**3.1. Growth signal activation and microRNAs**

**1. Introduction**

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oncogenes (6).

and progression.

**2. MicroRNA expression in glioblastoma**

Glioblastomas (GBMs) are the most common and intractable primary neoplasms of the central nervous system (CNS). Gross total surgical excision followed by radiotherapy up to a total dose of 60 Gy was the only accepted GBM management for decades because no chemothera‐ peutic agents significantly improved the survival of patients with GBM until the introduction of temozolomide, an oral alkylating agent (1). Despite aggressive treatments, recurrence is inevitable and fatal in GBMs. In a recent clinical study, the median survival was up to approximately 20 months and the two-year survival rate was approximately 30~40% (2, 3). Therefore, more efforts need to be made to change the poor prognosis of patients with GBM. MicroRNAs (miRNAs) are small (18–24 nt), noncoding RNAs that are transcribed from the intergenic or intronic regions of DNA. miRNAs regulate the expression levels or translational levels of messenger RNAs (mRNAs) post-transcriptionally and affect to various biological processes, such as cell proliferation, cell survival, differentiation, and metabolism (4). Primary miRNAs (pri-miRNAs), transcribed from DNA and have short stem-loop structures (SLSs), are processed into premature miRNAs (pre-miRNAs) and then finally processed into mature miRNAs (5). A single miRNA has many targets of mRNAs, binding its target sites mostly in the 3′ untranslated region (3′-UTR) of the mRNA. The main function of miRNAs is to down‐ regulate gene expression, and miRNAs can function either as tumor suppressors or as

A number of miRNAs are reported to display aberrant expression patterns in GBMs (7). In 2005, the first miRNA dysregulation was identified in GBMs (8). miR-21 detected by northern blot was overexpressed in GBM tissues when compared with non-neoplastic control tissues. On the other hand, a systemic screen for miRNA aberrations by miRNA microarray in GBM was performed by other researchers (7). Using miRNA microarray analysis, we previously reported that miR-10b, miR-21, miR-183, miR-92b, and miR-106b are highly expressed in GBMs compared with normal brain tissue (9). Several other reports have also identified these miRNAs as being upregulated in GBMs (8). Recently, we reported that the expression of miR-183 involved in HIF-1α expression and its downstream molecules (10). Since then, it has been documented that miRNA dysregulation could play an important role in development

Similar to other malignant tumors, GBM has the hallmarks of cancer: biological capabilities of sustaining proliferative signaling, inducing angiogenesis, evading growth suppressors, resistance to apoptosis, activating invasion and metastasis, genomic instability, reprogram‐ ming energy metabolism, limitless replicative potential, and evading immune destruction. It is generally accepted that hallmark features of GBM are not only a reflection of genetic abnormalities and aberrant signal transition but also the dysregulation of miRNA-mediated translational control. The miRNA-mRNA interactions transform the short "nonsense"

Cell proliferation in GBM could be triggered by somatic alterations within the receptor tyrosine kinase (RTK)-signaling pathways. The most important pathways in GBM are the phosphati‐ dylinositide 3-kinase (PI3K)/AKT and RAS/mitogen-activated protein kinase (MAPK) path‐ ways (14). Abnormally augmented signals downstream from RTKs enable tumor cells to sustain proliferation that is under tight control in normal cells. At least 90% of GBM cases harbor genetic alterations in RTK pathways. Epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor alpha polypeptide (PDGFRA), MET proto-oncogene (MET), and fibroblast growth factor receptor (FGFR) are among the most commonly dysre‐ gulated RTKs in GBM (15). EGFR and PDGFRA are well-established oncogenes in GBM (16), and thus, identification of their miRNA regulators following the discovery of functional implications of miRNAs in GBMs is warranted.

