Activation of nuclear factor‐κB in the angiogenesis of glioma: Insights into the associated molecular mechanisms and targeted therapies

Glioma is the most commonly observed primary intracranial tumour and is associated with massive angiogenesis. Glioma neovascularization provides nutrients for the growth and metabolism of tumour tissues, promotes tumour cell division and proliferation, and provides conditions ideal for the infiltration and migration of tumour cells to distant places. Growing evidence suggests that there is a correlation between the activation of nuclear factor (NF)‐κB and the angiogenesis of glioma. In this review article, we highlighted the functions of NF‐κB in the angiogenesis of glioma, showing that NF‐κB activation plays a pivotal role in the growth and progression of glioma angiogenesis and is a rational therapeutic target for antiangiogenic strategies aimed at glioma.


| INTRODUC TI ON
Glioma, which originates from the glial cells surrounding the neurons, is the most commonly observed intracranial tumour with the greatest degree of malignancy and accounts for approximately 80% of all brain malignancies. 1 The median survival of malignant glioma patients is only about 1 year, even after common treatments including surgical resection, radiotherapy and chemotherapy are performed. 2 Angiogenesis is among the factors vital to tumour development. 3 As is the case with most solid tumours, the survival and growth of fast-growing gliomas with an avascular area volume exceeding 2 mm 3 require newly generated blood vessels for the provision of the necessary oxygen, growth factors and nutrients. 4 Several studies have shown that angiogenesis has the strongest prognostic significance among all the clinical and pathological features of glioma, and that widespread angiogenesis tends to be associated with worse prognoses. 5 Based on the clinical significance and potentialities of the therapeutic interventions for glioma, it is necessary to identify the targets and underlying molecular mechanisms that regulate glioma angiogenesis.
Tumour necrosis factor (TNF, also referred to as TNF-α) exerts its function using two receptors-TNF receptor I (TNFR1, p55 receptor) and TNF receptor II (TNFR2, p75 receptor)-which are members of the TNF superfamily. 6 TNF plays a role in the promotion of tumour cell apoptosis through TNFR1 binding; however, it also promotes tumour cell growth through TNFR2. 7,8 TNFR2 activation leads to the recruitment of TNF receptor-associated factor 2 and motivates the pro-survival nuclear factor (NF)-κB pathway. 9,10 NF-κB regulates the genes involved in tumour microenvironment development and proangiogenic and pro-inflammatory cytokine synthesis. 11 Abnormal or constitutive NF-κB activity in glioma 12 and a remarkable correlation between NF-κB activation level and glioma grade have been previously demonstrated. 13 Furthermore, accumulating evidence shows that constitutive NF-κB activity could regulate the proangiogenic context of glioma. Notably, NF-κB restraint even led to the blocking of the angiogenesis of glioma in nude mice. 14 Herein, we sought to discuss the current understanding of the molecular mechanisms of NF-κB in diverse glioma microenvironments such as hypoxia, inflammation and oxidative stress, and its function as a therapeutic target for antiangiogenic strategies aimed at glioma.

