# Bmi-1 Regulates Snail Expression and Promotes Metastasis Ability in Head and Neck

Authors: Cheng-Chia Yu; Wen-Liang Lo; Yi-Wei Chen; Pin-I Huang; Han-Shui Hsu; Ling-Ming Tseng; Shih-Chieh Hung; Shou-Yen Kao; Charn-Jung  Chang; Shih Hwa Chiou
Journal: Journal of Oncology
 (2011)

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## 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-ALDH1+ cells remain unclear. Initially, HNSCC-ALDH1+ cells from HNSCC patient showed
cancer stemness properties, and high expression of Bmi1 and Snail. Functionally, tumorigenic properties
of HNSCC-ALDH1+ cells could be downregulated by knockdown of Bmi-1. Overexpression of Bmi-1 altered in
expression property ALDH1− cells to that of ALDH1+ cells. Furthermore, knockdown of Bmi-1 enhanced
the radiosensitivity of radiation-treated HNSCC-ALDH1+ cells. Moreover, overexpression of Bmi-1 in
HNSCC-ALDH1− cells increased tumor volume and number of pulmonary metastatic lesions by xenotransplant
assay. Importantly, knock-down of Bmi1 in HNSCC-ALDH1+ 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-ALDH1+ cells.


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## Body

## 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    [15–17]. 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 Table1. The dissociated cells from the samples of HNSCC patients were suspended at 1 × 106 cells/mL in 37°C 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 μmol/l per 1 × 106 cells) and incubated for 40 min at 37°C. 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].Table 1
Case description, tumorigenic characteristics and treatment effects of ALDH1+ and ALDH1-  HNSCC.
Number of cells injectedCaseAge/SexALDH+ (%)SpheresParentalALDH1+ALDH1+ (Sh-Bmi 1)ALDH1-ALDH1- (Bmi 1Over)Formation171/ M44.2Yes1,000 (0/3)1,000 (1/3)1,000 (0/3)1,000 (0/3)1,000 (1/3)3,000 (0/3)3,000 (3/3)3,000 (0/3)3,000 (0/3)3,000 (1/3)10,000 (1/3)10,000 (3/3)10,000 (2/3)10,000 (1/3)10,000 (2/3)273/ F24.7Yes1,000 (0/3)1,000 (1/3)1,000 (1/3)1,000 (0/3)1,000 (0/3)3,000 (0/3)3,000 (2/3)3,000 (2/3)3,000 (0/3)3,000 (1/3)10,000 (2/3)10,000 (2/3)10,000 (2/3)10,000 (0/3)10,000 (2/3)361 / F8.6Yes1,000 (0/3)1,000 (1/3)1,000 (1/3)1,000 (0/3)1,000 (0/3)3,000 (0/3)3,000 (3/3)3,000 (1/3)3,000 (0/3)3,000 (2/3)10,000 (0/3)10,000 (3/3)10,000 (2/3)10,000 (0/3)10,000 (3/3)471 / M1.2Yes1,000 (0/3)1,000 (0/3)1,000 (0/3)1,000 (0/3)1,000 (0/3)3,000 (0/3)3,000 (3/3)3,000 (0/3)3,000 (0/3)3,000 (2/3)10,000 (0/3)10,000 (3/3)10,000 (2/3)10,000 (0/3)10,000 (3/3)569 / M19.2Yes1,000 (0/3)1,000 (1/3)1,000 (0/3)1,000 (0/3)1,000 (0/3)3,000 (0/3)3,000 (3/3)3,000 (1/3)3,000 (0/3)3,000 (2/3)10,000 (1/3)10,000 (3/3)10,000 (2/3)10,000 (0/3)10,000 (3/3)672 / M5.5Yes1,000 (0/3)1,000 (1/3)1,000 (0/3)1,000 (0/3)1,000 (0/3)3,000 (0/3)3,000 (3/3)3,000 (1/3)3,000 (0/3)3,000 (2/3)10,000 (0/3)10,000 (3/3)10,000 (1/3)10,000 (0/3)10,000 (2/3)ALDH1+: ALDH1-positive HNSCC cells; ALDH1-: ALDH1-negative HNSCC cells.ALDH1+ or ALDH1+cells were injected into neck of SCID mice.


