Chromosomal instability (CIN) is widely considered a hallmark of cancer, but its precise roles in cancer stem cells (CSC) and malignant progression remain uncertain. BMI1 is a member of the Polycomb group of chromatin-modifier proteins that is essential for stem cell self-renewal. In human cancers, BMI1 overexpression drives stem-like properties associated with induction of epithelial–mesenchymal transition (EMT) that promotes invasion, metastasis, and poor prognosis. Here, we report that BMI1 mediates its diverse effects through upregulation of the mitotic kinase Aurora A, which is encoded by the AURKA gene. Two mechanisms were found to be responsible for BMI1-induced AURKA expression. First, BMI1 activated the Akt pathway, thereby upregulating AURKA expression through activation of the β-catenin/TCF4 transcription factor complex. Second, BMI1 repressed miRNA let-7i through a Polycomb complex-dependent mechanism, thereby relieving AURKA expression from let-7i suppression. AURKA upregulation by BMI1 exerts several effects, including centrosomal amplification and aneuploidy, antiapoptosis, and cell-cycle progression through p53 degradation and EMT through stabilization of Snail. Inhibiting Aurora A kinase activity attenuated BMI1-induced tumor growth in vivo. In clinical specimens of head and neck cancer, we found that coamplification of BMI1 and AURKA correlated with poorer prognosis. Together, our results link CSCs, EMT, and CIN through the BMI1–AURKA axis and suggest therapeutic use from inhibiting Aurora A in head and neck cancers, which overexpress BMI1. Cancer Res; 73(2); 953–66. ©2012 AACR.

Recently, one of the most important conceptual advances in cancer research is the discovery of a small population of cancer cells harboring stem cell properties, that is, cancer stem cells (CSC). CSCs are critically involved in the initiation, progression, recurrence, and therapeutic resistance of human cancers (1, 2). The regulatory mechanisms of CSCs have been extensively investigated. Among them, the role of Polycomb group proteins in promoting stem-like properties of cancer cells has been valued significantly (3). The Polycomb group proteins, including Polycomb repressive complex 1 and 2 (PRC1 and 2), are chromatin modifiers, which regulate the expression of a number of genes during stem cell self-renewal, tumor formation, and progression (4). BMI1 is a member of PRC1 and plays an essential role in maintaining self-renewal of stem cells through suppressing the INK4A-ARF locus (5–7). In human cancers, BMI1 is often overexpressed and acts as an oncogene to promote tumorigenesis (8), and overexpression of BMI1 is frequently observed in CSCs (9–11). Importantly, the role of BMI1 in epithelial–mesenchymal transition (EMT)–generated CSCs has been highlighted in pancreatic cancer, the EMT regulator ZEB1 inhibits the expression of the miRNA-200 family, leading to an increased expression of BMI1 and stem-like properties (12); in squamous cell carcinoma of the head and neck (HNSCC), BMI1 is essential for Twist1-induced EMT and stem-like properties (13); in nasopharyngeal carcinoma, BMI1 represses PTEN expression, resulting in Akt activation and EMT (14). However, whether BMI1 possesses a more extended function to facilitate cancer progression beyond INK4A-ARF suppression and EMT-generated CSCs, is unclear.

Chromosomal instability (CIN) is the inability to maintain a correct chromosome complement after mitosis. The chromosome aberrations in tumor cells have been discovered for decades, and CIN has been considered to be one of the hallmarks of tumor formation (15). However, the understanding of CIN in tumor progression is relatively limited. Emerging evidence suggests the involvement of CIN in cancer progression. CIN is associated with a poor prognosis in patients with lung and colon cancer (16, 17). Furthermore, overactivation of the mitotic checkpoint, a major mechanism for CIN acquisition, has been shown to inhibit certain tumor suppressive signal pathways (15). However, the molecular link between CIN and tumor progression, and whether CIN is related to CSCs, remain largely elusive.

Aurora A is a serine/threonine kinase that regulates mitotic processes in mammalian cells, including centrosome maturation, spindle assembly, and chromosome segregation (18, 19). Amplification of AURKA has been shown in various types of human cancers (20), and overexpression of Aurora A promotes CIN, cell-cycle progression, and therapeutic resistance (20, 21). In addition, Aurora A phosphorylates p53, which leads to ubiquitination and degradation of p53 and suppresses apoptosis (22). In this study, we discover a novel link between BMI1 and Aurora A through 2 PRC-mediated signal pathways. In HNSCC, BMI1 expression results in multifaceted changes through regulating Aurora A, including centrosomal amplification/aneuploidy, cell-cycle progression, antiapoptosis, and EMT. All these events act collaboratively to facilitate tumor progression.

Cell lines and plasmids

The human hypopharyngeal cancer cell line FaDu and the human embryonic kidney cell line HEK-293T were obtained from the Bioresource Collection and Research Center of Taiwan. The human oral cancer cell line OECM-1 was originally from Dr. Ching-Liang Meng of National Defense Medical College (Taipei, Taiwan; ref. 23). The human oral cancer cell lines SAS and CAL-27 were from Dr. Cheng-Chi Chang's laboratory (National Taiwan University, Taipei, Taiwan). The pCDH-BMI1 plasmid was generated by inserting a 981-bp fragment of the full-length human BMI1 cDNA into the NheI/BamHI sites of the pCDH lentiviral vector. pmCherry-let7i, pmCherry-spg-ctrl, and pmCherry-spg-let7i were previously described (24). The pcDNA3–β-catenin was generated by cloning the coding sequence of β-catenin into pcDNA3.1 vector. The pcDNA3-AURKA was generated by cloning the coding sequence of AURKA into pCDH-GPF-puro vector. The dominant-negative TCF4 plasmid pPGS-dnTcf4(ΔN31) and pcDNA3-HA-GSK-3β was purchased from Addgene. The plasmids for short hairpin RNA (shRNA) experiments were generated by inserting a specific shRNA target sequence or a scrambled sequence into the pSUPER.puro vector. The sequences used to generate the plasmid are listed in Supplementary Table S1. Stable cell lines were generated by transfection/infection of expression or shRNA plasmids into parietal cell lines and selected using the appropriate antibiotics.

