empty vector, which was named KYSE30‐EV, was also established and used as a control.

In vivo tumor growth assay

KYSE30‐SOX2 and KYSE30‐EV cells (1 × 106 cells suspended in 0.1 mL PBS) were injected s.c. into the backs of 8‐week‐old male BALB/c nude mice. Tumor dimensions were measured using calipers, and tumor volume was calculated using the following formula: (shortest diameter)2 × (longest diameter) × 0.5. Each group included five mice, which were housed in pathogen‐free conditions. After 24 days of observation, the mice were killed and tumors were excised. Tumor samples were immediately snap frozen in liquid nitrogen and stored at −80°C for immunoblotting sample preparation, or were fixed in 10% buffered formalin and embedded in paraffin for immunohistochemical analyses. All animal experiments fulfilled the requirements for humane animal care at the Kyoto Prefectural University of Medicine (Kyoto, Japan). The Ethics Committee reviewed and approved all experimental protocols.

Inhibition of PI3K/AKT

LY294002 was given i.p. to KYSE30‐SOX2 xenografted mice at a dose of 25 mg/kg twice a week, as described previously.19, 28, 29 Treatment with LY294002 was initiated on day 14, when tumors had reached a volume of approximately 200 mm3. The control mice were injected with the same volume of vehicle. Each group included four mice. After tumor cell transplantation, mice were euthanized at day 28, and tumors were collected for immunoblotting and immunohistochemical analyses. To validate the effects of LY294002, a final dose of LY294002 was given 8 h before the mice were killed and tumors were excised. Tumor size was measured with calipers twice a week, and tumor volume was calculated as described above.

Immunohistochemistry

Immunostaining of p‐AKT and Ki‐67 was carried out with the EnVision+ system (Dako), and immunostaining of SOX2 was carried out using the Histofine Simple Stain MAX (Nichirei, Tokyo, Japan), as described previously.13 Anti‐SOX2, anti‐p‐AKT, and anti‐Ki‐67 antibodies were used at dilutions of 1:100.

The proliferation index of tumor cells was determined by counting the number of nuclei positive for Ki‐67 antigen in 1000 cells. Results were expressed as a percentage of labeled cells.

TUNEL assay

Apoptosis was determined using the ApopTag Peroxidase In Situ Apoptosis Detection kit (Chemicon, Temecula, CA, USA), according to the manufacturer's instructions. The apoptosis index was determined by counting the number of immunoreactive cells per 1000 scored cells. Results were expressed as a percentage of labeled cells.

Tissue microarray analysis

The ESCC tissue microarray (US Biomax, Rockville, MD, USA) was analyzed for SOX2 and p‐AKT expression. Immunoreactivity was scored according to the estimated percentage of positive cells. Specifically, when <75% of cells were positive, it was scored as “low expression”, and when ≥75% of cells were positive, it was scored as “high expression”.

Statistical analysis

Differences between groups were determined with anova, Student's t‐test, or the χ2‐test, as appropriate. All statistical analyses were carried out with spss 15.0 software (SPSS Inc., Chicago, IL, USA). P‐values of <0.05 were considered statistically significant.

Results

SOX2 elevates AKT/mTORC1 and ERK1/2 signaling levels in ESCC cells

We previously showed that SOX2 is amplified and overexpressed in ESCC KYSE70 and KYSE140 cells, and that SOX2 promotes proliferation of these cells.13 To identify the signaling pathways through which SOX2 promotes the proliferation of these ESCC cells, we knocked down SOX2 expression with siRNA in KYSE70 and KYSE140 cells and assayed multiple signaling pathways using a phosphoprotein array (the PathScan RTK Signaling Antibody Array). This array revealed that p‐AKT (Thr308), p‐AKT (Ser473), p‐ERK1/2 (Thr202/Tyr204), and p‐S6 (Ser235/236) levels were reduced by half in KYSE70 and KYSE140 cells treated with SOX2 siRNA compared to those treated with control siRNA (Fig. 1a,b). However, the levels of other phosphorylated proteins were not markedly reduced by SOX2 knockdown (Table S1; Fig. 1a,b for p‐EGFR and p‐ERBB2).

Figure 1.

Figure 1

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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.

To confirm these findings, we knocked down the expression of SOX2 with siRNA in KYSE70 and KYSE140 cells and used immunoblot assays to assess levels of total and phosphorylated AKT and ERK1/2, p‐4E‐BP1, p‐p70‐S6K, and p‐S6. Both p‐4E‐BP1 and p‐p70‐S6K are direct targets of mTORC1, and p‐S6 is a downstream effector of p70‐S6K. Knockdown of SOX2 resulted in decreased levels of p‐AKT, p‐ERK1/2, p‐4E‐BP1, p‐p70‐S6K, and p‐S6, but not total AKT or ERK1/2 (Fig. 1c). Furthermore, p‐AKT and p‐ERK1/2 were detected in SOX2‐expressing KYSE70 cells with immunofluorescence. However, the expression levels of p‐AKT and p‐ERK1/2 were markedly decreased following knockdown of SOX2 in KYSE70 cells (Fig. 1d,e). Levels of p‐AKT were higher in KYSE70 and KYSE140 cells that overexpressed SOX2 than in KYSE30 cells, which barely expressed SOX2 (Fig. 1f). We identified no mutations in PIK3CA, PTEN, AKT1, KRAS, MEK1, or EGFR, which function upstream of AKT or ERK1/2, in KYSE70 and KYSE140 cells (data not shown). Taken together, these results suggest that overexpression of SOX2 increases AKT/mTORC1 and ERK1/2 activation.

