2.1 NF-κB/Rel protein family

In humans, the NF-κB superfamily comprises five transcription factors: NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, and REL (c-Rel).⁶ These proteins all contain a Rel homology domain (RHD), a conserved N-terminal domain, which is very important for DNA binding, dimerization, and nuclear localization. According to the similarity of amino acid sequence within the RHD in the C-terminal, the NF-κB superfamily is generally divided into two subfamilies: NF-κB subfamily (p50 and p52) and Rel subfamily (Rel, RelA, and RelB). NF-κB1 (p50) and NF-κB2 (p52) are produced by the processing of protein precursor, p105 and p100, respectively, with ankyrin (ANK) repeat domains in the C-terminal, which form dimers with other NF-κB family members. The inhibitor of the NF-κB (IκB) protein family controls the activation process of NF-κB by interacting with transcriptional factors. The proteins bind and persist in the cytoplasm, while the Rel protein (Rel, RelA, RelB) has no protein precursor but a conserved transactivation domain in the C-terminal.⁷ These members of the NF-κB family share a highly conserved 3000-amino-acid RHD, which plays a crucial role in their function.^8-10 In most cases, all five NF-κB proteins could form homodimers and heterodimers with each other to start gene transcription.¹¹ However, RelB is a special exception, which only forms heterodimers. The most common NF-κB heterodimer is mainly composed of NF-κB1 (p50) and RelA (p65) (Figure 1). Whether NF-κB dimer activates transcription or inhibits transcription depends on the DNA regions it binds to and the interaction with other transcription factors.^{12, 13}

2.2 The IκB protein family

The activity of NF-κB dimer is regulated by the IκB protein. The IκB family is characterized by ANK repeats that interact with the RHD domain of NF-κB protein.⁹ The IκB protein family has eight members including IκBα, IκBβ, IκBɛ, IκBζ, IκBNS, Bcl-3, p100, and p105, and every member contains a series of ANK repeat domain (ARD)¹⁴ (Figure 1). The ARD of IκBs forms a slightly curved cylindrical structure, which contains amino acid residues that specifically recognize and bind to NF-κB dimers. These repetitive sequences can interact with RHD, allowing IκB to bind to NF-κB, preventing NF-κB from entering the nucleus.^{11, 15, 16} The activation of NF-κB relies on the degradation of IκB protein. Upon activation, IκBα is phosphorylated by IκB kinase (IKK) complex and ubiquitinated and degraded through the proteasome-dependent pathway, releasing NF-κB into the nucleus for transcription to activate target genes. NF-κB interacts with newly synthesized IκB in the nucleus and exits the nucleus into the cytoplasm, forming the cycle of a non-phosphorylated nuclear-cytoplasmic shuttling and inactivation.^{10, 16, 17} It should be noted that the C-terminal sequence of the precursor protein p100/p105 of p50/p52 also contains the ANK repeat sequence like IκB, which allows p100/p105 to inhibit the activity of NF-κB like IκB.^{18, 19}

2.3 Activation of NF-κB

The NF-κB activation signaling pathways have been well studied. There are two types of NF­κB activation signaling pathways: the canonical and the noncanonical pathways,²⁰ relying on distinct molecular mechanisms to activate NF-κB, as well as the different sets of target genes involved in cell proliferation, differentiation, and immune response.^{16, 21}

The IKKs complex is a large multi-component protein kinases complex, including two homologous catalytically active subunits IKKα (IKK1) and IKKβ (IKK2) and an auxiliary subunit IKKγ (NEMO, NF-kappaB essential modulator).²² IKK can be activated by various stimuli such as growth factors, cytokines, stress factors, and microbial components.¹⁷ The two catalytically active IKK subunits participate in different pathways. The noncanonical NF-κB activation pathway is mainly regulated by IKKα, while the regulation of the canonical NF-κB activation pathway induced by pro-inflammatory cytokines and various microbial products requires the participation of IKKβ. The oligomerization of IKKα/β/γ can affect the activity of IKKβ.^{17, 23, 24} Although IKKγ has no catalytic activity, it is necessary for the canonical pathway. IKKγ can form an IKK complex with other IKKs through its N-terminal, and its C-terminal can mediate the interaction with the upstream signal adaptor.^{16, 22, 25}

In the canonical NF­κB pathway, the key event is the phosphorylation of IκB protein mediated by IKKs (Figure 2). The adaptor molecule, ubiquitin ligase, and protein kinase activate the IKK complex at different levels.^{22, 26} The canonical activation pathway of NF-κB is stimulated by various factors, such as tumor necrosis factor receptor (TNFR) superfamily members, ligands of various cytokine receptors, pattern recognition receptors (PRRs, like Toll-like receptor (TLR) ligands), as well as B-cell receptor (BCR) and T-cell receptor (TCR).^{16, 27, 28} After stimulation and upstream signal transduction, IKKs phosphorylate IκBs, which in turn are ubiquitinated by ubiquitin ligase and degraded by 26S proteasome, losing the ability to bind to NF-κB. When IκB is degraded, NF-κB is dissociated from the NF-κB/IκBs complex and transferred into the nucleus, binds to the target gene promoter region on DNA, and initiate the transcription of the target gene.^{5, 11, 29} In this activation process, p50/RelA and p50/c-Rel have mainly involved dimers. In addition, in the canonical NF-κB pathway, one of the target genes activated is the gene encoding IκBα (Nfkbia), so IκBα is quickly re-synthesized after degradation, and the newly synthesized IκBα can directly bind to NF-κB in the nucleus. In this way, NF-κB is dissociated from the κB-binding site of DNA. The NF-κB/IκBs complex is exported back to the cytoplasm to keep the original latent state, fulfilling the cycle of NF-κB activation and inactivation. Therefore, the activation of the canonical NF-κB pathway is usually robust and transient.⁹ The canonical NF-κB pathway plays an important role in cell survival and proliferation, tumor cell epithelial to mesenchymal transformation (EMT), angiogenesis, cancer metastasis, and inflammation.

