Skip to main content
NCBI home page
Acta Biochimica et Biophysica Sinica logoLink to Acta Biochimica et Biophysica Sinica
. 2016 Feb 4;48(3):229–237. doi: 10.1093/abbs/gmv134

β-Catenin is important for cancer stem cell generation and tumorigenic activity in nasopharyngeal carcinoma

Rui Jiang 1, Xiaoshuang Niu 1, Yuxiang Huang 1, Xiaosheng Wang 1,*
PMCID: PMC4885127  PMID: 26849897

Abstract

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors with poor prognosis and recurrence in South China. The hard eradication of NPC in clinic is predominantly due to cancer stem cells (CSCs). Increasing evidence revealed that the aberrant activation of Wnt/β-catenin was positively correlated with the produce of CSCs. To further investigate the effect of β-catenin on CSCs and tumorigenesis in NPC, a CNE2 cell line (pLKO.1-sh-β-catenin-CNE2) with stably suppressed expression of β-catenin was used in this study. The expressions of biomarkers in CSCs including c-myc, Nanog, Oct3/4, Sox2, EpCAM as well as adhesion-related proteins like E-cadherin and vimentin were analyzed by western blot analysis and immunofluorescent staining. The proliferation and migration abilities were investigated by MTT assay and Transwell assay, respectively. Cell cycle was analyzed by flow cytometry. Finally, xenograft was performed to determine the effect of β-catenin on oncogenesis in vivo. Results showed that the expressions of c-myc, Nanog, Oct3/4, Sox2, and EpCAM were all decreased in pLKO.1-sh-β-catenin-CNE2 cells. It was also found that vimentin was downregulated, while E-cadherin was upregulated. Results of MTT and Transwell assays suggested that the proliferation and migration abilities were impaired by silencing of β-catenin, and more cells were arrested in G1 phase when compared with the control. In vivo study indicated that the tumor growth was markedly suppressed in experimental group. Based on current findings, β-catenin may function as an essential protein for the maintenance of migration and proliferation abilities of NPC cells, and a complicated network consisting of c-myc, Nanog, Oct3/4, Sox2, EpCAM, E-cadherin, vimentin, and β-catenin may be involved in the inherent regulation mechanisms.

Keywords: CNE2 cell line, cancer stem cell, β-catenin, tumorigenic capability

Introduction

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors and reported as an endemic cancer with high prevalence in Southeast Asia, particularly in South China [1,2]. Thus far, the etiology and pathogenesis mechanism of NPC have not yet been elucidated thoroughly. Recent studies suggested that environmental factors, genetic susceptibility, and Epstein–Barr virus may play critical roles in its progression. Although the 5-year survival rate of NPC has been greatly improved through comprehensive treatment such as radiotherapy and chemotherapy [3], long-term prognosis remains unsatisfactory. About 55% of the NPC metastasis and recurrence occurred in 5 years after conventional treatment. Many patients developed chemotherapy resistance.

Cancer stem cell (CSC) theory suggests that CSC is related to tumor recurrence and metastasis as well as chemotherapy-related tolerance [4,5]. Since the existence of CSCs, the solid tumors cannot be eradicated. Although the size of metastatic tumor can be reduced after regular treatment, it can relapse quickly. Research showed that CSCs are a small population of cell subsets that have high proliferative capacity, self-renewal, and multi-directional differentiation potential, with the characteristics of resistance to radiotherapy and chemotherapy. These features are similar to ordinary stem cells and embryonic stem cells. Normally, the differentiation and self-renewal of stem cells are tightly regulated by signal transduction pathways. Once the related signal components become aberrant or are destroyed, dysregulated differentiation will occur and cells will grow indefinitely. Thus, CSCs occur, subsequently leading to tumor formation. Recent research suggested that the tumor cells, stem cells, and CSCs have some common signaling pathways, such as Wnt [6,7], Notch, SHH, BMP, PI3K/Akt [8,9], and Bmi1 signal pathway [10]. Ma et al. [11] reported that the effects of epidermal growth factor receptor (EGFR) on CSCs were mediated by β-catenin, demonstrating that β-catenin was a crucial downstream factor in signaling pathway concerning CSC generation. However, further and detailed studies about β-catenin were absent in their research. The essential effects of β-catenin on CSCs deserve more attention.

