Sex-Determining Region Y-box 2 Promotes Growth of Lung Squamous Cell Carcinoma and Directly Targets Cyclin D1

Abstract

Sex-determining region Y-box 2 (SOX2) is an oncogene known to be amplified and overexpressed in various human malignancies, including lung squamous cell carcinoma (SCC). However, the role played by SOX2 in lung SCC development remains to be elucidated. We measured the levels of SOX2 and cyclin D1 mRNA and protein expression in lung SCC tissues and a lung SCC cell line, and found that both levels were dramatically upregulated in specimens of lung SCC tissue when compared with their expression levels in samples of adjacent nonneoplastic tissue. The lung SCC cell line also showed higher levels of SOX2 and cyclin D1 expression than a normal human bronchial epithelium cell line. After using RNA interference to knock down SOX2 expression in NCI-H520 lung SCC cells, their proliferation was reduced. Furthermore, overexpression of SOX2 promoted the proliferation of normal human bronchial epithelium cells. To further determine whether cyclin D1 was downstream target gene of SOX2, we measured the levels of cyclin D1 expression that occurred when SOX2 was knocked down or overexpressed. SOX2 knockdown significantly decreased the levels of cyclin D1 mRNA and protein expression, while SOX2 overexpression upregulated the levels of cyclin D1. We used bioinformatics data to identify potential cyclin D1 promoter binding sites for SOX2. Results of luciferase reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays confirmed that cyclin D1 was a direct target of transcription factor SOX2 in human lung SCC cells.

Introduction

Lung cancer is one of the most common cancers worldwide, and a leading cause of cancer-related mortality among both women and men (Siegel et al., 2013, 2014). As a heterogeneous disease comprising tumors derived from phenotypically and functionally distinct cells, lung cancers can be roughly divided into two groups: small-cell lung cancer (SCLC) and non-SCLC (NSCLC). Lung squamous cell carcinoma (SCC) is the main type of NSCLC, and also the second-largest histologic lung cancer subtype. While the incidence of lung SCC has recently increased (Ren et al., 2015; Miller et al., 2016; Torre et al., 2016), it remains a challenge to diagnose early-stage lung cancer due to the lack of a standard screening program and the asymptomatic course of the disease during its early stages. Therefore, it is essential to identify new biomarkers and therapeutic targets for lung cancer, and better understand the mechanisms of lung carcinogenesis if new therapeutic strategies are to be developed (DeSantis et al., 2014).

The embryonic stem cell transcription factors SOX2 and cyclin D1 have recently received increased attention by researchers. The SOX2 gene is located on chromosome 3q26.3–q27, and encodes a transcription factor with three main domains: the N-terminal domain, HMG domain, and the transactivation domain (Boyer et al., 2005; Bass et al., 2009). These studies on SOX2 have primarily focused on its crucial roles in regulation of pluripotency in embryonic stem cells, lineage fate determinant, maintenance of homoeostasis in tracheobronchial epithelium, and reprogramming somatic cells back toward pluripotency (Adameyko et al., 2012). SOX2 is amplified and overexpressed in several human cancers, including lung cancer, glioma, breast cancer, pancreatic carcinoma, esophageal carcinoma, and ovarian carcinoma (Bass et al., 2009; Herreros-Villanueva et al., 2013; Zhang et al., 2013; Tian et al., 2014). Furthermore, SOX2 is more highly expressed in lung cancer tissue than in normal lung tissue and other tumor. In addition, the SOX2 gene is amplified to a greater extent in lung SCC than in lung adenocarcinoma tissue (Lu et al., 2010; Yuan et al., 2010). Accumulated evidence indicates that in cancer cells, SOX2 is involved in cellular proliferation, invasion, migration, self-renewal, and the maintenance of “steminess.” Although SOX2 is an oncogenic transcription factor, the downstream target genes of SOX2 in lung SCC cells remain to be identified.

