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MicroRNA-146a Inhibits Glioma Development by Targeting Notch1 12
Running title: miR-146a restricts gliomagenesis 34
Jie Mei1, Robert Bachoo2, and Chun-Li Zhang1,*56
1Department of Molecular Biology, 2Department of Neurology, University of Texas 7Southwestern Medical Center, Dallas, TX 75390-9148 8
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Keywords: miR-146a, Notch1, glioma, EGFR, PTEN, INK4A/ARF 111213
Word Count: Abstract (193), Materials and Methods (1,196), Introduction, Results and 14Discussion (2,911) 15
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*To whom correspondence may be addressed. 2021
Chun-Li Zhang 22Department of Molecular Biology 23University of Texas Southwestern Medical Center 24Dallas, TX 75390-9148 25
26Tel: 214-648-1670 27Fax: 214-648-1488 28Email: chun-li.zhang@utsouthwestern.edu29
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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.05821-11 MCB Accepts, published online ahead of print on 5 July 2011
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ABSTRACT 32
Dysregulated EGFR signaling through either genomic amplification or dominant active 33
mutation (EGFRvIII), in combination with dual inactivation of INK4A/ARF and PTEN, is a 34
leading cause of gliomagenesis. Our global expression analysis for microRNAs 35
revealed that EGFR activation induces miR-146a expression, which is further 36
potentiated by inactivation of PTEN. Unexpectedly, over-expression of miR-146a 37
attenuates proliferation, migration and tumorigenic potential of Ink4a/Arf-/-Pten-/-EgfrvIII38
murine astrocytes. Its ectopic expression also inhibits glioma development of a human 39
glioblastoma cell line in an orthotopic xenograft model. Such inhibitory function of miR-40
146a on gliomas is largely through down-regulation of Notch1, which plays a key role in 41
neural stem cell maintenance and is a direct target of miR-146a. Accordingly, miR-146a 42
modulates neural stem cell proliferation and differentiation, and reduces the formation 43
and migration of glioma stem-like cells. Conversely, knockdown of miR-146a by 44
microRNA sponge upregulates Notch1 and promotes tumorigenesis of malignant 45
astrocytes. These findings indicate that, in response to oncogenic cues, miR-146a is 46
induced as a negative feedback mechanism to restrict tumor growth by repressing 47
Notch1. Our results provide novel insights into the signaling pathways that link neural 48
stem cells to gliomagenesis and may lead to new strategies treating brain tumors.49
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INTRODUCTION51
Gliomas are the most frequently observed brain tumors, with glioblastoma multiforme 52
(GBM) being the most common and aggressive form in adults (35). Despite major 53
therapeutic improvements made by combining neurosurgery, chemotherapy and 54
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radiotherapy, the prognosis and survival rate for patients with GBM is still extremely 55
poor (7). The deadly nature of GBM originates from explosive growth and invasive 56
behavior, which are fueled by dysregulation of multiple signaling pathways. EGFR 57
activation, in cooperation with loss of tumor suppressor functions, such as mutations in 58
Ink4a/Arf and Pten genes, constitutes a lesion signature for GBM (1). Such 59
dysregulated genetic pathways are sufficient to transform neural stem cells (NSCs) or 60
astrocytes into cancer stem-like cells. This gives rise to high-grade malignant gliomas 61
with a pathological phenotype resembling human GBM (5, 59). However, the 62
downstream events underlying these genetic dysregulations in gliomagenic cells have 63
not been fully elucidated. 64
65
MicroRNAs are 20-22 nucleotide non-coding RNA molecules that have emerged as key 66
players in controlling NSC self-renewal and differentiation (11, 57). Aberrant expression 67
of miRNAs, such as miR-21, miR-124 and miR-137, is linked to glioma formation (49). 68
miR-199b-5p and miR-34a impair cancer stem-like cells through negative regulation of 69
several components of the Notch pathway in brain tumors (18, 21). The Notch pathway 70
is an evolutionarily conserved signaling pathway that plays an important role in 71
neurogenesis (4, 10, 23). Upon binding to its ligand, Delta, the Notch intracellular 72
domain (NICD, the activated form of Notch) is released from the membrane by 73
presenilin/ -secretase-mediated cleavage and translocates to the nucleus. In the 74
nucleus, NICD forms a complex with Rbpj and activates the expression of several 75
transcriptional repressors, such as Hes1 and Hes5, which inhibit neurogenesis (15, 27, 76
29). Thus, activation of the Notch pathway is essential to maintain both developing and 77
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adult NSCs (36). This property of the Notch pathway enables it to promote glioma 78
growth (30), and its inhibition by drugs could abolish glioma stem-like cells and reduce 79
tumorigenesis (17). 80
81
In this study, we show that miR-146a is specifically induced as a converging 82
downstream target of EGFR and PTEN signaling in immortalized Ink4a/Arf-/- astrocytes. 83
We further demonstrate that miR-146a acts as a native safeguarding mechanism to 84
restrict the formation of glioma stem-like cells and glioma growth by directly controlling 85
the expression of Notch1. 86
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MATERIALS AND METHODS 88
Cell culture, MTT and anchorage-independent growth assays. We isolated primary 89
NSCs by mechanical dissociation using 1 ml pipettes from embryonic day 14.5 (E14.5) 90
mouse forebrains in growth medium consisting of DMEM/F12, 1 mM L-glutamine, N2 91
