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Cell Transplantation, Vol. 22, pp. 741–753, 2013 0963-6897/13 $90.00 + .00Printed in the USA. All rights reserved. DOI: http://dx.doi.org/10.3727/096368912X655190Copyright 2013 Cognizant Comm. Corp. E-ISSN 1555-3892 www.cognizantcommunication.com

Online prepub date: November 1, 2012.1These authors contributed equally to this study.Address correspondence to Shinn-Zong Lin, M.D., Ph.D., Center for Neuropsychiatry and Department of Neurosurgery, China Medical University Hospital, Yuh-Der Road, Taichung 40447, Taiwan, ROC. E-mail: shinnzong@yahoo.com.tw or Shi-Hwa Chiou, M.D., Ph.D., Institute of Pharmacology, School of Medicine, National Yang-Ming University, 155, Sec 2, Linong Street, Taipei, 112, Taiwan, ROC. Tel: +886-2-28757394; E-mail: shchiou@vghtpe.gov.tw

Review

Deregulated MicroRNAs Identified in Isolated Glioblastoma Stem Cells: An Overview

Pei-Ming Chu,*†1 Hsin-I Ma,‡1 Li-Hsin Chen,§1 Ming-Teh Chen,¶# Pin-I Huang,¶** Shinn-Zong Lin,† and Shih-Hwa Chiou§¶**

*Department of Anatomy and Cell Biology, College of Medicine, China Medical University, Taichung, Taiwan, ROC†Center for Neuropsychiatry and Department of Neurosurgery, China Medical University Hospital, Taichung, Taiwan, ROC

‡Department of Neurological Surgery, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan, ROC§Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan, ROC

¶School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC#Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan, ROC**Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, ROC

Glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor, is extremely resistant to current treatment paradigms and has a high rate of tumor recurrence. Recent progress in the field of tumor-initiating cells suggests that GBM stem cells (GBMSCs) may be responsible for tumor progression, resistance to treat-ment, and tumor relapse. Therefore, understanding the biologically significant pathways involved in modulating GBMSC-specific characteristics offers great promise for development of novel therapeutics, which may improve therapeutic efficacy and overcome present drug resistance. In addition, targeting deregulated microRNA (miRNA) has arisen as a new therapeutic strategy in treating malignant gliomas. In GBMSCs, miRNAs regulate a wide vari-ety of tumorigenic processes including cellular proliferation, stemness maintenance, migration/invasion, apoptosis, and tumorigenicity. Nevertheless, the latest progress with GBMSCs and subsequent miRNA profiling is limited by the identification and isolation of GBMSCs. In this review, we thus summarize current markers and known features for isolation as well as the aberrant miRNAs that have been identified in GBM and GBMSCs.

Key words: Glioblastoma; GBM stem cells (GBMSCs); microRNA (miRNA); Therapeutic strategy

binding transcription factor 4 (Oct 4), and Musashi (41). These fundamental characteristics make the CSCs the prime candidate for tumor maintenance and reoccurrence. Up to now, however, it is unclear whether the brain tumor CSCs are derived from adult stem cells or if mutations in a progenitor or even a terminally differentiated cell lead to a cancer cell with stem cell characteristics.

Glioblastoma multiforme (GBM, World Health Organi- zation grade IV gliomas) is one of the most devastat-ing brain tumors in adults and has a very poor prognosis (104). GBM display considerable heterogeneity and high infiltration into the parenchyma, making it extremely

INTRODUCTION

A rare subpopulation of cells with chemo- and/or radioresistant properties in each malignancy has a greater potential for tumor initiation and displays accelerated regrowth after a sublethal treatment. This subpopulation was termed cancer stem-like cells (CSCs) due to isolated cells possessing the ability to self-renew, differentiate into multiple lineages, and initiate tumors that mimic the parent tumor (1,13,30,41,56,70,77,88,89,99,100). CSCs in brain tumors have been shown to express various spe-cific neural stem/progenitor cell (NSPC) proteins such as nestin, sex-determining region Y box 2 (Sox2), octamer-

742 CHU ET AL.

difficult to remove by traditional surgical resection, radiotherapy, and chemotherapy, which consequently contribute to the development of tumor recurrences or progressions. These cancer therapies, while killing the majority of tumor cells, ultimately fail in GBM treatment because they do not remove GBM stem cells (GBMSCs), which survive to repopulate new tumors. Therefore, all these characteristics indicate that GBMSCs are critical therapeutic targets and that the understanding of the bio-logical properties and roles of CSCs may provide new insights into the factors that drive tumor initiation and progression and may help to develop novel methods to overcome drug resistance and improve therapeutic effi-cacy in malignant gliomas.

