Molecular mechanisms and functions of guanylate‑binding protein 2 in inflammation and cancer (Review)

Introduction

Human guanylate-binding protein 2 (hGBP2) and murine (m)GBP2 were first isolated from human fibroblasts in 1982 (1) and macrophages in 1998 (2). GBP2 is a member of the IFN-inducible GTPase family that serves notable roles in cellular signaling. hGBP2 is reported to be highly expressed in various immune cells, including monocytes, lymphocytes and natural killer cells (3). Under basal conditions, hGBP2 and mGBP2 are primarily distributed diffusely in the cytoplasm and nucleus. However, upon stimulation by IFN-γ and in the GTP-bound state, such as hGBP1 and hGBP5, it can translocate to the Golgi apparatus (4,5). The genes encoding hGBP1-7 are located on human chromosome 1, whilst the genes encoding mGBP1-11 are located on mouse chromosome 3 (3), where mGBP2 is mapped to the distal end and is putatively associated with mGBP1 (2).

The inflammasome is a multi-protein complex that functions to eliminate abnormal cells and amplify inflammation. It is comprised of various sensor proteins, such as the NLR family pyrin domain-containing 3 [NLRP3; which primarily recognizes lipopolysaccharide (LPS)-induced signals], absent in melanoma 2 (AIM2; which detects intracellular double-stranded DNA), adaptor proteins, such as the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase effector proteins. It also senses pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), thereby triggering pyroptosis, a type of programmed cell death characterized by gasdermin-D-mediated plasma membrane perforation and release of inflammatory cytokines. hGBP2 activates inflammasome assembly and pyroptosis primarily by binding and delivering LPS into the cytoplasm (6). Additionally, hGBP2 facilitates inflammasome assembly through direct binding to ASC and cooperates with hGBP5 (7).

In the context of cancer, paclitaxel (PTX), which has become an important chemotherapeutic agent since its discovery in 1967, is a natural diterpenoid compound that can be isolated from Taxus brevifolia. hGBP2 cooperates with PTX through the MCL-1 apoptosis regulator, myeloid cell leukemia-1 (MCL-1)/Bcl-2 antagonist/killer 1 (Bak) pathway (8) to activate the vascular endothelial growth factor (VEGF) pathway, thereby promoting angiogenesis and oxygen supply to tumor. Immunologically ‘cold’ tumors are characterized by poor responses to immunotherapies (9) and host a tumor immunosuppressive microenvironment (TIME) lacking infiltrating immune cells. Such tumors typically exhibit rapid progression and poor prognosis (10), posing a major challenge in cancer immunotherapy. Notably, hGBP2 may offer a promising therapeutic strategy for ‘cold’ tumors by activating a number of immune checkpoints, such as programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) and lymphocyte activating 3 (LAG3) (11), thereby remodeling the TIME and inhibiting the activation of CD8+ T cells and CD4+ T cells (12).

Structure and hydrolase function of hGBP2

Structure of hGBP2

hGBP2 is a 67 kDa GTPase that is comprised of three distinct domains (13). The N-terminal region contains a large GTPase domain (LG; residues 1–309), followed by the middle domain (MD; residues 310–480) in the central region and the α12/13 helical domain (HD; residues 480–580) at the C-terminus. In its crystalline state, hGBP2 adopts a bi-molecular asymmetric unit configuration.

The LG domain serves as the nucleotide-binding site and is characterized by five conserved motifs involved in GTP binding and hydrolysis (Table I) (14). The G1/P-loop motif has a GxxxxGKS/T sequence (15) that encloses the β-phosphate of GTP. By contrast, the G2/SWITCH I motif serves a critical role in magnesium ion binding, which is a key feature for all GTPases, since it stabilizes the GTP molecule through coordination bonds with the phosphate groups. The G3/SWITCH II motif, with its conserved DTEG residue sequence, forms hydrogen bonds with the phosphate, aiding in its release. The G4 motif interacts specifically with the guanine base with its acidic Asp residue in the RDF residue sequence, facilitating the binding of GTP to the domain. The primary non-conserved region, located between residue 240 and 280, corresponds to a flexible region involved in membrane interactions and is situated within the G5 region. The G1-G5 motifs collectively constitute the ‘nucleotide-binding pocket’ for GTP (Table I).

Table I. Key motifs (G1-G5) of the human guanylate-binding protein 2 hydrolysis site.

Table I.

Key motifs (G1-G5) of the human guanylate-binding protein 2 hydrolysis site.

Motif designation	Sequence	Corresponding residue numbers
G1/P-loop	GLYRTGKS	45-52
G2/SWITCH I	TVKSHT	70-75
G3/SWITCH II	DTEG	97–100
G4	RDF	181-183
G5/Guanine cap	WPAPKKYLAHLEQLKEEE	236-255

The MD domain consists of 5 α-helices. The first helix group is formed of the N-terminal halves of α7, α8 and α9, where the second helix group is formed of the C-terminal half of α9, α10 and α11. By contrast, the α12/α13 domain contains a conserved CaaX sequence, where isoprenylation occurs, promoting the localization of hGBP2 on the membrane as a hydrophobic group. Another feature of α12/α13 domain is its interaction with LG domain. The α12/α13 domain is mainly negatively charged, while the LG domain mainly carries positive charge. This enables the two domains to contact through electrostatic interactions (13,16), where the α12/α13 domain is in a tightened state.

