Molecular Mechanisms and Strategies for Inducing Neuronal Differentiation in Glioblastoma Cells

Abstract

Glioblastoma multiforme (GBM) is a highly invasive brain tumor, and traditional treatments combining surgery with radiochemotherapy have limited effects, with tumor recurrence being almost inevitable. Given the lack of proliferative capacity in neurons, inducing terminal differentiation of GBM cells or glioma stem cells (GSCs) into neuron-like cells has emerged as a promising strategy. This approach aims to suppress their proliferation and self-renewal capabilities through differentiation. This review summarizes the methods involved in recent research on the neuronal differentiation of GBM cells or GSCs, including the regulation of transcription factors, signaling pathways, miRNA, and the use of small molecule drugs, among various strategies. It also outlines the interconnections between the mechanisms studied, hoping to provide ideas for exploring new therapeutic avenues for GBM and the development of differentiation-inducing drugs for GBM.

Introduction

Glioma is the most prevalent primary malignant brain tumor in adults, accounting for 60% of all primary brain tumors (Wang et al., 2023b). Among gliomas, glioblastoma multiforme (GBM) is the most common type, classified as a World Health Organization grade 4 glioma, which comprises 57% of all gliomas and 48% of all primary central nervous system malignancies (Yang et al., 2022). GBM is associated with a dismal prognosis, with an untreated median survival of only 3–4.5 months, and even with maximal treatment, survival can be extended to only 15–16 months, with 2- and 5-year survival rates ranging from 27%–31% and 17%–10%, respectively (McKinnon et al., 2021). Currently, the mainstay of GBM treatment involves surgery combined with radiotherapy and chemotherapy, yet tumor recurrence is often inevitable in the long run (Aldoghachi et al., 2022). Research has shown that glioma stem cells (GSCs) play a crucial role in GBM, being assDociated with resistance to radiotherapy and chemotherapy and contributing significantly to the high recurrence rate of GBM (Ramar et al., 2023). The gene expression profile of GSCs is similar to that of neuronal stem cells (NSCs), but GSCs exhibit differentiation impairment, enabling them to maintain their self-renewal capacity (Valor and Hervás-Corpión, 2020). Given the lack of proliferative potential in neurons, inducing the terminal differentiation of GSCs into neurons may represent an effective strategy against GBM. Furthermore, inducing the differentiation of GBM cells can lead to a reduction in their invasiveness and an increase in their sensitivity to temozolomide (Pinto et al., 2020). Several researchers have explored this approach and successfully induced GBM cells or GSCs to differentiate into neuron-like cells. This review summarizes the methods employed in recent studies on the induction of neuronal differentiation in GBM cells, encompassing strategies such as modulation of transcription factors, signaling pathways, miRNAs, and the utilization of small molecule compounds. Furthermore, it outlines the molecular interactions involved in these pathways, aiming to provide insights for the discovery of novel therapeutic approaches and the development of differentiation-inducing drugs for GBM.

Transcription Factors Regulation

Achaete-scute complex homolog 1

The basic helix-loop-helix (bHLH) transcription factors constitute a large superfamily of transcriptional regulators that play crucial roles in various developmental processes, including the development of the nervous system (Lee et al., 2022). Achaete-scute complex homolog 1 (ASCL1), belonging to the bHLH family of transcription factors, is a vital regulator of neurogenesis, directly modulating genes involved in multiple aspects of neurogenic processes such as neuronal differentiation, axon guidance, and synapse formation (Vasconcelos and Castro, 2014). In NSCs, ASCL1 expression is regulated by oscillatory or sustained patterns mediated by Hes family bHLH transcription factor 1 (Hes1). Low ASCL1 activity promotes NSC proliferation, whereas enhanced ASCL1 activity leads to cell cycle exit and differentiation toward neurons (Soares et al., 2022). Studies have demonstrated that overexpression of ASCL1 alone can transform human GBM cell line U251 into neurons. These transformed neurons express neuronal markers including microtubule-associated protein-2 (MAP2), neuronal nuclei antigen (NeuN), doublecortin (DCX), tubulin beta-3 (TUBB3) and synapsin 1 (SYN1) and exhibit neuronal-specific electrophysiological activities, with significant inhibition of proliferation. Furthermore, ASCL1-induced neurons possess characteristics of γ-aminobutyric acid (GABA)ergic neurons and glutamatergic neurons, including the expression of GABAergic neuron marker GABA and glutamatergic neuron marker vesicular glutamate transporter 1 (VGLUT1) (Cheng et al., 2019; Wang et al., 2021). While ASCL1 is expressed in some GBMs, it fails to participate in the neuronal differentiation process; instead, it maintains the stem cell properties of GSCs (Rheinbay et al., 2013). This phenomenon is primarily associated with the phosphorylation status of ASCL1 in GBM. The highly active rat sarcoma (Ras)/extracellular regulated protein kinases (ERK) signaling pathway and cyclin-dependent kinases in tumor cells promote ASCL1 phosphorylation, which favors cell proliferation and maintains GSC stemness. In contrast, dephosphorylated ASCL1 promotes neuronal differentiation (Azzarelli et al., 2022; Li et al., 2014; Woods et al., 2022).

Neurogenin 2

Neurogenin 2 (NGN2), another member of the bHLH family of transcription factors, serves as a crucial regulator of neurogenesis. It has been widely utilized for rapid neuronal differentiation from human pluripotent stem cells, and its expression is directly regulated by paired box gene 6 (Hulme et al., 2022). Studies (Cheng et al., 2019; Wang et al., 2021) have demonstrated that NGN2 overexpression alone can induce the differentiation of human GBM cell line U251 into neuron-like cells both and inhibit their proliferation. Similar to ASCL1-induced neurons, these transformed neurons express neuronal markers including MAP2, NeuN, DCX, TUBB3 and SYN1. However, the efficiency of NGN2-induced differentiation (8%∼10%) is lower than that of ASCL1 (65%∼70%). Besides, NGN2-induced neurons expressed glutamatergic neuron marker VGLUT1 but not GABAergic neuron marker GABA (Cheng et al., 2019; Wang et al., 2021). NGN2 overexpression can also induce neuronal differentiation in patient-derived GSCs with the expression of MAP2, DCX, and TUBB3 (Guichet et al., 2013).