| NF-κB in hypoxia-induced glioma angiogenesis
During the entire process of angiogenesis, new capillaries sprout from the current capillaries, and endothelial cells (ECs) are released from their stroma and migrate and transfer to areas without capillaries, thereby allowing them to differentiate into tubular structures.
The newly generated capillaries provide a large amount of necessary oxygen and nutrients for fast-growing malignant tumours that have an avascular area volume greater than 2 mm 315 .
Due to abnormalities in the structure of malignant tumours, local or temporary hypoxia and a lack of nutritional components may lead to EC apoptosis and tumour angiogenesis inhibition. 16,17 Nevertheless, migrating ECs often overcome these adverse conditions to boost tumour angiogenesis. ECs are stimulated by vascular endothelial growth factor (VEGF) or adhere to extracellular matrix (ECM) molecules, leading to the augmentation of anti-apoptotic genes via the phosphatidylinositol 3-kinase (PI3K)/Akt or NF-κB signalling pathways. 18,19 Akt induces the transcription function of NF-κB by stimulating the RelA/p65 transactivation subunit via IκB kinase and activation of the protein kinase p38. 20 Studies have reported that the induction of cell survival signals by PI3K/Akt partially mediates the activation of NF-κB transcription factors. 20 The VEGF released by glioma cells stimulates EC proliferation, resulting in angiogenesis. 21 Interestingly, TNF, which could induce the apoptosis of ECs, was detected in glioma but did not inhibit the associated angiogenesis. 22,23 It was reported that human umbilical vein ECs must activate NF-κB in order to avoid undergoing TNF-induced apoptosis. 24 Using a human brain microvascular endothelial cell (HBMVEC) and U251 glioma cell co-culture system, investigators found that EC apoptosis was induced by serum starvation and reversed by recombinant VEGF protein and a culture medium of hypoxic U251 glioma cells. In addition, hypoxia treatment activated TNF-induced VEGF and NF-κB to upregulate the antiapoptotic gene expressions, such as those of Bcl-2, Bcl-XL, survivin and X-chromosome-linked inhibitor of apoptosis protein (XIAP) in ECs 25 ( Figure 1). Therefore, it is clear that the hypoxic environment of glioma, in addition to not killing ECs, promotes NF-κB-dependent angiogenesis.
Galectin-3 (gal-3) is a b-galactoside binding protein that is involved in several types of pathological tumour progression, such as angiogenesis, cell proliferation and anti-apoptosis. [26][27][28] Evidence shows that gal-3 is visibly upregulated in a hypoxia-inducible factor (HIF)-1α-dependent manner in mouse fibroblasts and nucleus pulposus cells under hypoxic conditions. 29,30 In addition, HIF-1α is a pivotal transcriptional regulator of the hypoxic response, which upregulates its target genes including vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP) to boost tumour angiogenesis and invasion. 31 Hypoxia is a commonly observed feature of solid tumours such as gliomas, in which a high proportion of gal-3 accumulates. 32,33 Gal-3 is released by tumour cells for the induction of EC chemotaxis and motility and the stimulation of angiogenesis ( Figure 1). Gal-3 knockout U87 glioma cells implanted subcutaneously in nude mice blocked tumour growth in vivo. 34 In vitro, Ikemori et al found that gal-3 protected T98G glioma cells from apoptosis in the absence of oxygen and nutrition, and the knockdown of gal-3 induced double apoptosis. It is worth noting that the upregulation of gal-3 was dependent not only on HIF-1α but also on NF-κB. 35 Hypoxia-induced NF-κB was conducive to the regulation of the HIF-1α and gal-3 genes and prevention of cell death caused by hypoxia. 36,37 Based on the above-stated literature, it can be concluded that hypoxia in glioma protects both ECs and tumour cells against death, which facilitates angiogenesis and leads to tumour aggravation, directly or indirectly.
Therefore, NF-κB exerts multiple effects on the angiogenic system in glioma under a hypoxic microenvironment.

| NF-κB in inflammation-induced glioma angiogenesis
Some interactions exist between inflammation and angiogenesis in the course of glioma progression. 41 The  (Table 1). Therefore, the expression of IL-8 and IL-6 in the glioma-associated ECs induced by TNF is blocked by the NF-κBmediated pathway, which has important implications for anti-angiogenesis therapy.
As early as 1991, thalidomide was shown to be a potent TNF inhibitor that inhibited NF-κB activation with anti-inflammatory effects; in 1994, it was demonstrated to inhibit VEGF with antiangiogenic effects. 69,70 Investigators found that thalidomide inhibited the proliferation of ECs in vitro without affecting their viability, but did not suppress the proliferation of U251 glioma cells. 71 NF-κB also controlled the genes related to vascular endothelial growth factor receptor (VEGFR) expression, 72  In numerous types of tumours including gliomas, the interaction between inflammation and tumours has been recognized, and inflammation is considered the 'seventh sign of cancer'. 73, 74 Growing evidence shows that TNF is a key mediator of inflammation and tumour growth.
Furthermore, the NF-κB activated by TNF further releases pro-inflammatory and proangiogenic factors to promote tumour vessel formation and tumour cell survival. Thus, understanding the mechanisms of NF-κB in the interaction and mutual promotion between inflammation and angiogenesis will provide new ideas for glioma treatment.

