**3.3. Insensitivity to antigrowth signals and microRNAs**

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

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

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

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

treatments should also be taken into consideration either alone or in combination.

**3.2. Sustained angiogenesis and microRNAs**

32 Neurooncology - Newer Developments

event during glioma progression (50).

(57).

To proliferate without limit, GBM cells must circumvent biologically programmed pathways that negatively regulate cell proliferation. There are two major tumor suppressor genes, retinoblastoma 1 (RB1) and tumor protein p53. Similarly, a better understanding of the dysfunction of the RB1 and p53 pathways should also implicate the roles of dysregulated miRNAs. The mutation of p53 results in the inability to stop further cell-cycle progression triggered by oncogenic signals (63). miR-10b, one of the most studied miRNAs in GBMs, is highly upregulated in human GBM and pleiotropically regulates invasion, angiogenicity, and apoptosis of GBM cells. The pleiotropic effect of miR-10b is caused by its suppression of multiple tumor-suppressive genes, including p53 (7, 64, 65). miR-10b directly targets p53 in GBM, giving the tumors a way to evade growth control and enable persistent cell proliferation by perturbing the miRNAs expression.

Moreover, p53 signaling is under the precise control of the negative regulator mouse double minute 2 (MDM2). MDM2 regulates the ubiquitin-dependent degradation and transcriptional activity of p53 (66). MDM2 mRNA is upregulated in both GBM cell lines and samples (67), and the upregulation could be a consequence of the downregulation of miR-17, miR-181b, miR-25, or miR-32, which directly target MDM2 gene expression (46, 67, 68). It is worth noting that miR-25 and miR-32 are two miRNAs repressed by p53, suggesting a feedback circuit between p53 and MDM2 mediated by miRNAs (67). The feedback circuit can explain the overexpression of miR-25 in GBM reported by several separate studies, in which the miRNA is meant to be downregulated to increase MDM2 expression and thus inactivate p53 (7, 39, 69). In addition,

miR-181b is downregulated in GBM samples, further indicating that miRNA contributes to the complexity of the pathological progression of gliomas (68).

Another key tumor suppressive is p16INK4a, an important inhibitor of RB pathway (70), namely p16INK4a can bind specifically to CDK4/6 and inhibit the catalytic activity of CDK4/6 cyclin D complexes (71). Approximately, 80% of GBMs have one or more alterations affecting the RB1 function. In addition to targeting p53 in GBMs, miR-10b also targets p16INK4a, and the inhibition of miR-10b leads to cell cycle arrest (65). miR-26a also targets RB1 in GBMs. Additionally, CDK-cyclin complex-mediated phosphorylation is one of the main mechanisms by which RB1 protein is inactivated (72). The frequent gain-of-function mutations on CDK4/6 cyclin D complexes underscore their importance and potential in the development and progression of GBM. miR-124 is reported to radiosensitize human glioma cells by downregu‐ lating CDK4 (73), whereas CDK6 is a direct target of miR-138 (74) and miR-491-3p/5p (24). miR-195 inhibited glioma cell proliferation by downregulating expression of cyclin D1 and cyclin E1, via directly targeting cyclin D1 and cyclin E1 mRNA (75). Notably, those miRNAs that target cyclin-CDK complexes are all downregulated in GBM samples (69).

#### **3.4. Invasion in the brain and microRNAs**

The invasive propensity of glioma cells remains the major obstacle to improving the poor outcomes of patients with GBM. GBM cells prefer to migrate through the tortuous extracellular spaces of the brain. Therefore, it is supposed that the interaction of invading glioma cells with the extracellular matrix (ECM) is crucial in the initiation of invasion and migration. Generally, cell attachment is mediated by interactions between cell-cell and cell-ECM receptors, including integrins and cadherins, and degradation of ECM components by metalloproteinases is essential for cell detachment (76). The matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs) are two distinct types of metalloproteinases secreted by glioma cells to overcome the dense matrix, and the proteolytic activity can be blocked by endogenous metalloproteinase inhibitors, such as tissue inhibitors of metalloproteinase (TIMPs) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) (76).