In this study, we measured the levels of SOX2 expression in lung SCC tissue and an SCC cell line, and found that both SOX2 mRNA and protein levels were dramatically higher in SCC tissue when compared with their levels in adjacent nonneoplastic tissue. Furthermore, the lung SCC cell line had higher levels of SOX2 and cyclin D1 than a normal human bronchial epithelium cell line. We also investigated the role of SOX2 expression in the proliferation of SCC cells and normal human bronchial epithelial cells. The SOX2 knockdown inhibited the proliferation of lung SCC cells, while the SOX2 overexpression promoted the proliferation of normal human bronchial epithelial cells.

Cyclin D1 is a key regulator of the G0–G1 transition that occurs during cycling of both normal and cancer cells, and is thought to be a proto-oncogene involved in the initiation and progression of carcinoma (Quelle et al., 1993; Yu et al., 2001). In this study, we found that the levels of cyclin D1 mRNA and protein were both dramatically higher in samples of lung SCC tissue when compared with samples of adjacent nonneoplastic tissue. We also confirmed that SOX2 is involved in the proliferation of lung SCC cells by regulating the cell cycle. In addition, we investigated whether the cyclin D1 gene might be located downstream of SOX3 and performed a bioinformatics search to identify potential cyclin D1 promoter binding sites for SOX2. Finally, we confirmed that cyclin D1 was a direct target of transcription factor SOX2 in human lung SCC cells.

Materials and Methods

Cells and tissue samples

Human lung SCC cell line NCI-H520 and normal human bronchial epithelium cell line BEAS-B2 were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in the Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum. Ten samples of lung SCC tissue and adjacent nonneoplastic tissue were collected from 10 consecutive lung cancer patients at the First People's Hospital in Chenzhou City, China. The study protocol was approved by the Ethics Committee of Chenzhou First Peoples Hospital, and each patient signed a written Informed Consent document indicating their willingness to participate in the study. All of the enrolled patients were undergoing curative surgical resection without receiving chemotherapy or radiation therapy. The samples of bronchial epithelium collected from adjacent nonneoplastic lung tissue were used as control tissues.

CCK-8 assays

A commercial cell counting kit (CCK-8) (Dojindo; Kumamoto, Japan) was used to evaluate cell viability.

BrdU cell proliferation assay

Cells were seeded on cover slips (Fisher Scientific International, Hampton, NH), and then incubated with bromodeoxyuridine (BrdU) for 1 h, after which, they were stained with anti-BrdU antibody (Upstate) (Merck KGaA, Darmstadt, Germany) as described in the manufacturer's instructions. Hoechst staining solution was used to stain cell nuclei. Images were acquired with a confocal microscope (Olympus FV1000; Olympus, Tokyo, Japan).

Cell cycle analysis

Briefly, cells were trypsinized, harvested, washed with cold phosphate-buffered saline, and then fixed in 70% solution overnight at 4°C. On the following day, the cells were stained with RNase A (10 μg/mL) and propidium iodide (20 mg/mL) (Sigma, St Louis, MO) in the dark for 30 min. The cells were then analyzed for their DNA content with an Aria Calibur flow cytometer (BD Biosciences; Franklin Lakes, NJ) and Flowjo 7.6 software (BD Biosciences).

Western blot studies

Cells and tissues were lysed in the lysis buffer, and aliquots of their total proteins were separated on polyacrylamide gels containing 4–12% SDS. Next, the separated protein bands were transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were then blocked for 1 h in TBST containing 2% bovine serum albumin; after which, they were incubated with antibodies against SOX2 [1:1000, Cell Signaling Technology (CST); Danvers, MA], cyclin D1 (1:1000, CST), GAPDH (1:1000, CST), and HRP-conjugated goat anti-rabbit secondary antibodies (1:1000; Promab, Richmond, CA).