(Invitrogen), 20 ng/ml EGF and 20 ng/ml FGF2 (Peprotech). These cells were cultured 92
as free-floating neurospheres under 37°C and 5% CO2. For differentiation, we exposed 93
NSCs to DMEM/F12 media with N2 supplement, further supplemented with 5 μM 94
forskolin and 0.5% FBS (FSK), 1 μM retinoic acid and 0.5% (v/v) FBS (RA), or 0.5% (v/v) 95
FBS (-GF). This was done in 60-mm dishes or 8-well chamber slides coated with 96
laminin (5 μg/ml) and poly-L-ornithine (10 μg/ml). The Ink4a/Arf-/-, Ink4a/Arf-/-EGFRvIII97
and Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes, or human U87 glioma cells were cultured in 98
DMEM containing 10% FBS. Glioma stem-like cells from malignant astrocytes or U87 99
cells were enriched by culturing in NSC medium with growth factors. Cell growth was 100
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determined by the CellTiter 96® nonradioactive cell proliferation assay kit (MTT assay, 101
Promega) according to the manufacturer’s instructions. For anchorage-independent 102
growth by soft agar assay, 2,000 Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes transduced with 103
either wild-type or seed-region-mutated miR-146a were plated and cultured in 12-well 104
plates, as described previously (47). Five weeks later, cell colonies in the plates were 105
stained with 0.5 ml of 0.005% crystal violet and counted under an inverted microscope. 106
We conducted each experiment in triplicate. 107
108
miRNA microarray and quantitative RT-PCR (qPCR). We extracted total RNAs from 109
Ink4a/Arf-/- (I), Ink4a/Arf-/-EGFRvIII (IE), or Ink4a/Arf-/-Pten-/-EGFRvIII (IPE) astrocytes 110
using the miReasy mini kit (Qiagen). Biological replicates were collected. RNA quality 111
was examined by Bioanalyzer (Illumina). Five μg of each RNA sample was processed 112
for labeling and hybridization to GeneChip miRNA arrays (Affymetrix) by the Microarray 113
Core Facility at UT Southwestern Medical Center. After scanning and normalization to 114
control probes, the array data was analyzed by VAMPIRE software to identify 115
statistically significant differences in gene expression between sample groups 116
(http://genome.ucsd.edu/microarray). For qPCR, 1 μg of total RNA was reverse 117
transcribed using NCode miRNA First-Strand Synthesis and qRT-PCR Kits (Invitrogen). 118
qPCR reactions were performed in a 384-well plate using an ABI 7900HT instrument. 119
The PCR program consisted of 2 min at 50°C and 10 min at 95°C, followed by 40 cycles 120
of 15s at 95°C and 60s at 58°C. Primer quality was analyzed by dissociation curves. 121
The expression of miR-146a and Notch1 was normalized to that of U6 and Hprt,122
respectively.123
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Lentivirus production and transduction. A genomic fragment encompassing the miR-125
146a coding region or a seed-region-mutated version was cloned by PCR into pTomo 126
vector (provided by Dr. Inder Verma at Salk Institute). To construct the miRNA sponge, 127
we inserted the synthesized DNA fragment into the BamHI site of a pCSC-SP-PW-128
IRES/GFP lentiviral vector, and the empty vector as the control. We produced the 129
lentiviruses, determined their titer, and transduced cultured cells using previously 130
described methods (38, 48). Transduction efficiency was monitored by GFP expression. 131
All experiments were performed using stably transduced cells within 6 passages.132
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Cell migration assay. Migration of malignant astrocytes in culture was determined by 134
the ‘scratch’ assay. For this, cells were seeded into a 6-well tissue culture dish and 135
allowed to grow to 90% confluency in complete medium with 10% FBS. Cells in 136
monolayers were scratched in a single straight line using a pipette tip (1 mm in 137
diameter). Wounded monolayers were washed three times with culture medium to 138
remove cell debris and then incubated for another 24h. Migratory distance was 139
measured under a microscope equipped with a camera. Transwell migration assay was 140
conducted essentially as previously described (43). Invasive behavior of glioma stem-141
like cells in vitro was examined by measuring migration ability of cultured neurospheres. 142
Neurospheres with similar diameter were selected and plated onto 6-well culture plates 143
coated with laminin (5 μg/ml) and poly-L-ornithine (10 μg/ml) and cultured for 48h in 144
NSC growth medium. Cell migration and spreading was quantified by measuring the 145
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distance between the edge of the neurosphere and the periphery of radially migrating 146
cells. 147
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Luciferase reporter assay. The 3´UTR of the Notch1 gene, which contains one 149
putative miR-146a targeting site, was amplified by PCR and inserted into the SacI and 150
PmeI sites of pMIR-REPORT vector (Ambion). To express miR-146a, a 610-bp genomic 151
fragment encompassing the coding region was cloned by PCR and inserted into the 152
HindIII and BamHI sites of pCMV6 vector. A seed-sequence-mutated version of miR-153
146a was used as a control for all of the experiments. To create this mutant (pCMV6-154
miR-146a-mt), we replaced the seed sequence TGAGAACT with GCGGCCGC through 155
site-directed mutagenesis using the QuickChange kit (Stratagene). Similarly, the binding 156
site (AGTTCT) for miR-146a within the 3’UTR of Notch 1 gene was replaced with 157
ACGCGT to generate a control for the luciferase reporter assays. HEK293 cells were 158
transiently transfected using Fugene 6 reagent (Roche) in 48-well plates. The following 159
