Abstract

Glioblastoma multiforme (GBM) is the most aggressive and lethal primary tumor of the central nervous system, characterized by extensive molecular heterogeneity, intrinsic therapy resistance, and poor clinical outcome. Despite advances in surgical resection, radiotherapy, and chemotherapy, patient prognosis remains dismal, highlighting the urgent need for a deeper understanding of the molecular signaling networks driving GBM initiation and progression.\\nThis mini-review aims to summarize recent advancements in the molecular biology of GBM, with a specific focus on the functional interplay between the PI3K/AKT/mTOR and WNT/β-catenin signaling pathways. Both pathways are frequently dysregulated in GBM and play central roles in regulating cell proliferation, survival, metabolism, stemness, and therapeutic resistance. Rather than acting as independent oncogenic modules, accumulating evidence indicates that these signaling cascades engage in a dynamic molecular crosstalk, converging on shared effectors such as GSK3B, the DEPTOR-FZD-DVL axis, EIF4E, and PP2A\\nBy integrating current findings, this review provides insight into the reciprocal regulation between PI3K/AKT/mTOR and WNT/β-catenin signaling in GBM cells and glioma stem cells, emphasizing their cooperative contribution to tumor progression. Finally, we discuss the therapeutic implications of targeting pathway crosstalk rather than individual signaling nodes, highlighting this interconnected network as a promising vulnerability for future combinatorial treatment strategies in GBM.

Content

I. INTRODUCTION
Glioblastoma multiforme (GBM) represents the most aggressive and malignant form of glioma and remains one of the most challenging tumors to treat in neuro-oncology. Gliomas are primary brain tumors classified by the World Health Organization (WHO) into grades I–IV according to histopathological and molecular criteria, with GBM corresponding to grade IV. GBM displays an incidence rate of approximately 10 cases per 100,000 individuals and is associated with an extremely poor prognosis, with a median overall survival of 14–15 months and a five-year survival rate below 5% ¹.
At the molecular level, GBM is characterized by profound genetic and epigenetic heterogeneity. Several oncogenic drivers have been implicated in GBM pathogenesis, including mutations in metabolic enzymes such as IDH1 and IDH2 ^2,3, as well as aberrant activation of receptor tyrosine kinases (RTKs). Large-scale genomic analyses from The Cancer Genome Atlas (TCGA) have revealed that approximately 88% of primary GBM samples harbor alterations in the RTK/PI3K signaling axis, while frequent disruptions are also observed in key tumor suppressor pathways, including TP53 (87%) and RB1 (78%).
Within this complex oncogenic landscape, increasing attention has been directed toward the PI3K/AKT/mTOR and WNT/β-catenin CTNNB1 signaling pathways, both of which play pivotal roles in embryonic development, tissue homeostasis, and cancer. Dysregulation of these pathways has been extensively documented across multiple malignancies, including colorectal cancer, ⁴, pancreatic cancer ⁵, lung cancer ⁶, breast cancer ⁷, and hepatocellular carcinoma ⁸, as well as in GBM. Importantly, recent studies suggest that the oncogenic potential of these pathways in GBM cannot be fully explained by their independent activation alone, but rather by their ability to engage in a complex and coordinated molecular crosstalk.
In recent years, growing evidence has highlighted the reciprocal regulation between PI3K/AKT/mTOR and WNT/β-catenin signaling, revealing multiple points of convergence and shared downstream effectors. This bidirectional communication influences critical cellular processes such as proliferation, metabolic reprogramming, invasion, therapy resistance, and the maintenance of glioma stem cells (GSCs). Understanding how these pathways interact at the molecular level is therefore essential for unraveling the biological complexity of GBM.
GBM remains particularly difficult to treat due to both anatomical and biological constraints. The presence of the blood–brain barrier (BBB) severely limits the delivery of therapeutic agents to the tumor site, while intratumoral heterogeneity further complicates effective targeting. Moreover, GSCs—key drivers of tumor initiation, progression, invasiveness, recurrence, and therapeutic resistance—exhibit slow proliferation rates and enhanced adaptive capacity, distinguishing them from other cancer stem cell populations and rendering them less susceptible to conventional cytotoxic therapies ⁹.
Given these challenges, dissecting the signaling networks that regulate GBM growth, stemness, and survival is of paramount importance. In this context, the PI3K/AKT/mTOR and WNT/β-catenin pathways emerge not only as central oncogenic drivers but also as interconnected signaling hubs whose molecular crosstalk may represent an exploitable vulnerability. This review aims to provide an integrated overview of the mechanisms underlying their interaction in GBM and to discuss the therapeutic implications of targeting this signaling convergence.

