ABBREVIATIONS:
- ATM
- ataxia telangiectasia mutated kinase
- GBM
- glioblastoma
- GSC
- GBM stem cell
- HIF
- hypoxia-inducible transcription factor
- lncRNA
- long noncoding RNA
- mRNA
- messenger RNA
- miRNA
- microRNA
- RNAi
- RNA interference
- TF
- transcription factors
- TMZ
- temozolomide.
Glioblastoma (GBM), the most common malignant primary brain tumor in adults, is one of the most genomically defined forms of cancer.1-4 Mutational burden in coding genes that occur above background frequencies have likely been delineated.5 However, advances in the genomic characterization of GBM, including at the single cell level,6-9 have not resulted in an appreciable improvement in patient survival. Despite advances in multimodal therapeutic strategies encompassing surgical resection, focused radiation, and various cytotoxic chemotherapy regimens, the survival of most patients with GBM has not improved in the past 50 years, demonstrating the need for alternative therapeutic targets.
In GBM, significant progress has been made in defining the functions of protein-coding genes10 and microRNA (miRNA).11,12 However, the coding genome accounts for less than 2% of all DNA sequences.13-15 In fact, large-scale cancer genomics projects have elucidated that aberrations within the noncoding genome also play a significant role in driving cancer phenotypes. For example, several mutations and copy-number changes found in cancer are frequently located in noncoding DNA,16,17 with profound effects on the expression of noncoding RNAs.
Long noncoding RNA (lncRNAs) are functionally defined as greater than 200 base pairs (bp) in length with no protein coding potential. Although they do not encode protein, lncRNAs have been shown to regulate diverse biological processes, including chromatin structure remodeling and histone modifications,18 activation and repression of gene expression,19 messenger RNA (mRNA) stability,20 and modulation of signal transduction.21 For example, in GBM specifically, lncRNAS are known to produce noncoding transcripts that regulate multiple hallmark biological characteristics of cancer.22 Because the majority of lncRNAs are expressed in a tissue- and cell type–specific manner,23,24 they are evolving to be increasingly promising diagnostic markers and attractive targets for efficacious cancer treatment. Herein, we review the biological roles of lncRNAs in GBM and potential avenues for novel therapeutics.
MOLECULAR MECHANISMS OF lncRNAs
lncRNA functionality can be characterized depending on cellular localization. Nuclear lncRNAs are involved in chromatin interactions,25 transcriptional regulation,19,26-28 and RNA processing.29 Nuclear regulation by lncRNAs can be accomplished on multiple levels (Figure 1). First, lncRNAs can directly converge on chromatin structure to activate or silence multiple genes by acting as a scaffold and interacting with DNA and chromatin-modifying proteins, subsequently recruiting them to specific target regions (Figure 1A).25,30,31 Second, lncRNAs can bind DNA and recruit proteins to initiate DNA looping, resulting in superenhancer formation (Figure 1B).32 Third, antisense binding of lncRNAs to promoter regions can directly activate binding of transcriptional machinery (Figure 1C). Finally, lncRNAs can act as decoy molecules, by binding to transcription factors and thus suppress their transcriptional activity (Figure 1D).
;)
FIGURE 1.: Mechanisms of lncRNA regulation in the nucleus. A, lncRNAs can converge on chromatin structure and act as scaffold to recruit chromatin-modifying proteins to activate or silence gene expression. B, lncRNAs can bind to DNA enhancer regions and recruit proteins to initiate DNA looping and superenhancer formation to promote gene expression. C, Antisense binding of lncRNAs to promoter regions can activate binding of transcriptional machinery to induce gene expression of target genes. D, lncRNAs can act as decoy molecules by binding to TF and thus suppress their transcriptional activity. lncRNA, long noncoding RNA; TF, transcription factors.