Nasopharyngeal CSCs have been evaluated by analyzing the number of side population (SP) cells, which appear as a dimly stained subgroup by extruding the Hoechst 33342 dye [12–14], as well as the expressions of octamer-binding transcription factor 3/4 (Oct3/4), sex-determining region Y-box 2 (Sox2), c-myc, and Nanog [14]. Sufficient evidence has demonstrated that Oct3/4, Sox2, c-myc, and Nanog function as reprograming factors which can efficiently transform terminally differentiated cells into induced pluripotent stem cells [15]. Wnt/β-catenin signaling pathway exists not only in normal stem cells, playing an important role in maintaining cell self-renewal and inhibiting cell differentiation [6,16,17], proliferation, migration, and apoptosis, but also in immature CSCs, playing important roles in controlling drug resistance and tumor recurrence. Recent studies revealed that β-catenin signaling was important in the maintenance of CSC phenotype [17–19]. Knockdown of β-catenin led to the loss of CSC phenotype and tumor degeneration [20]. However, the Wnt/β-catenin signaling pathway is quiescent in normal epithelial cells. This difference may be used to kill CSCs and ultimately cure the cancer. NPC is a kind of malignant tumor occurred in nasopharyngeal epithelium. In our previous work [21], several siRNAs targeting β-catenin were designed and transfected into CNE1, CNE2, and 6–10B cell lines. A CNE2 cell line (pLKO.1-sh-β-catenin-CNE2) with stable downregulation of β-catenin was successfully established. In the present study, this cell line was used to investigate the role of Wnt/β-catenin signaling pathway in CSC of NPC. The results indicated that silencing the expression of β-catenin resulted in the elimination of CSC properties and inhibition of NPC in vitro and in vivo. Our studies may provide a novel approach for molecular targeted therapy for NPC patients.

Materials and Methods

Cell culture

A CNE2 cell line (pLKO.1-sh-β-catenin-CNE2) with stably suppressed β-catenin expression was established by RNA interference, and verified by western blot analysis [21]. A control cell line transfected with nonsense siRNA (pLKO.1-sh-ctr-CNE2) was simultaneously established. The untreated CNE2 cells were used as the parental cell. All the cell lines were cultured in RPMI-1640 medium (Gibco, Gaithersburg, USA) supplemented with 10% fetal bovine serum (FBS; Sigma, St Louis, USA) in a humidified incubator at 37°C in an atmosphere of 5% CO2.

Western blot analysis

Cells were harvested for western blot analysis after 3 days of incubation. Cell lysate was prepared in RIPA buffer containing 1% (v/v) phenylmethane sulfonyl fluoride (PMSF; Sigma). Total protein was quantified using a BCA Protein Assay kit (Pierce, Rockford, USA) according to the manufacturer's instructions. All the protein samples were boiled together with 6× loading buffer, and then an equal amount of each protein sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then the separated protein was transferred to a polyvinylidene fluoride membrane and probed with appropriate primary antibodies. The antibodies against β-catenin, c-myc, E-cadherin, vimentin, Oct3/4, and GAPDH as well as the corresponding FITC- and HRP-conjugated secondary antibodies were all purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Protein bands were detected using an Enhanced Chemiluminescence (ECL) Detection kit (Pierce).

Immunofluorescent staining

The pLKO.1-sh-β-catenin-CNE2, pLKO.1-sh-ctr-CNE2, and untreated cells were separately seeded on sterilized cover slides which were placed in six-well culture dishes, and maintained in 2 ml of RPMI-1640 medium supplemented with 10% new born calf serum (NBS; Sigma) in a humidified incubator at 37°C in an atmosphere of 5% CO2. After incubation for 6–12 h, medium were discarded and cells were washed twice with phosphate-buffered saline (PBS). Cells were then fixed with 3.5% paraformaldehyde for 7 min at room temperature. After rehydration, fixed cells were incubated with monoclonal antibodies against β-catenin, c-myc, Oct3/4, Sox2, Nanog, vimentin, and EpCAM at 4°C overnight, followed by incubation with fluorescein isothiocyanate-conjugated secondary antibodies for 30 min at room temperature. The nuclei were stained with 2,6-diaminopimelic acid (DIPA). Sections were examined with a Nikon Eclipse 80i fluorescent microscope (Nikon Instruments, Inc., Melville, USA).

Cell viability assay

The effects of suppressing β-catenin expression on cell proliferation were evaluated by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma). The pLKO.1-sh-β-catenin-CNE2, pLKO.1-sh-ctr-CNE2, and untreated cells were separately seeded in 96-well culture plates at the density of 1000 cells per well. After 24, 48, and 72 h of incubation, 5 mg/ml of MTT solution was added to each well and the absorbance was determined at 490 nm.

