TOPIC REVIEW

MicroRNAs involved in the EGFR/PTEN/AKT pathwayin gliomas

Yingyi Wang • Xiefeng Wang • Junxia Zhang •

Guan Sun • Hui Luo • Chunsheng Kang •

Peiyu Pu • Tao Jiang • Ning Liu • Yongping You

Received: 3 February 2011 / Accepted: 30 July 2011 / Published online: 13 August 2011

� Springer Science+Business Media, LLC. 2011

Abstract Gliomas are the most common type of malig-

nant primary brain tumor. Despite advances in surgery,

radiation therapy, and chemotherapy, the prognosis of

patients with gliomas has not significantly improved.

MicroRNAs (miRNAs), a class of non-coding RNAs,

21–25 nucleotides long, negatively regulate the expression

of target genes by interacting with specific sites in mRNAs,

and play a critical role in the development of gliomas. The

EGFR/PTEN/AKT pathway is a promising target for anti-

glioma therapy. Recent studies have showed that regulation

of the EGFR/PTEN/AKT pathway by miRNAs plays a

major role in glioma progression, indicating a novel way to

investigate the tumorigenesis, diagnosis, and therapy of

gliomas. Here, we focus on recent findings of miRNAs

with respect to the EGFR/PTEN/AKT pathway in gliomas.

Keywords MicroRNA � Glioma � EGFR � PTEN � AKT

Introduction

Gliomas are the most common primary brain tumors and

are associated with high mortality and morbidity. The

prognosis for malignant gliomas has not significantly

improved in the last four decades. A meta-analysis of 12

randomized clinical trials showed that the overall survival

rate of high-grade gliomas was 40% 1 year after surgical

removal and only slightly higher, 46%, after combined

radiotherapy and chemotherapy [1]. Fundamental genetic

alterations in gliomas cause oncogene amplification and

over-expression and/or tumor suppressor loss leading to

evasion from the normal regulatory mechanisms of the cell.

Three key molecular events are frequently detected in

gliomas: epidermal growth factor receptor (EGFR) ampli-

fication and over-expression, phosphatase, and tensin

homolog deleted on chromosome 10 (PTEN) loss, and

AKT activation. These events contribute to glioma pro-

gression and are promising targets for anti-glioma therapy.

It is critical to gain a deeper understanding of the molecular

mechanisms underlying gliomagenesis and to identify tar-

gets for therapeutic intervention to enable the development

of more optimized and effective treatment strategies.

In recent years, we have witnessed an explosion in the

literature regarding the role of microRNAs (miRNAs) in

tumor progression. miRNAs are small non-coding single-

stranded RNAs, 21–25 nucleotides in length, that regulate

Yingyi Wang, Xiefeng Wang, and Junxia Zhang contributed equally

to this work.

Y. Wang � X. Wang � J. Zhang � H. Luo � N. Liu � Y. You (&)

Department of Neurosurgery, The First Affiliated Hospital

of Nanjing Medical University, 300, Guangzhou Road,

210029 Nanjing, People’s Republic of China

e-mail: YYPL9@njmu.edu.cn

N. Liu

e-mail: liuning0853@126.com

J. Zhang � C. Kang � P. Pu

Department of Neurosurgery, Tianjin Medical University

General Hospital, and Laboratory of Neuro-Oncology,

Tianjin Neurological Institute, 300052 Tianjin,

People’s Republic of China

G. Sun

Department of Neurosurgery, Fourth Affiliated Hospital

of Nantong University, First Hospital of Yancheng,

224006 Nantong, People’s Republic of China

T. Jiang

Department of Neurosurgery, Tiantan Hospital, Capital Medical

University, 100050 Beijing, People’s Republic of China

123

J Neurooncol (2012) 106:217–224

DOI 10.1007/s11060-011-0679-1

the expression of target genes by interacting with specific

sites on mRNAs, thereby repressing protein translation [2].

miRNAs have important regulatory functions in basic

biological processes, such as development, cellular differ-

entiation, proliferation, and apoptosis. Extensive studies

have indicated that miRNAs are dysregulated in gliomas

and function as oncogenic miRNAs or as tumor-suppressor

miRNAs. Altered miRNA regulation is involved in glioma

pathogenesis via the modulation of oncogenes and tumor

suppressors that subsequently impact on downstream sig-

naling pathways [2–4]. This article will provide a brief

overview of recent evidence concerning miRNAs and the

EGFR/PTEN/AKT pathway in glioma progression to

highlight EGFR/PTEN/AKT signal regulation by miRNAs

in gliomas.