II. PI3K/AKT/mTOR SIGNALING PATHWAY
2.1 Physiological Signaling Pathway Breakdown
The PI3K/AKT/mTOR signaling pathway is a central regulator of cellular proliferation, growth, metabolism, migration, and survival, while actively suppressing apoptotic processes.¹⁰ Activation of this pathway is typically initiated by receptor tyrosine kinases (RTKs), which undergo ligand-induced dimerization and transphosphorylation. Upon activation, RTKs become phosphorylated on specific tyrosine residues that serve as docking sites for adaptor proteins such as GRB2 (Growth Factor Receptor-Bound Protein 2).
GRB2 binds to the activated receptor through its SH2 domain and recruits XYLT2 (Son of Sevenless), a guanine nucleotide exchange factor (GEF), via its SH3 domain. XYLT2 facilitates the exchange of GDP for GTP on RAS, a small GTPase associated with RTKs, thereby converting RAS into its active GTP-bound form. Activated RAS-GTP subsequently stimulates PIK3CA (Phosphatidylinositol-4,5-Bisphosphate 3-Kinase), a key upstream driver of this signaling cascade.
PIK3CA is a lipid kinase composed of a regulatory subunit (p85), which binds to phosphorylated tyrosine residues on RTKs, and a catalytic subunit (p110), which mediates downstream signaling once activated by RAS-GTP. Activated PIK3CA phosphorylates the membrane phospholipid PIP2 (Phosphatidylinositol-4,5-bisphosphate), converting it into PIP3 (Phosphatidylinositol-3,4,5-triphosphate).¹¹ PIP3 acts as a second messenger by recruiting and activating downstream kinases, including PDK1 and PDK2 ¹².
PDK1 and PDK2 phosphorylate AKT1/AKT1 (AKT serine/threonine kinase) at Thr308 and Ser473, respectively, leading to full activation of AKT. Once activated, AKT1/AKT1 functions as a central signaling hub, phosphorylating multiple downstream substrates involved in cell survival and growth, including IKK (NF-κB pathway), MDM2, CREB, and FOXO-1. Crucially, AKT inhibits the TSC1/TSC2 (Tuberous Sclerosis Complex 1/2), a GAP (GTPase-activating protein) for RHEB (Ras Homolog, mTORC1 binding), thereby promoting RHEB-mediated activation of MTOR (Mechanistic Target of Rapamycin Kinase).
MTOR is the third major effector of this pathway and exists within two distinct multiprotein complexes. The first, MTORC1, is activated downstream of AKT through inhibition of its negative regulator TSC1/TSC2, or through interactions involving RPTOR (Regulatory Associated Protein of mTOR Complex 1). MTORC1 promotes protein synthesis by phosphorylating EIF4EBP (Eukaryotic Translation Initiation Factor 4E Binding Protein), thereby releasing and activating EIF4E, and by activating RPS6K (Ribosomal Protein S6 Kinase), which phosphorylates ribosomal protein p70S6K and enhances mRNA translation. These events collectively promote cellular growth and proliferation.
The second complex, MTORC2, is directly activated by PIK3CA and by additional cellular stimuli, including growth factors, stress conditions, oxygen availability, and amino acids. MTORC2 primarily regulates cell survival, cytoskeletal organization, and lipid metabolism, further contributing to tumor progression.


Figure 1. PI3K/AKT/mTOR signaling pathway
In response to EGFR activation, PIK3CA is stimulated by RAS-GTP and catalyzes the conversion of PIP2 into PIP3, which recruits and activates PDK1 and PDK2. PDK1/PDK2 phosphorylate AKT1, leading to inhibition of the TSC1/TSC2 complex and consequent activation of RHEB. Activated RHEB promotes MTORC1 signaling. RPTOR regulates MTORC1 activity, which phosphorylates and inactivates EIF4EBP, thereby releasing EIF4E. This event enables RPS6K-mediated phosphorylation of the p70 ribosomal subunit, ultimately promoting protein synthesis and cell proliferation.

2.2 Mutations of PI3K/AKT/mTOR in GBM
Given its strong tumor-promoting activity, the PI3K/AKT/mTOR pathway is tightly regulated under physiological conditions. A key negative regulator is PTEN (Phosphatase and Tensin Homolog), a lipid phosphatase and tumor suppressor that antagonizes PI3K activity by dephosphorylating PIP3 to PIP2. In GBM cells, PTEN is frequently mutated or deleted, leading to loss of negative feedback control, accumulation of PIP3, and constitutive activation of AKT1/AKT1 ^13,14.
In addition to PTEN loss, GBM frequently exhibits amplification or overexpression of EGFR (Epidermal Growth Factor Receptor), an RTK whose aberrant activation further enhances PI3K/AKT/mTOR signaling. Together, these alterations contribute to sustained pathway activation, promoting tumor growth, survival, and therapeutic resistance.