Neuronal differentiation 1

The neuronal differentiation (NeuroD) family, a subgroup of bHLH transcription factors, includes NeuroD1, NeuroD2, NeuroD4, and NeuroD6. These factors play pivotal roles in neuronal progenitor differentiation and neuronal specification in various regions of the nervous system, such as the cerebral cortex, cerebellum, brainstem, and spinal cord (Tutukova et al., 2021). Overexpression of NeuroD1 can convert astrocytes into neurons (Ma et al., 2022). NeuroD1 can be activated by NGN2 (Hulme et al., 2022), and its overexpression in human GSCs exhibits similar neuronal differentiation-inducing effects as NGN2 overexpression (Guichet et al., 2013). Studies have shown that NeuroD1 promotes neuronal differentiation of human GBM cell lines LN229, T98G, U373, and U87, with the expression of neuronal markers MAP2, TUBB3, and NeuN (Jiang et al., 2024). NeuroD1-induced neurons also expressed glutamatergic neuron marker VGLUT1 (Wang et al., 2021).

Neuronal differentiation 4

NeuroD4, belonging to the same NeuroD family of transcription factors as NeuroD1, is also capable of inducing neuronal conversion of astrocytes (Rao et al., 2021). Studies have shown that NeuroD4 alone effectively reprogrammed human GBM cells U251 and KNS89 into nonproliferating neuron-like cells, which express neuronal markers TUBB3 and MAP2. This conversion resulted in a significant inhibition of tumor growth both in experimental models and cell cultures (Wang et al., 2023a).

SRY-related high-mobilityi group box 11

SRY-related high-mobility group box 11 (SOX11) belongs to the SOXC group of transcription factors and promotes neuronal differentiation. It is consistently expressed during the differentiation of neural progenitor cells into neurons but is not expressed in mature neurons (Kavyanifar et al., 2018). In gliomas, SOX11 expression is upregulated (Soukup et al., 2023; Tsang et al., 2020), independent of tumor histological grade and other clinicopathological features (Yang et al., 2019). However, downregulation of SOX11 mRNA is associated with a significant decrease in survival rate (Hide et al., 2009). SOX11 expression is absent in GSCs, while its overexpression can induce neuronal differentiation in human GBM cells U87 or human GSCs, with the expression of neuronal markers MAP2 and TUBB3, thereby inhibiting their proliferation, clone formation ability, and tumorigenicity in vivo (Fu et al., 2019; Hide et al., 2009).

Zic family member 1

Zic family member 1 (ZIC1) is a highly conserved zinc finger transcription factor that plays a key role in the establishment of the nervous system, primarily related to neuronal differentiation (Rahman et al., 2020). Overexpression of ZIC1 in human GBM cells U87 yields a neuronal differentiation-inducing effect similar to that of SOX11 overexpression. SOX11 upregulates ZIC1 expression by binding to its promoter, and ZIC1 partially mediates SOX11-induced neuronal differentiation in U87 cells (Fu et al., 2019).

NGN2 + SOX11

Su et al. (2014) found that NGN2 and SOX11 exhibit synergistic effects. Simultaneous overexpression of NGN2 and SOX11 in human GBM cells U251 significantly improved neuronal conversion efficiency (>95%) compared with individual overexpression of NGN2 (22.2%) or SOX11 (none). The synergistic action of NGN2 and SOX11 induced conversion of human GBM cells U87 and U251 into neuron-like cells. The converted cells exhibited neuronal morphology, expressed neuronal markers, and possessed electrophysiological properties. The expressed neuronal markers included MAP2, NeuN, TUBB3, SYN1, synaptotagmin-1, as well as inhibitory neuron markers GABA and glutamic acid decarboxylase 67 (GAD67) and excitatory neuron markers VGLUT1 and VGLUT2. This conversion process was accompanied by cell cycle exit and significantly inhibited the proliferation and tumor development of GBM cells after orthotopic transplantation.

ASCL1 + NGN2+ brain-2

Brain-2 (BRN2), a member of the class III POU (Pit-Oct-Unc) domain family of transcription factors, plays a central role in embryogenesis and is highly expressed in neural crest cells and the developing brain (Fane et al., 2019). BRN2 alone can convert mouse astrocytes into neurons (Zhu et al., 2018). Zhao et al. (2012) demonstrated that simultaneous overexpression of ASCL1, NGN2, and BRN2 in human GBM cell lines U87 and U251 as well as patient-derived GBM cells efficiently induced the generation of neuron-like cells, which expressed multiple neuronal markers including TUBB3, MAP2, neurofilament heavy, NeuN, synaptophysin (SYP) and exhibited electrophysiological properties. The proliferation of these cells was significantly inhibited both in vitro and in vivo. In addition, the induction efficiency of ASCL1 overexpression alone (<1%) is lower than that of the combination of three factors (31%∼39%).

Signaling Pathways Regulation

Cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate-responsive element binding protein signaling pathway

Cyclic adenosine monophosphate (cAMP), one of the earliest discovered second messengers, plays a crucial role in cellular signal transduction. cAMP activates cAMP-responsive element binding protein (CREB) through protein kinase A (PKA) phosphorylation, which forms homodimers or heterodimers with activating transcription factor 1 and binds to cAMP response elements (CREs) in target gene promoters, initiating gene transcription and regulating neuronal activities such as cell differentiation, proliferation, apoptosis, metabolism, glucose homeostasis, hematopoiesis, immune responses, memory, and learning (Zhang et al., 2020). Studies have shown that cAMP analogs like dbCAMP or cAMP activators like forskolin can induce neuronal differentiation in patient-derived GSCs, including the expression of neuronal markers MAP2 and TUBB3 and morphological changes, and inhibit GSC proliferation and self-renewal (Chen et al., 2022; Liu et al., 2021). PKA inhibitors and CREB1 knockout block cAMP-induced neuronal differentiation (Liu et al., 2021).