In the noncanonical NF-κB pathway, the key event is the processing of the precursor protein p100 of NF-κB2 rather than the degradation of IκBα (Figure 2). Compared with the canonical pathway with a wide variety of activators, the noncanonical pathway has relatively fewer activators. Noncanonical pathway selectively responds to specific stimuli, including ligands for part of TNF receptor family members, like lymphotoxin (LT) β receptor (LTβR), CD40, B-cell activation factor family (BAFF), and receptor activator of NF-κB (RANK).^30-34 Under the action of stimuli, the corresponding receptor is activated, leading to degradation of TNF receptor-associated factor 3 (TRAF3). The protein level of NF-κB-induced kinase (NIK) is mediated by TRAF3-cellular inhibitor of apoptosis (cIAP) under the steady-state condition. It is currently believed that NIK and IKKα mediate the conversion of p100 to p52, which are the key components of the noncanonical NF-κB pathway.³⁵ NF-κB2 exists in the form of precursor p100 under the non-stimulated condition. p100 functions as IκB protein, binding to RelB and retaining RelB in the cytoplasm.³⁶ Together with IKKα, NIK mediates the phosphorylation of p100, which is further conjugated with ubiquitin chains mediated by β-transducin repeats-containing proteins (β-TrCP) E3 ligase. Ubiquitinated p100 is processed to generate p52, forming p52/RelB heterodimer.^{30, 32, 37}

Two NF-κB pathways have shared characteristics: forming functional NF-κB dimer and translocating the dimer into nuclear. The difference between these two pathways is that the activation of the canonical pathway is to rely on the degradation of IκBα; meanwhile, in the noncanonical NF-κB pathway, processing of p100 into p52 does not only generate p52 but also liberate RelB from p100, forming p52/RelB dimer. The resynthesis of NIK is the key to start the downstream pathway, so the activating non-standard NF-κB pathway is much slower than the standard pathway.²¹ Optimizing target gene expression also requires the interaction of NF-κB with other transcription factors, such as activator protein 1 (AP1), signal transducer and activator of transcription (STAT) family members, and interferon regulatory factors (IRFs).^38-40

2.4 Regulation of NF-κB pathway

As mentioned in the previous chapter 2.3, NF-κB is often blocked in the cytoplasm by IκBs, keeping the entire pathway silent. However, once activated by stimuli (regardless of the various stimuli in the canonical pathway or a subset of specific stimuli in the noncanonical pathway), NF-κB is activated to induce the production of pro-inflammatory mediators and molecules leading to inflammation and activation and differentiation of the immune cell.²⁹ If the NF-κB activation is dysregulated, diseases such as chronic inflammation, tumors, and autoimmune diseases may occur. Therefore, NF-κB is strictly regulated to maintain homeostasis.

First of all, as an inhibitory protein of NF-κB, IκB plays a very important role in controlling the intensity and duration of NF-κB activity. The target genes of NF-κB include genes encoding IκB, such as Nfkbia (encoding IκBα) and Nfkbie (encoding IκBε). When the activated NF-κB dimer enters the nucleus to bind to the κB-binding site, target genes including Nfkbia are transcribed, and the newly generated IκBα and IκBε proteins bind and drive the NF-κB dimer from the nucleus to the cytoplasm. This dynamic negative feedback mechanism is critical for keeping the activation of NF-κB in check.^41-43 Although both IκBα and IκBε have regulatory functions, they function in different ways. Compared with the rapid production and inhibition of IκBα, IκBε provides slower negative feedback regulation with delayed expression.⁴⁴ In contrast, another member of the IκB family, IκBβ, directly binds to RelA and c-Rel in the nucleus to counteract the inhibitory function of IκBα. When the hypo-phosphorylated IκBβ binds to RelA and c-Rel, the inhibitory effect mediated by IκBα is impaired, and the continuous transcription of the target gene is maintained.^{24, 45} In addition to driving NF-κB back to the cytoplasm through the IκB protein, another way to downregulate NF-κB activation is to degrade NF-κB dimers in the nucleus directly. For example, elongin-B-elongin-C-cullin-SOCS1 (ECS) is a ubiquitin ligase complex that promotes ubiquitination and subsequent degradation of RelA in the nucleus.^46-48 Studies have also reported that the E3 ubiquitin ligase post-synaptic density-95, disks-large, and zonula occludens-1 and Lin-11, Isl1 and Mec-3 (LIM) domain protein 2 directly remove RelA from the DNA binding site and mediate its degradation to suppress NF-κB transcription activity.^{49, 50} Protein inhibitor of activated STAT 1 (PIAS1) also moves to the promoter region of NF-κB target genes after phosphorylation of IKKα, inhibiting the binding of RelA-containing dimers to DNA.^51-53

