The role of AEG-1 in the development of liver cancer

Abstract

AEG-1 is an oncogene that is overexpressed in all cancers, including hepatocellular carcinoma. AEG-1 plays a seminal role in promoting cancer development and progression by augmenting proliferation, invasion, metastasis, angiogenesis and chemoresistance, all hallmarks of aggressive cancer. AEG-1 mediates its oncogenic function predominantly by interacting with various protein complexes. AEG-1 acts as a scaffold protein, activating multiple protumorigenic signal transduction pathways, such as MEK/ERK, PI3K/Akt, NF-κB and Wnt/β-catenin while regulating gene expression at transcriptional, post-transcriptional and translational levels. Our recent studies document that AEG-1 is fundamentally required for activation of inflammation. A comprehensive and convincing body of data currently points to AEG-1 as an essential component critical to the onset and progression of cancer. The present review describes the current knowledge gleaned from patient and experimental studies as well as transgenic and knockout mouse models, on the impact of AEG-1 on hepatocarcinogenesis.

Practice points.

• Overexpression of AEG-1 induces aggressive, angiogenic and metastatic hepatocellular carcinoma (HCC) while knockdown of AEG-1 profoundly inhibits HCC initiation and progression. This observation holds true for a variety of other cancers.

• AEG-1 overexpression is associated with poor prognosis in HCC patients.

• AEG-1 is a scaffold protein and the oncogenic function of AEG-1 is predominantly mediated by interactions with diverse protein complexes.

• Overexpression of AEG-1 activates numerous protumorigenic signaling pathways.

• AEG-1 contributes to broad-spectrum resistance to various chemotherapeutics.

• AEG-1 is a positive regulator of NF-κB, a pivotal regulator of inflammation. Thus, AEG-1 is a fundamentally required for activation of inflammation.

• Targeted inhibition of AEG-1 might be an effective therapeutic strategy for HCC and other cancers.

Hepatocellular carcinoma: a cancer with increasing incidence & mortality

Hepatocellular carcinoma (HCC) is an epithelial tumor resulting from transformation of hepatocytes, the primary and most abundant cell type in the liver. Representing over 80% of all liver cancers, HCC is also one of the five most common cancers and the third most common cause of cancer-related deaths worldwide [1,2]. The disease is generally a consequence of chronic liver diseases. HCC predominates in central and south-east Asia, Sub-Saharan Africa and the Amazon [2]. Over half of the global HCC cases are diagnosed in China where approximately 90% of the afflicted individuals are infected with hepatitis B virus (HBV). HCC has largely been considered a rare cancer in North America and western Europe. However, the incidence of HCC is rising in western countries largely owing to increasing rates of hepatitis C virus (HCV) infection. Alcoholic liver disease and nonalcoholic fatty liver disease are also well-documented contributors leading to hepatocarcinogenesis in western countries [1]. HCC is a highly aggressive and vascular tumor characterized by rapid growth and early vascular invasion and thus the incidence and mortality of the disease run in parallel [1,3]. Standard treatment options for patients afflicted with localized forms of the disease include surgical resection, radiofrequency ablation or liver transplantation [4–7]. Most HCC patients however, present with advanced symptomatic tumors with underlying cirrhotic changes that are not amenable to surgical resection or transplantation [4]. Multiple etiologies have been linked to HCC, and therefore no consistent genomic abnormalities have been attributed to this disease. Identification of molecules involved in the genetic and subsequent molecular dysregulation is a critical step in the development of effective therapeutics for the treatment of HCC.

