MicroRNAs in glioblastoma multiforme pathogenesis and therapeutics

miRNAs in GBM

Many studies have begun to map out the expression profiles and functions of miRNAs in GBM, aiming to garner new insight that can be used to combat this insidious disease. A recent systematic review by Møller et al. 17 found that the most common aberration in miRNA expression with GBM is upregulation. Indeed, 256 miRNAs were found to be significantly overexpressed (notably miR-10b, miR-17–92 cluster, miR-21, and miR-93) and 95 miRNAs were significantly underexpressed (including miR-7, miR-34a, miR-128, and miR-137) in GBM as compared to normal brain tissue 17. Moreover, individual miRNAs have been correlated with different glioma stages. Investigating the miRNA expression profiles of WHO grade II gliomas that spontaneously progressed to WHO grade IV GBM, Malkorn et al. 18 identified 12 miRNAs (miR-9, miR-15a, miR-16, miR-17, miR-19a, miR-20a, miR-21, miR-25, miR-28, miR-130b, miR-140, and miR-210) that were upregulated and two (miR-184 and miR-328) that were downregulated during progression 18. Other studies have additionally found miRNAs that are differentially expressed in advanced clinical stages of GBM, including miR-182, which is upregulated and miR-137, which is downregulated in late stages 19, 20.

Importantly, miRNAs seem to have a predilection for targeting developmental genes, making them fundamental in the regulation of proliferation, differentiation, and apoptosis. In relation to cancer pathobiology, this means that miRNAs impact many of the hallmarks of cancer 21, 22. Indeed, miRNAs have been found to contribute to sustained proliferative signaling and evasion of growth suppressors, resistance to cell death, enabling of replicative immortality, induction of angiogenesis, and activation of invasion and metastasis. Further, miRNAs are also important regulators of drug resistance in GBM. Table 1 provides an overview of miRNAs that are known to participate in GBM, including which pathways they have been found to interact with. In the following sections, we briefly discuss the impact of various miRNAs on these pathways.

Enhancing cell proliferation

The ability to continuously proliferate is a fundamental characteristic of all cancers that is achieved through deregulation of cell signaling pathways. Importantly, miRNAs can influence the promotion of sustained signaling for proliferation as well as the ability to evade growth suppressors, enhancing the expansion capacities of cancer cells.

This is exemplified by survival and proliferation pathways modulated by EGFR and Akt. In GBM, where amplification of EGFR is a characteristic trait of primary tumors, miRNAs involved in EGFR regulation display corresponding disruptions in expression with disease progression. miR-7, which acts as a tumor suppressor, directly targets EGFR and can independently repress the Akt pathway through targeting of its upstream regulators. In GBM, miR-7 is frequently downregulated, allowing for enhanced activation of the Akt pathway, and thus increased viability and invasiveness of tumor cells 23, 24. These protumorigenic effects can be corrected with transfection of mimic miR-7 oligonucleotides, which results in decreased invasiveness and increased apoptosis of GBM cell lines 23, 24.

miR-128 also exerts an antiproliferative effect and is frequently downregulated with GBM 25. Its growth-suppressive functions are mediated through numerous pathways via targeting of (1) EGFR and growth factor receptor PDGFRA 25, (2) WEE1, a regulator of cell cycle progression that can block entry into mitosis through inhibition of CDK1 26, (3) MSI1, which regulates the expression of the NOTCH1 antagonist NUMB, influencing proliferation and the maintenance of stem cells 27, and (4) the transcription factor E2F3A, which is a cell cycle activator 28. Thus, repression of miR-128 in cancer cells can be an advantageous mechanism for overcoming normal proliferative signaling.

