Interferon-gamma signaling pathway: Modulation of key genes in the progression of glioblastoma

Abstract

The canonical signaling of interferon gamma (IFN-γ) through the Janus kinase 1 and 2–signal transducer and activator of transcription 1 (STAT1) axis leads to the expression of several interferon-stimulated genes (ISGs), which have diverse effects depending on the cellular context. In glioblastoma, a highly aggressive primary brain tumor in adults, elements of IFN-γ canonical signaling are deregulated, resulting in the overexpression of STAT1-target ISGs associated with tumor progression. This mini-review highlights key ISGs, including STAT1, interferon regulatory factor 1, programmed death-ligand 1, indoleamine 2,3-dioxygenase 1, and interferon-stimulated gene 15, involved in the pathology of glioblastoma. These genes may serve as valuable biomarkers and have therapeutic potential for targeting IFN-γ signaling in this malignancy.

Key Words: Glioblastoma; Interferon gamma; Janus kinases; Interferon-stimulated genes; Signal transducer and activator of transcription 1; Interferon regulatory factor 1; B7-H1 antigen; Indoleamine-2,3-dioxygenase; Interferon-stimulated gene 15; Signal transduction

Core Tip: This review examines the characteristics and functions of five genes induced by interferon-gamma—signal transducer and activator of transcription 1, interferon regulatory factor 1, programmed death-ligand 1, indoleamine 2,3-dioxygenase 1, and interferon-stimulated gene 15—that participate in various mechanisms that promote glioblastoma, a highly aggressive brain tumor; therefore, investigating these genes could contribute to the identification of potential biomarkers and therapeutic targets.

INTRODUCTION

Interferon gamma (IFN-γ) is responsible for orchestrating both adaptive and innate immune responses. IFN-γ can also play a dual role, pro- or anti-tumorigenic, depending on the tumor context[1]. Hence, IFN-γ is recognized for its connection to immunity and cancer; for example, IFN-γ can activate cytotoxic T lymphocytes and natural killer cells and contribute to the polarization of macrophages toward an M1 phenotype, thereby promoting apoptosis and necroptosis in tumor cells. Nevertheless, IFN-γ can also promote immune evasion when present in a chronic stimulus environment[2].

IFN-γ develops its function through its pathway and exerts its effects primarily by activating the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. Upon IFN-γ binding to its receptor, composed of IFNGR1 and IFNGR2 subunits, Janus kinase 1 (JAK1) and Janus kinase 2 (JAK2) become activated, leading to STAT1 activation[3-5], and its subsequent nuclear translocation, where it induces the transcription of interferon-stimulated genes (ISGs) that influence immune response, cell proliferation, apoptosis, and other cellular functions[6-8].

In cancer, the outcome of IFN-γ signaling is highly context-dependent. Although traditionally viewed as a tumor suppressor due to its cytotoxic and immune-activating properties, IFN-γ can paradoxically promote tumor progression in specific settings, including glioblastoma[9]. This dual nature is mediated by the altered expression and function of ISGs, which serve as downstream effectors of STAT1[10,11].

Glioblastoma is the most aggressive and lethal form of primary brain cancer and is characterized by high invasiveness, resistance to therapy, and poor prognosis[12,13]. Recent studies have shown that the canonical IFN-γ signaling pathway is not only active in glioblastoma cells but is also upregulated. Tumor sample analyses have revealed increased expression of IFNGR1, IFNGR2, and STAT1 in glioblastoma compared to healthy brain tissue[11]. This upregulation enhances the responsiveness of tumor cells to IFN-γ in the tumor microenvironment, which facilitates aberrant ISG expression[14-17] (Figure 1).

Figure 1 Interferon-gamma pathway in normal tissue and glioblastoma. A: Under physiological conditions, interferon-gamma binds to its receptor and activates the Janus kinases 1 and 2, promoting the phosphorylation and dimerization of the signal transducer and activator of transcription 1. The signal transducer and activator of transcription 1 (STAT1) dimer translocates to the nucleus, where it binds to gamma-activated sequence elements in the promoters of interferon-stimulated genes; B: In glioblastoma cells, the pathway is amplified. This is evidenced by increased interferon gamma receptor density on the plasma membrane and higher levels of STAT1, resulting in enhanced transcription of genes implicated in glioblastoma progression, including STAT1, interferon regulatory factor 1, programmed death-ligand 1, indoleamine 2,3-dioxygenase 1, and interferon-stimulated gene 15. JAK1: Janus kinases 1; JAK2: Janus kinases 2; STAT1: Signal transducer and activator of transcription 1; ISGs: Interferon-stimulated genes; PD-L1: Programmed death-ligand 1; IDO1: Indoleamine 2,3-dioxygenase 1; ISG15: Interferon-stimulated gene 15; GAS: Gamma-activated sequences; IRF1: Interferon regulatory factor 1.

