Advances in IKBKE as a potential target for cancer therapy

Abstract IKBKE (inhibitor of nuclear factor kappa‐B kinase subunit epsilon), a member of the nonclassical IKK family, plays an important role in the regulation of inflammatory reactions, activation and proliferation of immune cells, and metabolic diseases. Recent studies have demonstrated that IKBKE plays a crucial regulatory role in malignant tumor development. In recent years, IKBKE, an important oncoprotein in several kinds of tumors, has been widely found to regulate a variety of cytokines and signaling pathways. IKBKE promotes the growth, proliferation, invasion, and drug resistance of various cancers. This paper makes a detailed review that focuses on the recent discoveries of IKBKE in the malignant tumors, and puts forward that IKBKE is becoming an important therapeutic target for clinical treatment, which has been more and more realized.


| INTRODUCTION
The IKK (IκB kinase) family, whose members are key activators of the NF-κB signaling pathway, plays an important role in the regulation of NF-κB-mediated inflammatory reactions, immune cell activation, and tumorigenesis. The IKK family mainly consists of five protein factors: IKKα, IKKβ, IKKγ (also known as NEMO), IKBKE, and TBK1. Recent studies demonstrated that IKBKE, as an IKK family protein factor, plays an important regulatory role not only in the activation of inflammatory factors and the progression of metabolic diseases and cellular immunity but also in the development of various malignant tumors. IKBKE (IKKε, also known as IKK-i) is a serine/threonine protein kinase belonging to the IKK family and has a molecular weight of 80 kDa. The IKBKE gene is located at 1q32 and has 22 exons. In 1999, Shimada et al 1 first isolated a novel kinase called IKK-i that was induced by LPS (lipopolysaccharide) in mouse macrophage cell lines using the SSH (suppression subtraction hybridization) technique. Furthermore, IKK-i was found to be highly expressed in normal pancreatic tissue, thyroid tissue, spleen tissue, and peripheral blood leukocytes. IKBKE expression was upregulated by stimulation with LPS or other inflammatory cytokines, such as TNF-α, IL-1, IL-6, and IFN-γ. Subsequently, Peters et al 2 discovered the new protein IKBKE induced by PMA (phorbol 12-myristate 13-acetate) and confirmed it as IKK-i by analyzing its amino acid sequence, which is distinct from those of IKKα, IKKβ, and IKKγ. Nevertheless, determination of the amino acid sequence revealed 33% and 31% homology with IKKα and IKKβ, respectively, and 67% homology with TBK1 (TANK-binding kinase 1), 3 which has a similar HLH (helix-loop-helix) structure at the C-terminal region of the protein and has an LZ (leucine zipper) domain similar to the middle region of IKBKE. Ikeda et al 4 demonstrated that the adjacent kinase activity domain of IKBKE has a ULD (ubiquitin-like domain) motif similar to IKKβ to maintain downstream kinase activity and to regulate downstream signaling. However, IKBKE lacks the NBD (NEMO-binding domain) at the C terminus of the protein, unlike IKKα and IKKβ, and thus cannot form the IKK complex with IKKγ (NEMO) to induce activation of the NF-κB signaling pathway.
The classical NF-κB signaling pathway is triggered by a kinase complex that includes IKKα and IKKβ as catalytic subunits and the scaffold protein NEMO, which phosphorylates protein factors of the NF-κB signaling pathway. Similar to IKKα and IKKβ, IKBKE and TBK1 are required to form kinase complexes with the help of scaffold proteins, including TANK (TNF receptor-associated factor family member-associated NF-κB activator), NAP1 (NAK-associated protein 1) and INTBAD (similar to NAP and TBK1 adaptor), to effectively stimulate their substrates. [5][6][7] As a TRAF-interacting protein, TANK (also known as I-TRAF) synergizes with TRAF2 8 and IKBKE/TBK1 9 to trigger the NF-κB signaling pathway. TANK could be regulated by the kinase complex, including TRAF3, and be phosphorylated by IKBKE/TBK1, thereby inducing IKBKE/TBK1-mediated Lys 63 -linked polyubiquitination and then activating downstream kinase complexes or pathways. 