However, these pathways can also be targeted by other miRNAs. The E2F transcription factor family plays a pivotal role in the cell cycle. In response to mitogenic signaling, RB1 is phosphorylated by CDK/cyclin complexes leading to activation of E2F-responsive genes, thereby promoting cell cycle progression. This pathway can be suppressed by miR-137 and miR-124a, which inhibit CDK6 expression as well as phosphorylation of RB1. As both miR-137 and miR-124a are consistently downregulated in GBM 29, transfection of either miRNA can prevent proliferation of GBM cells. Further, forced miR-137 overexpression can also mediate MSI1 27 and PTGS2 30, which additionally function to reduce proliferation and invasion.

miR-34a is also persistently downregulated in GBM and has similar targets, repressing CDK6, CCND1, MET, NOTCH1, NOTCH2, and SIRT1 31-34. Of particular importance to proliferative signaling, targeting of CDK6 and CCND1 prevents the downstream prosurvival signaling of the cyclin/CDK pathway. Thus, restoration of miR-34a can reduce CDK6 protein expression, inhibiting cell survival, proliferation, and invasion as well as inducing apoptosis 31, 32.

RAS proteins, which process signals downstream of growth receptors, are also targeted by miRNAs and play a key role in the deregulation of proliferation pathways in many cancers, including GBM. let-7 is an miRNA which is decreased in gliomas and inversely correlates with the presence of RAS proteins. Restoration of let-7 reduces the expression of RAS, resulting in decreased proliferation and migration of tumor cells in vitro and inhibition of tumor growth in vivo. Importantly, transfection of let-7 has no effect on normal human astrocytes, indicating that normal cells are able to more effectively regulate miRNA activity 35.

In addition to miRNA modulation of RAS proteins, RAS proteins can conversely influence miRNA expression. Indeed, miR-21 is a transcriptional target of the AP-1 complex and RAS oncogenes are well-known inducers of AP-1 activity. AP-1 is a transcription factor that regulates a variety of target genes, leading to an increase in cell proliferation, invasion, and angiogenesis during tumor development. AP-1 induces miR-21, which downregulates tumor suppressors PDCD4 and PTEN. Inhibition of PDCD4 then contributes to an increase in AP-1 activity, revealing an AP-1 autoregulatory mechanism in RAS transformation 36. In addition to PDCD4 and PTEN, miR-21 also exerts its prooncogenic effect by downregulating numerous targets including ANP32A, SMARCA4, SPRY2, IGFBP3, LRRFIP1, and RECK. Through these targets, miR-21 can influence numerous biological processes in addition to the promotion of cell cycle progression, including promotion of invasion and metastasis and resistance to chemotherapeutics 37-44. Notably, inhibition of miR-21 expression can repress tumor growth 37, 43, 45.

Resisting cell death

In addition to sustained proliferation, the ability to evade apoptosis is an essential characteristic of cancer. Dysregulation of miRNA expression is one mechanism for allowing cancer cells to bypass signals for programmed cell death. Further, miRNA mediation of apoptosis is strongly linked to drug resistance, as many treatments aim to initiate apoptotic pathways by damaging cancer cells. Thus, the ability to override signals for cell death is critical for not only sustained proliferation but also for acquiring drug immunity.

miRNAs can have either pro- or antiapoptotic functions and are therefore differentially regulated during cancer progression. Antiapoptotic miRNAs target proapoptotic genes and are frequently upregulated with GBM. miR-21 is antiapoptotic and enhances tumor formation by targeting the signaling pathways of P53 and TGF-β as well as the mitochondrial apoptotic pathway 46. Inhibition of miR-21 results in activation of caspases, suspension of cell growth, reduced invasion, increased apoptosis, and enhanced chemosensitivity. These effects are mediated in part by decreased repression of targets including HNRPK, TAP63, and PDCD4 37, 44, 46. Further, miR-21 can modulate the extrinsic apoptotic pathway through downregulation of FASL, an ability that has been particularly evident in cancer stem cells 47. Thus, miR-21 has widespread effects on cell death pathways, making it a critical player in GBM pathogenesis and a promising target for therapeutic interventions.

