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Mol Cell Biol. Sep 2011; 31(17): 3584–3592.
PMCID: PMC3165557
MicroRNA-146a Inhibits Glioma Development by Targeting Notch1 [down-pointing small open triangle]
Jie Mei,1 Robert Bachoo,2 and Chun-Li Zhang1*
1Department of Molecular Biology
2Department of Neurology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148
*Corresponding author. Mailing address: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148. Phone: (214) 648-1670. Fax: (214) 648-1488. E-mail: chun-li.zhang/at/utsouthwestern.edu.
Received June 16, 2011; Revisions requested June 20, 2011; Accepted June 23, 2011.
Dysregulated epidermal growth factor receptor (EGFR) signaling through either genomic amplification or dominant-active mutation (EGFRvIII), in combination with the dual inactivation of INK4A/ARF and PTEN, is a leading cause of gliomagenesis. Our global expression analysis for microRNAs revealed that EGFR activation induces miR-146a expression, which is further potentiated by inactivation of PTEN. Unexpectedly, overexpression of miR-146a attenuates the proliferation, migration, and tumorigenic potential of Ink4a/Arf−/− Pten−/− EgfrvIII murine astrocytes. Its ectopic expression also inhibits the glioma development of a human glioblastoma cell line in an orthotopic xenograft model. Such an inhibitory function of miR-146a on gliomas is largely through downregulation of Notch1, which plays a key role in neural stem cell maintenance and is a direct target of miR-146a. Accordingly, miR-146a modulates neural stem cell proliferation and differentiation and reduces the formation and migration of glioma stem-like cells. Conversely, knockdown of miR-146a by microRNA sponge upregulates Notch1 and promotes tumorigenesis of malignant astrocytes. These findings indicate that, in response to oncogenic cues, miR-146a is induced as a negative-feedback mechanism to restrict tumor growth by repressing Notch1. Our results provide novel insights into the signaling pathways that link neural stem cells to gliomagenesis and may lead to new strategies for treating brain tumors.
Gliomas are the most frequently observed brain tumors, with glioblastoma multiforme (GBM) being the most common and aggressive form in adults (35). Despite major therapeutic improvements made by combining neurosurgery, chemotherapy, and radiotherapy, the prognosis and survival rate for patients with GBM is still extremely poor (7). The deadly nature of GBM originates from explosive growth and invasive behavior, which are fueled by dysregulation of multiple signaling pathways. Epidermal growth factor receptor (EGFR) activation, in cooperation with loss of tumor suppressor functions, such as mutations in Ink4a/Arf and Pten genes, constitutes a lesion signature for GBM (4). Such dysregulated genetic pathways are sufficient to transform neural stem cells (NSCs) or astrocytes into cancer stem-like cells. This gives rise to high-grade malignant gliomas with a pathological phenotype resembling human GBM (5, 59). However, the downstream events underlying these genetic dysregulations in gliomagenic cells have not been fully elucidated.
MicroRNAs are 20- to 22-nucleotide noncoding RNA molecules that have emerged as key players in controlling NSC self-renewal and differentiation (11, 57). Aberrant expression of miRNAs, such as miR-21, miR-124, and miR-137, is linked to glioma formation (49). miR-199b-5p and miR-34a impair cancer stem-like cells through the negative regulation of several components of the Notch pathway in brain tumors (18, 21). The Notch pathway is an evolutionarily conserved signaling pathway that plays an important role in neurogenesis (3, 10, 23). Upon binding to its ligand, Delta, the Notch intracellular domain (NICD; the activated form of Notch) is released from the membrane by presenilin/γ-secretase-mediated cleavage and translocates to the nucleus. In the nucleus, NICD forms a complex with Rbpj and activates the expression of several transcriptional repressors, such as Hes1 and Hes5, which inhibit neurogenesis (15, 27, 29). Thus, activation of the Notch pathway is essential to maintain both developing and adult NSCs (36). This property of the Notch pathway enables it to promote glioma growth (30), and its inhibition by drugs could abolish glioma stem-like cells and reduce tumorigenesis (17).
We show here that miR-146a is specifically induced as a converging downstream target of EGFR and PTEN signaling in immortalized Ink4a/Arf−/− astrocytes. We further demonstrate that miR-146a acts as a native safeguarding mechanism to restrict the formation of glioma stem-like cells and glioma growth by directly controlling the expression of Notch1.
Cell culture, MTT, and anchorage-independent growth assays.
We isolated primary NSCs by mechanical dissociation using 1-ml pipettes from embryonic day 14.5 (E14.5) mouse forebrains in growth medium consisting of Dulbecco modified Eagle plus F-12 (DMEM/F12) medium, 1 mM l-glutamine, N2 (Invitrogen), 20 ng of EGF/ml, and 20 ng of FGF2 (Peprotech)/ml. These cells were cultured as free-floating neurospheres under 37°C and 5% CO2. For differentiation, we exposed NSCs to DMEM/F12 medium with N2 supplement, further supplemented with 5 μM forskolin (FSK) and 0.5% fetal bovine serum (FBS), 1 μM retinoic acid (RA) and 0.5% (vol/vol) FBS, or 0.5% (vol/vol) FBS. This was done in 60-mm dishes or eight-well chamber slides coated with laminin (5 μg/ml) and poly-l-ornithine (10 μg/ml). The Ink4a/Arf−/−, Ink4a/Arf−/− EgfrvIII, and Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes or human U87 glioma cells were cultured in DMEM containing 10% FBS. Glioma stem-like cells from malignant astrocytes or U87 cells were enriched by culturing in NSC medium with growth factors. Cell growth was determined by using the CellTiter 96 nonradioactive cell proliferation assay kit (MTT assay; Promega) according to the manufacturer's instructions. For anchorage-independent growth by soft agar assay, 2,000 Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes transduced with either wild-type or seed region-mutated miR-146a were plated and cultured in 12-well plates, as described previously (47). Five weeks later, cell colonies in the plates were stained with 0.5 ml of 0.005% crystal violet and counted under an inverted microscope. We conducted each experiment in triplicate.
miRNA microarray and qRT-PCR.