Wnt/β-catenin signaling pathway

The Wnt/β-catenin pathway plays a pivotal role in embryonic development and adult tissue homeostasis. Activation of the canonical Wnt pathway induces glycogen synthase kinase 3β (GSK-3β) phosphorylation, stabilizing β-catenin and translocating it to the nucleus. β-Catenin binds to T cell factor (TCF), ultimately activating gene expression involved in cell proliferation, survival, differentiation, and migration (Liu et al., 2022; Yu et al., 2021). Wnt/β-catenin pathway activity is generally elevated in gliomas, contributing to their development (He et al., 2019). However, in NSCs, activation of the Wnt/β-catenin pathway activates NGN2 and promotes neuronal differentiation (Hulme et al., 2022; Wang et al., 2022a), while inhibition of this pathway hinders NSC neuronal differentiation (Rampazzo et al., 2013). Studies have shown that Wnt ligand treatment or β-catenin overexpression under hypoxic conditions significantly reduces the expression of NSC markers Nestin and CD133 in patient-derived GBM cells and mediates neuronal differentiation with the expression of TUBB3 and proliferation inhibition, which is related to hypoxia-inducible factor-1 (HIF-1α) activation of TCF1 and inhibition of TCF4. Simultaneous activation of the Wnt signal and silencing of TCF4 under normoxic conditions can also induce neuronal differentiation in GBM cells (Rampazzo et al., 2013; Zhang et al., 2022). In addition, the NeuroD1-induced transdifferentiation is regulated by Wnt signaling and markedly enhanced under a hypoxic condition in human GBM cell line U373 (Jiang et al., 2024).

ERK signaling pathway

ERK signaling is triggered by receptor-ligand interactions, activating receptor tyrosine kinases, which in turn activate Ras and phosphorylate downstream Raf-MEK-ERK1/2. Activated ERK translocates to the nucleus and activates various transcription factors. In NSCs, activation of the ERK signaling pathway promotes neuronal differentiation (Iroegbu et al., 2021). However, in gliomas, ERK activation is a tumor-promoting factor (Huang et al., 2023; Pan et al., 2021). Sabelström et al. (2019) showed that the MEK inhibitor PD0325901 can induce neuronal differentiation with the expression of neuronal marker TUBB3 in patient-derived GBM cells but only in those with high SOX9 expression. Inhibition of ERK1/2 induces miR-124 expression and simultaneously downregulates SOX9. Inhibition of ERK signaling-induced neuronal differentiation depends on the regulation of miR-124 and SOX9. Khan et al. (2023) showed that MEK inhibitor trametinib upregulated the expression of numerous neuronal differentiation marker genes in patient-derived GSCs detected by RNA-Seq.

cAMP pathway activation + wnt/β-catenin pathway inhibition

Chen et al. (2022) found that cAMP/PKA pathway activation directly phosphorylates and inactivates GSK-3β, stabilizing β-catenin. Activated β-catenin enters the nucleus and promotes the transcription of apelin receptor early endogenous ligand (APELA) and caspase recruitment domain family member 16 (CARD16), which are factors that hinder cAMP-induced GSC differentiation. Inhibition of β-catenin enhances cAMP-induced neuronal differentiation and proliferation inhibition in patient-derived GSCs. In vivo, β-catenin inhibitors synergize with cAMP activators to inhibit GSC tumor growth and trigger neuronal differentiation with the expression of neuronal mark MAP2.

cAMP pathway activation + histone deacetylase inhibition

Histone deacetylases (HDACs) are essential chromatin-modifying enzymes that deacetylate histone tail lysines, increasing their affinity for negatively charged DNA backbones and inhibiting transcription, thereby regulating numerous biological processes (King et al., 2021). HDAC inhibitors have been shown to enhance NSC neuronal differentiation (Shukla and Tekwani, 2020). Liu et al. (2023) demonstrated that the HDAC inhibitor MS-275 and cAMP activators synergistically and stably induce the differentiation of human GBM cell line U87 and patient-derived GSCs into neuron-like cells, accompanied by cell cycle arrest, abundant expression of neuronal marker TUBB3 and MAP2, and typical neuronal electrophysiological characteristics.

Combination of Transcription Factors and Signaling Pathways Regulation

ASCL1 + Notch pathway inhibition

Notch signaling exhibits diverse context-specific functions and plays a pivotal role in numerous processes. Typically, Notch pathways are activated through the binding of Notch ligands expressed on adjacent cells to Notch receptors (Wang et al., 2015). In neural progenitor cells, ASCL1, while driving neuronal differentiation, also directly activates the transcription of Notch ligands, thereby activating the Notch signaling pathway in neighboring progenitor cells. Activation of the Notch signal promotes Hes1 expression, which subsequently inhibits ASCL1 expression and neuronal differentiation (Vasconcelos and Castro, 2014). In GBM cells, ASCL1 activates the expression of NOTCH1 and NOTCH3, the genes encoding Notch receptors (Wang et al., 2021). Park et al. (2017) demonstrated that a subset of patient-derived GSCs expresses relatively high levels of ASCL1. Inhibition of Notch signaling in this ASCL1-high GSC subpopulation upregulates ASCL1 expression, subsequently reducing GSC self-renewal capacity and promoting neuronal differentiation with the expression of neuronal marker TUBB3. Conversely, GSCs with low ASCL1 expression cannot differentiate into neurons through Notch signaling inhibition.

