Bmi-1 promotes the aggressiveness of glioma via activating the NF-kappaB/MMP-9 signaling pathway
- Lili Jiang†1,
- Jueheng Wu†2, 3,
- Yi Yang3, 4,
- Liping Liu5,
- Libing Song5,
- Jun Li3, 6 and
- Mengfeng Li2, 3, 7Email author
© Jiang et al.; licensee BioMed Central Ltd. 2012
Received: 16 August 2012
Accepted: 31 August 2012
Published: 11 September 2012
The prognosis of human glioma is poor, and the highly invasive nature of the disease represents a major impediment to current therapeutic modalities. The oncoprotein B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) has been linked to the development and progression of glioma; however, the biological role of Bmi-1 in the invasion of glioma remains unclear.
A172 and LN229 glioma cells were engineered to overexpress Bmi-1 via stable transfection or to be silenced for Bmi-1 expression using RNA interfering method. Migration and invasiveness of the engineered cells were assessed using wound healing assay, Transwell migration assay, Transwell matrix penetration assay and 3-D spheroid invasion assay. MMP-9 expression and activity were measured using real-time PCR, ELISA and the gelatin zymography methods. Expression of NF-kappaB target genes was quantified using real-time PCR. NF-kappaB transcriptional activity was assessed using an NF-kappaB luciferase reporter system. Expression of Bmi-1 and MMP-9 in clinical specimens was analyzed using immunohistochemical assay.
Ectopic overexpression of Bmi-1 dramatically increased, whereas knockdown of endogenous Bmi-1 reduced, the invasiveness and migration of glioma cells. NF-kappaB transcriptional activity and MMP-9 expression and activity were significantly increased in Bmi-1-overexpressing but reduced in Bmi-1-silenced cells. The reporter luciferase activity driven by MMP-9 promoter in Bmi-1-overexpressing cells was dependent on the presence of a functional NF-kappaB binding site, and blockade of NF-kappaB signaling inhibited the upregulation of MMP-9 in Bmi-1 overexpressing cells. Furthermore, expression of Bmi-1 correlated with NF-kappaB nuclear translocation as well as MMP-9 expression in clinical glioma samples.
Bmi-1 may play an important role in the development of aggressive phenotype of glioma via activating the NF-kappaB/MMP-9 pathway and therefore might represent a novel therapeutic target for glioma.
KeywordsBmi-1 Glioma Invasion MMP-9 NF-kappaB
Glioma is a common type of primary brain tumor and represents one of the most aggressive and lethal human cancer types . Despite of enormous advances in surgical techniques and development of therapeutic agents, the mortality of glioma remains high, with a cumulative 1-year survival rate lower than 30% [2, 3]. The poor survival of glioma patients is largely attributed to the highly invasive phenotype of glioma cells, which has been associated with the widely recognized difficulty of performing complete surgical resection of gliomas . At the molecular level, tumor cell invasion is mediated by sets of factors that initiate or promote cell motility, matrix destruction, angiogenesis and other biological events [5–8]. Matrix metalloproteinase-9 (MMP-9), a member of the matrix metalloproteinase class of gelatinases, plays essential roles in the invasiveness of glioma cells, mainly by catalyzing the destruction of basal membrane and extracellular matrix [9–11].
B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) acts as a repressor of the expression of certain genes by forming complexes with multiple other Polycomb group (PcG) family members, such as RING1, HPC2 and Mph2. It has been reported that Bmi-1 represses cells senescence by acetylhydrolysis or deacetylhydrolysis of polycomb response elements in chromosomes [13, 14]. Numerous experimental studies have indicated that Bmi-1 plays an important role in the development and progression of cancer and essentially functions as an oncogene [15, 16]. Knocking down Bmi-1 induces cell-cycle arrest and rescues the mRNA levels of tumor-suppressive p16 INK4a homeobox A9 (HOXA9) and homeobox C13 (HOXC13) genes [14, 17]. In contrast, overexpression of Bmi-1 prevents cancer cell apoptosis, possibly by activation of nuclear factor kappaB (NF-kappaB) pathway signaling . Moreover, various studies have revealed that Bmi-1 is required for the maintenance of the self-renewing proliferation of several normal and cancer stem cells, including neural crest stem cells and mammary stem cells [19, 20].