Hydrolase function of hGBP2

The hydrolase function of hGBP2 is essential for a number of mechanisms and processes. The membrane localization of hGBP2 requires homodimerization, which is dependent on the transition state of GTP hydrolysis (17).

The hGBP2 hydrolysate, which contains >75% GDP (18,19), yields only a small amount of GMP as the final product (13,16), which is obtained by the hydrolysis of GTP instead of GDP. This distinct characteristic, differing from hGBP1, is primarily attributed to the LG domain of hGBP2 rather than changes in the MD or HD domains. Specifically, this is due to differences in the adjustment of the active site in the LG domain of hGBP2 following the first hydrolysis event. In terms of tetramerization, hGBP2, unlike hGBP1 but like mGBP2 (18), can extensively tetramerize, although the tetramerization of hGBP2 does not contribute to the formation of GMP (20,21).

When hGBP2 binds GTP, GTP enters the nucleotide binding pocket formed by the G1-G5 motif, where hGBP2 undergoes homodimerization at the same time with the participation of LG and MD domains (22,23). The LG domain of hGBP2 is dimerized only 50% of the time in the presence of GTP, suggesting that the C-terminal domain of hGBP2 also participates in dimerization by providing a dimerization interface or otherwise stabilizing hGBP2 dimers in the GTP state (16). In addition, residue T75 in the G2/SWITCH motif and S52 in the G1 motif are involved in Mg2+ coordination (13), implying that Mg2+ may enter the nucleotide binding pocket and bind to the phosphate group and surrounding amino acid residues at this stage, which are necessary for hGBP2 activity. It has previously been reported that Mg2+ is necessary for the activation of hGBP2 at Ph 8.0, but not pH 6.0 (16), which suggests that Mg2+ and H3O+ may have a functional substitution relationship. However, this interaction requires further validation.

In the hGBP2-GDP conformation following phosphate group dissociation, the N-O bond of the G1/P-loop motif interacts with the β-phosphate. Concurrently, residue S52 forms N-O bonds with both the α- and β-phosphates, facilitating nucleotide positioning and binding (24,25). The Y47 side chain undergoes movement and participates in dimer interface formation, whilst the displacement of Y47 and R48 in the G1 motif induces notable movement of the G2 motif, with some Cα atoms shifting >10 Å (26). The G4/RD motif interacts with the guanine moiety through hydrogen bonding. W238 in the G5 motif undergoes allosteric rearrangement and interacts with W238 from another hGBP2 molecule (13). Consequently, the guanine base and ribose are enclosed within the space formed by the G1, G4 and G5 motifs. At the opposite end of the GDP, the diphosphate group is tightly positioned within the pocket formed by the G1/P-loop, G2/SWITCH I and G3/SWITCH II motifs. The β-phosphate further coordinates with the conserved residues K51, S52 and E99. When the E99-K51 interaction is absent, SWITCH I remains open. However, when these residues engage through hydrogen bonding, SWITCH I closes, simultaneously positioning T75 in the G2 motif for optimal interaction with Mg2+ (16).

Regulation of hGBP2 and mGBP2

Structural foundation of hGBP2 and mGbp2 genes

The hGBP2 gene, as an IFN-stimulated gene (ISG), contains a promoter region featuring two transcription factor binding sequences, namely the IFN-stimulated response element (ISRE), located at +42 (27) and the γ-activated sequence (GAS) (27,28), located at +53 and at −532 (27). Notably, GAS exhibits binding affinity for all STAT factors except for STAT2. Both sequences can independently respond to IFN signaling, whilst their combined presence results in enhanced transcriptional activity (29). By contrast, the mGbp2 gene is also an ISG, which contains one ISRE, located at −2 (27) and two GASs, located at −487 and −735 (27). However, Ramsauer et al (28) previously reported that the mGbpP2 gene has two ISREs, located at −30 and −440 and one GAS located at −530 (28).

Molecular regulatory mechanisms

STAT1, STAT2 and IFN-regulatory factor (IRF) 9 can form four types of oligomers. They are STAT1-STAT1 (binding to GAS, referred to as U-STAT1), STAT1-STAT2-IRF9 (binding to GAS, referred to as U-ISGF3), STAT2-STAT2-IRF9 (binding to ISRE, referred to as U-ST2) and IRF1-IRF1 (binding to ISRE) (29). These oligomers subsequently localize to hGBP2 or mGbp2 genes and activate their transcription (Fig. 1). In mice, the IRF1 dimer has been demonstrated to directly bind to the transcription complex containing RNA polymerase II to activate mGbp2 gene transcription (28), whilst STAT1 dimers not only bind to GAS on the hGBP2 gene promoter but also promote the acetylation of histone 4 in the hGBP2 gene, thereby providing active chromatin. Additionally, previous studies have shown that p53 can form a complex with IRF1 to upregulate hGBP2 expression (30,31).