miR-7 is a common regulator of the PI3K/ATK and MAPK pathways, both of which are launched by EGFR through its two direct targets, the transcription factors PI3K and Raf-1, respectively (17). Human EGFR mRNA 3′-untranslated region contains three miR-7 target sites (18). Transient expression of miR-7 in GBM cells strongly inhibited *in vivo* GBM xenograft growth (17). Decreased expression of miR-128 correlates with aggressive human glioma subtypes, and miR-128 represses growth and mediates differentiation by targeting oncogenic EGFR and PDGFRA (19). The authors demonstrated that miR-128 suppresses glioma formation in a glioma mouse model, suggesting miR-128 as a glioma tumor suppressor that targets RTK signaling to repress gliomagenesis. miR-218 targets multiple components of RTK-signaling pathways, including the EGFR pathway, and miR-218 repression increases the abundance and activity of multiple RTK effectors (20). The expression of miR-218 is significantly decreased in the mesenchymal subtype of GBM, and the miR-218-RTK-HIF2α-signaling axis promotes GBM cell survival and tumor angiogenesis (20). A large-scale, genomewide miRNA expression analysis revealed miR-219 was downregulated in GBM, and exogenous overexpression of miR-219 in glioma cells inhibited proliferation and soft agar colony formation (21). In addition, overexpression of miR-219-5p reduced the activity of PI3 kinase and MAP kinase in glioma cell lines by targeting to EGFR (21). Moreover, overexpression of miR-133 decreased GBM cell growth and increased cell apoptosis, whereas depletion of miR-133 increased cell growth (22). The protein translation inhibition of EGFR by miR-133 was confirmed by a dual luciferase reporter assay (22). miR-340 overexpression suppressed several oncogenes, including EGFR (23). miR-340 induces glioma cells toward terminal differentiation and regulates glioma cell development by downregulating ROCK1 expression (23). miR-491 directly targets EGFR, CDK6, and Bcl-xL (24). Importantly, miR-491 is commonly codeleted with its adjacent CDKN2A on chromosome 9p21.3 in GBM. As a negative regulator of EGFR expression, miR-491 is a tumor suppressor gene. On the other hand, miR-148a expression was elevated in GBM specimens compared with normal human brain and astrocytes (25). miR-148a is a prognostic onco-miRNA that targets MIG6 and BIM to regulate EGFR and apoptosis in GBM (25). By inhibiting MIG6 expression, miR-148a reduced EGFR trafficking to Rab7-expressing compartments, including late endosomes and lysosomes. This process coincided with reduced degradation and elevated expression and activation of EGFR (25). Furthermore, the protein expression of EGFR decreased in cells with forced overexpression of miR-34a (26). Yin et al. reported that both deletion of miR-34a and amplification of EGFR were associated with significantly decreased overall survival of patients with GBM (26).

PDGFRA is a direct target of miR-34a, a downregulated miRNA in the proneural subtype compared with the mesenchymal subtype of GBM (27, 28). miR-34a specifically affects the growth of proneural glioma cells *in vitro* and *in vivo* (28). miR-34a repression in proneural gliomas is only modestly dependent on p53. Derepression of PDGFRA by the downregulation of miR-34a in the proneural subtype that results in a more proliferative phenotype may be responsible for the difference in response to clinical treatment for GBM. Gene mutation or amplification in MET is a relatively rare event in GBM (29). However, miR-34a expression inhibited c-Met reporter activities in glioma cells. In addition, miR-34a levels in human gliomas were inversely correlated with c-Met levels. Transient transfection of miR-34a into gliomas strongly inhibited cell proliferation, cell cycle progression, cell survival, and cell invasion (30). MET is highly expressed in the mesenchymal subtype of GBM (31). miR-182 sensitized glioma cells to TMZ-induced apoptosis, promoted glioma initiating cell (GIC) differentiation, and reduced tumor cell proliferation via knockdown of c-Met, Bcl2L12, and HIF2A (32). miR-144-3p specifically bound to the MET 3′-untranslated region (3′ UTR) and inhibited its expression. miR-144 strongly repressed GBM cell proliferation and invasion by suppressing MET *in vitro* and *in vivo* (33).