miR-221 and miR-222 are also overexpressed in GBM and have numerous targets involved in gliomagenesis, including in apoptotic pathways. miR-221/222 can regulate cell death through targeting of p53-upregulated modulator of apoptosis (PUMA). Under normal conditions, PUMA binds Bcl-2 and Bcl-x, rapidly inducing apoptosis. Thus, forced expression of miR-221/222 and the subsequent downregulation of PUMA, promotes cell survival. Therefore, the knockdown of miR-221/222 is able to induce cell death and decrease tumor growth 48, 49. miR-221/222 can also target the cell growth-suppressive CDK inhibitors P27 and P57 50 and thus are tightly linked to cell cycle checkpoints for initiation of S phase. When human glioblastoma U251 cells are treated with antisense miR-221/222, the cell cycle is arrested in G0 or G1 phase 51. Moreover, treatment with antisense oligonucleotides for miR-221/222 enhances the effects of both temozolomide (TMZ) and radiation 49, 52.

miR-335 is also antiapoptotic and is upregulated with GBM. miR-335 targets potential tumor suppressor, disheveled-associated activator of morphogenesis 1 (DAAM1), thereby promoting growth and invasion of astrocytoma cells. Thus, inhibition of miR-335 is able to effectively suppress growth and induce apoptosis of astrocytoma cells both in vitro and in vivo. Indeed, delivery of a miR-335 antagonist to rat glioma C6 cells resulted in growth arrest, activation of apoptosis, repression of invasion, and marked regression of astrocytoma xenografts 53. Contributing to its widespread influence on cellular function, miR-335 also regulates RB1 and controls cell proliferation in a p53-dependent manner 53.

On the opposing side of the spectrum are proapoptotic miRNAs, which target antiapoptotic genes and are often downregulated during GBM progression. miR-218 is proapoptotic and exerts its influence through targeting of CDK6. Thus, its downregulation corresponds with an increase in cellular proliferation with GBM. Exogenous administration of miR-218 can effectively repress expression of CDK6, inhibiting cell proliferation and inducing apoptosis of malignant glioma cells 54. Further, miR-218 was found to sensitize glioma cells to apoptosis by regulating epidermal growth factor receptor coamplified and overexpressed protein (ECOP), which can suppress the transcriptional activity of NFκB, thereby modulating the cellular apoptotic response 55.

miR-451 also acts as a tumor suppressor and is subject to downregulation in GBM tissue. Correcting the loss of miR-451 via administration of miR-451 mimics has been shown to inhibit cell growth, decrease invasive capacity, induce G0/G1 phase arrest, and increase apoptosis of GBM cells. This activity is predicted to result from modulation of the PI3K/Akt pathway as administration of miR-451 mimics results in decreased protein expression of Akt1, Cyclin D1, MMP-2, MMP-9, and Bcl-2 as well as increased expression of p27 56.

Activating invasion and metastasis

While metastasis to other parts of the body is rare, GBM is characterized by extensive, diffuse infiltration throughout the brain. This capacity for pervasive invasion and metastasis is enabled through various mechanisms including modulation of cell-to-cell and cell-to-matrix interactions, degradation and remodeling of the extracellular matrix, cytoskeletal reorganization, and gain of migratory behavior. Epithelial to mesenchymal transition (EMT) is an important part of this process. EMT is characterized by the loss of an epithelial phenotype, including expression of E-cadherin, and the acquisition of mesenchymal markers such as fibronectin, vimentin, and N-cadherin. Many distinct molecular processes occur during EMT including activation of transcription factors, expression of specific cell-surface proteins, reorganization of cytoskeletal proteins, and production of extracellular matrix-degrading enzymes 68. As a result, tumor cells develop increasing invasive and migratory potential. Importantly, these processes are accompanied by changes in expression of many miRNAs, which could serve as potential targets for inhibiting invasion and metastasis.

As previously noted, miR-21 is frequently overexpressed during gliomagenesis and contributes to a variety of protumorigenic pathways. Through the targeting of matrix metalloproteinase (MMP) inhibitors, such as RECK and TIMP3, as well as tumor suppressors including ANP32A and SPRY2, miR-21 can increase the expression and activity of various MMPs, facilitate Ras/Raf binding, and induce ERK phosphorylation, thereby enhancing the invasive potential of GBM cells 40, 69, 70. Administration of antisense oligonucleotides to miR-21 is able to ameliorate these effects, resulting in elevated levels of RECK and TIMP3, a reduction in MMP activity, and decreased migration and invasion of GBM cells 70.