We extracted total RNAs from Ink4a/Arf−/− (I), Ink4a/Arf−/− EgfrvIII (IE), or Ink4a/Arf−/− Pten−/− EgfrvIII (IPE) astrocytes using the miReasy minikit (Qiagen). Biological replicates were collected. RNA quality was examined by Bioanalyzer (Illumina). Portions (5 μg) of each RNA sample were processed for labeling and hybridization to GeneChip miRNA arrays (Affymetrix) by the Microarray Core Facility at UT Southwestern Medical Center. After scanning and normalization to control probes, the array data was analyzed by VAMPIRE software to identify statistically significant differences in gene expression between sample groups (http://genome.ucsd.edu/microarray). For quantitative reverse transcription-PCR (qRT-PCR), 1 μg of total RNA was reverse transcribed by using NCode miRNA first-strand synthesis and qRT-PCR kits (Invitrogen). qRT-PCRs were performed in a 384-well plate using an ABI 7900HT instrument. The PCR program consisted of 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 58°C. Primer quality was analyzed by dissociation curves. The expression of miR-146a and Notch1 was normalized to that of U6 and Hprt, respectively.
Lentivirus production and transduction.
A genomic fragment encompassing the miR-146a coding region or a seed region-mutated version was cloned by PCR into pTomo vector (provided by Inder Verma at Salk Institute). To construct the miRNA sponge, we inserted the synthesized DNA fragment into the BamHI site of a pCSC-SP-PW-IRES/GFP lentiviral vector and the empty vector as the control. We produced the lentiviruses, determined their titers, and transduced cultured cells according to previously described methods (38, 48). The transduction efficiency was monitored by determining the green fluorescent protein (GFP) expression. All experiments were performed using stably transduced cells within six passages.
Cell migration assay.
Migration of malignant astrocytes in culture was determined by the “scratch” assay. For this, cells were seeded into a six-well tissue culture dish and allowed to grow to 90% confluence in complete medium with 10% FBS. Cells in monolayers were scratched in a single straight line using a pipette tip (1 mm in diameter). Wounded monolayers were washed three times with culture medium to remove cell debris and then incubated for another 24 h. The migratory distance was measured under a microscope equipped with a camera. A Transwell migration assay was conducted essentially as previously described (43). Invasive behavior of glioma stem-like cells in vitro was examined by measuring the migration ability of cultured neurospheres. Neurospheres with similar diameters were selected and plated onto six-well culture plates coated with laminin (5 μg/ml) and poly-l-ornithine (10 μg/ml) and cultured for 48 h in NSC growth medium. Cell migration and spreading was quantified by measuring the distance between the edge of the neurosphere and the peripheries of radially migrating cells.
Luciferase reporter assay.
The 3′ untranslated region (3′UTR) of the Notch1 gene, which contains one putative miR-146a targeting site, was amplified by PCR and inserted into the SacI and PmeI sites of the pMIR-REPORT vector (Ambion). To express miR-146a, a 610-bp genomic fragment encompassing the coding region was cloned by PCR and inserted into the HindIII and BamHI sites of pCMV6 vector. A seed-sequence-mutated version of miR-146a was used as a control for all of the experiments. To create this mutant (pCMV6-miR-146a-mt), we replaced the seed sequence TGAGAACT with GCGGCCGC through site-directed mutagenesis using a QuikChange kit (Stratagene). Similarly, the binding site (AGTTCT) for miR-146a within the 3′UTR of the Notch1 gene was replaced with ACGCGT to generate a control for the luciferase reporter assays. HEK293 cells were transiently transfected using Fugene 6 reagent (Roche) in 48-well plates. The following plasmids were used: pMIR-REPORT was used with either pCMV6-miR-146a or its mutant, pCMV6-miR-146a-mt, and pCMV-LacZ was used as a control to monitor the transfection efficiency. The total plasmid amount (200 ng) was kept constant for each transfection with empty pCMV6 vector. After 48 h, the cells were lysed and assayed for luciferase and β-galactosidase activity using a Dual-Light system (Applied Biosystems). Relative reporter activities were determined by normalizing luminescence units to β-galactosidase expression.
Western blotting and immunocytochemistry.
Protein expression was examined by Western blotting, according to a standard procedure. Antibodies against the following proteins were used: β-Actin (Sigma), 1:8,000; GFAP and p-AKT-T308 (Cell Signaling Technology), 1:1,000; Sox2 (Chemicon), 1:1,000; Nestin (Aves), 1:10,000; and Notch1 (Santa Cruz), 1:1,000. To partially block processing of Notch1 protein by γ-secretase, cells were treated with 10 μM DAPT {N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester} (Sigma) for 36 h. The blots were scanned and quantified by NIH software ImageJ. To examine the proliferation and cell cycle exit, cultured cells were pulse-labeled with bromodeoxyuridine (BrdU) at 10 μM for 2 h and then fixed and processed for immunostaining, as previously described (56). Antibodies against the following proteins were used: BrdU (BD Pharmingen), 1:1,000; Ki67 (Novocastra), 1:500; cleaved caspase 3 (Cell Signaling Technology), 1:300; and Tuj1 (Covance), 1:500).