Experimentally, glioblastoma cell lines, such as LN-18, exhibit elevated STAT1 phosphorylation and increased expression of downstream targets, including interferon regulatory factor 1 (IRF1) and programmed death-ligand 1 (PD-L1), following IFN-γ treatment. These changes promote cell migration, a key trait associated with tumor aggressiveness, suggesting an enhancement of invasive potential[11]. This mini-review examines IFN-γ-regulated genes related to cancer progression, specifically in the context of glioblastoma. Therefore, this study focuses on the following ISGs that are involved in the progression of glioblastoma: STAT1, IRF1, PD-L1, indoleamine 2,3-dioxygenase 1 (IDO1), and interferon-stimulated gene 15 (ISG15).

ISGs ASSOCIATED WITH GLIOBLASTOMA PROGRESSION

STAT1

STAT1, as the central mediator of IFN-γ signaling, plays an essential role in cancer. The STAT1 protein has a modular structure composed of six highly conserved tandem functional domains that enable it to carry out its roles as a signal transducer and transcription activator[18,19]. The N-terminal domain (N) is involved in oligomerization and protein-protein interaction. The next domain, called the coiled-coil domain, is involved in receptor binding and protein–protein interactions[19]. The DNA-binding domain (DBD) enables STAT1 to recognize specific sequences, known as gamma-activated sequences (GAS), in gene promoters and participates in nuclear export and import[20]. The linker domain provides structural flexibility to the protein between its DBD and Src homology 2 (SH2) domains and is also involved in nuclear export and transcriptional activity[18,19]. Finally, the C-terminal region harbors the transactivation domain, which includes the Ser727 residue whose phosphorylation enhances STAT1’s transcriptional capacity[18,20].

The activation of STAT1 is modulated by the JAK-STAT signaling pathway[6]. The binding of the cytokine IFN-γ to its plasma membrane receptor, which consists of a heterotetramer composed of two IFNGR1 subunits and two IFNGR2 subunits, triggers the transphosphorylation and activation of JAK1/JAK2, which are associated with the intracellular domains of the receptor[21]. These kinases, in turn, phosphorylate tyrosine residues on the intracellular chain of the receptor, generating anchoring sites for STAT1 through its SH2 domain[22,23]. Upon recruitment, STAT1 is phosphorylated at Tyr701 by JAK1/JAK2, enabling dimerization into active homodimers. These dimeric complexes, which constitute the gamma interferon activation factor complex, translocate to the nucleus, where they bind to specific sequences known as GAS, the promoters of ISGs[18] (Figure 2).

Figure 2 The molecular function of signal transducer and activator of transcription 1 and its relationship with glioblastoma. The binding of interferon-gamma to its heterotetrameric receptor, composed of two dimers of IFNGR1 and IFNGR2, activates the Janus kinases 1 and 2, which leads to the phosphorylation and activation of the signal transducer and activator of transcription 1 (STAT1). Once phosphorylated, STAT1 forms dimers that translocate to the nucleus, where it binds to gamma-activated sequence elements in the promoters of interferon-stimulated genes, promoting their transcription. Sustained activation of this pathway induces the transcription of genes involved in cell proliferation, migration, and survival, contributing to the aggressive phenotype of glioblastoma. GAS: Gamma-activated sequences; STAT1: Signal transducer and activator of transcription 1; JAK1: Janus kinases 1; JAK2: Janus kinases 2; ISGs: Interferon-stimulated genes.

Interestingly, IFN-γ can contribute to the upregulation of STAT1. Therefore, the STAT1 transcription factor, in addition to being part of the IFN-γ pathway, is an ISG[24]. In glioblastoma, STAT1 acts as a key modulator of aggressive tumor behavior. Studies on human cell lines (e.g., U251 and SHG44) have demonstrated that reducing the expression of STAT1 significantly decreases proliferation, migration, and invasion, while its overexpression promotes epithelial–mesenchymal transition (EMT) and increases tumor aggressiveness[25]. In addition, a concomitant increase in STAT1/pSTAT1, IRF1, and PD-L1 was detected upon exposure to IFN-γ, along with an enhanced migratory capacity. Moreover, a poorer patient survival was associated with a sustained activation of the canonical IFN-γ pathway[11].