10 Through analysis of the amino acid sequence, NAP1 was determined to share 28% homology with TANK. Fujita et al 11 used an IP (immunoprecipitation) technique to demonstrate that NAP1 directly interacted with TBK1 and its family member IKBKE to form a protein kinase complex to effectively phosphorylate downstream factors of the NF-κB signaling pathway, thus protecting cells from apoptosis by promoting NF-κB activation. In contrast to TANK and NAP1, SINTBAD structurally binds to IKBKE/TBK1 but does not have kinase activity. Ryzhakov et al 12 also demonstrated a conserved TBK1/IKBKE-binding domain (TBD) in all three adaptor proteins, TANK, NAP1, and SINTBAD. In 2010, Koop et al 13 identified two novel splice variants of human IKBKE and designated them IKKε-sv1 and IKKε-sv2. The gene encoding IKKε-sv1 lacks exon 21, which leads to a deficiency of 25 amino acids at the C terminus. This mutation causes IKBKE to lose the ability to activate the downstream factor IRF-3, but IKBKE keeps the capacity to stimulate the NF-κB signaling pathway. The other mutation, IKKε-sv2, which lacks exon 20, results in a 13 amino acid loss at its C terminus, thus leading to a deficiency in IRF-3 stimulation and a loss of NF-κB signaling pathway activation. These findings highlight that the C-terminal region of IKBKE is required for the phosphorylation and activation of downstream molecules. 14 Previous studies have demonstrated that IKBKE/TBK1 is downstream of several factors or receptors, such as TLR3 (Toll-like receptor 3), RIG-1 (retinoic acid inducible gene 1), MDA5 (melanoma differentiation associated gene 5), and IFN-β (interferon-β). After viral infection and stimulation with double-stranded RNA (dsRNA), IKBKE/TBK1 was activated, and it then phosphorylated the C-terminal region of IRF-3 and IRF-7, thus triggering the formation of IRF-3 or IRF-7 homo-or heterodimers that could transplant into the nucleus to express targeted genes. [15][16][17] Fujii et al 18 used mass spectrometric analysis to demonstrate that Ser-386, Ser-396, and Ser-402 sites of IRF-3 were directly phosphorylated by activated IKBKE when innate immune cells were attached by virus, thereby promoting the transcription of related antiviral factors such as interferon type I. 19,20 Nevertheless, viral infection or dsRNA-induced expression of IFN-α and -β genes were intact in IKBKE-deficient MEFs (mouse embryonic fibroblast cells). 17 Meanwhile, McWhirter et al 21 showed that TBK1 played a key role in the activation and nuclear translocation of IRF-3 in MEF, considering that TBK1-deficient mice were deprived of the expression of IRF-3-dependent genes such as IFN-α, IFN-β, and IP-10. [20][21][22] This phenomenon was probably because the content of IKBKE in MEFs is far less than that of TBK1. In other words, the expression discrepancy between TBK1 and IKBKE in MEFs may be a major rationale for this phenomenon. 22 Interestingly, they also found that low IRF-3 expression in TBK1-deficient MEFs can be restored by implantation with wild-type IKBKE instead of IKBKE mutants lacking kinase activity. 22 This fact demonstrated the importance of IKBKE kinase activity in IRF-3 activation. Besides, Siednienko et al 23 demonstrated that a TLR (Toll-like receptor) named MyD88 can inhibit IKBKE-but not TBK1-induced activation of IRF-3. This study provided insight into the mechanism discrepancy between IKBKEand TBK1-mediated induction of IRF-3.
In recent years, an increasing number of studies have shown that the expression and regulation of IKBKE is not merely limited to the fields of innate immunity, inflammatory response, and metabolic diseases but that it also extends to the fields of oncogenesis, progression, transformation, and chemotherapeutic resistance of cancers.