plasmids were used: pMIR-REPORT with either pCMV6-miR-146a or its mutant, 160
pCMV6-miR-146a-mt. pCMV-LacZ was used as a control to monitor transfection 161
efficiency. Total plasmid amount (200 ng) was kept constant for each transfection with 162
empty pCMV6 vector. Forty-eight hours later, cells were lysed and assayed for 163
luciferase and -galactosidase activity using the Dual-Light system (Applied 164
Biosystems). Relative reporter activities were determined by normalizing luminescence 165
units to -galactosidase expression. 166
167
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Western blotting and immunocytochemistry. Protein expression was examined by 168
Western blotting, according to a standard procedure. Antibodies against the following 169
proteins were used: -Actin (Sigma, 1:8000), GFAP and p-AKT-T308 (Cell Signaling 170
Technology, 1:1,000), Sox2 (Chemicon, 1:1,000), Nestin (Aves, 1:10,000), Notch1 171
(Santa Cruz, 1:1,000). To partially block processing of Notch1 protein by –secretase,172
cells were treated with 10 M DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-173
phenylglycine t-butyl ester) (Sigma) for 36 h. Blots were scanned and quantified by NIH 174
software ImageJ. To examine proliferation and cell cycle exit, cultured cells were pulse-175
labeled with BrdU (10 μM) for 2 h and then fixed and processed for immunostaining, as 176
previously described (56). Antibodies against the following proteins were used: BrdU 177
(BD pharmingen; 1:1,000), Ki67 (Novocastra; 1:500), cleaved caspase 3 (Cell Signaling 178
Technology; 1:300) and Tuj1 (Covance; 1:500).179
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Intracranial and subcutaneous cell transplantation model for tumorigenesis.181
NOD/SCID and nude mice (nu/nu) were purchased from Harlan Laboratories. They 182
were housed in a pathogen-free facility under standard 12h light/dark cycles, controlled 183
temperature conditions and had free access to food and water. Experimental protocols 184
were approved by the Institutional Animal Care and Use Committee (IACUC) at UT 185
Southwestern Medical Center. Astrocytes (5X105 cells) or neurospheres (2X105 cells) 186
were respectively implanted into the right caudoputamen of the brain (for orthotopic 187
transplantation) or subcutaneous tissues of 5 to 6 weeks old NOD/SCID or nude mice 188
using a 25 gauge-needle. The injection sites were monitored frequently. Mice 189
transplanted with neurospheres were euthanized 3 weeks later. Tumors were finely 190
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dissected out, imaged, and measured according to the following formula: Volume of 191
tumor (mm3) = d2×D/2, where d and D are the shortest and the longest diameters, 192
respectively. For survival analysis, the mice were euthanized when they demonstrated 193
severe health deterioration according to IACUC guidelines. 194
195
Statistical analysis. Data are expressed as mean ± SD. Except for the survival studies, 196
all data were analyzed by two-tailed Student t tests. The Kaplan-Meier survival curves 197
were determined by using Graphpad Prism v5.0 (Graphpad Software, Inc.) and the Log-198
rank test. A p value of < 0.05 was considered significant. 199
200
RESULTS201
EGFR activation and PTEN inactivation synergistically induce expression of miR-202
146a. Loss of Ink4a/Arf results in immortal growth of primary murine astrocytes. 203
Subsequent activation of EGFR signaling through expression of a constitutively active 204
EGFRvIII mutant transforms these astrocytes into tumorigenic and neurosphere-forming 205
glioma stem-like cells when cultured in defined medium for NSCs (5, 24). In vivo,206
concomitant activation of EGFR and inactivation of Ink4a/Arf and Pten produce rapid-207
onset and fully penetrant high-grade gliomas that resemble GBM in humans (60), 208
suggesting cooperative actions of these signaling pathways on cell proliferation. To 209
confirm this, we performed MTT assays and direct cell counts to assess the growth rate 210
of Ink4a/Arf-/-, Ink4a/Arf-/-EGFRvIII, or Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes. Indeed, 211
constitutive activation of EGFR confers Ink4a/Arf-/- astrocytes with a growth advantage 212
that is further enhanced by ablation of Pten (Fig. 1A and B). 213
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To understand the downstream molecular events of this genetic cooperation and 215
because of the emerging role of microRNA in cancers, we performed global expression 216
analysis of microRNAs in Ink4a/Arf-/-, Ink4a/Arf-/-EGFRvIII or Ink4a/Arf-/-Pten-/-EGFRvIII217
astrocytes. We identified a total of 19 unique miRNAs whose expression is altered by 218
activation of EGFR alone or in combination with loss of Pten (Fig. 1C). Among these 219
miRNAs, miR-146a, miR-182, miR-183 and miR-199a-3p are shown to be enriched in 220
diverse cancers, whereas miR-127 and miR-140 are down-regulated in gliomas (32, 46). 221
In contrast to unchanged expression of miR-146b, miR-146a was significantly induced 222
by EGFRvIII alone or in combination with ablation of Pten (Fig. 1C). The change in 223
expression was subsequently confirmed by qPCR using independent RNA samples (Fig. 224
1D). We also examined the expression of miR-146a after acute deletion of Pten through 225
Cre-mediated recombination of floxed Pten alleles in immortalized astrocytes (Fig. 1E 226
and F). Pten loss resulted in more than a 7-fold induction of miR-146a expression. This 227
induction was further increased to 9-fold after activation of EGFR. These data indicate a 228
cooperative nature of EGFR, PTEN and INK4A/ARF signaling on cell proliferation and 229
miR-146a expression. 230