III. WNT/β-CATENIN SIGNALING PATHWAY
3.1 Physiological Signaling Pathway Breakdown
The WNT/β-catenin signaling pathway plays a fundamental role in cell proliferation, embryonic development, stem cell regulation, and tissue homeostasis. In the central nervous system (CNS), this pathway is also implicated in vascularization and the maintenance of neuronal integrity ¹⁰. The principal components of this pathway are WNT ligands and CTNNB1 (β-catenin). In mammals, WNT proteins are encoded by 19 distinct genes and are critical mediators of extracellular signaling, including communication via exosomes. β-Catenin is a cytoplasmic protein that interacts with cadherins, which are key regulators of cell–cell adhesion and are frequently exploited during cancer progression. Under physiological conditions, WNT ligands bind to transmembrane Frizzled (FZD) receptors and their co-receptors LRP5/6 (Low-Density Lipoprotein Receptor-Related Protein 5/6) ¹⁵.
Ligand binding triggers downstream signaling events that promote β-catenin stabilization and nuclear translocation, a process facilitated by RAC1 (Rac Family Small GTPase 1). Once in the nucleus, β-catenin interacts with transcriptional co-activators such as PYGO (Pygopus Family PHD Finger), BCL9 (BCL9 Transcription Coactivator), and CREBBP (CREB Binding Lysine Acetyltransferase), forming a transcriptional complex with TCF/LEF family transcription factors.¹⁶ This complex drives the expression of WNT target genes, including c-myc, c-jun, cyclin D1, MMP-7 (Matrix Metalloproteinase-7), and Axin-1.
In the absence of WNT signaling, β-catenin levels are tightly controlled by a cytoplasmic destruction complex composed of AXIN1, APC (APC Regulator of WNT Signaling Pathway), GSK3B (Glycogen Synthase Kinase 3 Beta), and CSNK1A1 (Casein Kinase 1 Alpha). This complex phosphorylates β-catenin,¹⁷ targeting it for ubiquitination and subsequent proteasomal degradation,¹⁸ thereby preventing its nuclear accumulation and transcriptional activity.

3.2 Mutations of WNT/β-Catenin in GBM
In GBM cells, the WNT/β-catenin pathway is frequently overactivated, although the precise molecular mechanisms underlying this upregulation remain incompletely understood. Sustained WNT signaling promotes cellular proliferation through activation of target genes and contributes to tumor progression via interactions between cytoplasmic β-catenin and cadherins ¹⁰.
As observed in other malignancies, activation of the FZD–LRP5/6 receptor complex in GBM leads to recruitment of DVL1 (Dishevelled Segment Polarity Protein), which inhibits the β-catenin destruction complex. This inhibition results in cytoplasmic accumulation of β-catenin, its translocation to the nucleus mediated by RAC1, and enhanced transcriptional activity. In parallel, increased β-catenin availability facilitates its interaction with cadherin proteins, further supporting tumor invasiveness and progression.

Figure 2.Canonical WNT/β-Catenin signaling activation
Upon WNT ligand binding to the FZD–LRP5/6 receptor complex, DVL1 is activated and inhibits the β-catenin destruction complex composed of AXIN1, APC, GSK3B, and CSNK1A1. As a consequence, β-catenin accumulates in the cytoplasm and translocates to the nucleus in a RAC1-GTP–dependent manner, where it forms a transcriptional complex with TCF/LEF factors, enabling the expression of WNT target genes.


Figure 3. Inactive WNT/β-Catenin signaling
In the absence of WNT stimulation, DVL1 remains inactive and the β-catenin destruction complex (AXIN1–APC–GSK3B–CSNK1A1) is fully functional. This complex phosphorylates cytoplasmic β-catenin, targeting it for ubiquitination and subsequent proteasomal degradation, thereby preventing nuclear translocation and transcriptional activation.