EGFR/PTEN/AKT pathway

EGFR is one of four members of the HER family of

receptors (EGFR, nue, HER3, and HER4) [5]. These four

receptors share a similar structure: an extracellular ligand-

binding domain, an intracellular tyrosine kinase domain,

and a transmembrane anchoring segment [5]. This struc-

ture transduces signals from the cell surface to the intra-

cellular domain, triggering a complex signaling cascade.

One of the key pathways involved in this cascade is the

PI3K/AKT pathway [6]. PTEN was originally identified in

1997 as a tumor-suppressor gene that is mutated in glio-

blastoma multiforme (GBM) [7]. It has homology to

protein phosphatases and can dephosphorylate serine,

threonine, and tyrosine residues in peptide substrates [8].

PTEN acts as a phosphatase for the lipid signaling inter-

mediate phosphatidylinositol-3,4,5-trisphosphate (PIP3),

removing the phosphate from the three position of the

inositol ring [9] creating phosphatidylinositol-4,5-bis-

phosphate (PIP2), thereby directly antagonizing signaling

through the PI3K pathway. The serine–threonine protein

kinase, AKT, is one of the most important downstream

targets of PI3K. There are multiple AKT isoforms encoded

by three separate genes designated as AKT1, AKT2, and

AKT3. AKT and the serine/threonine kinase phosphoino-

sitide-dependent kinase 1 (PDK1) are recruited to the

plasma membrane by the binding of their pleckstrin

homology (PH) domains to PIP3 [10]. AKT is then

phosphorylated in the kinase domain by PDK1. Phos-

phorylation within the carboxyl terminal hydrophobic

motif of AKT by PDK2 is also required for the full acti-

vation of AKT [11]. Once activated, AKT moves to the

cytoplasm and nucleus to phosphorylate, activate, or

inhibit many downstream targets, regulating various cel-

lular functions.

EGFR amplification

Amplification of the EGFR gene occurs in *40% of pri-

mary glioblastomas, but rarely in secondary glioblastomas

[12, 13]. Consequently, EGFR over-expression is found in

about 60% of primary glioblastomas and in only 10% of

secondary glioblastomas [14]. Clinical data suggest that

EGFR amplification is related to a worse prognosis and

decreased overall survival in patients with glioblastomas

[15]. In addition, EGFR contributes to resistance to radia-

tion therapy [16] and chemotherapy [17], explaining why

some patients with glioblastomas show a particularly poor

response to these treatment modalities. Thus, EGFR rep-

resents a particularly attractive therapeutic target in glio-

mas. Small-molecule tyrosine kinase inhibitors (TKIs) are

the most clinically advanced EGFR-targeted agents for the

treatment of gliomas. Gefitinib is a molecularly targeted

agent that has been tested in gliomas. Rich et al. [18]

reported the first clinical trial with gefitinib in the treatment

of glioblastomas, and found that gefitinib was well toler-

ated and that the median progression-free survival (PFS)

was 2 months, the PFS at 6 months was 13% and the

median overall survival (OS) was 10 months, with no

observed radiographic response. Further studies of EGFR

inhibitors are still required.

PTEN loss

PTEN mutations are frequent in primary glioblastomas, but

are rare (\10%) in secondary glioblastomas [19]. PTEN

gene aberrations are addressed as a potential prognostic

marker for glioma patients. The median survival times for

cases with and without PTEN mutation were 4.4 versus

34.4 months [20]. Patients whose tumors had low PTEN

transcript levels had significantly shorter survival times

than patients with high levels of PTEN mRNA [21].

Furthermore, PTEN can sensitize glioma cells to chemo-

therapy [22] and to radiation therapy and also to CD95L-

induced apoptosis [23].