AEG-1: an oncogene required for initiation & progression of HCC

First identified over a decade ago, AEG-1 was cloned by rapid subtraction hybridization method as a gene induced in primary human fetal astrocytes infected with HIV-1 or treated with TNF-α [8]. AEG-1 has since been studied extensively in cancer due to initial expression analysis revealing that AEG-1 was significantly elevated in subsets of breast carcinoma, melanoma and malignant glioma cell lines compared with normal cells [9]. Research groups around the globe have identified AEG-1 overexpression in cancers of diverse lineages as compared with matched normal tissue [10–12]. A comprehensive and convincing body of data currently points to AEG-1 as an essential component critical to the onset and progression of cancer. AEG-1, also known as metadherin and LYRIC, is now established as an oncogene that is overexpressed in all cancers [13]. AEG-1 overexpression is detected with the progression of cancer, especially in the aggressive metastatic stage, and negatively correlates with poor survival and overall adverse prognosis [13]. In vitro studies using nude mouse xenograft and metastatic models with diverse cancer cell lines and studies using transgenic and knockout models document that AEG-1 overexpression induces an aggressive, angiogenic and metastatic phenotype; whereas knockdown of AEG-1 inhibits proliferation and invasion and markedly abrogates tumor initiation, growth and metastasis [14–22]. Conversely, siRNA inhibition of AEG-1 was shown to effectively inhibit the growth of cancer cells in nude mouse xenograft and metastatic models, further implicating AEG-1 as an integral component of cancer pathogenesis [14,15].

AEG-1 plays an important role in regulating hepatocarcinogenesis. AEG-1 overexpression at both mRNA and protein levels has been identified in a high percentage (>90%) of HCC patients and a significant percentage of patients harbored genomic amplification of the AEG-1 locus in chromosome 8q22 [14]. Immunohistochemical analysis of 109 human HCC patient samples detected a progressive increase in the levels of AEG-1 that directly correlated with the stages of the disease based on the Barcelona Clinic Liver Cancer staging system [14]. These data were further substantiated by gene expression-microarray analysis of 132 human samples. The analysis compared normal liver, cirrhotic liver, low-grade dysplastic nodules, high-grade dysplastic nodules and HCV-related HCC tissue samples. AEG-1 expression was significantly increased in HCC as compared with the normal liver and cirrhotic tissue samples [14]. A statistically significant correlation was observed between AEG-1 copy number and AEG-1 expression level (r = 0.723). Among the 91 tumors with expression data, 24 had AEG-1 copy number >3 and 8 showed AEG-1 copy number of >4 indicating genomic amplification of AEG-1 gene. Microarray analysis has identified that overexpression of AEG-1 in human HCC cells results in profound modulation of expression in genes regulating chemoresistance, senescence, metastasis, angiogenesis and invasion [23]. These same cell lines also exhibit an AEG-1-mediated upregulation of several cell proliferation and prosurvival signaling cascades [24–27]. These findings suggest that a direct relationship exists between the level of AEG-1 expression and the stage of the disease. HCC with more microvascular invasion or pathologic satellites, poorer differentiation and tumor node metastasis stages II–III are prone to exhibit higher AEG-1 expression [28]. HCC patients with high AEG-1 expression documented higher recurrence and poor overall survival [28,29]. Overexpression of AEG-1 in a poorly aggressive HCC cell line HepG3, which expresses low level of AEG-1, significantly increases in vitro proliferation, invasion and anchorage-independent growth and in vivo tumorigenesis, angiogenesis and metastasis in nude mice [14]. These observations were further corroborated in a transgenic mouse with hepatocyte-specific overexpression of AEG-1 (Alb/AEG-1) that developed highly aggressive metastatic HCC in diethynitrosamine (DEN)-induced HCC model [20]. Conversely, knockdown of AEG-1 in highly aggressive QGY-7703 cells, expressing high levels of AEG-1, significantly abrogates in vivo tumorigenesis [14,30]. As a corollary AEG-1 knockout (AEG-1KO) mouse shows profound resistance to DEN/phenobarbital (PB)-induced hepatocarcinogenesis and metastasis [18].