RNA extraction and quantitative reverse transcription PCR

Total RNA was extracted from cells and tissues using Trizol Reagent (Life Technologies, Carlsbad, CA) following the manufacturer's protocols. RT-qPCR was performed using SYBR green PCR Mix (iTAP; Bio-Rad, Hercules, CA) in conjunction with an Applied Biosystems (ABI) step-one plus sequence detection system (Applied Biosystems, Foster City, CA). Fold changes were calculated using ΔΔCT values and the formula 2^−ΔΔCT.

Transient transfection and small interfering RNA

Small interfering RNA (siRNA) with the oligonucleotides 5′-GGT TGA CAC CGT TGG TAAT-3′ and targeting human SOX2 was purchased from RiboBio (Guangzhou, China). Nonspecific oligonucleotides were used as negative controls (NCs). Lipofectamine 2000 (Life Technologies) was used to transfect siRNA into the targeted cells. Following transfection, the cells were harvested at the specified time point and used for further assays.

Luciferase assays

Plasmid pCDNA3.1-SOX2 expressing SOX2 was constructed. The reporter construct was generated by ligating the putative cyclin D1 promoter region and mutations to pGL3-basic (pGL3-cyclinD1-WT and pGL3-cyclinD1-mut reporters). The region containing the putative cyclin D1 promoter mutations (pGL3-cyclinD1-mut reporter) was amplified using the following cloning primers: 5′-TTCTTGGAAATGCGCAATCCAACCCGGCTT-GGATATG-3′ and 5′-CATATCCAAGCCGGGTTGGATTGCGCATTTCCAAGAA-CTCTCA-3′. Plasmids pcDNA3.1 and pGL3-basic were used as controls. All vectors were sequenced to verify the presence of the correct insert. NCI-H520 cells cultured in 12-well plates were transfected using Lipofectamine 3000 (Life Technologies). After 48 h of transfection, the cells were lysed and assayed for the luciferase activity. Transfections performed for luciferase assays were carried at least four times, and the results were measured in duplicate. All measurements of luciferase activity were performed using a Promega Turner Biosystems Modulus Multimode Reader luminometer. The resultant data were analyzed using the two-way ANOVA test.

Immunofluorescence staining

NCI-H520 and BEAS-B2 cells were grown on round cover slips and processed for immunofluorescence staining as previously described (Khromov et al., 2011). SOX2 and cyclin D1 were detected by using a polyclonal antibody against SOX2 and cyclin D1 (CST). The secondary antibodies were conjugated with Alexa Fluorescence 568 and DAPI (1:1000; Sigma). The stained cells were visualized using an Olympus BX60 fluorescent microscope.

Electrophoretic mobility shift assays

Electrophoretic mobility shift assays (EMSAs) were performed using 5′-Cy5-labeled cognate double-stranded DNA elements containing the proper motif sequence. The nuclear extracts were prepared and EMSAs were performed as previously described (Li et al., 2013; Mistri et al., 2015). The following probes were used in the assays: (1) probe containing the Sox2 binding site sequence: 5′-TCGGGCAGCCATTGTGATGCATAT-3′ and (2) mutant probe sequence: 5′-TCGGGCAGCACGTGTGATGCATAT-3′, in which the mutant Sox2 consensus sequence is underlined. The gels were imaged using either a Typhoon 9140 Phosphor Imaging Scanner or a Typhoon FLA-7000 Phosphor Imager (FUJIFILM, Tokyo, Japan).

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were performed as previously described (Turner et al., 2006; Carey et al., 2009) using SOX2 (CST) and a matched IgG antibody (CST). The immunoprecipitated samples were analyzed by RT-qPCR, and the results were normalized using the percent input method. The Ct value of each chromatin immunoprecipitated DNA fraction was normalized to the Ct value of the input DNA fraction. The percent input (% input) was calculated for each ChIP fraction. The cyclin D1 primers used were as follows: forward, 5′-TGCCGGGCTTTGATCTTT-3′; reverse, 5′-CGGTCGTTGAGGAGGTTGG-3′.

Statistical analysis

All results are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. Student's t-test was used to assess differences between two groups. A p value <0.05 was considered statistically significant.

