About this Journal Submit a Manuscript Table of Contents
Journal of Oncology
Volume 2011 (2011), Article ID 609259, 16 pages
http://dx.doi.org/10.1155/2011/609259
Research Article

Bmi-1 Regulates Snail Expression and Promotes Metastasis Ability in Head and Neck Squamous Cancer-Derived ALDH1 Positive Cells

1Institute of Oral Biology and Biomaterial Science, Chung-Shan Medical University, Taichung 40201, Taiwan
2Department of Dentistry, Chung Shan Medical University Hospital, Taichung 40201, Taiwan
3Division of Oral and Maxillofacial Surgery, Department of Stomatology, Taipei Veterans General Hospital, Taipei 11217, Taiwan
4Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 11217, Taiwan
6Department of Surgery, Taipei Veterans General Hospital, Taipei 11217, Taiwan
5Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan
7Department of Pharmacy Practice, Tri-Service General Hospital and 114 National Defense Medical Center, Taipei, Taiwan

Received 28 May 2010; Accepted 15 August 2010

Academic Editor: Eric Deutsch

Copyright © 2011 Cheng-Chia Yu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Recent studies suggest that ALDH1 is a putative marker for HNSCC-derived cancer stem cells. However, the regulation mechanisms that maintain the stemness and metastatic capability of HNSCC- cells remain unclear. Initially, HNSCC- cells from HNSCC patient showed cancer stemness properties, and high expression of Bmi1 and Snail. Functionally, tumorigenic properties of HNSCC- cells could be downregulated by knockdown of Bmi-1. Overexpression of Bmi-1 altered in expression property cells to that of cells. Furthermore, knockdown of Bmi-1 enhanced the radiosensitivity of radiation-treated HNSCC- cells. Moreover, overexpression of Bmi-1 in HNSCC- cells increased tumor volume and number of pulmonary metastatic lesions by xenotransplant assay. Importantly, knock-down of Bmi1 in HNSCC- cells significantly decreased distant metastases in the lungs. Clinically, coexpression of Bmi-1/Snail/ALDH1 predicted the worst prognosis in HNSCC patients. Collectively, our data suggested that Bmi-1 plays a key role in regulating Snail expression and cancer stemness properties of HNSCC- cells.

1. Introduction

Head and neck squamous cell carcinoma (HNSCC), including oral squamous cell carcinoma (OSCC), is the sixth most prevalent type of malignancy worldwide and accounts for approximately 8% to 10% of all cancers in Southeast Asia [1, 2]. HNSCC-related mortality is mainly caused by cervical lymph node metastasis, and occasionally by distant organ metastasis [3].

The epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their polarity and adopt a mesenchymal phenotype [4]. This process is thought to be a critical step in the induction of tumor metastasis and malignancy [5]. Mani et al. demonstrated that induction of EMT results in cells that have stem cell properties and generates cells with properties similar to breast cancer stem cells [6]. Snail, a member of the zinc-finger transcription factor family, is one of the master regulators that promotes EMT and mediates invasiveness as well as metastasis in many different types of malignant tumors [7, 8]. The aldehyde dehydrogenase (ALDH) family of enzymes is comprised of cytosolic isoenzymes that oxidize intracellular aldehydes and contribute to the oxidation of retinol to retinoic acid in early stem cell differentiation [9]. Recently, ALDH has been reported to be a unique marker of head and neck cancer stem cells (CSC) [10, 11]. ALDH1 was also found to co-localize with other CSCs-related markers, including MMP-9, CD44, and CK14, at the invasive front of the tumor [12]. We previously reported the isolation of ALDH1-positive cells from patients with HNSCC [13]. These HNSCC-ALDH1+ cells displayed the radioresistance and represented a reservoir of cells that have the proliferative potential to generate tumors [13]. ALDH1+-lineage cells underwent EMT and endogenously co-expressed Snail [13]. These findings suggested that Snail expression may regulate the tumorigenesis, radiochemoresistance, and cancer stem cell properties of malignant HNSCC tumors [13]. However, the molecular mechanisms involved in mediating metastasis and tumor malignancy of HNSCC-CSC through the regulation of Snail remain unknown.

Bmi-1 is a member of the Polycomb (PcG) family of transcriptional repressors that mediate gene silencing by regulating chromatin structure [14]. Bmi-1 is essential for maintaining the ability of neural, hematopoietic, and intestinal stem cells to self-renew [1517]. Bmi-1 was identified as a proto-oncogene that cooperates with MYC to promote the generation of lymphoma [18]. Bmi-1 also inhibited MYC-induced apoptosis by repressing the Cdkn2a locus [19]. Additionally, Bmi-1 has been verified as a predictor of prognosis in bladder cancer [20], prostate cancer [21], brain cancer [22, 23], breast cancer [24], pancreatic cancer [25], and lung cancer [26]. Bmi-1 has been demonstrated to play a role in the tumorigenesis of HNSCC [27, 28]. Bmi-1 has also been reported to be involved in tumor metastasis [29, 30]. Recently, an elegant study by Song et al. showed that Bmi-1 can directly promote EMT and malignancy in nasopharyngeal carcinoma by regulating Snail [31]. The goal of this study was to clarify the relationship between Bmi-1, Snail, and ALDH1 in HNSCC or HNSCC-associated CSC and the involved molecular mechanisms.