Molecular mechanisms of the oncogenic activity of AEG-1

The AEG-1 protein containing 582 a.a. is highly basic with a transmembrane domain, multiple nuclear localization signals and has been identified as a putative scaffold protein [9]. AEG-1 is localized in the nucleus, cytoplasm, endoplasmic reticulum and cell membrane [9,14–16,18–20]. In each compartment AEG-1 interacts with diverse protein complexes, thereby modulating signal transduction pathways and gene expression, which contribute to its oncogenic function. In HCC, AEG-1 is a potent activator of multiple protumorigenic signal transduction pathways including NF-κB, PI3K/Akt, Wnt/β-catenin and MEK/ERK [14,20]. Pharmacological and genetic inhibition studies have elucidated the importance of these signaling pathways in mediating AEG-1-induced HCC [14]. In addition, AEG-1 modulates fundamental intracellular processes, such as transcription; translation and RNA interference in cancer cells and thus alters global gene and protein expression profiles [12,31–32].

• Activation of NF-κB by AEG-1

AEG-1 activates NF-κB by multiple mechanisms and this activation plays a critical role in mediating the oncogenic functions of AEG-1. Upon TNF-α treatment, AEG-1 translocates to the nucleus where it interacts with the p65 subunit of NF-κB and the CBP and functions as a bridging factor between NF-κB and basal transcriptional machinery. This interaction augments transcription of NF-κB downstream genes, leading to gains in angiogenesis, invasion and metastasis [33,34]. Although AEG-1 does not directly bind to DNA, chromatin immunoprecipitation assay has identified recruitment of AEG-1 along with NF-κB on the IL-8 promoter [11]. Another study documented that AEG-1, anchored on endoplasmic reticulum membrane, associates with upstream ubiquitinated activators of NF-κB, such as RIP1 and TRAF2, facilitating their accumulation and subsequent NF-κB activation by diverse stimuli [35]. NF-κB activation in hepatocytes and macrophages is an essential component of the inflammatory microenvironment that characterizes the onset and progression of HCC [36,37]. In a recent study we documented that germline knockout of AEG-1 in mice, markedly inhibits NF-κB activation conferring a profound resistance to DEN and DEN/PB-induced HCC, further confirming the importance of NF-κB activation in mediating AEG-1 function [18]. AEG-1 itself is induced by inflammatory cytokines such as TNF-α, LPS and IL-1β via NF-κB pathway [9,38–40]. Thus, a positive feedback loop exists between AEG-1 and NF-κB.

• AEG-1 activates PI3K/Akt pathway

The PI3K/Akt pathway is not only activated by AEG-1 it also plays an essential role in regulating AEG-1 expression thereby establishing a vicious cycle in which these oncogenic molecules augment each other [14,41]. AEG-1 activation of the PI3/Akt signaling pathway has been shown to downregulate proapoptotic factors such as Bad and p21, providing protection from serumstarvation-induced apoptosis, and induce angiogenesis by upregulation of Ang1, Tie2 and VEGF [14,17,42]. PI3K/AKT activation by AEG-1 was also shown to increase levels of MDR1 in human HCC cells leading to chemoresistance [12]. Recently it was documented that AEG-1 and Akt2 interact to create a unique protein–protein signaling complex critical to survival, proliferation and invasion in malignant glioma and this observation might also be relevant in liver cancer [43].

• Activation of Wnt/β-catenin pathway

Activation of the Wnt/β-catenin pathway is also important in the initiation and progression of HCC [24,26]. Microarray analysis identified that overexpression of AEG-1 results in marked upregulation of LEF-1, the integral transcription factor mediating the effects of Wnt signaling [14]. The transcriptional activity of LEF-1 requires its heterodimerization with β-catenin. It was documented that AEG-1 overexpression results in translocation of β-catenin from the cell membrane to the nucleus [14]. β-catenin is phosphorylated by GSK3β and undergoes proteasomal degradation, while phosphorylation of GSK3β inactivates and allows nuclear translocation of β-catenin. It was also documented that AEG-1-induced phosphorylation of ERK42/44 leads to GSK3β phosphorylation and inactivation facilitating β-catenin accumulation and nuclear translocation. These findings indicate that AEG-1 activates the Wnt signaling pathway by directly inducing LEF-1 levels and indirectly by activating ERK42/44 thus facilitating nuclear translocation of β-catenin. Inhibition of LEF-1 resulted in significant inhibition of AEG-1-induced invasion. The molecular mechanisms by which AEG-1 upregulates LEF-1 and induces ERK42/44 phosphorylation remain to be determined.