Figure 1. GBP2 gene regulation under basal conditions (without IFN-I/II stimulation). GBP2, guanylate-binding protein 2; GAS, γ-activated sequence; ISRE, IFN-stimulated response element; IFNAR, IFN-α/β receptor; IFNGR, IFN-γ receptor; IRF, IFN-regulatory factor; ISGF, IFN-stimulated gene factor.

Highly activated phase under stimulation by IFN-I, IFN-II and IRF1

Upon pathogen invasion or intracellular abnormalities, the corresponding PAMPs or DAMPs are recognized by a series of pattern recognition receptors (PRRs). This type of recognition triggers the phosphorylation and activation of IRFs, thereby promoting the expression of IFN-I and IFN-II (the former mainly includes subtypes IFN-α and IFN-β) through the classical secretory pathway. These IFNs are secreted extracellularly and bind to their corresponding respective receptors. Mitochondrial outer membrane permeabilization (MOMP) under radiation conditions and apoptosis can lead to the release of mitochondrial double-stranded (ds)DNA, which activates the cyclic GMP-AMP synthase/stimulator of IFN genes pathway, subsequently inducing IFN-β and upregulating mGBP2 expression (32).

IFN-I can bind to the ubiquitously expressed IFN-α/β receptor (consisting of the IFNAR1 and IFNAR2 subunits), leading to the dimerization of the receptor subunits. This dimerization, through juxtaposition and trans-phosphorylation, enhances the kinase activity of Janus kinase (JAK) 1 and tyrosine kinase (TYK) 2. Subsequently, JAK1 and TYK2 phosphorylate tyrosine residues on IFNAR1 and IFNAR2, which serve as binding sites for STAT1 and STAT2 (33). Phosphorylation of STAT1 at Tyr701 and STAT2 at Tyr690 then occurs. By contrast, IFN-II, specifically IFN-γ, interacts with a tetrameric receptor complex composed of two IFNGR1 subunits and two IFNGR2 subunits. This receptor is associated with JAK1 and JAK2 kinases, which exclusively phosphorylate the STAT1 protein.

Similar to the pathway under normal conditions, phosphorylated (p-)STAT1, p-STAT2 and IRF1 can form four types of oligomers: p-STAT1-p-STAT1 (also known as γ-IFN activation factor, binds to GAS), p-STAT1-p-STAT2-IRF9 (binds GAS), pSTAT1-pSTAT2-IRF9 (also known as IFN-stimulated gene factor, binds to ISRE) and IRF1-IRF1 (binds ISRE). These oligomers subsequently localize to the hGBP2 or mGbp2 gene and activate its transcription through a similar mechanism (Fig. 2), exhibiting higher activity compared with their non-phosphorylated counterparts.

Figure 2. GBP2 gene regulation upon IFN-I and IFN-II stimulation. GBP2, guanylate-binding protein 2; GAS, γ-activated sequence; ISRE, IFN-stimulated response element; IFNAR, IFN-α/β receptor; IFNGR, IFN-γ receptor; IRF, IFN-regulatory factor; ISGF, IFN-stimulated gene factor; JAK1, Janus kinase 1; TYK2, tyrosine kinase 2.

Function of hGBP2 and mGBP2 in inflammation

Activation of the inflammasome pathway by hGBP2 and mGBP2

The inflammasome pathway can be divided into two types, namely the canonical pathway and the non-canonical pathway. Their objective is to induce pyroptosis, which is mediated by the formation of plasma membrane pores through gasdermin-D. This leads to membrane rupture and alerting other cells, with the release of cytokines IL-1β and IL-18 through the gasdermin-D-formed membrane pores. hGBP2 and mGBP2 primarily serve supporting roles in inflammasome assembly.

In the canonical pathway, activation is primarily mediated by PRRs, such as nucleotide-binding oligomerization domain-like receptors, such as NLRP3, or AIM2-like receptors, including AIM. PAMPs or DAMPs are recognized by PRRs, such as NLRP3 or AIM2. During this process, mGBP2 can facilitate the release of dsDNA by lysing pathogen-containing vacuoles or pathogens, enabling AIM2 recognition (7,34,35). Upon activation, NLRP3 or AIM2 interacts with the adaptor protein ASC through their pyrin domains (36). In this step, mGBP2 and mGBP5 can form a heterocomplex, where mGBP2 binds ASC and mGBP5 binds NLRP3. This mGBP2-mGBP5 complex brings NLRP3 and ASC together, thereby promoting the assembly of the NLRP3 inflammasome (7). ASC in turn recruits caspase-1 through its caspase recruitment domain, forming the inflammasome. Subsequently, caspase-1 within the inflammasome cleaves gasdermin-D, releasing its N-terminal fragment to form plasma membrane pores and induce pyroptosis. Caspase-1 also processes pro-IL-1β and pro-IL-18 into their active forms, which are then released through the membrane pores to alert neighboring cells (7).