Secondly, the studies on NF-κB signaling pathways reveal that ubiquitination is essential for NF-κB activation. In the canonical NF-κB pathway, NF-κB activation mediated by multiple stimuli requires ubiquitination. For example, when interleukin-1 receptor (IL-1R) or TLR is activated, the ubiquitination of tumour necrosis factor receptor-associated factor 6 (TRAF6) requires TRAF6-regulated IKK activator 1 (TRIKA1, a dimeric ubiquitin-conjugating enzyme complex, composed of ubiquitin-conjugating enzyme 13 (Ubc13) and ubiquitin E2 variant 1Uev1A).^{54, 55} The function of TRIKA1 is to synthesize K63-linked polyubiquitin chains on IKKγ and TRAF6.^54-56 TRAF6 requires TRIKA2 (a complex composed of TAK1, TAB1, and TAB2) to activate IKK, and the activation of TRIKA2 requires ubiquitination of the adaptor protein TAB2/3.^{55, 57, 58} Similarly, when stimulation goes through TNFR, the activation of IKK depends on K63-ubiquitination of receptor-interacting protein 1 (RIP1), and this process is mediated by cIAP1/2.⁵⁹ In the noncanonical NF-κB pathway, p100 ubiquitination is the key event to regulate NF-κB activation. β-TrCP acts as a ubiquitin E3 ligase to mediate inducible p100 ubiquitination, another E3 ligase Fbxw7α requires glycogen synthase kinase 3 (GSK3)-mediated phosphorylation to mediate ubiquitination and degradation of p100.^{37, 60} Another molecule of interest is deubiquitinase (DUB) A20. A20 contains the DUB domain and the C2-C2 zinc finger E3 ubiquitin ligase domain, which inhibits K63 ubiquitination during the activation of NF-κB, leading to the decomposition of the IKK complex. A20 also mediates the K48 ubiquitination of RIP1 after inhibiting K63 ubiquitination, causing the degradation of RIP1 and suppressing the signaling pathway induced by TNFα.^61-64 As a key enzyme that inhibits ubiquitination in the canonical NF-κB pathway, A20 regulates NF-κB activity by coordinating with another E3 lineage such as Itch.⁶⁵ Meanwhile, the A20 encoding gene, Tnfaip3, is one of the NF-κB target genes to mediate the inactivation of NF-κB, suggestive of negative feedback regulation.⁶⁶ Similar to A20, the tumor suppressor protein cylindromatosis (CYLD) is also a DUB involved in the regulation of NF-κB activity. Its function is to remove K63 ubiquitin chain of key molecules upstream of IKK, including TRAF2/6 and IKKγ.^67-69 In noncanonical NF-κB pathway, ovarian tumor domain-containing protein 7B (OTUD7B) functions as the DUB of TRAF3 to stabilize TRAF3 protein upon stimulation.⁷⁰ OTUD7B deficiency leads to enhanced noncanonical NF-κB activity. Additionally, OTUD 1 is also a DUB, which inhibits the ubiquitination of p100, leading to the stabilization of p100 under stimulation condition and steady state.⁷¹

Up to date, all the noncanonical NF-κB-activating stimuli activate NIK, which means that NIK is essential for noncanonical NF-κB activation.⁷² Considering the importance of NIK, the regulatory factors of NIK are also considered to play an important role in the regulation of NF-κB. As a negative regulator of NIK, TRAF3 binds to NIK through the N-terminal region causing NIK degradation in a ubiquitination-dependent way and keeping NIK protein at a very low level.⁷³ This process also requires the participation of TRAF2 and ubiquitin E3 ligase cIAP1/2.⁷⁴ This constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3, and cIAP1/2 inhibits NIK.^{33, 74} NIK is also regulated by TBK1. TBK1 mediates the phosphorylation of NIK at Ser862, which is located in the degradation domain of NIK, making the phosphorylated NIK unstable.^{75, 76} Moreover, TBK1 forms a ternary complex with TRAF family member-associated NF-κB activator (TANK), and TRAF2 and plays a role upstream of the NIK and IKK complex, a substitute for the receptor signaling complex for TRAF-mediated NF-κB activation.⁷⁷ Kinase inactive TBK1 inhibits TANK-mediated NF-κB activation but does not block TNFα, IL-1, or CD40-mediated NF-κB activation.⁷⁷ IKKα also mediates the negative feedback regulation of NIK. IKKα phosphorylates the C-terminus of NIK and causes NIK degradation to prevent excessive accumulation of NIK.⁷⁸

In summary, most of the regulating mechanisms of the NF-κB pathway are based on the regulation of key molecules or processes of this pathway, such as IKKs, NIK, ubiquitination. These mechanisms also indicate that the activation of NF-κB is strictly regulated at multiple layers. Due to various stimuli and target genes involved in the NF-κB pathway, the precise and comprehensive understanding of regulating mechanisms of the NF-κB pathway requires additional intensive studies in vivo and in vitro.

3.1 NF-κB regulates innate immune response and inflammation

The innate immune cells including macrophages, dendritic cells (DCs), and neutrophils, are required to produce inflammatory mediators and regulators to eliminate pathogens, while at the same time avoiding a sustained inflammatory response through negative feedback mechanisms.⁸⁰ The PRRs expressed by these cells can detect various microbial components, so-called pathogen-associated molecular patterns (PAMPs). PRRs can also sense molecules released by necrotic cells and damaged tissues, called damage-associate molecular patterns (DAMPs).^{81, 82} The PRRs on innate immune cells induce the expression of pro-inflammatory cytokines such as TNF, IL-1, IL-6, IFN-I, chemokines, and antimicrobial proteins and mediate an inflammatory response to eliminate pathogens and repair damaged tissue.^{83, 84} Different PRRs families have different structural characteristics and respond to different PAMPs and DAMPs. Five PRR families have been found in mammals, including TLRs, NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs), and cytosolic DNA sensors.^{83, 85-87} The activation of the canonical NF-κB pathway through these PRRs induces pro-inflammatory cytokines, chemokines, and other inflammatory mediators.^{82, 88} Moreover, NF-κB plays an important role in the signal transduction mediated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and myeloid progenitor cell differentiation.⁸⁹ The development of innate immune cells from myeloid progenitor cells is specifically affected by different members of the NF-κB transcription factor family.⁹⁰