miR-7 is a common regulator of the PI3K/ATK and MAPK pathways, both of which are launched by EGFR through its two direct targets, the transcription factors PI3K and Raf-1, respectively (17). Human EGFR mRNA 3′-untranslated region contains three miR-7 target sites (18). Transient expression of miR-7 in GBM cells strongly inhibited *in vivo* GBM xenograft growth (17). Decreased expression of miR-128 correlates with aggressive human glioma subtypes, and miR-128 represses growth and mediates differentiation by targeting oncogenic EGFR and PDGFRA (19). The authors demonstrated that miR-128 suppresses glioma formation in a glioma mouse model, suggesting miR-128 as a glioma tumor suppressor that targets RTK signaling to repress gliomagenesis. miR-218 targets multiple components of RTK-signaling pathways, including the EGFR pathway, and miR-218 repression increases the abundance and activity of multiple RTK effectors (20). The expression of miR-218 is significantly decreased in the mesenchymal subtype of GBM, and the miR-218-RTK-HIF2α-signaling axis promotes GBM cell survival and tumor angiogenesis (20). A large-scale, genomewide miRNA expression analysis revealed miR-219 was downregulated in GBM, and exogenous overexpression of miR-219 in glioma cells inhibited proliferation and soft agar colony formation (21). In addition, overexpression of miR-219-5p reduced the activity of PI3 kinase and MAP kinase in glioma cell lines by targeting to EGFR (21). Moreover, overexpression of miR-133 decreased GBM cell growth and increased cell apoptosis, whereas depletion of miR-133 increased cell growth (22). The protein translation inhibition of EGFR by miR-133 was confirmed by a dual luciferase reporter assay (22). miR-340 overexpression suppressed several oncogenes, including EGFR (23). miR-340 induces glioma cells toward terminal differentiation and regulates glioma cell development by downregulating ROCK1 expression (23). miR-491 directly targets EGFR, CDK6, and Bcl-xL (24). Importantly, miR-491 is commonly codeleted with its adjacent CDKN2A on chromosome 9p21.3 in GBM. As a negative regulator of EGFR expression, miR-491 is a tumor suppressor gene. On the other hand, miR-148a expression was elevated in GBM specimens compared with normal human brain and astrocytes (25). miR-148a is a prognostic onco-miRNA that targets MIG6 and BIM to regulate EGFR and apoptosis in GBM (25). By inhibiting MIG6 expression, miR-148a reduced EGFR trafficking to Rab7-expressing compartments, including late endosomes and lysosomes. This process coincided with reduced degradation and elevated expression and activation of EGFR (25). Furthermore, the protein expression of EGFR decreased in cells with forced overexpression of miR-34a (26). Yin et al. reported that both deletion of miR-34a and amplification of EGFR were associated with

30 Neurooncology - Newer Developments

significantly decreased overall survival of patients with GBM (26).

PDGFRA is a direct target of miR-34a, a downregulated miRNA in the proneural subtype compared with the mesenchymal subtype of GBM (27, 28). miR-34a specifically affects the growth of proneural glioma cells *in vitro* and *in vivo* (28). miR-34a repression in proneural gliomas is only modestly dependent on p53. Derepression of PDGFRA by the downregulation of miR-34a in the proneural subtype that results in a more proliferative phenotype may be responsible for the difference in response to clinical treatment for GBM. Gene mutation or amplification in MET is a relatively rare event in GBM (29). However, miR-34a expression inhibited c-Met reporter activities in glioma cells. In addition, miR-34a levels in human gliomas were inversely correlated with c-Met levels. Transient transfection of miR-34a into gliomas strongly inhibited cell proliferation, cell cycle progression, cell survival, and cell invasion (30). RAS signaling, upstream of MAP kinase, is reported to be regulated by miRNAs. Although only 1% of GBMs have a RAS mutation or amplification, 10% of GBMs contain neurofibromin 1 (NF1) inactivating genetic alterations that lead to hyperactive RAS activity by enhancing the intrinsic GTPase activity (29). Two recent reports focused on miRNAs targeting RAS in GBMs and showed that miR-143 directly targets N-RAS (34) and that let-7a directly targets K-RAS (35). Low expression of let-7a was correlated with a poor prognosis of primary GBM patients (35). In addition, miR-124 governs glioma growth and angiogenesis and enhances chemosen‐ sitivity by targeting R-RAS and N-RAS (36). In addition, miR-124 inhibits the MAP kinase pathway by repressing SOS1 mRNA (37). Moreover, these miRNAs are all downregulated in GBM specimens, underlying the malignant transformation.