In the non-canonical pathway, activation in humans is primarily mediated by hGBPs through PRRs while in mice, it is mediated by caspase-11 with the assistance of mGBP2. In humans, when bacteria enter cells, hGBP1 binds bacterial LPS and recruits hGBP2, hGBP3 and hGBP4 to the surface of bacteria, especially gram-negative bacteria, forming a coating (6,37). hGBP2 and hGBP4 expose the lipid moiety of LPS, recruiting caspase-4 to the bacterial surface for LPS binding, while hGBP3 regulates caspase-4 activation (6,37). Activated caspase-4 then cleaves pro-IL-1β, pro-IL-18 and gasdermin-D, leading to pyroptosis and cytokine release.

In mice, caspase-11 directly recognizes the mGBP2-LPS complex, oligomerizes and activates, gaining the ability to cleave gasdermin-D and induce pyroptosis (38). Additionally, the formation of plasma membrane pores causes potassium ion efflux due to the intracellular potassium gradient, further promoting NLRP3 inflammasome assembly and creating a positive-feedback amplification loop (39). In this process, mGBP2 assists caspase-11 in LPS recognition. A previous study has reported that mGBP2 can interact with gasdermin-D and mGBP3, potentially serving as a novel component of this pathway, although the precise mechanisms remain unclear and require further investigation (7).

The severe consequences of pyroptosis, such as further inflammation cascade caused by the release of IL-18 and IL-1β (40), may explain the need for the continuity and regulation of the GBP2/caspase pathway. This type of regulation allows for the existence of multiple regulatory checkpoints before caspase activation, increasing the difficulty of activation and preventing unnecessary triggering (6).

Role of hGBP2 and mGBP2 in inflammatory diseases

The expression of hGBP2 is increased in the kidney tissues of patients with lupus nephritis, particularly in the glomeruli and renal tubulointerstitium (41). In diabetic nephropathy, macrophages at the injury site are predominantly of the M1 subtype, where hGBP2 can promote the polarization of macrophages toward the pro-inflammatory M1 subtype through the Notch 1 pathway. Through the hGBP2-mediated pathway, macrophages can be induced into the M1 phenotype by LPS or IFN-γ (42).

In allergic rhinitis (AR), mGBP2 can alleviate oxidative stress and abnormal lipid metabolism by inhibiting the hypoxia-inducible factor-1 (HIF-1) pathway. It can also inhibit mitochondrial fission whilst maintaining mitochondrial fusion to mitigate oxidative stress-induced damage to cells (43). A previous study has demonstrated that the overexpression of mGBP2 can notably reduce inflammatory cell infiltration into the nasal mucosa and markedly decrease the levels of various factors, such as total cholesterol, low-density lipoprotein-cholesterol, TNF-α, IL-5, IFN-γ and trimethylamine N-oxide, in AR mouse models whilst increasing high-density lipoprotein-cholesterol levels (44). However, the underlying mechanisms require further investigation.

LPS-induced macrophages-derived exosomes (L-Exo) can be transported from macrophages to lung epithelial cells, resulting in damage to the alveolar epithelial tissue. The mGBP2 content in L-Exo is higher compared with that in control Exo (45), leading to the hypothesis that the mGBP2 contained in L-Exo can trigger pyroptosis in the lung epithelial tissue, thereby causing injury. This may represent a potential therapeutic approach for sepsis-associated acute lung injury (46). In depression-like behaviors induced by neuroinflammation, reducing the expression of mGBP2 may also alleviate symptoms (47).

Role of hGBP2 and mGBP2 in oncogenesis and cancer

hGBP2 and mGBP2 regulate cancer progression through multiple signaling pathways

Previous studies have demonstrated that in glioblastoma (GBM) and low-grade glioma cells, hGBP2 can directly interact with kinesin family member 22 (KIF22) to post-transcriptionally regulate and elevate its levels, thereby enhancing KIF22-mediated EGFR internalization and signaling (Table II) (48–63). This in turn fosters cell proliferation (64). Unc-51 like autophagy activating kinase 1 (ULK1) is another critical regulator of autophagy (65), phosphorylating and inducing autophagy under low-nutrient conditions. hGBP2 can activate the ULK1 complex by suppressing PI3K/Akt/mTORC1 pathway (66), thereby enhancing cellular autophagy (66). Notably, the outcomes of this function may vary, where it may exert tumor-suppressive effects during early-stage cancer but potentially promote cancer progression at advanced stages (67).

Table II. GBP2 expression level across different cancer types and corresponding mechanisms.

Table II.

GBP2 expression level across different cancer types and corresponding mechanisms.