3.2 Functions of NF-κB in adaptive immunity

Inflammation also involves adaptive immunity. After activation, T cells and B cells proliferate and differentiate into effector cells. These effector cells mediate different immune responses, including the secretion of cytokines, cytotoxic T lymphocyte response, and the production of antibodies by B cells.²⁹ It is worthy of special attention that the activated CD4⁺ T cells can differentiate into effector T-cell subsets with different functions, including Th1, Th2, Th9, Th17, Tfh, and regulatory T (Treg) cells.^115-117 The differentiation of CD4⁺ T cells is regulated not only by the cytokines secreted by APCs and other innate immune cells but also by T-cell intrinsic factors.⁴ Th1 cells produce pro-inflammatory cytokines IL-12 and Interferon-γ (IFN-γ) to activate macrophages and mediate the immune response to intracellular pathogens and participate in inflammation.¹¹⁵ Th2 cells release IL-4, IL-5, and IL-13 and stimulate the response of mast cells, eosinophils and basophils to pathogens.^{118, 119} Th17 cells can produce IL-17 and IL-22 to recruit neutrophils and monocytes to the site of inflammation and mediate immune responses against pathogens or autoantigens.¹¹⁷ Treg cells are produced during the intrathymic development and can produce cytokines, such as IL-10 and transforming growth factor-β (TGF-β), to suppress the immune response.^{115, 117, 120} Similar to CD4⁺ T cells, activated naive CD8⁺ T cells can also proliferate and differentiate into various effector and memory cell types, including T effector cells, T memory stem cells, and T central memory cells, which are responsible for the elimination of tumor cells and viral infections cells.^121-123 While B lymphocytes develop in the bone marrow, they differentiate into plasma cells that can produce pathogen-specific antibodies after activation.¹²⁴ Memory B cell is formed in the germinal center after the initial infection. In the case of re-infection, memory B cells quickly produce antibodies and play an important role in the secondary immune response.¹²⁵

3.3 NF-κB is involved in inflammatory diseases

4.1 Activation of NF-κB in cancer

v-Rel is an oncogene of avian Rev-T retrovirus, isolated from a turkey liver lymphoma by Theilen and Robinson in 1958,²⁸⁰ which is the earliest evidence that NF-κB is related to cancer. However, the carcinogenicity of the rel gene is only found in birds, not in humans. Although the gene rel is not a bona fide oncogene, the constitutive-activated NF-κB has been examined in most tumors, which participates in a variety of cancer-related biological procedures.¹⁶

In most cancers, the activation of NF-κB is enhanced, and this enhancement in NF-κB activation is often due to increased stimuli of the NF-κB pathway, such as increased TNFα and IL-1 in the tumor microenvironment.^{281, 282} On the other hand, NF-κB has a certain tumor-suppressing function confirmed using tumor cell line and mouse models but not fully demonstrated in human cancers yet.^{283, 284} Mutations of NF-κB are found in many cancers, although the mutation frequency of RelA and RelB is much lower than that of REL, p50, and p52.⁷ It has been reported that REL gene amplification in lymphoma leads to an increased expression of REL protein, as well as a C-terminal truncated p100 protein that lacks the ANK repeat inhibitory sequence.^285-287 In addition to NF-κB family proteins, mutations have been found in the core components and regulators of the NF-κB pathway, which affect both canonical and noncanonical NF-κB activation. For example, studies have identified a loss-of-function mutation of IκB family proteins in lymphoma, glioblastoma, and nasopharyngeal carcinoma.^288-291 Meanwhile, studies have also reported that mutations of IKKα and IKKβ have been found in several cancers,^292-294 although the mutation of IKK is relatively rare. The reason may be that IKK also participates in many other signaling pathways except for NF-κB.²⁹⁵ The functional versatility of IKK may reduce the possibility of mutations. Mutations in critical regulating molecules that affect the activation and function of NF-κB are identified in tumor cells. Considering NF-κB as a family of the conserved transcription factor, the most common mutations that enhance NF-κB signaling may occur in the upstream molecules and downstream target genes.⁷ In diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM), proteins such as CD79 and MyD88 have been found to activate NF-κB through gain-of-function mutations. It was also found that loss-of-function mutations of CYLD, A20, TRAF3, and other negative regulators also cause abnormal activation of NF-κB.^296-301 Similarly, TNFα coded by an NF-κB target gene TNFA acts as a strong activator of NF-κB, functions in many tumor cells with constitutive activation of NF-κB in an autocrine manner (usually stromal).^{301, 302} Taking specific tumor types as an example, DLBCL is the most studied malignant tumor with NF-κB mutation.²⁹⁷ Studies have shown that mutations in the three proteins (REL, IκB, p300) are involved in the NF-κB activation in human B-cell lymphoma cell line RC-K8.^{287, 288} Most B-cell malignancies not only have an oncogenic driving force activated by mutated NF-κB but also have mutations in many other pathways. The subtype of DLBCL lymphoma with the activation of the canonical NF-κB pathway is named by the activated B-cell (ABC) subtype. Meanwhile, the abnormal activation of the noncanonical NF-κB pathway is also detected more in other DLBCL subtypes.^{299, 300} The mutations in the NF-κB signaling pathway involved in cancers are summarized in Table 1.

4.2 NF-κB and cancer cell survival and proliferation

One important function of the NF-κB is to regulate cell survival. In tumor cells, the activation of NF-κB usually leads to impaired cell apoptosis.³⁰³ The deletion or inactivation of genes encoding key molecules in the NF-κB pathway promotes cell apoptosis. For example, inactivation of the v-Rel oncogene leads to apoptosis of transformed lymphocytes, and KO of the Rela gene leads to apoptosis of mouse fibroblasts and embryonic hepatocytes under the TNFα stimulation.^{210, 304} NF-κB target genes encode anti-apoptotic molecules such as Bcl2, Bcl-xL, and inhibitor of apoptosis (IAP). Upregulation of NF-κB activity has been detected in various tumor cells, and the expression of target genes of anti-apoptotic NF-κB is also increased.⁷ It is noteworthy that NF-κB is not only anti-apoptotic, the activation of NF-κB is also required for cell apoptosis. Studies have found that inhibition of NF-κB reduced the apoptosis induced by drugs in many tumor cells.^305-307 Therefore, NF-κB may also be a cell type-specific repressor of anti-apoptotic genes or an activator of pro-apoptotic genes.^308-310 In addition, the recent researches suggest that many molecules (such as various anti-cancer drugs, non-coding RNA, microRNA, etc.) regulate the apoptosis of cancer cell through the NF-κB pathway and other coordinated pathways (such as AMPK, AKT, glycogen synthase kinase 3, metabolism pathways).^311-317