By contrast, cytoplasmic lncRNAs modulate mRNA stability,20 translation,33 and signal transduction (Figure 2).21 In post-transcriptional regulation, lncRNAs function as molecular sponges, sequestering miRNAs to reduce their regulatory effect on target mRNA. For instance, the critical tumor suppressor PTEN is regulated by the lncRNA PTENP1 through an RNA-dependent mechanism, in which binding and sequestering PTEN-targeting miRNA promotes PTEN expression.34-36 During post-translational regulation, lncRNAs bind directly to target mRNA transcripts, activating or reducing their ability to form ribosomal complexes required for translation. Furthermore, lncRNAs can also bind to individual proteins or protein complexes to mask regulatory phosphorylation sites or facilitate protein interactions leading to sustained signaling cascades. Thus, lncRNA molecular mechanisms have profound effects on key biological processes, critical in cancer pathogenesis.
FIGURE 2.: Mechanisms of lncRNA regulation in the cytoplasm. A, lncRNAs can function as molecular sponges by binding to and sequestering microRNAs, and thus reducing their regulatory effect on target mRNA. B, During post-translational regulation, lncRNAs bind directly to mRNA to recruit (top panel) or inhibit (bottom panel) binding of ribosomal complexes required for translation. C, lncRNAs can bind to target proteins to mask phosphorylation sites for protein kinases. D, lncRNAs can facilitate protein complex formation leading to sustained protein kinase signaling cascades. lncRNA, long noncoding RNA; mRNA, messenger RNA; miRNA, microRNA.
lncRNAs IN GBM INVASION AND PROLIFERATION
Infiltrative growth of malignant cells invading normal brain parenchyma is a hallmark of GBM.37 Targeting key molecular pathways that contribute to the proliferation and invasion of GBM, while sparing normal brain counterparts, may lead to improvement in patient survival. Several lncRNAs demonstrate critical roles in GBM tumor cell proliferation and invasion, the entirety of which are beyond the scope of this review. Herein, we include key examples of lncRNAS that have well-established roles in GBM proliferation and invasion: EGFR-AS1, NEAT1, ATB, HOTAIR, and HMMR-AS1.
EGFR-AS1 is a lncRNA transcribed from the antisense strand of the epidermal growth factor receptor (EGFR) gene, which is amplified, mutated, or overexpressed in up to 60% of GBMs.2 EGFR-AS1 acts as a molecular sponge for the miRNA miR-133b, which in turn downregulates the RACK1 protein. RACK1 is upregulated in multiple human tumors and has been shown to have a role in glioma progression. In preclinical in vivo models, targeting of EGFR-AS1 resulted in decreased invasion and proliferation of GBM cells.38
The lncRNA NEAT1 is known to promote tumor growth in several cancers.39 In GBM, NEAT1 promotes migration, invasion, and proliferation of GBM cells by suppressing expression of the miRNA mir-132, which negatively regulates sex determining region Y-box protein (SOX2).40 SOX2 is a transcription factor involved in pluripotency induction that is also critical for GBM stem cell (GSC) survival, proliferation, and invasion. NEAT1 also targets the miRNA let-7e, which in turn targets the oncogene NRAS. let-7e expression is downregulated in GBM. Importantly, restoration of let-7e suppresses GSC proliferation, migration, and invasion.41
Quite interestingly, glioma cell–derived exosomes shuttle the lncRNA ATB to astrocytes, which in turn promote the migration and invasion of glioma cells.42 Further study revealed ATB promotes glioma cell invasion through the NF-κB and P38/MAPK pathways.43
The lncRNA HOTAIR is overexpressed in GBM and it also controls GBM cell proliferation.44,45 A diagnostic and prognostic marker in GBM,46 HOTAIR primarily influences oncogenic programs through chromatin remodeling.47 Targeting of HOTAIR has been shown to lead to decreased GBM tumor growth in preclinical in vivo models.48
HMMR is a highly expressed oncogene in GBM that promotes tumor growth. The antisense lncRNA, HMMR-AS1, stabilizes the expression of HMMR oncogene.49 Targeting of the lncRNA HMMR-AS1 resulted in inhibition of cell migration and invasion, and decreased GBM cell growth in both in vitro and in vivo preclinical models. Importantly, targeting HMMR-AS1 also resulted in decreased expression of ataxia telangiectasia mutated kinase, RAD51, and BMI1, which are required for efficient homologous repair of DNA double-strand breaks.
