The role of lncRNAs in the interplay of signaling pathways and epigenetic mechanisms in glioma

ABSTRACT

Gliomas, highly aggressive tumors of the central nervous system, present overwhelming challenges due to their heterogeneity and therapeutic resistance. Glioblastoma multiforme (GBM), the most malignant form, underscores this clinical urgency due to dismal prognosis despite aggressive treatment regimens. Recent advances in cancer research revealed signaling pathways and epigenetic mechanisms that intricately govern glioma progression, offering multifaceted targets for therapeutic intervention. This review explores the dynamic interplay between signaling events and epigenetic regulation in the context of glioma, with a particular focus on the crucial roles played by non-coding RNAs (ncRNAs). Through direct and indirect epigenetic targeting, ncRNAs emerge as key regulators shaping the molecular landscape of glioblastoma across its various stages. By dissecting these intricate regulatory networks, novel and patient-tailored therapeutic strategies could be devised to improve patient outcomes with this devastating disease.

1. Introduction

Gliomas, particularly glioblastoma multiforme (GBM), are among the most aggressive central nervous system (CNS) tumors, that originate from the glial stem or progenitor cells [1]. According to the World Health Organization (WHO) classification of CNS tumors published in 2021 is grounded in molecular genetics, utilizing markers like mutations in the isocitrate dehydrogenase (IDH) gene or histone H3 lysine 27 mutant (H3K27M) and 1p/19q co-deletion to classify tumors. GBM falls under the adult-type diffuse gliomas representing the highest grade (WHO Grade 4) [2]. The classification further distinguishes between pediatric-type diffuse low-grade gliomas, pediatric-type diffuse high-grade gliomas, and circumscribed astrocytic gliomas [3].

Despite advances in therapeutic strategies, the prognosis for GBM remains dismal, with a median survival of approximately 15 months and a five-year survival rate of less than 5.5% [4,5]. The high degree of inter- and intra-tumoral heterogeneity, resistance to treatment, and aggressive growth complicate the development of effective therapies. This complexity arises from multiple factors, including genetic mutations, aberrant signaling pathways, and dysregulated epigenetic mechanisms [6]. A growing body of evidence has highlighted the critical role of signaling pathways in driving the tumorigenic behavior of gliomas, but emerging research also points to the importance of epigenetic mechanisms in regulating these pathways [7,8]. Epigenetic modifications, such as DNA methylation and histone modifications, do not change the DNA sequence but significantly alter gene expression, influencing key processes like cell proliferation, migration, and invasion. In gliomas, these epigenetic changes are often driven by or contribute to the dysregulation of key signaling pathways, such as the epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT), and Eph/ephrin pathways [9,10]. The convergence of signaling and epigenetic mechanisms forms a complex regulatory network that underpins glioma initiation and progression, and its elucidation could provide novel therapeutic opportunities.

This review aims to explore the intricate interplay between signaling pathways and epigenetic mechanisms in glioma, mainly focusing on GBM and the role of non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs). LncRNAs have emerged as pivotal regulators of both signaling and epigenetic landscapes, influencing gene expression through direct interactions with epigenetic modifiers and by modulating signaling pathways [11–13]. We suggest that dissecting these interactions provide new insights into glioma pathogenesis and identify potential therapeutic targets that finally lead to improvements in the clinical management of this devastating disease. To this end, this narrative review delves into the complex relationship between these key regulatory mechanisms, highlighting the emerging evidence that supports the synergistic role of signaling pathways and epigenetic factors in glioma initiation and progression, while also addressing existing controversies and knowledge gaps within the field.

2. Initiation of GBM and associated genetic causes

GBM is suggested to arise from an accumulation of somatic mutations in diverse genes [14,15] in line with the observed heterogeneity of GBM. Mutations in the cyclin-dependent kinase (CDK) inhibitor 2A (CDKN2A/P16), tumor protein P53 (TP53), neurofibromin 1 (NF1), phosphatase and tensin homolog (PTEN), EGFR, and platelet-derived growth factor receptor (PDGFR) genes amongst others have been detected [16,17]. These mutations can contribute to gliomagenesis either by resulting in a knockout of tumor suppressor genes (TP53, NF1, CDKN2A, PTEN, and retinoblastoma protein (RB1)) or overexpression of driver oncogenes (e.g., Rat sarcoma virus GTPase (RAS), EGFR, and PDGFR) [18]. Mutations in tumor suppressor genes such as PTEN and TP53, impair cell cycle regulation and promote resistance to apoptosis [19]. Moreover, mutations in TP53 result in increased genomic instability, leading to further mutations and genetic alterations that drive tumor progression in gliomas [20]. Tumor progression is further exacerbated by the amplification or overexpression of the EGFR gene which has been implicated in uncontrolled cell growth and division [21].

IDH mutations, a common hallmark of gliomas, lead to a gain of function that results in the production of 2-hydroxyglutarate (2-HG). This oncometabolite plays a crucial role in glioma pathogenesis by interfering with cellular processes like DNA methylation and histone modifications. These disruptions can lead to altered gene expression and promote tumor growth [22,23]. However, IDH mutations are more prevalent in lower-grade gliomas and less common in higher-grade gliomas like primary GBM [24].

Mutations in the telomerase reverse transcriptase gene (TERT) have been reported to be relevant for the initiation of glioma, particularly in the context of primary GBM [25]. TERT promoter mutations increase telomerase activity, preventing telomere shortening and enabling uncontrolled cell division which is key for tumor initiation and progression. TERT mutations often occur alongside other alterations like EGFR amplification or TP53 mutations, facilitating sustained tumor cell proliferation and malignant transformation [26,27]. Some cancer stem cells (CSCs) also use a telomerase-independent mechanism to elongate their telomeres. The alpha-thalassemia/mental retardation syndrome X-linked (ATRX) gene is a suppressor of these alternative mechanisms, and mutations in ATRX are also frequently identified providing further evidence of the association of telomere maintenance mechanisms with GBMs [28].