Results

SOX2 and cyclin D1 were overexpressed in lung SCC tissues and cell lines

We measured the levels of SOX2 and cyclin D1 expression in samples of lung SCC tissue and adjacent nonneoplastic tissue. As shown in Figure 1A and B, the levels of SOX2 and cyclin D1 mRNA were dramatically higher in lung SCC tissue when compared with their levels in adjacent nonneoplastic tissue. In addition, Western blot results showed that both SOX2 and cyclin D1 proteins were overexpressed in lung SCC tissues (Fig. 1C, E). We also examined SOX2 and cyclin D1 expression in a lung SCC cell line (NCI-H520) and a normal human bronchial epithelium cell line (BEAS-2B), and found that the lung SCC cell line showed higher levels of SOX2 and cyclin D1 expression than the normal human bronchial epithelium cell line (Fig. 1F, G). Moreover, amplification of SOX2 and cyclin D1 in the lung SCC cell line (NCI-H520) was further confirmed by immunofluorescence (Fig. 1H, I).

FIG. 1.

SOX2 and cyclin D1 were overexpressed in lung SCC tissues and cell lines. (A, B) SOX2 and cyclin D1 mRNA levels were significantly upregulated in samples of lung SCC tissue when compared with their levels in adjacent nonneoplastic tissues, n = 10. (C) The levels of SOX2 and cyclin D1 protein in lung SCC tissues and adjacent nonneoplastic tissues, n = 4. T represented lung SCC tissues (T1–T4) and A represented corresponding adjacent nonneoplastic lung tissues (A1–A4). (D, E) Densitometry plot of results shown in (C). The relative expression levels were normalized to GAPDH. Data represent the mean ± SEM, (n = 3). (F) qPCR analyses of SOX2 and cyclin D1 mRNA levels in a lung SCC cell line (NCI-H520) and normal human bronchial epithelium cell line (BEAS-2B). (G) Western blot analyses of SOX2 and cyclin D1 protein expression. (H, I) Immunofluorescence analyses of SOX2 and cyclin D1 expression in a lung SCC cell line (NCI-H520) and a normal human bronchial epithelium cell line (BEAS-2B). Data represent results from three independent experiments (mean and SEM for three samples). *p < 0.05, **p < 0.01, ***p < 0.001. qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; SCC, squamous cell carcinoma.

Knockdown of SOX2 inhibited the proliferation of lung SCC cells

To further study the role of SOX2 in lung SCC cell proliferation, we performed SOX2-silencing transfection studies using NCI-H520 cells. After transfection for 48 h, the levels of SOX2 mRNA and protein were detected to determine transfection efficiency (Fig. 2A–C). Cell viability was assessed after transfection, and found to be decreased by SOX2 knockdown in both NCI-H520 and BEAS-2B cells (Fig. 2D). However, the SOX2 knockdown produced no effect on the rates of apoptosis in NCI-H520 and BEAS-2B cells (Supplementary Fig. S1A; Supplementary Data are available online at www.liebertpub.com/dna). BrdU proliferation assays showed that the numbers of BrdU-positive cells were significantly decreased after siRNA-SOX2 transfection, suggesting that the knockdown of SOX2 had inhibited the proliferation of lung SCC cells (Fig. 2E). We also assessed the cell cycle of NCI-H520 cells and found that the SOX2 knockdown had increased the proportion of G1 phase cells and reduced the proportion of S phase cells (Fig. 2F), suggesting that the SOX2 knockdown had inhibited NCI-H520 cell proliferation by inducing G1 phase arrest. These results showed that the SOX2 knockdown inhibits the proliferation of lung SCC cells.

FIG. 2.