IV. MOLECULAR NODES OF CROSSTALK
4.1 GSK3B
GSK3B is a critical regulator of WNT/β-catenin signaling and a core component of the β-catenin destruction complex. Through phosphorylation of cytoplasmic β-catenin, GSK3B promotes its ubiquitination and proteasomal degradation, thereby suppressing WNT signal transmission. Consequently, inhibition of GSK3B represents a key event enabling activation of the WNT/β-catenin pathway. Beyond its role in WNT signaling, GSK3B also functions as an important node of interaction between the PI3K/AKT/mTOR and WNT/β-catenin pathways. Within the PI3K axis, GSK3B can be inhibited by phosphorylation at Ser9 mediated by AKT1/AKT1, resulting in reduced kinase activity ¹⁹. Under conditions of reduced AKT activity, GSK3B may also be inactivated by the p70S6K complex downstream of MTORC1 activation.
He et al. ²⁰ demonstrated that rapamycin, an inhibitor of MTORC1, induces nuclear translocation of GSK3B, leading to the activation of transcriptional targets such as IIF1 and FOXO1 ²¹, which are associated with inhibition of tumor proliferation. Although PI3K/AKT/mTOR signaling clearly contributes to GSK3B inhibition, this regulatory effect appears largely independent of canonical WNT/β-catenin signaling, as no consistent feedback regulation of WNT activity has been observed under these conditions.
Nevertheless, Ding et al. ²² reported that hyperactivation of AKT1/AKT1, resulting in sustained inhibition of GSK3B, may facilitate cooperative activation of the WNT/β-catenin pathway. Conversely, WNT/β-catenin signaling exerts regulatory control over the PI3K/AKT/mTOR pathway. Inactivation of GSK3B through WNT-dependent mechanisms—including DVL1 activation, disassembly of the destruction complex via APC loss, or LRP5/6 phosphorylation—also leads to inhibition of TSC2, thereby promoting MTORC1 activation.
In summary, although both PI3K/AKT/mTOR and WNT/β-catenin pathways converge on GSK3B inhibition, current evidence suggests that only WNT/β-catenin signaling can directly regulate PI3K/AKT/mTOR activity through this node. Further studies are required to clarify whether PIK3CA–GSK3B interactions exert a reciprocal regulatory influence on WNT signaling.


4.2 DEPTOR–FZD–DVL Axis
Studies in colorectal cancer (CRC) ⁴ have revealed an important reciprocal negative regulation between the PI3K/AKT/mTOR and WNT/β-catenin pathways. Specifically, WNT/β-catenin signaling has been shown to upregulate DEPTOR (DEP Domain Containing MTOR Interacting Protein), a negative regulator of MTORC1, thereby attenuating PI3K/AKT/mTOR pathway activity ²³.
Conversely, MTORC1 has been reported to downregulate the expression of FZD receptors through mechanisms involving DVL1. In this context, DVL1 emerges not only as a core component of WNT/β-catenin signaling but also as a critical mediator of FZD and LRP5/6 receptor turnover, further reinforcing the bidirectional regulatory loop between these pathways.

4.3 EIF4E
EIF4E represents a key shared effector of the PI3K/AKT/mTOR and WNT/β-catenin pathways and is subject to tight regulatory control due to its strong oncogenic potential. Phosphorylation of EIF4E at Ser209 has been associated with increased oncogenic activity and enhanced translational output. This phosphorylation is mediated by MKNK1/MKNK2 (MAPK Interacting Kinase 1 and 2), which are activated downstream of MAPK signaling ²⁴.
Notably, in the presence of rapamycin, an inhibitor of MTORC1, compensatory activation of MAPK signaling has been observed. This activation allows MKNK1/MKNK2 to phosphorylate EIF4E, thereby enhancing its oncogenic function. Phosphorylated EIF4E promotes increased nuclear translocation of β-catenin ^25,26, ultimately driving transcription of WNT target genes and tumor proliferation. Importantly, inhibition of PIK3CA has been shown to suppress this entire signaling cascade, highlighting EIF4E as a critical integrative node within the PI3K–WNT crosstalk network.

Fig.4A

Fig.4B

Fig.4C

Fig.4D

Figure 4. Molecular crosstalk between PI3K/AKT/mTOR and WNT/β-Catenin signaling
Panels (4a–4d) illustrate the principal mechanisms underlying the bidirectional interplay between the PI3K/AKT/mTOR and WNT/β-catenin pathways. (4a) Regulation of PI3K/AKT/mTOR signaling by WNT/β-catenin through inhibition of GSK3B. (4b) Regulation of WNT/β-catenin signaling by PI3K/AKT/mTOR via MTORC1-dependent downregulation of FZD receptors mediated by DVL1. (4c) Inhibition of PI3K/AKT/mTOR signaling by WNT/β-catenin through upregulation of DEPTOR and consequent inhibition of MTORC1. (4d) Pharmacological inhibition of PI3K/AKT/mTOR signaling by rapamycin leads to compensatory EIF4E phosphorylation mediated by MKNK1/MKNK2, resulting in enhanced WNT/β-catenin signaling.⁴.