2. Materials and Methods

2.1. Isolation and Cultivation of HNSCC-Derived ALDH1+ and ALDH1- Cells from HNSCC Patients

This study followed the tenets of the Declaration of Helsinki. All samples were obtained after patients provided informed consent. The study was approved by the Institutional Ethics Committee/Institutional Review Board of Taipei Veterans General Hospital. The information of HNSCC patients has been previously described in Table 1. The dissociated cells from the samples of HNSCC patients were suspended at 1 106 cells/mL in DMEM supplemented with 2% FCS. The identification of aldehyde dehydrogenase 1 (ALDH1) positive HNSCC cells was carried out using the Aldefluor assay (StemCell Technologies, Durham, NC, USA) and fluorescence-activated cell sorting. Cells were suspended in ALDEFLUOR assay buffer containing ALDH substrate (BAAA, 1  mol/l per 1 × 106 cells) and incubated for 40 min at . As a negative control, for each sample of cells, an aliquot was treated with 50 mmol/l diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor. The sorting gates were established using the cells stained with PI only as a negative control; the ALDEFLUOR-stained cells treated with DEAB and staining with a secondary antibody alone to test for viability. HNSCC-ALDH1+ cells were cultured in a medium consisting of serum-free DMEM/F12 (Gibco-BRL, Gaithersburg, MD), N2 supplement (R and D Systems Inc., Minneapolis), 10 ng/mL bFGF (R and D Systems), and 10 ng/mL EGF (R and D Systems) [13, 32].

tab1
Table 1: Case description, tumorigenic characteristics and treatment effects of ALDH1+ and ALDH1- HNSCC.
2.2. Quantitative Real-Time RT-PCR

Briefly, total RNA (1  g) of each sample was reverse-transcribed using 0.5  g oligo dT and 200 U Superscript II RT (Invitrogen). The primer sequences for real-time RT-PCR were listed in Table 2. The amplification was carried out in a total volume of 20  L containing 0.5  mol L-1 of each primer, 4 mmol L-1 MgCl2, 2  L LightCyclerTM-FastStart DNA Master SYBR green I (Roche Molecular Systems, Alameda, CA), and 2  L of 1 : 10 diluted cDNA. PCR reactions were prepared in duplicate and performed using the following program: for 10 min, followed by 40 cycles of denaturation at for 10 sec, annealing at for 5 sec, and extension at for 20 sec. Standard curves (cycle threshold values versus template concentration) were prepared for each target gene and for the endogenous reference gene (GAPDH) for each sample. Quantification of unknown samples was performed using LightCycler Relative Quantification Software version 3.3 (Roche).

tab2
Table 2: The sequences for the primers of quantitative RT-PCR.
2.3. Knockdown and Overexpression of Bmi-1 with Lentivirus

The pLVRNAi vector was purchased from Biosettia Inc. (Biosettia, San Diego CA). The oligonucleotide -AAAACCTAATACTTTCCAGATTGATTTGGAT CCAAATCAATCTGGAAAGTATTAGG- targeting human Bmi-1 (NM_005180, nt 1061–1081) was synthesized and cloned into pLVRNAi to generate the lentiviral expression vector, pLVRNAi/sh-Bmi1. The lentiviral expression vector carrying Bmi-1 full-length cDNA, pLV/Bmi-1 was obtained from Biosettia Inc. pCMVΔR8.9 and pMD.G, expressing GAG-POL and the vesicular stomatitis virus envelope, respectively, were provided by the consortium (Academia Sinica, Taipei, Taiwan). The lentiviruses were generated by cotransfecting 5 106 293FT cells per 10 cm plate with lentiviral vector and packaging plasmids using Lipofectamine 2000 (LF2000, Invitrogen). Supernatants were collected 48 hours after transfection and filtered. The 48-hour posttransduction viral titers were determined by FACS. Subconfluent cells were infected with lentivirus at a multiplicity of infection of 5 in the presence of 8  g/mL polybrene (Sigma-Aldrich) [13, 33].

2.4. Microarray Analysis and Bioinformatics

Total RNA was extracted from cells using Trizol reagent (Life Technologies, Bethesda, MD, USA) and the Qiagen RNAeasy (Qiagen, Valencia, CA, USA) column for purification. Affymetrix HG U133 Plus 2.0 microarrays containing 54,675 probe sets for 47,000 transcripts and variants, including 38,500 human genes. A typical probeset contains eleven 25-mer oligo nucleotide pairs (a perfect match and a mismatch control). For microarray analysis, sample labeling, hybridization, and staining were carried out by Affymetrix standard protocol with affyQCReport. Probeset was normalized with loess method of all microarrays. The average linkage distance was used to assess the similarity between two groups of gene expression profiles as described below. The difference in distance between two groups of sample expression profiles to a third was assessed by comparing the corresponding average linkage distances (the mean of all pairwise distances (linkages) between members of the two groups concerned). The error of such a comparison was estimated by combining the standard errors (the standard deviation of pairwise linkages divided by the square root of the number of linkages) of the average linkage distances involved. Classical multidimensional scaling (MDS) was performed using the standard function of the R program to provide a visual impression of how the various sample groups are related.

2.5. In Vivo Tumor Growth and Metastasis

All procedures involving animals were in accordance with the institutional animal welfare guidelines of Taipei Veterans General Hospital. Eight-week-old SCID mice and/or nude mice (BALB/c strain) were injected with 105 cells orthotopically. In vivo GFP imaging was performed using an illuminating device (LT-9500 Illumatool/TLS equipped with an excitation source (470 nm) and filter plate (515 nm)). Tumor size was measured with calipers and the tumor volume was calculated using the formula (Length Width2)/2. The integrated optical density of green fluorescence intensity was captured and analyzed using Image Pro-plus software [33, 34].

2.6. Statistical Analysis

The Statistical Package of Social Sciences software (SPSS, Inc., Chicago, IL) was used for statistical analysis. An independent Student’s -test was used to compare the continuous variables between groups. The Kaplan-Meier procedure was used to calculate survival probability estimates. A log-rank test was used to compare the cumulative survival durations in different patient groups. The statistical significance level was set at 0.05 for all tests.