AKT activation

Gliomas show high levels of activated AKT. As mentioned

above, PI3K regulates the single transmission of AKT

phosphorylation. A high frequency of mutations in

PIK3CA, which encodes the p110alpha subunit of PI3K, is

found in glioblastoma, indicating the therapeutic value of

this pathway [24, 25]. Several studies have shown that

PI3K inhibition sensitizes glioma cells to radiation and

chemical therapy [26, 27]. Our recent data have shown that

PI3K activity is greatly increased with ascending tumor

218 J Neurooncol (2012) 106:217–224

123

grade and is positively correlated with AKT2 expression

[28]. AKT2 expression is also associated with progression

of glioma malignancy and is required for cell survival and

invasion [29]. AKT also contributes to glioma cell migra-

tion, cell cycle progression, and apoptosis inhibition via

activation of BCL2, NFjB, mTOR, and the MMP2/9

pathway [29–31].

TCGA data

The Cancer Genome Atlas (TCGA) network recently cat-

aloged recurrent genomic abnormalities in GBM [32].

Although the analyses are still ongoing, they have already

uncovered new genetic alterations and provided pre-

liminary evidence that glioblastoma can be subdivided into

several subtypes. The first subtype was labeled ‘‘classical’’.

The tumors in this group always have an amplification of

the EGFR gene (with gene rearrangements in 50% of

cases) and loss of the PTEN and CDKN2A gene loci [32].

The second subtype was called ‘‘mesenchymal’’, with fre-

quent inactivation of the NF1 (37%), TP53 (32%), and

PTEN (32%) genes [32]. The third subtype, termed ‘‘pro-

neural’’, has an expression profile reminiscent of gene

activation in neuronal development, including a high level

of expression of oligodendrocytic (PDGFRA, OLIG2,

TCF3, and NKX2-2) and proneural (SOX, DCX, DLL3,

ASCL1, and TCF4) developmental genes [32].

miRNA biogenesis and mechanisms of action

miRNAs are generated by a multistep process. The primary

miRNA transcripts (pri-miRNAs) are transcribed from the

genome by RNA polymerase II and fold into a stem-loop

structure, which is essential for the maturation process. In

association with DGCR8/Pasha, Drosha cleaves pri-miR-

NAs to generate precursor miRNAs (pre-miRNAs) [33].

Then, pre-miRNAs are transported to the cytoplasm by the

RNA GTP-dependent transporter, exportin 5. In the cyto-

plasm, pre-miRNAs are recognized by Dicer and TAR

RNA-binding protein (TRBP/TARBP2). Dicer cleaves pre-

miRNAs, generating 21–25 nucleotide mature miRNA

duplexes. The mature miRNA ultimately gets integrated

into the RNA-induced silencing complex (RISC), which is

a trimeric complex composed of Dicer, TRBP and a protein

of the Argnaute superfamily (Ago2 in humans) [34].

The classical view of miRNA function was that mature

miRNAs allow RISC to recognize the 30-untranslated

region (30-UTR) of their mRNA targets through sequence

complementarity in two main ways: (1) perfect comple-

mentarity, followed by mRNA degradation, and (2)

imperfect complementarity, blockading the translation of

mRNA. Both lead to post-transcriptional repression of

target gene expression. However, recent studies have

reported that miRNAs also target the coding regions or the

50-UTR of mRNAs to regulate their expression. Zhou et al.

[35] found that there are abundant conserved miRNA target

sites in 50-UTRs and coding sequences; miR-148 targets

the human DNMT3b protein coding region [36]. Tsai et al.