In this review, we will summarize current data describ-ing recent progress as well as persistent problems that arise in studying GBMSCs. A critical aspect of GBMSC research is the ability to propagate cells that maintain the properties of both the stem cells and the original tumor. We will discuss the advantages and disadvantages for CSC research of serum cultures, serum-free neurosphere cultures, and the recently described serum-free adherent cultures. We will also examine the current methods used to identify and isolate GBM CSCs using surface markers and side populations. A current research focus is whether GBM CSCs are truly the population resistant to therapy. If CSCs evade treatment, an important question is whether future therapeutics can be directed against GBMSCs. Promising data have revealed that inhibition of stem cell pathways in brain tumors, such as Notch and Hedgehog signaling, may present a novel means to directly target the resistant CSC population. In addition, the discovery of microRNAs (miRNAs) opened up a new era of molecular and cellular biology. Aberrant miRNA expressions were found to be associated with a variety of physiological and pathological processes. Application of miRNA modula-tion on cancer thereby provides new insight into brain

tumor therapy, especially the most aggressive GBM. We therefore will subsequently discuss recent progress in the miRNAs identified in GBMSC. The possibility and strat-egy based on miRNA for future therapy and the limita-tions will also be included.

THE UTILITY AND LIMITATIONS OF ENRICHMENT MARKERS

FOR GBMSC ISOLATION

Due to the heterogeneous mix of neoplastic and non-neoplastic cells that comprise both native and recruited cells, it is difficult to purify CSCs, which significantly vary from tumor to tumor. Until now, CSCs have been consis-tently characterized only from a limited number of brain tumor types (30,41,46,99,100,116). Research on specific GBMSCs has developed using the acknowledged NSC markers, such as cluster of differentiation 133 (CD133) (98–100), stage-specific embryonic antigen 1 (SSEA1; CD15) (101), and L1 cell adhesion molecule (L1CAM; CD171) (3). Al though there are many criticisms of the CSC hypothesis, it is still attracting more and more interest. In addition, side population (SP), autofluorescence emission, and exploitation of in vitro culture conditions for NSCs have also proved useful for the enrichment of GBMSCs. Currently used approaches are summarized in Table 1.

NEUROSPHERE ASSAY (FLOATING SPHERES OF CELLS)

Studies using culture media containing growth factors similar to that used to culture NSCs (87) showed that CSCs are capable of proliferating and generating large clusters of cells, termed tumorospheres (30,31,41,46,99,100,122). When dissociated, these cultures are capable of serial plating, in which a small percentage of the cells can form secondary and tertiary tumorospheres. These spheroid approaches are carried out in selective serum-free media, in which stem-like cells are able to continually divide and

Table 1. The Isolation Methods for Brain Tumor Stem Cells

Selection Marker/Method Assessment Function Ref.

CD133 Magnetic sorting or FACS Unknown (4,11,41,62,99,100,107)L1CAM (CD171) FACS Cell motility (3)SSEA1 (CD15) FACS Cell adhesion and axon guidance (2,83,101,114)A2B5 FACS for A2B5 and CD133 expression

(A2B5+/CD133– cells)Unknown (71,108)

Integrin a6 FACS for a6 integrin expression alone or in combination with CD133

Cell adhesion (57)

ALDH1 activity FACS for ALDH1 activity Oxidation of intracellular aldehydes

(81)

Side population (SP) FACS based on exclusion of Hoechst 33342 N/A (12,20,39,40,55,74,102)Autofluorescence Intrinsic autofluorescence and morphology N/A (22)Sphere formation Serum-free medium supplemented with

EGF, FGFN/A (30,31,37,41,46,99,122)

N/A, not applicable.

TARGETING microRNAs IN GBM STEM CELL 743

form multipotent spheres, while the more differentiated cells incapable of self-renewal and multipotency die off with serial passages (15,85,110). Under these conditions, neurospheres demonstrate their high capacity for prolif-eration as well as self-renewal and facilitate studying the other properties of CSCs: multilineage differentiation into neuronal and glial lineages and tumorigenic capacity after transplantation in vivo (86,87). Broadley et al. showed that sphere formation in primary GBM cells and GBM cell lines could enrich for a stem-like phenotype of enhanced self-renewal gene expression in vitro and increased tumor initiation in vivo (14). Besides, GBMSCs in serum-free cultures have closely mirrored and retained the same phe-notype and genotype of the patient’s primary tumors more accurately than serum cultures (58). Except for GBM, other types of brain tumors can be cultured for short periods in neurosphere conditions, such as ependymoma, but long-term cell lines remain difficult to establish (30,41,107). Moreover, benign brain tumors show a low clonogenic sphere-forming ability from plating fresh cells (28).