Aberrant activation of NF-kappaB is observed in various types of human cancer, including glioma. Nuclear localization of p65, an indicator of NF-kappaB activation, has also been demonstrated in clinical specimens of glioblastoma multiform (GBM) [8, 18, 21]. The NF-kappaB signialing orchestrates several key biological processes during the development and progression of cancer by inducing transcription of a variety of target genes that regulate cell proliferation, survival, invasion and angiogenesis [8, 18, 22–25]. Inhibition of NF-kappaB activation enhances the radiosensitivity of human glioma cells, and also inhibits the proliferation and invasiveness of glioblastoma cells and glioblastoma-induced angiogenesis [26, 27]. Nevertheless, whether, and how, the biological functions of NF-kappaB is involved in the oncogenic role of Bmi-1 remains obscure.
In the present study, we observed that overexpression of Bmi-1 promoted, whereas knockdown of Bmi-1 inhibited, the invasion and migration of glioma cells. We also demonstrated that the ability of Bmi-1 to stimulate the invasive phenotype in glioma cells was mechanistically associated with activation of NF-kappaB and subsequent upregulation and activation of MMP-9.
Cells and cell treatments
Glioma cell lines LN229 and A172 were grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 100 units penicillin and 100 units streptomycin at 37°C in 5% CO2 atmosphere in a humidified incubator. The MMP inhibitor and NF-kappaB activation inhibitor II JSH-23 (EMD, La Jolla, CA) compounds were dissolved in dimethyl sulfoxide (DMSO) and used, respectively, at 50μM and 30μM. Treatment of cells with the MMP inhibitor was performed for indicated time lengths, and JSH-23 was used to treat cells for 11 h.
Vectors and retroviral infection
pMSCV/Bmi-1 overexpressing human Bmi-1 was constructed as previously described . To silence endogenous Bmi-1 expression, Bmi-1 RNA interference (RNAi) sequence (5’-ATGAAGAGAAGAAGGGATT-3’, synthesized by Invitrogen) was cloned into retroviral transfer vector pSuper-retro-puro. As described previously , retroviral particles were produced by cotransfection with pSuper-retro-Bmi-1-shRNA and PIK packaging plasmid into 293 T cells and collected 24 to 48 hrs after transfection to infect glioma cells. Stable cell lines expressing Bmi-1 or with Bmi-1 silenced were selected by treatment with 0.5 μg/ml puromycin for 10 days, beginning from 48 hours after infection . After selection for 10–14 days, the cell lysates prepared from the pooled population of cells in the sampling buffer were fractionated on SDS-PAGE for immunoblotting detection of Bmi-1 protein level.
Real-time RT-PCR and data analysis
Total cellular RNA was extracted using the Trizol reagent (Invitrogen) according to the manufacturer’s instruction. Two micrograms of RNA from each sample were used for cDNA synthesis primed with random hexamers. For PCR amplification of cDNA, an initial amplification using gene-specific primers was done with a denaturation step at 95°C for 10 minutes, followed by 28 cycles of denaturation at 95°C for 60 seconds, primer annealing at 58°C for 30 seconds, and primer extension at 72°C for 30 seconds. At completion of the cycling, a final extension at 72°C for 5 minutes was done before the reaction was terminated. Expression levels of genes were normalized to housekeeping gene GAPDH as the control. PCR primers were designed by employing the Primer Express version 2.0 software (Applied Biosystems, Foster City, CA). The Primers was shown as following: MMP9-up: ACGACGTCTTCCAGTACCGA; MMP9-dn: TTGGTCCACCTGGTTCAACT; CCND1-up: AACTACCTGGACCGCTTCCT; CCND1-dn: CCACTTGAGCTTGTTC ACCA; Bcl-xL-up: ATTGGTGAGTCGGATCGCAGC; Bcl-xL-dn: AGAGAAGGGGG TGGGAGGGTA; TNF alpha-up: CCAGGCAGTCAGATCATCTTCTC; TNF alpha-dn: AGCTGGTTATCTCTCAGCTCCAC; VEGFC-up: GTGTCCAGTGTAGATGAACTC; VEGFC-dn: ATCTG TAGACGGACACACATG; MYC-up: TTCGGGTAGTGGAAAACCAG; MYC-dn: CAGCAGCTCGAATTTCTTCC; GAPDH-up: GACTCATGACCACAGTCCATGC; GAPDH-dn: AGAGGCAGGGATGATGTTCTG.