Transwell migration assay

The migration ability of pLKO.1-sh-β-catenin-CNE2, pLKO.1-sh-ctr-CNE2, and untreated cells were measured by Transwell migration assay. A total of 5 × 104 cells were pelleted and resuspended in 500 μl serum-free RPMI-1640 medium, then seeded into the inserts of the Transwell chamber (Corning Costar, Corning, USA). The inserts were then put into the bottom chambers loaded with 500 μl RPMI-1640 medium supplemented with 10% NBS, and the cells were incubated in a humidified incubator at 37°C in an atmosphere of 5% CO2 for 24 h. The inserts were then taken out, and the cells remaining on the upper surface (non-migrant) were gently removed with a cotton swab. Cells on the lower surface of the inserts (migrant cells) were fixed with 500 μl methanol for 10 min, and stained with hematoxylin for 10 min at room temperature. Images of the migrant cells were captured using a photomicroscope (Axiovert 200 M; Carl Zeiss, Oberkochen, Germany). Three images per well were randomly selected for cell counting, and an average was finally calculated. Each assay was performed in triplicate.

Cell cycle and SP analysis by flow cytometry

The pLKO.1-sh-β-catenin-CNE2, pLKO.1-sh-ctr-CNE2, and untreated cells in exponential growth stage were collected and suspended in RPMI-1640 medium containing 2% FBS to 1 × 106 cells/ml, and subsequently stained with Hoechst33342 dye (1 mg/ml) for 90 min in a light-resistant container at 37°C with continuous mixing. After incubation, the cells were immediately washed with D-Hanks solution, stained with propidium iodide (PI, 1 mg/ml), and maintained on ice for flow cytometry analyses (FACS). Cells for cell cycle analysis were washed twice with D-Hanks solution and resuspended in 300 μl PBS. Then the cells were fixed with pre-cooled alcohol for 12 h at 4°C and washed again with D-Hanks solution, resuspended in D-Hanks solution containing 0.1 mg/ml RNase A, and then stained with PI for 30 min at room temperature for FACS analysis.

In vivo tumorigenicity assay

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Fudan University Shanghai Cancer Center. For xenograft study, the pLKO.1-sh-β-catenin-CNE2, pLKO.1-sh-ctr-CNE2, and untreated cells were washed with D-Hanks solution and resuspended in serum-free medium. A volume of 100 μl cell solution of the 3 cell lines containing 1 × 106 cells were separately transplanted into the backs of 5 BALB/c-nu mice (4–6 weeks of age) by subcutaneous injection. Mice transplanted with pLKO.1-sh-β-catenin-CNE2 cells were used as experimental group and pLKO.1-sh-ctr-CNE2 cells were used as control. After tumor formation, tumor volumes were measured every 5 days. Tumor volume was calculated as follows: V = 1/2(width2 × length). Four weeks later, all mice were sacrificed for tumor tissue collection. Tumor tissues were fixed in 4% formaldehyde and embedded in paraffin for immunohistochemistry and hematoxylin-eosin (HE) staining.

Immunohistochemistry and HE staining

Paraffin-embedded sections were deparaffinized in xylene and rehydrated in a graded alcohol series, then boiled in 10 mM sodium citrate buffer for antigen retrieval. Subsequently, sections were stained using a VECTASTAIN® ABC kit (Vector Laboratories, Burlingame, USA) according to the manufacturer's protocol. Diaminobenzidine (DBA) was chosen as the substrate for peroxidase. HE staining was performed after the sections were deparaffinized and rehydrated.

Statistical analysis

All data were expressed as the mean ± SD. Statistical evaluation of the data was conducted using SPSS version 19.0. Student's t-test was used to determine the significant differences. Graphs summarizing immunohistochemical staining results were analyzed using Fisher's exact test. P-values <0.05 were considered to be statistically significant.

Results

Low expression of β-catenin inhibits NPC cell proliferation

As is well known, β-catenin acts as a critical factor in Wnt/β-catenin transduction pathway, which is activated when the abnormal accumulation of β-catenin occurs in the cytoplasm. Previous researches have revealed that tumorigenesis of most organs and tissues is closely related to the aberrant activation of Wnt/β-catenin. In this study, the effect of β-catenin on NPC cell proliferation was investigated. Results of MTT assay indicated that proliferation of pLKO.1-sh-β-catenin-CNE2 cells was significantly suppressed after 2 days of incubation when compared with the parental cells (Fig. 1A; P = 0.001), while no significant difference was observed between the parental cells and control cells (P = 0.098). Cell cycle analysis demonstrated that the proportion of pLKO.1-sh-β-catenin-CNE2 cells in G1 phase was significantly higher than that of the control cells (P = 0.007) and parental CNE2 cells (P = 0.001) (Fig. 1B,C), meanwhile lower proportion of cells in S phase was observed in pLKO.1-sh-β-catenin-CNE2 cells (Fig. 1B,C; P < 0.05). These findings implied that most cells were arrested in G1 phase by knockdown of β-catenin gene expression. And, the results were further confirmed by evidence at the molecular level. C-myc plays a critical role in regulation of cell proliferation. Western blot analysis showed that expression of c-myc was significantly downregulated in pLKO.1-sh-β-catenin-CNE2, in which β-catenin was also downregulated (Fig. 2A,B).