3. Results

3.1. HNSCC-Derived ALDH1-Positive Cells Displayed Tumorigenic and Stemness Properties

Initially, parental, isolated ALDH1+, and ALDH1 cells were isolated from tissue samples of six HNSCC patients using the Aldefluor assay and the fluorescence-activated cell sorting (FACS) analysis (Figure 1(a) and Table 1) [13, 35]. It has been reported that cancer stem-like cells can be cultured in suspension to generate floating spheroid-like bodies (SB) under serum-free medium with bFGF and EGF [36]. Interestingly, ALDH1+ increased higher tumor spheres-forming capability than that of ALDH1- (Figure 1(b)). Furthermore, ALDH1+-derived spheres with regular 10% serum cultivation increased epithelial-attached cells and differentiation marker (CK18)(See Figure (a) in supplementary material available online at doi: 10.1155/2011/609259).To evaluate the enhancement of tumorigenicity of HNSCC-ALDH1+ cells, soft agar colony formation assays and Matrigel/Transwell-invasion and were examined. Compared with parental and ALDH1, ALDH1+ derived from HNSCC Patients no. 1 and no. 2 showed colony-forming ability and higher invasion activity (Figures 1(c) and 1(d)). To evaluate the in vivo tumor initiating capability of ALDH1+ and ALDH1, we injected 1000, 3000, and 104 cells into the neck of SCID mice. The results showed that 104 ALDH1 did not induce tumor formation but 3,000 ALDH1+ from the HNSCC tissues of six patients in xenotransplanted mice all resulted in the generation of visible tumors 6 weeks after injection (Table 1).The results of xenotransplanted analysis further showed that ALDH1+ demonstrated higher abilities to induce tumor growth (Figure 1(e)). Lastly, serial xenotransplanted analysis suggested that ALDH1+ had in vivo self-renewal ability (Supplementary Figure (b)). Based on these findings, the ALDH1+-lineage cells isolated from HNSCC patients presented the significant tumor-initiating abilities, especially in ALDH1+ cells from patients no. 1 and no. 2. Real-time RT-PCR data demonstrated that the stemness and EMT-related genes (especially in Bmi-1 and Snail) were significantly activated in HNSCC ALDH1+ (Table 2 and data not shown).

fig1
Figure 1: Isolation and Characterization of HNSCC-derived ALDH1-positive Cells. (a) Analyzing and sorting ALDH1+-positive and ALDH1--negative from HNSCC tissues via FACScan. DEAB, an inhibitor of ALDH1, was used for negative control. (b) Evaluation of sphere body formation in the parental cells, ALDH1 cells, and ALDH1+ cells. Sphere bodies were counted after 1 week. The numbers of resultant colonies (c) and invasion cells (d) from parental cells, ALDH1+ cells, and ALDH1cells were counted in vitro. (e) Macroscopic features of cells in a nude mouse at 6 weeks after xenotransplantation. Blue arrow indicates the site of injection of ALDH1cells. Red arrow indicates the site of injection of ALDH1+ cells. Yellow arrow indicates the site of injection of ALDH1+ cells. . Data shown here are the mean ± SD of three experiments.
3.2. Knockdown of Bmi-1 in HNSCC-ALDH1+ Cells Down-Regulates Snail and Lessens in vitro Tumorigenicity

To further investigate the role of Bmi-1 in maintaining the biological properties of HNSCC-ALDH1+, we used a loss-of-function approach, in which Bmi-1 was knocked down by small hairpin RNA (shRNA) in HNSCC-ALDH1+ cells. Stable knockdown of Bmi-1 in HNSCC-ALDH1+ cells was achieved by transduction with lentivirus that expressed shRNA targeting Bmi-1 (sh-Bmi-1). Lentivirus that expressed shRNA targeted against luciferase (sh-Luc.) was used as a control. Western blot analysis confirmed that shBmi-1 repressed Bmi-1 protein expression in HNSCC-ALDH1+ cells (Figure 2(a)). Importantly, silencing Bmi-1 expression led to downregulation of Snail and ALDH1 expression (Figure 2(a)). Additionally, our results showed that silencing of Bmi-1 in HNSCC-ALDH1+ cells inhibited the ability of the cells to form colonies on soft agar (Figure 2(b)) and migrate/invade (Figure 2(c)).

fig2
Figure 2: Overexpression of Bmi-1 in HNSCC-ALDH1 cells or knockdown of Bmi-1 in HNSCC-ALDH+ cells modulates Snail expression and tumorigenicity in vitro. (a) Down-regulation of Bmi-1 mediated by lentiviral shRNA and expression of Snail and ALDH1 in HNSCC-ALDH1+ cells was analyzed by western blot. Colony formation (b) and migration/invasion ability (c) of shLuc.-expressing and shBmi-1-expressing HNSCC-ALDH1+ cells was determined. (d) Total protein was prepared from control GFP–expressing andBmi-1-overexpressing HNSCC-ALDH1- cells and analyzed by immunoblotting with anti-Bmi-1, anti-Snail, anti-ALDH1, or anti-GAPDH antibodies as indicated. The amount of GAPDH protein from each crude cell extract was used as loading control. Colony formation (e) and migration/invasion ability (f) of Bmi-1-overexpressing and control-GFP-expressing HNSCC-ALDH1- were analyzed. . Data shown here are the mean ± SD of three experiments.
3.3. Overexpression of Bmi-1 in HNSCC-ALDH1 Cells Enhances Tumorigenic Properties by Upregulating Snail

To evaluate whether overexpression of Bmi-1 could enhance the tumorigenic properties of HNSCC-ALDH1- cells, we generated stable Bmi-1-overexpressing (Bmi-1Over) HNSCCs using lentiviral transduction (Figure 2(d)). Total proteins from HNSCC-ALDH1- overexpressing Bmi-1 exhibited elevated expression of Snail and ALDH1 (Figure 2(d)). In addition, overexpression of Bmi-1 significantly increased soft agar colony formation (Figure 2(e)), and migration/invasion of HNSCC-ALDH- cells (Figure 2(f)). Taken together, our results suggest that Bmi-1 modulates the in vitro tumorigenic properties in HNSCC-ALDH1+ or ALDH1 cells by regulating Snail.