Western blotting analysis
Western blotting analysis was performed according to standard methods as previously described .The membrane was probed with a 1:500-diluted rabbit anti-human Bmi-1 antibody (Cell Signaling, Danvers, MA). The membranes were stripped and re-probed with a mouse anti-β-actin monoclonal antibody (1:1,000; Sigma, Saint Louis, MI) as a loading control.
Wound healing assay
Cells were seeded on six-well plates with DMEM containing 10% FBS and grown to confluence. The cells were scratched with a sterile 200μL pipette tip to create artificial wounds. At 0 and 24 hr after wounding, respectively, phase-contrast images of the wound healing process were photographed digitally using an inverted Olympus IX50 microscope with 10× objective lens. Eight images per treatment were analyzed to determine averaging parameters of positioning of the migrating cells at the wound edges by digitally drawing lines using the Image-Pro Plus software (Media Cybernetics).
Transwell migration assay and Transwell matrix penetration assay
Cells (1 × 104) to be tested were plated on the top side of the polycarbonate Transwell filter without (for Transwell migration assay) or with Matrigel coating (for Transwell matrix penetration assay) in the upper chamber of the BioCoatTM Invasion Chambers (BD, Bedford, MA) and incubated at 37°C for 22 hrs, followed by removal of cells inside the upper chamber with cotton swabs. Migrated and invaded cells on the membrane bottom-surface were fixed in 1% paraformaldehyde, stained with hematoxylin, and counted (Ten random 200× fields per well). Cell counts were expressed as the mean number of cells per field of view. Three independent experiments were performed and the data are presented as mean ± standard deviation (SD).
3-D spheroid invasion assay
The Matrigel matrix (BD Biosciences, San Jose, CA) was used in 3-D spheroid invasion assay, which displays morphologies typical of highly aggressive invasiveness presenting more outward projections (Invadopodia or invasive feet) [29–33]. Indicated cells (1 × 104) were trypsinized and seeded in 24-well plates coated with Matrigel (2%, BD Biosciences), and medium was changed every other day. Pictures were taken under microscope at 2-day intervals for 2–3 weeks.
Immunohistochemical analysis (IHC)
IHC was performed according to standard methods as previously described . Sections were IHC analyzed using anti-Bmi-1, anti-MMP-9 and anti-NF-kappaB antibodies (Cell signaling, Danvers, MA,). Images were captured using the AxioVision Rel.4.6 computerized image analysis system (Carl Zeiss Co. Ltd., Jena).
Cells (1.5 × 104) were seeded in triplicates in 24-well plates and allowed to settle for 24 hrs. One hundred nanograms of luciferase reporter plasmid containing fragments of the MMP-9 promoter with serial deletions, pNF-kappaB-luc plasmid, or the control-luciferase plasmid, in combination with 1 ng of pRL-TK renilla plasmid (Promega,Madison, WI), were transfected into glioma cells using the Lipofectamine 2000 reagent (Invitrogen, Co., Carlsbad, CA) according to a protocol provided by the manufacturer. Luciferase and renilla signals were measured at 48 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI) according to the manufacturer’s instruction. Three independent experiments were performed and the data are presented as mean ± SD.
Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed using a commercial kit according to the manufacturer’s manual (Keygentec, Shanghai). Briefly, 100 μl of diluted standard and tested samples, namely, Bmi-1-overexpressing or -silencing cells, and the vector control cells, including a negative control, were added to the ELISA plate and incubated at 36°C for 90 min. After the unbound samples were washed off by DI water and PBS-Triton, specific antibody (anti-MMP-9, anti-MMP-2, or anti-MMP-7) was incubated with the plate at 36°C for 60 min. After further washing steps, 100 μl of second antibody was added and incubated for 60 min. Subsequently, the substrate was added and incubated at RT for 60 min before the reaction was stopped, followed by the results-reading with a microplate reader. Colorimetric measurement was recorded as OD450 readings.