ASCL1 + Notch and Wnt pathway inhibition

Rajakulendran et al. (2019) reported that in GSCs expressing high levels of ASCL1, both Wnt and Notch signaling pathways contribute to maintaining GSC self-renewal capacity and undifferentiated status. Dual inhibition of Wnt/β-catenin and Notch signaling pathways in ASCL1-high patient-derived GSCs synergistically induces stable neuronal differentiation and inhibits clonogenic potential. The transformed neurons expressed neuronal marker TUBB3. ASCL1-low GSCs, however, lack differentiation potential, possibly escaping these developmental signals through additional mutations or epigenetic changes.

miRNA

miR-124

miR-124 is one of the most abundant miRNAs in the central nervous system. It is highly expressed in neurons, participating in neuronal maturation and function, and its expression increases with neuronal maturation. Additionally, miR-124 expression is reduced in many tumor cells (Sanuki and Yamamura, 2021). Overexpression of miR-124 promotes neuronal differentiation of NSCs (Jiao et al., 2017). Studies have shown that overexpression of miR-124 can induce neuronal differentiation in patient-derived GSCs, with the expression of neuronal marker TUBB3, and induce cell cycle arrest in human GBM cell lines U87 and U251 (Silber et al., 2008). The differentiation-inducing effect of miR-124 is associated with downregulation of SOX9 (Sabelström et al., 2019).

miR-137

Similar to miR-124, miR-137 is a miRNA related to neurodevelopment and tumorigenesis, highly expressed in the brain and promoting neurodifferentiation of various stem cells. Dysfunction of miR-137 has been implicated in various malignancies, including GBM (Mahmoudi and Cairns, 2017). Both miR-124 and miR-137 are negatively regulated by RE-1 silencing transcription factor in gliomas (Marisetty et al., 2017; Wang et al., 2020). miR-137 targets in gliomas mainly include oncogenes and key genes in neurogenesis, with predicted extensive overlap with miR-124 targets (Tamim et al., 2014). Overexpression of miR-137 can also induce neuronal differentiation of patient-derived GSCs and cell cycle arrest in human GBM cell lines U87 and U251 and GSCs (Silber et al., 2008).

Other Targets

Polypyrimidine tract binding protein 1

Polypyrimidine tract binding protein 1 (PTBP1) is a posttranscriptional regulator of gene expression that binds to pyrimidine tracts within mRNA 3′ splice sites, preventing exon splicing. PTBP1 plays a crucial role in neuronal growth and differentiation and is also implicated in various tumors, including gliomas (Zhu et al., 2020). In mouse hair follicle stem cells, PTBP1 is identified as the target of miR-124, and miR-124 may promote neuronal differentiation by targeting PTBP1 (Mokabber et al., 2019). Wang et al., (2022b) showed that PTBP1 knockout induces neuronal differentiation in human GBM cell lines U251, U87, and KNS89, with cells exhibiting neuronal morphology and expressing neuronal marker TUBB3. PTBP1 knockout inhibits GBM cell proliferation and tumor growth in mice, partially through upregulation of the neuronal axon guidance receptor unc-5 netrin receptor B (UNC5B).

Soluble amyloid precursor protein α/β

There are two processing pathways for amyloid precursor protein (APP): amyloidogenic and nonamyloidogenic pathways. The amyloidogenic pathway produces the soluble N-terminal APP fragment soluble amyloid precursor protein β (sAPP-β), and the nonamyloidogenic pathway produces the soluble N-terminal APP fragment sAPP-α (Cho et al., 2022). Nonamyloidogenic proteogenesis is the predominant APP processing pathway in the healthy brain, and sAPP-α plays a role in synaptic growth and plasticity (Corbett and Hooper, 2018). Jiang et al. (2013) demonstrated that sAPP-α promotes the neuronal differentiation of human GBM cell line U251 and the differentiated cells exhibit cholinergic-like neuronal phenotypes, expressing neuronal marker TUBB3 and cholinergic neuron marker choline acetyltransferase (ChAT), whereas sAPP-β inhibits the neuronal differentiation of GBM cells toward neurons but promotes their differentiation to astrocytes.

Compounds

Sodium butyrate

Liu et al. (2019) showed that sodium butyrate induces differentiation of human GBM cell line U87 into cells with cholinergic neuronal characteristics, expressing neuronal markers MAP2, SYP and neurofilament medium, and cholinergic receptors M1, M4, and M5, accompanied by increased expression of the neurotransmitter acetylcholine and cell cycle arrest. Sodium butyrate-induced differentiation of GBM cells into cholinergic neurons partially occurs through activation of the Akt signaling pathway.

Isoliquiritigenin

Lin et al. (2018) reported that isoliquiritigenin inhibits GSCs (isolated from GBM cell line SHG44) growth and neurosphere formation in a dose-dependent manner while inducing differentiation into astrocytes and neurons. The transformed neurons expressed neuronal marker TUBB3. The differentiation-inducing effect of isoliquiritigenin may be related to downregulation of the Notch signaling pathway.

Forskolin + ISX9 + CHIR99021 + I-BET 151 + DAPT (FICBD)

Lee et al. (2018) showed that a small molecule cocktail consisting of forskolin, isoxazole 9 (ISX9), CHIR99021, I-BET 151, and DAPT reprogrammed human GBM cells U87 into differentiated neurons. The reprogrammed cells displayed morphological characteristics associated with neuronal phenotypes and expressed neuronal markers DCX, TUBB3, and MAP2. In addition, the chemical cocktail upregulated the expression of NGN2, ASCL11, and BRN2, which may play a role in the mechanism underlying neuronal differentiation induction.

Conclusion

Numerous studies on inducing neuronal differentiation in glioma cells have shown that differentiating GBM cells or GSCs into neuron-like cells inhibits cell proliferation, promotes cell cycle exit, reduces cell self-renewal capacity, and inhibits in vivo tumorigenicity or tumor growth, suggesting that neuronal differentiation induction is a promising therapeutic strategy for GBM. These studies involve regulating multiple targets and pathways, including single and combined targeting strategies. Many studies focus on transcription factor-induced neuronal differentiation, particularly those belonging to the bHLH family, with ASCL1 being the most studied. These transcription factors can induce neuronal differentiation in both NSCs and gliomas. Signaling pathway regulation mainly involves cAMP, Wnt, and Notch signaling, with some pathways exhibiting different roles in NSCs and gliomas.