IRF1

Human IRF1 is one of nine members of a family of transcription factors; it is linked to the regulation of IFN-responsive genes, whereas IRF2 acts as an antagonist of IRF1-mediated activation, competing for the same cis-promoter elements of IFN-I- and IFN-II-inducible genes[26-28].

The gene encoding IRF1 is located on human chromosome 5q31.1. This gene, which is 9165 bp in length, contains 10 exons and 9 introns. The IRF1 protein consists of 325 amino acids with a molecular weight of 45 kDa. The protein contains a DBD in its amino-terminal region (amino acids 1-113), a linker region (amino acids 114-164), followed by an interferon association domain type 1 (amino acids 165-263). The IRF1 transcription factor, through its DBD domain, recognizes interferon-stimulated response elements located on the promoters of various ISGs[26].

The IRF1 gene is expressed in many human cells at low basal levels, but its expression can be modified in response to stimuli such as IFNs, tumor necrosis factor, and interleukin-1[26]. It has been reported that IRF1 acts as a tumor suppressor, regulating genes essential to cell cycle control and apoptosis[29]. IFN-γ sensitizes resistant medulloblastoma cells to death-inducing ligand-induced apoptosis by upregulating caspase-8 through a STAT1/IRF1-dependent pathway[30].

Methionine deprivation imposes metabolic stress, called methionine stress, which inhibits mitosis and induces cell cycle arrest and apoptosis. Studies on the DAOY, a medulloblastoma cell line, have shown that this stress induces upregulation of genes such as IRF1, IFN-α, IFN-β, IRF3, IRF7, IFN-inducible protein kinases, IFN-γ receptor I, and IFN-γ-inducible protein (IP-30)[31].

In the case of other cerebral tumors, information about the function of IRF1 is scarce. Through in silico analysis, it has been shown that IRF1 and other members of the IRF gene family are significantly upregulated in glioma samples and that their mRNA levels correlate with poor survival and high-grade malignancy of glioma. Additionally, genetic mutations of IRF1 have been associated with improved survival outcomes in patients with glioma[32]. In addition, IRF1 is upregulated in glioblastoma cells through IFN-γ stimulation, suggesting that the IFN-γ/STAT1/IRF1 axis may promote tumor progression[11]. Regarding the contribution of IRF1 to glioblastoma chemoresistance, IRF1 is involved in regulating the response to bevacizumab treatment. In glioma-derived cells with IRF1 depletion and bevacizumab, the levels of apoptotic cells increase compared to those of control cells[33].

Therefore, IRF1 is a transcription factor directly regulated by STAT1 and, in consequence, IRF1 is responsible for secondary gene expression programs induced by IFN-γ. Hence, the IFN-γ/STAT1/IRF1 axis amplifies the IFN-γ response in glioblastoma cells, thereby further increasing the transcription of pro-tumorigenic target genes, such as PD-L1 and IDO1[15,34,35] (Figure 3).

Figure 3 The molecular function of interferon regulatory factor 1 and its relationship with glioblastoma. Interferon regulatory factor 1 protein has the molecular function of a transcription factor; through this mechanism, it can positively regulate the gene expression of programmed death-ligand 1 and indoleamine 2,3-dioxygenase 1, promoting immune evasion and cell migration. IRF1: Interferon regulatory factor 1; PD-L1: Programmed death-ligand 1; IDO1: Indoleamine 2,3-dioxygenase 1; ISRE: Interferon-stimulated response element.

PD-L1

The immune system, under physiological conditions, functions to protect the host against adverse events such as infectious diseases, autoimmunity, and allergies through a series of barriers that act as immune checkpoints[36]. Among these immune checkpoints are the axes of programmed cell death protein 1 (PD-1) and PD-L1, which play roles in immune homeostasis[37].

The PD-1 protein functions as a receptor, primarily expressed on memory T cells, and is expressed to a lesser extent by natural killer cells, activated monocytes, dendritic cells, and B cells[38]. PD-1 Ligands are PD-L1 and PD-L2 proteins; in this review, we focus on PD-L1 since it has been studied more extensively in the context of cancer[39]. The expression of PD-L1 is constitutive of a wide variety of cells, including macrophages, mesenchymal stem cells, and bone marrow-derived mast cells[40].