### 2.2. Quantitative Real-Time RT-PCR

Briefly, total RNA (1μg) of each sample was reverse-transcribed using 0.5 μg 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 μL containing 0.5 μmol·L-1 of each primer, 4 mmol·L-1 MgCl2, 2 μL LightCyclerTM-FastStart DNA Master SYBR green I (Roche Molecular Systems, Alameda, CA), and 2 μL of 1 : 10 diluted cDNA. PCR reactions were prepared in duplicate and performed using the following program: 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 55°C for 5 sec, and extension at 72°C 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).Table 2
The sequences for the primers of quantitative RT-PCR.
Gene(Accession No.)Primer Sequence (5′ to 3′)Product size (bp)Tm (°C)Oct-4(NM_002701)F: GTGGAGAGCAACTCCGATG8660R: TGCTCCAGCTTCTCCTTCTCNanog(NM_024865)F: ATTCAGGACAGCCCTGATTCTTC7660R: TTTTTGCGACACTCTTCTCTGCSOX-2(NM_003106)F: CGAGTGGAAACTTTTGTCGGA7460R: TGTGCAGCGCTCGCAGMusashi(NM_002442)F: TCCCTCGGCGAGCACA6460R: GACAGCCCCCCCACAAAc-Myc(NM_002467)F: GGAACGAGCTAAAACGGAGCT7155R: GGCCTTTTCATTGTTTTCCAACTβ-catenin(NM_001904)F: CCAGCCGACACCAAGAAG13055R: CGAATCAATCCAACAGTAGCCBmi1(NM_ 005180)F：AAATGCTGGAGAACTGGAAAG12450R：CTGTGGATGAGGAGACTGCNestin(NM_006617)F: AGGAGGAGTTGGGTTCTG11250R: GGAGTGGAGTCTGGAAGGSnail(NM_005985)F:GCTGCCAATGCTCATCTGGGACTCT30055R: TTGAAGGGCTTTCGAGCCTGGAGATSlug(NM_003068)F: GTGATTATTTCCCCGTATCTCTAT29250R: CAATGGCATGGGGGTCTGAAAGMDR-1 (NM_000927)F: TGGCAAAGAAATAAAGCGACTGA7660R: CAGGATGGGCTCCTGGGMRP-1(X60111)F: GCTTCCTCTTGGTGATATTCG17650R: GCAGTTCAACGCATAGTGGABCG2(NM_004827)F: CATGTACTGGCGAAGAATATTTGGT7460R: CACGTGATTCTTCCACAAGCCGAPDH(NM_002046)F: CATCATCCCTGCCTCTACTG18060R: GCCTGCTTCACCACCTTC


### 2.3. Knockdown and Overexpression of Bmi-1 with Lentivirus

The pLVRNAi vector was purchased from Biosettia Inc. (Biosettia, San Diego CA). The oligonucleotide5′-AAAACCTAATACTTTCCAGATTGATTTGGAT CCAAATCAATCTGGAAAGTATTAGG-3′ 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 μg/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 vivoGFP 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’st-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.


## 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 Table1. The dissociated cells from the samples of HNSCC patients were suspended at 1 × 106 cells/mL in 37°C 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 μmol/l per 1 × 106 cells) and incubated for 40 min at 37°C. 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].Table 1
Case description, tumorigenic characteristics and treatment effects of ALDH1+ and ALDH1-  HNSCC.
Number of cells injectedCaseAge/SexALDH+ (%)SpheresParentalALDH1+ALDH1+ (Sh-Bmi 1)ALDH1-ALDH1- (Bmi 1Over)Formation171/ M44.2Yes1,000 (0/3)1,000 (1/3)1,000 (0/3)1,000 (0/3)1,000 (1/3)3,000 (0/3)3,000 (3/3)3,000 (0/3)3,000 (0/3)3,000 (1/3)10,000 (1/3)10,000 (3/3)10,000 (2/3)10,000 (1/3)10,000 (2/3)273/ F24.7Yes1,000 (0/3)1,000 (1/3)1,000 (1/3)1,000 (0/3)1,000 (0/3)3,000 (0/3)3,000 (2/3)3,000 (2/3)3,000 (0/3)3,000 (1/3)10,000 (2/3)10,000 (2/3)10,000 (2/3)10,000 (0/3)10,000 (2/3)361 / F8.6Yes1,000 (0/3)1,000 (1/3)1,000 (1/3)1,000 (0/3)1,000 (0/3)3,000 (0/3)3,000 (3/3)3,000 (1/3)3,000 (0/3)3,000 (2/3)10,000 (0/3)10,000 (3/3)10,000 (2/3)10,000 (0/3)10,000 (3/3)471 / M1.2Yes1,000 (0/3)1,000 (0/3)1,000 (0/3)1,000 (0/3)1,000 (0/3)3,000 (0/3)3,000 (3/3)3,000 (0/3)3,000 (0/3)3,000 (2/3)10,000 (0/3)10,000 (3/3)10,000 (2/3)10,000 (0/3)10,000 (3/3)569 / M19.2Yes1,000 (0/3)1,000 (1/3)1,000 (0/3)1,000 (0/3)1,000 (0/3)3,000 (0/3)3,000 (3/3)3,000 (1/3)3,000 (0/3)3,000 (2/3)10,000 (1/3)10,000 (3/3)10,000 (2/3)10,000 (0/3)10,000 (3/3)672 / M5.5Yes1,000 (0/3)1,000 (1/3)1,000 (0/3)1,000 (0/3)1,000 (0/3)3,000 (0/3)3,000 (3/3)3,000 (1/3)3,000 (0/3)3,000 (2/3)10,000 (0/3)10,000 (3/3)10,000 (1/3)10,000 (0/3)10,000 (2/3)ALDH1+: ALDH1-positive HNSCC cells; ALDH1-: ALDH1-negative HNSCC cells.ALDH1+ or ALDH1+cells were injected into neck of SCID mice.