Figure 1.

Figure 1.

Open in a new tab

The inhibitory effect of silencing β-catenin on cell proliferation of CNE2 cells (A) Cell viability analysis by MTT. (B,C) Distribution of DNA content among G1, S and G2 phases in three cell lines. Data are presented as the mean ± SD (n = 3). **P < 0.01, *P < 0.05.

Figure 2.

Figure 2.

Open in a new tab

The inhibitory effect of silencing β-catenin on cell migration of CNE2 cells (A) Expression levels of β-catenin, Sox2, c-myc, E-cadherin, vimentin, and Oct3/4 determined by western blot analysis. GAPDH was used as reference. (B) Cellular level of c-myc detected by immunofluorescence staining (original magnification, ×200). (C) Silencing β-catenin reduced migration of CNE2 cells. Data are presented as the mean ± SD (n = 3). **P < 0.01, *P < 0.05.

Migration of NPC cells is compromised when β-catenin gene is silenced

It was reported that most tumor recurrences and poor prognosis were associated with the migration of tumor cells. In this study, the ability of cell migration was diminished by silencing the β-catenin gene. Notably, cells that penetrated the Transwell membrane were observed with comparatively lower counts in pLKO.1-sh-β-catenin-CNE2 cells (Fig. 2C; P < 0.001), while no significant difference was observed between the control cells and parental cells (P = 0.096). The increased expression of E-cadherin was found to be negatively correlated with cell migration (Fig. 2A). EpCAM, which contributes to cell adhesion, was decreased in pLKO.1-sh-β-catenin-CNE2 cells (Fig. 3A).

Figure 3.

Figure 3.

Open in a new tab

Expression levels of EpCAM and Nanog (A), vimentin and Oct3/4 (B) in three cell lines determined by immunofluorescence EpCAM, Nanog, vimentin, Oct3/4 were all decreased in pLKO.1-sh-β-catenin-CNE2 cell line (original magnification ×200).

β-Catenin is necessary for maintaining characteristics of CSCs in NPC

CSCs are reportedly responsible for the recurrence, drug resistance, and metastasis in most tumors. The impacts of β-catenin on the characteristics of CSCs in NPC were explored. C-myc, Nanog, Oct3/4, Sox2, and EpCAM were selected as biomarkers. The decreased expressions of c-myc, Sox2, and Oct3/4 in pLKO.1-sh-β-catenin-CNE2 cells were observed by western blot analysis (Fig. 2A). Furthermore, expressions of β-catenin, c-myc, Nanog, Oct3/4, Sox2, EpCAM, and vimentin were analyzed by immunofluorescence stain. The low expressions of c-myc and Oct3/4 were further verified in Figs. 2B and 3B, respectively. Additionally, expressions of Nanog, Sox2, EpCAM, and vimentin were also suppressed in pLKO.1-sh-β-catenin-CNE2 (Figs. 3 and 4). The low expression levels of these biomarkers indirectly demonstrated that the CSCs in NPC were reduced by downregulating β-catenin. In order to directly verify that CSCs were decreased, SP cells, a subpopulation of CSCs, were analyzed by FACS. Results showed that the percentage of SP cells were lower than that of the parental and control cells (Fig. 5; P < 0.05), and the difference between parental and control cells was not statistically significant (P = 0.255). The above findings indicated that decreased expression of β-catenin exerted an inhibitory effect on the generation of CSCs. Neoplasm recurrence was reported to be linked to the existence of CSCs in tumor cells. To evaluate the influence of β-catenin on tumor formation ability of CSCs, pLKO.1-sh-β-catenin-CNE2, pLKO.1-sh-ctr-CNE2, and parental cells were delivered subcutaneously into nude mice. Tumor growth was monitored by measuring the volume of solid tumors. As shown in Fig. 6A, the size of tumors in mice transplanted with pLKO.1-sh-β-catenin-CNE2 was evidently smaller than that in control and parental groups (P < 0.001), while the difference between the control group and parental group was not statistically significant (P = 0.882). After 21 days of growth, the average tumor volume was 70.342 ± 46.448 mm3 in the experimental group and 1014.91 ± 430.624 and 1185.77 ± 364.965 mm3 in the control group and parental group, respectively (Fig. 6A). Histological analysis of subcutaneous tumors by HE staining demonstrated more seriously disorganized tumor cells, hyperchromatic nuclei, and higher nuclear–cytoplasmic ratio in control and parental groups than in the experimental group (Fig. 6B), indicating a more advanced stage of NPC in the control and parental groups than in the experimental group. Immunohistochemistry analysis showed a relatively lower expression of Sox2 in pLKO.1-sh-β-catenin-CNE2 group (Fig. 4A), indicating a reduced generation of CSCs. Therefore, the limited tumor formation ability of NPC cells might lead to the decreased CSCs due to low endogenous expression of β-catenin.