Given the similarities between NSCs and GSCs, many studies have successfully applied factors promoting NSC neuronal differentiation to GSCs or GBM cells. However, there are exceptions. For instance, RAS/ERK signal activation promotes NSC neuronal differentiation but acts as an oncogenic signal in gliomas, where excessive RAS/ERK signaling promotes ASCL1 phosphorylation, abolishing its differentiation-inducing effect. Inhibiting RAS/ERK signaling can induce neuronal differentiation in GBM cells but only in SOX9-high cells. Wnt signal activation promotes NSC neuronal differentiation but acts as an oncogenic signal in gliomas, where Wnt inhibition can induce neuronal differentiation only under hypoxic conditions. These findings indicate that differentiation induction is a complex regulatory process requiring tailored differentiation schemes based on specific gene expression profiles.

For certain targets or pathways, while inducing neuronal differentiation in GBM cells or GSCs, they may also activate factors inhibiting differentiation. For example, ASCL1 induces neuronal differentiation but simultaneously activates Notch signaling to counteract differentiation, with ASCL1 and Notch inhibition exhibiting synergy. Activation of cAMP signaling induces neuronal differentiation but also activates Wnt/β-catenin signaling to hinder differentiation, with cAMP activation and Wnt inhibition working synergistically. Therefore, thoroughly understanding the mechanisms or gene expression changes induced by regulating a specific target/pathway is crucial for identifying efficient differentiation-inducing schemes.

Although neuronal differentiation has demonstrated inhibitory effects on GBM cell and GSCs, many studies are conducted under in vitro conditions, and its in vivo efficacy remains to be further validated. Additionally, many methods for inducing differentiation rely on genetic manipulation or compounds in preclinical stage, indicating a long distance from clinical application. Since neuronal differentiation merely inhibits cell proliferation and stemness, it may not serve as a standalone therapeutic approach for glioma. However, in combination with surgery, it may exhibit promising effects in preventing recurrence.

In summary, cellular differentiation is a complex regulatory process. We have summarized the molecules involved in neuronal differentiation in GBM cells and their interactions in Figure 1 and approaches for neuronal differentiation induction in Table 1. Although significant progress has been made in inducing neuronal differentiation in glioma cells, there is still a long way to go before clinical application. Future research should further elucidate the biological processes of GSC neuronal differentiation, delve into the specific molecular mechanisms of these differentiation-inducing strategies, and discover more efficient methods for inducing differentiation, ultimately leading to the identification or development of suitable differentiation-inducing drugs based on these mechanisms.

FIG. 1.

Molecules/pathways associated with neuronal differentiation in glioblastoma multiforme (GBM).

Table 1.

Approaches for Neuronal Differentiation Induction in Human GBM Cells or GSCs

Approaches for neuronal differentiation induction	Cell lines	Neuronal markers expression	References
ASCL1 ↑	U251	MAP2, NeuN, DCX, TUBB3, SYN1, GABA, VGLUT1	Cheng et al., 2019; Wang et al., 2021
NGN2 ↑	U251	MAP2, NeuN, DCX, TUBB3, SYN1, VGLUT1	Cheng et al., 2019; Wang et al., 2021
NGN2 ↑	patient-derived GSCs	MAP2, DCX, TUBB3	Guichet et al., 2013
NeuroD1 ↑	LN229, T98G, U373, U87	MAP2, TUBB3, NeuN	Jiang et al., 2024
NeuroD4 ↑	U251, KNS89	TUBB3, MAP2	Wang et al., 2023a
SOX11 ↑	U87, patient-derived GSCs	MAP2, TUBB3	Fu et al., 2019; Hide et al., 2009
ZIC1 ↑	U87	MAP2, TUBB3	Fu et al., 2019
NGN2+SOX11 ↑	U87, U251	MAP2, NeuN, TUBB3, SYN1, SYT1, GABA, GAD67, VGLUT1, VGLUT2	Su et al., 2014
ASCL1+NGN2+BRN2 ↑	U87, U251, patient-derived GSCs	TUBB3, MAP2, NEFH, NeuN, SYP	Zhao et al., 2012
cAMP/PKA/CREB ↑	patient-derived GSCs	MAP2, TUBB3	Chen et al., 2022; Liu et al., 2021
Wnt/β-catenin ↑ (hypoxia)	patient-derived GSCs	TUBB3	Rampazzo et al., 2013; Zhang et al., 2022
ERK inhibition ↓	patient-derived GSCs	TUBB3	Sabelström et al., 2019
cAMP ↑ + Wnt/β-catenin ↓	patient-derived GSCs	MAP2	Chen et al., 2022
cAMP ↑ + HDAC ↓	U87, patient-derived GSCs	TUBB3, MAP2	Liu et al., 2023
ASCL1 ↑ + Notch ↓	patient-derived GSCs	TUBB3	Park et al., 2017
ASCL1 ↑ + Notch ↓ + Wnt/β-catenin ↓	patient-derived GSCs	TUBB3	Rajakulendran et al., 2019
miR-124 ↑	patient-derived GSCs	TUBB3	Silber et al., 2008
miR-137 ↑	patient-derived GSCs	TUBB3	Silber et al., 2008
PTBP1 ↓	U251, U87, KNS89	TUBB3	Wang et al., 2022a
sAPP-α ↑	U251	TUBB3, ChAT	Jiang et al., 2013
Sodium butyrate	U87	MAP2, SYP, HEFM, M1, M4, M5	Liu et al., 2019
Isoliquiritigenin	SHG44-derived GSCs	TUBB3	Lin et al., 2018
Forskolin + ISX9 + CHIR99021 + I-BET 151 + DAPT (FICBD)	U87	DCX, TUBB3, MAP2	Lee et al., 2018

ASCL1, achaete-scute complex homolog 1; BRN2, brain-2; GBM, glioblastoma multiforme; cAMP, cyclic adenosine monophosphate; CREB, cAMP-responsive element binding protein; DCX, doublecortin; GABA, γ-aminobutyric acid; GAD67, glutamic acid decarboxylase 67; GSC, glioma stem cell; HDAC, histone deacetylase; HEFM, neurofilament medium; MAP2, microtubule-associated protein-2; NEFH, neurofilament heavy; NeuN, neuronal nuclei antigen; NeuroD, neuronal differentiation; NGN2, neurogenin 2; PKA, protein kinase A; PTBP1, polypyrimidine tract binding protein 1; sAPP, soluble amyloid precursor protein; SOX11, SRY-related high-mobility group box 11; SYN1, synapsin 1; SYT, synaptotagmin-1; TUBB3, tubulin beta-3; VGLUT1, vesicular glutamate transporter 1; ZIC1, Zic family member 1.