miR-218 expression has been found to be significantly downregulated in GBM tissue samples (77). Several studies have shown that miR-218 is a negative regulator of invasion in GBM through various pathways (77, 78). miR-218 has been reported to target the mRNA of Lef1, the transcription factor that is upregulated by β-catenin (77). Suppression of Lef1 leads to the reduction of MMP-2, MMP-7, and MMP-9 activity and inhibition of invasion *in vitro* (79). Another mechanism through which miR-218 negatively regulates GBM invasion is targeting IKKβ mRNA along with reducing the transcription of NF-kB. NF-kB is a transcription factor that is important in many cellular processes and has been strongly linked to the migration and invasion of various cancer cells (78). NF-kB's target genes include MMP-9; therefore, its inhibition by miR-218 causes a decrease in invasion.

MMP-9 is a direct target of miR-491-5p, which is upregulated in GBM (24). miR-491-5p expression has been reported to reduce cell proliferation and invasion by targeting MMP9 (80). miR-491-3p also reduces the invasiveness of GBM cells by targeting the mRNA of insulin-like growth factor-binding protein 2 (IGFBP2). MMP-9 is also a direct target of miR-211 (81). MMP-3 is targeted by miR-152 (82). Additionally, ADAM17, a non-MMP, is under the direct regulation of miR-145 (83). It is clear that if glioma cells lose the control of MMPs and ADAMs by miRNAs, ECM homeostasis is compromised and the combined activity of these proteases remodels the ECM to favor tumor invasion. miR-101 is a tumor suppressor that is downregulated in GBM, as well as other cancers. (58). It has been shown to downregulate the invasion of glioma cells, as well as proliferation and migration, by targeting the transcription factor Kruppel-like factor 6 (KLF6). This suppression of KLF6 reduced the expression of chitinase-3-like protein 1 (CHI3L1) and inactivated MEK1/2 and PI3K signaling (72). miR-101 downregulation has been shown to result in EZH2-induced proliferation, migration, and angiogenesis in GBM (74).

miR-181b is downregulated in GBM samples, further indicating that miRNA contributes to

Another key tumor suppressive is p16INK4a, an important inhibitor of RB pathway (70), namely p16INK4a can bind specifically to CDK4/6 and inhibit the catalytic activity of CDK4/6 cyclin D complexes (71). Approximately, 80% of GBMs have one or more alterations affecting the RB1 function. In addition to targeting p53 in GBMs, miR-10b also targets p16INK4a, and the inhibition of miR-10b leads to cell cycle arrest (65). miR-26a also targets RB1 in GBMs. Additionally, CDK-cyclin complex-mediated phosphorylation is one of the main mechanisms by which RB1 protein is inactivated (72). The frequent gain-of-function mutations on CDK4/6 cyclin D complexes underscore their importance and potential in the development and progression of GBM. miR-124 is reported to radiosensitize human glioma cells by downregu‐ lating CDK4 (73), whereas CDK6 is a direct target of miR-138 (74) and miR-491-3p/5p (24). miR-195 inhibited glioma cell proliferation by downregulating expression of cyclin D1 and cyclin E1, via directly targeting cyclin D1 and cyclin E1 mRNA (75). Notably, those miRNAs

that target cyclin-CDK complexes are all downregulated in GBM samples (69).

The invasive propensity of glioma cells remains the major obstacle to improving the poor outcomes of patients with GBM. GBM cells prefer to migrate through the tortuous extracellular spaces of the brain. Therefore, it is supposed that the interaction of invading glioma cells with the extracellular matrix (ECM) is crucial in the initiation of invasion and migration. Generally, cell attachment is mediated by interactions between cell-cell and cell-ECM receptors, including integrins and cadherins, and degradation of ECM components by metalloproteinases is essential for cell detachment (76). The matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs) are two distinct types of metalloproteinases secreted by glioma cells to overcome the dense matrix, and the proteolytic activity can be blocked by endogenous metalloproteinase inhibitors, such as tissue inhibitors of metalloproteinase

(TIMPs) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) (76).