## 2.2. Quantitative Real-Time RT-PCR

Briefly, total RNA (1μg) of each sample was reverse-transcribed using 0.5 μg 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 μL containing 0.5 μmol·L-1 of each primer, 4 mmol·L-1 MgCl2, 2 μL LightCyclerTM-FastStart DNA Master SYBR green I (Roche Molecular Systems, Alameda, CA), and 2 μL of 1 : 10 diluted cDNA. PCR reactions were prepared in duplicate and performed using the following program: 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 55°C for 5 sec, and extension at 72°C 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).Table 2
The sequences for the primers of quantitative RT-PCR.
Gene(Accession No.)Primer Sequence (5′ to 3′)Product size (bp)Tm (°C)Oct-4(NM_002701)F: GTGGAGAGCAACTCCGATG8660R: TGCTCCAGCTTCTCCTTCTCNanog(NM_024865)F: ATTCAGGACAGCCCTGATTCTTC7660R: TTTTTGCGACACTCTTCTCTGCSOX-2(NM_003106)F: CGAGTGGAAACTTTTGTCGGA7460R: TGTGCAGCGCTCGCAGMusashi(NM_002442)F: TCCCTCGGCGAGCACA6460R: GACAGCCCCCCCACAAAc-Myc(NM_002467)F: GGAACGAGCTAAAACGGAGCT7155R: GGCCTTTTCATTGTTTTCCAACTβ-catenin(NM_001904)F: CCAGCCGACACCAAGAAG13055R: CGAATCAATCCAACAGTAGCCBmi1(NM_ 005180)F：AAATGCTGGAGAACTGGAAAG12450R：CTGTGGATGAGGAGACTGCNestin(NM_006617)F: AGGAGGAGTTGGGTTCTG11250R: GGAGTGGAGTCTGGAAGGSnail(NM_005985)F:GCTGCCAATGCTCATCTGGGACTCT30055R: TTGAAGGGCTTTCGAGCCTGGAGATSlug(NM_003068)F: GTGATTATTTCCCCGTATCTCTAT29250R: CAATGGCATGGGGGTCTGAAAGMDR-1 (NM_000927)F: TGGCAAAGAAATAAAGCGACTGA7660R: CAGGATGGGCTCCTGGGMRP-1(X60111)F: GCTTCCTCTTGGTGATATTCG17650R: GCAGTTCAACGCATAGTGGABCG2(NM_004827)F: CATGTACTGGCGAAGAATATTTGGT7460R: CACGTGATTCTTCCACAAGCCGAPDH(NM_002046)F: CATCATCCCTGCCTCTACTG18060R: GCCTGCTTCACCACCTTC


## 2.3. Knockdown and Overexpression of Bmi-1 with Lentivirus

The pLVRNAi vector was purchased from Biosettia Inc. (Biosettia, San Diego CA). The oligonucleotide5′-AAAACCTAATACTTTCCAGATTGATTTGGAT CCAAATCAATCTGGAAAGTATTAGG-3′ 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 μg/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 vivoGFP 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’st-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 1(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 1(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).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 ALDH1−cells were countedin vitro. (e) Macroscopic features of cells in a nude mouse at 6 weeks after xenotransplantation. Blue arrow indicates the site of injection of ALDH1−cells. Red arrow indicates the site of injection of ALDH1+cells. Yellow arrow indicates the site of injection of ALDH1+cells. *P<.05. Data shown here are the mean ± SD of three experiments.
(a)(b)(c)(d)(e)


### 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)).Overexpression of Bmi-1 in HNSCC-ALDH1− cells or knockdown of Bmi-1 in HNSCC-ALDH+ cells modulates Snail expression and tumorigenicityin 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. *P<.05. Data shown here are the mean ± SD of three experiments.
(a)(b)(c)(d)(e)(f)