Apart from the multiple oncogenic mutations, which in concert can lead to glioma initiation, the complexity of the underlying mechanisms becomes further apparent when examining the diverse cellular origins potentially involved in tumor development. Dissecting the cellular origin of GBM is essential, as tumors with different cellular origins exhibited different features in GBM mouse models, which could be relevant for GBM progression and therapy in humans [29]. Numerous oncogenic mutations underlying gliomagenesis could accumulate during the self-renewal of neural progenitor cells (NPCs), thus making NPCs, neural stem cell (NSC)-derived astrocytes and oligodendrocyte precursor cells (OPCs) attractive candidates for being the source of GBM [28,30, p.53]. However, the cell type of origin is an ongoing matter of debate. Diverse lines of evidence such as cell surface markers, expression profiles, cell morphology in GBM tissue as well as genetic modeling of GBM in mice [31] exist for the three aforementioned cell types. For more details, we refer to other reviews addressing the aspect of the origin of GBM [32–34].

3. The role of signaling pathways in glioma progression and metastasis

At the cellular level, certain tumorigenic processes linked to the development and progression of gliomas are regarded as hallmarks: (a) enhanced proliferation, (b) high and reversible stem cell plasticity, (c) motility and aggressive invasion into surrounding normal brain tissue, and (d) the adeptness to modulate the local microenvironment [35].

Key signaling pathways, such as those mediated by growth factors, play central roles in these processes. Epidermal growth factor (EGF), fibroblast growth factor (FGF), and insulin-like growth factor (IGF), among others, activate receptor tyrosine kinases to trigger downstream cascades, including the mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways, which drive glioma and also GBM proliferation and metastasis [36,37]. Dysregulation of these pathways, particularly through mutations in EGFR and its epidermal growth factor receptor variant III (EGFRvIII), results in aberrant MAPK signaling, contributing to uncontrolled cellular proliferation and enhanced survival [38,39]. As vital components of the MAPK signaling pathway, extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2) play an important role in the regulation of tumor proliferation. In human glioma samples, levels of active (phosphorylated) ERK1 and ERK2 are significantly higher than in healthy tissues, suggesting abnormal activation or overexpression of ERK1/2 to be capable of malignant progression [40].

Approximately 50% of GBM cases with an ERK hyperactivation are associated with EGFR mutations, leading to uncontrolled proliferation and metastasis of glioma cells [41]. For instance, inactivating mutations of EGFRvIII results in abnormally high levels of ligand-independent activation, leading to a hyperactivation of the MAPK signaling pathway, and, through this, increased proliferation, survival, and tumorigenicity [39]. Similar to the EGF/EGFR axis, elevated levels of the insulin-like growth factors IGF1 and IFG2 were shown to promote GBM growth by interacting with their cognate receptors IGFIR and IGFIIR, which resulted in the activation of MAPK and PI3K/AKT signaling pathways [42].

Aberrant focal adhesion kinase (FAK) signaling has further been observed in glioma [43], e.g., due to mutations in PTEN. FAK is a cytoplasmic tyrosine kinase that regulates signaling cascades emanating from integrins and growth factor receptors. The activation of FAK potentiates proliferation by increasing the expression levels of cyclin-D1 (CCND1) and reducing the expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A/p21), thereby accelerating the G1 to synthesis (S)-phase transition [44]. When phosphorylated at Y397, FAK is targeted and dephosphorylated by PTEN, which diminishes its kinase activity and attenuates the G1-to-S phase transition [45]. Mutations in PTEN dysregulate FAK signaling, enhancing proliferation through increased CCND1 expression and reduced CDKN1A/p21 levels, accelerating the G1-to-S-phase transition [44,46,47]. Apart from regulating FAK signaling, PTEN mutations can promote gliomagenesis via the PI3K/AKT pathway. Mutations in PTEN that interfere with its dephosphorylating activity cause abnormally high levels of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which is then used by PI3K to phosphorylate AKT. This in turn leads to AKT hyperactivation and uncontrolled cell proliferation, ultimately aiding the progression of gliomas [48].

Membrane-bound erythropoietin-producing hepatocellular (Eph) receptors and their ephrin ligands represent another signaling axis involved in glioma and GBM progression, particularly in processes like the epithelial-to-mesenchymal transition (EMT), invasion, and metastasis [43]. The Eph/ephrin system is classified into A and B groups based on sequence homology and binding affinities. Ephrin‑A ligands are membrane-anchored via glycosylphosphatidylinositol (GPI), while ephrin‑B ligands are transmembrane proteins. Although EphA receptors primarily bind ephrin‑A ligands and EphB receptors bind ephrin‑B, cross-group interactions also occur, such as ephrin-A5 binding to EphB2 and EphA4 binding to ephrin‑B ligands [49–51]. Forward signaling via Eph receptors depends on their tyrosine kinase domain, which triggers the phosphorylation of downstream effectors like Ras, Rho GTPases, and Src kinases [52]. Ephrin ligands can mediate reverse signaling through transmembrane proteins or PSD‑95/Discs-large/ZO‑1 (PDZ)-binding sites, leading to phosphorylation and recruitment of Src homology 2 (SH2) domain proteins, such as growth factor receptor-bound protein 4 (GRB4), regulating neuronal migration and synaptic development [53–55]. This complex bidirectional signaling system activates downstream pathways, including the MAPK and the PI3K/AKT signaling pathways, to modulate important cellular events such as proliferation, migration, and cell survival [56].

In GBM, EphB2 is dysregulated, and its expression levels have been correlated with tumor aggressiveness and poor prognosis [57,58]. EphB2 signaling has been linked to increased cell migration and invasion, which are key features of mesenchymal cells. Through its effects on cytoskeletal dynamics and cell motility, EphB2 can promote the invasive behavior of GBM cells, facilitating tumor spread [59,60]. EphA receptors are predominantly expressed in stem cells and undifferentiated cells, with EphA2 and EphA3 being associated with tumor stem cell self-renewal in glioblastomas. Considering the role of stem cells in gliomagenesis coupled with the high expression of EphA receptors found in these cells, further research targeting EphA receptors in GBM is warranted [61].