IKBKE IN MALIGNANCIES
Recent studies have shown that IKBKE is highly expressed in a variety of malignant tumors (Table 1), and the difference in expression is related to the prognosis of patients. In most cases, high expression of IKBKE is associated with poor prognosis, but in a few cases, IKBKE may indicate a slightly better prognosis. Generally, IKBKE plays an important role in tumorigenesis, antichemotherapeutic properties, tumor metastasis and tumor microenvironment.

| BREAST CANCER
Eddy et al 24 showed that IKBKE was highly expressed in human breast cancer specimens and several breast cancer cell lines, which hinted that IKBKE might regulate the growth of breast cancer in some ways. Boehm et al 25 found that IKBKE was highly expressed in 30% of breast cancer specimens and 16.3% of breast cancer cell lines through analysis of integrative genomic approaches, thus first identifying IKBKE as a new oncogene in breast cancer. They also found that the tumor cell death rate was increased when they silenced IKBKE expression in breast cancer cell lines that harbor IKBKE amplifications, which highlighted the regulatory role of IKBKE in tumor cell proliferation. In addition, increased IKBKE expression was observed at the transcriptional and translational levels in numerous breast cancer cell lines and breast cancer specimens, but expression was not absolutely correlated with DNA copy number changes. This suggested that multiple mechanisms regulated IKBKE expression. Furthermore, Qin et al 26 showed that IKBKE knockdown in human breast cancer cells using siRNA resulted in an obvious reduction of tumor cell proliferation, migration, and invasion. In addition, the tumor cell cycle was arrested at the G 0 /G 1 phase after silencing IKBKE by flow cytometry analysis, while IKBKE knockdown did not seem to significantly affect apoptosis in breast cancer cells. Barbie et al 27 found that IKBKE was overexpressed in breast cancer cell lines, including TNBC (triplenegative breast cancer) cell lines that were negative for ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2), and that knocking down IKBKE in TNBC cell lines decreased tumor cell proliferation and colony formation. Furthermore, treatment of IKBKE-driven breast cancer cells with a potent inhibitor of TBK1/IKBKE and JAK signaling impaired proliferation and colony formation of TNBC cells, whereas inhibition of JAK alone did not, suggesting that IKBKE regulated tumor cell progression. Li et al 28 demonstrated that IKBKE interacted with ER-α36 (estrogen receptor-α), a novel variant of ER-α, and increased its expression in breast cancer cells, enhancing ER-α36-mediated mitogenic and nongenomic estrogen signaling. In addition, knocking down IKBKE also inhibited the proliferation of TNBC cells. Tang et al 29 showed that the upregulation of IKBKE suppressed taxol-induced apoptosis and led to increased resistance to taxol (paclitaxel). Moreover, the expression of IKBKE was positively correlated with T stage, lymph node metastasis, and clinical stage.

| GLIOMA
Guan et al 30 demonstrated that IKBKE was highly expressed in glioma cell lines and human primary glioma tissues at mRNA and protein levels but that IKBKE expression was not associated with the pathological grade of gliomas by ICH (immunohistochemical) analysis. They testified that the expression of IKBKE was closely associated with the apoptotic markers caspase-3 and Bcl-2. IKBKE knockdown in glioma cells decreased Bcl-2 expression and promoted cleavage and activation of caspase-3, which suggested that IKBKE induced glioma cell resistance to apoptosis. Subsequently, Li et al 31 confirmed that the overexpression of IKBKE in gliomas was positively related to the grade of glioma by ICH analysis. Silencing of IKBKE in human glioma cells using siRNA showed significant inhibition of cell growth, migration, and invasion and arrested tumor cells at the G 0 /G 1 phase. However, notable apoptosis was not observed. Furthermore, when nude mice were treated with IKBKE siRNA in vivo, the tumor growth of established subcutaneous gliomas was significantly attenuated. Lu et al 32 also demonstrated that IKBKE promoted glioma cell proliferation, migration, invasion, and EMT (epithelial-mesenchymal transition). A recent study also pointed out that IKBKE influenced glioblastoma chemosensitivity via the NF-κB pathway. 33 Collectively, these results suggest that the overexpression of IKBKE plays a critical role in the elevated proliferation and malignant invasion of glioma cells.

SIGNALING PATHWAYS ASSOCIATED WITH MALIGNANCY
IKBKE plays an important role in multiple signaling pathways, including NF-κB, Akt, STAT (signal transducer and activator of transcription), Hippo and the Wnt/β-catenin signaling pathways, and participates in the progression of tumors, inflammation-driven cancer development, tumor microenvironment regulation and so on ( Figure 1). Here, we highlight the association between IKBKE and multiple cytokines, miRNA, and signaling pathways in tumors.