Tumor type	Cells or databases involved	Species	GBP2 expression level	Mechanisms	(Refs.)
GBM	Glioblastoma cell lines, U87, U251, SNB19, GSC11 and G91; database, TCGA	Human	Upregulated	Expression of various carcinogenesis-related genes in GBM, particularly in mesenchymal subtype, including fibronectin 1, MMP-1, MMP-3, MMP-14, urokinase, secreted protein acidic and rich in cysteine, TGFB1, macrophage colony-stimulating factor 1, CD44, IL-8, monocyte chemoattractant protein 1 and IL6, are elevated with hGBP2 involvement. Furthermore, cell proliferation is accelerated, the block of G0/G1 phase of cell cycle is prevented and apoptosis is decreased, due to hGBP2-activated kinesin family member 22/EGFR signaling pathway	(48–51)
BC, particularly triple-negative BC	Primary BC cells isolated from tumor tissues from patients with BC and normal breast tissue	Human	Downregulated	The methylation of hGBP2 gene promoter decreases the levels of hGBP2, inhibiting the promotion of the immune response of CD8+ T cells, thus facilitating the development of BC	(52–54)
SKCM	Human melanoma cell lines, A2058, A375; mouse melanoma cell lines, B16 and B16F10; human epidermal cell line, NHEK	Human and mouse	Downregulated	It has been revealed that overexpressing hGBP2 can upregulate E-cadherin and downregulate N-cadherin and vimentin, inhibiting the EMT, invasion and proliferation of SKCM. While whether hGBP2 downregulation, which occurs in SKCM cells, can facilitate EMT remains unstudied.hGBP2 also markedly downregulates the Wnt/β-catenin signaling pathway and related proteins, including transcription factor 4, c-Myc and cyclin D1, thus inhibiting cell proliferation, migration, invasion and promoting cell apoptosis. The mechanisms of mGBP2 in SKCM is unstudied	(51,55)
ccRCC	Primary ccRCC cells isolated from patient tumor tissue and normal kidney tissue; monocyte cell line, THP-1; common RCC cell lines, 786-O, 769-P, CAKI-1, A498 and ACHN; renal tubular epithelium cell line, HK-2; databases, Clinical Proteomic Tumor Analysis Consortium and TCGA	Human	Upregulated	hGBP2 activates the polarization of M0 macrophages toward the M2 macrophages through JAK/STAT3-dependent upregulation on IL-18 secretion. Simultaneously, M2 macrophages can induce the expression of GBP2 in tumor cells by secreting IL-10 and TGF-β, which in turn activates the expression of hGBP2, forming a loop. hGBP2 can also enhance the levels of regulatory T cells and exhausted T cells, facilitating ccRCC tumor immune evasion and proliferation	(51,56,57)
PAAD	Databases, TCGA and GEO; primary PAAD cells isolated from tumor tissue from patients with PAAD and adjacent normal tissue	Human	Upregulated	hGBP2 levels are markedly elevated in active CD4 memory T cells, resting dendritic cells and M1 macrophages, indicating that these three types of cells can possibly induce the expression of hGBP2. Subsequently, hGBP2 can further regulate the TIME	(11,51)
Thyroid carcinoma	Database, TCGA	Human	Upregulated	Remains unstudied	(51)
Uveal melanoma	Database, TCGA	Human	Upregulated	Remains unstudied	(51)
Hepatocellular carcinoma	Databases, TCGA and GEO	Human	Upregulated	hGBP2 can induce tumor cell infiltration, especially by upregulating macrophages and downregulating Th17 and neutrophils in the TIME	(51,58)
ESCC	Human ESCC cell lines, TE-1, TE-7, TE-10 and TE-13	Human	Upregulated	hGBP2 is involved in a p53/IRF-1/hGBP2 pathway and is thus overexpressed in ESCC. Downstream mechanisms remain unstudied	(30)
CRC	Databases: GEO, UCSC Xena Browser, TCGA; human MSS CRC cell lines, HT29 and SW480; murine MSS CRC cell line, CT26	Human and mouse	The level of hGBP2 varies: Upregulated in MSS CRC of IC and dMMR/MSI CRC; downregulated in pMMR/MSS CRC of non-IC	hGBP2 can activate STAT1 by competing with SHP1, which inhibits the phosphorylation and activation of STAT1, for binding to STAT1	(59,60)
Gastric cancer	Databases: GEO, TIDE, TCGA, UCSC Xena Browser, KEGG	Human	Upregulated	Remains unstudied	(61)
HNSCC	Databases, ONCOMINE, TCGA, HPA	Human	Stably upregulated across different stages of HNSCC	Remains unstudied	(31)
OC	Human cervical cancer cell line, HeLa; murine breast cancer cell line, 4T1; murine macrophage cell line, RAW264.7; murine preadipocyte cell line, 3T3-L1; murine ovarian cancer cell line, ID8	Human and mouse	Downregulated	mGBP2/Pin1/NF-κB pathway, which is possibly inhibited in OC, induces the polarization of M0 to M1, thus the anticancer macrophage subtype M1 is at low levels in OC	(62)
Prostate cancer	Databases, KEGG, GEO, TCGA	Human	Upregulated	Remains unstudied	(63)