Eph/ephrin signaling interacts with other pathways initiated by EGFR and fibroblast growth factor receptor (FGFR) as well as adhesion molecules such as integrins and cadherins, thereby mediating effects on cellular proliferation and migration [62]. Ephrin‑A5, capable of binding different Eph receptors (EphA2, EphA3, EphA4, EphA7, and EphB2), was found to reduce tumor growth in glioma as well as glioma cell migration and metastasis by negatively regulating the oncoprotein EGFR, hence acting as a tumor suppressor [63]. The EphA4 receptor was described to increase GBM aggressiveness by forming an EphA4:FGFR1 complex, potentiating cellular proliferation and migration, accompanied by the phosphorylation of MAPK and AKT [64]. Ligand-independent EphA2 signaling downstream of the ERK pathway promotes glioma progression, whereas downregulating the expression of EphA2 by exposing it to its ligand ephrin‑A1 leads to the inhibition of progression [65]. The expression of ephrin‑B3 has been demonstrated to be upregulated in migrating glioma cells, with ephrin‑B3 being found to promote glioma invasion through the activation of Ras-related C3 botulinum toxin substrate 1 (RAC1) [66]. In line with this, another study has demonstrated the phosphorylation of ephrin‑B2 to promote glioma cell migration and invasion [67], while overexpression of EphB2 was found to restrict cellular adhesion and potentiate invasion in glioma cells [67,68].

In addition to the activation of signaling pathways, emerging evidence suggests that ephrin-mediated Eph receptor activation can also act on cell migration by influencing epigenetic remodeling and, through this, gene expression [56,69,70]. This could represent another mechanism of how the Eph/ephrin system and potentially other signaling systems could be implicated in GBM initiation and progression. Indeed, several lines of evidence propose epigenetic mechanisms and signatures to be implicated in different types of glioma, as discussed in more detail in the next paragraphs.

4. Epigenetics of glioma

4.1. Epigenetic regulation of gene expression

Histone variants, post-translational modifications, DNA methylation, and ncRNAs dynamically regulate gene expression in brain development, with dysregulations being implicated in disease. Nucleosomes are composed of histone octamers around which DNA is wrapped, playing a crucial role in the organization and regulation of genetic material. The canonical histones found within these octamers can be replaced by diverse histone variants which influence transcription in distinct ways, thereby affecting gene expression and cellular functions. Furthermore, histones undergo post-translational modifications like methylation and acetylation, which are reversible and impact gene accessibility and expression [71,72]. These histone modifications are catalyzed by two antagonistic sets of enzyme complexes that either attach (writers) or remove (erasers) the respective chemical groups. For example, histone acetyltransferases (HATs) add acetyl groups to lysine residues leading to active transcription while histone deacetylases (HDACs) remove these groups [71]. Depending on the methylated site as well as the degree of methylation, histone methylation can either be associated with transcriptional repression or activation. While the histone H3 lysine 4 trimethylation (H3K4me3) results in open chromatin, histone 3 lysine 27 trimethylation (H3K27me3) and H3K9me3 leads to chromatin condensation associated with gene repression [73].

DNA methylation, primarily regulated by DNA methyltransferases (DNMTs), controls transcription by affecting enhancer and promoter accessibility. The often-reported transcriptionally repressive effect of promoter methylation can result from physically impeding the binding of transcription factors either directly or indirectly by methyl-CpG-binding domain proteins (MBDs) that interact with methylated DNA [74]. These MBDs can recruit other chromatin and nucleosome remodeling proteins concertedly leading to chromatin compaction [75]. DNA methylation is a dynamic regulatory process, similar to histone post-translational modifications. While methylation signatures can be passively removed during DNA replication in dividing progenitors, the ten-eleven translocation (TET) enzyme family can actively trigger the conversion to unmethylated cytosines in non-dividing cells by catalyzing the oxidation of 5-methylcytosines (5mC) to 5-hydroxymethylcytosine (5hmC), followed by successive oxidation steps [76]. This ultimately leads to the recovery of cytosine by thymine DNA glycosylase (TDG)-mediated base excision repair [77], also evident in neurons [78].

Another group of epigenetic effectors, the ncRNAs, have been in spotlight for the past decade due to their multifaceted functional spectrum. Based on their size, biogenesis, and function, they can be classified into small non-coding RNAs (sncRNAs) and lncRNAs, respectively. SncRNAs, which include microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs), are mainly involved in post-transcriptional regulation in the cytoplasm [79,80]. Conversely, lncRNAs are exceptionally diverse in their function, as they can modulate transcriptional, post-transcriptional, and even translational processes [81]. In concert, these different epigenetic mechanisms can modulate chromatin structure and gene expression by crosstalking with one another. For instance, while specific histone modifications can prime genomic loci for DNA methylation, certain methylation signatures can act as recognition sites for the deposition of histone marks [73]. This crosstalk is further enriched by DNA methyltransferase 1 (DNMT1) which has been shown to not only interact with histone-modifying complexes such as the polycomb repressive complex 2 (PRC2) at the protein level but also modulate the expression of genes encoding for enzymes within said complexes [82–84]. Furthermore, the multi-faceted lncRNAs are proposed to mediate the target specificity in these events, as they have been described to recruit or evict epigenetic modifiers, such as DNMTs and histone modifiers [79,85]. Therefore, it is of utmost importance to acknowledge the complexity of the epigenomic landscape where different epigenetic regulatory mechanisms intricately orchestrate the regulation of distinct physiological processes.

4.2. DNA methylation and histone modifications in glioma

GBM is widely known for its intricate molecular and genetic heterogeneity, a characteristic that is also reflected at the epigenetic level. Unraveling this heterogeneity is a prerequisite for the development of targeted therapies capable of addressing the diverse molecular landscapes inherent to GBM [86,87].

The exploration of DNA methylation signatures emerges as a promising avenue in this quest. These epigenetic markers not only hold potential as diagnostic tools but also as prognostic indicators for GBM. By analyzing DNA methylation patterns, researchers aim to classify tumors, predict patient outcomes, and ultimately pave the way for personalized treatment strategies tailored to the specific molecular characteristics of the tumor [88].

In the context of cancer cells, abnormal or altered DNA methylation can activate proto-oncogenes while silencing tumor suppressor genes. These aberrant gene expression patterns result in genomic instability and alterations in chromatin structure that are involved in or causative for tumorigenic processes [89,90].