[37] confirmed that miR-346 targets the 50-UTR of recep-

tor-interacting protein 140 (RIP140) mRNA and upregu-

lates its protein expression. Through the inhibition of

mRNAs, miRNAs regulate the processes of cell develop-

ment, such as proliferation, differentiation, and apoptosis

and play a crucial in role in glioma biology.

miRNA involvement in gliomas

An increasing body of literature has identified that a group

of miRNAs are dysregulated in gliomas and are involved in

the modulation of glioma development. The first study,

reported by Ciafre et al. in 2005 [38], used microarray

technology to investigate miRNA expression profiles of

gliomas and found that some miRNAs were significantly

altered in both glioblastoma tissues and glioblastoma cell

lines; miR-221 and miR-222 were upregulated, while miR-

181a and miR-181b were downregulated . In recent years,

there has been increasing interest in exploring the biolog-

ical significance of miRNAs in glioma progression. Here,

we have summarized the miRNAs expressed in gliomas

identified from other independent expression profiling

studies (Table 1) [38–42]. Several altered miRNAs that

have been characterized with regard to their biological

function and mechanism in gliomagenesis are discussed in

the following sections.

miR-21

miR-21, one of the most commonly upregulated miRNAs,

has been identified as a key oncogenic miRNAs in gli-

omagenesis. Compared to normal brain tissue, miR-21

expression was 7- to 11-fold higher in low grade astrocy-

tomas, anaplastic astrocytomas, and glioblastomas [43].

Our study and others have confirmed over-expression of

this miRNA in gliomas using miRNA oligonucleotide

arrays, northern blot analysis, and quantitative RT-PCR,

thereby indicating a critical role of miR-21 in glioma

progression. In the first study exploring miR-21 function in

gliomas, Chan et al. [44] demonstrated that knockdown of

miR-21 in cultured glioblastoma cell lines triggered cas-

pase activation and associated apoptotic cell death. Our

recent data showed that reduction of miR-21 levels led to

caspase 9- and 3-mediated mitochondrial apoptosis in gli-

oma cells [45]. Further study in glioblastoma cells showed

J Neurooncol (2012) 106:217–224 219

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that miR-21 repressed p53-mediated apoptosis in response

to chemotherapeutic agents, such as doxorubicin, and

induced DNA damage, contributing to drug resistance [46].

Knockdown of miR-21 enhanced the chemo-sensitivity of

human glioblastoma cells to taxol by inhibiting expression

and phosphorylation of STAT3 [47]. In addition, miR-21

also promoted glioma cell migration and invasion by

activating matrix metalloproteinases (MMPs) [48]. These

studies suggest that targeting miR-21 has great therapeutic

potential for glioblastomas.

Recent studies have confirmed that miR-21 negatively

regulates some specific targets that function as tumor

suppressors to modulate glioma pathogenesis.

1. PDCD4 (Programmed cell death 4): Chen et al. [49]

showed that in glioma cells reducing levels of miR-21

increases levels of PDCD4 and over-expression of

miR-21 inhibits PDCD4-dependent apoptosis by tar-

geting the PDCD4 30-UTR.

2. PTEN: Zhou et al. [50] identified PTEN as a target of

miR-21 in gliomas. Furthermore, comparing the

response of U251 (mutant PTEN) and LN229 (wild-

type PTEN) cells to antisense miR-21 using the MTT

assay, showed similar growth and inhibition of EGFR/

AKT signaling in both lines, suggesting that miR-21

can regulate the EGFR/AKT pathway in a PTEN-

independent manner; however, this warrants further

investigation.

3. LRRFIP1: Li et al. [51] revealed that miR-21

contributed to VM-26 resistance through repression

of LRRFIP1 (an inhibitor of NF-kappaB signaling),

leading to a reduction in the cytotoxicity of chemo-

therapy drugs.

4. RECK and TIMP-3: miR-21 regulated MMP activities

by targeting MMP inhibitors RECK and TIMP-3,

thereby contributing to the glioma malignant phenotype

[48]. Other targets of miR-21 validated in other cancers

include TPM1 [52], FasL [53], and MARCKS [54].

Regulation of miR-21 expression involves upstream

factors that affect levels of mature miRNAs. Computa-

tional analysis has identified several conserved enhancer

elements in the consensus sequence upstream of the tran-

scription start site of pri-miR-21 including sites for Foxo3a,

STAT3, activator protein-1, CAAT/enhancer-binding pro-

tein-a, and p53. Direct transcriptional regulation of miR-21

by Foxo3a, STAT3, and activator protein-1 has been

reported. In glioma cells, STAT3 negatively regulated

miR-21 transcription in response to IFN-b treatment.