Intracranial and subcutaneous cell transplantation model for tumorigenesis.
NOD/SCID and nude mice (nu/nu) were purchased from Harlan Laboratories. They were housed in a pathogen-free facility under standard 12-h light/dark cycles and controlled temperature conditions and had free access to food and water. Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the UT Southwestern Medical Center. Astrocytes (5 × 105 cells) or neurospheres (2 × 105 cells) were respectively implanted into the right caudoputamen of the brain (for orthotopic transplantation) or subcutaneous tissues of 5 to 6 weeks old NOD/SCID or nude mice using a 25-gauge needle. The injection sites were monitored frequently. Mice transplanted with neurospheres were euthanized 3 weeks later. The tumors were finely dissected, imaged, and measured according to the following formula: volume of tumor (in mm3)=(d2 × D)/2, where d and D are the shortest and longest diameters, respectively. For survival analysis, the mice were euthanized when they demonstrated severe health deterioration according to IACUC guidelines.
Statistical analysis.
Data are expressed as means ± the standard deviations. Except for the survival studies, all data were analyzed by two-tailed Student t tests. The Kaplan-Meier survival curves were determined by using GraphPad Prism v5.0 (GraphPad Software, Inc.) and the log-rank test. A P value of <0.05 was considered significant.
EGFR activation and PTEN inactivation synergistically induce expression of miR-146a.
Loss of Ink4a/Arf results in immortal growth of primary murine astrocytes. Subsequent activation of EGFR signaling through expression of a constitutively active EGFRvIII mutant transforms these astrocytes into tumorigenic and neurosphere-forming glioma stem-like cells when cultured in defined medium for NSCs (5, 24). In vivo, concomitant activation of EGFR and inactivation of Ink4a/Arf and Pten produce rapid-onset and fully penetrant high-grade gliomas that resemble GBM in humans (60), suggesting cooperative actions of these signaling pathways on cell proliferation. To confirm this, we performed MTT assays and direct cell counts to assess the growth rate of Ink4a/Arf−/−, Ink4a/Arf−/− EgfrvIII, or Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes. Indeed, constitutive activation of EGFR confers Ink4a/Arf−/− astrocytes with a growth advantage that is further enhanced by ablation of Pten (Fig. 1 A and B).
Fig. 1.
Fig. 1.
Induction of miR-146a expression by EGFR and PTEN signaling in immortalized Ink4a/Arf−/− astrocytes. (A and B) Proliferation of Ink4a/Arf−/− (I), Ink4a/Arf−/− EgfrvIII (IE), or Ink4a/Arf−/− (more ...)
To understand the downstream molecular events of this genetic cooperation and because of the emerging role of microRNA in cancers, we performed global expression analysis of microRNAs in Ink4a/Arf−/−, Ink4a/Arf−/− EgfrvIII, or Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes. We identified a total of 19 unique miRNAs whose expression is altered by activation of EGFR alone or in combination with loss of Pten (Fig. 1C). Among these miRNAs, miR-146a, miR-182, miR-183, and miR-199a-3p are shown to be enriched in diverse cancers, whereas miR-127 and miR-140 are downregulated in gliomas (32, 46). In contrast to the unchanged expression of miR-146b, miR-146a was significantly induced by EGFRvIII alone or in combination with ablation of Pten (Fig. 1C). The change in expression was subsequently confirmed by qPCR using independent RNA samples (Fig. 1D). We also examined the expression of miR-146a after acute deletion of Pten through Cre-mediated recombination of floxed Pten alleles in immortalized astrocytes (Fig. 1E and F). Pten loss resulted in a >7-fold induction of miR-146a expression. This induction was further increased to 9-fold after activation of EGFR. These data indicate a cooperative nature of EGFR, PTEN, and INK4A/ARF signaling on cell proliferation and miR-146a expression.
miR-146a inhibits the proliferation and oncogenic potential of malignant astrocytes.
The induction of miR-146a by EGFR and/or PTEN signaling in Ink4a/Arf−/− astrocytes suggests that miR-146a may act as an onco-miR by transducing oncogenic signals to control cell behavior. To test this hypothesis, we performed overexpression experiments to examine whether exogenous miR-146a could further enhance proliferation and tumorigenesis of malignant astrocytes. Due to the extremely low efficiency of transient transfections in these astrocytes, we used a lentivirus-mediated expression system, in which the cytomegalovirus (CMV) promoter drives the expression of both miR-146a and GFP. As a control, we mutated eight nucleotides within the seed region of miR-146a (Ctrl, Fig. 2 A). Such a mutation presumably abolishes the interactions of miR-146a with its targets. Examination of GFP expression showed that nearly 100% of the cells were transduced by lentivirus. qRT-PCR analysis demonstrated a 26-fold increase of wild-type miR-146a in Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes after lentiviral transduction (Fig. 2B). Since miR-146a had no effect on apoptosis (Fig. 2E), we examined cell growth by MTT assays and direct cell counting. Interestingly, overexpression of miR-146a inhibited proliferation of Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes (Fig. 2C and D), indicating that this microRNA may instead function as a tumor suppressor. Indeed, overexpression of miR-146a resulted in a 25% reduction in the diameter (Fig. 2F) and a 35% decrease in the number (Fig. 2G) of colonies in an anchorage-independent growth assay, which is an in vitro model for cellular transformation.
Fig. 2.
Fig. 2.
miR-146a inhibits the cellular transformation and migration of malignant astrocytes. (A) Schema of miR-146a mutant (Ctrl). The seeding sequence of miR-146a is replaced with a NotI digestion site. (B) qRT-PCR analysis of miR-146a expression in lentivirus-transduced (more ...)