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miR-146a inhibits proliferation and oncogenic potential of malignant astrocytes. 232
The induction of miR-146a by EGFR and/or PTEN signaling in Ink4a/Arf-/- astrocytes 233
suggests that miR-146a may act as an onco-miR by transducing oncogenic signals to 234
control cell behavior. To test this hypothesis, we performed overexpression experiments 235
to examine whether exogenous miR-146a could further enhance proliferation and 236
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tumorigenesis of malignant astrocytes. Due to extremely low efficiency of transient 237
transfections in these astrocytes, we employed a lentivirus-mediated expression system, 238
in which the CMV promoter drives expression of both miR-146a and GFP. As a control, 239
we mutated eight nucleotides within the seed region of miR-146a (Ctrl, Fig. 2A). Such a 240
mutation presumably abolishes the interactions of miR-146a with its targets. 241
Examination of GFP expression showed that nearly 100% of the cells were transduced 242
by lentivirus. qPCR analysis demonstrated a 26-fold increase of wild-type miR-146a in 243
Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes after lentiviral transduction (Fig. 2B). Since miR-244
146a had no effect on apoptosis (Fig. 2E), we examined cell growth by MTT assays and 245
direct cell counting. Interestingly, overexpression of miR-146a inhibited proliferation of 246
Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes (Fig. 2C-D), indicating that this microRNA may 247
instead function as a tumor suppressor. Indeed, overexpression of miR-146a resulted in 248
a 25% reduction in the diameter (Fig. 2F) and a 35% decrease in the number (Fig. 2G) 249
of colonies in an anchorage-independent growth assay, which is an in vitro model for 250
cellular transformation. 251
252
High-grade gliomas exhibit aggressive behavior, which is manifested by rapid cellular 253
migration under culture conditions in vitro (12, 50). Using a standard ‘scratch’ assay 254
after the cells become confluent in culture dishes, we found that enhanced expression 255
of miR-146a significantly reduced the rate of migration of Ink4a/Arf-/-Pten-/-EGFRvIII256
astrocytes when compared to the same cells transduced with a control lentivirus (Fig. 257
2H). This observation was further confirmed by a Matrigel transwell migration assay, 258
which showed a 50% reduction of migrating malignant astrocytes upon ectopic 259
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expression of miR-146a (Fig. 2I). Finally, we examined the in vivo function of miR-146a 260
during glioma development after cell transplantation into the right caudoputamen of 261
NOD/SCID mice. For this, we grafted an equal number (5X105) of Ink4a/Arf-/-Pten-/-262
EGFRvIII astrocytes that were transduced with lentiviruses expressing either wild-type 263
miR-146a or seed-region-mutated miR-146a (Ctrl). Mice were monitored daily for 264
morbidity. A subset of mice was also sacrificed around 3 weeks post transplantation to 265
examine tumor burden by histology (Fig. 3A). Ectopic expression of miR-146a but not 266
the Ctrl significantly reduced the tumor burden and prolonged the survival of mice that 267
were transplanted with Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes (Fig. 3B). Together, these 268
data suggest that miR-146a acts as a tumor suppressor of glioma development. 269
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miR-146a inhibits formation of glioma stem-like cells from malignant astrocytes. 271
Accumulating evidence suggests that self-renewable stem-like cells within the bulk of 272
brain tumors are the driving force for initiation and maintenance of aggressive gliomas 273
(13). These cells share common features with NSCs, including the ability to form 274
neurospheres and the expression of stem cell markers, such as Sox2 and Nestin (5, 21). 275
In fact, neurosphere formation is routinely used to enrich glioma stem-like cells from 276
primary human brain tumors (31, 41). The finding that miR-146a inhibits tumorigenesis 277
raised that possibility that it may negatively control the behavior of glioma stem-like cells. 278
We enriched these cells from Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes by culturing them 279
under serum-free conditions in the presence of 20 ng EGF and 20 ng FGF. After 7 days 280
in culture, control-virus-transduced cells readily formed large free-floating spheres, 281
robustly expressing Nestin and Sox2 (Fig. 4A-D). Additionally, these neurospheres were 282
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able to differentiate into GFAP-positive astrocytes and Tuj1-positive neurons in 283
response to 1% fetal bovine serum and 5 μM forskolin treatment, respectively (Fig. 4C). 284
In sharp contrast, ectopic expression of miR-146a markedly diminished the ability of 285
Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes to form neurospheres, as indicated by a 40% 286
reduction of sphere size and more than a 30% decline in sphere number (Fig. 4A). miR-287
146a also impaired the self-renewal ability of these neurospheres, demonstrated by a 288
significant reduction of secondary and tertiary sphere formation upon serial passages 289
(Fig. 4B). Furthermore, exogenous miR-146a led to a considerable decrease in the 290
expression of Nestin and Sox2. This was accompanied by a sharp increase in the level 291
of GFAP expression, indicating enhanced glial differentiation (Fig. 4D). The invasive 292
behavior of glioma stem-like cells is evidenced by rapid cellular migration from 293
neurospheres when attached to the coated plates in culture. We selected neurospheres 294
with similar diameters from either control or miR-146a virus-transduced malignant 295