However, the role of STAT3 activation is debatable

because its over-activation has been reported to be onco-

genic in glioma cell lines [55, 56]. Loffler et al. [57]

showed that IL-6-dependent STAT3 activated the tran-

scription of miR-21 in multiple myeloma cells. This dis-

crepancy may arise from the difference in cytokine

stimulus and cell type. Thus, the functional identification of

regulatory genes, which are responsible for controlling the

spatial and temporal expression of specific miRNAs is in

its early stages.

miR-221/222

miR-221 and miR-222, located in a cluster on chromosome

Xp11.3, are over-expressed in glioblastomas [58]. Our and

other studies showed that single suppression of miR-221 or

miR-222 in vivo induced lower glioma growth inhibition

than co-suppression of miR-221/222. miR-221/222 share

the same ‘seed’ sequence, short regions at their 50 ends

through, which they bind their target sites in the 30-UTRs

Table 1 Important miRNAs in gliomas

Upregulation Downregulation

miRNA Chrom. miRNA Chrom. miRNA Chrom. miRNA Chrom. miRNA Chrom.

miR-123 1 miR-210 11 miR-34a 1 miR-29b 7 miR-203 14

miR-10b 2 miR-16 13 miR-101 1 miR-129 7 miR-299 14

miR-26a 3 miR-21 17 miR-137 1 miR-124 8 miR-323 14

miR-425 3 miR-451 17 miR-181a 1 miR-7 9 miR-190 15

miR-9-2 5 miR-516-3p 19 miR-181b 1 miR-31 9 miR-328 16

miR-25 7 miR-519d 19 miR-128-1 2 miR-511-1 10 miR-132 17

miR-182 7 miR-125b-2 21 miR-149 2 miR-139 11 miR-133a 18

miR-383 8 miR-155 21 miR-153 2 miR-326 11 miR-187 18

miR-486 8 miR-185 22 miR-128-2 3 miR-483 11 miR-181c 19

miR-107 10 miR-221 X miR-138 3 miR-17-92 13 miR-330 19

miR-125b-1 11 miR-222 X miR-218 4 miR-154 14 miR-185 22

miR-130a 11 miR-133b 6

220 J Neurooncol (2012) 106:217–224

123

of mRNAs; therefore, they have the same targets and

synergistically regulate the same pathway. According to

bioinformatic analysis, using three different target predic-

tion programs (PicTar, TargetScan, and miRanda) and

Pathway Studio soft, about 70 common target genes of

miR-221/222 were identified and 16 of them represented

direct or indirect interaction with AKT [59]. Several genes

have been confirmed as targets, such as p27 [58], p57 [60],

kit [61], and PTEN [62]. Recently, we have shown that

miR-221/222 inhibited cell apoptosis by targeting the

proapoptotic gene, PUMA, in human glioma cells, indi-

cating that PUMA is a novel target of miR-221/222 [63]. In

addition, over-expression of miR-221/222 cooperated to

enhance the malignant phenotype of U251 and C6 glioma

cells by activating the AKT pathway. These findings sug-

gest that the modulation of miR-221/222 in gliomas could

be used as a therapeutic strategy.

miR-7

miR-7 is also a tumor suppressor and has been shown to be

downregulated in glioma tissue by Kefas et al. [64]. miR-7

expression is decreased in glioblastomas through reduced

processing of precursor miR-7 [64]. Furthermore, miR-7

reduced the viability and invasiveness of glioblastoma cells

by directly targeting EGFR. miR-7 also suppressed AKT

pathway activation by repressing IRS-1. IRS-2 proved to

be another direct target of miR-7 independent of its EGFR

inhibition.

miR-451

miR-451 is downregulated in gliomas and is also involved

in the oncogenesis of gliomas. In human glioblastoma

cells, miR-451 could inhibit tumor growth of glioblastoma

stem cells [65]. Godlewski et al. [66] found that miR-451 is

an inhibitor of the LKB1/AMPK pathway and that it reg-

ulates LKB1 activity through direct targeting of CAB39, a

component of the active LKB1 complex. miR-451 also

plays a key role in the response of glioma cells to glucose

deprivation by regulating the balance of glioma cell pro-

liferation, migration, and survival in response to metabolic

alterations. Recently, our research showed that miR-451

could inhibit human glioma cell proliferation, invasion, and

apoptosis through the AKT pathway and might be a tumor-

suppressor factor in human gliomas [67]; however, the

direct targets of miR-451 still need to be explored.