lncRNAs IN GBM ANGIOGENESIS
Angiogenesis, characterized by the formation of new blood vessels from pre-existing ones, involves the migration, proliferation, and differentiation of endothelial cells.50 As a prerequisite for tumor progression and invasion, angiogenesis fulfills the nutrient and oxygen requirements of tumors.22,51 Tumor angiogenesis is a complex process triggered by vascular endothelial growth factor (VEGF).52,53 Hypoxia-inducible transcription factors (HIFs), which act upstream of VEGF, are critical in the physiological adaptation to differential oxygenation states in tumorigenesis.54 HIF1-α also plays a pivotal role in tumor-associated angiogenesis in GBM.55 While angiogenesis can develop from oncogene activation, lncRNAs have been found to serve crucial downstream regulatory functions of gene expression during angiogenesis in GBM.
In addition to promoting GBM proliferation, migration, and invasion, the lncRNA HOTAIR plays a role in the regulation of GBM angiogenesis by directly targeting the VEGF promoter and inducing VEGF transcription.56 Inhibition of HOTAIR has been shown to impede glioma-induced endothelial cell proliferation, migration, and tube formation. HOTAIR may also regulate angiogenesis by transmission into endothelial cells via GBM cell–derived extracellular vesicles. Likewise, lncRNA H19 was found to directly bind the miRNA miR-138, which normally inhibits HIF1 and VEGF, thus inhibiting its function and promoting GBM angiogenesis.57 First-line use of bevacizumab, a VEGF inhibitor, does not improve overall survival in patients with newly diagnosed GBM.58 However, targeting lncRNAs involved in angiogenesis, in addition to other key aspects of GBM pathophysiology, may provide a superior therapeutic alternative in patients with GBM.
lncRNAs IN GSCs
Although defined by a heterogeneous population of tumor cells, GBM is postulated to be driven by a subpopulation of GBM cells, known as GSCs. GSCs retain stem-cell characteristics including self-renewal and are potentially responsible for tumor maintenance, recurrence, and resistance to treatment.59 Bulk tumor transcriptomic characterization of GBM resulted in the identification of several distinct cellular subtypes, known as mesenchymal (M), proneural (P), neural (N), and classical (C).3 Two distinct subtypes of GSCs are thought to be derived from the proneural and mesenchymal subtypes.60-62 Importantly, mesenchymal GSCs are considered the most malignant and therapy resistant. The lncRNA LINC01057, which is overexpressed in GBM, is critical in maintaining the mesenchymal subtype transcriptomic signature.63 Targeting LINC01057 suppresses GBM proliferation and invasion, whereas its overexpression promotes mesenchymal differentiation in preclinical models of GBM. Further studies have also elucidated the functional roles of other lncRNAs within GSCs in the tumor microenvironment. The lncRNA HIF1A-AS2, for instance, was found to be upregulated in mesenchymal GSCs, and its inhibition decreased GSC growth, self-renewal, and hypoxia-dependent molecular reprogramming.64 This suggests that HIF1A-AS2 acts in a GBM anatomic site–dependent fashion to control adaptation to hypoxic niches within the tumor.