astrocytes and plated them onto laminin- and polyornithine-coated chamber slides. 296
Forty-eight hours later, control-virus-transduced cells rapidly migrated from the edge of 297
the attached spheres and spread out onto the coated culture surfaces. In contrast, 298
ectopic expression of miR-146a caused a 38% reduction of migratory distance from the 299
edge of the spheres (Fig. 4E). These data indicate that miR-146a controls not only the 300
number of glioma stem-like cells but also their invasive behavior in vitro.301
302
miR-146a targets Notch1. How does miR-146a impose an inhibitory function on glioma 303
stem-like cells and tumorigenesis? Using the Targetscan bioinformatics algorithm, we 304
searched for direct targets of miR-146a with an emphasis on those genes that were 305
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shown to play a role in cancer stem cells, tumorigenesis or NSCs. Among several 306
potential candidates, Notch1 is most prominent due to its established function in 307
maintaining NSCs (23, 27) (Fig. 5A). Notch1 also interacts with the EGFR and AKT 308
pathways to positively regulate the proliferation of glioma stem-like cells and 309
tumorigenesis (54). To confirm the regulation of Notch1 by miR-146a, we first performed 310
a luciferase reporter assay by linking the 3’UTR of Notch1 to the firefly luciferase gene. 311
Luciferase activity in COS7 cells was significantly reduced (by 34-57%) when the 312
reporter was cotransfected with increasing amount of plasmids expressing wild-type 313
miR-146a. Such reduction is specific since a control plasmid expressing seed-region-314
mutated miR-146a (Ctrl) has little effect on reporter activity. Moreover, wild type miR-315
146a did not change the activity of a luciferase reporter when the binding site for miR-316
146a in 3’UTR of Notch1 was mutated (Fig. 5B). Western blot analysis further showed 317
that ectopic miR-146a in malignant astrocytes induced a dramatic reduction of Notch1 318
protein, especially the processed intracellular NICD, which is the most predominant 319
form in these glia cells (Fig. 5C). When these cells were treated with DAPT, an inhibitor 320
for -secretase, to partially block Notch1 processing, miR-146a significantly reduced the 321
appearance of both full-length Notch1 and NICD (Fig. 5D). The inhibitory role of miR-322
146a on Notch1 is mainly through posttranscriptional control since it does not change 323
the mRNA level of Notch1 (Fig. 5D). Furthermore, phosphorylated AKT, a known 324
downstream target of Notch1 signaling (22, 58), was also markedly reduced (Fig. 5C). It 325
is known that individual microRNAs can target numerous mRNAs (6), raising the 326
possibility that Notch1 may not be the major target of miR-146a in glioma stem-like cells. 327
We examined this possibility by performing a rescue experiment. Interestingly, ectopic 328
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expression of NICD completely reversed the inhibitory effect of miR-146a on the 329
formation of glioma stem-like cells. This was indicated by a respective 50% and 60% 330
increase in the number and size of neurospheres when compared to miR-146a-331
expressing cells (Fig. 5F). Importantly, overexpressing NICD did not change the level of 332
miR-146a, suggesting that the rescue was not due to down-regulation of miR-146a 333
expression (data not shown). 334
335
miR-146a regulates NSC proliferation and differentiation. Notch signaling plays an 336
essential role in maintaining NSCs (2, 23). Its down-regulation promotes neurogenesis 337
during development or in the adult stage (40, 55). Our finding that miR-146a targets 338
Notch1 expression suggests that miR-146a may regulate NSC behavior. Indeed, qPCR 339
analysis showed over 12-fold induction of miR-146a expression after 4 days of culturing 340
primary mouse E14.5 NSCs under differentiation conditions (Fig. 6A). Ectopic 341
expression of miR-146a through lentiviral transduction enhanced neuronal 342
differentiation, indicated by a 5-fold increase of Tuj1+ cells compared to control-virus-343
transduced NSCs under low EGF and FGF concentrations (1 ng/ml) (Fig. 6B and C). 344
Exogenous miR-146a also inhibited NSC proliferation, demonstrated by a 50% 345
reduction of either BrdU+ or Ki67+ cells (Fig. 6D and E). Such reduction of proliferation 346
was accompanied by increased cell cycle exit, which was shown by a higher ratio of 347
BrdU+Ki67- cells over the total number of BrdU+ cells after 2 hr of BrdU pulse (Fig. 6F). 348
In contrast, miR-146a had no effect on apoptosis, since an equal number of active 349
caspase 3-positive cells were observed (Fig. 6D). Together, these results indicate that 350
miR-146a potentiates neuronal differentiation of NSCs by promoting cell cycle exit. 351
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352
miR-146a prolongs the survival of mice bearing human glioblastoma cells. We 353
next examined whether the biological role of miR-146a is evolutionarily conserved in 354
humans. Similar to murine Notch1 gene, the 3’UTR of its human ortholog also harbors a 355
potential binding site for miR-146a, albeit not a perfect match (Fig. 7A). Western blotting 356
analysis showed that ectopic miR-146a markedly reduced the level of both full-length 357
and the active form (NICD) of human Notch1 protein in U87 glioblastoma cells (Fig. 7B). 358
miR-146a also diminished the ability of U87 cells to form glioma stem-like cells. This 359
was indicated by a more than 50% decline in sphere number, a 31% reduction of 360
sphere size (Fig. 7C), and a near 40% decrease on the appearance of secondary and 361