The structures of PD-1/PD-L1 proteins comprise a signal sequence, an IgV-like domain, a transmembrane domain, and a cytoplasmic domain; additionally, PD-L1 has an extra domain called the IgC-like domain[36]. The receptor/Ligand function of PD-1/PD-L1 proteins blocks the T-cell receptor and CD28 receptors, thereby decreasing proliferation, activation, cytokine production (e.g., IL-2 and IFN-γ), and cytotoxic function[41]. The physiological function of this axis is essential for maintaining immune homeostasis, as PD-1-deficient mice have been shown to develop an autoimmune disease similar to systemic lupus erythematosus[42]. In addition, PD-L1 inhibition has been shown to affect fetomaternal tolerance[43].

In the context of cancer, the function of PD-L1 has been described as an immunosuppressor in the immune response against tumor cells, as it can block the cytotoxic activity of CD8+ lymphocytes, thereby protecting cells that express PD-L1 from immune attack[44]. However, this is not its only function in cancer; it has been shown that in the context of ovarian cancer and melanoma, PD-L1 can promote the generation of tumor-initiating cells[45]. PD-L1 can also stimulate DNA repair[46] and promote mTOR activation and autophagy in bladder cancer[47]. Furthermore, high levels of PD-L1 have been associated with lung cancer, and signaling implicated in promoting EMT[48].

In glioblastoma, it is well established that PD-L1 expression is associated with immunosuppression and poor patient survival[49]. In the case of glioblastoma, one study found that stimulating glioblastoma-derived cells with IFN-γ increased STAT1 levels and upregulated IRF1 and PD-L1[11]. Additionally, PD-L1 knockdown suppresses the formation of xenografts in nude mice. In the glioblastoma-derived cell line (U251), PD-L1 can alter the expression of transcriptome associated with proliferation, migration, and invasion pathways. PD-L1 promotes migration through EMT activation via MEK-ERK-dependent signaling, in which PD-L1 binds to Ras and activates it[50].

IFN-γ-induced PD-L1 expression in glioblastoma cells has been well documented and is associated with poor prognosis, suggesting that PD-L1 can play a role as both a biomarker and a target for immunotherapy[16,51,52] (Figure 4). Similarly, PD-L1 is one of the genes stimulated by the canonical IFN-γ signaling pathway via JAK-STAT1 signaling, which has been studied in different tumor contexts, such as colorectal cancer-derived cells. Moreover, patients expressing high levels of PD-L1 have worse survival[53]. Gastric cancer-derived cell lines treated with IFN-γ significantly increase PD-L1 expression as well as components of the IFN-γ pathway[33].

Figure 4 The molecular function of programmed death-ligand 1 and its relationship with glioblastoma. Glioblastoma tumor cells can evade the immune response through the overexpression of programmed death-ligand 1 on their surface. This ligand binds to the programmed cell death protein 1 receptor present on T cells, inhibiting T cell receptor-mediated signaling, even in the presence of tumor antigens. As a result, T cells are not properly activated, promoting an immunosuppressive microenvironment that allows tumor progression. This regulatory axis represents a critical point in glioblastoma immune evasion. PD-L1: Programmed death-ligand 1; TCR: T cell receptor; PD-1: Programmed cell death protein 1.

IDO1

The cytosolic enzyme IDO1 catalyzes the degradation of tryptophan, an essential amino acid necessary for protein synthesis along the kynurenine pathway (KP). First, L-tryptophan is converted to N-formylkynurenine by IDO1. Subsequently, N-formylkynurenine is catabolized to kynurenine (KYN), which is then processed by other enzymes to generate final products[54]. It is also related to the endogenous production of nicotinamide adenine dinucleotide. Moreover, it has been reported that IDO1, through its phosphorylation, can act as a signaling molecule in plasmacytoid dendritic cells by transforming growth factor-β (TGF-β)[55].

Research has proposed that the enzymatic or signaling function of IDO1 depends on cellular context and subcellular localization. For example, in signaling events, IDO1 is primarily located in early endosomes, but its enzymatic action occurs in the cytosol[56]. Under physiological conditions, IDO1 is poorly expressed in most tissues; however, IDO1 expression can be induced in response to stress conditions and pathological processes[57].