### 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 (P*<.05; 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 (P*<.05; Table 2 and Figure 3(d)).Table 3
The expression profiling of up-regulated genes in ALDH1-/Bmi1-overexpressed as compared to ALDH1- HNSCC.
Probe set IDGene symbolGene title217757_atA2Malpha-2-macroglobulin209459_s_atABAT4-aminobutyrate aminotransferase213353_atABCA5ATP-binding cassette, sub-family A (ABC1), member 5209993_atABCB1(MDR1)ATP-binding cassette, sub-family B (MDR/TAP), member 1214033_atABCC6ATP-binding cassette, sub-family C (CFTR/MRP), member 6210246_s_atABCC8ATP-binding cassette, sub-family C (CFTR/MRP), member 8204567_s_atABCG1ATP-binding cassette, sub-family G (WHITE), member 1209735_atABCG2ATP-binding cassette, sub-family G (WHITE), member 2204151_x_atAKR1C1aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase)209699_x_atAKR1C2aldo-keto reductase family 1, member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein; 3-alpha hydroxysteroid dehydrogenase, type III)212224_atALDH1A1aldehyde dehydrogenase 1 family, member A1204446_s_atALOX5arachidonate 5-lipoxygenase205216_s_atAPOHapolipoprotein H (beta-2-glycoprotein I)39248_atAQP3aquaporin 3 (Gill blood group)218501_atARHGEF3Rho guanine nucleotide exchange factor (GEF) 3219087_atASPNAspirin201242_s_atATP1B1ATPase, Na+/K+ transporting, beta 1 polypeptide200921_s_atBTG1B-cell translocation gene 1, anti-proliferative228067_atC2orf55chromosome 2 open reading frame 55206488_s_atCD36CD36 molecule (thrombospondin receptor)208783_s_atCD46CD46 molecule, complement regulatory protein1553970_s_atCELcarboxyl ester lipase (bile salt-stimulated lipase)203854_atCFIcomplement factor I205043_atCFTRcystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)204260_atCHGBchromogranin B (secretogranin 1)221188_s_atCIDEBcell death-inducing DFFA-like effector b203953_s_atCLDN3claudin 3221042_s_atCLMNcalmin (calponin-like, transmembrane)1567081_x_atCLN6ceroid-lipofuscinosis, neuronal 6, late infantile, variant208791_atCLUClusterin229831_atCNTN3contactin 3 (plasmacytoma associated)205615_atCPA1carboxypeptidase A1 (pancreatic)206212_atCPA2carboxypeptidase A2 (pancreatic)205509_atCPB1carboxypeptidase B1 (tissue)201117_s_atCPEcarboxypeptidase E224829_atCPEB4cytoplasmic polyadenylation element binding protein 4204920_atCPS1carbamoyl-phosphate synthetase 1, mitochondrial201990_s_atCREBL2cAMP responsive element binding protein-like 2205971_s_atCTRB1 /// CTRB2chymotrypsinogen B1 /// chymotrypsinogen B2214411_x_atCTRB2chymotrypsinogen B2209774_x_atCXCL2chemokine (C-X-C motif) ligand 2205765_atCYP3A5cytochrome P450, family 3, subfamily A, polypeptide 5228391_atCYP4V2cytochrome P450, family 4, subfamily V, polypeptide 2228739_atCYS1cystin 1222925_atDCDC2doublecortin domain containing 2205311_atDDCdopa decarboxylase (aromatic L-amino acid decarboxylase)210397_atDEFB1defensin, beta 1221081_s_atDENND2DDENN/MADD domain containing 2D214787_atDENND4ADENN/MADD domain containing 4A205684_s_atDENND4CDENN/MADD domain containing 4C214079_atDHRS2dehydrogenase/reductase (SDR family) member 2222850_s_atDNAJB14DnaJ (Hsp40) homolog, subfamily B, member 14225415_atDTX3Ldeltex 3-like (Drosophila)225645_atEHFEts homologous factor210080_x_atELA3Aelastase 3A, pancreatic201510_atELF3E74-like factor 3 (ets domain transcription factor, epithelial-specific)206191_atENTPD3ectonucleoside triphosphate diphosphohydrolase 3220012_atERO1LBERO1-like beta (S. cerevisiae)210103_s_atFOXA2forkhead box A2235201_atFOXP2forkhead box P2226847_atFSTFollistatin205674_x_atFXYD2FXYD domain containing ion transport regulator 2205890_s_atGABBR1 /// UBDgamma-aminobutyric acid (GABA) B receptor, 1 /// ubiquitin D205848_atGAS2growth arrest-specific 2216733_s_atGATMglycine amidinotransferase (L-arginine:glycine amidinotransferase)204965_atGCgroup-specific component (vitamin D binding protein)219508_atGCNT3glucosaminyl (N-acetyl) transferase 3, mucin type225853_atGNPNAT1glucosamine-phosphate N-acetyltransferase 1212950_atGPR116G protein-coupled receptor 116212070_atGPR56G protein-coupled receptor 56203924_atGSTA1glutathione S-transferase A1221942_s_atGUCY1A3guanylate cyclase 1, soluble, alpha 3228697_atHINT3histidine triad nucleotide binding protein 3209558_s_atHIP1Rhuntingtin interacting protein 1 related207062_atIAPPislet amyloid polypeptide213620_s_atICAM2intercellular adhesion molecule 2203828_s_atIL32interleukin 32205945_atIL6Rinterleukin 6 receptor206598_atINSInsulin226535_atITGB6integrin, beta 6226189_atITGB8integrin, beta 8210078_s_atKCNAB1potassium voltage-gated channel, shaker-related subfamily, beta member 1219564_atKCNJ16potassium inwardly-rectifying channel, subfamily J, member 16205303_atKCNJ8potassium inwardly-rectifying channel, subfamily J, member 8212531_atLCN2lipocalin 2235970_atLCORLligand dependent nuclear receptor corepressor-like1554006_a_atLLGL2lethal giant larvae homolog 2 (Drosophila)225996_atLONRF2LON peptidase N-terminal domain and ring finger 2242931_atLONRF3LON peptidase N-terminal domain and ring finger 3226748_atLYSMD2LysM, putative peptidoglycan-binding, domain containing 2213975_s_atLYZlysozyme (renal amyloidosis)222670_s_atMAFBv-maf musculoaponeurotic fibrosarcoma oncogene homolog B (avian)223577_x_atMALAT1metastasis associated lung adenocarcinoma transcript 1 (non-protein coding)220945_x_atMANSC1MANSC domain containing 1204388_s_atMAOAmonoamine oxidase A235077_atMEG3maternally