Figure 4.

Figure 4.

Open in a new tab

Expression of Sox2 in tumor tissues determined by immunohistochemistry (A) and in cells by immunofluorescence (B) Sox2 was downregulated both in vivo and in vitro experiments (original magnification ×100).

Figure 5.

Figure 5.

Open in a new tab

SP cells analysis of three CNE2 cell lines by FACS SP cells were obviously reduced in pLKO.1-sh-β-catenin-CNE2 cells, indicating the decreased production of CSC.

Figure 6.

Figure 6.

Open in a new tab

The attenuation effect of downregulated β-catenin on tumor growth in nude mice (A) The tumor growth was significantly suppressed by inoculating pLKO.1-sh-β-catenin-CNE2 cells. (B) HE staining of tumor tissues among three groups (original magnification ×100). Data are presented as the mean ± SD (n = 3). **P < 0.01, *P < 0.05.

Discussion

CSC, as a subset cell within the majority tumors including brain tumor, breast cancer, ovarian cancer, and NPC, has long been suggested to be involved in tumor relapse, metastasis, and resistance to chemoradiotherapy [22,23]. β-Catenin, which acts as a pivotal protein in Wnt/β-catenin pathway, was found to be overexpressed in CSCs in many tumors including NPC, and it was determined with lower level in cells from normal tissues. Jang et al. [24] demonstrated that blocking Wnt/β-catenin signaling would reverse CSC-like phenotypes in breast cancer. In previous studies, cell lines with overexpression of β-catenin were constructed to assess the effect of β-catenin on the fate of CSCs and tumorigenesis, and the results showed that up-regulated endogenous β-catenin may promote the proliferation, migration, and tumor formation of CSC and also promote tumorigenesis in mice. Increasing evidence demonstrated that β-catenin was of great importance in the maintenance of CSC properties in NPC, as well as in the development of cancers. However, whether the high expression level of β-catenin is necessarily required for stemness maintenance of CSC is still not clear. Some studies have suggested that the CSC characteristics in NPC cells with high level of β-catenin are attenuated when the Wnt/β-catenin signaling pathway is blocked by inhibitors targeting β-catenin [16,25]. In addition, the antineoplastic activities of some microRNAs like miR-200a were reported to be mediated by their inhibitory effect on β-catenin pathway [26–28].

In the present study, whether β-catenin is necessarily involved in the stemness maintenance of CSCs in NPC was studied by silencing β-catenin in CNE2 cells (pLKO.1-sh-β-catenin-CNE2), and we hypothesized that the lower level of β-catenin would reverse the CSC traits of NPC cells. Subsequently, the proliferation and migration of CNE2 cells in which β-catenin had been silenced were reduced as expected. C-myc, one of numerous downstream proliferation signals activated by β-catenin, was also found to be downregulated in pLKO.1-sh-β-catenin-CNE2 cells by western blot analysis and immunofluorescent staining. And, the cell line possessed a relatively higher ratio of cells that were arrested in G1 phase compared with the control cells. These findings may partly explain a relatively weak proliferation of cells with reduced β-catenin expression at molecular level. Simultaneously, E-cadherin was inversely up-regulated in pLKO.1-sh-β-catenin-CNE2 cells. E-cadherin was reported to contribute to the cell adhesion and was negatively correlated with tumor cell migration [29,30]. E-cadherin and the interaction between its intracellular domain and cytoplasmic β-catenin are essential for cell adhesion. However, the expression of E-cadherin was found to be negatively correlated with intracellular β-catenin expression in this study. The inhibitory effect of E-cadherin expression was potentially caused by hypomethylation of E-cadherin gene resulted from activation of signaling pathway, such as JNK pathway [10,31]. The translocation of β-catenin to the nucleus had a validated negative relationship with E-cadherin expression, and the intracellular β-catenin localized in the nucleus of pLKO.1-sh-β-catenin-CNE2 cell was markedly reduced. Fang et al. [1] found that when E-cadherin and β-catenin were up-regulated by (−)-epigallocatechin-3-gallate, the nuclear translocation of β-catenin was suppressed. More efforts should be made to figure out the detailed underlying mechanisms between E-cadherin expression and nuclear translocation of β-catenin. Reduced ability of cell migration in cells with stably suppressed expression of β-catenin was presumably attributed to the changes of E-cadherin expression. The results implied that the inhibitory effect of β-catenin via indirectly suppressing E-cadherin expression was the main influence of cellular β-catenin on cell migration, and the regulatory mechanisms of cell migration in NPC mediated by β-catenin were more complicated than expected. Vimentin (an intermediate filament protein) and EpCAM (a transmembrane protein) [32], two proteins related to cell adhesion and migration, were both decreased in β-catenin-silenced cells. Vimentin expression was speculatively correlated with the progression of NPC, which was supported by an investigation in 122 NPC patients showing that high nuclear vimentin expression was correlated significantly with advanced clinical stage [33]. Up-regulated vimentin and downregulated E-cadherin are the typical phenotypes of epithelial–mesenchymal transition (EMT) which contributes to tumor invasion and metastasis [34]. Involvement of E-cadherin, vimentin, EpCAM, and β-catenin in cell adhesion and migration implied an intricate mechanism in tumor invasion and metastasis. More investigations are needed to further understand the regulatory mechanisms of cell migration in NPC.