Phosphatase and tensin homolog (PTEN) and NF1 are the most important negative regulators of cell proliferative pathways. The inactivated genetic mutations of NF1 and PTEN are found in 10 and 41% of GBM cases, respectively (29). NF1 is targeted by miR-9, which is an miRNA upregulated in GBM and promotes the proliferation and migration of glioma cells (38, 39). In addition, NF1 is a direct target of miR-514a, and over-expression of miR-514a inhibited NF1 expression, which correlated with increased survival cells (40). The protein phosphatase activity of PTEN is involved in cell proliferation, preventing cells from growing. miR-26a, frequently amplified at the DNA level in human gliomas, is identified as a direct regulator of PTEN expression (41). miR-26a-mediated PTEN repression in a murine glioma model enhan‐ ces *de novo* glioma formation (41). Overexpression of miR-26a in PTEN-competent and PTENdeficient GBM cells promoted tumor growth *in vivo* and further increased growth in cells overexpressing CDK4 or CENTG1 (42). Guo et al. reported that c-Myc modulates genes associated with oncogenesis in GBM through deregulation of miRNAs via the c-Myc-miR-26a-PTEN-signaling pathway (43). CREB, a proto-oncogenic transcription factor that is overex‐ pressed in gliomas, can promote gliomagenesis by modulating the expression of oncogenic miR-23a, which represses PTEN directly (44). miR-23a-mediated suppression of PTEN led to the activation of AKT/ERK pathways and epithelial-mesenchymal transition (EMT) (45). miR-17-5p (46), and miRNA-1908 (47) directly target PTEN in GBMs. Overexpression of miR-17 prolongs GBM cell survival and increases cell motility, and induces HIF-1α activation in response to stress by targeting PTEN. (46). miR-1908 promotes proliferation, invasion, and sphere formation in GBM cells by targeting PTEN, and PTEN levels are inversely correlated with miR-1908 levels in GBM tissues (47). The expression of PTEN-targeting miR-17-5p, miR-19a, miR-19b, miR-21, miR-130b, miR-221, and miR-222 was significantly higher in irradiated glioma cells than in nonirradiated cells, and the PTEN expression levels were lower in the irradiated glioma cells than in the nonirradiated cells (48).

In recent years, molecular target therapy-targeting RTK pathways has been one of the most exciting developments in cancer therapy, and certain molecular targeted drugs have been clinically validated. However, for GBMs, no effective molecular targeted drug has yet been developed. By understanding the roles of miRNAs in growth signal activation, miRNA-based treatments should also be taken into consideration either alone or in combination.

#### **3.2. Sustained angiogenesis and microRNAs**

New growth in the vascular network is important because the proliferation, as well as metastatic spread, of tumor cells depends on an adequate supply of oxygen and nutrients and the removal of waste products. GBMs have a strong angiogenetic property because GBMs possess glomeruloid microvascular proliferation, a hallmark of GBMs (49). GBMs stimulate new blood vessel formation through processes driven primarily by vascular endothelial growth factor A (VEGF-A), the most established proangiogenic protein in the VEGF family. The overexpression of VEGF-A, and subsequent activation of its receptors, is an important event during glioma progression (50).