expressed 3229254_atMFSD4major facilitator superfamily domain containing 4219797_atMGAT4Amannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isozyme A204259_atMMP7matrix metallopeptidase 7 (matrilysin, uterine)227747_atMPZL3myelin protein zero-like 3204438_atMRC1 /// MRC1L1mannose receptor, C type 1 /// mannose receptor, C type 1-like 1203037_s_atMTSS1metastasis suppressor 1212093_s_atMTUS1mitochondrial tumor suppressor 1213693_s_atMUC1mucin 1, cell surface associated213375_s_atN4BP2L1NEDD4 binding protein 2-like 1220184_atNANOGNanog homeobox209107_x_atNCOA1nuclear receptor coactivator 11556057_s_atNEUROD1neurogenic differentiation 1206915_atNKX2-2NK2 homeobox 2225911_atNPNTNephronectin205259_atNR3C2nuclear receptor subfamily 3, group C, member 2212768_s_atOLFM4olfactomedin 4203845_atPCAFp300/CBP-associated factor240317_atPCDHB4protocadherin beta 4212593_s_atPDCD4programmed cell death 4 (neoplastic transformation inhibitor)213228_atPDE8Bphosphodiesterase 8B225207_atPDK4pyruvate dehydrogenase kinase, isozyme 4205380_atPDZK1PDZ domain containing 11553589_a_atPDZK1IP1PDZK1 interacting protein 1226459_atPIK3AP1phosphoinositide-3-kinase adaptor protein 1220954_s_atPILRBpaired immunoglobulin-like type 2 receptor beta219584_atPLA1Aphospholipase A1 member A206311_s_atPLA2G1Bphospholipase A2, group IB (pancreas)221529_s_atPLVAPplasmalemma vesicle associated protein205912_atPNLIPpancreatic lipase211766_s_atPNLIPRP2pancreatic lipase-related protein 2208286_x_atPOU5F1(Oct4)POU class 5 homeobox 1 /// POU class 5 homeobox 1B /// POU class 5 homeobox 1 pseudogene 3 /// POU class 5 homeobox 1 pseudogene 4228469_atPPIDPeptidylprolyl isomerase D (cyclophilin D)210670_atPPYpancreatic polypeptide242482_atPRKAR1Aprotein kinase, cAMP-dependent, regulatory, type I, alpha (tissue specific extinguisher 1)227629_atPRLRProlactin receptor228656_atPROX1prospero homeobox 1205869_atPRSS1protease, serine, 1 (trypsin 1)205402_x_atPRSS2protease, serine, 2 (trypsin 2)213421_x_atPRSS3protease, serine, 3203317_atPSD4pleckstrin and Sec7 domain containing 4203029_s_atPTPRN2protein tyrosine phosphatase, receptor type, N polypeptide 2219562_atRAB26RAB26, member RAS oncogene family226436_atRASSF4Ras association (RalGDS/AF-6) domain family member 4223322_atRASSF5Ras association (RalGDS/AF-6) domain family member 5235638_atRASSF6Ras association (RalGDS/AF-6) domain family member 6204364_s_atREEP1receptor accessory protein 1209752_atREG1Aregenerating islet-derived 1 alpha (pancreatic stone protein, pancreatic thread protein)205886_atREG1Bregenerating islet-derived 1 beta (pancreatic stone protein, pancreatic thread protein)205815_atREG3Aregenerating islet-derived 3 alpha1554003_atRGNEFRho-guanine nucleotide exchange factor219263_atRNF128ring finger protein 128221614_s_atRPH3ALrabphilin 3A-like (without C2 domains)213939_s_atRUFY3RUN and FYVE domain containing 3210592_s_atSAT1spermidine/spermine N1-acetyltransferase 1203408_s_atSATB1SATB homeobox 1204035_atSCG2secretogranin II (chromogranin C)205697_atSCGNsecretagogin, EF-hand calcium binding protein229620_atSEPP1Selenoprotein P, plasma, 1202833_s_atSERPINA1serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1202376_atSERPINA3serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3209443_atSERPINA5serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 5213572_s_atSERPINB1serpin peptidase inhibitor, clade B (ovalbumin), member 1227627_atSGK3serum/glucocorticoid regulated kinase family, member 3219256_s_atSH3TC1SH3 domain and tetratricopeptide repeats 1204019_s_atSH3YL1SH3 domain containing, Ysc84-like 1 (S. cerevisiae)213464_atSHC2SHC (Src homology 2 domain containing) transforming protein 2205799_s_atSLC3A1solute carrier family 3 (cystine, dibasic and neutral amino acid transporters, activator of cystine, dibasic and neutral amino acid transport), member 1223044_atSLC40A1solute carrier family 40 (iron-regulated transporter), member 1228221_atSLC44A3solute carrier family 44, member 3213139_atSNAI2(Slug)snail homolog 2 (Drosophila)1560228_atSNAI3(Snail)snail homolog 3 (Drosophila)213721_atSOX2SRY (sex determining region Y)-box 2200795_atSPARCL1SPARC-like 1 (mast9, hevin)206239_s_atSPINK1serine peptidase inhibitor, Kazal type 1213921_atSSTsomatostatin216905_s_atST14suppression of tumorigenicity 14 (colon carcinoma)230285_atSVIPsmall VCP/p97-interacting protein227134_atSYTL1synaptotagmin-like 1202286_s_atTACSTD2tumor-associated calcium signal transducer 2205513_atTCN1transcobalamin I (vitamin B12 binding protein, R binder family)203887_s_atTHBDthrombomodulin209937_atTM4SF4transmembrane 4 L six family member 4226403_atTMC4transmembrane channel-like 4223503_atTMEM163transmembrane protein 163218345_atTMEM176Atransmembrane protein 176A220532_s_atTMEM176Btransmembrane protein 176B200847_s_atTMEM66transmembrane protein 66202687_s_atTNFSF10tumor necrosis factor (ligand) superfamily, member 10203824_atTSPAN8tetraspanin 8229169_atTTC18tetratricopeptide repeat domain 18209660_atTTRtransthyretin (prealbumin, amyloidosis type I)231008_atUNC5CLUnc-5 homolog C (C. elegans)-like226344_atZMAT1zinc finger, matrin type 1206059_atZNF91zinc finger protein 91Stemness 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-/Bmi1over cells. *P<.05. (d) Transcripts of Oct-4, Nanog, Sox2, KLF4, Lin28, Snail, Slug, MDR-1, and ABCG2 in HNSCC-ALDH1- and HNSCC-ALDH1-/Bmi-1over cells (*P<.05: ALDH1- versus Bmi-1-overexpressing ALDH1-).
(a)(b)(c)(d)