IDO1 is inducible by IFN-γ, and its expression can result in an immunosuppressive mechanism through rapid tryptophan metabolism; its role in maternal T-cell tolerance is also crucial[58]. IFN-γ exposure increases IDO1 expression in glioblastoma cells and extracellular vesicles (EVs). These EVs induce myeloid-derived suppressor cells and non-classical monocytes in the tumoral microenvironment, promoting an immunosuppressed environment[59]. Moreover, EVs have been engineered as drug carriers: For example, biomimetic extracellular vesicles derived from chimeric antigen receptor monocytes effectively deliver therapeutic payloads via the intranasal route to glioblastoma models[60]. Hence, IDO1 has a critical role in the immune-inflammatory pathogenesis and progression of glioblastoma via the KP. Enzymes related to KP, including IDO1, are increased in glioblastoma cells. IDO1 promotes the accumulation of toxic metabolites such as KYN and quinolinic acid, which suppress T-cell proliferation[61] (Figure 5). Additionally, alterations in tryptophan metabolism have been reported in glioblastoma[62].

Figure 5 The molecular function of indoleamine 2,3-dioxygenase 1 and its relationship with glioblastoma. Indoleamine 2,3-dioxygenase 1 is involved in the degradation of tryptophan into kynurenine and other toxic metabolites such as quinolinic acid; these metabolites function to suppress T-cell proliferation, thereby creating an immunosuppressed environment in glioblastoma. IDO1: Indoleamine 2,3-dioxygenase 1.

Increased levels and gene expression of IDO1 are associated with a poor prognosis and shorter survival in glioblastoma patients compared to normal tissues[63,64]. IDO1 expression increases with glioma grade and IDH status and varies between molecular subtypes[64]. The activation of KP by chitinase-3-like protein 1, a mesenchymal marker, results in the regulation of IDO1 via aryl hydrocarbon receptor, contributing to an inhibitory immune microenvironment[65]. It has also been suggested that IDO-1 expression is upregulated by other cells in the tumor microenvironment, contributing to an enhanced immunosuppressive effect[66]. Overexpression of IDO1 in glioblastoma cell lines suppresses ferroptotic cell death through regulation of the cystine/glutamate antiporter SLC7A11. Higher IDO1 levels reduce the generation of peroxides and other reactive oxygen species and mitochondrial damage[67]. Additionally, angiogenesis mediated by vascular endothelial growth factor A (VEGF-A) contributes to the malignancy of glioblastoma. Patients exhibit a positive correlation between the high expression of IDO1, VEGF-A, and CD34, a marker of vascular density[68]. Tryptophan deficiency caused by IDO1 overexpression activates the GCN2 (general control nonderepressible 2) pathway, which is involved in autophagy, and promotes angiogenesis through amino acid deficiencies[69].

ISG15

The ISG15 corresponds to the family of ubiquitin-like proteins (UBLs) with 156 amino acids and a weight of 15 kDa. This protein is composed of a hinge sequence that connects the N-terminal UBL domain to the C-terminal UBL domain, presenting a motif with leucine, arginine, leucine, arginine, glycine, and glycine residues[70-72]. Through this sequence, ISG15 covalently binds to other proteins at their lysine residues, a process known as ISGylation[73-76]. E1-activating enzyme (UBE1 L), E2-conjugating enzyme (UBCH8), and E3 ligases (HERC5, HHARI, and TRIM25) are the three enzyme types that carry out protein ISGylation (Figure 6). ISG15 has also been detected when it is not conjugated to its target proteins and is known as free ISG15[77-80].

Figure 6 The molecular function of interferon-stimulated gene 15 and its relationship with glioblastoma. Interferon-stimulated gene 15 (ISG15) carries out its function through a post-translational modification similar to ubiquitination, known as ISGylation, which involves three steps: Activation, conjugation, and ligation of ISG15 to its target. The figure shows that this post-translational modification is reversible by ubiquitin-specific protease 18. ISG15 can be found both free and conjugated to its substrate, and, in the context of glioblastoma, it functions to enhance proliferation, migration, and invasion. ISG15: Interferon-stimulated gene 15; USP18: Ubiquitin-specific protease 18.