tertiary spheres (data not shown). When U87 glioblastoma cells were transplanted into 362
the caudoputamen of NOD/SCID mice, miR-146a reduced tumor burden and 363
significantly extended the survival of tumor-bearing mice (Fig. 7D-E). Together, these 364
data suggest that miR-146a function is evolutionarily conserved from mice to human 365
and is able to modulate the behavior of glioma cells both in vitro and in vivo.366
367
Down-regulation of miR-146a function enhances tumorigenesis. miR-146a is 368
induced by oncogenic EGFR and PTEN signaling (Fig. 1). However, its overexpression 369
inhibits the behavior of glioma stem-like cells and tumorigenesis by targeting Notch1. 370
These seemingly paradoxical data suggest that miR-146a may act as a feedback 371
mechanism to restrict tumorigenesis (see below). To vigorously test this hypothesis, we 372
used an ‘miRNA sponge' (14) to knock down miR-146a function in Ink4a/Arf-/-Pten-/-373
EGFRvIII astrocytes. This sponge was designed to have an imperfect binding site near 374
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the miR-146a seed region – with a bulge from positions 10 to 13 (Fig. 8A). This design 375
is presumed to be more effective and stable for inhibiting miR-146a function. We first 376
evaluated its efficacy by using a Notch1 3’UTR-containing luciferase reporter and found 377
that the sponge completely reversed the inhibitory effect of miR-146a on this reporter 378
(Fig. 8B). We also examined this miRNA sponge on the expression of Notch1 through 379
Western blotting. As expected, transduction of Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes with 380
sponge-expressing lentiviruses totally rescued Notch1 protein level that was inhibited by 381
ectopic miR-146a (Fig. 8C). Moreover, this sponge also enhanced the expression of 382
endogenous Notch1, most likely by releasing the inhibition of miR-146a that is induced 383
by EGFRvIII and Pten-loss (Fig. 8D). These data indicate that the use of a miRNA 384
sponge is an effective and specific way to down-regulate miR-146a function. We next 385
examined the impact of knocking down miR-146a on the behavior of glioma stem-like 386
cells and tumorigenesis. As expected if miR-146a has an inhibitory role, its down-387
regulation promoted efficient formation of glioma stem-like cells from Ink4a/Arf-/-Pten-/-388
EGFRvIII astrocytes by significantly increasing the number and the size of neurospheres 389
(Fig. 8E and F). When neurospheres (2X105 cells) from lentivirus-transduced malignant 390
astrocytes were grafted into the flanks of female nude mice, knocking-down miR-146a 391
function using a miRNA sponge caused more than an 80% increase in tumor size 28 392
days post transplantation (Fig. 8G). Therefore, these results indicate that miR-146a is 393
induced as a feedback mechanism to restrict the oncogenic potential of EGFR and 394
PTEN signaling. 395
396
DISCUSSION 397
18
Here, we reveal that constitutively active EGFRvIII cooperates with the loss of Pten to 398
synergistically induce expression of miR-146a in Ink4a/Arf-/- astrocytes. 399
Counterintuitively, upregulation of miR-146a inhibits tumor growth and the formation and 400
migration of glioma stem-like cells by both malignant murine Ink4a/Arf-/-Pten-/-EGFRvIII401
astrocytes and human glioblastoma cells. We further show for the first time that miR-402
146a directly downregulates Notch1 and potentiates differentiation of normal NSCs. 403
Knocking down miR-146a function enhances glioma stem-like cell formation and 404
exacerbates tumor burden. These data suggest that miR-146a integrates oncogenic 405
cues to restrict tumor development as a feedback mechanism (Fig. 8H). 406
407
Previously, miR-146a was shown to be induced by endotoxin (lipopolysaccharide) 408
through two consensus NF- B binding sites in the promoter region (9). This region is 409
highly homologous between human and mouse, suggesting an evolutionarily conserved 410
regulatory mechanism for controlling miR-146a expression. NF- B is constitutively 411
activated in glioma and many other cancer cells, in which it promotes survival and 412
metastatic potential of these cells as well as tumorigenesis (28, 52). One of the key 413
pathways that controls NF- B activity in gliomas is the PI3K pathway (16, 39), which is a 414
converging point of EGFR- and PTEN-dependent signal transduction. Activation of 415
receptor tyrosine kinase EGFR leads to recruitment and activation of PI-3 kinase, which 416
phosphorylates phosphoinositol lipids to generate phosphatidylinositol-3-phosphates. 417
These lipids in turn, recruit and activate AKT in the plasma membrane through PDK1. 418
The function of PI3K is antagonized by PTEN, a lipid phosphatase that 419
dephosphorylates phosphatidylinositol 3,4,5-trisphosphate. Activated AKT further 420
19
phosphorylates IKK, thus promoting NF- B activation. The synergistic induction of miR-421
146a by constitutively active EGFRvIII and inactivation of Pten in Ink4a/Arf-/- astrocytes 422
may reflect a convergence of these two signaling pathways on NF- B activity. Activated 423
AKT also promotes Notch1 expression, which subsequently modulates transcription of 424
Egfr through p53 (44, 45). These data indicate that miR-146a belongs to an integrated 425
genetic circuit consisting of the PTEN, EGFR, NF- B and Notch pathways. The results 426
from our current study reveal that miR-146a regulates the activity of this circuit by 427
targeting Notch1 expression. 428
429
Recent studies indicate that certain miRNAs (such as miR-124, miR-137, miR-128, miR-430
7) function as tumor suppressors (19, 49). These miRNAs are rarely expressed in 431
gliomas; however, their overexpression restrains proliferation and self-renewal of glioma 432