lncRNAs IN RADIATION AND TEMOZOLOMIDE RESISTANCE IN GBM
The current standard of care for GBM is maximal safe surgical resection, followed by radiation therapy and concomitant temozolomide (TMZ) chemotherapy.65,66 Despite initial response in the majority of patients, acquired resistance to the radiation and TMZ treatment is inevitable, after which patient survival decreases to less than 6 months.67 Several factors have been implicated in the mechanism of GBM radioresistance, including tumor microenvironment, hypoxia, metabolic alteration, glioma stem cells, tumor heterogeneity, miRNAs, cell cycle, and DNA damage and repair.68 TMZ induces tumor cell death by DNA alkylation of the O6 position of guanine resulting in cross-linking between adjacent strands of DNA and subsequent inhibition of DNA replication. The canonical mechanism of resistance to TMZ involves the expression and activity of the DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). MGMT inhibits the activity of TMZ by removing alkylation at the O6 position of guanine. Silencing of MGMT expression by promoter methylation is a predictor of response to TMZ in GBM.69
Several lncRNAs are involved in resistance to radiation and TMZ therapy. The lncRNAs HMMR-AS1 and TALNEC2 confer radiation resistance, which can be rescued by silencing of these transcripts in preclinical models of GBM.49,70 The lncRNA AHIF, found to be upregulated both in response to radiation and radioresistant GBM cells, both confers resistance to radiation and transfers resistance to neighboring tumor cells through exosome transmission.71 Glioma stem cells, thought to be the primary source of radiation resistance, enhance radioresistance through the lncRNA PCAT1.72
TMZ resistance is regulated by several lncRNAs in GBM. NEAT1 lncRNA, in addition to promoting invasion and proliferation, also promotes TMZ resistance through downregulating the miRNA let-7g-5p, which activates the MAP3K1 pathway that is known to regulate tumorigenesis and drug resistance.73 The lncRNA TP73-AS1 promotes TMZ resistance in GSCs through the regulation of therapy resistance marker ALH1A1.74 TMZ-resistant GBM cells also show an upregulation of lncRNA MALAT1; silencing of the MALAT1 transcript rescues TMZ sensitivity.75 Finally, the transcriptional coactivator, TRIM24, promotes glioma progression through activation of the PI3K pathway and increases chemoresistance in GBM.76 The lncRNA NCK1-AS1 targets the miRNA miR-137, which normally downregulates TRIM24 expression, thereby inducing PI3K activation and TMZ resistance.77
lncRNAs IN GBM IMMUNE MODULATION
lncRNAs have significant roles in controlling innate and adaptive immune response, as well as immune cell differentiation.78 It is well established that the GBM tumor microenvironment, which includes immune infiltrating cells, constitutes a critical role in supporting its malignant growth and progression.79,80 An in silico analysis of GBM patient RNA from the Tumor Cancer Genome Atlas identified a 6-lncRNA signature related to immunity that stratified patients into high- and low-risk groups with significantly different survival.81 This 6-lncRNA signature was found to be a positive prognostic indicator and significantly enriched in immune-related pathways. A similar study of Tumor Cancer Genome Atlas low-grade glioma patient samples found 16 immune-related lncRNAs, involved in chemokine signaling pathways and cytokine-cytokine receptor interactions, which strongly correlated with positive patient prognosis.82 Additional studies have started to characterize the role of specific lncRNAs involved in GBM immune processes. For instance, the DGCR5 lncRNA independently predicted worse prognosis, because it was found to be downregulated in GBM and significantly associated with immune infiltration.83 The recently identified lncRNA, INCR1, was shown to regulate interferon gamma signaling and inhibit T-cell–mediated tumor cell death in GBM.84 Taken together, these studies link GBM lncRNAs to patient survival and immune-related biological processes and provide the basis for future studies into immunotherapy-related targets and novel prognostic biomarkers that may predict patient response to immunotherapy.
CLINICAL IMPLICATIONS OF lncRNAs IN GBM
The tissue-specific expression of lncRNAs makes them attractive targets for clinical translation as GBM biomarkers and potential therapeutic targets. For example, a systematic, large-scale clustered regularly interspaced short palindromic repeats interference screen identified nearly 500 lncRNA loci required for both cancer and induced pluripotent stem-cell growth.85 Approximately 90% of the lncRNA genes identified altered growth in a single cell type and, importantly, no single lncRNA was required for growth in all cell types tested. By contrast, similar large-scale screening for protein-coding genes showed that an indispensible gene in a particular cell lineage is likely to be required in other cell lineages.86,87 Improved clinical responses may be observed by targeting lncRNAs that are implicated in multiple aspects of GBM pathophysiology (Table 1). For instance, targeting HOTAIR would potentially result in decreases in GBM tumor growth and angiogenesis. In an analogous fashion, targeting NEAT1 may result in decreases in invasion, proliferation, and resistance to TMZ. Taken together, the exquisite cell type specificity and roles of lncRNA function have clear implications for targeted therapy.
TABLE 1. -
lncRNAs Involved in Key Aspects of
GBM Pathophysiology
| LncRNA |
Role(s) in GBM |
Mechanism |
| EGFR-AS1 |
• Invasion and proliferation |
• Binds miR-133b as a molecular sponge, inhibiting miR-133b from downregulating RACK1, which promotes invasion and proliferation.