Knockdown of SOX2 inhibited the proliferation of lung SCC cells. (A) After transfection with siRNA-SOX2 for 48 h, the SOX2 mRNA levels were tested to determine transfection efficiency. (B) Western blot analyses of SOX2 protein levels after siRNA-SOX2 transfection. (C) Densitometry plot of results shown in (B). The relative expression levels were normalized to GAPDH. Data represent the mean ± SEM, (n = 3). (D) Approximately 3 × 10⁴ cells/well were cultured in 96-well plates and transfected with siRNA-SOX2 (100 nM) or a negative control siRNA (100 nM) using Lipofectamine 2000. NCI-H520 and BEAS-2B cells were transfected for 24 or 48 h, after which, CCK-8 assays were performed to determine their viability. (E) BrdU assays were performed to quantitate NCI-H520 cell proliferation. (F) Flow cytometry assays were performed to analyze the cell cycle of NCI-H520 cells after they were transfected with siRNA-SOX2 for 48 h. Data represent results from three independent experiments (mean and SEM for three samples). *p < 0.05, **p < 0.01, ***p < 0.001.

SOX2 overexpression promoted the proliferation of normal human bronchial epithelial cells

As SOX2 expression in BEAS-2B cells was lower than that in NCI-H520 cells, we chose BEAS-2B cells for use in performing further studies on cell proliferation. SOX2 was highly expressed in BEAS-2B cells after they had been transfected with pcDNA3.1-SOX2 plasmid for 48 h (Fig. 3A). As shown in Figure 3B and C, the SOX2 protein was also more highly expressed in BEAS-2B cells transfected with pcDNA3.1-SOX2 plasmid than in cells transfected with the empty vector. The BrdU proliferation assay revealed that overexpression of SOX2 significantly increased the numbers of BrdU-positive cells, suggesting that SOX2 overexpression had promoted BEAS-2B cell proliferation (Fig. 3D). In addition, we found that SOX2 overexpression decreased the proportion of cells in G1 phase and increased the proportion of cells in S phase (Fig. 3E). Our studies also showed that knockdown of SOX2 inhibited the migration/invasion capabilities of NCI-H520 cells, while SOX2 overexpression increased those capabilities (Supplementary Fig. 1C, D).

FIG. 3.

SOX2 overexpression promoted the proliferation of lung SCC cells. (A) After transfection with pcDNA3.1-SOX2 or pcDNA3.1-Vector plasmids for 48 h, the levels of SOX2 in BEAS-2B cells were tested to confirm the efficiency of transfection. (B, C) Western blot analyses of SOX2 protein levels in BEAS-2B cells after transfection. Densitometry plot of results shown in (B, C). The relative expression levels were normalized to GAPDH. Data represent the mean ± SEM, (n = 3). (D) BrdU assays were performed to quantitate BEAS-2B cell proliferation. (E) Flow cytometry assays were performed to analyze the cell cycle of BEAS-2B cells after transfection for 48 h. Data represent the results from three independent experiments (mean and SEM for three samples). *p < 0.05, **p < 0.01, ***p < 0.001.

Cyclin D1 was a direct target of transcription factor SOX2 in human lung SCC cells

We confirmed that SOX2 was involved in the proliferation of lung SCC cells through its ability to regulate the cell cycle. As a key regulator of the G1 progression step within the cell cycle, cyclin D1 plays a pivotal role in the lung SCC cell proliferation process. To further examine whether cyclin D1 might be a downstream target gene for SOX2, transfections using siRNA-SOX2 and pcDNA3.1-SOX2 plasmids were performed. The SOX2 knockdown significantly decreased the levels of cyclin D1 mRNA, while SOX2 overexpression upregulated the cyclin D1 mRNA levels (Fig. 4A, B). Moreover, similar results were found when investigating the corresponding changes in cyclin D1 protein expression, Figure 4C–E. We used the Jaspar database (http://jaspar.genereg.net) to identify potential SOX2 and cyclin D1 promoter binding sites. Luciferase reporter assays revealed that the luciferase activity was significantly enhanced in pCDNA3.1 and pGL3-cyclinD1-WT cotransfected cells, when compared with its activity in empty vector cotransfected cells. Furthermore, the luciferase activity was significantly lower in pCDNA3.1 and pGL3-cyclinD1-mut cotransfected cells when compared with its activity in pCDNA3.1 and pGL3-cyclinD1-WT cotransfected cells (Fig. 4F). An EMSA Super-shelf assay was performed and verified the presence of SOX2-binding sites in the cyclin D1 promoter region (Fig. 4G). Finally, we used ChIP-PCR to confirm that SOX2 bound to the cyclin D1 promoter binding sites (Fig. 4H). We assessed the proliferation of SOX2 overexpressing lung squamous cells after their cyclin D1 expression had been blocked by miRNA and found that the proliferation-promoting effect of SOX2 was inhibited by interference with cyclin D1 expression (Supplementary Fig. S1B). These results indicate that cyclin D1 is a direct target of transcription factor SOX2 in human lung SCC cells.