In addition to apoptosis, proliferation is also the fundamental cellular procedure. NF-κB activates genes that regulate cell proliferation, such as cyclin D1/D2/D3.^318-320 In addition to cyclins, NF-κB also regulates cell proliferation by induction of critical enzymes. The expression of E3 ubiquitin ligase mouse double minute 2 protein is NF-κB-dependent, which affects the stability of p53 and cell proliferation.³²¹ In inflammatory immune cells such as macrophages and neutrophils, the activation of NF-κB activates the expression of inflammatory cytokines such as TNFα, IL-1β, and IL-6, thereby promoting the proliferation of malignant cells and tumor stromal cells.^{16, 322, 323} Taken together, it has been well-accepted that NF-κB plays an important role in the regulation of tumor cell proliferation and apoptosis, and its functions and mechanisms are multi-faceted.

4.3 NF-κB and tumorigenesis

The journal from tumorigenesis to tumor progression is a long-term procedure controlled by multiple factors. Most tumors usually require multiple gene mutations.³²⁴ Inflammation and NF-κB affect the tumor occurrence by promoting the production of reactive oxygen species and reactive nitrogen species, leading to DNA damage and carcinogenic mutations.²⁸² Chronic inflammation and NF-κB also cause chromosomal instability, aneuploidy, and epigenetic changes, resulting in the occurrence and progression of tumors.²⁸² In addition, one of the mechanisms by which NF-κB promotes tumorigenesis is that when cells with damaged DNA strands enter the cell cycle, NF-κB activation integrates mutations into the two DNA strands and transmits them to daughter cells.^{325, 326} Another mechanism by which NF-κB stimulates tumorigenesis is to induce mutant-related enzymes such as activation-induced cytidine deaminase that deaminates cytosine residues and causes the conversion of cytosine to thymine, increasing mutations probability.³²⁷ Furthermore, by preventing p53-dependent apoptosis, NF-κB activation increases the number of DNA-damaged cells that accumulate oncogenic mutations.³²⁸ In addition to gene mutation, viral infection is the risk factor leading to tumorigenesis, such as hepatitis B virus (HBV), human papillomavirus, and Epstein–Barr virus (EBV), and so forth. Some viruses promote tumorigenesis through chronic NF-κB-dependent inflammation or the continuous activated transcriptional activity induced by viral genes. Viruses also directly encode NF-κB-stimulating factors. EBV that causes B-cell lymphoma and nasopharyngeal carcinoma encode LMP1 protein that functions as CD40 homolog to prevent apoptosis of EBV-infected B-cell by upregulating NF-κB activation.³²⁹ Kaposi's sarcoma-associated herpesvirus that causes sarcoma and lymphoma encodes viral FLICE inhibitory protein (vFLIP) that hijacks both canonical and noncanonical NF-κB pathways promoting cell survival and proliferation.³²⁹ Besides direct transcribing cell proliferation-related genes, NF-κB also induces the expression of proinflammatory cytokines such as IL-1β, IL-6, and TNFα, which play a key role in the NF-κB-dependent tumor cell proliferation.³³⁰ Meanwhile, NF-κB also regulates apoptosis of tumor cells. NF-κB blocks the death of tumor cells induced by the activation of oncogenes Ras.³³¹ NF-κB-dependent Rac guanosine triphosphatase effectively inhibits the p53-independent apoptosis response induced by high levels of Ras activity. Rac mutants that could not activate NF-kB are defective in inhibiting Ras-induced apoptosis. In particular, under hypoxic conditions, NF-κB enhances the expression of hypoxia-inducible factor 1α, thereby enhancing the early survival of tumor cells.^{332, 333}

NF-κB is important for tumor angiogenesis.³³⁴ Among the singling pathways contributing to tumor angiogenesis, NF-κB is critical to activate angiogenesis-related genes. Study has confirmed that B7-H3 activates NF-κB pathway to upregulate the expression of vascular endothelial growth factor-A (VEGFA) in colorectal cancer and promotes angiogenesis.³³⁵ In contrast, NF-κB interacting long noncoding RNA (NKILA) was found to inhibit the IL-6 production and angiogenesis through NF-κB.³³⁶ NF-κB in cancer cells promotes EMT.³³⁷ The expression of major EMT molecules (including Twist, Zinc finger transcription factor Snail2 (Slug) and Smad-interacting protein1) is NF-κB-dependent, which initiate EMT and identify malignant phenotypes by enhancing the stemness and migration of cancer cells.^{338, 339} Additionally, NF-κB activation also promotes EMT through other mechanisms, such as the matrix-degrading enzyme.³⁴⁰ MMPs induced by NF-κB promote the release of TGF-β, EMT process, and hypoxia that contribute to metastatic dissemination.^341-343 In addition, NF-κB and cytokines (such as VEGF and TGF-β) can directly stimulate the expression of metastasis-related genes and promote tumor metastasis, such as exercise-inducing factors and chemokines.^{344, 345} Inflammation and NF-κB can also directly stimulate metastatic dissemination through EMT, increase the extravasation of cancer cells into the blood and lymphatic vessels, and prevent the death of connective tissue tumor cells.³⁴⁶ Taking liver cancer as an example, the functions of NF-κB in tumorigenesis are schematically shown in Figure 3.