### 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 thein 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 vivoGFP imaging system. Firstly, the results showed that 1 × 104ALDH1− 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 (P<.05; 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 (P<.05; 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 sizein 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.Determination of the role of Bmi-1 onin 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.
(a)(b)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. (*P<.05: ALDH1- versus Bmi-1-overexpressing ALDH1-; #P<.05: ALDH1+ versus shBmi-1 treated HNSCC-ALDH1+).
(a)(b)(c)


### 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.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 (−). (*, P<.05; **, P<.01; ***, P<.001).
(a)(b)(c)(d)(e)


## 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 1(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 1(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).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 ALDH1−cells were countedin vitro. (e) Macroscopic features of cells in a nude mouse at 6 weeks after xenotransplantation. Blue arrow indicates the site of injection of ALDH1−cells. Red arrow indicates the site of injection of ALDH1+cells. Yellow arrow indicates the site of injection of ALDH1+cells. *P<.05. Data shown here are the mean ± SD of three experiments.
(a)(b)(c)(d)(e)


## 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)).Overexpression of Bmi-1 in HNSCC-ALDH1− cells or knockdown of Bmi-1 in HNSCC-ALDH+ cells modulates Snail expression and tumorigenicityin 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. *P<.05. Data shown here are the mean ± SD of three experiments.
(a)(b)(c)(d)(e)(f)