ISGylation begins when UBE1 L facilitates the formation of an adenosine triphosphate-dependent thioester bond with ISG15, allowing ISG15 to be transferred from UBE1 L to UBCH8, forming a thioester bond between ISG15 and UBCH8. E3 Ligases then promote the transfer and covalent conjugation of ISG15 to the lysine residue of target proteins[73,76,79]. Protein ISGylation is regulated by a des-ISGylase enzyme called ubiquitin-specific peptidase 18 (USP18), which separates ISG15 from target proteins, decreasing ISGylation and increasing free ISG15 levels[73,76,81]. Although type I IFNs α and β are the classic inducers of ISG15 expression, IFN-γ has also been shown to induce ISG15 expression, depending on the cell type[73].

In cancer, ISG15 has been observed to have a dual function. In most types of cancer, including breast, colon, pancreatic ductal adenocarcinoma, hepatocellular, prostate, and oral and nasopharyngeal cancer, ISG15 has a pro-tumor function[73,74,76,79]. In contrast, it has also been observed that in ovarian, cervical, and leukemia cancers, ISG15 can have an anti-tumor function[70,76,77,81].

The expression of ISGylation enzymes and USP18 is crucial for determining the relationship between ISGylation and free ISG15 levels. Therefore, the proportion of free and conjugated ISG15, as well as its regulation, may be different for each cell type[70,72,78,81]. In glioblastoma, high levels of free ISG15 and ISGylation have been observed in response to IFN-γ[73,82]. An increase in ISG15 expression levels is associated with poor survival. Moreover, ISG15 protein levels are higher in grade III astrocytomas than in grade I/II astrocytomas and healthy tissue. In addition, studies with samples from glioblastoma patients showed that those who expressed higher levels of ISG15 had lower survival rates compared to glioblastoma patients with lower levels of ISG15[82].

Studies have indicated that octamer-binding transcription factor 4 (Oct4) interacts directly with ISG15 via the ISGylation of its lysine 284, resulting in the increased stability of the protein. This suggests that the ISGylation of Oct4 is crucial for the pluripotency of glioma cells and, consequently, their tumor progression[83]. Thus, ISG15 overexpression can result in the activation or impairment of various molecular pathways, making it a potential biomarker candidate in glioblastoma[82].

ISG INDUCED BY IFN-γ: IMPLICATIONS IN GLIOBLASTOMA

The IFN-γ canonical signaling pathway primarily impacts the transcriptome to generate a cellular response in different contexts, including cancer. The gene profile associated with the actions of IFN-γ, pro- or anti-tumorigenic, is not entirely known in all cancer types. However, specific key genes, such as STAT1, IRF1, PD-L1, IDO1, and ISG15, suggest several mechanisms through which IFN-γ promotes the progression of glioblastoma (Figure 7), as each is associated with a defined function. STAT1 is a transcription factor central to the IFN-γ signaling pathway, but it also appears to be also involved in other signaling pathways[84]. Interestingly, STAT1 overexpression in glioblastoma cells has been associated with more aggressive phenotypes, including increased migration, invasion, and resistance to therapy[53,85]. Unphosphorylated STAT1 also participates in gene regulation through mechanisms independent of its canonical activation, and these may also have an impact on tumor plasticity and adaptation to the microenvironment[86,87]. Hence, the increase in STAT1 by IFN-γ appears to be a pro-tumorigenic axis in glioblastoma. Similarly, IRF1 is another transcription factor induced by IFN-γ that promotes, secondarily, the expression of IFN-γ-induced ISG. IRF1 is upregulated in glioblastoma, and its target genes may be involved in tumor progression[11]; however, further studies are required in the context of glioblastoma. Both STAT1 and IRF1 can directly modulate the transcriptome; consequently, their modulation by IFN-γ is crucial in the pro-tumorigenic axis and crosstalk with other signaling pathways in glioblastoma cells.

Figure 7 Key interferon-stimulated genes involved in glioblastoma pathology. The binding of interferon-gamma to its receptor activates the Janus kinase/signal transducer and activator of transcription 1 (STAT1) pathway, promoting the transcription of interferon-stimulated genes. In glioblastoma, this activation is associated with the overexpression of genes such as programmed death-ligand 1 (PD-L1), indoleamine 2,3-dioxygenase 1 (IDO1), interferon regulatory factor 1 (IRF1), interferon-stimulated gene 15 (ISG15), and STAT1, which contribute to the establishment of an immunosuppressive and aggressive tumor microenvironment. PD-L1 and IDO1 promote immune evasion, while IRF1 is associated with poor survival. Meanwhile, STAT1 and ISG15 are linked to processes of cell proliferation, migration, and invasion, all of which are related to an aggressive tumor. PD-L1: Programmed death-ligand 1; ISG15: Interferon-stimulated gene 15; IRF1: Interferon regulatory factor 1; STAT1: Signal transducer and activator of transcription 1; JAK: Janus kinase; IFN-γ: Interferon-gamma; IDO1: Indoleamine 2,3-dioxygenase 1.