38
|
| NEAT1 |
• Invasion and proliferation
• TMZ resistance |
• Suppresses miRNA mir-132, which negatively regulates GSC transcription factor SOX2.
40
• Downregulates the miRNA let-7e, which targets the oncogene NRAS for proliferation and invasion.
41
• Suppresses the miRNA let-7g-5p, which activates the MAP3K1 pathway.
73
|
| ATB |
Invasion |
• Shuttled via exosomes to activate astrocytes to promote invasion.
42
• Promotes invasion by activating the NF-κB and P38/MAPK pathways.
43
|
| HOTAIR |
• Invasion and proliferation
• Angiogenesis |
• Promotes proliferation and invasion via chromatin remodeling.
47
• Induces VEGF mRNA expression to induce angiogenesis.
56
|
| HMMR-AS1 |
• Promotes tumor growth
• Radiation resistance |
• Stabilizes HMMR oncogene mRNA
• Promotes ATM, RAD51, and BMI1 expression to repair DNA double-stranded breaks.
49
|
| H19 |
• Angiogenesis |
• Sequesters miR-138 promoting HIF1 and VEGF expression.
57
|
| LINC01057 |
• GSC maintenance |
• Promotes mesenchymal GSC differentiation by activating NF-kB signaling.
63
|
| HIF1A-AS2 |
• GSC growth and adaptation to hypoxia |
• Interacts with IGF2BP2 and DHX9 to maintain expression of HMGA1.
64
|
| TALNEC2 |
• GSC maintenance and radiation resistance |
• Regulates expression of several miRNA to maintain GSCs and response to radiation.
70
|
| AHIF |
• Invasion and radiation resistance |
• Promotes invasion factors VEGF and angiogenin, and decreases expression of anti-apoptotic factors Bcl-2, Bcl-xl, and Mcl-1.
71
|
| PCAT1 |
• Radiation resistance |
• Binds miR-129-5p to promote HMGB1 expression.
72
|
| TP73-AS1 |
• TMZ resistance |
• Regulates transcriptional profile related to metabolism, mitochondria, and nucleotide metabolism.
74
|
| MALAT1 |
• TMZ resistance |
• Directly binds and suppresses mir-101. Mir-101 reverses TMZ resistance by inhibition of GSK3β in GBM.
75
|
| NCK1-AS1 |
• TMZ resistance |
• Targets miR-137, which downregulates TRIM24, thereby activating PI3K activation and TMZ resistance.
77
|
| INCR1 |
• Immune modulation |
• Regulates interferon gamma signaling to inhibit T-cell–mediated GBM cell death.
84
|
ATM, ataxia telangiectasia mutated kinase; GBM, glioblastoma; GSC, GBM stem cell; lncRNA, long noncoding RNA; mRNA, messenger RNA; miRNA, microRNA; TMZ, temozolomide; VEGF, vascular endothelial growth factor.
lncRNAs can be therapeutically targeted using an RNA interference (RNAi) approach, which uses chemically synthesized short sequences of complementary RNA that bind targets provoking RNA degradation. Synthetic RNAs are relatively inexpensive to produce, in contrast to chemotherapy drugs, and thus can be developed more rapidly. Recent advances in both our understanding of lncRNA biology and the chemical stabilization of RNA and nanodelivery methods provide the landscape for a panoply of actionable therapeutic targets. Key obstacles will be in determining the safety, method, and location to deliver RNAi oligonucleotides targeting lncRNAs in patients with GBM. Should lncRNAs be delivered by peripheral injection, into the resection cavity, or via intraventricular injection? Recent work suggests that oligonucleotides can be delivered to orthotopic infiltrating GBM in preclinical mouse models via intratumoral, systemic intravenous, and subcutaneous injections.88-90 Of note, the overall efficacy of RNAi-based therapy depends largely on the invasiveness, vascularization, and cellular properties of any given preclinical orthotopic GBM model. Appropriate pharmacokinetic and toxicology preclinical studies will be required before the clinical translation of RNAi targeting lncRNAs in GBM.