MMPs are also targeted by other miRNAs, including miR-146b. miR-146b inhibits expression of MMP16, an enzyme that functions in proteolysis of extracellular matrix components and is therefore critical for the migration and invasion properties of tumor cells. Thus, miR-146b is often decreased during gliomagenesis, allowing for upregulation of MMP16 71.

miR-10b can also modulate MMPs, indirectly targeting MMP14 as well as urokinase receptor (uPAR) and RhoC through direct suppression of their upstream target HOXD10. Augmented miR-10b expression levels are correlated with higher grade gliomas, 72, 73 and indeed, GBM cells display reduced growth, invasion, and angiogenesis, as well as enhanced cell death when treated with antisense oligonucleotides to miR-10b 73. Further, in an orthotopic human glioma mouse model, inhibition of miR-10b diminished the growth, invasiveness, and angiogenicity of glioma cells in the brain, significantly prolonging the survival of glioma-bearing mice 74.

miR-7 also has many targets involved in metastasis including FAK, PI3K, EGFR, and RAF1 23, 24, 75, 76. As indicated by its targets, miR-7 functions as a tumor suppressor and is downregulated in GBM. Its overexpression can inhibit metastasis and invasion of GBM cells by directly repressing FAK, a mediator of cell-extracellular matrix signaling, as well as by reducing the expression of MMP2 and MMP9, thereby decreasing the ability of GBM cells to move through extracellular matrix 75. Further, by suppressing EGFR expression and inhibiting the Akt pathway, miR-7 can decrease the viability and invasiveness of GBM cells 23.

Inducing angiogenesis

GBM is distinguished from lower grade gliomas by excessive microvascular proliferation, which results in highly vascularized tumors. Enhanced vascularization allows for increased proliferative and invasive capacity of cancer cells due to the greater availability of oxygen and nutrients. Additionally, blood vessels can aid in cell migration, supporting diffuse infiltrative migration of single cells into the brain parenchyma as well as perivascular migration of cells along the microvasculature 77.

A group of miRNAs now termed angiomiRs, have recently been identified as important contributors to neovascularization in GBM 78. These molecules can act on either tumor cells or neighboring tumor-associated cells. miR-296 is an angiomiR that has been found to be upregulated in endothelial cells with the presence of glioma cells or angiogenic growth factors such as VEGF. Augmented expression of miR-296 is associated with increased endothelial cell tube formation and enhanced vascularization of tumors, while knockdown of miR-296 results in reduced tumor angiogenesis 79.

miR-125b is an angiomiR that has conversely been found to be repressed in GBM-associated endothelial cells. This downregulation results in increased expression of its target, myc-associated zinc finger protein (MAZ), a transcription factor that regulates VEGF. Decreased expression of miR-125b in target cells promotes vascularization of tumors 80.

Within GBM tumor cells, hypoxia can also influence miRNA expression as a mechanism for augmenting angiogenesis. Exemplifying this, hypoxia induces expression of miR-210-3p, which directly targets HIF3A, a negative regulator of hypoxic response that acts through downregulation of VEGF. Thus, miR-210-3p overexpression induces HIF, VEGF, and CA9 transcriptional activity, enhancing vasculogenesis. However, inhibition of miR-210-3p under hypoxic conditions prevents HIF-mediated induction of VEGF and CA9, reducing vascular density, and abating growth of tumors in vivo 81.

miR-128 is also able to modulate angiogenesis, functioning through suppression of P70S6K1, a kinase upstream of HIF-1α and VEGF. miR-128 is decreased in gliomas, which promotes angiogenesis, cell proliferation, and tumor growth. Restoration of miR-128 is able to attenuate these effects, while forced expression of P70S6K1 can partly rescue the inhibitory function of miR-128 on cancer growth 82.