Gelatin zymography assay
Cells were seeded in 48-well culture plates at a density of 3 × 104/well and incubated for 24 h before the medium was replaced with serum-free medium (Invitrogen, Carlsbad, CA), followed by collection of conditioned medium and quantification for protein contents. Samples containing equal amounts of protein (1 μg/μl) mixed with 4 × sampling buffer (3:1) were run on 9% polyacrylamide gels containing 0.2% gelatin (Sigma, Saint Louis, MI). After electrophoresis, the gel was washed twice in wash buffer (2.5% Triton X-100, 50 mM Tris–HCl, 1 μM ZnCl2, pH 7.6) for 45 min/each time, followed by two rinses with the wash buffer (without Triton X-100) and subsequent incubation at 37°C in 50 mM Tris–HCl (pH7.6), 5 mM CaCl2, 1 μM ZnCl2 and 0.02% Brij-35 for 16 h. The gels were stained with 0.1% Coomassie brilliant blue R-250 and then de-stained with de-staining solution (40% methanol, 10% acetic acid in distilled water). A protein marker was used to measure the molecular weights of proteins in SDS-PAGE before the staining and washing step of the Zymography Assay. Meanwhile, the MMP-9 recombinant protein was also used as the standard to confirm the band of MMP-9.
Tissue specimens and patient information
Paraffin-embedded, archived glioma specimens were histopathologically diagnosed at the First Affiliated Hospital of Sun Yat-sen University from 2000 to 2005. The clinical information is described in Additional file 1: Table S1. The use of the clinical specimens was approved by the local Institutional Review Board, the Ethical Committee of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China, and conformed to the ethical guidelines of the Helsinki Declaration.
Statistical analyses were performed using the SPSS 11.0 statistical software package. Data represent mean ± SEM. P values of 0.05 or less were considered statistically significant.
Bmi-1 induced the aggressive phenotype in glioma cells in vitro
Bmi-1 increased MMP-9 expression and activity in glioma cells
Silencing Bmi-1 reduced glioma cell invasiveness and MMP-9 expression
Bmi-1 induced expression of MMP-9 via activation of the NF-κB pathway
Moreover, the luciferase activity of a reporter gene driven by the MMP-9 promoter containing the NF-kappaB binding site increased significantly in Bmi-1-overexpressing cells and decreased in Bmi-1-silenced cells. Mutating the NF-kappaB binding site in the MMP-9 promoter abrogated luciferase activity, and a MMP-9 promoter fragment lacking the NF-kappaB binding site displayed no significant change in the luciferase activity in Bmi-1 overexpressing glioma cells (Figure 5C). Taken together, these results indicated that Bmi-1 promoted the transactivation activity of the NF-kappaB binding site present in the MMP-9 promoter in glioma cells.
Bmi-1 induced aggressiveness in glioma cells via the NF-kappaB/MMP-9 pathway
Collectively, these results indicated that the overexpression of Bmi-1 induced an aggressive phenotype in glioma cells by activating NF-kappaB signaling, leading to the upregulation of the NF-kappaB target gene MMP-9.
Clinical relevance of Bmi-1 triggered NF-kappaB/MMP-9 activation in human gliomas
The key finding of this study is that Bmi-1 may induce an aggressive phenotype in glioma via modulation of the NF-kappaB signaling. Previous studies have indicated that Bmi-1 is overexpressed and associated with poorer overall survival in glioma . Our current study used glioma cell lines expressing intermediate levels of endogenous Bmi-1 as an experimental model, and by examining the effect of knocking down endogenous Bmi-1, or overexpressing ectopic Bmi-1, on the phenotype of glioma cells, we have identified that Bmi-1 activates NF-kappaB and subsequently upregulates MMP-9 expression, leading to increased migration and invasion of glioma cells.