Ability of SOX2 to enhance cell viability is dependent on AKT activity

To examine whether AKT and ERK1/2 were involved in the ability of SOX2 to enhance proliferation of ESCC cells, we carried out in vitro inhibition assays using LY294002, an inhibitor of PI3K, and U0126, an inhibitor of MEK1/2. We confirmed that LY294002 and U0126 successfully reduced the levels of p‐AKT (Fig. 2a) and p‐ERK1/2 (Fig. 2b), respectively, in KYSE70 and KYSE140 cells. As a result, LY294002 significantly decreased cell viability and reduced p‐S6 levels in a dose‐dependent manner in KYSE70 and KYSE140 cells (Fig. 2a). Conversely, U0126 did not decrease cell viability in KYSE70 and KYSE140 cells (Fig. 2b).

Figure 2.

Figure 2

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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.

Interestingly, we observed that U0126 treatment increased p‐AKT levels in KYSE70 and KYSE140 cells. To investigate whether SOX2‐induced AKT activation may be involved in the resistance of cells to U0126 treatment, we first knocked down SOX2 expression with siRNA in KYSE70 and KYSE140 cells and then treated these cells with U0126. Due to SOX2 knockdown and subsequent suppression of p‐AKT levels, U0126 decreased cell viability (Fig. 2c).

We next examined whether effects of SOX2 knockdown can be reversed with constitutive activation of AKT using knockdown of PTEN. This knockdown restored the reduced p‐AKT level and the decreased cell viability that were induced by SOX2 knockdown in KYSE70 cells (Fig. 2d).

Furthermore, we assessed effects of rapamycin, an inhibitor of mTORC1, on KYSE70 cells. Rapamycin successfully reduced levels of p‐p70S6K and p‐S6 and significantly decreased cell viability in KYSE70 cells (Fig. 2e). Taken together, these findings suggest that the ability of SOX2 to enhance cell viability is, at least in part, dependent on the activation of the AKT/mTORC1 pathway.

SOX2 promotes in vivo tumor growth of ESCC

To examine whether SOX2 promoted in vivo tumor growth of ESCC, we used KYSE30 cells to establish an independent clone (KYSE30‐SOX2) that stably expressed SOX2 and a control clone (KYSE30‐EV) carrying an empty vector. KYSE30‐SOX2 or KYSE30‐EV cells were injected s.c. into nude mice. Tumors were significantly larger in mice injected with KYSE30‐SOX2 cells than in those with KYSE30‐EV cells (Fig. 3a,b). Immunoblotting analysis (Fig. 3c) and immunohistochemistry (Fig. 3d) confirmed SOX2 expression in the xenograft tumors derived from KYSE30‐SOX2 cells and revealed that xenograft tumors derived from KYSE30‐SOX2 cells had higher levels of p‐AKT than those derived from KYSE30‐EV cells. Levels of p‐4E‐BP1, p‐p70‐S6K, and p‐S6 were also higher in xenograft tumors derived from KYSE30‐SOX2 cells than those derived from KYSE30‐EV cells (Fig. 3c).

Figure 3.

Figure 3

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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.

Phosphatidylinositol 3‐kinase inhibitor suppresses the ability of SOX2 to enhance ESCC tumor growth in vivo

We also examined the effects of a PI3K inhibitor on the ability of SOX2 to enhance tumor growth in mouse xenografts. KYSE30‐SOX2 cells were injected s.c. into nude mice, and LY294002 or vehicle was then given i.p. Tumors were significantly smaller in mice given LY294002 than those given vehicle alone (Fig. 4a,b). Immunoblotting analysis confirmed that LY294002 reduced p‐AKT and p‐S6 levels in xenograft tumors (Fig. 4c). Additionally, xenograft tumor sections were stained with Ki‐67, a marker of cell proliferation, and TUNEL, a marker of apoptosis. The proportion of Ki‐67‐positive cells was significantly lower in tumors treated with LY294002 than in those treated with vehicle alone (Fig. 4d,e). However, there were no differences in the percentage of TUNEL‐positive cells between tumors treated with LY294002 and those treated with vehicle alone (Fig. 4d,e). These findings suggest that the PI3K inhibitor suppressed the ability of SOX2 to enhance tumor growth by reducing cell proliferation, but not by enhancing apoptosis. Taken together, these data indicate that SOX2 promotes in vivo tumor growth of ESCC through activation of the AKT/mTORC1 pathway, which promotes cell proliferation.

Figure 4.

Figure 4

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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.