[i] GBP2, guanylate-binding protein 2; h, human; GBM, glioblastoma; TCGA, The Cancer Genome Atlas; BC, breast cancer; SKCM, skin cutaneous melanoma; EMT, epithelial-mesenchymal transition; ccRCC, clear cell renal cell carcinoma; JAK, Janus kinase; GEO, Gene Expression Omnibus; PAAD, pancreatic adenocarcinoma; TIME, tumor immunosuppressive microenvironment; ESCC, esophageal squamous cell carcinoma; CRC, colorectal cancer; MSS, microsatellite stable; MSI, microsatellite instability; dMMR, deficient DNA mismatch repair; pMMR, proficient DNA mismatch repair; IC, immune class; SHP1, tyrosine-protein phosphatase non-receptor type 6; TIDE, Tumor Immune Dysfunction and Exclusion; KEGG, Kyoto Encyclopedia of Genes and Genomes; HNSCC, head and neck squamous cell carcinoma; HPA, Human Protein Atlas; OC, ovarian cancer; Pin1, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1.

hGBP2 can induce the assembly of inflammasomes, leading to a positive feedback loop of inflammatory responses. This forms the hGBP2/STAT3/fibronectin (FN1) pathway (49,53), where hGBP2 induces FN1, which is an extracellular glycoprotein involved in cell migration (68), on both transcriptional and translational levels to enhance glioblastoma migration and invasion without influencing proliferation in vitro (49). In addition, STAT3 contributes to maintaining the mesenchymal subtype of GBM (50) and is indispensable for hGBP2-induced FN1 elevation (49). FN1 can also promote MMP-9 secretion in a focal adhesion kinase (FAK)- and Ras-dependent manner (69) or through the FN1/MMP-2/MMP-9 pathway (70). However, hGBP2 can induce FN1 in vivo but lacks this capability in vitro (49). Furthermore, MMP-9 is an inducer of epithelial-mesenchymal transition (EMT) (71). Thus, hGBP2 should activate, rather than inhibit, MMP-9 through the STAT3/FN1/FAK or STAT3/FN1/MMP-2 signaling pathways. However, the regulatory effect of hGBP2 on migration has not been experimentally demonstrated.

Bak is a member of the Bcl-2 family that is crucial for the activation of apoptosis. It promotes MOMP, thereby triggering the apoptotic process (72). hGBP2 acts as a dual stimulator of Bak. It can not only release Bak but can also promote its expression (8). The LG domain of hGBP2 specifically binds to the BH3 domain of Mcl-1, a member of the Bcl-2 family, thereby preventing the interaction between Bak and Mcl-1. This then releases Bak, which oligomerizes to induce MOMP, ultimately promoting apoptosis (8). Simultaneously, hGBP2 upregulates the expression of Bak through its inhibitory effect on the PI3K/Akt pathway (8). In summary, hGBP2 can promote MOMP by activating Bak and enhancing its expression, thereby facilitating apoptosis. PTX, as an anticancer drug, occupies the upstream position in the hGBP2/Mcl-1/Bak pathway. Through this pathway, hGBP2 enhances cellular sensitivity to PTX (8,66). Notably, hGBP2 upregulation is a common feature of PTX treatment and other chemotherapeutic agents for hematologic malignancies (such as doxorubicin, cytarabine, vincristine, etoposide and IFN-γ). However, among these antineoplastic agents, only PTX possesses the unique ability to elevate Bak levels (8).

In breast cancer (BC) cells, hGBP2 has been demonstrated to directly interact with dynamin-related protein 1 (Drp1) in a K51-dependent manner, preventing its translocation from the cytoplasm to the mitochondria. This interaction inhibits Drp1-dependent mitochondrial fission and elongation, thereby blocking mitochondrial division and cell metastasis in cancer cells (73). Notably, the activity of hGBP2 binding to Drp1 is influenced by all three major structural domains of hGBP2, where it is possible that hGBP2 and Drp1 bind to each other to form hetero-oligomers, although further investigations are required (73). hGBP2 also exerts its anticancer effects by inhibiting the Wnt/β-catenin/EMT pathway (55,71,74) and cancer cell metastasis and invasion, which has been demonstrated in cancers such as skin cutaneous melanoma (SKCM) and colon cancer, but the specific molecular mechanisms remain unclear. PTX primarily inhibits tumor metastasis and progression by blocking angiogenesis in tumor tissues, whilst hGBP2 enhances the sensitivity of colon cancer cells to PTX by inhibiting the Wnt/β-catenin pathway (75,76). Additionally, a previous study has indicated that inhibiting the Wnt/β-catenin pathway in BC and triple-negative BC (TNBC) can promote ferroptosis in BC cells and suppress cell cycle and growth regulatory proteins, such as cyclin D1 and c-Myc. Therefore, it can be speculated that this may be a potential function of hGBP2, warranting further investigation.