Tumorospheres enriched from brain tumor stem cells (BTSCs) could be reformed even in the persistent differ-entiation condition if resubjected to defined serum-free medium (122,124). The rate of tumorosphere formation is also enhanced at higher passages. However, there still are several limitations to using the spheroid assay to identify stem cells. Among them, neurospheres are highly motile structures prone to aggregate and fuse with one another when plated at higher densities (97) that likely contain mixtures of small numbers of stem cells, larger numbers of immature progenitors, and small numbers of differenti-ated cells. Up to now, the conditions for sustaining long-term growth of CSCs in vitro have not been optimized. Spontaneous differentiation and cell death are the biggest problems with tumorospheres. Recently, a new methodol-ogy for expanding GBM culture with a high percentage of CSCs, grown as adherent cultures on laminin-coated plates under the same serum-free conditions used for neu-rosphere formation, has been proposed (76). These adher-ent GBMSCs were less heterogeneous than neurosphere cultures, with most of the cells expressing stem cell- associated genes, and they formed tumors when only 100 cells were intracranially injected into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice.

MARKER-SPECIFIC IDENTIFICATION

Nestin is expressed in NSCs and was the first widely used NSC marker (59). Unfortunately, nestin is an inter-mediate filament protein, which makes it unhelpful as a means for sorting the CSC population. Therefore, cell surface markers are commonly used to isolate and char-acterize stem cells. An excellent set of surface markers found in leukemia have been used as the basis for well-accepted models of CSC research. Several identifiable

markers, explored for NSC enrichment, have been stud-ied in the field of GBMSC biology. Presently, there are currently no universally accepted markers for enrich-ment of a pure population of GBMSCs. Here, we will review the most commonly used markers for GBMSC collection: CD133, L1CAM, CD15/SSEA-1, A2B5, and more recently integrin α6.

CD133

CD133 (also known as Prominin-1), a pentaspan mem-brane protein with unknown function, is a surface marker commonly found in stem cells and progenitor cells in the hematopoietic and central nervous systems (CNS) (42,109,117). By fluorescence activated cell sorting (FACS) or immunomagnetic beads, CD133 was prospec-tively used to isolate population of CSCs with enhanced stem cell phenotypes from specimens of brain tumors (4,62,99,100,107). As compared with CD133-negative (CD133-), CD133-positive (CD133+) cells from GBMs had higher colony formation efficiency, multilineage differentiation capacity, and increased serially trans-plantable xenograft tumors in NOD-SCID mouse brain (99,100). CD133+ cells also express significantly higher levels of stem cell genes, such as nestin and musashi. Surprisingly, injection of only 100 CD133+ cells results in a tumor mass capable of recapturing the features of the original tumors regarding morphology and lineage marker expression and propagating both CD133+ and CD133- cells, while 105 CD133- injected cells do not. In addition, CD133+ cells were more likely to be G

0/G

1

phase cells, and more resistant to hypoxia, irradiations and some chemotherapeutics than CD133- cells (80). Moreover, increased frequencies of CD133-positive cells were demonstrated to increase with tumor grade (84) and significantly correlated with the poor prognosis and the decreased survival of the patient (27,123).

Reports that up to 40% of freshly dissociated GBM specimens do not express CD133 highlight the limits of using CD133 to select for GBMSCs (9,32,101). Nishide et al. generated a glioma-initiating mouse cell line whose CD133+ cells could be eliminated conditionally by a Cre-inducible diphtheria toxin fragment A (DTA) gene on the CD133 locus. After induction of the DTA gene, the cell line depleted of CD133+ cells maintained the capacity to form neurospheres in vitro and drove tumor progression in vivo (69). Hence, in this mouse model, gliomagen-esis was independent of CD133 expression. Otherwise, the CD133-low GBMs showed more aggressive mor-phologies as determined by magnetic resonance imaging (MRI) scans and gene expression profiles characteristic of mesenchymal or proliferative subtypes, whereas the CD133-high GBMs showed features of well-demarcated tumors and gene expressions typical of a proneuro-nal subtype (50). Joo et al. indicated that CD133- cells