High-grade gliomas exhibit aggressive behavior, which is manifested by rapid cellular migration under culture conditions in vitro (12, 50). Using a standard “scratch” assay after the cells become confluent in culture dishes, we found that enhanced expression of miR-146a significantly reduced the rate of migration of Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes compared to the same cells transduced with a control lentivirus (Fig. 2H). This observation was further confirmed by a Matrigel transwell migration assay, which showed a 50% reduction of migrating malignant astrocytes upon ectopic expression of miR-146a (Fig. 2I). Finally, we examined the in vivo function of miR-146a during glioma development after cell transplantation into the right caudoputamen of NOD/SCID mice. For this, we grafted an equal number (5 × 105) of Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes that were transduced with lentiviruses expressing either wild-type miR-146a or seed region-mutated miR-146a (Ctrl). Mice were monitored daily for morbidity. A subset of mice was also sacrificed around 3 weeks posttransplantation to examine tumor burden by histology (Fig. 3 A). Ectopic expression of miR-146a but not the Ctrl significantly reduced the tumor burden and prolonged the survival of mice that were transplanted with Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes (Fig. 3B). Together, these data suggest that miR-146a acts as a tumor suppressor of glioma development.
Fig. 3.
Fig. 3.
miR-146a decreases the tumor burden induced by intracranial transplantation of Ink4a/Arf−/− Pten−/− EgfrvIII murine astrocytes. (A) Representative histological analysis of brain gliomas in NOD/SCID mice 3 weeks posttransplantation. (more ...)
miR-146a inhibits the formation of glioma stem-like cells from malignant astrocytes.
Accumulating evidence suggests that self-renewable stem-like cells within the bulk of brain tumors are the driving force for initiation and maintenance of aggressive gliomas (13). These cells share common features with NSCs, including the ability to form neurospheres and the expression of stem cell markers, such as Sox2 and Nestin (5, 21). In fact, neurosphere formation is routinely used to enrich glioma stem-like cells from primary human brain tumors (31, 41). The finding that miR-146a inhibits tumorigenesis raised that possibility that it may negatively control the behavior of glioma stem-like cells. We enriched these cells from Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes by culturing them under serum-free conditions in the presence of 20 ng of EGF and 20 ng of FGF. After 7 days in culture, control-virus-transduced cells readily formed large free-floating spheres, robustly expressing Nestin and Sox2 (Fig. 4 A to D). In addition, these neurospheres were able to differentiate into GFAP-positive astrocytes and Tuj1-positive neurons in response to 1% FBS and 5 μM FSK treatment, respectively (Fig. 4C). In sharp contrast, ectopic expression of miR-146a markedly diminished the ability of Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes to form neurospheres, as indicated by a 40% reduction of sphere size and a >30% decline in sphere number (Fig. 4A). miR-146a also impaired the self-renewal ability of these neurospheres, demonstrated by a significant reduction of secondary and tertiary sphere formation upon serial passages (Fig. 4B). Furthermore, exogenous miR-146a led to a considerable decrease in the expression of Nestin and Sox2. This was accompanied by a sharp increase in the level of GFAP expression, indicating enhanced glial differentiation (Fig. 4D). The invasive behavior of glioma stem-like cells is evidenced by rapid cellular migration from neurospheres when attached to the coated plates in culture. We selected neurospheres with similar diameters from either control or miR-146a virus-transduced malignant astrocytes and plated them onto laminin- and polyornithine-coated chamber slides. After 48 h, control-virus-transduced cells rapidly migrated from the edge of the attached spheres and spread out onto the coated culture surfaces. In contrast, ectopic expression of miR-146a caused a 38% reduction of migratory distance from the edge of the spheres (Fig. 4E). These data indicate that miR-146a controls not only the number of glioma stem-like cells but also their invasive behavior in vitro.
Fig. 4.
Fig. 4.
miR-146a reduces the formation of glioma stem-like cells and their migration. (A) Glioma stem-like cells were established by culturing 5,000 Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes in NSC medium. The number of spheres (more ...)
miR-146a targets Notch1.
How does miR-146a impose an inhibitory function on glioma stem-like cells and tumorigenesis? Using the Targetscan bioinformatics algorithm, we searched for direct targets of miR-146a with an emphasis on those genes that were shown to play a role in cancer stem cells, tumorigenesis or NSCs. Among several potential candidates, Notch1 is most prominent due to its established function in maintaining NSCs (23, 27) (Fig. 5 A). Notch1 also interacts with the EGFR and AKT pathways to positively regulate the proliferation of glioma stem-like cells and tumorigenesis (54). To confirm the regulation of Notch1 by miR-146a, we first performed a luciferase reporter assay by linking the 3′UTR of Notch1 to the firefly luciferase gene. The luciferase activity in COS7 cells was significantly reduced (by 34 to 57%) when the reporter was cotransfected with increasing amount of plasmids expressing wild-type miR-146a. Such reduction is specific since a control plasmid expressing seed region-mutated miR-146a (Ctrl) has little effect on reporter activity. Moreover, wild-type miR-146a did not change the activity of a luciferase reporter when the binding site for miR-146a in 3′UTR of Notch1 was mutated (Fig. 5B). Western blot analysis further showed that ectopic miR-146a in malignant astrocytes induced a dramatic reduction of Notch1 protein, especially the processed intracellular NICD, which is the most predominant form in these glia cells (Fig. 5C). When these cells were treated with DAPT, an inhibitor for γ-secretase, to partially block Notch1 processing, miR-146a significantly reduced the appearance of both full-length Notch1 and NICD (Fig. 5D). The inhibitory role of miR-146a on Notch1 is mainly through posttranscriptional control since it does not change the mRNA level of Notch1 (Fig. 5E). Furthermore, phosphorylated AKT, a known downstream target of Notch1 signaling (22, 58), was also markedly reduced (Fig. 5C). It is known that individual microRNAs can target numerous mRNAs (6), raising the possibility that Notch1 may not be the major target of miR-146a in glioma stem-like cells. We examined this possibility by performing a rescue experiment. Interestingly, ectopic expression of NICD completely reversed the inhibitory effect of miR-146a on the formation of glioma stem-like cells. This was indicated by a respective 50 and 60% increase in the number and size of neurospheres compared to miR-146a-expressing cells (Fig. 5F). Importantly, overexpressing NICD did not change the level of miR-146a, suggesting that the rescue was not due to downregulation of miR-146a expression (data not shown).