4.4 NF-κB and cancer stem cells (CSCs)

CSCs are malignant cells that have the ability to self-renew and differentiate into highly differentiated malignant cells. CSCs are the major reason for cancer recurrence, metastasis, and treatment resistance.^{347, 348} CSCs were first identified in leukemia and then isolated as CD34⁺CD38^– cells in the 1990s.^{349, 350} The following studies indicate that in other hematologic malignancies and solid tumors, CSCs express other different surface markers (such as CD133, nestin, and CD44).^{351, 352} The activities of cancer stem cells are controlled by many intracellular and extracellular factors and various signaling pathways. The functions of NF-κB pathway in CSCs are complicated. NF-κB is constitutively activated in various CSCs (including leukemia, glioblastoma, prostate, ovary, breast, pancreatic, and colon cancer), which mediates inflammation, cell proliferation, survival, maintenance, and expansion.³⁵³ NF-κB could work alone or synergize with other signaling pathways to induce and promote the self-renewal, proliferation, and metastasis of CSCs by mediating the expression of stem-cell-related transcriptional factors and genes (such as Nanog, Sox2, Olig2, CD44, NKX3.1 and Krüppel-like factor 4 (KLF4)).³⁵⁴ In breast cancer, NF-κB activation causes CSC proliferation by activating Notch signaling in a cell-involuntary way.³⁵⁵ In addition, high levels of NIK also induce the activation of the noncanonical NF-κB pathway to regulate the self-renewal and metastasis of breast CSCs.³⁵⁶ Therefore, drugs targeting NF-κB pathway could be used to inhibit the proliferation and metastasis of cancer stem cells. The study has shown that in breast cancer stem cells, the anti-alcoholism drug disulfiram inhibits TGF-β through the extracellular regulated protein kinases(ERK)/NF-κB/Snail pathway to induce tumor metastasis.³⁵⁷ Sulforaphane also inhibits the self-renewal of triple-negative breast cancer stem cells by inhibiting NF-κB p65 subunit translocation and down-regulating p52 and its transcriptional activity.³⁵⁸ The canonical NF-κB pathway enhances the expression of the transcription factor Sry-related HMG box 9 (Sox9), which is associated with enhanced pancreatic CSCs and invasiveness.³⁵⁹ In ovarian cancer, CD44⁺ CSCs enhance self-renewal and metastasis by upregulating the expression of RelA, RelB, and IKKα and enhancing the activation of p50/RelA dimer.³⁶⁰ In colorectal cancer, the inflammatory mediator prostaglandin E2 activates NF-κB through prostaglandin E Receptor 4-phosphoinositide 3-kinase to promote the formation, maintenance, and metastasis of CSCs.³⁶¹ The transcription factor Foxp3 also interacts with NF-κB, to inhibit the expression of the NF-κB target gene COX2, and affect the self-renewal and metastasis of colorectal CSCs.³⁶²

4.5 NF-κB and tumor microenvironment

Tumorigenesis’ progress and metastasis rely on the environment where the tumor cells are located. The tumor microenvironment is composed of tumor cells, a variety of stromal cells, and immune cells, together with cytokines, chemokine, and other mediators, especially endothelial cells, cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs), and other numerous immune cells are critical for the tumor microenvironment.^363-365 NF-κB functions as the key regulator in many types of cells to shape the tumor microenvironment.

Cytokines produced by stromal cells, cancer cells, and immune cells are the key soluble factors for tumorigenesis, metastasis, and inflammation.³³⁰ Tumorigenesis is associated with tumor-promoting cytokines, so it is important to understand the correlation between NF-κB and pro-tumorigenic cytokines. Tumor necrosis factor α (TNFα) is also one of the well-studied tumor-promoting cytokines, and its expression is enhanced in many cancers, which often indicates a poor prognosis.^{302, 366, 367} TNFα is mainly produced by activated neutrophils and macrophages, which induces other proinflammatory cytokines, including IL-6 and IL-1β, and accelerates tumorigenesis.³⁰² Similar to TNFα, IL-6 is another important and abundant proinflammatory cytokine in the tumor microenvironment. The functions of this NF-κB-dependent cytokine on inflammation and tumorigenesis have been well studied.^{229, 367, 368} IL-6, mainly produced by malignant tumor cells and activated immune cells, initiates cancer-related inflammation and stem cell expansion in an autocrine manner.^{367, 369} In addition, IL-1α/β are also key proinflammatory cytokines produced by cancer cells and immune cells, which are NF-κB-dependent and promote the activation of NF-κB and MAPK pathways and tumorigenesis.^{370, 371} IL-17A activates NF-κB and MAPK signaling pathways, thereby promoting tumorigenesis.^372-374 The other IL-1 family cytokine, IL-33 is mainly expressed by epithelial cells, fibroblasts, and tumor cells, with a crucial function in allergy, autoimmunity, inflammation, and cancer.^{375, 376} Another important cytokine is TGF-β, which is produced by a variety of cells, including fibroblasts, myeloid cells, T cells, and cancer cells. TGF-β is essential for the differentiation of Treg and TH17 cells, making it a powerful inducer of tumor invasion and metastasis.^{330, 343}