744 CHU ET AL.

showed more proliferative and angiogenic features com-pared to CD133+ cells (50). The relevance of CD133 as a reliable marker in defining the GBMSC population was doubted, since growing studies suggest that CD133- cells also have the ability to form tumors when transplanted into immunocompromised mice (9,112) and can also give rise to CD133+ cells. This suggestion that only CD133+ cells are capable of self-renewal and recapitulation of the parental tumor must be viewed with caution. CD133 does not consistently distinguish between tumorigenic and nontumorigenic glioma cells, and both CD133+ and CD133- subtypes are able to form tumors upon trans-plantation into immunocompromised mice. By the same token, long-term cultured cells, enriched with either CD133+ or CD133- tumor cells, were equally resistant to temozolomide (75).

Recent research by Griguer and coworkers indicated that CD133 expression might be regulated by environ-mental conditions (hypoxia) and mitochondrial dysfunction (35). The authors demonstrated that CD133 expression most likely reflects the bioenergetics stress of the cells rather than their stem-like characteristics. Beier et al. and others provided first evidence that CD133+ CSCs maintain only a subset of primary glioblastomas. The remainder stems from unknown CD133- tumor cells with apparent stem cell-like properties, but distinct molecular profiles and growth characteristics in vitro and in vivo (9,63). The discrepancies in this research could be explained, in part, by the poor reliability of surface markers detecting GBMSC populations. Future studies may refine the util-ity of CSC surface markers and other techniques for the study of BTSCs through the identification of improved markers and/or additional new methodologies.

L1CAM

The neural cell adhesion/recognition protein L1 (L1CAM; CD171) was originally identified as a cell adhe-sion molecule in the nervous system during CNS devel-opment (65,90). It plays critical roles in regulating neural cell adhesion, survival, migration, and invasion. Ectopic expression of L1CAM in malignant gliomas has been correlated with increased tumor progression and metas-tasis (82). Bao et al. indicated that L1CAM as a cell sur-face molecule is preferentially overexpressed in GBMSCs and is critical in maintaining the cell survival and tumori-genic potential of GBMSCs (3). Interestingly, L1CAM knockdown resulted in effective abrogation of tumor sphere formation in vitro. Targeting L1CAM remark-ably suppressed the tumor growth and increased the survival of mice bearing intracranial xenografts. Most recently, Cheng et al. demonstrated that L1CAM regu-lates the DNA damage checkpoint response and enhances radio resistance of GBMSCs through nuclear transloca-tion of the L1CAM intracellular domain (19). Targeting

L1CAM not only attenuated DNA damage checkpoint activation and repair but also sensitized GBMSCs to radi-ation. Accordingly L1CAM overexpression in GBMSCs may be worth exploring as a CSC marker.

SSEA-1

The stage-specific embryonic antigen-1 (SSEA-1, also known as CD15 or Lewis X antigen) is a cell sur-face carbohydrate that is highly expressed in multipotent NSCs originating from embryonic stem cells (ESCs) and the adult subventricular zone (SVZ) (5,78). It has been proposed as an enrichment marker for CSCs in GBMs (66,101) and medulloblastoma (83,114), although the function of SSEA-1 in normal stem cells and CSCs remains poorly understood. During the analysis of pri-mary cells from fresh GBM specimens, 96% of these CSCs were found to express SSEA-1 as opposed to only 54% that expressed CD133 (101). The SSEA-1+ cells established from glioblastomas have increased expression of stem-associated genes and are capable of self-renewal and multilineage differentiation. The SSEA-1+ cells that are devoid of CD133+ are 100-fold more tumorigenic in a mouse xenograft model in comparison to SSEA-1- cell types, which displayed limited tumor formation. Like CD133+ GBMSCs, SSEA-1+ cells could generate both SSEA-1+ and SSEA-1- cells in vitro and in vivo (101), suggesting a hierarchical lineage and the capacity to cre-ate tumor heterogeneity. Most importantly, SSEA-1+ cells sorted from these intracranial tumors could initiate and recapitulate original tumors with the same characteristics. Taken together, these results suggest that SSEA-1 is a use-ful enrichment marker for CD133- GBMSCs, but precise characterization of these subpopulations and any poten-tial functional properties warrant further investigation.