Interestingly, IFN-γ signaling has pro-tumor actions due to its inhibition of anti-tumor immune defenses. For example, PD-L1 is a key protein that has been identified in the immunosuppression of various types of tumors, including glioblastoma[44,45,48]. Moreover, PD-L1 is induced by IFN-γ in glioblastoma, resulting in cell inhibition, specifically T cells; other studies have also suggested that PD-L1 can be involved in the proliferation, migration, and invasion of cancerous cells[16,49-51]. Another case is IDO1, which is upregulated by IFN-γ and encodes for a cytosolic enzyme that plays a crucial role in KP and catalyzes the degradation of tryptophan[54]. Interestingly, IDO1 is associated with an immunosuppressive microenvironment and hallmarks of cancer, such as angiogenesis[68]. Therefore, IFN-γ, through the modulation of PD-L1 and IDO1, favors resistance to immune defense, generating an immunosuppressor microenvironment that contributes to tumor progression.

ISG15 is a protein modifier via the ISGylation system. A lower survival rate is associated with higher ISG15 expression in glioblastoma[82]. Although ISGylome in glioblastoma cells has not been identified, it has been demonstrated that the Oct4 transcription factor is ISGylated, which confers protein stability and consequently promotes tumor development[83]. Thus, IFN-γ induces ISG15 expression, but ISG15 can affect the proteome via protein ISGylation, which seems to generate pro-tumorigenic responses in glioblastoma.

The deregulation of IFN-γ-induced ISGs in glioblastoma offers a promising avenue for biomarker development. STAT1, IRF1, and PD-L1 expression levels could serve as prognostic indicators or predictors of immunotherapy response[11]. These IFN-γ-induced ISGs can also be helpful as a target for therapy. For example, IDO1 inhibitors are already in clinical trials, and PD-L1 checkpoint inhibitors are being explored for the treatment of glioblastoma despite their current limited efficacy[88]. Principally, IDO1 inhibitors, such as RY103, have been evaluated and shown to inhibit tumor growth and reduce tumor weight, accompanied by a decrease in the expression and levels of angiogenic markers[68]. The dual inhibitor of IDO1/tryptophan 2,3-dioxygenase, AT-0174, combined with temozolomide, reduces tumor growth and CD4+ Treg infiltration and promotes CD8+ T-cell expression[89]. Additionally, KHK2455, a long-acting selective IDO1 inhibitor, combined with mogamulizumab, is being evaluated in a first-in-human phase 1 clinical trial[90]. The use of PROTAC, NU223612, promotes the degradation of IDO1 via the ubiquitin-proteasome system in glioblastoma cell lines[91]. In addition to chemical inhibitors and PROTACs, EV-based nanocarriers are emerging as minimally invasive delivery systems. Notably, chimeric antigen receptor monocyte-derived EVs have been shown to penetrate the blood–brain barrier and deliver cargo intranasally, offering a promising adjunct to conventional immunotherapies[60].

To date, therapeutic approaches to ISG15 as a target are unavailable. Thus, therapeutic strategies might aim to disrupt the pro-tumorigenic branch of IFN-γ signaling selectively. Additionally, negative regulators of the pathway, such as SOCS1, SHP2, and PIAS1, may offer control points for pro-tumorigenic ISG induced by IFN-γ[8,92].

Nevertheless, several yet unidentified ISGs may be critical in the pro-tumorigenic effect mediated by IFN-γ in glioblastoma, suggesting that further investigation is required to elucidate the molecular mechanisms induced by IFN-γ to promote the development and progression of glioblastoma.

CONCLUSION

In glioblastoma, canonical IFN-γ signaling via the JAK1/2-STAT1 axis is aberrantly activated, driving the expression of ISGs associated with tumor progression, immune evasion, and reduced patient survival. Key ISGs, such as STAT1, IRF1, PD-L1, IDO1, and ISG15, represent potential biomarkers and therapeutic targets. A better understanding of the molecular context governing these responses is essential for designing effective interventions against glioblastoma.