In mouse dendritic cells, mGBP2 has been reported to interact with the Akt-p110 complex, inhibiting the phosphorylation and activation of Akt (77,78). This in turn prevents the phosphorylation of TSC complex subunit 2, disrupting its ability to form a complex with tuberous sclerosis 1 that inhibits mTORC1 (77–80). VEGF promotes endothelial cell proliferation and migration by binding to VEGFRs on endothelial cells, thereby stimulating angiogenesis. Under basal conditions, the HIF-1α subunit is hydroxylated by prolyl hydroxylases and recognized by the von Hippel-Lindau protein complex, leading to its ubiquitination and degradation. Under hypoxic conditions, however, the HIF-1α subunit is stabilized, where it translocates to the nucleus and dimerizes with the HIF-1β subunit to form the HIF-1 transcription factor, which then translocates to the nucleus and binds to hypoxia-response element in the promoter of VEGF gene (81,82), regulating VEGF transcription (43). In oxygen-induced retinopathy mice model, mGBP2 has been shown to inhibit the HIF-1α/VEGF pathway by suppressing mTORC1 (78), thereby attenuating angiogenesis. This reduces blood supply to cancer lesions, maintaining hypoxic conditions and potentially curbing cancer progression.

mGBP2 can activate MMP-9 through distinct pathways, thereby influencing collagen degradation near the basement membrane (33), extracellular matrix remodeling, angiogenesis in cancer lesions and cancer cell invasion and metastasis (77). Previous studies in NIH 3T3 fibroblasts have shown that mGBP2 can inhibit Rac, disrupting its regulation of the cytoskeleton. This inhibition suppresses the TNF-α-mediated activation of the NF-κB pathway, thereby reducing NF-κB-induced MMP-9 transcription (83). mGBP2 has been demonstrated in NIH 3T3 fibroblasts to directly interact with the p65 (RelA) protein in the NF-κB pathway, preventing its binding to the MMP-9 promoter and subsequently lowering MMP-9 expression (83). In ovarian cancer, mGBP2 promotes the recruitment of the peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein, which activates the NF-κB pathway (62). Based on this, it can be hypothesized that mGBP2 may upregulate MMP-9 expression through the Pin1/NF-κB and Rac/NF-κB pathways, although further experimental validations are required.

hGBP2 and mGBP2 modulate cancer progression by regulating the TIME

hGBP2 exerts a dual influence on the TIME. It can promote immune activation, transforming the TIME into a ‘hot’ state, thereby addressing the immunologically ‘cold’ tumors, such as TNBC and proficient mismatch repair/microsatellite stable colorectal cancer (CRC) of the immune class (59). Simultaneously, it can also reduce immune cell infiltration through certain mechanisms, enhancing tumor immune evasion. Additionally, the expression of immunotherapy biomarkers is positively associated with hGBP2 expression (61). In CRC, hGBP2 expression is positively associated with CD8+ T cell infiltration, CD8, PD-L1, C-X-C motif chemokine ligand (CXCL) 9, CXCL10, CXCL11, CXCL13, HLA-I expression, antigen processing and presentation machinery and a variety of antitumor immunity steps, including the release of cancer cell antigens, cancer antigen presentation, priming and activation and trafficking of immune cells to tumors (59). By contrast, it is negatively associated with the cell count of cytokeratin (59). However, specific mechanisms of the regulation of hGBP2 in CRC require further study.

In gastric cancer, hGBP2 is reported to be positively associated with an inflamed TIME, immunophenoscore (IPS), abundance of PD-1+ cells and the expression of immunotherapy biomarkers, such as PD-L1, PD-L2, IFN-γ, CD8A, secreted and transmembrane protein 1 and IFN-induced transmembrane protein 3 (61). In GBM, it has been demonstrated that urokinase, secreted protein acidic and rich in cysteine, TGFB1, FN1 and colony-stimulating factor 1 are induced by elevated hGBP2 expression (49). In SKCM, previous studies have demonstrated that hGBP2 expression is positively associated with the infiltration of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and dendritic cells (11,12,57,61), This may serve a role in therapies that block PD-1/PD-L1 interactions to prevent tumor immune evasion, since CD8+ T cells are the primary effector cells in tumor killing (84). In BC, hGBP2 is positively associated with T cell infiltration levels and can serve as a marker for T cell infiltration (53). In clear cell renal cell carcinoma (ccRCC), hGBP2 promotes the infiltration of CD8+ T cells, regulator T cells and both M1 and M2 macrophages (57).