A2B5

Adult gliomas mostly occur in the subcortical white matter, and several studies show that human gliomas express A2B5 (neural cell surface antigen), a commonly used glial progenitor cell marker (23,54). A2B5 ganglio-side has been reported to be expressed in human BTSCs (121). In Ogden’s study, A2B5-positive cells were present in every glioma sample tested, whereas CD133-expressing cells were less abundant and less consistent in several GBMs (71). Both A2B5+/CD133+ and A2B5+/CD133- cell populations were capable of generating tumors in a series of xenograft experiments in nude rats (71,108), whereas A2B5-/CD133- cells did not. These results suggest that the A2B5 antigen is more reliable than CD133 as a poten-tial GBMSC marker regardless of CD133 expression. The authors also demonstrated that A2B5 reactivity recognizes glioma cells with increased tumorigenicity compared with cells without A2B5 reactivity. Moreover, Xia et al. revealed that the A2B5+ lineage has a significantly higher

TARGETING microRNAs IN GBM STEM CELL 745

recurrence rate than the tumors of the A2B5- lineage in astrocytomas (118). Taken together, the above findings clearly indicate that these A2B5+ cells are BTSCs. Further work will be required to fully understand the role of A2B5 as a GBMSC marker.

INTEGRIN a6

Integrin a6 subunit associates with b1 or b4 subunits to form functional heterodimers that selectively bind lami-nins. Integrin a6b1 plays an important role in anchoring NSCs to the niche vasculature and in regulating the SVZ lineage proliferation in vivo (93). Besides, adult NSCs highly express the laminin receptor a6b1 integrin, which disappeared as the cells differentiated (38). Gingras and colleagues demonstrated that a6b1 integrin was strongly expressed in biopsy samples from GBM patients, whereas it was only slightly expressed in normal brain (33). In glioma cells, stable expression of a6b1 integrin leads to increased tumor growth and progression in immunocom-promised mice receiving either subcutaneous or intracra-nial inoculations (26). Moreover, the authors demonstrated that a6b1 integrin is involved in GBM cell migration as well as invasion and acted on the balance between pro-liferation and apoptosis. Additionally, integrin a6+ cells were localized in close proximity to the perivascular microenvironment, a region enriched in GBMSCs, and they often coexpressed the stem cell markers CD133 and nestin. Based on integrin a6 selection alone or in com-bination with CD133 to enrich GBMSCs, Lathia et al. showed effective integrin a6 expression isolation for GBMSCs in tumors that lack CD133 expression and that combining CD133 and integrin a6 expression results in a higher enrichment of GBMSCs than CD133 expres-sion alone (24,57). Altogether, these results demonstrate that integrin a6 is a modern GBMSC enrichment marker and also suggests that integrin a6 expression can be capable of isolating GBMSCs in tumors with low or little CD133 expression.

ALDEHYDE DEHYDROGENASE 1Aldehyde dehydrogenase 1 (ALDH1) is a detoxify-

ing enzyme responsible for the oxidation of intracellular aldehydes and regulates early differentiation of stem cells through its role in oxidizing retinol to retinoic acid (21). A fluorescent ALDH substrate, Aldefluor, is a cell viability dye, which is therefore more reliable than surface marker selection-based methods that do not discriminate between viable and nonviable cells (25,92). Numerous studies indicated that ALDH activity may provide a novel marker for both normal and malignant stem and progenitor cells (21,42,44,48,105). Corti et al. showed that the ALDH1+ cells isolated from primordial NSCs are capable of self-renewal, and able to differentiate into both neurons and glial cells (25). These ALDH1+ cells express high levels

of stem cell markers such as nestin and musashi. After transplantation into the neonatal brain, ALDH1+ cells displayed high engraftment and neurogenic potential and fully integrated into the host architecture.

To further characterize GBMSCs, Rasper and col-leagues examined well-established glioma cell lines and primary GBM cells for ALDH1 expression to determine if it correlated with stem cell capacity (81). Using the neuro- sphere assay as a functional method to identify BTSCs, the authors demonstrated that high protein levels of ALDH1 facilitate tumorosphere formation in established GBM cells. Besides, the inhibition of ALDH1 leads to a prominent decrease in tumorosphere formation as well as clonogenic capacity, and cells express high levels of b-III-tubulin suggesting a higher grade of neuronal dif-ferentiation compared with untreated controls. The results claim that high ALDH1 activity in GBM cells is involved in the maintenance of an undifferentiated stem cell-like phenotype.