| NF-κB in oxidative stress-induced glioma angiogenesis
Interactions between inflammation and angiogenesis have been observed in the course of pathological progression. 41 One of the characteristics of the cellular inflammatory process is a respiratory burst, which generates and accumulates a large amount of extracellular reactive oxygen species (ROS), thereby preventing the invasion of pathogens. 75,76 However, the excessive accumulation of extracellular ROS leads to an imbalance of aerobic cells and tissues, called 'oxidative stress', which is related to ageing and several heart and vascular diseases. 77,78 Intracellular and extracellular ROS are involved in the angiogenesis process in many pathophysiological processes. 79,80 Intracellular ROS plays a crucial role in VEGF signalling in ECs. 81 In the tumour microenvironment, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family, plasma membrane-bound enzymes that generate superoxide, is a major source of ROS. 82 ROS affect the angiogenesis of tumours in numerous ways, the most important of which is the regulation of NF-κB transcriptional activation. Since NF-κB facilitates the synthesis of proangiogenic factors including IL-6 and IL-8, ROS could promote glioma angiogenesis. 46,47 The regulation of the NF-κB signalling pathway by ROS is very complex and depends on multiple processes. However, the main regulatory mechanism is the phosphorylation and direct oxidation of the NF-κB subunit. Since antioxidants have been shown to decrease Ser-276 phosphorylation to inhibit p65 transcriptional activity and oxidation of p50 by ROS suppress its DNA-binding ability, increased intracellular ROS reduce the p50 subunit activation and increase p65 subunit activation.. 11,83,84 The mutation status of the tumour suppressor gene p53 results in further NF-κB activation commands. 11,85 P53-mutated tumours tend to show a greater degree of malignancy, with enhanced invasion and reduced sensitivity to apoptotic signals. 86 Mateusz et al stated that graphite nanoparticles and graphene oxide nanoplatelets could reduce intracellular ROS-induced angiogenesis via the downregulation of NF-κB-dependent proangiogenic cytokines including IL-6, IL-8, growth-regulated oncogene α (GROα) and monocyte chemotactic protein 1 (MCP-1) in a p53wt glioma cell line (U87); however, they had no effect in a p53mut cell line (U118) 84 (Figure 3 and Table 1).
In this regard, oxidative stress-induced angiogenesis depends on p53 mutation status and NF-κB regulation, which provides novel strategies in the field of nanoparticle treatment for glioma angiogenesis.

| Caspase in NF-κB-dependent glioma angiogenesis
Caspase-8 was initially identified as participating in death receptorinduced apoptosis. 87 Apoptosis signalling is usually absent in cancer, and caspase-8 expression is also suppressed. 88,89 However, caspase-8 shows high expression in glioma and may be associated with poorer prognoses. In glioma models, caspase-8 could facilitate the expression of NF-κB-dependent proangiogenic cytokines and tumour promoters. 90 Further, it has been confirmed that it exerts growth-promoting F I G U R E 3 Role of NF-κB in glioma angiogenesis under a microenvironment of oxidative stress. The ROS produced by NADPH oxidase and TNFR2 mediates the activation of NF-κB and the main regulatory mechanism of ROS is the phosphorylation and direct oxidation of the NF-κB subunit (reduce p50 activation and increase p65 activation), and the activation of NF-κB further depends on the p53 mutation status. The transcriptional activity of NF-κB for IL-6, IL-8, GROα and MCP-1 promotes glioma angiogenesis. NF-κB: nuclear factor-κB; ROS: reactive oxygen species; IL: interleukin; TNFR: tumour necrosis factor receptor; NADPH: nicotinamide adenine dinucleotide phosphate; GRO: growthregulated oncogene; MCP: monocyte chemotactic protein effects in several conditions, such as fibrosis, 91 wound healing, tissue regeneration 92 and tumour reunion. 93 Feng et al found that dying glioma cells, following radiation, built a proangiogenic microenvironment by the caspase 3-dependent NF-κB/COX-2/PGE axis. 94 These results demonstrate that certain cancers such as glioma may reverse caspase-8 or the caspase-3 pro-apoptotic function that is dependent on NF-κB, leading to the promotion of blood vessel formation (Figure 4).

| Matrix metalloproteinase
Angiogenesis is related to invasion and is used for glioma grading. 99 A recent study showed similar molecular mechanisms for angiogenesis and invasion. 100 The new formation of blood vessels can be considered to an invasive course in which activated ECs proliferate, adhere to the ECM molecules and migrate. 101 MMPs are involved in angiogenesis, invasion and ECM degradation for the promotion of tumour development. [102][103][104] Among MMPs, MMP-2 and MMP-9 have been indicated as having an upregulated expression in glioma.
The upregulation and activation of MMP-2 in association with HIF-1α expression enhance tumour cell infiltration and blood-brain barrier permeability. 105 MMP-1 and MMP-3 levels also increase as the tumour grade increases. 106,107 It is also to be noted that the NF-κB binding sites in the MMPs promoter regions are closely related to tumour cell invasion and angiogenesis. 108 Therefore, effective MMP inhibitors may show promise for use in therapeutic strategies for glioma angiogenesis ( Table 1).
The use of tumour treating field (TTF) therapy, entailing an alternating electric field with an intermediate-frequency  for tumour treatment, led to glioma suppression. 109

| Vascular endothelial growth factor
ECs are stimulated by VEGF or adhere to ECM molecules, leading to the augmentation of anti-apoptotic genes via the PI3K/Akt or NF-κB signalling pathways. 18,19 Therefore, anti-angiogenesis therapy, that is, the inhibition of tumour-associated ECs, has become a major strategy for tumour treatment. 88 (Table 1).
These findings indicate that Bmi-1 could promote angiogenesis in glioma via NF-κB/VEGF-C, further suggesting that NF-κB/VEGF-Cdependent Bmi-1 may represent a novel therapeutic target for antiangiogenic strategies aimed at glioma.