Fig. 5.
Fig. 5.
Notch1 is a direct target of miR-146a. (A) Sequence alignments between 3′UTR of mouse Notch1 and miR-146a. (B) miR-146a suppresses the activity of a luciferase reporter that is linked to the 3′UTR of Notch1, but not mutant 3′UTR (more ...)
miR-146a regulates NSC proliferation and differentiation.
Notch signaling plays an essential role in maintaining NSCs (1, 23). Its downregulation promotes neurogenesis during development or in the adult stage (40, 55). Our finding that miR-146a targets Notch1 expression suggests that miR-146a may regulate NSC behavior. Indeed, qRT-PCR analysis showed >12-fold induction of miR-146a expression after 4 days of culturing primary mouse E14.5 NSCs under differentiation conditions (Fig. 6 A). Ectopic expression of miR-146a through lentiviral transduction enhanced neuronal differentiation, indicated by a 5-fold increase of Tuj1+ cells compared to control-virus-transduced NSCs under low EGF and FGF concentrations (1 ng/ml) (Fig. 6B and C). Exogenous miR-146a also inhibited NSC proliferation, as demonstrated by a 50% reduction in either BrdU+ or Ki67+ cells (Fig. 6D and E). Such reduction of proliferation was accompanied by increased cell cycle exit, which was shown by a higher ratio of BrdU+ Ki67 cells over the total number of BrdU+ cells after 2 h of BrdU pulse (Fig. 6F). In contrast, miR-146a had no effect on apoptosis, since an equal number of active caspase 3-positive cells was observed (Fig. 6D). Together, these results indicate that miR-146a potentiates neuronal differentiation of NSCs by promoting cell cycle exit.
Fig. 6.
Fig. 6.
miR-146a regulates normal NSCs. (A) Induction of miR-146a after culture of NSCs under differentiation conditions for 4 days. GF, growth factor bFGF and EGF; RA, retinoic acid; FSK, forskolin. (B and C) Promotion of neuronal differentiation of normal NSCs (more ...)
miR-146a prolongs the survival of mice bearing human glioblastoma cells.
We next examined whether the biological role of miR-146a is evolutionarily conserved in humans. Similar to murine Notch1 gene, the 3′UTR of its human ortholog also harbors a potential binding site for miR-146a, albeit not a perfect match (Fig. 7 A). Western blotting showed that ectopic miR-146a markedly reduced the level of both the full-length and the active forms (NICD) of human Notch1 protein in U87 glioblastoma cells (Fig. 7B). miR-146a also diminished the ability of U87 cells to form glioma stem-like cells. This was indicated by a >50% decline in sphere number, a 31% reduction in sphere size (Fig. 7C), and a nearly 40% decrease in the appearance of secondary and tertiary spheres (data not shown). When U87 glioblastoma cells were transplanted into the caudoputamen of NOD/SCID mice, miR-146a reduced the tumor burden and significantly extended the survival of tumor-bearing mice (Fig. 7D and E). Together, these data suggest that miR-146a function is evolutionarily conserved from mice to humans and is able to modulate the behavior of glioma cells both in vitro and in vivo.
Fig. 7.
Fig. 7.
miR-146a promotes the survival of mice transplanted with human glioblastoma cells. (A) A recognition site for miR-146a in the 3′UTR of human Notch1 gene. (B) miR-146a targets human Notch1. Protein levels were examined by Western blotting of lysates (more ...)
Downregulation of miR-146a function enhances tumorigenesis.
miR-146a is induced by oncogenic EGFR and PTEN signaling (Fig. 1). However, its overexpression inhibits the behavior of glioma stem-like cells and tumorigenesis by targeting Notch1. These seemingly paradoxical data suggest that miR-146a may act as a feedback mechanism to restrict tumorigenesis (see below). To vigorously test this hypothesis, we used an “miRNA sponge”' (14) to knock down miR-146a function in Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes. This sponge was designed to have an imperfect binding site near the miR-146a seed region, with a bulge from positions 10 to 13 (Fig. 8 A). This design is presumed to be more effective and stable for inhibiting miR-146a function. We first evaluated its efficacy by using a Notch1 3′UTR-containing luciferase reporter and found that the sponge completely reversed the inhibitory effect of miR-146a on this reporter (Fig. 8B). We also examined this miRNA sponge on the expression of Notch1 through Western blotting. As expected, transduction of Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes with sponge-expressing lentiviruses totally rescued Notch1 protein level that was inhibited by ectopic miR-146a (Fig. 8C). Moreover, this sponge also enhanced the expression of endogenous Notch1, most likely by releasing the inhibition of miR-146a that is induced by EGFRvIII and Pten-loss (Fig. 8D). These data indicate that the use of a miRNA sponge is an effective and specific way to downregulate miR-146a function. We next examined the impact of knocking down miR-146a on the behavior of glioma stem-like cells and tumorigenesis. As expected if miR-146a has an inhibitory role, its downregulation promoted efficient formation of glioma stem-like cells from Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes by significantly increasing the number and the size of neurospheres (Fig. 8E and F). When neurospheres (2 × 105 cells) from lentivirus-transduced malignant astrocytes were grafted into the flanks of female nude mice, knocking down miR-146a function using a miRNA sponge caused a >80% increase in tumor size by 28 days posttransplantation (Fig. 8G). Therefore, these results indicate that miR-146a is induced as a feedback mechanism to restrict the oncogenic potential of EGFR and PTEN signaling.