Sorting of CD44+ HNSCC cells

For CD44+cell isolation, cells were suspended in 100 μL 1× PBS and stained with 5 μL CD44-AlexaFluor488 (Clone 156-3C11, Cat# 3516, Cell Signaling Technology, Inc.), or AlexaFluor488 mouse IgG2a (Clone MOPC-173, Cat# 558055, BD Biosciences) as isotype control. The stained cells were separated by FACSAria (BD Biosciences).

Luciferase reporter assay

The wild-type human AURKA firefly luciferase promoter construct (pGL-1486) was a gift from Dr. Ishigatsubo (Yokohama City University, Yokohama, Japan; ref. 25). The pGL-1486 mutant construct was generated by mutating the TCF4-binding site on the AURKA promoter. The full-length 3′-untranslated region (UTR) of AURKA was cloned into the pMIR-REPORTER to generate the pMIR-AURKA-wt construct, and the putative let-7i-binding site was mutated to generate pMIR-AURKA-mut. A plasmid expressing the renilla luciferase gene (pRL-TK) was cotransfected into each transfection experiment as a control for transfection efficiency. Cells were harvested after 48 hours of transfection, and luciferase activity was assayed. The relative promoter activities were expressed as the fold-change of luciferase activity after normalization to renilla luciferase activity.

miRNAs microarray analysis

The Agilent human miRNA array (V2) was conducted in FaDu cells transfected with a BMI1-expressing vector (FaDu-BMI1) or a control vector. The microarray data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database with the accession number of GSE 31910. The miRNA microarray data for OECM1-sh-BMI1 versus OECM1-sh-scr have been published (24) and deposited in the NCBI GEO database with the accession number of GSE 29586.

Immunofluorescence staining

Cells were seeded in poly-l-lysine–coated slides and fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 10% FBS. The primary antibody used was against γ-tubulin for evaluating the number of centrosomes, or E-cadherin for evaluating the adherent junctions. The secondary antibody used was fluorescein isothiocyanate (FITC)–conjugated against mouse immunoglobulin G (IgG; Cat# AP124F, Millipore Corp.) for detecting γ-tubulin, and rhodamine-conjugated against rabbit IgG (Cat# AP132R, Chemicon International Inc.) for detecting E-cadherin. 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. The slide images were captured on an Olympus 1000i (Olympus Corporation).

Karyotype analysis

Cells were treated with colcemid at 37°C for 2 hours. Cells were harvested, swollen in warm 0.075 mol/L KCl, fixed in cold 3:1 methanol/glacial acetic acid, dropped onto slides, and dried at room temperature. The chromosome images were captured on the Olympus BX51 High Class System Microscope (Olympus Corporation).

Cell-cycle analysis

Cells were treated with the Aurora kinase inhibitor III (Cat# C1368, Sigma-Aldrich Corp.) at 3 μmol/L for 24 hours. The cells were fixed with 75% ethanol at −20°C overnight and were stained in propidium iodide (PI; 50 μg/mL; Sigma-Aldrich Corp.), RNase A (0.02 μg/μL), and 1× PBS at 37°C for 30 minutes in the dark. The stained cells were analyzed by flow cytometry.

Apoptosis analysis

For evaluating the effect of Aurora kinase inhibition on apoptosis, cells were treated with 5 μmol/L Aurora kinase inhibitor III (AKI III; Cat# C1368, Sigma-Aldrich Corp.) or the vehicle control for 48 hours. Then the cells were suspended in 100 μL 1× binding buffer containing 10 mmol/L HEPES at pH 7.4, 140 mmol/L NaCl, and 2.5 mmol/L CaCl2, and stained with 5 μL allophycocyanin-conjugated Annexin V (Cat# 550475, BD Biosciences) and 5 μL of 50 μg/mL PI for 15 minutes. The stained cells were analyzed by flow cytometry.

In vivo drug sensitivity assay

All animal protocols were carried out in accordance with the institutional animal welfare guidelines of Taipei Veterans General Hospital (Taipei, Taiwan). A total of 1 × 107 of each stable cell lines were injected into the subcutaneous area of the 6-week-old BALB/C nude mice. After the tumor volume reached 200 mm3, we started to treat the mice with the Aurora kinase inhibitor VX-680 at the dose of 0, 25, or 50 mg/kg, 4 times a week for 3 weeks. The mice were sacrificed after the 3-week treatment, and the xenotransplanted tumors were collected for analysis. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay by using the In Situ Cell Death Detection Kit (No. 11-684-817-910, Roche Applied Science) was applied for detecting the apoptotic cells in tumors.

Immunohistochemistry

Immunohistochemistry (IHC) for detecting BMI1 and Aurora A was conducted, and the slides were independently scored by 2 individuals according to the immunoreactive score (IRS; ref. 26). For BMI1, only nuclear expression was considered positive, whereas both nuclear and cytoplasmic expression were considered positive for Aurora A. A moderate to strong level (IRS > 4) was considered positive, and weak or absent expression (IRS, 0–4) was considered negative.

Please see Supplementary Methods for the other methods used in this study.