Promoter hypermethylation of tumor suppressor genes like CDKN2A, CDKN2B, and Ras association domain-containing protein 1 (RASSF1A) is common in gliomas, contributing to tumor initiation and progression by disrupting cell cycle control and promoting proliferation [91,92]. Similarly, hypermethylation of retinoic acid receptor beta (RARB) silences its tumor-suppressive function, enabling uncontrolled glioma cell growth [93]. Silencing of tissue inhibitors of metalloproteinases 3 (TIMP3) through hypermethylation enhances angiogenesis by promoting endothelial proliferation, fueling tumor growth [94]. Methylation of SFRP1 and SFRP2 activates aberrant Wnt signaling in gliomas [95]. In addition to hypermethylation, global hypomethylation in GBM affects genomic stability and can increase invasiveness. Specific hypomethylation signatures have also been identified as poor prognostic markers [96–98]. Similar to DNA methylation, histone modifications play a crucial role in glioblastoma tumorigenesis [99,100]. The balance between HATs and HDACs influences chromatin accessibility and gene expression. Histone deacetylation affects gene expression by changing chromatin conformation and reducing the expression of tumor suppressors [101]. Overexpression of various HDACs has been shown to cause resistance against therapies based on genotoxicity, making them promising targets for GBM therapy [102]. In recent years, HDAC inhibitors have been applied in pre-clinical and clinical trials as mono- and combination therapies against GBM [103].

HDAC6, involved in microtubule stability and EGFR turnover, promotes GBM progression [102–105]. Enhancer of zeste homolog 2 (EZH2), part of the PRC2 complex, suppresses gene expression via H3K27 methylation and is linked to poor survival in glioma. Its activity, regulated by AKT, suggests a connection between EZH2 and PI3K signaling, positioning EZH2 as a therapeutic target, particularly in H3K27-altered diffuse midline glioma [106–111].

4.3. NcRNAs in glioma and treatment resistance

Over the past decade, ncRNAs, particularly lncRNAs and miRNAs, have attracted growing interest in cancer research due to their critical roles in gene expression regulation. By influencing key processes such as cell proliferation, apoptosis, and metastasis, ncRNAs contribute to tumor development and progression. This highlights their potential as biomarkers and therapeutic targets, offering valuable insights into cancer biology and treatment. LncRNAs are particularly notable for their ability to regulate gene transcription directly or through various epigenetic mechanisms, such as DNA methylation and histone modifications. Additionally, lncRNAs can modulate translation by acting as sponges for miRNAs or serving as miRNA precursors. As lncRNA-mediated miRNA sponging is implicated in glioma chemoresistance [112,113], targeting dysregulated ncRNAs with antagonists or mimics holds promise for reversing chemoresistance, although miRNA-based therapies face significant challenges, such as adverse effects and delivery obstacles. While therapies targeting lncRNAs and circRNAs are still in early development, the use of siRNA and shRNA as delivery systems shows potential. Identifying key ncRNA targets and developing safe and effective delivery methods are crucial for advancing these therapies [114]. Utilizing ncRNAs for early drug sensitivity screening and combining them with chemotherapeutics are promising research avenues, as ncRNA therapy holds potential for improving glioma prognosis by addressing chemoresistance [115].

Cancer susceptibility candidate 2 (CASC2) is a lncRNA that acts as a tumor suppressor in various cancers, including gliomas. It modulates the PI3K/AKT/mTOR signaling pathway, which is often dysregulated in malignancies [116]. CASC2 interacts with miR-181a to promote glioma growth and resistance to temozolomide (TMZ) by upregulating PTEN and inhibiting the AKT pathway. Additionally, CASC2 downregulates miR‑193a‑5p, reducing autophagy and enhancing TMZ efficacy by upregulating mTOR [117].

The differentiation antagonizing non-protein coding RNA (DANCR) is a promising tumor-associated lncRNA, upregulated in various cancers and serving as a potential biomarker [118]. It enhances the expression of anaplastic lymphoma receptor tyrosine kinase (AXL), a receptor that activates the PI3K/AKT/NF-κB pathway, by competitively binding to miR‑33a‑5p, miR‑33b‑5p, miR‑1‑3p, miR‑206, and miR‑613. This interaction contributes to resistance against cisplatin therapy in gliomas [119]. The lncRNA small nucleolar host gene 15 (SNHG15) is overexpressed in various cancers, including gliomas, also making it a promising target for research [120]. It modulates the Yes-associated protein 1 (YAP1)-Hippo pathway and facilitates oncogene expression, promoting tumorigenesis [121,122]. Additionally, SNHG15 is highly expressed in vascular endothelial cells of glioma tumors, where its downregulation restricts proliferation and migration in vitro. It also contributes to GBM tumor resistance against TMZ [123].

The lncRNA small nucleolar host gene 16 (SNHG16) is overexpressed in gliomas and linked to poor prognosis and adverse clinicopathologic features. It enhances cell migration, invasion, and proliferation [124]. Silencing SNHG16 in glioma cells reduces viability and invasion by regulating the PI3K/AKT pathway. Additionally, SNHG16 sponges miR‑4518 to upregulate protein arginine methyltransferase 5 (PRMT5), a key protein involved in histone modifications and signaling, with its overexpression associated with poor survival [125].

ADAMTS9 Antisense RNA 2 (ADAMTS9-AS2) is an oncogenic lncRNA upregulated in GBM and other cancers, involved in PI3K/AKT and MEK/ERK signaling and interacting with miRNAs [126]. Its expression can serve as a biomarker for cancer diagnosis and prognosis. In GBM, ADAMTS9-AS2 levels correlate with tumor grade and patient survival, suggesting it could enhance the accuracy of glioma diagnosis and prognosis when used with other biomarkers [127].

The antisense noncoding RNA from the INK4 locus (ANRIL) is an oncogenic lncRNA dysregulated in GBM patients’ blood. It modulates gene expression and acts as a scaffold for PRC2, promoting tumorigenesis by enhancing cell proliferation, migration, and invasion while suppressing apoptosis [128]. ANRIL expression, influenced by inflammation, correlates with poor prognosis, tumor grade, size, and metastasis, suggesting its potential as a biomarker for GBM screening [129].

Beyond miRNAs, small nucleolar RNAs (snoRNAs), piRNAs, and circular RNAs (circRNAs) are promising future biomarkers for gliomas. SnoRNAs, which play roles in various biological processes, exhibit differential expression in pediatric gliomas [130]. Specific snoRNAs such as SNORD, SNORD76 as well as snoRNA host genes such as SNHG18 are associated with EMT, TMZ sensitivity, cell proliferation, and radioresistance. Additionally, scaffold RNAs (ScaRNAs), circRNAs, and piRNAs may influence glioma development through miRNA sponging and epigenetic modifications [131].