miR-93, a member of the miR-17 family and part of the miR-106b-25 cluster, is upregulated in GBM and enhances cell survival, sphere formation, and tumor growth, in part through promotion of angiogenesis. miR-93-expressing cells induce blood vessel formation, potentially through suppression of integrin-β8, a protein involved in cell–cell and cell–matrix interactions. Fang et al. 83 found that vasculogenesis could be enhanced by overexpressing miR-93 in the human glioblastoma cell line U87 and then coculturing the GBM cells with endothelial cells. This resulted in an increase in endothelial cell proliferation and tube formation in vitro and highly increased blood vessel formation in GBM xenograft tumors in mice in vivo 83. These studies illustrate the important role that miRNAs play in the cross-talk between cells, serving as critical mediators in tumor cell modulation of their microenvironment.

miRNAs in GBM drug resistance

Currently, the standard treatment for newly diagnosed GBM is cytoreductive surgery followed by concurrent radiation and chemotherapy with TMZ 2. However, even the most successful treatment plans are only palliative, aiming to delay relapse for as long as possible. A major challenge is that the response to treatments is highly variable across patients. This is largely due to the heterogeneity of tumors, including differences in genotypes and miRNA profiles between each tumor. However, an advantage is that the correlation between miRNA expression levels and GBM progression can be exploited and miRNAs can be used as predictors of treatment response or overall survival. Indeed, Srinivasan et al. 84 found a 10-miRNA signature of GBM tumors that was associated with patient survival. The signature included three miRNAs (miR-20a, miR-106a, and miR-17-5p) that were protective and seven (miR-31, miR-222, miR-148a, miR-221, miR-146b, miR-200b, and miR-193a) that were risky with respect to the association between their expression and patient survival. Additionally, other studies have found that high expression of miR-21, miR-182, and miR-196 as well as low expression of miR-181b and miR-106a are associated with poor patient outcomes 19, 85, 86.

As the standard GBM treatment utilizes radiation and TMZ, many studies have focused on the potential role of miRNAs in resistance to these therapeutics. To identify miRNAs involved in TMZ resistance in GBM, Ujifuku et al. 87 used miRNA microarrays to perform a comprehensive analysis of miRNA expression in a TMZ-sensitive GBM cell line. They found that miR-195, miR-455-3p, and miR-10a^∗ were the three most upregulated miRNAs in resistant cells. Of the three, knockdown of miR-195 had the greatest effect on initiating tumor cell death, significantly enhancing the effectiveness of TMZ. Slaby et al. 88 also examined the correlation between expression levels of selected miRNAs and TMZ resistance in GBM, but used 22 primary GBM tumors instead of cell lines. They found that miR-221, miR-222, miR-181b, miR-181c, and miR-128 were significantly downregulated with GBM, while miR-21 was overexpressed. Downregulation of miR-181b and miR-181c had the strongest correlation with responsiveness to TMZ treatment, indicating that their presence could serve as a predictive marker for response to TMZ therapy.

As miR-21 is consistently upregulated in GBM and targets numerous pathways including those involved in survival, proliferation, invasion, and apoptosis, it is unsurprising that it has also been found to play a role in drug resistance. Shi et al. 89 determined that overexpression of miR-21 significantly inhibited the effect of TMZ on apoptosis, which was mediated through downregulation of proapoptotic proteins Bax and caspase-3 as well as upregulation of antiapoptotic protein Bcl-2. Moreover, numerous other studies have investigated the impact of miR-21 on drug resistance in GBM, finding that inhibiting miR-21 can enhance the chemosensitivity of human GBM cells to TMZ, paclitaxel, sunitinib, doxorubicin, and VM-26 39, 90-94.

miRNAs can also influence drug resistance by targeting the multidrug resistance protein ABCG2 (ATP-binding cassette subfamily G member 2), a transporter which regulates shuttling of substrates across the cellular membrane. miR-328 directly targets ABCG2 and is underexpressed in many cancers, including GBM. This allows for elevated expression of ABCG2, enhancing chemoresistance 95. Moreover, high expression of miR-328 is a protective factor in GBM, while low levels are associated with shorter survival times 96. Thus, restoration of miR-328 may be an effective option for combination therapy with radiation or chemotherapeutics.

Extracellular miRNAs and GBM

miRNAs are present and stable at high levels in body fluid, including serum, plasma, saliva, urine, and milk. Additionally, miRNA profiles and concentrations in body fluids are correlated with pathological conditions such as cancer, suggesting that miRNAs could serve as promising noninvasive prognostic and diagnostic biomarkers 97, 98. Thus, in tumors such as GBM, where it is often difficult to obtain tissue samples, miRNA profiling via liquid biopsies could provide a promising alternative.