BMI1 upregulates the expression of Aurora A and both of them are coamplified in HNSCC stem-like cells

We started this study by aiming to understand whether the expression of the mitosis/CIN–related genes could be regulated by the “stemness factor” BMI1, to provide a molecular connection between CSCs and CIN. For the major role of BMI1 in the CSCs of HNSCC (11, 13), we used HNSCC cell lines as the platform to investigate the mechanism. To this end, 2 HNSCC cell lines were applied for manipulating the expression of BMI1 and examined the change of the expression of mitosis-related genes. FaDu cells were selected for ectopic expression of BMI1 (FaDu-BMI1) because of the low endogenous BMI1 level, and OECM-1 cells was selected as the parental cell line to generate the BMI1-knockdown clones (OECM1-sh-BMI1) owing to the high endogenous BMI1 expression levels (24). Quantitative reverse transcription PCR (qRT-PCR) analysis of a panel of mitosis/CIN–related genes (AURKA, AURKB, BUB1, BUB1B, BUB3, CDC20, FZR1, CENPE, CCNB1, NDC80, MAD1L1, PTTG1, PLK1, and PLK4; with reference to ref. 15) showed that AURKA was the only candidate both upregulated in FaDu-BMI1 and downregulated in OECM1-sh-BMI1 (Fig. 1A). The protein level of Aurora A was also upregulated in FaDu-BMI1 and downregulated OECM1-sh-BMI1 (Fig. 1B). Because BMI1 is directly regulated by Twist1 (13), we examined if Twist1 upregulates Aurora A. The result showed that ectopic Twist1 augmented the expression of BMI1 as well as Aurora A in FaDu cells (Supplementary Fig. S1A), and repression of Twist1 in OECM-1 cells attenuated both BMI1 and Aurora A (Supplementary Fig. S1B).

Figure 1.

BMI1 upregulates Aurora A in cancer cells. A, a heatmap summarizing the results of relative mRNA levels of the mitotic-related genes in FaDu cells transfected with a BMI1-expressing vector (FaDu-BMI1) versus control vector (FaDu-CDH), or OECM-1 cells transfected with a shRNA against BMI1 (OECM1-sh-BMI1) versus a scrambled sequence (OECM1-sh-scr). B, top, Western blot analysis of BMI1 and Aurora A in FaDu and OECM-1 clones. Bottom, quantification of the Western blot analysis results. C, correlation between the relative expression levels of BMI1, CDKN2A, and AURKA in cancer cell lines from the NCI-60 panel. D, top, relative mRNA levels of BMI1 and AURKA in HNSCC cell lines. Bottom, Western blot analysis of BMI1 and Aurora A in HNSCC cell lines. E, representative result of flow cytometry for sorting the CD44+ cells. F, relative mRNA levels of CD44, BMI1, and AURKA in CD44+ versus CD44 FaDu cells. FSC-A, forward scatter. In each experiment, data represent mean ± SEM (n = 3). **, P < 0.01.

Figure 1.

BMI1 upregulates Aurora A in cancer cells. A, a heatmap summarizing the results of relative mRNA levels of the mitotic-related genes in FaDu cells transfected with a BMI1-expressing vector (FaDu-BMI1) versus control vector (FaDu-CDH), or OECM-1 cells transfected with a shRNA against BMI1 (OECM1-sh-BMI1) versus a scrambled sequence (OECM1-sh-scr). B, top, Western blot analysis of BMI1 and Aurora A in FaDu and OECM-1 clones. Bottom, quantification of the Western blot analysis results. C, correlation between the relative expression levels of BMI1, CDKN2A, and AURKA in cancer cell lines from the NCI-60 panel. D, top, relative mRNA levels of BMI1 and AURKA in HNSCC cell lines. Bottom, Western blot analysis of BMI1 and Aurora A in HNSCC cell lines. E, representative result of flow cytometry for sorting the CD44+ cells. F, relative mRNA levels of CD44, BMI1, and AURKA in CD44+ versus CD44 FaDu cells. FSC-A, forward scatter. In each experiment, data represent mean ± SEM (n = 3). **, P < 0.01.

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To further confirm the relationship between AURKA and BMI1, we investigated the correlation between AURKA and BMI1 in microarray datasets of the NCI-60 panel (27). We selected the cell lines from the NCI-60 panel in which the expression of BMI1 was inversely correlated with that of CDKN2A, a known target suppressed by BMI1 (correlation coefficient r < −0.7), indicating the functional significance of the BMI1 in these cell lines. Then, we observed the correlation between BMI1 and AURKA. Among these cell lines, the expression of BMI1 was positively correlated with that of AURKA (correlation coefficient r = 0.422; P < 0.05; Fig. 1C). Because the NCI-60 panel does not contain HNSCC cell lines, we compared the mRNA and protein levels of BMI1 and Aurora A in 4 HNSCC cell lines. Among these cell lines, OECM-1 and SAS cells had a significant higher expression of BMI1 and Aurora A in both mRNA and protein levels. In contrast, FaDu and CAL-27 had a lower expression of BMI1 and Aurora A (Fig. 1D). Furthermore, BMI1 and AURKA were coamplified in CD44+ population of the HNSCC cell lines and a primary HNSCC culture (Fig. 1E and F; Supplementary Fig. S1C and S1D), indicating the activation of BMI1–AURKA signal in HNSCC stem-like cells. Taken together, the earlier findings indicate that in HNSCC, BMI1 upregulates the expression of Aurora A, and both of them are coamplified in the stem-like population.

BMI1 increases AURKA through activating Akt–β-catenin pathway

Next, we sought to find out the mechanism about how BMI1 regulates AURKA expression. Because BMI1 suppresses target genes expression through chromatin silencing (28, 29), we speculated that BMI1 indirectly enhances Aurora A expression. BMI1 was shown to suppress PTEN and activate Akt pathway (14). Furthermore, β-catenin, an oncoprotein that is phosphorylated and destabilized by glycogen synthase kinase-3β (GSK-3β; ref. 30), was reported to upregulate AURKA (31). We therefore hypothesized that BMI1 induces the expression of AURKA through activation of Akt, which in turns results in the phosphorylation and inactivation of GSK-3β, leading to accumulation of β-catenin and transactivation of AURKA. To confirm this notion, we examined the change of phospohrylated Akt, phosphorylated GSK-3β, β-catenin, and Aurora A in BMI1 knocked down OECM-1 cells versus control cells, and BMI1-overexpressed FaDu cells versus control cells. Knockdown of BMI1 in OECM-1 reduced the phosphorylation of Akt at serine 473 and GSK-3β at serine 9, and decreased the expression of β-catenin and Aurora A (Fig. 2A). Consistently, overexpression of BMI1 in FaDu cells enhanced Akt and GSK-3β phosphorylation and increased β-catenin and Aurora A expression. Inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway by a PI3K inhibitor (LY294002) partially attenuated the BMI1-induced Aurora A upregulation (Fig. 2B and C). Ectopic β-catenin upregulated Aurora A in FaDu cells (Fig. 2D). These results indicate that BMI1 upregulates Aurora A through activation of the Akt–β-catenin pathway.