Reduced production of CSC may also contribute to the impaired proliferation and migration. The lower expressions of c-myc, Nanog, Oct3/4, Sox2, and EpCAM in β-catenin-silenced cells suggested that the production of CSC was suppressed. The enrichment of Sox2 in CSC-like population in NPC has already been reported, and Sox2 has also found to be involved in regulating the growth of the CSC-like population in NPC [4,35]. In the present study, we demonstrated that silencing β-catenin would inversely influence the expression of Sox2 both in vitro and in vivo. EpCAM is a tumor-associated protein involved in regulating the functions of cell adhesion, migration, and proliferation [36]. In our study, low β-catenin level resulted in decreased EpCAM expression, which was in agreement with the fact found in breast cancer cells that high EpCAM expression was closely related to the activation of the Wnt pathway [31]. Nanog, Sox2, and Oct3/4, which function as critical transcriptional factors in cell self-renewal and pluripotency, were downregulated when β-catenin was decreased. According to recent studies, Nanog was positively regulated by Sox2 and Oct3/4 in NPC, and the inhibitory effect of reduced β-catenin on Nanog was potentially mediated by Sox2 and Oct3/4 [17]. Nanog was also found to be associated with chemoresistance in head and neck cancer cells [37], and reduced Nanog expression implied a weakened resistance in β-catenin-silenced cells. Therefore, low levels of c-myc, Nanog, Oct3/4, Sox2, and EpCAM proved that the production of CSCs was indeed reduced, which was also confirmed by SP analysis. Based on the above findings, the complicated regulation network consisting of Nanog, Oct3/4, Sox2, and β-catenin was of great significance for the maintenance of pluripotency in CSCs. Since CSCs were responsible for tumor recurrence and metastasis, and the biomarkers of CSC were found to be highly expressed in most patients with advanced-stage NPC or recurrence, decreased CSCs caused by silencing β-catenin would limit the oncogenesis. Our xenograft experiment results showed that the growth of tumors was indeed limited. In addition, prevention of Wnt/β-catenin transduction pathway can also inhibit the expressions of angiogenesis-related genes, such as MMP9, MMP2, and VEGF [25,38], which were very important in solid tumor growth. Some studies have already proved that suppressing the function or expression of β-catenin by corresponding inhibitors and microRNA ultimately leads to the limited growth of tumors in mice. Therefore, β-catenin may be a potential target for the treatment of NPC and its recurrence. However, our understanding of the pathologies of neoplasm recurrence and metastasis is still limited, and more efforts are needed for the final cure of NPC.

The poor prognosis of NPC found in patients was mostly associated with CSCs. The migration and proliferation of CSC can assist the growth and invasion of tumors. The activation of Wnt/β-catenin signal pathway by accumulation of endogenous β-catenin was partly responsible for the characteristics of CSC. Our study demonstrated that β-catenin acts as an indispensable factor for the maintenance of migration and proliferation abilities in NPC cells. Reduced CSC capacities in NPC cells were ascribed to the decreased CSC production caused by downregulated β-catenin. Furthermore, the inhibitory effect on CSC was presumably regulated by a comprehensive signal network consisting of c-myc, Nanog, Oct3/4, Sox2, EpCAM, E-cadherin, vimentin, and β-catenin. It has been proposed that β-catenin may be a promising target for tumor therapy, and the anti-tumor effect of reduced expression of β-catenin was verified in vivo in this study. The development of specific inhibitors of β-catenin and drugs that can suppress β-catenin expression may potentially improve the therapeutic effects and prognosis of NPC.

Funding

This work was supported by a grant from the Shanghai Committee of Science and Technology (No. 124119a6202).