Aberrant miRNA expression plays a critical role in GBM pathophysiology. For example, miR‑422a suppresses glioma cell proliferation and invasion by targeting IGF1 and IGF1R [132–134]. miR‑422a is inhibited by the lncRNA circNT5E, through which tumorigenesis is promoted [135]. Other miRNAs, such as miR‑7, miR‑128, and miR‑219‑5p, downregulate EGFR, which is often overexpressed in GBM and fuels tumorigenesis through pyruvate kinase M2 (PKM2) [136–138]. Additionally, miR‑21 overexpression indirectly enhances EGFR signaling via signal transducer and activator of transcription 3 (STAT3), creating a positive feedback loop [139,140]. The silencing of miR‑51 by SNHG15 is linked to VEGFA overexpression, driving angiogenesis in GBM [121]. MiR‑7 and miR‑542‑3p have also been shown to deregulate PI3K/AKT signaling, affecting GBM metabolism [141–144].

As is evident from previous points, lncRNAs and miRNAs are heavily involved in the epigenetic regulation of genes implicated in tumorigenic processes of GBM. Importantly, their intricate regulatory roles extend beyond direct modulation of gene expression to encompass indirect mechanisms, such as recruitment or targeting of epigenetic modifiers. Through these multifaceted actions, lncRNAs and miRNAs wield significant influence over the epigenetic landscape, shaping the aberrant gene expression profiles characteristic of tumorigenesis. The relationships between lncRNA expression and glioma characteristics are summarized in Table 1.

Table 1.

Role of long non-coding RNAs in glioma: expression patterns, pathways, and clinical implications.

LncRNA	LGG Expression	HGG Expression	Pathways Involved	Role in Glioma	Post-Transcriptional Processes	Clinical Implications	Epigenetic Interactions	Ref
CASC2	Downregulated; associated with better overall survival	Upregulated; correlates with aggressive disease phenotypes	PI3K/Akt pathway, TGF-β signaling	Acts as a tumor suppressor; inhibits cell proliferation	May interact with miRNAs to regulate gene expression	Potential prognostic marker: low expression linked to better outcomes	Histone modification may influence CASC2 expression	[224,225]
DANCR	Upregulated; linked with higher tumor grades.	Upregulated; associated with aggressive behavior	Wnt signaling pathway, MAPK pathway	Promotes tumor growth and metastasis; functions as an oncogene	Interacts with miR-203, modulating gene expression involved in cancer progression	High expression correlates with poor prognosis; potential therapeutic target	DNA methylation may impact DANCR expression levels	[226,227]
SNHG15	Upregulated; higher expression associated with advanced grades of LGG	Upregulated; correlates with poor prognosis and advanced tumor characteristics in HGG	PI3K/Akt pathway, Wnt/β-catenin pathway, and mTOR pathway	Promotes cell proliferation, migration, and invasion; functions as an oncogene in glioma	Acts as a sponge for miR-132, enhancing the expression of target genes like CDK6	Potential biomarker for diagnosis and prognosis; high expression indicates poor outcomes	DNA methylation may regulate SNHG15 expression, impacting glioma progression	[228,229]
SNHG16	Upregulated; associated with higher tumor grades in LGG, indicating a more aggressive phenotype	Upregulated; linked to poor prognosis and aggressive behavior in HGG	Wnt/β-catenin signaling, TGF-β signaling, and EMT pathways	Promotes glioma cell proliferation and invasion; involved in the regulation of stemness traits	Interacts with miRNAs such as miR-195, affecting the expression of target genes related to cell cycle regulation	Potential prognostic marker; could be targeted for therapeutic strategies in glioma treatment	Histone modifications and chromatin remodeling influence SNHG16 expression in glioma cells	[124,230]
HOTAIR	Upregulated; associated with increased tumor aggressiveness in LGG	Upregulated; correlates with poor prognosis and aggressive tumor behavior in HGG	Polycomb repressive complex, Wnt/β-catenin pathway, and epithelial-mesenchymal transition (EMT)	Promotes tumor invasion and metastasis; acts as an oncogene	Interacts with PRC2 to regulate gene expression; influences splicing of specific mRNAs	High levels are indicative of poor prognosis; potential therapeutic target.	DNA methylation and histone modifications can regulate HOTAIR expression, contributing to glioma progression	[231–233]
MALAT1	Upregulated; associated with advanced tumor stages and poor clinical outcomes in LGG	Highly expressed; linked to aggressive HGG phenotypes and poor survival rates	Regulation of alternative splicing, Wnt signaling, and cellular stress response pathways	Involved in cell proliferation, migration, and invasion; acts as an oncogene in glioma	Stabilizes mRNAs and regulates their splicing; interacts with various RNA-binding proteins	Considered a prognostic marker; potential target for therapeutic interventions.	Histone modifications and chromatin remodeling influence MALAT1 expression in glioma cells.	[234–236]
ANRIL	Upregulated - Elevated levels correlate with increased tumor size and malignancy in LGG	Upregulated - Strongly associated with poor prognosis and aggressive HGG phenotype	p53 signaling, NF-κB pathway, and cellular senescence	Promotes tumor growth and invasion; acts as an oncogene in glioma by modulating key signaling pathwa	Stabilizes various mRNAs involved in cell cycle regulation; interacts with the RNP complex to influence splicing	Considered a potential therapeutic target; high levels indicate poor prognosis and may guide treatment strategies	Histone modifications (e.g., H3K27me3) promote ANRIL expression, linking it to chromatin remodeling processes in glioma	[237,238]

Abbreviations: Long Non-Coding RNA (lncRNA), Low-Grade Glioma (LGG), High-Grade Glioma (HGG), Phosphoinositide 3-Kinase (PI3K), Protein Kinase B (AKT), Transforming Growth Factor Beta (TGF-β), Wingless/Integration Site (Wnt), Mitogen-Activated Protein Kinase (MAPK), Epithelial-Mesenchymal Transition (EMT), Polycomb Repressive Complex 2 (PRC2), MicroRNA (miRNA), Cyclin-Dependent Kinase 6 (CDK6), Phosphatase and Tensin Homolog (PTEN), Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NF-κB), Ribonucleoprotein (RNP).