Wang et al. 99 characterized plasma-derived miRNA expression profiles, finding that with GBM, miR-21 was upregulated while miR-128 and miR-342-3p were downregulated. Further, expression levels correlated with glioma stage and could be used to distinguish gliomas from other brain tumors such as pituitary adenomas and meningiomas. Roth et al. 100 also explored the blood-derived miRNA profiles of GBM patients, substantiating a decrease in miR-342-3p, but conversely finding that expression of miR-128 was significantly increased with disease. However, a more recent study investigating miR-128 supported Wang et al., finding that it was significantly decreased in the serum of glioma patients prior to surgery, and was elevated post surgery (although still not reaching normal levels) 101.

miR-205 has also been identified as a potential biomarker due to its significantly lower levels in the serum of GBM patients as compared to controls. In a study of 64 glioma patients, serum miR-205 levels displayed a stepwise decrease with ascending tumor grade and additionally increased following surgery and decreased again during tumor recurrence. Further, patients with advanced pathological grade demonstrated longer overall survival times when serum miR-205 levels were elevated 101, 102. Thus, serum miRNAs could serve as critical indicators of disease progression and outcome.

The mechanisms for release of miRNAs from cancer cells as well as for crossing the blood–brain barrier to enter systemic circulation are not completely understood. Potential means of release and stabilization include binding of miRNAs to protein complexes or packaging within extracellular vesicles. Studies investigating the presence of extracellular miRNAs have found that a vast majority of those present in blood or cell culture media are independent of exosomes and are bound to Ago2, which enhances their stability 103, 104. However, the selection mechanism for release of miRNAs into extracellular space has yet to be elucidated. Turchinovich et al. 103 proposed that instead of being actively released, large portions of circulating miRNAs could be derived from dead or dying cells.

In addition to protein attachment, horizontal transfer of miRNAs from cancer cells could be facilitated by extracellular vesicles (EV), which are able to protect proteins and nucleotides from degradation 105. Although it has been suggested that a minority of miRNAs in circulation are found in EVs, these vesicles may represent a potent means of paracrine signaling to nearby cells. Cancer cells are able to use EVs to export selected molecules as a mechanism for modulating the tumor microenvironment and establishing premetastatic niches, thereby facilitating dissemination and distant engraftment. The extracellular presence of miRNAs suggests that they may be involved in this process, contributing to the metastatic potential of cancer cells and the mediation of communication between cancer cells and their environment.

Extracellular vesicles such as exosomes and microvesicles, are among the most well-studied mechanisms of lateral transfer. While microvesicles bud directly from the plasma membrane, exosomes are formed from intraluminal vesicles, which bud into early endosomes and then form multivesicular endosomes (MVE). MVEs are then released by either fusion with the plasma membrane or direct release from the plasma membrane 106. Importantly, the release of miRNAs via exosomes is selective, based on factors such as cell of origin and malignancy. For example, while EVs derived from dendritic cells contain costimulatory proteins necessary for T-cell activation, the content of vesicles from tumor cells more frequently aid in tumor growth and invasiveness 107. Additionally, Pigati et al. 108 found that approximately 30% of the miRNAs released into exosomes did not reflect the intracellular profile. In particular, a majority of the miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, while the majority of these same miRNAs produced by nonmalignant mammary epithelial cells were retained, indicating that there is a selection mechanism for miRNA release 108. Moreover, selectively exported miRNAs from malignant cells may be packaged in structures that differ from normal cells and further, some miRNAs exclusively associate with certain vesicles 109.