SIDE POPULATION

ATP-binding cassette (ABC) transporters, includ-ing those encoded by the multidrug-resistant (MDR) gene 1, the MDR protein (MRP), and the breast cancer- resistant protein 1 (BCRP1/ ABCG2), play a pivotal role in drug resistance by using ATP hydrolysis to expel vari-ous endogenous compounds and xenobiotics from the cell (106). A Hoechst-based technique, via dual-wave-length flow cytometry sorting to select cells that exhibit low red and blue fluorescence (16), can be used to assess the frequency of side population (SP). With the renais-sance of the CSC hypothesis, numerous studies have demonstrated the presence of stem cell-enriched SPs in glioma cells, such as rat C6 (55), human T98G, U87-MG, U373-MG (20,74), and platelet-derived growth factor (PDGF)-induced (12) as well as transgenic glioma mouse models (40). In the C6 glioma cells, for example, only SP cells endowed with the characteristics of GBMSCs (mul-tipotentiality and self-renewal) were observed, compared with non-SP cells from the same counterparts. Qiang et al. found that the ABCG2 transporter was highly expressed in CD133+ cells, which was thought to contribute to the SP phenotype of CSCs (80). Importantly, the expression level of ABC transporters is positively correlated with the pathological grading of glioma (49), implying that it may act as a predictor of patient outcome.

Hoechst 33342 is harmful to the clonogenicity and pro-liferation of single C6 cells (126). Therefore, the non-SP cells may be deprived of their stem cell characteristics in the process of Hoechst 33342 staining and FACS. In addition to being a highly variable technique, SP cells are heterogeneous and express both endothelial and stem cell markers. Moreover, Broadley et al. recently showed that purified SP cells neither have a self-renewing stem-like

746 CHU ET AL.

phenotype nor did they initiate tumors more efficiently. Given that GBMSC spheres lack SP cells regardless of whether they were derived from immortalized cell lines or from tumor-derived primary cells, tumorige-nicity is therefore unlikely to be solely dependent on their presence.

MARKER-INDEPENDENT APPROACH OR MARKER-INDEPENDENT ISOLATION

PROCEDURES

Autofluorescence Emission

Based on morphological aspects and autofluorescence emission in FACS, Clement and coworkers reported a marker-independent method to enrich and identify a sub-population of glioma cells with the characteristics of GBMSCs (22). By avoiding the usage of surface mark-ers and relying on universal phenotypic properties, the approach offers a simple avenue to enrich GBMSCs with high nuclear: cytoplasmic ratio. These autofluorescent cells (FL1+) are capable of self-renewal over serial pas-sages in clonogenic assays and possess tumor-propagating capacity in mice even if only 103 glioma cells were injec- ted. On the contrary, non-autofluorescent cells (FL10) are not tumor inducing cells (TICs) since they apparently do not have the properties of GBMSCs.

MicroRNA IN GBM AND GBMSCs

MicroRNAs (miRNA) are small noncoding regulatory RNA molecules with a size of about 18–25 nucleotides (nts), with a profound impact on a wide range of biologi-cal processes. miRNA modulates protein expression at the posttranslational level primarily through near-perfect or partial complementarity against the coding region or 3¢ untranslated region (3¢UTR) of target mRNA and lead to translation repression and/or degradation thus regulating gene expression. miRNAs are frequently deregulated in a wide variety of human cancers where they play impor-tant roles in regulating the expression of oncogenes or tumor suppressors.

In brain tumors, miRNAs are involved in many aspects of brain tumor progression including the malignant pro-gression of gliomas. Several miRNAs such as miR-7, miR-21, miR-26a, miR-124, miR-137, miR-184, and miR-221/222 have been implicated in GBM pathogen-esis (7,17,18,45,53,60,64). Furthermore, these miRNAs have been reported to be involved in the controlling sig-naling pathways of GBM. For example, several studies identified mRNA targets of miR-21 among critical com-ponents of the epidermal growth factor receptor (EGFR) signaling pathway (94). Another study showed miR-451 affects glioblastoma cells via regulation of the phosphati-dylinositol 3-kinase/protein kinase B (AKT) [PI3K/AKT] signaling pathway (68). Raf-1, another member of the EGFR signaling pathway, is reported to be a direct target

of miR-7 in cancer cells (115). Papagiannakopoulos et al. recently reported that p53, transforming growth factor (TGF)-b and mitochondrial apoptotic networks are de-repressed in response to miR-21 knockdown (73). Using miRNA microarray analysis of glioma stem cells trans-fected with Notch-1 small interfering RNA (siRNA), Kefas et al. reported that miR-326 was one of the miRNAs sig-nificantly increased in Notch-1 knockdown cells when compared to control transfected cells, suggesting miR-326 is suppressed by Notch activity (52).

