**3.8. Reprogramming energy metabolism and microRNAs**

Cellular metabolism of malignant tumor is considerably different from that of normal cell, because of limitless proliferation and motility. (119). GBM cells uptake a large amount of glucose, and aerobic glycolysis generates various substrates, such as fatty acids and nucleoti‐ des that are required for rapidly proliferating cells, and is associated with a survival advantage in the tumor cells (Warburg effect). MR spectroscopy analyses have shown that as much as 90% of the glucose converts into lactate by aerobic glycolysis in GBM cells. Clinically, it is known that the levels of lactate are elevated in GBM tissue compared with those in contralateral normal brain tissue. In addition, lactate was shown to be high-grade gliomas than for lowgrade gliomas, and glutamate levels were significantly elevated for GBMs. Pretreatment F18-2 fluoro-2-deoxy-D-glucose (FDG)-PET provides significant additional prognostic information in high-grade gliomas (120).

In normal cells, the 5'-adenosine monophosphate-activated protein kinase (AMPK) pathway is the major cellular sensor of energy availability (121), but its function in cancer is not clear. AMPK is activated by metabolic stress to promote energy conservation and glucose uptake, allowing cells to survive periods of low-energy availability. Allosteric interaction with elevated intracellular AMP, which acts to inhibit dephosphorylation of AMPK (122), and phosphory‐ lation at Thr172 by the protein kinase LKB1 are necessary for AMPK activation under condi‐ tions of bioenergetic stress (123).

miR-146a is known as miRNA associated with the innate immune response to microbial infection (112). Promotor analysis revealed that miR-146a is a NF-kB-dependent miRNAs (112). miR-146a has complementary sequences in the mRNA of the TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK1), key adaptor molecules downstream of toll-like and cytokine receptors (113). miR-146a overexpression enhanced apoptosis and suppressed NF-kB activation in TMZ-treated GBM cells (114). Increased expression of miR-146a was observed in glioneuronal lesions (115), and miR-146a expression in human glial cells was strongly induced by IL-1β (115). miR-146a expression by transfection in astrocytes regulated many mRNA expression levels, including those of IRAK-1, IRAK-2, and TRAF-6. In

Recent evidence suggests that the signal transducer and activator of transcription (STAT) family proteins play a crucial role in selectively inducing and maintaining a procarcinogenic inflammatory microenvironment, both upon the initiation of malignant transformation and during cancer progression (116). Upon upregulating miR-124, a miRNA downregulated in GBM, specifically in glioma cancer stem cells (gCSC), the STAT3 pathway was inhibited, and miR-124 reversed gCSC-mediated immunosuppression of T cell proliferation and induction of regulatory T cells (Treg) (117). Systemic administration of miR-124-transfected T cell transfers exerted potent antiglioma therapeutic effects in murine models of GBM (117).

Human glioma immune activation is potently elicited by a cytokine combination. Cytokines such as IL-1β and TNFα induce the expression of miR-155 and miR-155\*, the miRNAs crucial in immunity and inflammation-induced oncogenesis, and this expression is dose dependently suppressed by IRF3. Importantly, IRF3 also inhibits glioma proliferation, migration, and

Cellular metabolism of malignant tumor is considerably different from that of normal cell, because of limitless proliferation and motility. (119). GBM cells uptake a large amount of glucose, and aerobic glycolysis generates various substrates, such as fatty acids and nucleoti‐ des that are required for rapidly proliferating cells, and is associated with a survival advantage in the tumor cells (Warburg effect). MR spectroscopy analyses have shown that as much as 90% of the glucose converts into lactate by aerobic glycolysis in GBM cells. Clinically, it is known that the levels of lactate are elevated in GBM tissue compared with those in contralateral normal brain tissue. In addition, lactate was shown to be high-grade gliomas than for lowgrade gliomas, and glutamate levels were significantly elevated for GBMs. Pretreatment F18-2 fluoro-2-deoxy-D-glucose (FDG)-PET provides significant additional prognostic information

In normal cells, the 5'-adenosine monophosphate-activated protein kinase (AMPK) pathway is the major cellular sensor of energy availability (121), but its function in cancer is not clear. AMPK is activated by metabolic stress to promote energy conservation and glucose uptake, allowing cells to survive periods of low-energy availability. Allosteric interaction with elevated intracellular AMP, which acts to inhibit dephosphorylation of AMPK (122), and phosphory‐

addition, the expression of IL-6 and COX-2 was suppressed by miR-146a (115).

**3.8. Reprogramming energy metabolism and microRNAs**

invasion (118).

44 Neurooncology - Newer Developments

in high-grade gliomas (120).

Godlewski et al. identified miR-451 as a glioma-expressed miRNA that regulates the balance of proliferation and migration in glioma cells in response to changes in glucose levels (124). miR-451 regulates LKB1 activity through direct targeting of CAB39, a component of the active LKB1 complex. Glucose deprivation was shown to reduce miR-451 levels (124). Thus, miR-451 plays an essential role in LKB1/AMPK signaling in glioma cells. When glucose is sufficient, elevated miR-451 levels lead to reduced LKB1/AMPK pathway activation, which facilitates cell proliferation by allowing unrestrained mTOR activity and reducing apoptosis. In contrast, when glucose is limiting, miR-451 levels decline, allowing for increased CAB39 expression and activation of AMPK by LKB1-mediated phosphorylation. This effect promotes cell survival in response to metabolic stress and activates pathways involved in glioma motility.