miR-218 expression has been found to be significantly downregulated in GBM tissue samples (77). Several studies have shown that miR-218 is a negative regulator of invasion in GBM through various pathways (77, 78). miR-218 has been reported to target the mRNA of Lef1, the transcription factor that is upregulated by β-catenin (77). Suppression of Lef1 leads to the reduction of MMP-2, MMP-7, and MMP-9 activity and inhibition of invasion *in vitro* (79). Another mechanism through which miR-218 negatively regulates GBM invasion is targeting IKKβ mRNA along with reducing the transcription of NF-kB. NF-kB is a transcription factor that is important in many cellular processes and has been strongly linked to the migration and invasion of various cancer cells (78). NF-kB's target genes include MMP-9; therefore, its

MMP-9 is a direct target of miR-491-5p, which is upregulated in GBM (24). miR-491-5p expression has been reported to reduce cell proliferation and invasion by targeting MMP9 (80). miR-491-3p also reduces the invasiveness of GBM cells by targeting the mRNA of insulin-like growth factor-binding protein 2 (IGFBP2). MMP-9 is also a direct target of miR-211 (81). MMP-3

**3.4. Invasion in the brain and microRNAs**

34 Neurooncology - Newer Developments

inhibition by miR-218 causes a decrease in invasion.

the complexity of the pathological progression of gliomas (68).

Similarly, miR-152 is a tumor suppressor that is often downregulated in cancers, including in GBM. It was shown to suppress the invasion of glioma stem cells, as well as cell proliferation, migration, invasion, and apoptosis. It has been reported that miR-152 exerts its tumorsuppressing effects by targeting the transcription factor Kruppel-like factor 4 (KLF4). This suppression of KLF4 causes the transcriptional downregulation of galectin-3 (LGALS3) and the inactivation of MEK1/2 and PI3K signaling (75).

Because the MMPs and ADAMs each comprises more than 20 members, targeting a single target appears to be nonessential in cancerous diseases, which provides miR-21 with an opportunity to broadly inhibit metalloproteinase function. miR-21 regulates multiple genes associated with glioma cell migration and invasion, including the RECK and TIMP3 genes, which are inhibitors of matrix metalloproteinases (84).

In addition, interactions with the ECM are mostly mediated by integrins, which enable cells to sense the extracellular environment and adjust their behavior to environmental cues. miR-124, frequently downregulated in GBM, targets β1 integrin and is shown to affect glioma cell migration and invasion *in vitro* (85). PPP1R13L (protein phosphatase 1, regulatory subunit 13 like), an inhibitory member of the apoptosis-stimulating protein of p53 family (IASPP), was found to be a direct target of miR-124 in GBM cells. miR-124-mediated PPP1R13L regulates invasion of GBM cells (86). Cai et al. found that miR-542-3p expression was decreased in GBM cells, and miR-542-3p suppressed GBM cell invasion by not only targeting AKT1 but also directly downregulating its two important upstream regulators, ILK (integrin-linked kinase) and PIK3R1 (87). miR-181 family is downregulated in GBM and is inversely correlated with activities of NF-kB-targeting genes. Furthermore, miR-181b was shown to suppress epithelialmesenchymal transition (EMT) by targeting KPNA4 (88). miR-181c overexpression inhibits TGF-β signaling by downregulating TGFBR1 (transforming growth factor, beta receptor 1), TGFBR2 and TGFBRAP1 (transforming growth factor, beta receptor-associated protein 1) expression. miR-181c expression levels are correlated with poor prognosis of patients with GBM.

miR-125a are downregulated in GBMs and directly target the 3′-UTR of podoplanin (PDPN) and inhibit invasion, apoptosis, and proliferation of GBMs. In addition, miR-125a inhibits Nrg1, one of the most active members of the EGF-like family, and suppresses the proliferation and migration of GBM cells *in vitro* and *in vivo* (89). miR-203 expression is decreased in anaplastic astrocytoma and GBM tissues. Forced expression of miR-203 was shown to suppress glioma cell proliferation, migration, and invasion, by disrupting the Robo1/ERK/MMP-9signaling axis (90). In addition, miR-203 inhibits the proliferation and invasion by directly targeting phospholipase D2 (PLD2) (91). miR-29b expression, downregulated in GBMs, was inversely proportional to that of BCL2-like 2 (BCL2L2) mRNA or protein (92). Interestingly, BCL2L2 mRNA is highly expressed in the mesenchymal type of GBM. BCL2L2 repression is of central importance to miR-29b antitumor activity in migration, invasion, and angiogenesis. Moreover, miR-29b regulates PDPN, which promotes glioma invasion.