Expression profiling of miRNA has also been per-formed in cancer stem cells originated from various types of cancer, including GBM. Recent studies have revealed that miRNAs play vital roles in oncogenesis by regulat-ing a variety of various cellular functions, such as cel-lular proliferation, apoptosis, cell cycle progression, and migration/invasion. Accordingly, miRNA-based molecular cancer therapy should provide a promising tool to eliminate the tumorigenetic ability of cancer cells. From the aspect of cancer stem cell theory, miRNAs are involved in the regulation of the self-renewal capabili-ties, stemness maintenance, and stress resistance charac-teristics of GBMSCs and therefore are potential targets for anticancer therapeutics. Delivering antagomirs can correct oncogenic miRNA dysregulated in GBM CSCs and miRNA mimics are able to restore miRNAs acting as tumor suppressors in cancer stem cells. To date, some miRNAs have been found to be up- or downregulated in GBM and GBMSCs. Here, recent progress of aberrant miRNAs observed in GBMSCs will be summarized.

A common strategy for researchers who intend to search for certain miRNA dysregulations in GBMSCs is to isolate a subpopulation, the so-called CSCs (or tumor initi-ated cells), within GBM samples or cell lines and compare their miRNA profile with non-CSC populations. Based on this approach, less than a dozen miRNAs were identified (Table 2). Except for the opposite results of miR-125b, there are reports suggesting up- and downregulation of miRNAs (94,111). Among all of the miRNAs so far dis-covered, only two miRNAs, miR-9 and miR-9*, were found to be upregulated in GBMSCs. These two miRNAs inhibit differentiation and maintain the stemness of GBMSCs by targeting calmodulin binding transcription activator 1 (CAMTA1), a transcription factor that induces the expression of the antiproliferative cardiac hormone natriuretic peptide A (NPPA) (91). Schraivogel et al. con-firmed the significance of CAMTA1, whose upregulation inhibits neurosphere formation, CD133-positive percent-ages, and tumor growth (91). Nevertheless, more experi-ments need to be conducted to evaluate whether inhibitors of miR-9/9* could inhibit glioma progression in vivo.

MiRNAs downregulated in GBMSCs are potential tumor suppressors. Overexpression of miR-124 or knock-down of its target SNAI2 (Slug), a transcription factor

TARGETING microRNAs IN GBM STEM CELL 747

belonging to the Snail family, inhibited tumorigenicity and invasion in vivo (119). SNAI2 has been implicated in epithelial-mesenchymal transition (EMT) and tumor metastasis (31–33). Previous reports revealed that breast cancer cells overexpressed SNAI2 acquired CD44+/CD24- phenotypes with enhanced mammosphere form-ing ability (10). Despite the relationship between EMT and stemness remaining unclear, the inhibitory effect of miR-124 on gliomas may be impaired by its downregula-tion and thereby leading to the elevated invasive growth.

Several signaling pathways important for GBMSC self-renewal have been addressed, including Notch, Wnt, and Hedgehog (120). Three miRNAs, miR-34a, miR-146a, and miR-326, listed in Table 2, have one or more targets involved in Notch pathway. The Notch signaling pathway is highly conserved from Drosophila to mam-mals and is involved in brain development and mainte-nance of neural progenitor cells (8). While it is still not clear whether Notch signaling pathway plays a role in glioma tumorigenesis, some studies have revealed its functional significance in glioma (51,79) and glioma stem cells (43,113,125). Elevated expression of any of three miRNAs reduced glioma tumor size in vivo (52,67,95). Nevertheless, the xenografts were established

from transplantation of parental mixture, such as U87 glioma cell line, instead of the CSC subpopulation. It is not known whether the rescue effect is attributed to their inhibitory effects on CSCs or on the entire tumor.

Although a number of publications regarding the role of miRNAs in GBMSCs have emphasized their significance in oncogenic properties, the majority of them used CD133 or tumor spheres to identify and isolate the subpopula-tion within the whole mixture. Nevertheless, controver-sies of these two characteristics have been addressed. As mentioned previously, up to 40% primary GBM samples do not express CD133. For miRNAs identified from cell lines, it is questionable whether their significance could be reproduced in clinical specimens and whether it repre-sents the same therapeutic potential in GBMSCs isolated using other markers. While the isolation markers remain debatable, the overall concept of target therapy for GBM-CSCs is to target CSC-specific capability, stemness main-tenance, and resistance to present therapeutics. Although some miRNAs modulate self-renewal and affect dif-ferentiation, such as miR-9/9*, miR-137, miR-128, and miR-146a, some miRNAs simply regulate proliferation, cell cycle progression, and migration/invasion (Fig. 1). For those miRNAs that target non-CSC-specific pathways,