Footnotes

Authors’ Contributions

Z.-Q.T.: Conceptualization, writing—original draft. Y.-R.Y.: Writing—original draft. Y.S.: Writing—review and editing.

Author Disclosure Statement

The authors have no relevant financial or nonfinancial interests to disclose.

Funding Information

This work was financially supported by the Natural Science Foundation of Xiamen, China (grant number 3502Z202372068).

References

Aldoghachi

, Aldoghachi

, Breyne

, et al. Recent advances in the therapeutic strategies of glioblastoma multiforme. Neuroscience, 2022; 491:240–270; doi: j.neuroscience.2022.03.030

Azzarelli

, McNally

, Dell’Amico

, et al. ASCL1 phosphorylation and ID2 upregulation are roadblocks to glioblastoma stem cell differentiation. Sci Rep, 2022; 12(1):2341; doi: 10.1038/s41598-022-06248-x

Chen

, Zhong

, Chen

, et al. Disruption of β-catenin-mediated negative feedback reinforces cAMP-induced neuronal differentiation in glioma stem cells. Cell Death Dis, 2022; 13(5):493; doi: 10.1038/s41419-022-04957-9

Cheng

, Tan

, Huang

, et al. Inhibition of glioma development by ASCL1-Mediated direct neuronal reprogramming. Cells, 2019; 8(6):571; doi: 10.3390/cells8060571

Cho

, Bae

, Okun

, et al. Physiology and pharmacology of amyloid precursor protein. Pharmacol Ther, 2022; 235:108122; doi: 10.1016/j.pharmthera.2022.108122

Corbett

, Hooper

. Soluble amyloid precursor protein α: Friend or foe? Adv Exp Med Biol, 2018; 1112:177–183; doi: 10.1007/978-981-13-3065-0_13

Fane

, Chhabra

, Smith

, et al. BRN2, a POUerful driver of melanoma phenotype switching and metastasis. Pigment Cell Melanoma Res, 2019; 32(1):9–24; doi: 10.1111/pcmr.12710

, Chen

, Hu

, et al. A single factor induces neuronal differentiation to suppress glioma cell growth. CNS Neurosci Ther, 2019; 25(4):486–495; doi: 10.1111/cns.13066

Guichet

, Bieche

, Teigell

, et al. Cell death and neuronal differentiation of glioblastoma stem-like cells induced by neurogenic transcription factors. Glia, 2013; 61(2):225–239; doi: 10.1002/glia.22429

10.

, Zhou

, Zeng

, et al. Wnt/β-catenin signaling cascade: A promising target for glioma therapy. J Cell Physiol, 2019; 234(3):2217–2228; doi: 10.1002/jcp.27186

11.

Hide

, Takezaki

, Nakatani

, et al. Sox11 prevents tumorigenesis of glioma-initiating cells by inducing neuronal differentiation. Cancer Res, 2009; 69(20):7953–7959; doi: 10.1158/0008-5472.CAN-09-2006

12.

Huang

, Liu

, Luo

, et al. Par6 enhances glioma invasion by activating MEK/ERK pathway through a LIN28/let-7d positive feedback loop. Mol Neurobiol, 2023; 60(3):1626–1644; doi: 10.1007/s12035-022-03171-0

13.

Hulme

, Maksour

, St-Clair Glover

, et al. Making neurons, made easy: The use of Neurogenin-2 in neuronal differentiation. Stem Cell Reports, 2022; 17(1):14–34; doi: 10.1016/j.stemcr.2021.11.015

14.

Iroegbu

, Ijomone

, Femi-Akinlosotu

, et al. ERK/MAPK signalling in the developing brain: Perturbations and consequences. Neurosci Biobehav Rev, 2021; 131:792–805; doi: 10.1016/j.neubiorev.2021.10.009

15.

Jiang

, Wang

, Hou

, et al. Distinct roles of sAPP-α and sAPP-β in regulating U251 cell differentiation. Curr Alzheimer Res, 2013; 10(7):706–713; doi: 10.2174/15672050113109990141

16.

Jiang

, Yu

, Estaba

, et al. Reprogramming glioblastoma cells into non-cancerous neuronal cells as a novel anti-cancer strategy. Cells, 2024; 13(11):897; doi: 10.3390/cells13110897

17.

Jiao

, Liu

, Yao

, et al. miR-124 promotes proliferation and differentiation of neuronal stem cells through inactivating Notch pathway. Cell Biosci, 2017; 7:68; doi: 10.1186/s13578-017-0194-y

18.

Kavyanifar

, Turan

, Lie

. SoxC transcription factors: Multifunctional regulators of neurodevelopment. Cell Tissue Res, 2018; 371(1):91–103; doi: 10.1007/s00441-017-2708-7

19.

Khan

, Martinez-Ledesma

, Dong

, et al. Neuronal differentiation drives the antitumor activity of mitogen-activated protein Kinase Kinase (MEK) inhibition in glioblastoma. Neurooncol Adv, 2023; 5(1):vdad132; doi: 10.1093/noajnl/vdad132 vdad132

20.

King

, Patel

, Chandrasekaran

. Metabolism, HDACs, and HDAC inhibitors: A systems biology perspective. Metabolites, 2021; 11(11):792; doi: 10.3390/metabo11110792

21.

Lee

, Robinson

, Willerth

. Direct reprogramming of glioblastoma cells into neurons using small molecules. ACS Chem Neurosci, 2018; 9(12):3175–3185; doi: 10.1021/acschemneuro.8b00365

22.