NF-κB drives chemokine expression in tumor cells, stromal cells, and immune cells in the tumor microenvironment, especially CAFs.^377-379 The activation of NF-κB and other transcription factors (such as AP1 and STAT3) induces the expression of chemokines, which recruit more immune cells and further aggravate the inflammatory response.^{380, 381} Meanwhile, chemokines are important to stimulate the growth and metastasis of primary tumors, synthesizing growth factors, and angiogenesis. Although the functions of chemokines are complicated and uncertain in tumorigenesis and metastasis, chemokines still are important targets for cancer immunotherapy.^{364, 382, 383} For example, chemokine (C-C motif) ligand 5 (CCL5) induces the formation of eukaryotic initiation factor 4F translation initiation complex in an mechanistic target of rapamycin (mTOR)-dependent way, which mediates the quick upregulation of cyclin D1, c-Myc, and defender against cell death-1 (Dad-1) protein expression to promote tumor cell proliferation in breast cancer.³⁸⁴ In addition, in human pancreatic cancer and mouse pancreatic tumor models (Pan02), the CCL5 level on tumor cells is elevated, while CD4⁺Foxp3^– effector T cells preferentially express C-C chemokine receptor 5 (CCR5). When the CCR5/CCL5 signal is disturbed, either by inhibiting CCL5 expression on tumor cells or systemically administrating CCR5 inhibitors, the migration of Treg cells to the tumor microenvironment is reduced, which indicates that the chemokine CCL5 is required for Treg cells migration.³⁸⁵ Tumor-related cytokines are mainly expressed by inflammatory cells, such as tumor-associated macrophages (TAMs) and neutrophils. NF-κB is mainly activated in these cells to induce the expression of cytokines (TNFα, IL-1β, IL-6, etc.), thereby promoting tumor survival and proliferation.^{16, 323} As the most abundant immune cells in the microenvironment, macrophages produce inflammatory cytokines and chemokines and produce proteases such as cysteine cathepsins, involved in activating cytokines to promote tumor development.^{330, 365} M1 macrophages activated by IFN-γ and LPS can produce proinflammatory cytokines, chemokines, and enzymes to promote inflammation. In contrast, M2 macrophages induced by IL-4, IL-10, and IL-13 can inhibit inflammation by releasing anti-inflammatory mediators.^{92, 386, 387} M1 macrophages promote inflammation, while M2 macrophages create an immunosuppressive tumor microenvironment. The increase in p50 activity determines the M1 to M2- polarization of macrophages.^387-389 Inhibiting NF-κB transforms M2 macrophages to M1 macrophages because the polarization of M1 macrophages through IL-1R and MyD88 into M2 macrophages requires IKKβ-mediated activation of NF-κB. Moreover, the impaired NF-κB activation in TAMs also promotes the tumor-killing activity of macrophages. When NF-κB signaling is inhibited in TAMs, they become M1 phenotype with anti-tumor cytotoxicity.³⁹⁰ NF-κB could be a target for manipulating the phenotype of macrophages in the tumor microenvironment.

DCs are also important immune cells modulating anti-tumor immunity in the tumor microenvironment. The canonical NF-κB pathway in DCs can be inhibited by signals from the immune checkpoint molecule programmed cell death protein 1 (PD-1), thereby inhibiting the production of cytokines.³⁹¹ In contrast, the ligand of checkpoint like programmed cell death 1 ligand 1 (PD-L1) is an NF-κB target gene. The upregulation of PD-L1 expression in cancer cells relies on NF-κB activation triggered by many stimuli and activators, including oncogenes, stress, inflammatory cytokines, and chemotherapeutic drugs.³⁹² In addition to macrophages and DCs, it has also been reported that IL-1β induces NF-κB activation in MDSCs to suppress the function of tumor microenvironment (TME), leading to tumor proliferation.³⁹³ In natural killer (NK) cells that directly kill tumor cells to exert anti-tumor activity, the expression of cytotoxic effector molecules (such as perforin and granzyme B) ia also affected by NF-κB.^394-396 The activation of NF-κB in NK cells can be stimulated by exogenous anti-cancer drugs, like paclitaxel.³⁹⁷

As the important components of the tumor microenvironment, T cells and B cells in the tumor microenvironment could have tumor-promoting or anti-tumor functions.³⁹⁸ As mentioned above, the important stimuli of the NF-κB pathway include TCR and BCR. The activation of conventional T cells requires the participation of the canonical NF-κB pathway, which is required for CD8⁺ T cell proliferation and anti-tumor immune response.^{399, 400} The development of Treg cells requires the participation of p65 and Rel. Rel is not only essential for the optimal steady-state expansion of Treg cells in peripheral blood¹⁵⁰ but also for the development of thymic Treg cells. p65 is crucial for Tregs maturation, and the maintenance of immune tolerance⁴⁰¹ IKKβ-dependent NF-κB is activated in B cells that induce a cytokine LT, a heterotrimeric member of the TNF family that activates IKKα.⁴⁰² The LT produced by B cells can activate IKKα in tumor cells. IKKα phosphorylates a site of the transcription factor E2F transcription factor 1 (E2F1) to promote E2F1 translocation and recruit to the target of the Bmi1 gene. The IKKα–E2F1–Bmi1 cascade activated by B cells controls prostate regeneration and tumor recurrence and regulates the renewal function of tumor stem cells.⁴⁰³

The component that cannot be ignored in the tumor microenvironment is fibroblasts. Normal fibroblasts undergo changes in a self-intrinsic way, and under the stimulation of tumor-induced alterations tissue structure, TGF-β, and hypoxia, they become tumor-associated fibroblasts (CAFs) and express a large number of inflammatory factors and chemokines.^{378, 379} For example, IL-1β is induced by the NF-κB signaling pathway to promote inflammation, cancer cell proliferation, angiogenesis, and tumor metastasis.⁴⁰⁴ For example, in pancreatic ductal adenocarcinoma, CAFs mediate the exosomal metastasis and paracrine of tumor cells through producing cytokines (such as GM-CSF and IL-6), and this process requires the participation of NF-κB. In breast cancer, locked nucleic acid (LNA)-i-miR-221 inhibits miR-221-mediated NF-κB activation to reduce the secretion of tumor-promoting cytokines of CAFs.⁴⁰⁵ In brief, NF-κB has complex functions on the different types of cells in the tumor microenvironment and might become a potentially effective therapeutic target. The function of the NF-κB in the tumor microenvironment is shown in Figure 4.