References

  • 1.Fang CY, Wu CC, Hsu HY, Chuang HY, Huang SY, Tsai CH, Chang Y et al. EGCG inhibits proliferation, invasiveness and tumor growth by up-regulation of adhesion molecules, suppression of gelatinases activity, and induction of apoptosis in nasopharyngeal carcinoma cells. Int J Mol Sci 2015, 16: 2530–2558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Zhang LF, Li YH, Xie SH, Ling W, Chen SH, Liu Q, Huang QH et al. Incidence trend of nasopharyngeal carcinoma from 1987 to 2011 in Sihui County, Guangdong Province, South China: an age-period-cohort analysis. Chin J Cancer 2015, 34: 15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Hong JS, Tian J, Han QF, Ni QY. Quality of life of nasopharyngeal cancer survivors in China. Curr Oncol 2015, 22: e142–e147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Chan KC, Chan LS, Ip JC, Lo C, Yip TT, Ngan RK, Wong RN et al. Therapeutic targeting of CBP/β-catenin signaling reduces cancer stem cell population and synergistically suppresses growth of EBV-positive nasopharyngeal carcinoma cells with cisplatin. Sci Rep 2015, 5: 9979–9991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Woodward WA, Chen MS, Behbod F, Alfaro MP, Buchholz TA, Rosen JM. WNT/β-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci USA 2007, 104: 618–623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Cheng Y, Phoon YP, Jin X, Chong SYS, Ip JC, Wong BW, Lung ML. Wnt-C59 arrests stemness and suppresses growth of nasopharyngeal carcinoma in mice by inhibiting the Wnt pathway in the tumor microenvironment. Oncotarget 2015, 6: 14428–14439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature 2005, 434: 843–850. [DOI] [PubMed] [Google Scholar]
  • 8.Meng F, Li H, Shi H, Yang Q, Zhang F, Yang Y, Kang L et al. MACC1 down-regulation inhibits proliferation and tumourigenicity of nasopharyngeal carcinoma cells through Akt/β-catenin signaling pathway. PLoS ONE 2013, 8: e60821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Yip WK, Seow HF. Activation of phosphatidylinositol 3-kinase/Akt signaling by EGF downregulates membranous E-cadherin and β-catenin and enhances invasion in nasopharyngeal carcinoma cells. Cancer Lett 2012, 318: 162–172. [DOI] [PubMed] [Google Scholar]
  • 10.Chou J, Lin Y-C, Kim J, You L, Xu Z, He B, Jablons DM. Nasopharyngeal carcinoma—review of the molecular mechanisms of tumorigenesis. Head Neck 2008, 30: 946–963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ma L, Zhang G, Miao X-B, Deng XB, Wu Y, Liu Y, Jin ZR et al. Cancer stem-like cell properties are regulated by EGFR/AKT/β–catenin signaling and preferentially inhibited by gefitinib in nasopharyngeal carcinoma. FEBS 2012, 280: 2027–2041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Tabor MH, Clay MR, Owen JH, Bradford CR, Carey TE, Wolf GT, Prince ME. Head and neck cancer stem cells: the side population. Laryngoscope 2011, 121: 527–533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Wang J, Guo LP, Chen LZ. Identification of cancer stem cell–like side population cells in human nasopharyngeal carcinoma cell line. Cancer Res 2007, 67: 3716–3724. [DOI] [PubMed] [Google Scholar]
  • 14.Hoe SL, Tan LP, Jamal J, Peh SC, Ng CC, Zhang WC, Ahmad M et al. Evaluation of stem-like side population cells in a recurrent nasopharyngeal carcinoma cell line. Cancer Cell Int 2014, 14: 101–110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Kulcenty K, Wróblewska J, Mazurek S, Liszewska E, Jaworski J. Molecular mechanisms of induced pluripotency. Contemp Oncol 2015, 19: A22–A29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Guan Z, Zhang J, Wang J, Wang H, Zheng F, Peng J, Xu Y et al. SOX1 down-regulates β-catenin and reverses malignant phenotype in nasopharyngeal carcinoma. Mol Cancer 2014, 13: 257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Cheng Y, Cheung AK, Ko JM, Phoon YP, Chiu PM, Lo PH, Waterman ML et al. Physiological β-catenin signaling controls selfrenewal networks and generation of stem-like cells from nasopharyngeal carcinoma. BMC Cell Bio 2013, 14: 1471–2121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Teng Y, Wang X, Wang Y, Ma D. Wnt/β-catenin signaling regulates cancer stem cells in lung cancer A549 cells. Biochem Biophys Res Com 2010, 392: 373–379. [DOI] [PubMed] [Google Scholar]