ASSOCIATED PATHWAYS
Xie et al 73 demonstrated that IKBKE activated AKT by directly phosphorylating AKT at Ser473 and Thr308 and then accelerated the activation of the downstream signaling pathway and promoted the oncogenic function of IKBKE. Meanwhile, this research revealed that the activation of AKT induced by IKBKE required the involvement of PI3K (phosphatidylinositol-3 kinase). Mahajan et al 74 reported that the activation of AKT induced by IKBKE did not require the involvement of PI3K. Recently, Zubair et al 39 demonstrated that IKBKE increased c-Myc in a manner associated with enhanced signaling through an AKT/GSK3β/c-Myc phosphorylation cascade that promoted c-Myc nuclear translocation and then triggered the transcriptional activation of c-Myc to increase glucose uptake, tumorigenesis and metastasis of PDAC cells. In breast cancer cell lines, Krishnamurthy et al 75 found that AKT could regulate TNF-dependent IKBKE expression levels. When AKT2 was knocked down, decreased TNF-induced IKBKE expression was observed, whereas AKT2 overexpression increased IKBKE expression. Furthermore, they also demonstrated that IKBKE functions downstream of AKT2 to promote breast cancer cell survival. Rajurkar et al 40 showed that IKBKE promoted the reactivation of AKT postinhibition of mTOR in PDAC (pancreatic ductal adenocarcinoma) cells. Furthermore, Guo et al 76 reported that IKBKE regulated FOXO3a (forkhead box O3), the downstream AKT pathway, through direct phosphorylation of FOXO3a at serine 644 and then induced its degradation and nuclear-cytoplasmic translocation. Previous studies had shown that Akt inhibited FOXO3a by phosphorylation of Ser32, Ser253, and Ser315. 77 However, the activity of FOXO3a-A3, which had three serine residues converted to alanine residues and could not be phosphorylated by Akt, was inhibited by IKBKE. After phosphorylation by IKBKE, FOXO3a was deprived of the ability to induce tumor cell apoptosis due to its retention and degradation in the cytoplasm, thus accelerating the progression of NSCLC and breast cancer cells.

PATHWAYS
The Hippo pathway mainly regulates cell proliferation, apoptosis, differentiation, and stemness in response to changes in the intracellular and extracellular microenvironment, including cell contact, cell polarity, mechanotransduction, and G-proteincoupled receptor (GPCR) signaling. 78,79 The core Hippo pathway is composed of a series of serine/threonine kinases, such as MST (mammalian Ste2-like kinases), LATS (large tumor suppressor kinases), MOB (MOB kinase activator), and SAV (Salvador). Normally, the Hippo pathway is activated. MST directly phosphorylates and activates LATS with the help of SAV. Then, LATS directly phosphorylates and restricts the activity of two transcriptional co-activators, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ binding motif). Phosphorylated YAP1 induced by LATS, mainly at the serine 127 site, is ubiquitinated by 14-3-3 and degrades in the cytoplasm. 80 Recently, Lu et al 32 discovered that IKBKE promoted the expression of YAP1 and TEAD2 (transcriptional enhancer activation domain 2), which are two important downstream factors of the Hippo pathway in glioma cell lines, thus accelerating the EMT (epithelial-mesenchymal transition) of cancers. IKBKE could directly combine with YAP1 and TEAD2, which were detected by endogenous IP (immunoprecipitation). Zhang et al 81 reported that IKBKE increased the expression of YAP1, promoted YAP1 translocation into the nucleus and decreased the expression of p-YAP1 (Ser127), which was a degradative marker of YAP1, thereby promoting the progression of glioblastoma. Liu et al 82 demonstrated that direct interaction of IKBKE with LATS induced degradation of LATS, thereby inhibiting the activity of the Hippo pathway and facilitating glioblastoma cell line development.