3.4. Overexpression of Bmi-1 in HNSCC-ALDH1- Cells Promotes Stemness Properties

To explore molecules governing stemness and tumorigenicity in HNSCC-CD44-ALDH1- cells treated with Bmi1-overexpressing lentivirus, we examined their transcriptome profile using gene expression microarray analysis (Figure 3(a)). Principle component analysis (PCA) further showed that the transcriptome profile of HNSCC-ALDH1 cells overexpressing Bmi-1 demonstrated higher expression levels of embryonic stem cells (ESCs) transcriptomes (Table 3 and Figure 3(b)). Multidimensional scaling analysis further demonstrated that HNSCC-ALDH1+ cells and HNSCC-ALDH1 cells overexpressing Bmi-1 are more similar to ESCs than HNSCC-ALDH1 cells ( ; Figure 3(c)). To validate the microarray analysis results, real-time PCR was performed to confirm that the mRNA expression levels of the embryonic genes (Oct-4, Nanog, Sox2, KLF4, and Lin28), EMT-related genes (Snail and Slug), and drug-resistant-related genes (MDR-1 and ABCG2) in Bmi-1-overexpressing ALDH1- cells were significantly higher than those in ALDH1 cells ; Table 2 and Figure 3(d)).

tab3
Table 3: The expression profiling of up-regulated genes in ALDH1-/Bmi1-overexpressed as compared to ALDH1- HNSCC.
fig3
Figure 3: Stemness properties were enhanced in HNSCC-ALDH1- cells when Bmi-1 was overexpressed. (a) Gene expression microarray analysis (Gene tree) for altered genes differentially expressed in Bmi-1-overexpressing HNSCC-ALDH1- cells compared to HNSCC-ALDH1- cells by a hierarchy heat map. The time dependent changes of altered genes are presented on a log scale of expression values provided by GeneSpring GX software. (b) Principle component analysis (PCA) demonstrated that overexpression of Bmi-1 in HNSCC-ALDH1- cells could enhance the gene signature of embryonic stem cells (ESCs) in HNSCC-ALDH1- cells. (c) Multidimensional scaling analysis. Average lineage transcriptome distances between HNSCC-ALDH1+, HNSCC-ALDH1-, HNSCC-ALDH1+/sh-Bmi-1, and HNSCC-ALDH1-/ cells. . (d) Transcripts of Oct-4, Nanog, Sox2, KLF4, Lin28, Snail, Slug, MDR-1, and ABCG2 in HNSCC-ALDH1- and HNSCC-ALDH1-/Bmi- cells ( : ALDH1- versus Bmi-1-overexpressing ALDH1-).
3.5. Elevation of In Vivo Tumor Growth, Metastatic Activity, and Radioresistance in HNSCC-ALDH1- Cells by Overexpression of Bmi-1

We next sought to determine if Bmi-1 expression could modulate the in vivo tumor initiating activity in immunocompromised nude mice. To monitor the in vivo growth of ALDH1+, ALDH1, and Bmi-1-overexpressing ALDH1 cells, these cells were transfected using a lentivector combined with the green fluorescent protein gene (GFP) and followed by in vivo GFP imaging system. Firstly, the results showed that 1 104 ALDH1 cells did not induce tumor formation in nude mice, but 1000 ALDH1+ cells generated visible tumors 6 weeks after injection (Table 1). In contrast to ALDH1 cells, one of three (33.3%) nude mice was detected with the tumor formation after 6-week transplantation of 3000 Bmi-1-overexpressing ALDH1 cells. Furthermore, tumor volumes in HNSCC-ALDH1+ transplanted mice were significantly decreased when mice were treated with sh-Bmi-1 (Table 1; Figure 4(a)). Overexpression of Bmi-1 enhanced in vivo tumor growth in HNSCC-ALDH1 (Table 1; Figure 4(a)). Furthermore, we investigated the role of Bmi-1 in the radio sensitivity of HNSCC-ALDH1 and HNSCC-ALDH1+ treated with sh-Bmi-1 and Bmi-1 overexpressing. An ionizing radiation (IR) dose of 0 to 10 Gy was applied to these cells, and HNSCC-ALDH1+ cells showed greater radioresistance than the ALDH1 cells ( ; Figure 4(b)). Knockdown of BMI-1 in ALDH1+ cells results in significant inhibition of radioresistance while overexpression of BMI-1 in ALDH- cells promotes radioresistant properties ( ; Figure 4(b)). Moreover, to confirm that Bmi-1 is crucial for metastasis in vivo, mice were injected with different numbers of ALDH1+, ALDH1+/sh-Bmi-1, ALDH1/Bmi-1over or control GFP-expressing ALDH1 cells. 5x105 Bmi-1-overexpressing ALDH1 cells significantly increased local invasion, distant metastasis to the lungs and tumor size compared with control ALDH1 cells (Figures 5(a) and 5(b)). In addition, silencing Bmi-1 in ALDH1+ cells effectively reduced the number of lung metastases and tumor size in vivo (Figures 5(a) and 5(b)). Taken together, our results reveal a crucial role for Bmi-1 signaling in the maintenance of in vivo tumorigenicity and metastasis of HNSCC-ALDH1+ and -ALDH1- cells.