The formation of EVs to transport cellular information has been confirmed in GBM. Cultured primary cells from GBM tumors were found to be covered with microvesicles of various sizes, containing both RNA and proteins 107. Moreover, the exosomes from GBM tumors were internalized by endosome-like structures of brain endothelial cells, where they were able to stimulate tubule formation. This confirmed that exosomes can serve as a means of intercellular communication and thus are a critical tool for tumor cells to use in influencing proximal cells 107. GBM microvesicles were also able to stimulate the proliferation of human glioma cell lines, indicating a potential self-promoting function 107.

miRNAs released from tumor cells have also been shown to modulate the tumor microenvironment. Tominaga et al. 110 discovered that miR-181c was significantly upregulated in EVs from breast cancer cells, particularly in brain metastatic breast cancer. Further, miR-181c was able to trigger the breakdown of an in vitro model of the blood–brain barrier. EVs from the cancer cells were selectively incorporated into endothelial cells (although not into pericytes or astrocytes), where miR-181c suppressed PDPK1, initiating downregulation of phosphorylated cofilin. This led to abnormal localization of actin and thus weakening of the blood–brain barrier 110. However, miR-181c has also been shown to act as a tumor suppressor, undergoing downregulation in GBM 111. This suggests that miRNAs may have different functions depending on the cancer type and microenvironment.

An additional example of miRNA regulation of the tumor microenvironment is demonstrated by miR-105. miR-105 released from cancer cells is able to destroy tight junctions through inhibition of tight junction protein ZO-1. Downregulation or loss of tight junctions contributes to cancer progression by altering cell migration, proliferation, polarity, and differentiation. Further, overexpression of miR-105 in nonmetastatic cancer cells induces metastasis as well as vascular permeability in distant organs. Fittingly, miR-105 can be detected in circulation at the premetastatic stage. Moreover, its levels in the blood and tumor are associated with ZO-1 expression and metastatic progression in early-stage breast cancer 112.

It is important to note that exosomal transfer of miRNAs is not exclusively involved in cancer promotion and can also serve as a physiological mechanism for suppressing cancer proliferation. Kosaka et al. 113 demonstrated that noncancerous cells can secrete and transfer antiproliferative miR-143 exclusively to cancer cells where miR-143 is deregulated, thereby suppressing their proliferation. As cells with normal levels of miR-143 were not affected by its overexpression, it appears that there may be a threshold for miRNA activity or that normal cells are more effectively able to regulate miRNAs.

miRNA-based therapeutics

Increasing evidence demonstrates that aberrant miRNA expression profiles and signaling pathways are present in many cancers. As these molecules have the ability to target several genes within the same pathway or even multiple oncogenic pathways, miRNAs represent promising therapeutic targets with potential for more comprehensive benefits than other targets with more limited activities. Importantly, miRNAs can function as either oncogenes or tumor suppressors, which then correspond to two different approaches to miRNA-targeted therapy. These methods aim to either (1) reduce the expression of target miRNAs via antisense technologies or miRNA sponges or (2) restore miRNA expression using synthetic mimics or gene replacement therapy. There are currently a multitude of different strategies being investigated under each category.

Antisense oligonucleotides

Overexpressed intracellular miRNAs can be inhibited via administration of synthetic antisense, single-stranded RNA-based oligonucleotides, termed antagomirs or antimiRs. Antagomirs are complementary to mature endogenous miRNAs, allowing for binding and silencing of their targets (Fig. 2A). Efficient inhibition by antagomirs requires optimization of the oligonucleotides for high binding affinity, high resistance to nuclease degradation, low toxicity, and efficient in vivo delivery. These factors can be modified through alterations of the sugar, the nucleobase, the internucleotide linkages, or with the addition of nonnucleotide modifiers 114.

Phosphorothioate oligodeoxynucleotides were the most common type of first-generation antisense oligonucleotides, having high resistance to nucleases but low binding stability and high toxicity. These were followed by 2′-O-methyl-modified oligonucleotides, which displayed increased binding stability and reduced nonspecific effects, but suboptimal efficiency 115. 2′-O-methyl-modified oligonucleotides were further improved on by the development of locked nucleic acid (LNA)-modified oligonucleotides, which have higher stability, efficacy, and specificity as well as lower toxicity 116, 117.