From the perspective of reducing immune infiltration and promoting tumor immune evasion, hGBP2 can exert oncogenic effects by increasing the phosphorylation of STAT2 and STAT3, modulating the JAK/STAT signaling pathway and reducing tumor immune infiltration (51). hGBP2 can also induce immune checkpoints, such as PD-1/PD-L1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoreceptor with Ig and ITIM domains, LAG3, indoleamine 2,3-dioxygenase 2 and V-domain Ig suppressor of T cell activation (VISTA) (11), which are inhibitory immune checkpoints. Additionally, hGBP2 can promote PD-L1 expression on the transcriptional level by binding to and phosphorylating STAT1, thereby reducing tumor immune infiltration and facilitating immune evasion (85). hGBP2 can also induce PD-L2; however, the relevant mechanisms remain unstudied (61). Based on these functions, hGBP2 will likely be a crucial target for restoring the TIME for the treatment of immunologically ‘cold’ tumors, such as TNBC.

mGBP2 has been found to upregulate the secretion of IL-6, IL-12 and TNF-α (77). Furthermore, mGBP2 promotes the maturation of dendritic cells and enhances their antigen-presenting capacity, thereby boosting T cell activation (77). mGBP2 also promotes the polarization of macrophages into M1 and M2 subtypes by promoting STAT3 pathway and by activating the NF-κB signaling pathway (62), while hGBP2 promotes polarization by stimulating the secretion of IL-18 (56). M2 macrophages, in turn, can enhance the migration and invasion of tumor cells by secreting IL-10 and TGF-β, which upregulate the hGBP2/STAT3 and ERK axes (part of the MAPK signaling pathway) as demonstrated in ccRCC (56).

Notably, a number of downstream factors promoted by hGBP2 can exhibit both pro-tumor and antitumor effects. CD80 in pancreatic adenocarcinoma can bind to either CD28 (activates T cells) or CTLA-4 (inhibits T cells), thereby bidirectionally modulating immune responses (11). Notably, hGBP2 is reported to promote the polarization of M0 to M2 in ccRCC by activating the secretion of IL-18 (56) and to M1 in ovarian cancer (62) and in diabetic nephropathy by activating the Notch 1 signaling pathway (42). By contrast, mGBP2 has only been reported to promote the polarization of M0 to M1 under the activation of the PLGA-CpG@ID8-M nano vaccine (62). The underlying mechanisms of these outcomes and whether mGBP2 can also promote the polarization of M0 to M2 require further investigation.

Regulation of hGBP2 in cancer

In primary CRC, hGBP2 has been found to exhibit a high mutation rate alongside genes such as G protein subunit β1 and GATA zinc finger domain containing 2A. However, only hGBP2 showed an even higher mutation rate in CRC with liver metastasis (60). Additionally, in CRC with liver metastasis, the hGBP2 gene undergoes methylation at four specific sites (m1A, m5C, m6A and m7G) (60). Peroxisome proliferator-activated receptor α, an anticancer factor, can inhibit hGBP2 expression (58). In BC (52) and SKCM (12), the methylation level of hGBP2 increases, leading to a decrease in hGBP2 expression.

Future directions

Prior reviews on GBP2 have predominantly emphasized their roles in host defense against bacterial and viral pathogens (6,86,87), with limited attention to the regulatory mechanisms of GBP2 expression or its functions in cancer and oncogenesis. Furthermore, species-specific distinctions-particularly the differences between hGBP2 and mGBP2-have been overlooked (86,87). This review bridges these gaps by providing a comprehensive analysis of the regulation of GBP2 expression and by systematically comparing hGBP2 and mGBP2 functions in oncogenesis. Whether a potential functional substitution relationship between Mg2+ and H3O+ during hGBP2 activation exists and its possible mechanisms could offer further insight into the hydrolysis of hGBP2, and requires further investigation. Although hGBP2 can form tetramers extensively, this tetramerization cannot facilitate the hydrolysis from GTP to GMP, rendering the physiological relevance of its tetramerization unknown. Moreover, while the context-dependent dual role of hGBP2 in cancer and oncogenesis has been extensively documented, the underlying mechanisms have not yet been systematically elucidated. For instance, although hGBP2 drives M0-to-M1 polarization via the Notch1 pathway in diabetic nephropathy, it mediates an M0-to-M2 polarization in ccRCC; the determinants of these opposing outcomes warrant further investigation.

Clinically, agonistic monoclonal antibodies which promote hGBP2 oligomerization and activation could be exploited to convert immunologically ‘cold’ tumors into ‘hot’ ones, thereby augmenting existing immunotherapies. Across multiple cancer types, hGBP2 abundance is positively associated with levels of immune-related biomarkers, such as PD-1/PD-L1 and VISTA, immune-cell infiltration and IPS, revealing its potential as a robust tumor biomarker for assessing the activation level of TIME and prognosis. Additionally, the methylation status of the hGBP2 promoter also has also emerged as a promising diagnostic indicator in SKCM and BC.

Acknowledgements

Not applicable.

Funding

The present study was Supported by the National Natural Science Foundation of China (grant no. 82104603), Beijing Natural Science Foundation (grant no. 7204322), Peking University People's Hospital Research and Development Fund (grant nos. RDZH2022-04, RS2021-12 and RDX2021-08).

Availability of data and materials

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Authors' contributions

ZL performed project administration, methodology, data curation and wrote the draft. SP performed visualization and reviewed the manuscript. JO conceived and designed the review, and was involved in funding acquisition, supervision, proofreading and revision of the manuscript. All authors read and approved the final version of the manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

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Patient consent for publication

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Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References