FIG. 4.

Cyclin D1 is a downstream target gene for transcription factor SOX2. (A) RT-qPCR analyses of cyclin D1 mRNA levels in BEAS-2B cells after transfection with siRNA-SOX2 for 48 h. (B) RT-qPCR analyses of cyclin D1 mRNA levels in BEAS-2B cells after transfection with pcDNA3.1-SOX2 or the pcDNA3.1-Vector plasmid for 48 h. (C) Western blot analyses of cyclin D protein levels in BEAS-2B cells after transfection with siRNA-SOX2 for 48 h. (D) Western blot analyses of cyclin D protein levels in BEAS-2B cells after transfection with pcDNA3.1-SOX2 or pcDNA3.1-Vector plasmid for 48 h. (E) Densitometry plot of results shown in (C, D). The relative expression levels were normalized to GAPDH. Data represent the mean ± SEM, (n = 3). (F) Plasmids pcDNA3.1, pGL3-basic, pGL3-cyclinD1-WT, and pGL3-cyclinD1-mut reporter were cotransfected after which, the luciferase activity was measured. (G) An EMSA Super-shelf assay was performed to identify SOX2 binding sites in the cyclin D1 promoter region. Nuclear protein extracts were prepared from NCI-H520 cells and then incubated with a biotin-labeled DNA probe, followed by chemiluminescent EMSA. The unlabeled probe served as a cold competitor, and its mutant served as a negative control. The specificity of the SOX2 binding motif was confirmed by the ability of a SOX2-specific antibody to block the DNA complex shift. An arrow indicates the SOX2 DNAbinding complex. (H) RT-qPCR analyses of ChIP DNA derived from the IgG and Sox2 ChIP of NCI-H520 cells. The analyses were performed using promoter-specific primers. Sox2 ChIP-qPCR signals were normalized to IgG signals. Data represent results from three independent experiments (mean and SEM for three samples). *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

As Sox2 is highly expressed in conjunction with cyclin D1 in most human lung SCC cells, the downstream targets of SOX2 have been widely investigated. As a transcription factor, SOX2 exerts a tumorigenic effect by regulating several downstream genes in different types of cancers. SOX2 amplifications and overexpression are generally associated with tumor progression in the esophagus, lungs, retina, skin, and pituitary. Tian et al. (2014) showed that SOX2 activated AKT signaling and thereby promoted gastric cancer progression. It was reported that the tumorigenic effect of SOX2 in lung SCC cells is partially mediated by suppression of CDKN1A (Fukazawa et al., 2016). However, the role of Sox2 in gastric cancer is still controversial. SOX2 was progressively downregulated during gastric cancer development and inhibited cell proliferation and metastasis by regulating PTEN (Wang et al., 2015). Otsubo et al. (2008) reported that SOX2 played important roles in growth inhibition through cell cycle arrest and apoptosis in gastric epithelial cells. Recently, Sox2 was reported as a tumor suppressor by modulating Wnt-related and intestinal genes (Sarkar et al., 2016). Our data, as well as data from other investigators, clearly indicate that inhibition of SOX2 suppresses lung SCC cell growth in vitro. Even though SOX2 is recognized as a lineage-specific oncogenic transcription factor in lung cells, the downstream genes that SOX2 targets in lung SCC cells have not been identified. These results indicate that Sox2's effect on cell proliferation and tumorigenesis is highly context dependent.