fig4
Figure 4: Determination of the role of Bmi-1 on in vivo tumor growth and radioresistance in HNSCC-ALDH1+ cells. (a) Tumor volume was measured after injection of either HNSCC-ALDH1+, sh-Bmi-1 treated HNSCC-ALDH1+, HNSCC-ALDH1-, or Bmi-1-overexpressing HNSCC- ALDH1- cells into the neck of SCID mice. Error bars correspond to SD. (b) To determine the radiation effect on the cell survival rate, an ionizing radiation (IR) dose from 0 to 10Gy was used to treated with ALDH1+/vector, ALDH1+/sh-Bmi-1, ALDH1- /vector, or Bmi-1-overexpressing HNSCC- ALDH1- HNSCC cells.
fig5
Figure 5: Elimination of metastatic activity in HNSCC-ALDH1+ cells treated with shBmi-1. (a) Summary of the in vivo metastasis ability of different numbers of HNSCC-ALDH1+, sh-Bmi-1 treated HNSCC-ALDH1+, HNSCC- ALDH1-, or Bmi-1-overexpressing HNSCC- ALDH1- cells examined by xenotransplantation analysis. (b) The average numbers of metastatic foci (left panel) and total weight (right panel) in the lungs of mice treated with either HNSCC-ALDH1+, sh-Bmi-1 treated HNSCC-ALDH1+, HNSCC- ALDH1-, or Bmi-1-overexpressing HNSCC-ALDH1- cells are shown. ( : ALDH1- versus Bmi-1-overexpressing ALDH1-; : ALDH1+ versus shBmi-1 treated HNSCC-ALDH1+).
3.6. Coexpression of Bmi-1, Snail, and ALDH1 in HNSCC Tissues Correlates with Poor Overall Survival Rate of HNSCC Patients

Elevated Snail protein expression in HNSCC is correlated with the development of metastasis and poor survival [37]. Elevated expression of ALDH1 also correlates with poor prognosis for HNSCC patients [13]. To investigate whether there is a positive correlation between Bmi-1, Snail, and ALDH1 in head and neck cancers, we studied the expression of Bmi-1, Snail, and ALDH1 by immunohistochemical (IHC) staining of a panel of specimens array from 93 HNSCC patients. The IHC results showed that elevated expression of Bmi-1, Snail, and ALDH1 was positively associated with high-grade, poorly differentiated HNSCC (Figure 6(a)). Our results also showed a significant positive correlation between ALDH-1, Bmi-1 (Figure 6(b)); ALDH-1 and Snail (Figure 6(c)); Bmi-1 and Snail (Figure 6(d)) in HNSCC tissues. This is consistent with previous studies that reported that HNSCC-ALDH1+ cells have elevated Bmi-1 and Snail expression [13, 38]. To determine the prognostic significance of Bmi-1, Snail, and ALDH1 coexpression in patients with HNSCC, Kaplan-Meier survival analysis was performed. Patients who were triple positive for Bmi-1, Snail, and ALDH1 were predicted to have the worst survival rate compared with other head and neck cancer patients (Figure 6(e); Bmi-1+/Snail+/ALDH1+ versus other groups). Overall, these data indicate that expression of Bmi-1, Snail, and ALDH1 in HNSCC patients could be a critical factor in predicting disease progression and clinical outcomes.

fig6
Figure 6: Coexpression of Bmi-1, Snail, and ALDH1 in HNSCC patient specimen and prediction of survival of the HNSCC patients. (a) Representative pictures of triple positive (upper panel) and triple negative (lower panel) HNSCC cases. Coexpression of Bmi-1 and ALDH1 (b), Bmi-1 and Snail (c) or Snail and Bmi-1 (d) of 93 HNSCC patient samples were examined immunohistochemically. (e) Kaplan-Meier analysis of overall survival of HNSCC patients according to expression of ALDH1 (+) Bmi-1 (+) Snail (+), ALDH1 (+) Bmi-1 (+) Snail (−), ALDH1 (−) Bmi-1 (+) Snail (+) or ALDH1 (−) Bmi-1 (−) Snail (−). (*, ; **, ; ***, ).

4. Discussion

A recent study demonstrated that Bmi-1 mRNA and protein overexpressed in a subpopulation of tumor initiating cells in CD44+ HNSCC, which possessed self-renewal and tumor formation ability [39]. Zhang et al. also reported that there are side populations of oral squamous cell carcinomas that express high levels of ABCG2, ABCB1, CD44, Oct-4, Bmi-1, NSPc1, and CK19 [28]. Our previous work showed that HNSCC-ALDH1+ cells have high levels of Bmi-1. The ability to self-renew and radiochemoresistance were significantly suppressed in Bmi-1-silenced HNSCC-ALDH1+ cells [38]. Using microarray, western-blotting, and immunofluorescent assays, Chen et al. confirmed that ALDH1+-lineage cells underwent epithelial-mesenchymal transition (EMT) and endogenously co-expressed Snail [13]. In the current study, our data demonstrated that HNSCC-ALDH1+ cells had high levels of Bmi-1, at both the mRNA and protein levels (Figure 2). Using a lentiviral vector expressing shRNA targeting Bmi-1, we observed that the level of ALDH1 expression and tumorigenic properties of HNSCC-ALDH1+ could be down-regulated by knockdown of Bmi-1 (Figure 2). Importantly, overexpression of Bmi-1 could turn HNSCC-ALDH1 into cancer stem cell-like HNSCC-ALDH1+ cells (Figure 3). Consistent with these findings, the immunohistochemical survey of 93 HNSCC patient tissues showed a positive correlation between expression of Bmi-1, Snail, or ALDH1 and tumor stage (Figure 6). Similar results were noted in other malignancies [40]. Kaplan-Meier analysis demonstrated that patients expressing Bmi-1, Snail, and ALDH1 were predicted to have the worst survival prognosis of HNSCC patients (Figure 6(e)). However, a recent study showed a significant correlation between negative Bmi-1 protein expression and the recurrence of tongue cancer. Their results showed Snail and c-myc expression did not correlate with prognosis [41]. The divergence from our results may be due to the different pathophysiology of HNSCC. Most HNSCC patients in Taiwan consume alcohol, chew betel quid and smoke cigarettes. Tongue cancer patients, especially female tongue cancer patients, usually do not have these habits [3]. The close relationship between tongue cancer and human papillomavirus has been explored by many researchers [4245]. The correlation between cancer stem cells and the virus infection remains to be discovered.