## 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 (P*<.05; 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 (P*<.05; Table 2 and Figure 3(d)).Table 3
The expression profiling of up-regulated genes in ALDH1-/Bmi1-overexpressed as compared to ALDH1- HNSCC.
Probe set IDGene symbolGene title217757_atA2Malpha-2-macroglobulin209459_s_atABAT4-aminobutyrate aminotransferase213353_atABCA5ATP-binding cassette, sub-family A (ABC1), member 5209993_atABCB1(MDR1)ATP-binding cassette, sub-family B (MDR/TAP), member 1214033_atABCC6ATP-binding cassette, sub-family C (CFTR/MRP), member 6210246_s_atABCC8ATP-binding cassette, sub-family C (CFTR/MRP), member 8204567_s_atABCG1ATP-binding cassette, sub-family G (WHITE), member 1209735_atABCG2ATP-binding cassette, sub-family G (WHITE), member 2204151_x_atAKR1C1aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase)209699_x_atAKR1C2aldo-keto reductase family 1, member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein; 3-alpha hydroxysteroid dehydrogenase, type III)212224_atALDH1A1aldehyde dehydrogenase 1 family, member A1204446_s_atALOX5arachidonate 5-lipoxygenase205216_s_atAPOHapolipoprotein H (beta-2-glycoprotein I)39248_atAQP3aquaporin 3 (Gill blood group)218501_atARHGEF3Rho guanine nucleotide exchange factor (GEF) 3219087_atASPNAspirin201242_s_atATP1B1ATPase, Na+/K+ transporting, beta 1 polypeptide200921_s_atBTG1B-cell translocation gene 1, anti-proliferative228067_atC2orf55chromosome 2 open reading frame 55206488_s_atCD36CD36 molecule (thrombospondin receptor)208783_s_atCD46CD46 molecule, complement regulatory protein1553970_s_atCELcarboxyl ester lipase (bile salt-stimulated lipase)203854_atCFIcomplement factor I205043_atCFTRcystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)204260_atCHGBchromogranin B (secretogranin 1)221188_s_atCIDEBcell death-inducing DFFA-like effector b203953_s_atCLDN3claudin 3221042_s_atCLMNcalmin (calponin-like, transmembrane)1567081_x_atCLN6ceroid-lipofuscinosis, neuronal 6, late infantile, variant208791_atCLUClusterin229831_atCNTN3contactin 3 (plasmacytoma associated)205615_atCPA1carboxypeptidase A1 (pancreatic)206212_atCPA2carboxypeptidase A2 (pancreatic)205509_atCPB1carboxypeptidase B1 (tissue)201117_s_atCPEcarboxypeptidase E224829_atCPEB4cytoplasmic polyadenylation element binding protein 4204920_atCPS1carbamoyl-phosphate synthetase 1, mitochondrial201990_s_atCREBL2cAMP responsive element binding protein-like 2205971_s_atCTRB1 /// CTRB2chymotrypsinogen B1 /// chymotrypsinogen B2214411_x_atCTRB2chymotrypsinogen B2209774_x_atCXCL2chemokine (C-X-C motif) ligand 2205765_atCYP3A5cytochrome P450, family 3, subfamily A, polypeptide 5228391_atCYP4V2cytochrome P450, family 4, subfamily V, polypeptide 2228739_atCYS1cystin 1222925_atDCDC2doublecortin domain containing 2205311_atDDCdopa decarboxylase (aromatic L-amino acid decarboxylase)210397_atDEFB1defensin, beta 1221081_s_atDENND2DDENN/MADD domain containing 2D214787_atDENND4ADENN/MADD domain containing 4A205684_s_atDENND4CDENN/MADD domain containing 4C214079_atDHRS2dehydrogenase/reductase (SDR family) member 2222850_s_atDNAJB14DnaJ (Hsp40) homolog, subfamily B, member 14225415_atDTX3Ldeltex 3-like (Drosophila)225645_atEHFEts homologous factor210080_x_atELA3Aelastase 3A, pancreatic201510_atELF3E74-like factor 3 (ets domain transcription factor, epithelial-specific)206191_atENTPD3ectonucleoside triphosphate diphosphohydrolase 3220012_atERO1LBERO1-like beta (S. cerevisiae)210103_s_atFOXA2forkhead box A2235201_atFOXP2forkhead box P2226847_atFSTFollistatin205674_x_atFXYD2FXYD domain containing ion transport regulator 2205890_s_atGABBR1 /// UBDgamma-aminobutyric acid (GABA) B receptor, 1 /// ubiquitin D205848_atGAS2growth arrest-specific 2216733_s_atGATMglycine amidinotransferase (L-arginine:glycine amidinotransferase)204965_atGCgroup-specific component (vitamin D binding protein)219508_atGCNT3glucosaminyl (N-acetyl) transferase 3, mucin type225853_atGNPNAT1glucosamine-phosphate N-acetyltransferase 1212950_atGPR116G protein-coupled receptor 116212070_atGPR56G protein-coupled receptor 56203924_atGSTA1glutathione S-transferase A1221942_s_atGUCY1A3guanylate cyclase 1, soluble, alpha 3228697_atHINT3histidine triad nucleotide binding protein 3209558_s_atHIP1Rhuntingtin interacting protein 1 related207062_atIAPPislet amyloid polypeptide213620_s_atICAM2intercellular adhesion molecule 2203828_s_atIL32interleukin 32205945_atIL6Rinterleukin 6 receptor206598_atINSInsulin226535_atITGB6integrin, beta 6226189_atITGB8integrin, beta 8210078_s_atKCNAB1potassium voltage-gated channel, shaker-related subfamily, beta member 1219564_atKCNJ16potassium inwardly-rectifying channel, subfamily J, member 16205303_atKCNJ8potassium inwardly-rectifying channel, subfamily J, member 8212531_atLCN2lipocalin 2235970_atLCORLligand dependent nuclear receptor corepressor-like1554006_a_atLLGL2lethal giant larvae homolog 2 (Drosophila)225996_atLONRF2LON peptidase N-terminal domain and ring finger 2242931_atLONRF3LON peptidase N-terminal domain and ring finger 3226748_atLYSMD2LysM, putative peptidoglycan-binding, domain containing 2213975_s_atLYZlysozyme (renal amyloidosis)222670_s_atMAFBv-maf musculoaponeurotic fibrosarcoma oncogene homolog B (avian)223577_x_atMALAT1metastasis associated lung adenocarcinoma transcript 1 (non-protein coding)220945_x_atMANSC1MANSC domain containing 1204388_s_atMAOAmonoamine oxidase A235077_atMEG3maternally expressed 3229254_atMFSD4major facilitator superfamily domain containing 4219797_atMGAT4Amannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isozyme A204259_atMMP7matrix metallopeptidase 7 (matrilysin, uterine)227747_atMPZL3myelin protein zero-like 3204438_atMRC1 /// MRC1L1mannose receptor, C type 1 /// mannose receptor, C type 1-like 1203037_s_atMTSS1metastasis suppressor 1212093_s_atMTUS1mitochondrial tumor suppressor 1213693_s_atMUC1mucin 1, cell surface associated213375_s_atN4BP2L1NEDD4 binding protein 2-like 1220184_atNANOGNanog homeobox209107_x_atNCOA1nuclear receptor coactivator 