Previous reports indicate that Myc is frequently upregulated or amplified in gliomas. As a transcriptional factor, Myc interacts with many tumor-related oncogenes in glioma cells, such as SPARC (125), GAS1 (126), and VEGF (127). c-Myc has been shown to stimulate glutamine metabolism by increasing the expression of amino acid transporters and glutaminase (128). Mechanistically, c-Myc expression has been shown to be suppressed by miR-145, which results in reduced Lin28/Lin28b transcription (129). miR-145 is one of the miRNAs significantly downregulated during malignant transformation in GBMs (130). miR-145 overexpression suppresses the activity of oncogenic proteins Sox9, leading to reduction of cell proliferation and invasion of GBM cells (130). Reduced levels of miR-145 may lead to metabolic remodeling in glioma cells via Sox9, because Sox9 is known as a regulator of c-myc.

mTORC2 regulates c-Myc and glycolysis through FoxO acetylation, and mTORC2 controls FoxO acetylation through class IIa HDACs, independently of Akt. FoxOs antagonize c-Myc (131) by increasing the expression of miR-145 (132) and miR-34c (133), limiting c-Myc mRNA stability and translation. In GBM cells, miR-34c levels were suppressed by FoxO1/FoxO3 knockdown, and miR-34c regulated c-Myc levels in GBM cells (134). The let-7 family has nine members, and previous studies have identified the let-7 miRNA as a tumor suppressor that regulates many important target genes during tumor development (135), such as RAS, Myc, CCND1, LIN28, and HMGA2, which are involved in cell cycle progression and cell stemness. The majority of let-7a miRNA functions in glioma malignancy are believed to involve K-Ras, suggesting that let-7a-mediated manipulation of K-Ras may also be involved in regulating glucose metabolism and GBM cell growth. The M2 isoform of pyruvate kinase (PK) is upre‐ gulated in most cancers, including GBM. The regulation of PKM2 was shown to occur via miR-326 (136). There are four isoforms of PK in mammals, and PKM2 is highly expressed in undifferentiated tissues and tumors. Because PKM2 is regulated by tyrosine phosphorylation and is overexpressed in malignant tumors, PKM2 is considered as key molecule of aerobic glycolysis. In addition, Zhou et al. reported that the human Nin one binding protein (NOB1), which is required for the biogenesis and function of the 26S proteasome and plays a role in RNA metabolism, is a direct target of miR-326 (137).

## **3.9. Evading immune destruction and microRNAs**

In the past, the infiltration of innate and adaptive immune cells into the tumor microenviron‐ ment was considered an immune attack against tumors. However, now, it is widely accepted that immune cells also promote tumor initiation, progression, and metastasis (138). Tumorassociated macrophages (TAMs) control the majority of immunological processes within tumors exerting both regressive (M1) and progressive (M2) effects on tumor development (139). However, the majority of TAMs exhibit an M2-like phenotype. Remarkably, miRs-29b, miR-125a, miR-146a, and miR-155 are involved in the differentiation of TAMs (140). Overex‐ pression of miRNA-155 was shown to attenuate the production of cytokines (IL-6 and TNFα) by suppressing C/EBPβ expression, which led to an inversion of M2 into M1 macrophages (141). M2 TAMs are capable of releasing anti-inflammatory cytokines such as the immuno‐ suppressive cytokine IL-10, which promotes tumor growth (142).

Natural killer T (NKT) cells are a subfraction of T cells. A large number of published studies have demonstrated that NKT cells have miscellaneous functions in immune regulation, one of which is that NKT cells are tumor cell killers, based on the production of antitumor cytokines. In addition to contributing to immune protection, NKT cells are involved in immune tolerance in the body. Tang et al. showed that glioma cells can induce immune-tolerant IL-6+ and IL-10+ NKT cells via miR-92a.

Another study revealed miR-221 and miR-222 as possible regulators of IFN pathways. Type-I IFN receptor activation triggers the JAK-mediated tyrosine phosphorylation of STAT family proteins. Zhang et al. found that STAT1 and STAT2 expression and phosphorylation were upregulated after repression of miR-221/222 in U251 cells (143). Upregulation of STAT pathway is controlled by the IFN-α activation after knockdown of miR-221/222 cluster in U251 glioma cells (143).

NKG2D is one of the major activating receptors of natural killer (NK) cells and binds to several ligands (NKG2DL). NKG2D recognizes different MHC class I-homologous ligands (NKG2DL), including the MHC class I-chain-related molecules A (MICA) and B (MICB) and the UL16- binding proteins (ULBP)1-6 (144), which are also present on the surface of glioma cells (145). Codo et al. reported that miR-20a, miR-93 or miR-106b regulates NKG2DL expres‐ sion in glioma cells (146), suggesting that the expression of miRNA-targeting NKG2DL may contribute to the immune evasion of glioma cells at the level of the NKG2D recognition pathway.

In addition, miRNA regulates immunocheckpoint molecules. miR-138 could bind the 3′-UTR of CTLA-4 and PD-1, and transfection of human CD4+ T cells with miR-138 suppressed expression of CTLA-4 and PD-1 (147). *In vivo* treatment in immunocompetent mice using miR-138 revealed marked tumor regression and prolonged survival time. Moreover, inocu‐ lated tumors showed decrease in intratumoral regulatory T cell, CTLA-4, and PD-1 expression (147). miR-138 exerts antiglioma efficacy by targeting immune checkpoints that may have rapid translational potential as novel immunotherapeutic agents.

On the other hand, miRNA expression is transcriptionally regulated by various cytokines. Ohno et al. analyzed the effect of IFN-β treatment on miR-21 expression in glioma cells and intracranial glioma xenografts (148). Systematic delivery of IFN-β markedly reduced the level of miR-21 in glioma cells 6 hours after the addition of IFN-β.