stem-like cells by promoting neural differentiation (20, 21). miR-146a is unique in that it 433
is significantly enriched in human tissues of skin (melanoma), cervical, breast, pancreas 434
and prostate cancers, compared to the same non-cancerous tissues (42, 51, 53). 435
Similarly, miR-146a was also up-regulated in human glioblastoma tissues and in both 436
human and mouse primary glioma cell lines (32). In this regard, increased expression of 437
miR-146a can be viewed as a biomarker for cancers. Unexpectedly, our study reveals 438
that, instead of promoting gliomagenesis through its upregulation, miR-146a rather 439
plays an inhibitory role in restricting the formation of glioma stem-like cells and tumor 440
burden. This result is consistent with a demonstrated role of miR-146a in other cancers, 441
such as pancreatic and breast cancers, where it inhibits cancer progression and 442
invasion (8, 26, 33). A most recent report also showed a positive correlation of miR-443
20
146a expression with the survival time of gastric cancer patients (25). Furthermore, 444
overexpression of miR-146a inhibits proliferation and survival of breast, prostate and 445
pancreatic cancer cells through down-regulation of other targets in these cells, including 446
ROCK1, EGFR, and MTA-2 (8, 33, 34). Our study adds Notch1 as a major target of 447
miR-146a in glioma cells. Supporting these cell culture models of tumorigenesis, it was 448
recently reported that mice with a deletion of miR-146a spontaneously developed 449
subcutaneous flank tumors (37). This data clearly indicates that miR-146a serves as a 450
native molecular brake for oncogenesis (3). 451
452
In summary, our current results and other emerging data indicate that miR-146a 453
constitutes an endogenous feedback system to counteract the oncogenic potential of 454
dysregulated signaling pathways, such as activation of EGFR and inactivation of Pten in 455
gliomas. By regulating multiple targets, including key neural stem cell factor Notch1, a 456
miR-146a-mediated innate regulatory mechanism provides an opportunity to devise 457
novel therapeutic strategies against aggressive and deadly brain tumors. 458
459
Acknowledgements. We thank the Microarray Core Facility, Yuhua Zou and Wenze 460
Niu for technical help, Rhonda Bassel-Duby and Eric Olson for discussions, Hiroyoshi 461
Tanda for careful reading of the manuscript, Pamela Jackson for administrative 462
assistance, and Dr. Inder Verma (Salk Institute) for providing pTomo lentiviral vector.463
C-L.Z. is a W. W. Caruth, Jr. Scholar in Biomedical Research. This work was supported 464
by the Whitehall Foundation (2009-12-05), the Welch Foundation (I-1724-01), the NIH 465
21
(1DP2OD006484 and R01NS070981) and a Startup Fund from UT Southwestern 466
Medical Center to C-L.Z. 467
468
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672
FIGURE LEGENDS673
FIG. 1. Induction of miR-146a expression by EGFR and PTEN signaling in immortalized 674
Ink4a/Arf-/- astrocytes. (A-B) Proliferation of Ink4a/Arf-/- (I), Ink4a/Arf-/-EGFRvIII (IE), or 675
Ink4a/Arf-/-Pten-/-EGFRvIII (IPE) astrocytes determined by MTT assay (A) or by cell 676
counting (B) (n=3). (B) Heat map representation of differentially expressed miRNAs 677
identified by microarray analysis. (C) qPCR analysis to confirm miR-146a expression 678
induced by EGFR and PTEN signaling using independent RNA samples. (D) Effects of 679
acute Pten loss on expression of miR-146a in Ink4a/Arf-/- Ptenf/f (IPf/f) or Ink4a/Arf-/- 680
Ptenf/f EGFRvIII (IPf/fE) astrocytes. These cells were transduced with adenoviruses 681
expressing either GFP or GFP-Cre. (F) Acute deletion of Pten in Ink4a/Arf-/-Ptenf/f and682
Ink4a/Arf-/-Ptenf/fEGFRvIII astrocytes. Astrocytes were transduced with adenoviruses 683
expressing GFP (Ad-GFP) or GFP-Cre (Ad-GFP-Cre). Using primers located in the 684
floxed exon 5 of the Pten gene, a 157-bp PCR product can be detected in Ad-GFP-685
transduced but not Ad-GFP-Cretransduced astrocytes. Expression of Hprt was used as 686
a loading control. Each experiment was performed in triplicate, except for microarrays 687
that were in duplicate (*p < 0.05, **p < 0.01, and ***p < 0.001). 688
689
31
FIG. 2. miR-146a inhibits cellular transformation and migration of malignant astrocytes. 690
(A) Schema of miR-146a mutant (Ctrl). The seeding sequence of miR-146a is replaced 691
with a Not1 digestion site. (B) qPCR analysis of miR-146a expression in lentivirus-692
transduced astrocytes. Experiments were performed in triplicate. (C-D) Growth curves 693
of Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes transduced with lentiviruses expressing either 694
miR-146a or its mutant (Ctrl) by MTT assay (C) or by cell counting (D) (n=3). (E) 695
Apoptotic cells were detected by an antibody recognizing cleaved caspase 3 and nuclei 696
were shown by DAPI-staining. (F-G) Ectopic expression of miR-146a restrains 697
clonogenic growth of Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes in a soft-agar assay (n=3).698
Representative images (100× magnification) are shown. (H) A scratch assay to examine 699
the effect of miR-146a on migration of malignant astrocytes (n=3). Representative 700
images (40× magnification) at 0 and 24 hours are shown. (I) Transwell migration assay 701
of Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes transduced with lentiviruses expressing either 702
miR-146a or its mutant (Ctrl) (n=3) (*p < 0.05, **p < 0.01, and ***p < 0.001). 703
704
FIG. 3. miR-146a decreases tumor burden induced by intracranial transplantation of 705
Ink4a/Arf-/-Pten-/-EGFRvIII murine astrocytes. (A) Representative histological analysis of 706
brain gliomas in NOD/SCID mice 3 weeks post transplantation. In each experiment, a 707