  • 19.Kanwar SS, Yu Y, Nautiyal J, Patel BB, Majumdar AP. The Wnt/β-catenin pathway regulates growth and maintenance of colonospheres. Mol Cancer 2010, 9: 212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Huang WS, Wang JP, Wang T, Fang JY, Lan P, Ma JP. ShRNA-mediated gene silencing of β-catenin inhibits growth of human colon cancer cells. World J Gastroentero 2007, 13: 6581–6587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Jiang R, Zhang G, Ma L, Li XQ, Yao K. Construction of a stable human nasopharyngeal carcinoma cell line with down-regulated expression of β-catenin. J Trop Med 2011, 11: 520–523. [Google Scholar]
  • 22.Song J, Chang I, Chen Z, Kong M, Wang C-Y. Characterization of side populations in HNSCC: highly invasive, chemoresistant and abnormal Wnt signaling. PLoS ONE 2010, 5: e11456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Malanchi I, Peinado H, Kassen D, Hussenet T, Metzger D, Chambon P, Huber M et al. Cutaneous cancer stem cell maintenance is dependent on β-catenin signalling. Nature 2008, 452: 650–653. [DOI] [PubMed] [Google Scholar]
  • 24.Jang GB, Kim JY, Cho SD, Park KS, Jung JY, Lee HY, Hong IS et al. Blockade of Wnt/beta-catenin signaling suppresses breast cancer metastasis by inhibiting CSC-like phenotype. Sci Rep 2015, 5: 12465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Chen J, Kwong DLW, Zhu CL, Chen LL, Dong SS, Zhang LY, Tian J et al. RBMS3 at 3p24 inhibits nasopharyngeal carcinoma development via inhibiting cell proliferation, angiogenesis, and inducing apoptosis. PLoS ONE 2012, 7: e44636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Xia H, Cheung WKC, Sze J, Lu G, Jiang S, Yao H, Bian XW et al. miR-200a regulates epithelial-mesenchymal to stem-like transition via ZEB2 and β-catenin signaling. J Bio Chem 2010, 285: 36995–37003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Xia MH. Great potential of microRNA in cancer stem cell. J Cancer Mol 2008, 4: 79–89. [Google Scholar]
  • 28.Liu J, Ruan B, You N, Huang Q, Liu W, Dang Z, Xu W et al. Downregulation of miR-200a induces EMT phenotypes and CSC-like signatures through targeting the β-catenin pathway in hepatic oval cells. PLoS ONE 2013, 8: e79409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA. Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res 2008, 68: 3645–3654. [DOI] [PubMed] [Google Scholar]
  • 30.Zhang S, Li J, Liu P, Xu J, Zhao W, Xie C, Yin Z et al. Pygopus-2 promotes invasion and metastasis of hepatic carcinoma cell by decreasing E-cadherin expression. Oncotarget 2015, 6: 11074–11086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Asiaf A, Ahmad ST, Aziz SA, Malik AA, Rasool Z, Masood A, Zargar MA. Loss of expression and aberrant methylation of the CDH1 (E-cadherin) gene in breast cancer patients from Kashmir. Asian Pac J Cancer P 2014, 15: 6397–6403. [DOI] [PubMed] [Google Scholar]
  • 32.Zhou FQ, Qi YM, Xu H, Wang QY, Gao XS, Guo HG. Expression of EpCAM and Wnt/β-catenin in human colon cancer. Genet Mol Res 2015, 14: 4485–4494. [DOI] [PubMed] [Google Scholar]
  • 33.Luo W, Fang W, Li S, Yao K. Aberrant expression of nuclear Vimentin and related epithelial-mesenchymal transition markers in nasopharyngeal carcinoma. Int J Cancer 2012, 131: 1863–1873. [DOI] [PubMed] [Google Scholar]
  • 34.Han XY, Wei B, Fang JF, Zhang S, Zhang FC, Zhang HB, Lan TY et al. Epithelial-mesenchymal transition associates with maintenance of stemness in spheroid-derived stem like colon cancer cells. PLoS ONE 2013, 8: e73341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Yang CH, Wang HL, Lin YS, Kumar KP, Lin HC, Chang CJ, Lu CC et al. Identification of CD24 as a cancer stem cell marker in human nasopharyngeal carcinoma. PLoS ONE 2014, 9: e99412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Guerra E 1, Lattanzio R, Sorda RL, Dini F 1, Tiboni GM, Piantelli M 1, Alberti S. mTrop1/Epcam knockout mice develop congenital tufting enteropathy through dysregulation of intestinal E-cadherin/β-catenin. PLoS ONE 2012, 7: e49302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Kumar B, Yadav A, Lang JC, Teknos TN, Kumar P. Suberoylanilide hydroxamic acid (SAHA) reverses chemoresistance in head and neck cancer cells by targeting cancer stem cells via the downregulation of nanog. Genes Cancer 2015, 6: 169–181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Song Y, Yang QX, Zhang F, Meng F, Li H, Dong Y, Han A. Suppression of nasopharyngeal carcinoma cell by targeting β-catenin signaling pathway. Cancer Epidemiol 2012, 36: e116–e121. [DOI] [PubMed] [Google Scholar]

Articles from Acta Biochimica et Biophysica Sinica are provided here courtesy of Acta Biochimica et Biophysica Sinica Editorial Office

RESOURCES