Figure 2.

Activation of Akt pathway by BMI1 enhances AURKA promoter activity through β-catenin/TCF-4 complex. A and B, Western blot analysis of BMI1, total Akt, phosphorylated Akt (serine 473), total GSK-3β, phosphorylated GSK-3β (serine 9), β-catenin, and Aurora A in OECM1-sh-BMI1 versus OECM1-sh-scr (A) and FaDu-CDH versus FaDu-BMI1 (B) with or without LY294002 treatment. Dimethyl sulfoxide (DMSO) was a vehicle control. C, quantification of the Western blot analysis results in B. D, Western blot analysis of β-catenin and Aurora A in FaDu cells transfected with pcDNA3–β-catenin or an empty vector (EV). E, schematic representation of the wild-type (left) or TCF4-binding site–mutated (right) AURKA promoter construct used in luciferase reporter assay. +1 indicates the transcription start site. F and G, luciferase reporter assay. Data represent mean ± SEM (n = 3). *, P < 0.05.

Figure 2.

Activation of Akt pathway by BMI1 enhances AURKA promoter activity through β-catenin/TCF-4 complex. A and B, Western blot analysis of BMI1, total Akt, phosphorylated Akt (serine 473), total GSK-3β, phosphorylated GSK-3β (serine 9), β-catenin, and Aurora A in OECM1-sh-BMI1 versus OECM1-sh-scr (A) and FaDu-CDH versus FaDu-BMI1 (B) with or without LY294002 treatment. Dimethyl sulfoxide (DMSO) was a vehicle control. C, quantification of the Western blot analysis results in B. D, Western blot analysis of β-catenin and Aurora A in FaDu cells transfected with pcDNA3–β-catenin or an empty vector (EV). E, schematic representation of the wild-type (left) or TCF4-binding site–mutated (right) AURKA promoter construct used in luciferase reporter assay. +1 indicates the transcription start site. F and G, luciferase reporter assay. Data represent mean ± SEM (n = 3). *, P < 0.05.

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For T-cell factors (TCF) are the major binding partners of β-catenin to activate target genes transcription (32), we investigated whether BMI1 activates AURKA promoter through the β-catenin/TCF4 complex. We first confirmed the transactivation of AURKA by β-catenin/TCF4. The results showed that β-catenin activated AURKA promoter, and the dominant-negative TCF4 abrogated the β-catenin–induced AURKA activation. Mutation of the TCF4-binding site also abolished the AURKA transactivation (Fig. 2E and F). We next examined whether BMI1 could activate AURKA promoter through β-catenin/TCF4. Ectopic BMI1 activated AURKA promoter, and either transfecting the dominant-negative TCF4 or mutating the TCF4-binding sequence on AURKA promoter abrogated the BMI1-induced AURKA activation (Fig. 2G). Taken together, these results suggest that BMI1 upregulates Aurora A through the activation of Akt pathway, leading to the recruitment of β-catenin/TCF4 complex to the promoter of AURKA to activate its transcription.

Repression of let-7i by BMI1 contributes to BMI1-induced Aurora A expression

Because suppression of Akt activity only partially abrogated BMI1-induced Aurora upregulation, we hypothesized that there may exist another mechanism to mediate BMI1-induced Aurora A expression. We reasoned that BMI1-repressed miRNA(s) may participate in this regulation as miRNAs are able to suppress target gene expression. In this scenario, BMI1 represses the expression of miRNA(s) that target(s) AURKA, resulting in upregulation of Aurora A. To this end, miRNA microarray analysis was conducted on BMI1-overexpressing FaDu cells versus a control cells (Supplementary Table S3). The result was compared with the miRNA profile in BMI1 knocked down OECM-1 cells versus control cells (24). Both let-7i and miR-15b were candidates repressed by BMI1 as they were downregulated in FaDu-BMI1 and upregulated in OECM1-sh-BMI1 (Fig. 3A). let-7i was more probable to be the target of BMI1 as let-7i ranked first in the miRNAs downregulated in FaDu-BMI1 (Supplementary Table S3), and ranked third in the miRNAs upregulated in OECM1-sh-BMI1 (24). qRT-PCR validation confirmed that let-7i, but not miR-15b, was consistently repressed by BMI1 in both FaDu and OECM1 systems (Fig. 3B). For the Polycomb group proteins repress target genes expression through forming PRC on the regulatory area containing Polycomb responsive element (PRE; ref. 33), we analyzed the sequence of let-7i promoter and 2 highly conserved PREs were found (Fig. 3C). Quantitative chromatin immunoprecipitation (qChIP) assays showed the enrichment of the binding of BMI1 as well as EZH2, a PRC2 subunit that methylates lysine 27 of histone 3 (H3K27; ref. 34), and trimethylated H3K27 (H3K27me3) on PREs of let-7i promoter (Fig. 3D). These results indicated that BMI1 represses let-7i expression through the PRC-mediated chromatin silencing.

Figure 3.