4.4. Modulation of epigenetic writers by lncRNAs in glioma

Since miRNAs are known to regulate DNA methylation and histone modifications by targeting the mRNA of key enzymes, the ability of lncRNAs to modulate miRNA activity places them in a central position within this regulatory network. This implies that lncRNAs could indirectly affect DNA methylation patterns and other epigenetic modifications by altering miRNA-mediated pathways. Thus, lncRNAs not only participate in gene expression regulation but also have the potential to shape epigenetic landscapes through their interactions with miRNAs, further highlighting their pivotal role in complex regulatory networks [11].

MiRNAs regulate epigenetic modifiers at the post-transcriptional level. miR‑152‑3p acts as a tumor suppressor in glioma by influencing cell viability; reduced levels of this miRNA are linked to increased DNMT1 expression, validated by its upregulation and direct inhibition of DNMT1 mRNA [145]. Another miRNA, miR‑185, decreases DNMT1 mRNA in glioma cells without affecting DNMT3A or DNMT3B [146]. miR‑29c targets DNMT3A/3B and indirectly suppresses the DNA repair protein MGMT which is downregulated in GBM and implicated in TMZ resistance [147,148]. Additionally, miR‑129‑5p targets DNMT3A, with its overexpression inhibiting glioma cell proliferation and inducing cell cycle arrest [149].

MiRNAs influence TET function and DNA demethylation. Specifically, miR‑10b‑5p disrupts TET2-mediated suppression of programmed death-ligand 1 (PD-L1) transcription, promoting immune evasion and tumor progression in glioblastoma [150].

MiRNAs also regulate histone modification writers in GBM by influencing their expression. miR‑101‑3p and miR‑137 are direct or indirect regulators of EZH2, which catalyzes H3K27 trimethylation [151]. Some miRNAs target EZH2 mRNA for degradation or inhibit its translation, while others indirectly regulate EZH2 through other molecules. By modulating EZH2, miRNAs affect the epigenetic landscape and gene expression in GBM cells [151]. Additionally, miRNAs like miR‑34a, miR‑132, and miR‑217 target HDACs, specifically regulating NAD-dependent deacetylase sirtuin-1 (SIRT1) during gliomagenesis and progression, thus modulating deacetylation [152].

Glioma research indicates a significant interplay between ncRNAs and epigenetic writers. Notably, the lncRNAs HOTAIR and MALAT1 influence glioma pathogenesis by interacting with histone-modifying enzymes EZH2 and lysine-specific histone demethylase 1A (LSD1), thereby regulating histone modifications [153–155]. AC016405.3 is a lncRNA with reduced expression in GBM, acting as a tumor suppressor. Its decreased levels are linked to increased cell proliferation and invasion, attributed to TET2 modulation from the lack of miR‑19a‑5p sponging [156]. SNHG15 inhibits the transcription factor Kruppel-like factor 2 (KLF2) by binding to EZH2, leading to repressive H3K27 trimethylation at the KLF2 locus [157].

SNHG15 can also indirectly affect epigenetic modifiers by targeting miRNAs, which enhances proliferation, migration, and angiogenesis in glioma [120]. For instance, it has been shown to downregulate miR‑627 which is involved in the translational regulation of the histone demethylase jumonji domain-containing 1A (JMJD1A). Additionally, SNHG15 has been reported to suppress miR‑153, a tumor suppressor in GBM which was found to be downregulated due to a hypermethylation [123,158]. This suggests that SNHG15 may transcriptionally control miR‑153 by recruiting DNA methyltransferases, a mechanism noted in its murine ortholog [70].

5. The intersection between signaling networks and epigenetic signatures in glioma

Glioma initiation and progression are driven by complex signaling pathways that regulate processes like proliferation and metastasis. Multiple pathways are dysregulated in GBM and other glioma types, including EGFR, PDGFR, transforming growth factor beta (TGFβ), PI3K/AKT, cell cycle pathways (CDK4/6, CDKN2A/B), P53, retinoblastoma protein (pRB), RAS/MAPK, and STAT3 signaling [159]. Our understanding of oncogenic signaling pathways in human cancers has advanced significantly in recent decades [160,161]. However, this knowledge continues to grow, revealing that the transcriptional output of these pathways is influenced by a cell’s epigenetic state, which is regulated by epigenetic readers, writers, and erasers [162,163]. While mutations can lead to aberrant signaling, the biological response to oncogenic signals is also shaped by the epigenome [164]. In glioma, signaling pathways interact with epigenetic mechanisms, reflecting the complex interplay between genetic and epigenetic regulation of tumorigenesis, contributing to glioblastoma heterogeneity [165] (see Figure 1). Epigenetic signatures, including chromatin modifications from extrinsic signals, can affect the binding of epigenetic modifiers and transcription factors, ultimately altering the gene expression profile of cancer cells [166].

Figure 1.

Schematic representation of key signaling pathways regulating cellular hallmarks of gliomas, including their modulation via non-coding RNAs. numerous oncogenic receptors and integrins can activate downstream signaling cascades, mediated by FAK, MAPK, and AKT, leading to an aberrant activation of tumorigenic processes. Hyperactivated FAK (1) has been implicated in glioma growth, migration as well as vasculogenesis, while abnormal MAPK and AKT activity (2–3) has been shown to promote glioma cell proliferation, stem cell renewal, and invasion. Non-coding RNAs (ncRnas), including lncRNAs and miRnas, can modulate the expression of these receptors as well as effectors of the downstream signaling cascades. LncRNAs can act as adaptors for epigenetic modifiers (4) to silence or limit the transcription of oncogenes such as xyz. MiRNAs can bind to transcripts of oncogenes (5) to inhibit their translation to proteins. Moreover, miRNAs and lncRNAs can bind to and inhibit each other (6). Created in BioRender. Yildiz, C.B. (n.D.) https://biorender.com/g75e522.