The prognosis of HNSCC patients with distant metastases in the lung, liver, and bone is very poor [3, 46]. In this study, we found that Bmi-1 can regulate Snail and ALDH1; change the EMT-related genotypes of the ALDH1- cells; and modulate distant lung metastases (Figure 5). Distant metastases have been reported to be associated with Bmi-1 expression in breast cancer [4749], melanoma [50], gastric cancer [51], and colon cancer [30]. Microarray analysis revealed that eleven gene signatures, which were correlated to the Bmi-1-driven pathway, were closely related to distant lung metastases [40]. Bmi-1 is the target gene of SALL4 in human hematopoietic as well as leukemic cells and is down-regulated if SALL4 is knocked down by the siRNA in the HL-60 leukemia cell line [52, 53]. Recently, researchers employed microRNA profiling to gain insight into the role of Bmi-1 in regulating EMT. Overexpression of miR-200c decreased Bmi-1 expression in breast cancer stem cells (BCSCs) and inhibited the formation of mammary ducts as well as tumors by normal mammary stem cells and BCSCs [54]. Bhattacharya et al. found that miR-15a and miR-16 directly targeted the Bmi-1 untranslated region and correlated with Bmi-1 protein levels in ovarian cancer patients and cell lines [55]. Further research effort is needed in this area. Together, our research shows that the Bmi-1 signaling pathways play a major role in the maintenance of stemness and the metastatic ability of HNSCC-CSC by regulating of Snail expression. Additionally, we demonstrate coexpression of Bmi-1, Snail, and ALDH1 in HNSCC patients was positively correlated with tumor grade and the worst prognosis.

Acknowledgments

This study was supported by research Grants from the National Science Council (NSC-97-3111-B-075-001-MY3/98-2314-B-075-008-MY3), the Taipei Veterans General Hospital (V97B1-006/E1-008/F-001/F-010), the National Yang-Ming University (Ministry of Education, Aim for the Top University Plan), the Chung Shan Medical University Hospital (CSH-2010-C-025), the Technology Development Program for Academia (98-EC-17-A-19-S2-0107), and Department of Industrial Technology, Ministry of Economic Affairs, Taiwan.