Suppression of let-7i by BMI1 upregulates Aurora A expression. A, schema for identifying the BMI1-downregulated miRNAs. B, expression of let-7i and miR-15b in FaDu and OECM-1 clones. C, organization of the MIRLET7I promoter and sequence alignment of the 2 PREs (PRE1 and PRE2). qChIP indicates the amplified sequences in qChIP. +1 indicates the transcription start site. D, qChIP assay. E, left, expression of let-7i in OECM-1 cells transfected with a let-7i–expressing vector (OECM1-let7i) or a control vector (OECM1-mCherry). Right, Western blot analysis of Aurora A. F, left, expression of let-7i in FaDu cells transfected with a sponge vector for neutralizing let-7i (FaDu-spg-let-7i) or a control vector (FaDu-spg-ctrl). Right, Western blot analysis of Aurora A. G, schematic representation of the wild-type or let-7i-binding site–mutated 3′-UTR reporter constructs of AURKA. H, 3′-UTR luciferase reporter assay. I, Western blot analysis of BMI1 and Aurora A in FaDu-CDH, FaDu-BMI1 transfected with an empty vector (EV) or a let-7i–expressing vector. In each experiment, data represent mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

Suppression of let-7i by BMI1 upregulates Aurora A expression. A, schema for identifying the BMI1-downregulated miRNAs. B, expression of let-7i and miR-15b in FaDu and OECM-1 clones. C, organization of the MIRLET7I promoter and sequence alignment of the 2 PREs (PRE1 and PRE2). qChIP indicates the amplified sequences in qChIP. +1 indicates the transcription start site. D, qChIP assay. E, left, expression of let-7i in OECM-1 cells transfected with a let-7i–expressing vector (OECM1-let7i) or a control vector (OECM1-mCherry). Right, Western blot analysis of Aurora A. F, left, expression of let-7i in FaDu cells transfected with a sponge vector for neutralizing let-7i (FaDu-spg-let-7i) or a control vector (FaDu-spg-ctrl). Right, Western blot analysis of Aurora A. G, schematic representation of the wild-type or let-7i-binding site–mutated 3′-UTR reporter constructs of AURKA. H, 3′-UTR luciferase reporter assay. I, Western blot analysis of BMI1 and Aurora A in FaDu-CDH, FaDu-BMI1 transfected with an empty vector (EV) or a let-7i–expressing vector. In each experiment, data represent mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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We next aimed to determine whether AURKA is a target of let-7i. Ectopic let-7i downregulated Aurora A in OECM-1 cells (Fig. 3E), and neutralization of let-7i enhanced Aurora A expression in FaDu cells (Fig. 3F). In contrast, miR-15 did not have impact on Aurora A expression (Supplementary Fig. S2A). A reporter assay showed that ectopic let-7i suppressed the activity of the AURKA 3′-UTR reporter, and mutation of the let-7i–binding site abrogated the repression (Fig. 3G and H). Reconstitution of let-7i in FaDu-BMI1 attenuated Aurora A expression (Fig. 3I). We further investigated whether let-7i repression and Akt–β-catenin pathway act independently to mediate BMI1-induced Aurora A upregulation. The result showed that suppression of Akt activity did not have significant impact on restoring let-7i expression in FaDu-BMI1 cells (Supplementary Fig. S2B). The regulation of Aurora A by BMI1 through both Akt pathway and let-7i repression was confirmed in another HNSCC cell line SAS: knockdown of BMI1 in SAS cells repressed Aurora A expression, reduced the levels of phosphorylated Akt, phosphorylated GSK-3β, and β-catenin, and upregulated let-7i expression (Supplementary Fig. S2C and S2D). Taken together, these data suggest that Aurora A is a bona fide target repressed by let-7i. Akt–β-catenin pathway and let-7i repression are 2 independent mechanisms induced by BMI1 to regulate Aurora A expression in HNSCC cells.

BMI1 promotes chromosomal instability, antiapoptosis, and cell-cycle progression through Aurora A

Next, we investigated whether BMI1 could acquire the Aurora A–induced phenotype through regulating AURKA. In cancer cells, one of the major functions of Aurora A is to induce CIN (18, 19). We examined the impact of BMI1–AURKA axis on centrosome number and karyotype in FaDu and OECM-1 stable cell lines. In FaDu cells, ectopic BMI1 increased the percentage of cells with more than 2 centrosomes, and repression of Aurora A in BMI1 transfectants abrogated such effect (Fig. 4A). In OECM-1 cells, knockdown of either BMI1 or AURKA caused a reduction of cells with more than 2 centrosomes (Supplementary Fig. S3A and S3B). Karyotype analysis showed that overexpression of BMI1 increased the proportion of aneuploidy in FaDu cells, and silencing Aurora A in BMI1 transfectants reversed it (Fig. 4B). Suppression of either BMI1 or Aurora A expression in OECM-1 cells significantly decreased the percentage of cells with aneuploidy (Supplementary Fig. S3C and S3D).

Figure 4.

BMI1–AURKA axis promotes CIN, antiapoptosis, and cell-cycle progression. A, top, representative results of immunoflouresent staining in FaDu clones. The arrows indicate centrosomes. Scale bar, 20 μm. Bottom, quantification of centrosome analysis (100 cells were counted for each clone). B, top, representative pictures of karyotype analysis in FaDu clones. The number of chromosomes was shown in each panel. Bottom, quantification of the percentage of cells with aneuploidy (30 cells were counted in each experiment to determine the percentage of aneuploidy). C, top, Western blot analysis of BMI1, Aurora A, and p53 in FaDu clones. Bottom, Western blot analysis for detecting p53 serine 315 phosphorylation in the above cells treated with MG132. D, left, representative results of flow cyotmetry for detecting annexin V or/and PI-positive cells. Right, quantification of the early apoptotic cells (annexin V+PI). E, top, flow cytometry for cell-cycle analysis. Bottom, histograms showing the results of cell-cycle analysis. In each experiment, data represent mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01.

Figure 4.

BMI1–AURKA axis promotes CIN, antiapoptosis, and cell-cycle progression. A, top, representative results of immunoflouresent staining in FaDu clones. The arrows indicate centrosomes. Scale bar, 20 μm. Bottom, quantification of centrosome analysis (100 cells were counted for each clone). B, top, representative pictures of karyotype analysis in FaDu clones. The number of chromosomes was shown in each panel. Bottom, quantification of the percentage of cells with aneuploidy (30 cells were counted in each experiment to determine the percentage of aneuploidy). C, top, Western blot analysis of BMI1, Aurora A, and p53 in FaDu clones. Bottom, Western blot analysis for detecting p53 serine 315 phosphorylation in the above cells treated with MG132. D, left, representative results of flow cyotmetry for detecting annexin V or/and PI-positive cells. Right, quantification of the early apoptotic cells (annexin V+PI). E, top, flow cytometry for cell-cycle analysis. Bottom, histograms showing the results of cell-cycle analysis. In each experiment, data represent mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01.