seed region-mutated version of miR-146a was used as a control (Ctrl). (B) Kaplan-Meier 708
survival curve of mice transplanted with Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes (n=11 and 709
8 for the control and miR-146a group, respectively; p=0.002 by Log-rank Test). 710
711
32
FIG. 4. miR-146a reduces formation of glioma stem-like cells and their migration. (A) 712
Glioma stem-like cells were established by culturing 5,000 Ink4a/Arf-/-Pten-/-EGFRvIII713
astrocytes in NSC medium. The number of spheres and their diameter were determined 714
7 days later. A representative morphology of the spheres is shown in the left panels at 715
×100 magnification (n=3). (B) Number counts of primary, secondary and tertiary 716
neurospheres established by culturing 2,500 Ink4a/Arf-/-Pten-/-EGFRvIII murine717
astrocytes in NSC medium (n=3). (C) In the absence of growth factor, neurospheres 718
(derived from Ink4a/Arf-/-Pten-/-EGFRvIII astrocytes) were differentiated into GFAP-719
positive astrocytes and Tuj1-positive neurons in response to 1% fetal bovine serum and 720
5 M forskolin (FSK) treatment, respectively. (D) Ectopic expression of miR-146a 721
induces glial marker GFAP and suppresses expression of stem cell markers Sox2 and 722
Nestin. Actin was used as a loading control for Western blotting. Relative protein level is 723
shown. (E) miR-146a inhibits spreading and migration of glioma stem-like spheres. 724
Representative photographs were taken at ×100 magnification. Distance was measured 725
from the edge of the sphere to the periphery of the migrating cells (n=3; *p < 0.05, **p <726
0.01 and ***p < 0.001). 727
728
FIG. 5. Notch1 is a direct target of miR-146a. (A) Sequence alignments between 3’UTR 729
of mouse Notch1 and miR-146a. (B) miR-146a suppresses the activity of a luciferase 730
reporter that is linked to the 3’UTR of Notch1, but not mutant 3’UTR of Notch1. After 731
normalization to -galactosidase expression, which was used as a control for 732
transfection efficiency, the data was presented as the ratio to that of empty-vector–733
transfected cells. (C) miR-146a blocks protein expression of Notch1 and its downstream 734
33
target, pAKT, in Ink4a/Arf-/-Pten-/-EGFRVIII astrocytes. Protein loading was monitored by 735
-actin. Relative protein level is indicated. (D) Blocking Notch1 processing by DAPT, a 736
-secretase inhibitor, reveals the inhibitory effect of miR-146a on full-length Notch1 737
protein in murine astrocytes. (E) miR-146a has no effect on the expression of Notch1738
transcripts, determined by qPCR. (F) NICD rescues the inhibitory effect of miR-146a on 739
the formation of glioma stem-like cells from Ink4a/Arf-/-Pten-/-EGFRVIII astrocytes (*p < 740
0.05 and ***p < 0.001). 741
742
FIG. 6. miR-146a regulates normal NSCs. (A) Induction of miR-146a after culturing 743
NSCs under differentiation conditions for 4 days. GF, growth factor bFGF and EGF; RA, 744
retinoic acid; FSK, forskolin. (B) and (C) Promoting neuronal differentiation of normal 745
NSCs by miR-146a. Neurons were stained with an antibody against Tuj1 and nuclei 746
were shown by DAPI staining. Representative photographs were taken at ×400 747
magnification. (D) - (F) miR-146a inhibits NSC proliferation by inducing cell cycle exit. 748
(D) Proliferating cells were examined by BrdU incorporation and Ki67 staining, which 749
are shown in merged panels with phase-contrast images. Apoptotic cells were stained 750
with an antibody for cleaved caspase 3 (Casp3). Representative photographs were 751
taken at ×200 magnification. (E) Quantification of proliferating NSCs (BrdU+ or Ki67+). (F) 752
miR-146a enhances cell cycle exit, which was measured by the ratio of BrdU+Ki67- cells 753
to total number of BrdU+ cells. Experiments were performed in triplicate (**p < 0.01 and 754
***p < 0.001). 755
756
34
FIG. 7. miR-146a promotes survival of mice transplanted with human glioblastoma cells. 757
(A) A recognition site for miR-146a in the 3’UTR of human Notch1 gene. (B) miR-146a 758
targets human Notch1. Protein levels were examined by Western blotting analysis of 759
lysates from human U87 glioblastoma cells. Protein loading was monitored by -actin.760
Relative protein level is indicated. (C) miR-146a inhibits the formation of glioma stem-761
like cells from U87. These cells were enriched in NSC medium. The number of spheres 762
and their diameter were measured 7 days later. (D) and (E) miR-146a promotes survival 763
of tumor-bearing mice by reducing glioma burden. (D) Histological analysis of brain 764
tumors 3 weeks post intracranial transplantation. (E) Kaplan-Meier survival curve of 765
mice transplanted with human U87 glioblastoma cells (n=6 for each group; p=0.006 by 766
Log-rank Test).767
768
FIG. 8. Knocking down miR-146a function promotes tumorigenesis. (A) Design of 769
miRNA sponge. (B) Sponge suppresses miR-146a’s function on activity of a luciferase 770
reporter that is linked to the 3’UTR of mouse Notch1 gene. The data was presented as 771
the ratio to that of miR-146a mutant–transduced cells. (C) miRNA sponge reverses miR-772
146a-mediated down-regulation of Notch1 expression. Relative protein level is indicated. 773
(D) Enhancing endogenous Notch1 expression by miRNA sponge in Ink4a/Arf-/-Pten-/-774
EGFRVIII astrocytes. (E) and (F) miRNA sponge against miR-146a promotes formation 775
of glioma stem-like cells from malignant astrocytes (n=3). (G) Knocking down miR-146a 776
function by sponge exacerbates tumor burden. The volume of subcutaneous tumors 777
was measured after microdissection (n=5; *p < 0.05, **p < 0.01, and ***p < 0.001). (H) A 778
diagram showing miR-146a-involved signaling network. 779