SOX2 expression levels correlate with p‐AKT expression levels in primary ESCC

Based on the findings in ESCC cell lines, we examined the relationship between expression levels of SOX2 and p‐AKT using immunohistochemistry on tissue microarrays containing 61 primary ESCC tumors. Representative immunostaining of SOX2 and p‐AKT proteins is shown in Figure 5. Of 61 tumors, there was a high expression of SOX2 and p‐AKT detected in 36 (59%) and 38 (62%) tumors, respectively. The expression levels of p‐AKT significantly correlated with those of SOX2 (P < 0.01; Table 1).

Figure 5.

Figure 5

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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.

Table 1.

Correlation between phosphorylated AKT (p‐AKT) and SOX2 expression levels in 61 primary esophageal squamous cell carcinoma tumors

Protein expression p‐AKT P‐value
High Low
SOX2
High 28 8 <0.01
Low 10 15
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High expression, more than 75% of positive cells; low expression, <75% of positive cells.

Discussion

In the present study, we showed that SOX2 activates AKT/mTORC1 signaling in vitro and in vivo in ESCC cells. SOX2 promotes in vivo tumor growth of ESCC. The ability of SOX2 to enhance tumor growth is dependent on AKT activation. Furthermore, high expression of SOX2 is associated with high expression of p‐AKT in primary ESCC tumors.

First, we found that SOX2 activates the AKT/mTORC1 signaling pathway in ESCC cells. Although several previous reports have shown overexpression of SOX2 in malignant tumors, little is known about the downstream targets of SOX2 in cancer cells. Together with β‐catenin, SOX2 promotes cell proliferation and tumorigenesis by facilitating the G1/S transition and transcriptional regulation of CCND1 in breast cancer cells.30 However, in our preliminary experiments, we did not detect SOX2‐induced modulation of CCND1 mRNA expression in ESCC cells (data not shown). This finding could indicate that SOX2 must pair up with cell‐specific partners to regulate gene transcription, and therefore, SOX2 appears to regulate different sets of target genes depending on the cell type.3, 31 In the present study, we assayed multiple signaling pathways using a phosphoprotein array and found that the AKT/mTORC1 signaling pathway was activated.

Second, using PI3K and MEK1/2 inhibitors, we showed that SOX2 promoted in vitro cell proliferation and in vivo tumor growth of ESCC through the PI3K/AKT pathway, but not the MAPK/ERK pathway. The PTEN knockdown and rapamycin treatment experiments confirmed that the ability of SOX2 to enhance cell proliferation is, in part, dependent on activation of the AKT/mTORC1 pathway.

Finally, we also showed that high expression of p‐AKT was associated with high expression of SOX2 in primary ESCC tumors. Our previous study showed that elevated expression of SOX2 is associated with histologically poor differentiation of ESCC, but not with the TNM stage.13 Unfortunately, no detailed clinicopathological data for the specimens on the ESCC tissue microarrays examined in the present study were available. Overactivation of AKT is induced by several genetic alterations. However, we detected no mutations in PIK3CA, PTEN, AKT1, KRAS, or EGFR in KYSE70 and KYSE140 cells in our analyses. Additionally, amplification of AKT1, KRAS, EGFR, and PIK3CA was not detected in these cells.13

Although our results suggest that SOX2 overexpression leads to AKT activation in ESCC cells, the mechanisms by which SOX2 activates AKT remain unclear. Our results suggest that activation of AKT by SOX2 was, in part, dependent on the activation of PI3K. However, receptor tyrosine kinases that are known to be molecules that are upstream of PI3K were not activated by SOX2 (Table S1). Other molecules upstream of PI3K or unknown factors may lead to activation of the PI3K/AKT pathway.

Our findings suggest that inhibition of p‐AKT suppresses the ability of SOX2 to enhance tumor growth by reducing cell proliferation in mouse xenografts. Therefore, p‐AKT may be an appropriate and important target for the development of novel therapies for the treatment of SOX2‐overexpressing ESCC. For example, small‐molecule AKT inhibitors32 or an inhibitor of mTOR may be effective against SOX2‐overexpressing ESCC.

In conclusion, we showed that SOX2 promotes in vitro cell proliferation and in vivo tumor growth of ESCC, at least in part, through the AKT/mTORC1 pathway. Although further studies are needed to examine the mechanisms by which SOX2 activates the AKT/mTORC1 pathway, our results indicate that molecular targeted therapy against the AKT/mTORC1 pathway could be effective against ESCC tumors that overexpress SOX2.

Disclosure Statement

The authors have no conflict of interest.

Supporting information

Table S1. Expression levels of phosphorylated proteins in SOX2 siRNA‐transfected KYSE70 and KYSE140 esophageal squamous cell carcinoma cells.

Click here for additional data file. (29KB, xls)

Acknowledgments

This study was supported by a Grant‐in‐Aid for Young Scientists (No. 24790339) (to Y.G.) and a Grant‐in‐Aid for Scientific Research (No. 23590470) (to K.Y.) from the Japan Society for the Program of Science.

(Cancer Sci 2013; 104: 810–816)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1. Expression levels of phosphorylated proteins in SOX2 siRNA‐transfected KYSE70 and KYSE140 esophageal squamous cell carcinoma cells.

Click here for additional data file. (29KB, xls)

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