Table 2. Dysregulated miRNAs in GBMSCs

miRNA Function of miRNACell Line(s) or Primary Tumor

Selection Phenotype(s) of GBMSC

Expression in GBMSCs

Potential Target(s) Ref.

miR-9/9* Inhibit differentiation and maintain stemness

Cell line R11 CD133 Upregulated CAMTA1 (91)

miR-9* N.D. Cell lines CD133 N.D. Sox2 (47)miR-125b Promote invasion# Cell lines SU3

and SU2CD133 Upregulated MMP9# (111)

Inhibit proliferation and cell cycle progression

Cell line U251 CD133 Downregulated CDK6#CDC25A#

(94)

miR-124 Inhibit invasion, tumorigenic-ity, and sphere formation

Primary tumors and cell lines

Spheres/CD133

Downregulated SNAI2 (96,119)

miR-34a Inhibit proliferation, cell cycle progression, and migration/invasion

Cell lines 0308 and 1228

Spheres Downregulated c-MetNotch1Notch2

(36,61,103)

miR-137 Promote differentiation and cell cycle arrest

Primary tumors and cell lines

Spheres/CD133

Downregulated N.D. (96)

miR-451 Inhibit proliferation and sphere formation

Primary tumors and A172 cell

CD133 Downregulated N.D. (29)

miR-128 Inhibit proliferation and self-renewal; promote differentiation

Primary tumors and cell lines

Spheres Downregulated Bmi-1EGFRPDGFRa

(34,72)

miR-146a Inhibit proliferation and promote differentiation

Primary cells and U87 cell

Spheres N.D. Notch1 (67)

miR-326 Reduce growth, invasion, metabolic activity, and tumorigenicity

Cell lines N/A N.D. and Down-regulated

PKM2Notch1Notch2

(52)

#No direct evidence. N.D., not determined; N/A, not applicable.

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it is not known whether they possess higher potential in GBM therapy or whether this dampens the possibility of clinical application. Another question also arises as most research assesses miRNA function in GBMSCs through evaluating self-renewal ability. Recently, Barrett and col-leagues had provided evidence showing that self-renewal does not correlate with tumorigenic ability. In contrast, cells with lower self-renewal capacity are much more efficient at forming tumors following transplantation into mice in their studies (6). Simply focusing on self-renewal may severely underscore the importance of cells lack-ing sphere-forming ability. It therefore brings challenges for GBMSC-specific miRNAs. Whether the evaluation approaches are suitable for identification of potential miRNAs and whether the result represents their signifi-cance in vivo is to be determined. The lack of a corre-lation between miRNA expression and clinical outcome further retards progress. As more details and mechanisms about GBMSCs are uncovered, it is likely that we could apply this knowledge towards future therapies.

CONCLUDING REMARKS

Despite the advances in multimodel therapy, the prog-nosis of patients with GBM remains unfavorable. The cytoreductive (debulking) strategy is believed to provide survival benefit for GBM patients despite the diffuse nature of the disease. In the past few years, growing evi-dence has indicated that current therapies preferentially target the bulk of the tumor except the GBMSCs, which possess a higher resistance to radiation therapy and che-motherapy and are responsible for tumor initiation and

propagation. Eliminating these small subpopulations that survive over the conventional therapy may improve ther-apy in clinical settings. Nevertheless, successful target therapy relies on the identification of unique markers and signaling pathways in GBMSCs that can distinguish them from the non-CSCs and normal stem/progenitor cells. Although great efforts have been made to target against specific molecular candidates that are assumed to link to poor prognosis, such as EGFR and vascular endothelial growth factor (VEGF), translation from laboratory find-ings to clinical practice has so far fallen short of expec-tations. While the functional significance of miRNA in GBM emerges, we are just beginning to investigate its role in the hallmarks of GBM and to appreciate its potential as a therapeutic target. Integration of multiple markers or modulators involved in regulating tumorigenic properties of GBMSCs might be the key to providing opportunities for achieving therapeutic success. Of course, additional research is required to confirm that the clinical relevance does exist and could be applied for practical use.

ACKNOWLEDGMENT: The authors declare no conflicts of interest.

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Figure 1. MicroRNAs identified in GBMSCs. A summary of deregulated microRNAs regulating cellular physiology was listed.

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