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For Aurora A phosphorylates p53 at serine 315, leading to ubiquitination and degradation of p53 (22), we investigated whether Aurora A is important for BMI1-induced cell antiapoptosis and cell-cycle progression through degrading p53. The results showed that in FaDu cells, ectopic BMI1 upregulated Aurora A, enhanced p53 serine 315 phosphorylation and decreased total p53. Repression of AURKA in FaDu-BMI1 reduced p53 serine 315 phosphorylation and restored total p53 level (Fig. 4C). In OECM-1 cells, silencing AURKA did not alter the level of BMI1, which confirms that Aurora A is located downstream to BMI1. Knockdown of either AURKA or BMI1 reduced p53 serine 315 phosphorylation and increased total p53 level (Supplementary Fig. S3E and S3F). For analyzing the apoptosis, we found that ectopic BMI1 repressed apoptosis as expected. Treatment of the cells with an Aurora A inhibitor (AKI III) increased the proportion of apoptotic cells (Fig. 4D). For cell-cycle analysis, overexpression of BMI1 enforced the cells into cell cycle by reducing the proportion of cells in G0–G1 and increasing the proportion of cells in S-phase. In FaDu-BMI1 cells, knockdown of AURKA increased sub-G1 and G2–M populations. Treatment with the Aurora A inhibitor AKI III had a more prominent effect than AURKA knockdown in enriching sub-G1 population (Fig. 4E). Collectively, these results suggest that BMI1 promotes CIN through regulating Aurora A. Furthermore, Aurora A is crucial for BMI1-induced p53 stabilization, antiapoptosis, and cell-cycle progression.

Aurora A is critical in BMI1-induced EMT through stabilization of Snail

For BMI1 is a major determinant of EMT and stem-like phenotype of cancer cells (12–14), we investigated whether Aurora A plays a role in BMI1-induced EMT. In FaDu cells, overexpression of BMI1 induced EMT as expected, that is, upregulation of the epithelial markers E-cadherin and γ-catenin, downregulation of mesenchymal markers N-cadherin and vimentin, dissociation of E-cadherin, and enhanced migration and invasion. Interestingly, knockdown of AURKA in FaDu-BMI1 restored the epithelial phenotype (Fig. 5A and B). Overexpression of BMI1 enhanced both migration and invasion ability of FaDu cells. Either knockdown of AURKA or treatment with AKI III abrogated BMI1-induced migration and invasion (Fig. 5C). Because the role of Aurora A in EMT is not clearly defined yet, we investigated whether Aurora A is capable of inducing EMT in HNSCC cells. In OECM-1 cells, repression of Aurora A reverted the cells to epithelial phenotype and reduced migration and invasion (Supplementary Fig. S4A–S4D). Ectopic expression of Aurora A in FaDu cells induced EMT (Supplementary Fig. S4E–S4H). These results suggest that Aurora A is critical in BMI1-induced EMT, and Aurora A itself is sufficient to induce EMT in HNSCC cells.

Figure 5.

Aurora A contributes to BMI1-induced EMT through stabilizing Snail. A, Western blot analysis of BMI1, Aurora A, epithelial markers (E-cadherin and γ-catenin), and mesenchymal markers (N-cadherin and vimentin) in FaDu clones. B, immunofluorescent staining of FaDu clones. Scale bar, 20 μm. C, top 2 panels, fold-change of migrated cells in wound-healing assay. Bottom 2 panels, fold-change of invaded cells in invasion assay. D, Western blot analysis of BMI1, Aurora A, and Snail in FaDu clones. E, relative SNAI1 mRNA expression in FaDu clones. F, Western blot analysis of BMI1, Aurora A, and Snail in FaDu clones after cyclohexamide (CHX) treatment for 0, 2, and 4 hours. G, Western blot analysis of BMI1, Aurora A, total GSK-3β, and serine 9-phosphorylated GSK-3β in FaDu clones. H, immunoprecipitation by an anti-Aurora A antibody and Western blot analysis for detecting pulled down Aurora A and serine 9-phosphorylated GSK-3β in OECM-1 cells. The arrows indicate the immunoprecipitated bands. I, coimmunoprecipitation assay in HEK-293T cells. The transfection and immunoprecipitation condition was indicated in the panel. The arrows indicate the immunoprecipitated bands. In C and E, data represent mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01.

Figure 5.

Aurora A contributes to BMI1-induced EMT through stabilizing Snail. A, Western blot analysis of BMI1, Aurora A, epithelial markers (E-cadherin and γ-catenin), and mesenchymal markers (N-cadherin and vimentin) in FaDu clones. B, immunofluorescent staining of FaDu clones. Scale bar, 20 μm. C, top 2 panels, fold-change of migrated cells in wound-healing assay. Bottom 2 panels, fold-change of invaded cells in invasion assay. D, Western blot analysis of BMI1, Aurora A, and Snail in FaDu clones. E, relative SNAI1 mRNA expression in FaDu clones. F, Western blot analysis of BMI1, Aurora A, and Snail in FaDu clones after cyclohexamide (CHX) treatment for 0, 2, and 4 hours. G, Western blot analysis of BMI1, Aurora A, total GSK-3β, and serine 9-phosphorylated GSK-3β in FaDu clones. H, immunoprecipitation by an anti-Aurora A antibody and Western blot analysis for detecting pulled down Aurora A and serine 9-phosphorylated GSK-3β in OECM-1 cells. The arrows indicate the immunoprecipitated bands. I, coimmunoprecipitation assay in HEK-293T cells. The transfection and immunoprecipitation condition was indicated in the panel. The arrows indicate the immunoprecipitated bands. In C and E, data represent mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01.