| Platelet-derived growth factor (PDGF)
PDGF, a proangiogenic factor, is the major mitogen for many mesenchymal-derived cell types, such as fibroblasts and pericytes. 121 PDGF-mediated endothelial-mesenchymal transformation (EMT) reduced the expression of VEGFR-2 in ECs. With the loss of VEGFR-2 expression, ECs convert to a VEGF-independent state for the maintenance of their growth and survival in glioma, leading to the resistance of ECs to anti-VEGF treatment. 122 The expression of snail, a pivotal downstream regulator of EMT in the glioma environment, is regulated by NF-κB. 123 In addition, PDGF induces NF-κB-dependent snail expression, resulting in resistance to anti-VEGF treatment with the downregulation of VEGFR-2. The inhibition of PDGF receptor sensitized VEGF/VEGFR-2 targeted therapy in glioma-bearing mice model (Table 1). Collectively, targeting NF-κB/snail-dependent PDGFs may serve as a promising strategy for cases with resistance to anti-VEGF in glioma.

| Epidermal growth factor receptor (EGFR)
The amplification of the EGFR gene occurs in almost half of all glioblastoma cases and is related to gene rearrangement. 124 The rearrangement is often related to activating mutations such as the loss of exons 2-7 (EGFRvIII or EGFRde2-7). EGFRvIII overexpression in human glioma cells or primary mouse astrocytes can lead to the significantly faster formation of tumours in animal models by intracranial or subcutaneous injection than in the control group, demonstrating that EGFRvIII enhances the carcinogenic capacity. 125 (Table 1). In conclusion, EGFRvIII facilitates

| Other NF-κB -targeted therapeutic strategies for glioma angiogenesis
Parthenolide reportedly has the potential to cross the blood-brain barrier and alleviate brain inflammation. 127  The transcriptional activity of NF-κB can be induced by IκB cytoplasmic segregation and RelA/p65 phosphorylation. 129,130 Using the phage display technique to frame a single-chain fragment of anti-p65 antibody variable region (scFv), investigators cloned the scFv-encoding sequence into the mammalian nuclear-targeting vector, pCMV/ myc/nuc, to fabricate an anti-p65 intrabody construct (pFv/nu).
U251 and U87 glioma cells transfected with pFv/nu dramatically suppressed the expression of p65, and NF-κB-dependent genes such as MMP-9, VEGF, urokinase-type plasminogen activator receptor and urokinase-type plasminogen activator. Additionally, U251 and U87 glioma cells transfected with pFv/nu-bearing intracranial tumours were almost restrained. 131 Thus, inhibition of the transcriptional activity of NF-κB by nuclear-targeting intrabody could serve as a promising antiangiogenic strategy for glioma.
The ketogenic diet (KD) is a novel high-fat, low-carbohydrate, protein-rich diet that targets tumour metabolism and has been used in non-drug therapy for intractable epilepsy. Of note, first, mouse glioma models fed a KD showed higher survival values than those on a normal diet. 132 Glioma models fed a KD at will demonstrated observable reductions in NF-κB activation and reductions in the levels of NF-κB-mediated regulators in the hypoxic context, such as carbonic anhydrase IX (CA IX) and HIF-1α. 36 Second, the KD inhibits the levels of ROS, which boosts angiogenesis by the activation of NF-κB transcription in tumours. 25 Third, the KD blocks the expression of VEGFR2, the major receptor involved in tumour angiogenesis regulation 133,134 (Table 1). KD therapy that targets tumour metabolism and represses the NF-κB-mediated hypoxic response may provide a low-toxic, easy-to-implement method for glioma aimed at angiogenesis inhibition.

| CLINI C AL RELE VAN CE AND FUTURE PER S PEC TIVE S
Bevacizumab, a recombinant humanized anti-VEGF monoclonal antibody, is the only FDA-approved anti-glioblastoma angiogenesis drug. 135 Although it has been used in clinical treatment, it usually causes serious adverse reactions, and its clinical efficacy remains controversial. 136 Bevacizumab increases the hypoxic area and boosts the rate of MMP-2 activation, resulting in a more invasive, treatment-resistant glioma state. 137

| CON CLUS IONS
Angiogenesis in glioma accelerates tumour growth and increases the degree of malignancy. NF-κB plays a pivotal role in the growth and progression of glioma angiogenesis. Interference with the transcriptional activity of NF-κB that leads to alterations in the proangiogenic context and the inhibition of proangiogenic gene expression may be promising therapeutic strategies aimed at glioma angiogenesis blocking.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflicts of interest.

AUTH O R CO NTR I B UTI O N
JJT and YLF drafted the manuscript. DFH, XWT, HFJ, XG, XMW, WMH and WW revised the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
No new data generated.