LNAs are modified, conformationally locked nucleotide analogs with a high level of affinity to DNA and RNA nucleotides and high nuclease resistance 118. LNA-linked oligonucleotides are currently the most widely used class of antagomirs and have shown promise for treating various conditions. Indeed, a PBS-formulated LNA antagomir was found to be effective at reducing serum cholesterol levels in African green monkeys. As miR-122 plays a critical role in fat and lipid metabolism, the study utilized an LNA-antagomir against liver-expressed miR-122. Upon administration, the antagomir was taken into the cytoplasm of hepatocytes, where it was able to successfully deplete miR-122. This was accompanied by effective lowering of plasma cholesterol levels and no evidence of toxicity or histopathological changes 119.

LNA-based antagomirs have also been utilized in studies targeting hepatitis C virus (HCV), which requires miR-122 for replication. Treatment with LNA-based anti-miR-122 oligonucleotides was able to suppress viremia and improve HCV-induced liver pathology in chimpanzees, also without negative side effects 120. Subsequent studies resulted in the development of miravirsen, an LNA-modified antisense oligonucleotide against miR-122, which was the first miRNA-targeting drug in clinical use. Phase I and II clinical trials evaluated the safety and efficacy of miravirsen, finding that patients had a dose-dependent reduction in HCV levels and no adverse side effects 121. However, as miRNAs have widespread impact and can function in various pathways, the long-term effects of antagomirs must be more thoroughly investigated. Notably, in other contexts, miR-122 has been shown to function as a tumor suppressor, including in GBM, where decreased expression of miR-122 is associated with decreased patient survival 122. Thus, antagonizing miRNAs likely has different consequences depending on the disease context.

Beyond determining long-term effects, one of the greatest challenges with miRNA-based therapeutics is finding effective delivery systems, particularly to the brain. There are various obstacles to efficient delivery including cellular resistance to the uptake of oligonucleotides, sequestration of treatments in the liver, and the blood–brain barrier, which prevents the delivery of most drugs to the brain. A recent study by Song et al. 123 addressed the first issue, finding that R3V6 peptides were able to protect anti-miR-21 oligonucleotides from nucleases, while delivering the treatment into the cells more efficiently than other modifications, including polyethylenimine. The peptide was also effective at reducing miR-21 levels and inducing apoptosis in GBM cells, suggesting that the R3V6 peptide may be a useful carrier for antisense oligonucleotides 123. Other modifications have also been tested, including cholesterol, which allows for injection of antagomirs. However, cholesterol-conjugated miRNAs are not able to access all tissues, primarily accumulating in the liver 124. Thus, much work remains to be done on optimization of delivery strategies.

Challenges

Despite advances in understanding of both miRNAs and GBM, there are still major difficulties to overcome before widespread application of miRNA-based treatments becomes feasible. The lack of an effective delivery system with enough specificity and efficacy is one of the greatest challenges in utilizing miRNAs for therapeutics. This is particularly relevant for GBM, where both the blood–brain and blood–tumor barriers prevent the accumulation of therapeutic drug concentrations in brain tumors. However, with the development of more accurate in vitro models of the blood–brain barrier, we have the opportunity to more rapidly and easily screen new delivery mechanisms for potential in penetrating the blood–brain barrier and specifically targeting tumor sites 140. Alternatively, emphasis on the development of more effective local delivery techniques that can bypass the blood–brain barrier could also offer more efficacious solutions 141.

Additional challenges could arise from the ability of miRNAs to engage with multiple targets. While offering many advantages, this ability is a double-edged sword. The potential for widespread effects increases the risk of deleterious repercussions from impact on unintended targets. Additionally, as mRNAs can be targeted by numerous different miRNAs, compensation mechanisms through alternative pathways could compromise treatment outcomes.

Finally, tumor heterogeneity is a major challenge as there are substantial variations across individuals as well as between different cells within tumors. This includes the presence of GSCs, which can dramatically impact tumor aggressiveness and resistance to treatments. Thus, each tumor may respond differently to the same therapeutic strategies, necessitating a need for more individualized treatment options. With the increasing ease in obtaining genomic information for each patient, miRNA profiling could be used as a critical factor for evaluating response to treatment. Thus, more comprehensive characterization of potential predictive and prognostic miRNA markers is essential to optimizing personalized treatments for GBM.