miR-10b is overexpressed in GBM and induces glioma cell invasion by modulating expression of RhoC, MMP-14, and uPAR (urokinase-type plasminogen activator receptor) via HOXD10 (93). Multifocal lesions of malignant gliomas were associated with higher expression levels of miR-10b (9). Dong et al. reported that not only miR-10b inhibition but also miR-21 inhibition could exert synergistic inhibition of the invasion of glioma cells. miR-10b pleiotropically regulates invasion, angiogenicity, and apoptosis of glioma cells by the suppression of multiple tumor suppressors, including p53, FOXO3, HOXD10, and NOTCH1 (64). miR-155 has been identified to be an oncomiR and is highly expressed in several solid cancers, including GBM. Knockdown of miR-155 sensitizes glioma cells to the chemotherapy of temozolomide by targeting p38 isoforms of mitogen-activated protein kinase 13 (MAPK13) and MAPK14 (94).

In sum, the roles of miRNAs in glioma cell invasion or migration further our understanding of the genesis of the aggressive glioma phenotype. Some of these miRNAs have been discov‐ ered, whereas more still remain to be found. The blockade of excessive pro-invasive miRNAs or the restoration of weakened anti-invasive miRNAs will provide extra treatment options for advanced-stage gliomas that are marked by poor prognoses for decades (**Table 1**).



signaling axis (90). In addition, miR-203 inhibits the proliferation and invasion by directly targeting phospholipase D2 (PLD2) (91). miR-29b expression, downregulated in GBMs, was inversely proportional to that of BCL2-like 2 (BCL2L2) mRNA or protein (92). Interestingly, BCL2L2 mRNA is highly expressed in the mesenchymal type of GBM. BCL2L2 repression is of central importance to miR-29b antitumor activity in migration, invasion, and angiogenesis.

miR-10b is overexpressed in GBM and induces glioma cell invasion by modulating expression of RhoC, MMP-14, and uPAR (urokinase-type plasminogen activator receptor) via HOXD10 (93). Multifocal lesions of malignant gliomas were associated with higher expression levels of miR-10b (9). Dong et al. reported that not only miR-10b inhibition but also miR-21 inhibition could exert synergistic inhibition of the invasion of glioma cells. miR-10b pleiotropically regulates invasion, angiogenicity, and apoptosis of glioma cells by the suppression of multiple tumor suppressors, including p53, FOXO3, HOXD10, and NOTCH1 (64). miR-155 has been identified to be an oncomiR and is highly expressed in several solid cancers, including GBM. Knockdown of miR-155 sensitizes glioma cells to the chemotherapy of temozolomide by targeting p38 isoforms of mitogen-activated protein kinase 13 (MAPK13) and MAPK14 (94).

In sum, the roles of miRNAs in glioma cell invasion or migration further our understanding of the genesis of the aggressive glioma phenotype. Some of these miRNAs have been discov‐ ered, whereas more still remain to be found. The blockade of excessive pro-invasive miRNAs or the restoration of weakened anti-invasive miRNAs will provide extra treatment options for

advanced-stage gliomas that are marked by poor prognoses for decades (**Table 1**).

miR-128 EGFR, PDGFRAdown downregulate PI3K/ATK

miR-218 EGFR down downregulate PI3K/ATK

miR-219 EGFR down downregulate PI3K/ATK

miR-133 EGFR down downregulate PI3K/ATK

**miRNAs Targets Expression Function Ref**

down downregulate PI3K/ATK and MAPK pathways

> and MAPK pathways

> and MAPK pathways

> and MAPK pathways

> and MAPK pathways

miR-340 EGFR down induce terminal differentiation Huang (23)

Liu (17), Webster (18)

(19)

Mathew (20)

Rao (21)

Xu (22)

Papagiannakopoulos

**Hallmarks of glioblastoma**

36 Neurooncology - Newer Developments

**Growth signal activation**

miR-7 EGFR, PI3K, Raf-1

Moreover, miR-29b regulates PDPN, which promotes glioma invasion.