Fig. 8.
Fig. 8.
Knocking down miR-146a function promotes tumorigenesis. (A) Design of miRNA sponge. (B) Sponge suppresses miR-146a's function on activity of a luciferase reporter that is linked to the 3′UTR of the mouse Notch1 gene. The data are presented as (more ...)
We reveal here that constitutively active EGFRvIII cooperates with the loss of Pten to synergistically induce expression of miR-146a in Ink4a/Arf−/− astrocytes. Counterintuitively, upregulation of miR-146a inhibits tumor growth and the formation and migration of glioma stem-like cells by both malignant murine Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes and human glioblastoma cells. We further show for the first time that miR-146a directly downregulates Notch1 and potentiates differentiation of normal NSCs. Knocking down miR-146a function enhances glioma stem-like cell formation and exacerbates tumor burden. These data suggest that miR-146a integrates oncogenic cues to restrict tumor development as a feedback mechanism (Fig. 8H).
Previously, miR-146a was shown to be induced by endotoxin (lipopolysaccharide) through two consensus NF-κB binding sites in the promoter region (9). This region is highly homologous between human and mouse, suggesting an evolutionarily conserved regulatory mechanism for controlling miR-146a expression. NF-κB is constitutively activated in glioma and many other cancer cells, in which it promotes survival and metastatic potential of these cells as well as tumorigenesis (28, 52). One of the key pathways that controls NF-κB activity in gliomas is the phosphatidylinositol 3-kinase (PI3K) pathway (16, 39), which is a converging point of EGFR- and PTEN-dependent signal transduction. Activation of receptor tyrosine kinase EGFR leads to recruitment and activation of PI-3 kinase, which phosphorylates phosphoinositol lipids to generate phosphatidylinositol-3-phosphates. These lipids in turn recruit and activate AKT in the plasma membrane through PDK1. The function of PI3K is antagonized by PTEN, a lipid phosphatase that dephosphorylates phosphatidylinositol 3,4,5-trisphosphate. Activated AKT further phosphorylates IKK, thus promoting NF-κB activation. The synergistic induction of miR-146a by constitutively active EGFRvIII and the inactivation of Pten in Ink4a/Arf−/− astrocytes may reflect a convergence of these two signaling pathways on NF-κB activity. Activated AKT also promotes Notch1 expression, which subsequently modulates transcription of Egfr through p53 (44, 45). These data indicate that miR-146a belongs to an integrated genetic circuit consisting of the PTEN, EGFR, NF-κB, and Notch pathways. The results from our present study reveal that miR-146a regulates the activity of this circuit by targeting Notch1 expression.
Recent studies indicate that certain miRNAs (such as miR-124, miR-137, miR-128, and miR-7) function as tumor suppressors (19, 49). These miRNAs are rarely expressed in gliomas; however, their overexpression restrains proliferation and self-renewal of glioma stem-like cells by promoting neural differentiation (20, 21). miR-146a is unique in that it is significantly enriched in the human tissues of skin (melanoma), cervical, breast, pancreas and prostate cancers compared to the same noncancerous tissues (42, 51, 53). Similarly, miR-146a was also upregulated in human glioblastoma tissues and in both human and mouse primary glioma cell lines (32). In this regard, increased expression of miR-146a can be viewed as a biomarker for cancers. Unexpectedly, our study reveals that, instead of promoting gliomagenesis through its upregulation, miR-146a rather plays an inhibitory role in restricting the formation of glioma stem-like cells and tumor burden. This result is consistent with a demonstrated role of miR-146a in other cancers, such as pancreatic and breast cancers, where it inhibits cancer progression and invasion (8, 26, 33). A recent report also showed a positive correlation of miR-146a expression with the survival time of gastric cancer patients (25). Furthermore, the overexpression of miR-146a inhibits the proliferation and survival of breast, prostate, and pancreatic cancer cells through the downregulation of other targets in these cells, including ROCK1, EGFR, and MTA-2 (8, 33, 34). Our study adds Notch1 as a major target of miR-146a in glioma cells. Supporting these cell culture models of tumorigenesis, it was recently reported that mice with a deletion of miR-146a spontaneously developed subcutaneous flank tumors (37). These data clearly indicate that miR-146a serves as a native molecular brake for oncogenesis (2).
In summary, our current results and other emerging data indicate that miR-146a constitutes an endogenous feedback system to counteract the oncogenic potential of dysregulated signaling pathways, such as activation of EGFR and inactivation of Pten in gliomas. By regulating multiple targets, including key neural stem cell factor Notch1, a miR-146a-mediated innate regulatory mechanism provides an opportunity to devise novel therapeutic strategies against aggressive and deadly brain tumors.
ACKNOWLEDGMENTS
We thank the Microarray Core Facility, Yuhua Zou and Wenze Niu for technical help, Rhonda Bassel-Duby and Eric Olson for discussions, Hiroyoshi Tanda for careful reading of the manuscript, Pamela Jackson for administrative assistance, and Inder Verma (Salk Institute) for providing pTomo lentiviral vector.