Substantial evidence indicates that transient kinase-mediated signals can initiate cascades that phosphorylate histones and non-histone nuclear proteins, leading to long-lasting chromatin modifications and altered gene expression [167–170]. For example, phosphorylation of serine 10 on histone H3 (H3S10) activates downstream targets like ribosomal protein S6 kinase alpha-3 (Rsk2), mitogen-activated protein kinase-activated protein kinase 1/2 (MSK1/2), inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα), Aurora B, and PIM1 and proviral insertion site in moloney murine leukemia virus 1 (PIM1) [171]. Low H3S10 levels have been linked to increased GBM survival rates, suggesting its potential as a biomarker [172]. Additionally, EGF-activated PKM2 phosphorylates histone H3 at T11, causing HDAC3 dissociation and promoting H3K9 acetylation, which activates transcription [173]. EGF-induced PKM2 activity has also been shown to enhance GBM tumorigenesis [138], while hyperacetylation of H3K9 triggers the expression of GDNF, promoting GBM initiation and progression [174]. Signaling cascades can directly modulate transcriptional programs by allowing downstream effectors to bind to chromatin and influence chromatin-modifying proteins. For example, Janus kinase (JAK)2-dependent phosphorylation of STAT5 promotes its dimerization, nuclear translocation, and binding to cis-regulatory elements [175].

STAT5 activation through EGFRvIII signaling enhances GBM cell migration and survival [176], suggesting JAK-independent STAT5 signaling as a potential vulnerability in GBM [177]. Additionally, inhibiting JAK2 with small molecules has been shown to reduce tumorigenic potential in a GBM cell model [178]. JAK2 acts as a histone tyrosine kinase, phosphorylating histone 3 tyrosine 41 (H3Y41) and disrupting the binding of gene repressor HP1α [179,180], which is crucial for cancer cell proliferation in GBM [181]. The enzymatic activity of the H3K27 methyltransferase EZH2, part of the PRC2 complex, is influenced by various signaling pathways [182]. PI3K/AKT signaling leads to the phosphorylation of EZH2 on serine 21, reducing PRC2 affinity for histone H3 and increasing H3K27-specific acetyltransferase activity of P300. This causes a transition from repressive to active chromatin states [183,184]. Interestingly, mitogen-activated protein kinase 14 (MAPK14)-mediated phosphorylation of EZH2 at Thr376 has been shown to retain EZH2 in the cytosol and diminish its nuclear activity, while enhancing its binding to vinculin and several other cytoskeletal modulators of cellular migration and invasion in breast cancer [110]. In GBM, while a short-term inhibition of EZH2 activity was found to arrest tumor growth, a prolonged depletion was associated with increased proliferation and tumor progression [185].

DNA methylation dynamics respond to certain signaling pathways influenced by DNMT or TET enzyme activity [186]. For instance, defects in the MAPK pathway, common in GBM, increase DNMT1 expression and enhance DNA methylation [187]. Additionally, JAK2-mediated phosphorylation affects TET2 activity [188]. Abnormal DNA methylation signatures are linked to disrupted gene expression and cellular phenotypes in GBM, suggesting that targeting these pathways may offer new therapeutic opportunities [189,190].

Signaling pathways can influence the expression and function of lncRNAs, known to influence the expression and targeting of epigenetic writers, with implications in regulating tumor growth and metastasis [191]. For example, activation of the PI3K/AKT/mTOR pathway leads to the upregulation of various effectors, including lncRNAs such as HOTAIR, which acts as a scaffold and interacts with chromatin-modifying complexes such as PRC2 [192]. In glioma cells, HOTAIR expression increases with the PI3K/AKT/mTOR activation [193], resulting in enhanced PRC2 activity and H3K27me3 deposition, which represses tumor suppressor genes and promotes glioma progression and invasion [194]. The lncRNA imprinted maternally expressed transcript H19 is often found upregulated in glioma and associated with the growth and invasion of tumor cells [195].

The expression of H19 has been reported to be regulated by P53 and hypoxia-inducible factor 1α (HIF-1α)-mediated signaling events [196] which have been found severely dysregulated in GBM [197–199]. Additionally, ncRNAs promote glioma cell proliferation and migration through the Wnt/β-catenin and PI3K/AKT pathways [200]. Recently, bidirectional Eph/ephrin signaling has gained attention in cancer research for its interactions with both the PI3K/AKT and Wnt/β-catenin pathways [201–203].

While specific interactions between lncRNAs and ephrins in gliomas are still being elucidated, there is emerging evidence that has pointed to a crosstalk between them. In a medulloblastoma cell model, ephrinA5 was found to regulate the expression of SNHG15, which interacts with DNMT1. Downregulation of SNHG15 by ephrinA5 reduces this interaction, leading to increased expression and lower methylation of neural cell adhesion molecule 1 (Ncam1), ultimately restricting cellular motility [69,70]. These findings indicate a possible therapeutic use for ephrinA5 as well as for targeting lncRNAs in preventing tumor invasion and metastasis.

GBM-relevant signaling pathways can converge on lncRNA function and expression. The ncRNAs in turn can also modulate the expression of genes related to signaling pathways that are dysregulated in GBM, proposing a complex interplay. For example, a study found that miR‑141 targets EphA2 expression leading to an inhibition of vasculogenic mimicry (VM) [204]. The extent of VM seems to correlate with increasing malignancy of glioma [205], in which the Eph/ephrin system has been described to play a critical role [59,206]. As miR‑141-mediated modulation of EphA2 expression inhibits VM, this miRNA appears to be a promising anti-VM therapeutic agent in glioma [204]. Of note, while EphA2 expression is often found to be enhanced in IDH-wildtype GBM tissue [207], it has been shown to have both oncogenic and tumor-suppressive functions [208,209]. GBM-associated angiogenesis relies on VEGFA signaling, with the lncRNA H19 promoting angiogenesis by sponging miR‑138, leading to increased VEGF expression, similar to the SNHG15/miR‑51 axis [123,210].