References

  1. R. I. Haddad and D. M. Shin, “Recent advances in head and neck cancer,” The New England Journal of Medicine, vol. 359, no. 11, pp. 1143–1096, 2008. View at Publisher · View at Google Scholar · View at Scopus
  2. Y.-J. Chen, J. T.-C. Chang, C.-T. Liao et al., “Head and neck cancer in the betel quid chewing area: recent advances in molecular carcinogenesis,” Cancer Science, vol. 99, no. 8, pp. 1507–1514, 2008. View at Publisher · View at Google Scholar · View at Scopus
  3. W.-L. Lo, S.-Y. Kao, L.-Y. Chi, Y.-K. Wong, and R. C.-S. Chang, “Outcomes of oral squamous cell carcinoma in Taiwan after surgical therapy: factors affecting survival,” Journal of Oral and Maxillofacial Surgery, vol. 61, no. 7, pp. 751–758, 2003. View at Publisher · View at Google Scholar · View at Scopus
  4. J. P. Their, “Epithelial-mesenchymal transitions in tumor progression,” Nature Reviews Cancer, vol. 2, no. 6, pp. 442–454, 2002. View at Scopus
  5. J. P. Thiery and J. P. Sleeman, “Complex networks orchestrate epithelial-mesenchymal transitions,” Nature Reviews Molecular Cell Biology, vol. 7, no. 2, pp. 131–142, 2006. View at Publisher · View at Google Scholar · View at Scopus
  6. S. A. Mani, W. Guo, M.-J. Liao et al., “The epithelial-mesenchymal transition generates cells with properties of stem cells,” Cell, vol. 133, no. 4, pp. 704–715, 2008. View at Publisher · View at Google Scholar · View at Scopus
  7. E. Batlle, E. Sancho, C. Franci et al., “The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells,” Nature Cell Biology, vol. 2, no. 2, pp. 84–89, 2000. View at Publisher · View at Google Scholar · View at Scopus
  8. B. P. Zhou, J. Deng, W. Xia et al., “Dual regulation of Snail by GSK-3β-mediated phosphorylation in control of epithelial-mesenchymal transition,” Nature Cell Biology, vol. 6, no. 10, pp. 931–940, 2004. View at Publisher · View at Google Scholar · View at Scopus
  9. A. Yoshida, “Molecular genetics of human aldehyde dehydrogenase,” Pharmacogenetics, vol. 2, no. 4, pp. 139–147, 1992. View at Scopus
  10. M. R. Clay, M. Tabor, J. H. Owen, et al., “Single-marker identification of head and neck squamous cell carcinoma cancer stem cells with aldehyde dehydrogenase,” Head Neck, vol. 32, no. 9, pp. 1195–1201, 2010.
  11. Z. G. Chen, “The cancer stem cell concept in progression of head and neck cancer,” Journal of Oncology, vol. 2009, Article ID 894064, 8 pages, 2009. View at Publisher · View at Google Scholar
  12. C. M. Sterz, C. Kulle, B. Dakic, et al., “A basal-cell-like compartment in head and neck squamous cell carcinomas represents the invasive front of the tumor and is expressing MMP-9,” Oral Oncology, vol. 46, no. 2, pp. 116–122, 2010. View at Publisher · View at Google Scholar
  13. Y.-C. Chen, Y.-W. Chen, H.-S. Hsu et al., “Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer,” Biochemical and Biophysical Research Communications, vol. 385, no. 3, pp. 307–313, 2009. View at Publisher · View at Google Scholar · View at Scopus
  14. V. Pirrotta, “Polycombing the genome: PcG, trxG and chromatin silencing,” Cell, vol. 93, no. 3, pp. 333–336, 1998. View at Publisher · View at Google Scholar · View at Scopus
  15. A. V. Molofsky, R. Pardal, T. Iwashita, I.-K. Park, M. F. Clarke, and S. J. Morrison, “Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation,” Nature, vol. 425, no. 6961, pp. 962–967, 2003. View at Publisher · View at Google Scholar · View at Scopus
  16. I.-K. Park, D. Qian, M. Kiel et al., “Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells,” Nature, vol. 423, no. 6937, pp. 302–305, 2003. View at Publisher · View at Google Scholar · View at Scopus
  17. E. Sangiorgi and M. R. Capecchi, “Bmi1 is expressed in vivo in intestinal stem cells,” Nature Genetics, vol. 40, no. 7, pp. 915–920, 2008. View at Publisher · View at Google Scholar · View at Scopus
  18. Y. Haupt, W. S. Alexander, G. Barri, S. P. Klinken, and J. M. Adams, “Novel zinc finger gene implicated as myc collaborator by retrovirally accelerated lymphomagenesis in E μ-myc transgenic mice,” Cell, vol. 65, no. 5, pp. 753–763, 1991. View at Scopus
  19. J. J. L. Jacobs, B. Scheijen, J.-W. Voncken, K. Kieboom, A. Berns, and M. Van Lohuizen, “Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-induced apoptosis via INK4a/ARF,” Genes and Development, vol. 13, no. 20, pp. 2678–2690, 1999. View at Publisher · View at Google Scholar · View at Scopus
  20. Z.-K. Qin, J.-A. Yang, Y.-L. Ye et al., “Expression of Bmi-1 is a prognostic marker in bladder cancer,” BMC Cancer, vol. 9, article 61, 2009. View at Publisher · View at Google Scholar · View at Scopus
  21. C. S. Cooper and C. S. Foster, “Concepts of epigenetics in prostate cancer development,” British Journal of Cancer, vol. 100, no. 2, pp. 240–245, 2009. View at Publisher · View at Google Scholar · View at Scopus
  22. V. Häyry, O. Tynninen, H. K. Haapasalo et al., “Stem cell protein BMI-1 is an independent marker for poor prognosis in oligodendroglial tumours,” Neuropathology and Applied Neurobiology, vol. 34, no. 5, pp. 555–563, 2008. View at Publisher · View at Google Scholar · View at Scopus
  23. R. Tirabosco, G. De Maglio, M. Skrap, G. Falconieri, and S. Pizzolitto, “Expression of the polycomb-group protein BMI1 and correlation with p16 in astrocytomas. An immunohistochemical study on 80 cases,” Pathology Research and Practice, vol. 204, no. 9, pp. 625–631, 2008. View at Publisher · View at Google Scholar · View at Scopus
  24. J. B. Arnes, K. Collett, and L. A. Akslen, “Independent prognostic value of the basal-like phenotype of breast cancer and associations with EGFR and candidate stem cell marker BMI-1,” Histopathology, vol. 52, no. 3, pp. 370–380, 2008. View at Publisher · View at Google Scholar · View at Scopus
  25. W. Song, K. Tao, H. Li, et al., “Bmi-1 is related to proliferation, survival and poor prognosis in pancreatic cancer,” Cancer Science, vol. 101, no. 7, pp. 1754–1760, 2010. View at Publisher · View at Google Scholar
  26. K. Vrzalikova, J. Skarda, J. Ehrmann et al., “Prognostic value of Bmi-1 oncoprotein expression in NSCLC patients: a tissue microarray study,” Journal of Cancer Research and Clinical Oncology, vol. 134, no. 9, pp. 1037–1042, 2008. View at Publisher · View at Google Scholar · View at Scopus
  27. M. Brunner, D. Thurnher, J. Pammer et al., “Expression of VEGF-A/C, VEGF-R2, PDGF-α/β, c-kit, EGFR, Her-2/Neu, Mcl-1 and Bmi-1 in Merkel cell carcinoma,” Modern Pathology, vol. 21, no. 7, pp. 876–884, 2008. View at Publisher · View at Google Scholar · View at Scopus