Previous studies reported that upregulated SOX2 expression is associated with a poor clinical outcome among lung SCC patients (Sholl et al., 2010). In this study, we found dramatically upregulated levels of SOX2 and cyclin D1 mRNA and protein expression in lung SCC tissue when compared with those levels in samples of adjacent nonneoplastic tissue. Moreover, similar results were found when examining SOX2 and cyclin D1 expression in lung SCC and normal human bronchial epithelium cell lines.

To understand the role of SOX2 in lung SCC and normal human bronchial epithelium cells, we knocked down SOX2 expression in lung SCC cells and induced its overexpression in normal human bronchial epithelium cells. The knockdown of SOX2 inhibited the proliferation of lung SCC cells, while SOX2 overexpression promoted the proliferation of normal human bronchial epithelium cells. Downregulation of SOX2 was previously shown to inhibit proliferation and induce apoptosis in two nonsmall cell lung cancer cell lines (A549 and H460) (Chen et al., 2012). Other studies have focused on the downstream pathways of SOX2 in lung cancer. For example, SOX2 knockdown was shown to induce apoptosis in NSCLC cells by activating the MAP4K4 pathway (Chen et al., 2014). Another study reported that the knockdown of SOX2 inhibited the viability and colony formation of lung SCC cell lines H520 and H226. SOX2 was also reported to sustain the proliferation of lung SCC cells by suppressing of CDKN1A (Fukazawa et al., 2016). Our results are consistent with those in previous studies. SOX2 is highly expressed in isolated cancer stem-like cells and helps to maintain their “stemness” by regulating complex transcriptional networks. Numerous studies have revealed that SOX2 regulates the transcriptional networks of various oncogenes, including WNT1, WNT2, NOTCH1, and c-MYC, and promotes tumor formation by human lung cancer cells (Chen et al., 2012).

Cyclin D1 is an important regulator of the cell cycle and is overexpressed in a large number of human cancers, including lung cancer. Moreover, cyclin D1 is suspected to play a pivotal role in processes involved in carcinogenesis and cancer progression. Cyclin D1 is amplified in both NSCLC and preinvasive bronchial lesions; generally by one parental allele. Furthermore, cyclin D1 expression is highly associated with the clinical outcomes of lung cancer patients (Tsuruta et al., 1993; Zhang et al., 1993; Shinozaki et al., 1996).

We hypothesized that SOX2 might promote the proliferation of lung SCC cells and normal human bronchial epithelial cells by activating a downstream target. In our study, the knockdown of SOX2 significantly decreased cyclin D1 expression, while SOX2 overexpression upregulated cyclin D1 levels. We searched the Jaspar database to identify potential binding sites for SOX2 in the cyclin D1 promoter region, and then performed luciferase reporter assays and EMSA combined with ChIP assays to confirm that cyclin D1 was a direct target for transcription factor SOX2 in human lung SCC cells. Previous studies showed that cyclin D1 was downregulated at the transcription level in a SOX2-dependent manner in human glioma stomal cells (Xu et al., 2016). Sox2 was also shown to enhance the proliferation of cervical squamous cancer cells by upregulating cyclin D1 expression (Ji et al., 2014).

In summary, we confirmed that both SOX2 and cyclin D1 are highly expressed in lung SCC tissue and lung SCC cells. In addition, we showed that SOX2 promotes the proliferation of lung SCC and normal human bronchial epithelium cells. Finally, we provided evidence that cyclin D1 is a direct target of transcription factor SOX2 in human lung SCC cells.

Footnotes

Acknowledgment

This work was supported by the Chenzhou First People's Hospital.

Disclosure Statement

No competing financial interests exist.

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