miRNA-205, an miRNA significantly downregulated in GBMs, can specifically suppress expression of VEGF-A directly (51). Moreover, miRNA-205 induces apoptosis and depresses the invasion of glioma cells *in vitro* (51). VEGF-A upregulation can be induced by hypoxia inducible factor 1 alpha subunit (HIF-1α), which is negatively regulated by the von Hippel-Lindau (VHL) tumor suppressor (52). HIF-1α levels were upregulated in glioma cells following transfection with miR-183 mimic RNA, and VEGF-A and glucose transporter 1 (GLUT1), which are downstream molecules of HIF-1α, were upregulated in cells transfected with miR-183 (10). miR-21, miR-23b, and miR-566 are reported to target VHL and decrease the production of the VHL protein, upregulating VEGF-A expression. miR-21 directly targets VHL and peroxisomeproliferator-activated receptor a (PPARa), and miR-21 regulates EGFR/AKT signaling through VHL/β-catenin and the PPARa/AP-1 axis (53). miR-21 significantly colocalized with the hypoxia- and angiogenesis-associated markers HIF-1α and VEGF (54). Downregulation of miR-23b triggered growth inhibition, induced apoptosis, and suppressed invasion of glioma *in vitro* (55). miR-23b deletion decreased HIF-1α/VEGF expression and suppressed β-catenin/ Tcf-4 transcription activity by targeting VHL (55). Inhibition of miR‑566 expression increases the expression levels of VHL, decreases the expression levels of VEGF, and inhibits the invasive and migratory abilities of GBM cells (56). Moreover, miR-7 downregulates the expression of O-linked *N*-acetylglucosamine transferase (OGT) involved in the VEGF-signaling pathway, leading to a profound reduction in vascularization, similar to the antiangiogenic drug sunitinib (57).

miR-101 is downregulated in GBMs and targets EZH2, a histone methyltransferase affecting gene expression profiles in an epigenetic manner. Inhibition of EZH2 by miR-101 attenuated GBM cell growth, migration, and GBM-induced endothelial tubule formation (58). Ectopic expression of miR-137 inhibited angiogenesis in a SCID mouse xenograft model. EZH2 was identified as a direct target of miR-137, and EZH2 overexpression can rescue the inhibitory effect of miR-137 on cell proliferation and angiogenesis (59). A key step in angiogenesis is the upregulation of growth factor receptors on endothelial cells. miR-296 level is increased in primary tumor endothelial cells isolated from GBM tissues (60). Overexpressed miR-296 enhances angiogenesis by directly targeting the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), results in upregulation of the VEGF receptoe-2 and PDGF receptor-β (60). miR-93 promotes angiogenesis by suppressing integrin-β8 (61). *In vivo* studies revealed that miR-93-expressing cells induced blood vessel formation, allowing blood vessels to extend to tumor tissues in high densities (61). Angiogenesis promoted by miR-93 in return facilitated cell survival, resulting in enhanced tumor growth. miR-17∼92 promotes angiogenesis and tumor growth by downregulation of clusterin (62). Specifically, miR-17-5p and miR-20 reduce the expression of the type-II TGFβ receptor and miR-18 limits the expression of Smad4, namely miR-17∼92 attenuates the TGFβ-signaling pathway to shut down clusterin expression, thereby stimulating angiogenesis and tumor cell growth.

Because angiogenesis is a hallmark of GBM, targeting therapies against angiogenesis using the VEGF antibody was previously considered to be promising in GBM. However, recently, two phase-III studies revealed that the addition of the VEGF antibody bevacizumab to radiother‐ apy-temozolomide did not improve survival in patients with GBM, although improved progression-free survival and maintenance of baseline quality of life and performance status were observed with bevacizumab (2, 3). Therefore, new therapeutic targets or strategies need to be developed. To make progress, a better understanding of the miRNAs contributing to angiogenesis will lead to more effective antiangiogenic therapy for patients with GBM.