11556057_s_atNEUROD1neurogenic differentiation 1206915_atNKX2-2NK2 homeobox 2225911_atNPNTNephronectin205259_atNR3C2nuclear receptor subfamily 3, group C, member 2212768_s_atOLFM4olfactomedin 4203845_atPCAFp300/CBP-associated factor240317_atPCDHB4protocadherin beta 4212593_s_atPDCD4programmed cell death 4 (neoplastic transformation inhibitor)213228_atPDE8Bphosphodiesterase 8B225207_atPDK4pyruvate dehydrogenase kinase, isozyme 4205380_atPDZK1PDZ domain containing 11553589_a_atPDZK1IP1PDZK1 interacting protein 1226459_atPIK3AP1phosphoinositide-3-kinase adaptor protein 1220954_s_atPILRBpaired immunoglobulin-like type 2 receptor beta219584_atPLA1Aphospholipase A1 member A206311_s_atPLA2G1Bphospholipase A2, group IB (pancreas)221529_s_atPLVAPplasmalemma vesicle associated protein205912_atPNLIPpancreatic lipase211766_s_atPNLIPRP2pancreatic lipase-related protein 2208286_x_atPOU5F1(Oct4)POU class 5 homeobox 1 /// POU class 5 homeobox 1B /// POU class 5 homeobox 1 pseudogene 3 /// POU class 5 homeobox 1 pseudogene 4228469_atPPIDPeptidylprolyl isomerase D (cyclophilin D)210670_atPPYpancreatic polypeptide242482_atPRKAR1Aprotein kinase, cAMP-dependent, regulatory, type I, alpha (tissue specific extinguisher 1)227629_atPRLRProlactin receptor228656_atPROX1prospero homeobox 1205869_atPRSS1protease, serine, 1 (trypsin 1)205402_x_atPRSS2protease, serine, 2 (trypsin 2)213421_x_atPRSS3protease, serine, 3203317_atPSD4pleckstrin and Sec7 domain containing 4203029_s_atPTPRN2protein tyrosine phosphatase, receptor type, N polypeptide 2219562_atRAB26RAB26, member RAS oncogene family226436_atRASSF4Ras association (RalGDS/AF-6) domain family member 4223322_atRASSF5Ras association (RalGDS/AF-6) domain family member 5235638_atRASSF6Ras association (RalGDS/AF-6) domain family member 6204364_s_atREEP1receptor accessory protein 1209752_atREG1Aregenerating islet-derived 1 alpha (pancreatic stone protein, pancreatic thread protein)205886_atREG1Bregenerating islet-derived 1 beta (pancreatic stone protein, pancreatic thread protein)205815_atREG3Aregenerating islet-derived 3 alpha1554003_atRGNEFRho-guanine nucleotide exchange factor219263_atRNF128ring finger protein 128221614_s_atRPH3ALrabphilin 3A-like (without C2 domains)213939_s_atRUFY3RUN and FYVE domain containing 3210592_s_atSAT1spermidine/spermine N1-acetyltransferase 1203408_s_atSATB1SATB homeobox 1204035_atSCG2secretogranin II (chromogranin C)205697_atSCGNsecretagogin, EF-hand calcium binding protein229620_atSEPP1Selenoprotein P, plasma, 1202833_s_atSERPINA1serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1202376_atSERPINA3serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3209443_atSERPINA5serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 5213572_s_atSERPINB1serpin peptidase inhibitor, clade B (ovalbumin), member 1227627_atSGK3serum/glucocorticoid regulated kinase family, member 3219256_s_atSH3TC1SH3 domain and tetratricopeptide repeats 1204019_s_atSH3YL1SH3 domain containing, Ysc84-like 1 (S. cerevisiae)213464_atSHC2SHC (Src homology 2 domain containing) transforming protein 2205799_s_atSLC3A1solute carrier family 3 (cystine, dibasic and neutral amino acid transporters, activator of cystine, dibasic and neutral amino acid transport), member 1223044_atSLC40A1solute carrier family 40 (iron-regulated transporter), member 1228221_atSLC44A3solute carrier family 44, member 3213139_atSNAI2(Slug)snail homolog 2 (Drosophila)1560228_atSNAI3(Snail)snail homolog 3 (Drosophila)213721_atSOX2SRY (sex determining region Y)-box 2200795_atSPARCL1SPARC-like 1 (mast9, hevin)206239_s_atSPINK1serine peptidase inhibitor, Kazal type 1213921_atSSTsomatostatin216905_s_atST14suppression of tumorigenicity 14 (colon carcinoma)230285_atSVIPsmall VCP/p97-interacting protein227134_atSYTL1synaptotagmin-like 1202286_s_atTACSTD2tumor-associated calcium signal transducer 2205513_atTCN1transcobalamin I (vitamin B12 binding protein, R binder family)203887_s_atTHBDthrombomodulin209937_atTM4SF4transmembrane 4 L six family member 4226403_atTMC4transmembrane channel-like 4223503_atTMEM163transmembrane protein 163218345_atTMEM176Atransmembrane protein 176A220532_s_atTMEM176Btransmembrane protein 176B200847_s_atTMEM66transmembrane protein 66202687_s_atTNFSF10tumor necrosis factor (ligand) superfamily, member 10203824_atTSPAN8tetraspanin 8229169_atTTC18tetratricopeptide repeat domain 18209660_atTTRtransthyretin (prealbumin, amyloidosis type I)231008_atUNC5CLUnc-5 homolog C (C. elegans)-like226344_atZMAT1zinc finger, matrin type 1206059_atZNF91zinc finger protein 91Stemness 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-/Bmi1over cells. *P<.05. (d) Transcripts of Oct-4, Nanog, Sox2, KLF4, Lin28, Snail, Slug, MDR-1, and ABCG2 in HNSCC-ALDH1- and HNSCC-ALDH1-/Bmi-1over cells (*P<.05: ALDH1- versus Bmi-1-overexpressing ALDH1-).
(a)(b)(c)(d)


## 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 thein 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 vivoGFP imaging system. Firstly, the results showed that 1 × 104ALDH1− 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 (P<.05; 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 (P<.05; 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 sizein 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.Determination of the role of Bmi-1 onin 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.
(a)(b)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. (*P<.05: ALDH1- versus Bmi-1-overexpressing ALDH1-; #P<.05: ALDH1+ versus shBmi-1 treated HNSCC-ALDH1+).
(a)(b)(c)


## 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.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 (−). (*, P<.05; **, P<.01; ***, P<.001).
(a)(b)(c)(d)(e)


## 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    [42–45]. 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    [47–49], 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 3′ 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.



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*Source: 609259-2010-09-27.xml*