Signaling pathways converge on epigenetic signatures that alter gene transcription in GBM, indicating that regulatory circuits and feedback loops mediate its features. Evidence shows that miRNAs, which are regulated by lncRNAs, and signaling pathways sustain GBM progression. Notably, the Wnt signaling pathway serves as a key nexus for extracellular ligands and diverse intracellular responses, ranging from survival signals to drug susceptibility [211,212]. In glioma, miR‑122 targets Wnt1 and inhibits Wnt/β-catenin signaling, while the Wnt/β-catenin pathway represses miR‑122, suggesting a potential autoregulation mechanism [213,214]. Interestingly, the lncRNA HOTAIR, which is overexpressed in GBM, has been shown to suppress miR‑122 expression via DNA methylation in vitro [215], highlighting how lncRNAs can regulate miRNAs and indirectly their target signaling pathways. GBM cell invasion is suppressed by miR‑218 targeting lymphoid enhancer-binding factor 1 (LEF1), a key oncogenic transcription factor in the Wnt pathway, thus acting as a tumor suppressor [216,217]. The lncRNA LINC00511, commonly upregulated in cancers including GBM, targets miR‑218 to promote proliferation, invasion, and epithelial-mesenchymal transition [218,219]. The overexpression of LINC00511 has been attributed to activation by the HIF‑1α [220], described to be a downstream effector of Wnt/ß-catenin and PI3K/AKT signaling pathways [221,222]. HIF-1α has further been implicated in the transcriptional regulation of the lncRNA low expression in tumor (LET) via HDAC3, resulting in a hypoxia-induced cancer cell invasion in many cancers [223]. Considering the hypoxic conditions observed in the cores of GBM tumors, targeting the expression of lncRNAs such as LINC00511 or LET as well as their upstream inducers such as HIF-1α could hold promise as a potential therapeutic strategy to attenuate GBM cell proliferation and invasion.

5.1. Future perspective

In sum, this review emphasizes the central role of lncRNAs in the intricate interplay between signaling pathways and epigenetic mechanisms in glioblastoma. As key regulators, lncRNAs modulate gene expression, chromatin structure, and cellular signaling through interactions with both ncRNAs and epigenetic modifiers. This positions lncRNAs as pivotal drivers of tumor initiation, progression, and therapeutic response, underscoring their importance in glioblastoma’s broader epigenetic landscape.

The convergence of epigenetic and signaling-mediated regulatory networks, involving both coding and non-coding elements, provides a complex framework for understanding glioblastoma pathogenesis and discovering novel therapeutic targets. By unraveling these molecular interactions, researchers have uncovered promising opportunities to disrupt aberrant signaling, modulate epigenetic regulators, and target specific ncRNAs.

These targeted strategies offer significant potential to improve patient outcomes and survival in glioblastoma. Continued investigation into the dynamic interplay of epigenetic modifications, signaling pathways, and ncRNAs is crucial for advancing precision medicine and developing tailored therapeutics based on individual molecular profiles. Incorporating these insights into clinical practice opens new pathways for personalized interventions, ultimately improving prognosis and quality of life for glioblastoma patients.

Funding Statement

This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation): [368482240/GRK2416] to Geraldine Zimmer-Bensch and ZI 1224/13–1 to Geraldine Zimmer-Bensch.

Article highlights

1. Introduction

• Gliomas, especially GBM, are aggressive CNS tumors originating from glial cells.

• Classification is based on molecular genetics, including IDH mutations and histone modifications.

• Prognosis remains poor despite advancements in treatment.

2. Initiation of GBM and Associated Genetic Causes

• GBM arises from accumulated somatic mutations in various genes (e.g., TP53, EGFR).

• IDH mutations lead to the production of the oncometabolite 2-HG, influencing tumor growth through epigenetic changes.

• TERT promoter mutations enhance telomerase activity, facilitating uncontrolled cell division.

3. The Role of Signaling Pathways in Glioma Progression

• Key signaling pathways like EGF/EGFR, PI3K/AKT, and FAK are crucial for glioma proliferation and invasion.

• Aberrant activation of these pathways contributes to tumor aggressiveness.

4. Epigenetics of Glioma 4.1. Epigenetic Regulation of Gene Expression

• Histone modifications and DNA methylation play vital roles in gene expression regulation.

• ncRNAs significantly influence chromatin structure and gene accessibility.

4.2. DNA Methylation and Histone Modifications in Glioma

• Abnormal DNA methylation patterns activate oncogenes while silencing tumor suppressor genes.

• Common hypermethylated genes include CDKN2A and RARB, contributing to glioma progression.

4.3. NcRNAs in Glioma and Treatment Resistance Long Non-Coding RNAs (lncRNAs)

• CASC2: Acts as a tumor suppressor; modulates PI3K/AKT/mTOR pathway; enhances TMZ sensitivity by upregulating PTEN.

• DANCR: Upregulated in cancers; enhances AXL expression; involved in cisplatin resistance by activating PI3K/AKT/NF-kB pathway.

• SNHG15: Overexpressed; modulates YAP1-Hippo pathway; contributes to TMZ resistance by regulating autophagy.

• SNHG16: Linked to poor prognosis; enhances migration/invasion through PI3K/AKT modulation.

• ADAMTS9-AS2: Oncogenic lncRNA; correlates with tumor grade/survival; involved in signaling pathways like PI3K/AKT.

MicroRNAs (miRNAs)

• miR422a: Suppresses proliferation/invasion by targeting IGF1/IGF1R; inhibited by circNT5E promoting tumorigenesis.

• miR21: Enhances EGFR signaling indirectly via STAT3 feedback loop; promotes survival signals in GBM cells.

5. Modulation of Epigenetic Writers by lncRNAs in Glioma

• LncRNAs can influence miRNA activity affecting DNA methylation patterns:

• HOTAIR & MALAT1: Interact with histone-modifying enzymes like EZH2, regulating histone modifications critical for glioma pathogenesis.

• SNHG15: Suppresses miR627, which is involved in translational regulation of histone demethylase JMJD1A; this downregulation leads to increased DNMT1 expression. Murine Snhg15 interacts with DNMT1 suggesting a role in modulating DNA methylation patterns, thereby influencing gene expression related to tumor progression.

6. The Intersection Between Signaling Networks and Epigenetic Signatures

• Dysregulated signaling pathways impact the expression/function of lncRNAs:

• H19: Upregulated by P53/HIF signaling events; promotes angiogenesis via miRNA sponging mechanisms affecting VEGFA levels.

Author contributions

Can Bora Yildiz., Conceptualization, Original draft preparation, Writing – Review & Editing, Figure illustration; Jian Du., Writing, Review & Editing, K. Naga Mohan., Streamlining, Review & Editing; Geraldine Zimmer-Bensch., Conceptualization, Writing – Review & Editing; Sara Abdolahi., Conceptualization, Original draft preparation, Writing – Review & Editing.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.