• AEG-1 interacts with SND1

Multiple approaches identified SND1 as an interacting partner of AEG-1 in a variety of cancers [14,22,44–45]. SND1, a multifunctional protein, serves as a nuclease in the RISC facilitating RNAi-mediated mRNA degradation [46]. Both AEG-1 and SND1 are overexpressed in HCC and facilitate increased RISC activity [32]. As a consequence, there is increased oncomiR-mediated degradation of tumor-suppressor mRNAs such as PTEN, CDKN1C, CDKN1A, p21 and TGFBR2 which might contribute to HCC [32]. It was documented that AEG-1 facilitates stabilization of SND1 in breast epithelial cells upon oncogenic stress leading to expansion and activation of tumor initiating cells further establishing the importance of AEG-1/SND1 interaction in oncogenesis [22].

• AEG-1 interacts with & inactivates RXR

Yeast two-hybrid assay identified RXR as a novel AEG-1-interacting partner [19]. RXR, a ligand-dependent transcription factor, heterodimerizes with a variety of nuclear receptors, and mediates the function of hormones, vitamins and lipids functioning as a key regulator of cell growth, differentiation, metabolism and development [47,48]. Overexpression of AEG-1 in tumor cells results in significant cytoplasmic accumulation of AEG-1 [20]. Cytoplasmic AEG-1 entraps RXR precluding its nuclear translocation and inhibiting transcriptional activation of target genes [19]. In addition, ERK, activated by AEG-1 in many tumor cells, phosphorylates RXR [19]. Phosphorylated RXR acts dominant negatively on normal (unphosphorylated) RXR abrogating heterodimerization and coactivator recruitment leading to functional inactivation and attenuation of ligand-dependent transactivation [49,50]. AEG-1 harbors an ‘LXXLL’ motif which is used by co-activators to interact with transcription factors [51]. In the nucleus, interaction of AEG-1 with RXR prevents co-activator recruitment thereby further inhibiting RXR-dependent transcription [19]. These scenarios favor unrestrained cancer cell proliferation and might be another mechanism by which AEG-1 promotes HCC. By heterodimerizing with RAR, RXR mediates the function of retinoic acid (RA), an anticancer agent used clinically is a first-line therapy for acute promyelocytic leukemia [52,53]. However, evaluation of retinoids as candidates for chemoprevention has been abandoned because retinoid signaling is often lost or compromised during hepatocarcinogenesis [50]. We hypothesized that AEG-1 overexpression might contribute to resistance to RA in clinical settings. Indeed, AEG-1 overexpression profoundly inhibited RA-mediated killing and a combination of AEG-1 inhibition and all-trans retinoic acid synergistically inhibited human HCC xenografts in nude mice. Thus, the AEG-1/RXR interaction might be exploited to develop novel combinatorial therapeutic regimen.