4.4 PP2A and Regulation of Active β-Catenin (ABC)
In 2002, Staal and colleagues ²⁷ demonstrated that β-catenin dephosphorylated at Ser37 and Thr41 residues exhibits enhanced transcriptional activity. This specific molecular species was termed ABC (Active β-Catenin). The dephosphorylation of these critical residues requires the activity of a phosphatase, which was initially hypothesized and subsequently confirmed to be PTPA (Protein Phosphatase 2A). This hypothesis was supported by experimental evidence showing that the regulatory subunit of PTPA inhibits WNT signaling, whereas the catalytic subunit promotes activation of the WNT/β-catenin pathway by increasing intracellular levels of ABC. Importantly, PTPA activity has been shown to be positively regulated by AKT1/AKT1. In cancer cells characterized by loss or mutation of PTEN, increased AKT1/AKT1 activity results in enhanced activation of PTPA, leading to elevated nuclear accumulation of ABC ²⁸.
Consistent with this mechanism, studies conducted in melanoma, prostate, and breast cancer models have demonstrated that reintroduction of functional PTEN or pharmacological inhibition of the PI3K/AKT pathway leads to reduced PTPA activity and a consequent decrease in nuclear ABC levels. These findings support a model in which PI3K/AKT signaling exerts indirect but critical control over β-catenin transcriptional output via modulation of PTPA activity.
Collectively, these observations indicate that while the WNT/β-catenin pathway primarily regulates the cytoplasmic stability and accumulation of β-catenin, the PI3K/AKT/mTOR pathway plays a dominant role in controlling the generation and nuclear availability of its transcriptionally active form (ABC) through PTPA-dependent mechanisms.


Figure 5. PP2A-mediated regulation of active β-Catenin (ABC)
This schematic illustrates the role of PTPA in regulating the transcriptionally active form of β-catenin (ABC). PTPA is activated downstream of AKT1, a key effector of the PI3K/AKT/mTOR pathway. Activated PTPA dephosphorylates β-catenin at Ser37 and Thr41 residues, promoting its nuclear translocation. In its dephosphorylated active form (ABC), β-catenin enhances assembly of the TCF/LEF transcriptional complex, thereby driving expression of WNT target genes ²⁸.

V. CONCLUSIONS
In summary, this review has critically analyzed recent advances in the understanding of the molecular mechanisms underlying Glioblastoma Multiforme (GBM), with a specific focus on the intricate crosstalk between the PI3K/AKT/mTOR and WNT/β-catenin signaling pathways. Although these pathways possess distinct oncogenic drivers and regulatory architectures, mounting evidence indicates that they are intimately interconnected through multiple shared molecular effectors, including GSK3B, the DEPTOR-FZD-DVL1 axis, EIF4E, and PTPA.
This analysis highlights that while the canonical WNT/β-catenin pathway primarily governs the stability and cytoplasmic accumulation of β-catenin, the PI3K/AKT/mTOR cascade is essential for regulating its transcriptionally active form (ABC), largely through mechanisms involving PTPA and its regulatory subunits. Furthermore, emerging evidence suggests that the reciprocal regulation between these signaling networks not only sustains GBM proliferation and invasiveness but also contributes to the complex resistance mechanisms characteristic of this aggressive tumor type.
Indeed, standard therapeutic approaches such as temozolomide (TMZ), an alkylating agent that induces DNA damage, have demonstrated limited long-term efficacy in GBM, in part due to compensatory activation of the PI3K/AKT/mTOR and WNT/β-catenin pathways and their downstream effectors ^10,29. These adaptive signaling responses underscore the limitations of monotherapies and the necessity for more integrated treatment strategies.
Given the intrinsic challenges associated with GBM treatment, a comprehensive understanding of these molecular interactions is essential. The interplay between the PI3K/AKT/mTOR and WNT/β-catenin pathways represents a promising therapeutic target landscape. Future research efforts should focus on elucidating the context-dependent dynamics of this crosstalk and on developing combinatorial therapeutic strategies capable of simultaneously disrupting multiple signaling nodes, thereby overcoming resistance mechanisms and improving clinical outcomes.
In conclusion, detailed dissection of these interconnected signaling pathways may pave the way for the development of innovative targeted therapies that transcend the limitations of current treatment modalities and offer renewed hope for patients with GBM.