Lee

, Kim

, Baek

. The bHLH transcription factors in neural development and therapeutic applications for neurodegenerative diseases. Int J Mol Sci, 2022; 23(22):13936; doi: 10.3390/ijms232213936

23.

, Mattar

, Dixit

, et al. RAS/ERK signaling controls proneural genetic programs in cortical development and gliomagenesis. J Neurosci, 2014; 34(6):2169–2190; doi: 10.1523/JNEUROSCI.4077-13

24.

Lin

, Sun

, Dang

, et al. Isoliquiritigenin inhibits the proliferation and induces the differentiation of human glioma stem cells. Oncol Rep, 2018; 39(2):687–694; doi: 10.3892/or.2017.6154

25.

Liu

, Xia

, Wang

, et al. Differentiation of human glioblastoma U87 cells into cholinergic neuron. Neurosci Lett, 2019; 704:1–7; doi: 10.1016/j.neulet.2019.03.049

26.

Liu

, Xiao

, et al. Wnt/β-catenin signalling: Function, biological mechanisms, and therapeutic opportunities. Signal Transduct Target Ther, 2022; 7(1):3; doi: 10.1038/s41392-021-00762-6

27.

Liu

, Tian

, Yu

, et al. miR-221/222 suppression induced by activation of the cAMP/PKA/CREB1 pathway is required for cAMP-induced bidirectional differentiation of glioma cells. FEBS Lett, 2021; 595(22):2829–2843; doi: 10.1002/1873-3468.14208

28.

Liu

, Guo

, Leng

, et al. Differential regulation of H3K9/H3K14 acetylation by small molecules drives neuron-fate-induction of glioma cell. Cell Death Dis, 2023; 14(2):142; doi: 10.1038/s41419-023-05611-8

29.

, Puls

, Chen

. Transcriptomic analyses of NeuroD1-mediated astrocyte-to-neuron conversion. Dev Neurobiol, 2022; 82(5):375–391; doi: 10.1002/dneu.22882

30.

Mahmoudi

, Cairns

. MiR-137: An important player in neural development and neoplastic transformation. Mol Psychiatry, 2017; 22(1):44–55; doi: 10.1038/mp.2016.150

31.

Marisetty

, Singh

, Nguyen

, et al. REST represses miR-124 and miR-203 to regulate distinct oncogenic properties of glioblastoma stem cells. Neuro Oncol, 2017; 19(4):514–523; doi: 10.1093/neuonc/now232

32.

McKinnon

, Nandhabalan

, Murray

, et al. Glioblastoma: Clinical presentation, diagnosis, and management. Bmj, 2021; 374:n1560; doi: 10.1136/bmj.n1560

33.

Mokabber

, Najafzadeh

, Mohammadzadeh Vardin

. miR-124 promotes neural differentiation in mouse bulge stem cells by repressing Ptbp1 and Sox9. J Cell Physiol, 2019; 234(6):8941–8950; doi: 10.1002/jcp.27563

34.

Pan

, Chen

, Sun

, et al. Effect of Ras-guanine nucleotide release factor 1-mediated H-Ras/ERK signaling pathway on glioma. Brain Res, 2021; 1754:147247; doi: 10.1016/j.brainres.2020.147247

35.

Park

, Guilhamon

, Desai

, et al. ASCL1 reorganizes chromatin to direct neuronal fate and suppress tumorigenicity of glioblastoma stem cells. Cell Stem Cell, 2017; 21(2):209–224.e7; doi: 10.1016/j.stem.2017.06.004

36.

Pinto

, Costa

ÂM

, Andrade

, et al. Brachyury is associated with glioma differentiation and response to temozolomide. Neurotherapeutics, 2020; 17(4):2015–2027; doi: 10.1007/s13311-020-00911-9

37.

Rahman

, Kim

, Ahn

, et al. PR domain containing protein 12 (prdm12) is a downstream target of the transcription factor zic1 during cellular differentiation in the central nervous system: PR domain containing protein is the right form. Int J Dev Neurosci, 2020; 80(6):528–537; doi: 10.1002/jdn.10048

38.

Rajakulendran

, Rowland

, Selvadurai

, et al. Wnt and Notch signaling govern self-renewal and differentiation in a subset of human glioblastoma stem cells. Genes Dev, 2019; 33(9–10):498–510; doi: 10.1101/gad.321968.118

39.

Ramar

, Guo

, Hudson

, et al. Progress in glioma stem cell research. Cancers (Basel), 2023; 16(1):102; doi: 10.3390/cancers16010102

40.

Rampazzo

, Persano

, Pistollato

, et al. Wnt activation promotes neuronal differentiation of glioblastoma. Cell Death Dis, 2013; 4(2):e500; doi: 10.1038/cddis.2013.32

41.

Rao

, Wang

, Li

, et al. Molecular mechanisms underlying ascl1-mediated astrocyte-to-neuron conversion. Stem Cell Reports, 2021; 16(3):534–547; doi: 10.1016/j.stemcr.2021.01.006

42.

Rheinbay

, Suvà

, Gillespie

, et al. An aberrant transcription factor network essential for Wnt signaling and stem cell maintenance in glioblastoma. Cell Rep, 2013; 3(5):1567–1579; doi: 10.1016/j.celrep.2013.04.021

43.

Sabelström

, Petri

, Shchors

, et al. Driving neuronal differentiation through reversal of an ERK1/2-miR-124-SOX9 axis abrogates glioblastoma aggressiveness. Cell Rep, 2019; 28(8):2064–2079.e11; doi: 10.1016/j.celrep.2019.07.071

44.

Sanuki

, Yamamura

. Tumor suppressive effects of miR-124 and its function in neuronal development. Int J Mol Sci, 2021; 22(11):5919; doi: 10.3390/ijms22115919

45.

Shukla

, Tekwani

. Histone deacetylases inhibitors in neurodegenerative diseases, neuroprotection and neuronal differentiation. Front Pharmacol, 2020; 11:537; doi: 10.3389/fphar.2020.00537

46.