| IKBKE AND STAT
Recently, Zhang et al 58 demonstrated that IKBKE played an important role in maintaining the activity of STAT3 in lymphoma cell lines, thereby accelerating lymphoma growth and malignant progression. Interestingly, after silencing IKBKE, the expression levels of IKKα, IKKβ, p65, p50, p100, p52, and p-p65 were negligibly changed, suggesting that IKBKE had no significant effect on the NF-κB pathway in lymphoma. Similarly, Barbie et al 27 confirmed that the overexpression of IKBKE was not only associated with NF-κB activation by measuring the expression level of p-p105 at Ser933 but also associated with the activation of STAT3 by analyzing the expression of p-STAT3 at Thr705. They also confirmed that the activation of NF-κB and STAT3 induced by IKBKE was associated with the expression of CCL5 (chemokine ligand 5) and promoted tumorigenicity and progression of breast cancer through these pathways. Guo et al 54 confirmed that IKBKE was a direct downstream target of STAT3 through ChIP (chromatin immunoprecipitation) and luciferase reporter assay analysis. Meanwhile, overexpression of IKBKE induced by STAT increased chemoresistance in NSCLC (non-small cell lung cancer) cells.

| IKBKE AND EGFR
Williams et al 83 identified a novel association between IKBKE and EGFR expression (P = .0011) using ICH (immunohistochemistry) analysis in breast cancer specimens, and knockdown of IKBKE using siRNA decreases the expression of EGFR. Challa et al 56 testified that both wild-type and mutant EGFR directly interacts with IKBKE, whereas only mutant EGFR, which tended to develop resistance to therapeutic EGFR inhibitors, phosphorylated IKBKE on Tyr153 and Tyr179 residues to promote proliferation and invasion of NSCLC in vitro and in vivo.

ASSOCIATED PATHWAYS
Chen et al 43 testified that IKBKE physically interacted with β-catenin by co-IP (co-immunoprecipitation) and that IKBKE phosphorylated β-catenin, probably at Ser680 and Ser681, to restrain its hyperactivation, thereby promoting colorectal cancer (CRC) cell proliferation. In addition, Göktuna et al 42 discovered that IKBKE established a proinflammatory signature in the intestine upon constitutive Wnt signaling and that genetic ablation of IKBKE in β-catenin-driven models of intestinal cancer-reduced tumor incidence and consequently extended survival. These results indicated that IKBKE influenced the β-catenin-associated pathway; however, the specific mechanism remained to be elucidated.
Since IKBKE has become an increasingly important therapeutic target for numerous malignancies, a series of micro-RNAs have been identified as vital regulators to control the expression of IKBKE, thus promoting tumor progression. Yuan et al 84

PATHWAYS
Li et al 28 demonstrated that IKBKE interacted with ERα-36, which lacks intrinsic transcriptional activity and, in contrast with ERα, mediated mainly nongenomic estrogen signaling; it also has increased expression in breast cancer cells. Then, ERα-36 promoted cell proliferation through the MAPK/ERK pathway in ER-negative breast cancers. Peant et al 53 also confirmed that IKBKE overexpression induced NF-κB-independent stimulation of IL-6 expression through the activation and nuclear translocation of transcription factor C/ EBP-β in prostate cancer cells. Meanwhile, Zhu et al 85 showed that the upregulation of IKBKE promoted KRAS-driven tumorigenesis and metastasis by regulating CCL5 and IL-6 in NSCLC cell lines. Liu et al reported that IKBKE could phosphorylate YB-1, an oncogenic gene regulator, and then increased its activity to enhance MYC gene transcription. 60 Rajurkar et al 40 demonstrated that the Gli transcription factor upregulated IKBKE expression at both the mRNA and protein levels, thereby increasing the activity of the NF-κB pathway to maintain the transformation and survival of Kras-induced pancreatic cancer cells. More recently, Rajurkar et al 41 confirmed that IKBKE was co-expressed with Gli and that IKBKE promoted the nuclear translocation of Gli as measured by immunofluorescence staining and western blotting. Li et al demonstrated that MEF2D, a member of the MEF2 family, could directly target the IKBKE promoter to control its translation to enhance tumor chemotherapeutic resistance in ovarian carcinoma. 86 Cheng et al 87 demonstrated that zinc finger protein (ZNF382), which functioned as a tumor suppressor, was methylated in multiple primary tumors, including nasopharyngeal, esophageal, colon, gastric, and breast cancers, thereby suppressing the NF-κB pathway and AP-1 signaling through downregulating IKBKE.