As a member of the PcG family, Bmi-1 is overexpressed in various tumor types, including acute myeloid leukemia, lung cancer, ovarian cancer, nasopharyngeal carcinoma, breast cancer and colon cancer, suggesting that Bmi-1 represents a potential oncogene [16, 35–39]. Furthermore, p16INK4a and p14ARF are targets of Bmi-1 suppression [40, 41], and Bmi-1 has been found to promote cell proliferation by suppressing the p16/Rb and/or p14ARF/MDM2/p53 pathways [42, 43]. Upregulation of Bmi-1 also induces the epithelial-mesenchymal transition (EMT), enhances the aggressiveness of human nasopharyngeal carcinoma cells and stabilizes Snail, a transcriptional repressor associated with EMT, via modulation of the PI3K/Akt/GSK-3β pathway . Moreover, it has been reported that Bmi-1 can downregulate transcription of the tumor suppressor phosphatase and tensin homolog deleted on chromosome ten (PTEN) via a direct association with the PTEN gene locus . Our current study indicates that Bmi-1 modulates the NF-kappaB/MMP-9 signaling pathway to mediate an aggressive phenotype in human glioma, suggesting that Bmi-1 may represent a potential therapeutic target for the treatment of glioma.
Activating mutations or amplification of oncogenes, such as epidermal growth factor receptor (EGFR) and phosphatidylinositol 3-kinase (PI-3 K), or loss of function in tumor suppressor genes, such as p53 and PTEN, are involved in oncogenesis and the progression of glioma. The molecular mechanisms that mediate the aggressive phenotype in gliomas, however, are incompletely understood [44–46]. Furthermore, it is now well recognized that the low survival rate of glioma patients can be largely attributed to the highly invasive nature of glioma cells, which results in the destruction of surrounding brain tissue, and the invasiveness of glioma cells correlates with patient prognosis [2, 3, 44–47]. Most current therapies for the treatment of glioma are ineffective against invading cells. Characterization of the molecular mechanisms mediating invasion may provide a foundation for the development of new anti-glioma strategies. MMP-9, one member of the MMP family, is upregulated and associated with progression and poor prognosis in glioma . Interestingly, numerous genes that promote the aggressiveness of glioma, including astrocyte elevated gene-1 (AEG-1), are also involved in the modulation of MMP-9 transcription . Furthermore, multiple transcription factor-binding sites have been characterized in the upstream regulatory region of the MMP-9 gene, including binding sites for the AP-1 and NF-kappaB transcription factors [8, 49, 50]. Moreover, AP-1 and NF-kappaB transcription factors can induce expression and activation of MMP-9 by interacting with these binding sites, and consequently promote tumor progression [48, 51]. The present study demonstrates that Bmi-1 induces MMP-9 expression and activity via a mechanism associated with NF-kappaB activation, whereas blocking the activity of NF-kappaB drastically reduces the pro-invasive effect of Bmi-1 and preventes upregulation of MMP-9. Taken together, our data provide new insights in the development of novel strategies to prevent tumor invasion in glioma by inhibiting the expression of Bmi-1.
In conclusion, this study demonstrates that Bmi-1 is upregulated and promotes an aggressive phenotype in glioma via activation of the NF-kappaB signaling pathway, leading to increased MMP-9 expression and activity. Bmi-1 may therefore represent a potential therapeutic target for improved treatment of human gliomas.
B-cell-specific Moloney murine leukemia virus integration site 1
Nuclear factor kappaB
Fetal bovine serum
Enzyme-linked immunosorbent assay
Short hairpin RNA
Epidermal growth factor receptor
- PI-3 K:
Astrocyte elevated gene-1
Endothelial growth factor-C
Polymerase chain reaction
Phosphatase and tensin homolog deleted on chromosome ten.
Supported by the Natural Science Foundation of China (No. 81071780, 81030048, 81071762, 81101680, 30900415); National Science and Technique Major Project (201005022–2, 2012ZX09102101-017); High-Tech Research (863) projects (2011AA09070201); The Science and Technology, Department of Guangdong Province, China (No. S2011020002757); The Key Science and Technique Research Project of Guangdong Province (2010B030600003); Guangdong Recruitment Program of Creative Research Groups.
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