Hallmarks of cancer modulated by AEG-1: relevant for HCC

• Regulation of angiogenesis by AEG-1

Angiogenesis is a fundamental event in the development and maintenance of solid tumors and their metastases [54–56]. We first documented the dominant positive role AEG-1 plays in regulating oncogenic angiogenesis [17]. Subcutaneous injection of AEG-1 overexpressing normal immortal cloned rat embryo fibroblasts in nude mice resulted in the formation of aggressive highly vascularized tumors with increased expression of molecular markers of angiogenesis, including angiopoietin-1, matrix metalloprotease-2 and hypoxia inducible factor 1-α [17]. Human umbilical vein endothelial cells (HUVECs) differentiation assays revealed that overexpression of AEG-1 significantly increased tube formation by HUVEC through PI3K/Akt signaling [17]. As previously mentioned, overexpression of AEG-1 in nontumorigenic HCC HepG3 cells also results in highly aggressive, angiogenic and metastatic tumors in subcutaneous xenografts models [14]. Conditioned media from Alb/AEG-1 hepatocytes but not WT hepatocytes induced a significant angiogenic response, as determined by HUVEC differentiation and chicken chorioallantoic membrane assays [20]. Interestingly, mass spectrometric analysis of conditioned media collected from Alb/AEG-1 hepatocytes revealed upregulation of several key components of the coagulation pathway, including fibrinogen α and β chains, plasminogen, factor XII (FXII) and prothrombin, all of which play significant roles in cancer angiogenesis, metastasis and invasion. Interestingly, FXII, known to demonstrate angiogenic activity independent of its function in coagulation, was upregulated a striking 56-fold in Alb/AEG-1 hepatocytes over WT hepatocytes [57]. AEG-1-induced upregulation of FXII protein, while modestly affecting the mRNA levels by facilitating the association of FXII messenger RNA with polysomes, resulting in increased translation. Short interfering RNA–mediated knockdown of FXII resulted in profound inhibition of AEG-1-induced angiogenesis, suggesting that FXII may play a pivotal role in mediating AEG-1 induced angiogenesis.

• AEG-1: a key promoter of metastasis

AEG-1 has been shown to facilitate invasion and metastasis [14,16–17,34,58]. It was initially identified as a membrane protein promoting metastasis of breast cancer cells and hence given the name metadherin [16]. The importance of AEG-1 in metastasis is highlighted by its inclusion in MammaPrint, the only US FDA-approved individualized metastasis risk assessment assay for breast cancer that includes a unique 70-gene signature [59]. As mentioned previously, overexpression of AEG-1 is detected with the progression of cancer, especially in the aggressive metastatic stage, and negatively correlates with poor survival and overall adverse prognosis [13]. Alb/AEG-1 mice develop highly aggressive metastatic HCC with significantly accelerated kinetics upon treatment with DEN when compared with their WT counterparts [20]. In vitro studies have shown increased anchorage-independent growth, migration and invasion properties of HeLa, human glioma, melanoma, neuroblastoma, breast cancer as well as HCC cells when AEG-1 is overexpressed [14–17,60–63]. Epithelial–mesenchymal transition (EMT) is the process by which a cell changes from a cobblestone shape with tight cell to cell contact into a spindle-shaped fibroblast-like cell, losing cell to cell contact and cell polarity [64]. Highly metastatic HCC cells overexpressing AEG-1 also expressed high levels of N-cadherin and snail, and low levels of E-cadherin and β-catenin, which are markers indicating EMT [28]. Knockdown of AEG-1 in these cells resulted in downregulation of N-cadherin and snail and upregulation of E-cadherin, indicating that AEG-1 plays an important role in promoting the EMT process. Induction of EMT has also been documented in Alb/AEG-1 hepatocytes [20]. Another factor contributing to development of tumor metastasis is anoikis resistance, the process that allows circulating tumor cell survival in the blood stream [65]. Recent reports suggested that elevated endogenous AEG-1 levels contribute to anoikis resistance in HCC cells through the activation of the PI3K/Akt signaling pathway and contribute to orientation chemotaxis in HCC cells via the CXCR4/CXCL12 axis [66].