Silber

, Lim

, Petritsch

, et al. miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells. BMC Med, 2008; 6:14; doi: 10.1186/1741-7015-6-14

47.

Soares

, Homem

CCF

, Castro

. Function of proneural genes ascl1 and asense in neurogenesis: How similar are they? Front Cell Dev Biol, 2022; 10:838431; doi: 10.3389/fcell.2022.838431

48.

Soukup

, Gerykova

, Rachelkar

, et al. Diagnostic utility of immunohistochemical detection of MEOX2, SOX11, INSM1 and EGFR in gliomas. Diagnostics (Basel), 2023; 13(15):2546; doi: 10.3390/diagnostics13152546

49.

, Zang

, Liu

, et al. Reprogramming the fate of human glioma cells to impede brain tumor development. Cell Death Dis, 2014; 5(10):e1463; doi: 10.1038/cddis.2014.425

50.

Tamim

, Vo

, Uren

, et al. Genomic analyses reveal broad impact of miR-137 on genes associated with malignant transformation and neuronal differentiation in glioblastoma cells. PLoS One, 2014; 9(1):e85591; doi: 10.1371/journal.pone.0085591

51.

Tsang

, Oliemuller

, Howard

. Regulatory roles for SOX11 in development, stem cells and cancer. Semin Cancer Biol, 2020; 67(Pt 1):3–11; doi: 10.1016/j.semcancer.2020.06.015

52.

Tutukova

, Tarabykin

, Hernandez-Miranda

. The role of neurod genes in brain development, function, and disease. Front Mol Neurosci, 2021; 14:662774; doi: 10.3389/fnmol.2021.662774

53.

Valor

, Hervás-Corpión

. The epigenetics of glioma stem cells: A brief overview. Front Oncol, 2020; 10:602378; doi: 10.3389/fonc.2020.602378

54.

Vasconcelos

, Castro

. Transcriptional control of vertebrate neurogenesis by the proneural factor Ascl1. Front Cell Neurosci, 2014; 8:412; doi: 10.3389/fncel.2014.00412

55.

Wang

CUI

, Chen

J-C

, Xiao

H-H

, et al. Jujuboside a promotes proliferation and neuronal differentiation of APPswe-overexpressing neural stem cells by activating Wnt/β-catenin signaling pathway. Neurosci Lett, 2022a;772:136473; doi: 10.1016/j.neulet.2022.136473

56.

Wang

, Zang

, Liu

, et al. The role of Notch receptors in transcriptional regulation. J Cell Physiol, 2015; 230(5):982–988; doi: 10.1002/jcp.24872

57.

Wang

, Zhao

, Zhang

, et al. NeuroD4 converts glioblastoma cells into neuron-like cells through the SLC7A11-GSH-GPX4 antioxidant axis. Cell Death Discov, 2023a;9(1):297; doi: 10.1038/s41420-023-01595-8

58.

Wang

, Pan

, Zhao

, et al. PTBP1 knockdown promotes neural differentiation of glioblastoma cells through UNC5B receptor. Theranostics, 2022b;12(8):3847–3861; doi: 10.7150/thno.71100

59.

Wang

, Englander

, Miller

, et al. Malignant Glioma. Adv Exp Med Biol, 2023b;1405:1–30; doi: 10.1007/978-3-031-23705-8_1, 37452933

60.

Wang

, Pei

, Hossain

, et al. Transcription factor-based gene therapy to treat glioblastoma through direct neuronal conversion. Cancer Biol Med, 2021; 18(3):860–874; doi: 10.20892/j.issn.2095-3941.2020.0499

61.

Wang

, Chen

, Zhou

, et al. miR-137: A novel therapeutic target for human glioma. Mol Ther Nucleic Acids, 2020; 21:614–622; doi: 10.1016/j.omtn.2020.06.028

62.

Woods

, Ali

, Gomez

, et al. Elevated ASCL1 activity creates de novo regulatory elements associated with neuronal differentiation. BMC Genomics, 2022; 23(1):255; doi: 10.1186/s12864-022-08495-8

63.

Yang

, Wu

, Zhang

, et al. Glioma targeted therapy: Insight into future of molecular approaches. Mol Cancer, 2022; 21(1):39; doi: 10.1186/s12943-022-01513-z

64.

Yang

, Jiang

, Lu

, et al. SOX11: Friend or foe in tumor prevention and carcinogenesis? Ther Adv Med Oncol, 2019; 11:1758835919853449; doi: 10.1177/1758835919853449

65.

, Yu

, Li

, et al. Wnt/β-catenin signaling in cancers and targeted therapies. Signal Transduct Target Ther, 2021; 6(1):307; doi: 10.1038/s41392-021-00701-5

66.

Zhang

, Kong

, Wang

, et al. Complex roles of cAMP-PKA-CREB signaling in cancer. Exp Hematol Oncol, 2020; 9(1):32; doi: 10.1186/s40164-020-00191-1

67.

Zhang

, Hu

, Jiang

, et al. Actin Alpha 2 downregulation inhibits neural stem cell proliferation and differentiation into neurons through canonical Wnt/β-Catenin signaling pathway. Oxid Med Cell Longev, 2022; 2022:7486726; doi: 10.1155/2022/7486726

68.

Zhao

, He

, Zhou

, et al. Neuronal transcription factors induce conversion of human glioma cells to neurons and inhibit tumorigenesis. PLoS One, 2012; 7(7):e41506; doi: 10.1371/journal.pone.0041506

69.

Zhu

, Zhou

, Rong

, et al. Roles of PTBP1 in alternative splicing, glycolysis, and oncogensis. J Zhejiang Univ Sci B, 2020; 21(2):122–136; doi: 10.1631/jzus.B1900422

70.

Zhu

, Zhou

, Jin

, et al. Brn2 alone is sufficient to convert astrocytes into neural progenitors and neurons. Stem Cells Dev, 2018; 27(11):736–744; doi: 10.1089/scd.2017.0250