4.6 NF-κB and inflammation in cancer

The relationship between inflammation and tumor has become an important field of cancer research. It is well-studied that inflammation is associated with autoimmune diseases, infection, and cancers.⁵ The well-established examples of inflammation leading to cancer are HBV infection and hepatocellular carcinoma (HCC), and chronic Helicobacter pylori infection and mucous MALT lymphoma and gastric cancer.^{25, 330} NF-κB is essential for the activation, differentiation, and effector functions of inflammatory T cells and innate immune cells and participate in the regulation of inflammasomes.⁴⁰⁶

In addition to NF-κB, the activation of other transcription factors such as AP1 and STAT3 can also induce the expression of chemokines, which recruit more immune cells (including T cells, B cells, macrophages, neutrophils, etc.) to aggravate the inflammatory response further.^{380, 407} As early as 2004, two studies showed the key roles of NF-κB in inflammation-driven colitis-associated cancer and HCC.^{229, 408} These results indicate that tumorigenic pathogens cause chronic infection and inflammation, and chronic inflammation sequentially leads to malignant tumors by increasing cellular stress response and recruiting inflammatory immune cells.^{5, 409} However, chronic inflammation does not necessarily lead to cancer. As a chronic inflammatory disease, IBD is associated with colorectal cancer, but RA and psoriasis are chronic inflammatory diseases without obvious tumor outcomes.⁴¹⁰ Another relevant evidence is that the loss of IKKβ in myeloid cells suppresses experimental colitis and colitis-related cancers.²²⁹ Another area worthy of attention is tertiary lymphoid tissues. Clinical studies have shown that there are tertiary lymphoid tissues in the tumor microenvironment, and the presence of tertiary lymphoid tissues suggests a better prognosis.^{411, 412} The formation of tertiary lymphoid tissues is achieved by LTβR-mediated noncanonical NF-κB activation pathway to induce the expression of adhesion molecules and chemokines (like CCL19, CCL21, CXCL12, and CXCL13).^{413, 414} In general, the relationship between the NF-κB signaling pathway and inflammation is profound and complex, and the precise mechanisms of NF-κB, inflammation, and cancer in the current context need to be further studied. The functions of NF-κB in the cross-talking between immune cells and tumor cells are shown in Figure 5.

4.7 The therapeutic application of NF-κB in cancer

With emerging research on NF-κB and tumors, it is generally believed that NF-κB is a valuable target for human cancer treatment. Considering the tumor-promoting effects of NF-κB, the current therapeutic strategy is inhibiting the activation of NF-κB. These inhibitors include microbial and viral proteins, microRNA, non-coding RNA, antioxidants, engineered active peptides, and various natural products to downregulate the canonical and noncanonical NF-κB pathways.⁴¹⁵ Due to the complexity of upstream stimulators and downstream target genes of NF-κB, more than 1000 inhibitors could block the NF-κB signaling pathway.⁴¹⁶ Most of these inhibitors are found to have certain anti-tumor effects using tumor cell models or animal models; their effects on human cancer treatment are unknown. Efforts have been made to develop specific and effective inhibitors with few side effects. A good example is bortezomib, a proteasome inhibitor, which has shown the inhibitory effect in MM.⁴¹⁷ As mentioned earlier, NF-κB is continuously activated in myeloma. Therefore, as a proteasome inhibitor, bortezomib was conceived to inhibit the degradation of ubiquitinated IκB, allowing IκB to bind to NF-κB dimers and stay in the cytoplasm to inhibit transcription factor activity. In addition to bortezomib, carfilzomib and ixazomib are also approved as proteasome inhibitors for MM treatment by inhibiting NF-κB.²⁹⁶ Two glucocorticoids prednisone and dexamethasone are also approved for the clinical treatment of MM. The treatment mechanism is to interfere with the phosphorylation of RNA polymerase II required for NF-κB-dependent transcription initiation.⁴¹⁸ Except for inhibiting critical components of the NF-κB pathway, blocking its downstream target genes or upstream stimuli is also a practicable therapeutic strategy. For example, denosumab, a monoclonal antibody that inhibits RANK ligand, and immunomodulatory drugs thalidomide, lenalidomide, and pomalidomide, which inhibit TNFα and IL-1β, are also approved for the treatment of MM.^{419, 420} Similarly, in some DLBCL subtypes that exhibit continuous NF-κB activation, BTK inhibitor has also been shown to have therapeutic value as a key molecule for BCR to activate the NF-κB pathway.⁴²¹ Other anti-TNFα antibodies, including infliximab, adalimumab, and golimumab, have also been found to improve the therapeutic effect in breast cancer patients.⁴²²

Considering that these drugs do not directly target the core molecules of the NF-κB pathway, but their upstream and downstream molecules, it is difficult to clarify how much the inhibitor functions on NF-κB per se. Except for MM and some DLBCL subtypes, the strategy of directly inhibiting NF-κB seems unsuccessful in other cancers. The success of the drugs mentioned above in MM and DLBCL cannot be replicated in other tumor treatments, suggesting NF-κB has universal and complicated functions that could not be a fixed therapeutic target like PD-1/PD-L. Inhibition of NF-κB may enhance the cancer treatment response to most traditional treatments and treatments developed in recent years such as immune checkpoint inhibitors; however, long-term inhibition of the NF-κB pathway perhaps causes serious side effects, especially impaired immune response and compensated anti-tumor immune function.⁴²³ Given that NF-κB regulates many physiological functions, the balance between the anti-cancer effect and the side effects that impair normal physiological functions should be considered. Moreover, under drug-mediated pressure, NF-κB might cooperate with other signaling pathways to regulate pro-survival genes (such as cyclin D1 and Bcl-2) and upregulate the transcription of the genes, which helps tumor cells to develop drug resistance. Therefore, inhibiting NF-κB alone without affecting other signaling pathways seems to be an unachievable presence.

One alternative strategy is to combine NF-κB inhibitors with other treatments (such as chemotherapy and radiotherapy). For example, pentoxifylline (an REL inhibitor) combined with PD-1 immunotherapy enhances the therapeutic effect.⁴²⁴ The combined usage of multiple NF-κB inhibitors is also one of the feasible strategies. The study has reported using multiple NF-κB inhibitors in combination and found that it improves the clinical treatment effect in MM.⁴²⁵ At present, there are many studies on NF-κB inhibitors, which will not be summarized and discussed in this review, but most of the studies are conducted on in vitro cell models and animal models, and their value of human cancer treatment warrants further investigation. Recent clinical trials of cancer treatment targeting the NF-κB signaling pathway are summarized in Table 2.