• AEG-1 positively regulates chemoresistance

Chemoresistance is a major effect attributed to AEG-1 expression. AEG-1 contributes to broad-spectrum resistance to various chemotherapeutics including 5-fluorouracil (5-FU), doxorubicin, paclitaxel, cisplatin and 4-hydroxycyclophosphamide (4-HC) [12,14,30,61,67]. Doxorubicin is the most common chemotherapeutic used in the treatment of HCC and development of resistance to doxorubicin is very common [68,69]. The most common mechanism of doxorubicin resistance is the enhanced efflux of drug by cancer cells [70]. By activating the PI3K/Akt pathway, AEG-1 has been shown to facilitate association of MDR1 mRNA to polysomes. This association increases the expression of MDR1 protein in HCC cells resulting in increased efflux and decreased accumulation of doxorubicin, culminating in doxorubicin resistance [12]. In nude mice xenograft studies, a lentivirus expressing AEG-1 short hairpin RNA (lenti.AEG-1sh), in combination with doxorubicin dramatically reduced growth of aggressive human HCC cells compared with either agent alone [12]. AEG-1 also confers resistance to 5-FU, another chemotherapeutic for HCC. Gene expression profiles of AEG-1-overexpressed human HCC cells identified several upregulated genes implicated in chemoresistance, including drug-metabolizing enzymes, such as DPYD and the transcription factor LSF/TFCP2 [14]. AEG-1 increases LSF, which in turn induces thymidylate synthase, the substrate for 5-FU. The dual effects of increase in 5-FU substrate and increase in the 5-FU-catabolizing enzyme dihydropyrimidine dehydrogenase by AEG-1 results in profound resistance to 5-FU therapy, which might be overcome by lenti.AEG-1sh [30]. The high expression of AEG-1 in HCC and its ability to confer chemoresistance would likely explain the almost inherent ability of HCC to resist chemotherapy. In light of these findings, localized inhibition of AEG-1 might be an effective means of sensitizing HCC patients to chemotherapeutics.

• Inflammation, a key event in the initiation of HCC, is promoted by AEG-1

Although numerous etiologies are associated with the onset and progression of HCC. It is clear that the majority of documented risk factors result in long-term liver inflammation or cirrhotic liver damage. Most cases of HCC arise in a setting of chronic inflammation, such as infection with HBV or HCV, alcoholic liver disease and nonalcoholic fatty liver disease [2]. The chronic inflammation associated with HCC is characterized by the continued expression of cytokines and recruitment of immune cells to the liver. Activated inflammatory cells release free radicals such as reactive oxygen species and nitric oxide reactive species, which can result in DNA damage, lead to gene mutations and ultimately neoplastic transformation [71–73]. NF-κB is a master regulator of inflammation [74]. Crosstalk between tumor cells and their surrounding microenvironment is required for HCC development and studies show that NF-κB activation in hepatocytes and macrophages is necessary in inflammation-induced HCC [36,37]. NF-κB-regulated expression of the STAT3-activating cytokine IL-6 plays a pivotal role in HCC [37]. We recently documented that while WT mice (16 months old) show signs of aging-associated inflammation, no such changes are observed in their AEG-1KO counterparts (littermates) [18]. Infiltration of macrophages observed in aged WT liver and spleen is not seen in AEG-1KO mice. Moreover, AEG-1 knockout mice exhibit complete protection from aging-associated, spontaneous tumor formation, when compared with WT mice. Additionally, AEG-1KO mice are profoundly resistant to DEN/PB-induced HCC. While WT mice showed activation of NF-κB, induction of IL-6 and activation of STAT3 in the liver upon DEN injection no such change was observed in AEG-1KO livers. In vitro studies showed that NF-κB activation was markedly abrogated in AEG-1KO hepatocytes and macrophages versus WT. Furthermore, analysis of global gene expression changes revealed inhibition of both myeloid cell migrations and granulocyte activation in AEG-1KO mice [18]. These findings indicate that AEG-1 is essential for NF-κB activation and inhibition of this activation in macrophages and hepatocytes confers protection to AEG-1KO mice from hepatocarcinogenesis. The observation that AEG-1 is required for NF-κB activation in macrophages has profound implications in cancer as well a host of additional physiological and pathological states.