miR-26a

miR-26a is another oncogenic miRNA expressed in glio-

mas that targets critical cancer signaling pathways. Huse

et al. [68] identified miR-26a over-expression in a subset of

high-grade gliomas. It has been shown that over-expression

of miR-26a in gliomas was primarily a consequence of

amplification at the miR-26a-2 locus, a genomic event

strongly associated with monoallelic PTEN loss. Further-

more, miR-26a reduced PTEN levels and facilitated glioma

formation in a well-characterized murine model system,

and functionally substituted for loss of heterozygosity at

the PTEN locus. AKT was also activated due to an

upstream signal of PTEN.

miR-128

miR-128 is enriched in brain but downregulated in glioma

tissues and cell lines. Zhang et al. [69] demonstrated that

miR-128 inhibited the proliferation of glioma cells through

negatively regulating one of its targets, E2F3a. Moreover,

knocking down E2F3a had a similar effect as over-

expression of miR-128, and over-expression of E2F3a

could partly rescue the proliferation inhibition caused by

miR-128. In addition, Godlewski et al. [41] showed that

miR-128 caused a striking decrease in the expression of the

oncogene, Bmi-1, by direct regulation of the Bmi-1 mRNA

30-UTR, with AKT phosphorylation, and upregulation of

p21 levels.

miR-124 and miR-137

Silber et al. [70] reported that the expression levels of

miRNA-124 and miRNA-137 were significantly decreased

in anaplastic astrocytomas and in glioblastoma multiforme.

Transfection of miRNA-124 or miRNA-137 into glioma

cells induced G1 cell cycle arrest by inhibiting CDK6

expression.

Other miRNAs

Our data showed that miR-181a and miR-181b were

downregulated in human glioma tissues and cell lines

(U87, TJ950, and U251), and that they functioned as

tumor suppressors to exert a significant effect on glioma

cell growth, invasion, and apoptosis [71]. Gal et al.

demonstrated that transfection of glioblastoma cells by

miR-451 can inhibit cell growth [65]. miR-10b is over-

expressed in malignant glioma and is associated with

tumor invasion factors, uPAR, and RhoC [72]. miR-10a is

also upregulated in glioblastomas [38, 70]. However, the

function of these altered miRNAs remains unknown, and

the expression levels of several miRNAs in gliomas

should be validated on larger, more representative cohorts

of glioma patients.

J Neurooncol (2012) 106:217–224 221

123

Concluding remarks

In conclusion, since the discovery of miRNAs as a new

class of gene regulators, numerous studies have correlated

glioma with altered expression levels and functions of

particular miRNAs. Interestingly and importantly, further

analyses have identified that key signaling components of

the EGFR/PTEN/AKT pathway are direct targets or

downstream molecules of these specific miRNAs, indicat-

ing that these miRNAs participate in regulating the EGFR/

PTEN/AKT pathway, one of the most important signaling

pathways involved in gliomas (Fig. 1). However, molecu-

lar regulation of miRNAs in glioma, including upstream

and downstream miRNA regulators, and the miRNA sig-

naling network, is still unclear and a massive wealth of

information is waiting to be discovered. Overall, we

believe that these miRNAs demonstrate a unique potential

to identify a novel aspect of glioma progression and,

therefore, a novel therapeutic approach, driving the field

closer to the ultimate goal of improving patient survival

rates and quality of life.

Acknowledgments This work was supported by the China Natural

Science Foundation (30872657, 30971136, and 81072078), Natural

Science Foundation of Jiangsu Province (2008475 and 2010580),

Scientific Program of Ministry of Health (W2011BX009), Program

for Development of Innovative Research Team in the First Affiliated

Hospital of NJMU, and A Project Funded by the Priority Academic

Program Development of Jiangsu Higher Education Institutions.

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