Close modal

To determine the mechanism of BMI1-induced Aurora A in promoting EMT, we examined the protein levels of different EMT regulators in established stable cell lines. Among the EMT regulators, only Snail was consistently regulated by BMI1–AURKA axis in different cell lines. In FaDu cells, ectopic BMI1 upregulated Snail expression, and knockdown of AURKA abrogated the BMI1-induced Snail expression (Fig. 5D); the other EMT regulators was not consistently affected (Supplementary Fig. S5A). Silencing AURKA reduced Snail expression in OECM-1 (Supplementary Fig. S5B), and overexpression of Aurora A upregulated Snail but not other EMT regulators in FaDu (Supplementary Fig. S5C). However, there was no difference between the mRNA level of SNAI1 in FaDu-CDH, FaDu-BMI1-sh-scr, and FaDu-BMI1-sh-AURKA (Fig. 5E), and ectopic Aurora A also did not change the mRNA level of SNAI1 (Supplementary Fig. S5D). Furthermore, BMI1 increased Snail stability, and knockdown of AURKA in FaDu-BMI1 reduced Snail stability (Fig. 5F). These results indicate that BMI1-regulated Aurora A induces EMT through stabilization of Snail.

Because GSK-3β phosphorylates Snail to promote degradation (35), and Aurora A enhances GSK-3β phsophorylation to repress its activity (36), we investigated whether Aurora A interacts with GSK-3β and increases its phosphorylation under BMI1-expressing situation. In FaDu cells, overexpression of BMI1 enhanced GSK-3β serine 9 phosphorylation, and knockdown of AURKA in FaDu-BMI1 abrogated this phosphorylation (Fig. 5G). Repression of Aurora A in OECM-1 cells attenuated GSK-3β serine 9 phosphorylation (Supplementary Fig. S6A). Coimmunoprecipitation experiments confirmed the physical association of Aurora A and GSK-3β among different HNSCC cell lines (Supplementary Fig. S6B–S6E). Serine 9-phsophorylated GSK-3β was present in the immunoprecipitates obtained with an anti-Aurora A antibody from OECM-1 cells (Fig. 5H). Ectopic Aurora A pulled down endogenous GSK-3β, and ectopic GSK-3β also pulled down endogenous Aurora A (Fig. 5I). Collectively, these data suggest that Aurora A is important in BMI1-induced EMT through interaction with GSK-3β, leading to phosphorylation of GSK-3β at serine 9 and inactivation. The stability of Snail is thereby increased.

Significance of BMI1–AURKA axis in vivo and in head and neck cancer patients

To determine the effect of BMI1-regulated Aurora A in vivo, we injected the stable cell lines FaDu-CDH and FaDu-BMI1 into the subcutaneous area of nude mice, and treated the mice with different doses of the Aurora kinase inhibitor VX-680 or a vehicle control to observe the impact of suppressing Aurora kinase activity in BMI1-expressing HNSCC cells (Fig. 6A). The result showed that ectopic BMI1 in FaDu cells increased the volume of xenotransplanted tumors. Treating mice with VX-680 25 mg/kg repressed the in vivo growth of FaDu-BMI1 to a similar extent of FaDu-CDH, and 50 mg/kg of VX-680 treatment further reduced the volume of FaDu-BMI1–formed tumors (Fig. 6B and C). IHC of the harvested tumor samples confirmed upregulation of Aurora A by BMI1 in vivo. BMI1 promoted nuclear translocation of β-catenin, reduced p53 expression, and suppressed apoptosis. Inhibition of Aurora kinase activity by VX-680 decreased nuclear β-catenin, restored p53 expression and enhanced apoptosis (Fig. 6D). Finally, we confirmed the clinical significance of BMI1–AURKA axis in 46 patients with HNSCC. A positive correlation was found between the mRNA levels of BMI1 and AURKA (Fig. 6E). The dCT value of AURKA was lower in the BMI1high group as compared with the BMI1low group, indicating a significant higher AURKA level in the BMI1high group (Fig. 6F). Patients with both high BMI1 and AURKA levels had a worse survival (Fig. 6G). The correlation between the protein levels of BMI1 and Aurora A was confirmed by IHC in 33 HNSCC samples (P = 0.015; the representative IHC results shown in Fig. 6H).

Figure 6.

Inhibition of Aurora kinase activity attenuates BMI1-induced tumor progression in vivo, and clinical significance of BMI1–AURKA axis in HNSCC. A, schema of the animal experiment. i.p., intraperitoneal injection. B, representative photos of xenotransplanted tumors. C, tumor volume curves. Data represent mean ± SEM (n = 6). *, P < 0.05 (compared with FaDu-CDH). D, top, IHC of BMI1, Aurora A, β-catenin, p53, and TUNEL assay in xenotransplanted tumors. Scale bar, 50 μm. The insets show the magnification of each image. Bottom, quantification of the IHC results by percentage of positive cells. E, qRT-PCR in HNSCC samples (n = 46), and correlation between the dCT values of BMI1 and AURKA. F, the dCT value of AURKA in BMI1low versus BMI1high patients with HNSCC. G, survival analysis in BMI1highAURKAhigh versus the residual cases. H, representative IHC results of HNSCC cases. HPF, high-power field. Scale bar, 200 μm in the first and third column, and 50 μm in the second and fourth column (HPF images).

Figure 6.