C.-L.Z. is a W. W. Caruth, Jr., Scholar in Biomedical Research. This study was supported by the Whitehall Foundation (2009-12-05), the Welch Foundation (I-1724-01), the National Institutes of Health (1DP2OD006484 and R01NS070981), and a Startup Fund from UT Southwestern Medical Center to C.-L.Z.
Footnotes
[down-pointing small open triangle]Published ahead of print on 5 July 2011.
1. Ables J. L., et al. 2010. Notch1 is required for maintenance of the reservoir of adult hippocampal stem cells. J. Neurosci. 30:10484–10492. [PMC free article] [PubMed]
2. Ameres S. L., Fukunaga R. 2010. Riding in silence: a little snowboarding, a lot of small RNAs. Silence 1:8. [PMC free article] [PubMed]
3. Androutsellis-Theotokis A., et al. 2006. Notch signaling regulates stem cell numbers in vitro and in vivo. Nature 442:823–826. [PubMed]
4. 2008. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455:1061–1068. [PMC free article] [PubMed]
5. Bachoo R. M., et al. 2002. Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis. Cancer Cell 1:269–277. [PubMed]
6. Bartel D. P. 2009. MicroRNAs: target recognition and regulatory functions. Cell 136:215–233. [PMC free article] [PubMed]
7. Behin A., Hoang-Xuan K., Carpentier A. F., Delattre J. Y. 2003. Primary brain tumours in adults. Lancet 361:323–331. [PubMed]
8. Bhaumik D., et al. 2008. Expression of microRNA-146 suppresses NF-κB activity with reduction of metastatic potential in breast cancer cells. Oncogene 27:5643–5647. [PMC free article] [PubMed]
9. Cameron J. E., et al. 2008. Epstein-Barr virus latent membrane protein 1 induces cellular MicroRNA miR-146a, a modulator of lymphocyte signaling pathways. J. Virol. 82:1946–1958. [PMC free article] [PubMed]
10. Chambers C. B., et al. 2001. Spatiotemporal selectivity of response to Notch1 signals in mammalian forebrain precursors. Development 128:689–702. [PubMed]
11. Cheng L. C., Pastrana E., Tavazoie M., Doetsch F. 2009. miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. Nat. Neurosci. 12:399–408. [PMC free article] [PubMed]
12. Chuang Y. Y., et al. 2004. Role of synaptojanin 2 in glioma cell migration and invasion. Cancer Res. 64:8271–8275. [PubMed]
13. Das S., Srikanth M., Kessler J. A. 2008. Cancer stem cells and glioma. Nat. Clin. Pract Neurol. 4:427–435. [PubMed]
14. Ebert M. S., Neilson J. R., Sharp P. A. 2007. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat. Methods 4:721–726. [PMC free article] [PubMed]
15. Ehm O., et al. 2010. RBPJκ-dependent signaling is essential for long-term maintenance of neural stem cells in the adult hippocampus. J. Neurosci. 30:13794–13807. [PubMed]
16. Fan X., et al. 2002. Genetic profile, PTEN mutation and therapeutic role of PTEN in glioblastomas. Int. J. Oncol. 21:1141–1150. [PubMed]
17. Fan X., et al. 2010. NOTCH pathway blockade depletes CD133-positive glioblastoma cells and inhibits growth of tumor neurospheres and xenografts. Stem Cells 28:5–16. [PMC free article] [PubMed]
18. Garzia L., et al. 2009. MicroRNA-199b-5p impairs cancer stem cells through negative regulation of HES1 in medulloblastoma. PLoS One 4:e4998. [PMC free article] [PubMed]
19. Godlewski J., Newton H. B., Chiocca E. A., Lawler S. E. 2010. MicroRNAs and glioblastoma; the stem cell connection. Cell Death Differ. 17:221–228. [PubMed]
20. Godlewski J., et al. 2008. Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal. Cancer Res. 68:9125–9130. [PubMed]
21. Guessous F., et al. 2010. microRNA-34a is tumor suppressive in brain tumors and glioma stem cells. Cell Cycle 9:1031–1036. [PMC free article] [PubMed]
22. Guo D., et al. 2009. Notch-1 regulates Akt signaling pathway and the expression of cell cycle regulatory proteins cyclin D1, CDK2, and p21 in T-ALL cell lines. Leukoc. Res. 33:678–685. [PubMed]
23. Hitoshi S., et al. 2002. Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev. 16:846–858. [PubMed]
24. Holland E. C., Hively W. P., DePinho R. A., Varmus H. E. 1998. A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice. Genes Dev. 12:3675–3685. [PubMed]
25. Hou Z., Xie L., Yu L., Qian X., Liu B. MicroRNA-146a is down-regulated in gastric cancer and regulates cell proliferation and apoptosis. Med. Oncol., in press. [PubMed]
26. Hurst D. R., et al. 2009. Breast cancer metastasis suppressor 1 up-regulates miR-146, which suppresses breast cancer metastasis. Cancer Res. 69:1279–1283. [PMC free article] [PubMed]
27. Imayoshi I., Sakamoto M., Yamaguchi M., Mori K., Kageyama R. 2010. Essential roles of Notch signaling in maintenance of neural stem cells in developing and adult brains. J. Neurosci. 30:3489–3498. [PubMed]
28. Inoue J., Gohda J., Akiyama T., Semba K. 2007. NF-κB activation in development and progression of cancer. Cancer Sci. 98:268–274. [PubMed]
29. Kageyama R., Ohtsuka T., Shimojo H., Imayoshi I. 2008. Dynamic Notch signaling in neural progenitor cells and a revised view of lateral inhibition. Nat. Neurosci. 11:1247–1251. [PubMed]