  28. P. Zhang, Y. Zhang, L. Mao, Z. Zhang, and W. Chen, “Side population in oral squamous cell carcinoma possesses tumor stem cell phenotypes,” Cancer Letters, vol. 277, no. 2, pp. 227–234, 2009. View at Publisher · View at Google Scholar · View at Scopus
  29. Y. Jiang, B. Su, X. Meng, et al., “Effect of siRNA-mediated silencing of Bmi-1 gene expression on HeLa cells,” Cancer Science, vol. 101, no. 2, pp. 379–386, 2010. View at Publisher · View at Google Scholar
  30. D. W. Li, H. M. Tang, J. W. Fan, et al., “Expression level of Bmi-1 oncoprotein is associated with progression and prognosis in colon cancer,” Journal of Cancer Research and Clinical Oncology, vol. 136, no. 7, pp. 997–1006, 2010. View at Publisher · View at Google Scholar
  31. L. B. Song, J. Li, W. T. Liao, et al., “The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells,” The Journal of Clinical Investigation, vol. 119, no. 12, pp. 3626–3636, 2009.
  32. C. C. Yu, G. Y. Chiou, Y. Y. Lee, et al., “Medulloblastoma-derived tumor stem-like cells acquired resistance to TRAIL-induced apoptosis and radiosensitivity,” Child's Nervous System, vol. 26, no. 7, pp. 897–904, 2010. View at Publisher · View at Google Scholar
  33. S.-H. Chiou, C.-L. Kao, Y.-W. Chen et al., “Identification of CD133-positive radioresistant cells in atypical teratoid/rhabdoid tumor,” PLoS ONE, vol. 3, no. 5, article e2090, 2008. View at Publisher · View at Google Scholar · View at Scopus
  34. S.-H. Chiou, C.-C. Yu, C.-Y. Huang et al., “Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma,” Clinical Cancer Research, vol. 14, no. 13, pp. 4085–4095, 2008. View at Publisher · View at Google Scholar · View at Scopus
  35. Y. C. Chen, C. J. Chang, H. S. Hsu, et al., “Inhibition of tumorigenicity and enhancement of radiochemosensitivity in head and neck squamous cell cancer-derived ALDH1-positive cells by knockdown of Bmi-1,” Oral Oncology, vol. 46, no. 3, pp. 158–165, 2010. View at Publisher · View at Google Scholar
  36. M. Baumann, M. Krause, and R. Hill, “Exploring the role of cancer stem cells in radioresistance,” Nature Reviews Cancer, vol. 8, no. 7, pp. 545–554, 2008. View at Publisher · View at Google Scholar · View at Scopus
  37. M.-H. Yang, M.-Z. Wu, S.-H. Chiou et al., “Direct regulation of TWIST by HIF-1α promotes metastasis,” Nature Cell Biology, vol. 10, no. 3, pp. 295–305, 2008. View at Publisher · View at Google Scholar · View at Scopus
  38. Y. C. Chen, C. J. Chang, H. S. Hsu, et al., “Inhibition of tumorigenicity and enhancement of radiochemosensitivity in head and neck squamous cell cancer-derived ALDH1-positive cells by knockdown of Bmi-1,” Oral Oncology, vol. 46, no. 3, pp. 158–165, 2010.
  39. M. E. Prince, R. Sivanandan, A. Kaczorowski et al., “Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 3, pp. 973–978, 2007. View at Publisher · View at Google Scholar · View at Scopus
  40. G. V. Glinsky, O. Berezovska, and A. B. Glinskii, “Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer,” Journal of Clinical Investigation, vol. 115, no. 6, pp. 1503–1521, 2005. View at Publisher · View at Google Scholar · View at Scopus
  41. V. Häyry, L. K. Mäkinen, T. Atula et al., “Bmi-1 expression predicts prognosis in squamous cell carcinoma of the tongue,” British Journal of Cancer, vol. 102, no. 5, pp. 892–897, 2010. View at Publisher · View at Google Scholar
  42. P. Attner, J. Du, A. Näsman et al., “The role of human papillomavirus in the increased incidence of base of tongue cancer,” International Journal of Cancer, vol. 126, no. 12, pp. 2879–2884, 2010. View at Publisher · View at Google Scholar
  43. A. Salem, “Dismissing links between HPV and aggressive tongue cancer in young patients,” Annals of Oncology, vol. 21, no. 1, pp. 13–17, 2010.
  44. X.-H. Liang, J. Lewis, R. Foote, D. Smith, and D. Kademani, “Prevalence and significance of human papillomavirus in oral tongue cancer: the Mayo Clinic experience,” Journal of Oral and Maxillofacial Surgery, vol. 66, no. 9, pp. 1875–1880, 2008. View at Publisher · View at Google Scholar · View at Scopus
  45. L. Dahlgren, H. Dahlstrand, D. Lindquist et al., “Human papillomavirus is more common in base of tongue than in mobile tongue cancer and is a favorable prognostic factor in base of tongue cancer patients,” International Journal of Cancer, vol. 112, no. 6, pp. 1015–1019, 2004. View at Publisher · View at Google Scholar · View at Scopus
  46. D. Sano and J. N. Myers, “Metastasis of squamous cell carcinoma of the oral tongue,” Cancer and Metastasis Reviews, vol. 26, no. 3-4, pp. 645–662, 2007. View at Publisher · View at Google Scholar · View at Scopus
  47. J. H. Kim, S. Y. Yoon, S.-H. Jeong et al., “Overexpression of Bmi-1 oncoprotein correlates with axillary lymph node metastases in invasive ductal breast cancer,” Breast, vol. 13, no. 5, pp. 383–388, 2004. View at Publisher · View at Google Scholar · View at Scopus
  48. J. Silva, V. Garcia, J. M. Garcia, et al., “Circulating Bmi-1 mRNA as a possible prognostic factor for advanced breast cancer patients,” Breast Cancer Research, vol. 9, no. 4, p. R55, 2007.
  49. M. J. Hoenerhoff, I. Chu, D. Barkan et al., “BMI1 cooperates with H-RAS to induce an aggressive breast cancer phenotype with brain metastases,” Oncogene, vol. 28, no. 34, pp. 3022–3032, 2009. View at Publisher · View at Google Scholar · View at Scopus
  50. D. Mihic-Probst, A. Kuster, S. Kilgus et al., “Consistent expression of the stem cell renewal factor BMI-1 in primary and metastatic melanoma,” International Journal of Cancer, vol. 121, no. 8, pp. 1764–1770, 2007. View at Publisher · View at Google Scholar · View at Scopus
  51. J.-H. Liu, L.-B. Song, X. Zhang et al., “Bmi-1 expression predicts prognosis for patients with gastric carcinoma,” Journal of Surgical Oncology, vol. 97, no. 3, pp. 267–272, 2008. View at Publisher · View at Google Scholar · View at Scopus
  52. J. Yang, L. Chai, C. Gao et al., “SALL4 is a key regulator of survival and apoptosis in human leukemic cells,” Blood, vol. 112, no. 3, pp. 805–813, 2008. View at Publisher · View at Google Scholar · View at Scopus
  53. J. Yang, L. Chai, F. Liu et al., “Bmi-1 is a target gene for SALL4 in hematopoietic and leukemic cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 25, pp. 10494–10499, 2007. View at Publisher · View at Google Scholar · View at Scopus
  54. Y. Shimono, M. Zabala, R. W. Cho et al., “Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells,” Cell, vol. 138, no. 3, pp. 592–603, 2009. View at Publisher · View at Google Scholar · View at Scopus
  55. R. Bhattacharya, M. Nicoloso, R. Arvizo, et al., “MiR-15a and MiR-16 control Bmi-1 expression in ovarian cancer,” Cancer Research, vol. 69, no. 23, pp. 9090–9095, 2009.