Inhibition of Aurora kinase activity attenuates BMI1-induced tumor progression in vivo, and clinical significance of BMI1–AURKA axis in HNSCC. A, schema of the animal experiment. i.p., intraperitoneal injection. B, representative photos of xenotransplanted tumors. C, tumor volume curves. Data represent mean ± SEM (n = 6). *, P < 0.05 (compared with FaDu-CDH). D, top, IHC of BMI1, Aurora A, β-catenin, p53, and TUNEL assay in xenotransplanted tumors. Scale bar, 50 μm. The insets show the magnification of each image. Bottom, quantification of the IHC results by percentage of positive cells. E, qRT-PCR in HNSCC samples (n = 46), and correlation between the dCT values of BMI1 and AURKA. F, the dCT value of AURKA in BMI1low versus BMI1high patients with HNSCC. G, survival analysis in BMI1highAURKAhigh versus the residual cases. H, representative IHC results of HNSCC cases. HPF, high-power field. Scale bar, 200 μm in the first and third column, and 50 μm in the second and fourth column (HPF images).

Close modal

We propose a model to summarize our finding (Fig. 7). In HNSCC cells, BMI1 upregulates Aurora A expression through 2 PRC-mediated pathways, that is, enrichment of BMI1, EZH2, and H3K27me3 on the promoter of target genes. First, BMI1 suppresses PTEN and activates the Akt signaling pathway, leading to transactivation of AURKA by β-catenin/TCF4 complex. Second, BMI1 inhibits the expression of let-7i, resulting in a release of Aurora A from let-7i suppression. Upregulation of Aurora A by BMI1 leads to several key events in HNSCC: CIN, cell-cycle progression, and antiapoptosis through degrading p53, and EMT through stabilizing Snail. All these events contribute to the progression of HNSCC.

Figure 7.

A model depicting the BMI1–AURKA axis-centered signal network during HNSCC progression.

Figure 7.

A model depicting the BMI1–AURKA axis-centered signal network during HNSCC progression.

Close modal

Although mitotic aberration and CIN are critically involved in tumorigenesis and they have been considered as the hallmark of cancer, the role of CIN in cancer progression is unclear. For CSCs, extensive studies support their role in both tumorigenesis and late-stage progression. Emerging evidence implicates a possible link between CIN and CSCs (37–39), however, the association between CIN and stem-like properties of cancer cells remains largely elusive. In this study, we have shown the mechanism linking CSCs to CIN through the BMI1–AURKA axis. For the promising antitumor efficacy of Aurora kinase inhibitors in different human cancers especially HNSCC (40, 41), our study provides a solid rationale for applying Aurora A inhibitors to disrupt the “evil molecular link” in advanced HNSCC.

In this study, we discovered that repression of let-7i by BMI1 contributes to the enhancement of Aurora A expression. let-7i is a member of the let-7 miRNA family, which functions as a tumor suppressor and represses self-renew of stem cells (42, 43). We previously discovered that Twist1 and BMI1 act cooperatively to repress the expression of target genes (CDH1, CDKN2A, and let-7i) through the Twist1-binding sites (13, 24). Here, we found that BMI1 itself is capable of repressing let-7i through PREs, suggesting the independent role of BMI1 on regulating let-7i. The independency versus cooperation between Twist1 and BMI1 during cancer progression deserves further investigation.

We here showed that under the BMI1-overexpressing situation, the β-catenin/TCF4 complex regulates the transcription of AURKA, which indicates that β-catenin is located upstream to Aurora A. However, results from the in vivo study revealed that inhibition of the Aurora kinase activity also caused a decrease of nuclear β-catenin expression in xenotransplanted tumors. A possible explanation is that Aurora A phosphorylates GSK-3β and reduces it activity, leading to stabilization and nuclear translocation β-catenin. These results indicate that when BMI1 is overexpressed, Aurora A and β-catenin form a positive feedback loop to amplify the signal.

In summary, our study identifies a novel signaling network in HNSCC, which links 3 major events during cancer progression, that is, CSCs, CIN, and EMT, through the BMI1–AURKA axis. Scientifically, this finding provides a mechanism for understanding how cancer cells develop multifaceted changes during progression through a network governed by a pivotal oncoprotein, BMI1. Clinically, this study suggests Aurora A as an ideal target for inhibiting BMI1-induced malignant progression of HNSCC cells, which will be valuable for the future development of personalized medicine in advanced HNSCC.

No potential conflicts of interest were disclosed.

Conception and design: C.-H. Chou, N.-K. Yang, T.-Y. Liu, D.S.-S. Hsu, C.-C. Chang, C.-H. Tzeng, M.-H. Yang

Development of methodology: C.-H. Chou, N.-K. Yang, T.-Y. Liu, D.S.-S. Hsu, C.-C. Chang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.-H. Chou, N.-K. Yang, T.-Y. Liu, S.-K. Tai, D.S.-S. Hsu, Y.-W. Chen, C.-H. Tzeng

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.-H. Chou, N.-K. Yang, T.-Y. Liu, S.-K. Tai, D.S.-S. Hsu, M.-H. Yang

Writing, review, and/or revision of the manuscript: C.-H. Chou, N.-K. Yang, T.-Y. Liu, D.S.-S. Hsu, M.-H. Yang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.-H. Chou, N.-K. Yang, S.-K. Tai, D.S.-S. Hsu, Y.-J. Chen, C.-H. Tzeng

Study supervision: C.-C. Chang, C.-H. Tzeng, M.-H. Yang

The authors thank Dr. Ishigatsubo (Yokohama City University, Japan) for the generous gift of the AURKA promoter plasmid pGL-1486.

This work was supported by National Science Council (100-2321-B-010-015 to M.-H. Yang; 99-2811-B-010-014 to T.-Y. Liu), National Health Research Institutes (NHRI-EX101-10037BI to M.-H. Yang), Taipei Veterans General Hospital (100-C1-088 and 101-C-005 to M.-H. Yang), Veterans General Hospitals University System of Taiwan Joint Research Program (VGHUST101-G7-4-1 to M.-H. Yang), Taichung Veterans General Hospital–National Yang-Ming University Joint Research Program (TCVGH-YM1000302 to M.-H. Yang), a grant from Ministry of Education, Aim for the Top University Plan, and a grant from Department of Health, Center of Excellence for Cancer Research (DOH101-TD-C-111-007 to M.-H. Yang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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