30. Kanamori M., et al. 2007. Contribution of Notch signaling activation to human glioblastoma multiforme. J. Neurosurg. 106:417–427. [PubMed]
31. Laks D. R., et al. 2009. Neurosphere formation is an independent predictor of clinical outcome in malignant glioma. Stem Cells 27:980–987. [PMC free article] [PubMed]
32. Lavon I., et al. 2010. Gliomas display a microRNA expression profile reminiscent of neural precursor cells. Neurol. Oncol. 12:422–433. [PMC free article] [PubMed]
33. Li Y., et al. 2010. miR-146a suppresses invasion of pancreatic cancer cells. Cancer Res. 70:1486–1495. [PMC free article] [PubMed]
34. Lin S. L., Chiang A., Chang D., Ying S. Y. 2008. Loss of mir-146a function in hormone-refractory prostate cancer. RNA 14:417–424. [PubMed]
35. Louis D. N., et al. 2007. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 114:97–109. [PMC free article] [PubMed]
36. Louvi A., Artavanis-Tsakonas S. 2006. Notch signaling in vertebrate neural development. Nat. Rev. Neurosci. 7:93–102. [PubMed]
37. Lu L. F., et al. 2010. Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses. Cell 142:914–929. [PMC free article] [PubMed]
38. Marumoto T., et al. 2009. Development of a novel mouse glioma model using lentiviral vectors. Nat. Med. 15:110–116. [PMC free article] [PubMed]
39. Mischel P. S., Cloughesy T. F. 2003. Targeted molecular therapy of GBM. Brain Pathol. 13:52–61. [PubMed]
40. Oya S., et al. 2009. Attenuation of Notch signaling promotes the differentiation of neural progenitors into neurons in the hippocampal CA1 region after ischemic injury. Neuroscience 158:683–692. [PubMed]
41. Pellegatta S., et al. 2006. Neurospheres enriched in cancer stem-like cells are highly effective in eliciting a dendritic cell-mediated immune response against malignant gliomas. Cancer Res. 66:10247–10252. [PubMed]
42. Philippidou D., et al. 2010. Signatures of microRNAs and selected microRNA target genes in human melanoma. Cancer Res. 70:4163–4173. [PubMed]
43. Piao Y., Lu L., de Groot J. 2009. AMPA receptors promote perivascular glioma invasion via β1 integrin-dependent adhesion to the extracellular matrix. Neurol. Oncol. 11:260–273. [PMC free article] [PubMed]
44. Purow B. W., et al. 2008. Notch-1 regulates transcription of the epidermal growth factor receptor through p53. Carcinogenesis 29:918–925. [PMC free article] [PubMed]
45. Rajasekhar V. K., et al. 2003. Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes. Mol. Cell 12:889–901. [PubMed]
46. Rao S. A., Santosh V., Somasundaram K. 2010. Genome-wide expression profiling identifies deregulated miRNAs in malignant astrocytoma. Modern Pathol. 23:1404–1417. [PubMed]
47. Rich J. N., et al. 2001. A genetically tractable model of human glioma formation. Cancer Res. 61:3556–3560. [PubMed]
48. Scherr M., et al. 2007. Lentivirus-mediated antagomir expression for specific inhibition of miRNA function. Nucleic Acids Res. 35:e149. [PMC free article] [PubMed]
49. Silber J., et al. 2008. miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells. BMC Med. 6:14. [PMC free article] [PubMed]
50. Soroceanu L., Manning T. J., Jr., Sontheimer H. 1999. Modulation of glioma cell migration and invasion using Cl and K+ ion channel blockers. J. Neurosci. 19:5942–5954. [PubMed]
51. Volinia S., et al. 2006. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl. Acad. Sci. U. S. A. 103:2257–2261. [PubMed]
52. Wang H., Zhang W., Huang H. J., Liao W. S., Fuller G. N. 2004. Analysis of the activation status of Akt, NFκB, and Stat3 in human diffuse gliomas. Lab. Invest. 84:941–951. [PubMed]
53. Wang X., et al. 2008. Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth. PLoS One 3:e2557. [PMC free article] [PubMed]
54. Xu P., et al. 2010. The oncogenic roles of Notch1 in astrocytic gliomas in vitro and in vivo. J. Neuro-Oncol. 97:41–51. [PubMed]
55. Yoon K., Gaiano N. 2005. Notch signaling in the mammalian central nervous system: insights from mouse mutants. Nat. Neurosci. 8:709–715. [PubMed]
56. Zhang C. L., Zou Y., He W., Gage F. H., Evans R. M. 2008. A role for adult TLX-positive neural stem cells in learning and behaviour. Nature 451:1004–1007. [PubMed]
57. Zhao C., et al. 2010. MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling. Proc. Natl. Acad. Sci. U. S. A. 107:1876–1881. [PubMed]
58. Zhao N., Guo Y., Zhang M., Lin L., Zheng Z. 2010. Akt-mTOR signaling is involved in Notch-1-mediated glioma cell survival and proliferation. Oncol. Rep. 23:1443–1447. [PubMed]
59. Zheng H., et al. 2008. p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation. Nature 455:1129–1133. [PubMed]
60. Zhu H., et al. 2009. Oncogenic EGFR signaling cooperates with loss of tumor suppressor gene functions in gliomagenesis. Proc. Natl. Acad. Sci. U. S. A. 106:2712–2716. [PubMed]
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