**Hallmarks of glioblastoma**

38 Neurooncology - Newer Developments

**Insensitivity to antigrowth signals**

**Invasion and metastasis**

miR-491- 3p/5p

miR-195 cyclin D1, cyclin E1

**miRNAs Targets Expression Function Ref**

miR-566 VHL up inhibit invasion and migration Xiao (56) miR-7 OGT down reduce vascularization Babae (57)

invasion, induce apoptosis

migration/invasion

angiogenesis

angiogenesis

proliferation

CDK6 down cell cycle arrest Li (24)

miR-218 IKKβ, Lef1 down inhibit cell invasion Liu (77), Song (78)

invasion

stem cells

angiogenesis

stem cells

suppress invasion of glioma

miR-211 MMP9 down reduce cell invasion Asuthkar (81)

miR-491 IGFBP2, MMP9 down reduce cell proliferation and

miR-101 KLF6, EZH2 down reduce invasion of glioma

miR-152 MMP3, KLF4 down reduce cell invasion and

down cell cycle arrest Hui (75)

miR-26a RB1 up promote cell proliferation Lundberg (72) miR-124 CDK4 down cell cycle arrest Deng (73) miR-138 CDK6 down cell cycle arrest Qiu (74)

miR-10b p53, p16INK4a up promote cell cycle Gabriely (65)

miR-17-3p MDM2 inhibit tumor progression Li (46) miR-181b MDM2 down inhibit tumor progression Suh (67) miR-25-3p MDM2, p53 up inhibit cellular proliferation Suh (67)

Chen (55)

Smits (58)

Sun (59)

Fang (61)

Suh (67)

Yan (80)

Smits (58), Qiu (74)

Zheng (82), Hui (75)

miR-23b VHL up inhibit tumor growth and

miR-101 EZH2 down attenuate GBM growth and

miR-137 EZH2 down inhibit cell proliferation and

miR-93 Integrin-β8 up promote tumor growth and

miR-32-5p MDM2, p53 up inhibit cellular



**Table 1.** Hallmarks of glioblastoma and microRNAs.

**Hallmarks of glioblastoma**

40 Neurooncology - Newer Developments

**Tumorpromoting inflammation**

**Reprogramming**

**energy metabolism** **miRNAs Targets Expression Function Ref**

mitosis

mitosis

regulation

regulation

damaged DNA

down DNA damage response

molecules

down DNA repair regulate non-

pathway

miR-124 down inhibit STAT3 pathway Wei (117)

regulate NF kB activation

signaling activity

stability and translation

down reduce Lin28/Lin28b transcription

up DNA mismatch repair Liu (94)

spindle assembly checkpoint

down upregulate p53 Park (106)

upregulate p53 downstream

homologous end joining of DNA double strand breaks Liu (101)

Liu (101)

Gillies (103)

Gillies (103)

He (107)

Yan (109)

Li (111)

Taganov (112), Park (113)

Godlewski (124)

Rani (130), Gan (132)

Masui (134)

Wan (104), Le (105)

miR-17-92 up DNA replication and

miR-20 DNA replication and

miR-221 p27 up Cell cycle chechpoint

miR-222 p27 up Cell cycle chechpoint

miR-125b p53, MXD1 down cell cycle arrest to repair

miR-21 LRRFIP1 up regulate NF-kB signaling

miR-451 CAB39 up regulate LKB1/AMPK

miR-34c c-Myc regulate c-Myc mRNA

miR-155 MLH1, MSH2, MSH6

miR-29 PIK3R1,

miR-34 CCND1,

miR-101 ATM,

miR-146a TRAF6,

mir-145 c-Myc, Sox9, ADD3

CDC42

CCNE2, CDK4, MET, MYC, SNAI1, and SIRT1

PRKDC

IRAK1