CONCLUSION
lncRNAs, despite the relative infancy of their study, are becoming increasingly recognized as key factors in regulating several hallmarks of GBM pathophysiology including proliferation, invasion, radiation and TMZ resistance, and immune modulation (Figure 3, Table 1). Tissue-specific expression of lncRNAs, and their diverse functional repertoire, renders them exciting putative candidates for biomarker development and therapeutic targeting. Although our understanding of lncRNA biology in GBM is increasing, gene therapy targeting lncRNAs is still in its infancy. lncRNA-targeted drug design provides a promising opportunity to transform the care of patients with GBM. The specificity of lncRNAs provides myriad translational opportunities; however, safety and efficient delivery are challenges that will need to be overcome. Nevertheless, the data are emerging that targeting lncRNAs will enable new options for precision medicine in GBM.
FIGURE 3.: lncRNA involvement in key aspects of
GBM pathophysiology. (1) lncRNAs promote angiogenesis through transcriptional activation of VEGF and miRNA regulation. (2) Invasion and proliferation of
GBM cells is promoted by lncRNAs via miRNA regulation, activation of NF-kB and MAPK signaling pathways, mRNA stabilization, and chromatin remodeling. (3)
GBM stem cells are maintained by lncRNAs by activation of NF-kB signaling and maintenance of HMGA1 mRNA expression. (4) Immune modulation by INCR1
lncRNA occurs by regulation of interferon gamma signaling to inhibit T-cell–mediated
GBM cell death. (5) Radiation and temozolomide resistance is mediated by lncRNAs via regulation of miRNAs and transcriptional regulation of DNA repair enzymes. See Table
1 for additional details.
GBM,
glioblastoma;
lncRNA,
long noncoding RNA; mRNA, messenger RNA; miRNA, microRNA; TMZ, temozolomide; VEGF, vascular endothelial growth factor.
Funding
This study did not receive any funding or financial support. Genaro Rodriguez Villa is supported by NREF & StacheStrong Research Fellowship Grant on behalf of the AANS/CNS Section on Tumors.
Disclosures
Ennio Antonio Chiocca is an inventor on a patent application covering the use of the long noncoding RNA, NEAT1, as a method for predicting treatment outcome to checkpoint inhibitors in cancer. The other author has no personal, financial, or institutional interest in any of the drugs, materials, or devices described in this article.
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COMMENTS
New biological understanding provides potent insight into pathobiology and potential putative therapeutics for a number neurological disorders, including glioblastoma (GBM). Recently, long noncoding RNAs (lncRNAs) have been shown to play a potentially important role in GBM pathobiology and therapeutic resistance. The authors of this review provide a clear, succinct, and neurosurgeon-directed description of the molecular mechanisms of lncRNAs, as well as the impact that lncRNAs may play in GBM pathogenicity, progression, angiogenesis, tumor-associated pluripotent cells, and therapeutic resistance. Finally, the authors define critical clinical implications for lncRNAs in GBM. Understanding of the impact of ribonucleic acids in both normal and disease states is continuing to rapidly expand. While understanding of microRNA1a,2a and short-interfering RNAs3a,4a continues to emerge, lncRNA molecules are an exciting new focus for cancer researchers, including those who study GBM. The translation of lncRNA biological knowledge into putative therapeutics has not yet been realized because of the novelty of understanding and because of the diverse roles they play in GBM tumorigenesis. lncRNAs impact multiple facets of GBM biology, including invasiveness, aggressiveness, and chemoresistance, and are clearly potential targets for future therapies.5a-7a Targeting these molecules may be beneficial as either monotherapy for GBM, or in combinatory therapies. Given the difficulty in improving long-term survival in patients with GBM, new avenues of research are important to explore. This review provides an important update for neurosurgeons to understand the emerging impact of lncRNAs on GBM pathogenesis and potential directed therapies. By concisely appraising the literature, this review offers the neurosurgeon a rapid review of the most important feature of lncRNAs in GBM.
Daniel Kreatsoulas
Russell R. Lonser
Columbus, Ohio, USA
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