AEG-1 co-operates with c-Myc to promote HCC: novel insights into AEG-1 function

c-Myc overexpression is a very common event in HCC, and studies utilizing c-Myc overexpression mouse models indicate that c-Myc is sufficient to induce HCC and is required to maintain the neoplastic state [75–80]. AEG-1 is transcriptionally regulated by c-Myc and induces c-Myc by a variety of mechanisms [42,81]. It was recently demonstrated that hepatocyte-specific AEG-1 and c-Myc transgenic mice (Alb/AEG-1/c-Myc) develop highly aggressive, metastatic HCC, both spontaneously and following DEN-exposure, when compared with transgenic mice expressing either oncogene alone [82]. Alb/AEG-1/c-Myc exhibited robust and sustained activation of numerous prosurvival signaling pathways as well as many positive modulators of EMT. RNA sequencing analysis revealed that Alb/AEG-1/c-Myc mice mimic human HCC and show a distinct gene signature demonstrating upregulation of cell cycle, cell division and metabolic processes. Robust upregulation of several noncoding RNAs, including Rian, Mirg and Meg3, was observed only in the double transgenic mice, indicating that noncoding RNAs, induced by AEG-1 and c-Myc, provide both proliferative and invasive advantages. Genomic amplification of AEG-1 and c-Myc is detected in HCC and other cancers [14,15]. Thus, Alb/AEG-1/c-Myc mice will be valuable models in which to analyze the consequences of oncogene activation and tumor suppressor mutations during hepatocarcinogenesis and to evaluate the efficacy of novel therapeutic modalities targeted to HCC.

Conclusion & future perspective

The role of AEG-1 as an oncogene is well established [13–16,30,61]. AEG-1 has an essential role in the regulation of tumor cell proliferation, invasion, metastasis and inflammation (Figure 1) [14,16,18,20,61], and positively contributes to tumor-associated angiogenesis, chemoresistance and protection from apoptosis [12–13,17,30,41]. Experimental evidence gathered from both in vivo and in vitro methods suggests that AEG-1 affects these processes by modulating diverse intracellular processes predominantly by functioning as a scaffold protein in protein–protein interactions. Subcellular localization of AEG-1 is a major determinant of AEG-1 interactions and subsequently its activity. For instance, AEG-1 interacts with the p65 subunit of the NF-κB in the nucleus while it interacts with SND1 in the cytoplasm [11,33]. Monoubiquitination of AEG-1 is one mechanism shown to induce cytoplasmic localization [20,81], however the specific mechanisms driving subcellular localization of AEG-1 require additional investigation. The discovery that AEG-1 plays a fundamental role in regulating NF-κB activation and hence inflammation has opened new avenues for AEG-1 studies. Investigating the role of AEG-1 in other inflammatory cancers as well as inflammatory diseases will provide important insights into the molecular pathogenesis of these disease processes and facilitate development of AEG-1 inhibitory strategies as potential therapy. NF-κB plays a pivotal role in regulating immune cell function and as such, analysis of AEG-1 function in immune diseases is another avenue of study. Transgenic and AEG-1KO mice will significantly aid in these studies. Since AEG-1 is a scaffold protein, small molecule inhibitors of AEG-1 might be difficult to develop. Strategies to block the interaction of AEG-1 with specific proteins may however garner success employed in combination with other regimens for therapeutic purposes. Additionally nanoparticle-mediated targeted delivery of AEG-1 might be a strategy worth exploring, particularly in the context of HCC since intravenous delivery of payload will first go to the liver. There are multitudes of AEG-1 studies, both mechanistic and translational, that are ongoing and impending that will provide a better understanding of the molecular mechanism of HCC allowing us to better treat this fatal disease.

Figure 1. . The mechanism by which AEG-1 augments the onset and progression of hepatocellular carcinoma.

EMT: Epithelial–mesenchymal transition; HCC: Hepatocellular carcinoma.