THERAPEUTIC TARGET
As the regulatory mechanism of IKBKE in tumors has gradually been revealed in recent years, its critical role in tumorigenesis and development of malignant tumors has been increasingly recognized. The research of small molecule inhibitors targeting IKBKE has also become a hot topic.
Using an in vitro kinase assay, Zhu et al 85 indicated that CYT387, a small molecule inhibitor, not only inhibits JAK/ STAT signaling pathway activation but also inhibits IKBKE kinase activity. CYT387 significantly reduced tumorigenesis of NSCLC through interrupting the IKBKE-induced autocrine cytokine feedback loop required for KRAS-driven lung tumorigenesis. Furthermore, Barbie et al 27 found that treatment of IKBKE-driven breast cancer cells with CYT387, a potent inhibitor of TBK1/IKBKE and JAK signaling, reduced the proliferation and migration of TNBC cells, whereas inhibition of JAK alone does not. A combination of CTY387 with a MEK inhibitor is definitely an effective therapy for abrogating the growth of patient-derived xenografts. In addition, Hu et al 88 demonstrated that CYT387 was highly effective in combination with EGFR inhibitor against NSCLC tumors, particularly with EGFR inhibitor tumors that have intrinsic resistance. Liu also showed that CYT387 reduced the viability and clonogenicity of primary AML cells and demonstrated efficacy in a murine model of AML. 60 Recently, Li et al 89 reviewed that targeting IKBKE with three TBK1/IKBKE dual inhibitors, including WO2009032861, SAR and Domainex, could powerfully inhibit cell viability and tumor development in human breast, prostate, and oral cancers both in vivo and in vitro. These inhibitors' anticancer functions were partially owing to their suppression of TBK1/IKBKE-mediated AKT phosphorylation and VEGF (vascular endothelial cell growth factor) expression. Liu et al 90 showed that MCCK1, a specific and effective IKKε inhibitor, enhanced the anticancer effect of temozolomide in glioblastoma, suggesting that IKKε was associated with chemotherapeutic resistance of glioblastoma.
In addition to these studies, emerging evidence has demonstrated that amlexanox, a small molecule regulator used to treat ulcers and asthma, can selectively inhibit IKBKE kinase activation through competing with IKBKE on the ATP-binding site. 91 Furthermore, Challa et al 56 confirmed that combining amlexanox with the MEK inhibitor AZD6244 inhibits the in vivo growth of xenografted NSCLC cells targeting activating EGFR mutations, including EGFR T790M . IKBKE may be a direct target for reversing EGFR-TKIresistance in NSCLC. In addition, Liu et al 82 certified that amlexanox, as an IKBKE inhibitor, suppresses glioblastoma cell growth and development in vivo and in vitro. More recently, Cheng et al 92 showed that amlexanox inhibits the mobility, migration, metastasis, and EMT of prostate tumors in vitro and in vivo by the IKBKE/TBK1/NF-κB pathway.

| CONCLUSIONS AND PERSPECTIVES
Recently, IKBKE was defined as a new oncogene in breast cancer and was subsequently found to be overexpressed in various kinds of tumors, including female reproductive system tumors, lung cancer (especially NSCLC), gastrointestinal tumors, male urological tumors and gliomas. Expanded research revealed that the tumorigenic functions of IKBKE were not limited to the NF-κB signaling pathway but also extended to other signaling pathways, including AKT, STAT3, Hippo, and EGFR, mainly binding a large crosstalk network with numerous cytokines. Meanwhile, an increasing number of studies have suggested that the tumorigenic effect of IKBKE is related to its role in facilitating the secretion of associated inflammatory cytokines, thereby affecting the tumor microenvironment and accelerating tumor development.
Overall, IKBKE is closely related to tumorigenesis and the development of cancers. In addition, emerging evidence demonstrated that combining a small molecule inhibitor of IKBKE with other inhibitors suppresses the growth of xenografted tumor cells. In the future treatment of malignant tumors, IKBKE may be a therapeutic target and an important candidate for clinical evaluation.