Tumor progression and metastasis are complex processes involving intricate interplay among multiple gene products. Astrocyte Elevated Gene (AEG)-1 was cloned as an HIV-1- and tumor necrosis factor α (TNF-α)-inducible transcript in primary human fetal astrocytes by a rapid subtraction hybridization approach. AEG-1 downregulates the expression of the glutamate transporter EAAT2, thus it is implicated in glutamate-induced excitotoxic damage to neurons as evident in HIV-associated neurodegeneration. Interestingly, AEG-1 expression is elevated in subsets of breast cancer, glioblastoma multiforme and melanoma cells and AEG-1 cooperates with Ha-ras to augment the transformed phenotype of normal immortal cells. Moreover, AEG-1 is overexpressed in >95% of human malignant glioma samples when compared with normal human brain. Overexpression of AEG-1 increases and siRNA inhibition of AEG-1 decreases migration and invasion of human glioma cells, respectively. AEG-1 contains a lung-homing domain facilitating breast tumor metastasis to lungs. These findings indicate that AEG-1 might play a pivotal role in the pathogenesis, progression and metastasis of diverse cancers. Our recent observations indicate that AEG-1 exerts its effects by activating the NF-κB pathway and AEG-1 is a downstream target of Ha-ras and plays an important role in Ha-ras-mediated tumorigenesis. These provocative findings are intensifying interest in AEG-1 as a crucial regulator of tumor progression and metastasis and as a potential mediator of neurodegeneration. In this review, we discuss the cloning, structure and function(s) of AEG-1 and provide recent insights into the diverse actions and intriguing properties of this molecule.
AEG-1; Progression; Metastasis; Ha-ras oncogene; Glutamate excitotoxicity; AEG-1 promoter
Aggressive tumor growth, diffuse tissue invasion and neurodegeneration are hallmarks of malignant glioma. Although glutamate excitotoxicity is considered to play a key role in glioma-induced neurodegeneration, the mechanism(s) controlling this process is poorly understood. AEG-1 is an oncogene overexpressed in multiple types of human cancers including >90% of brain tumors. AEG-1 also promotes gliomagenesis particularly in the context of tumor growth and invasion, two primary characteristics of glioma. In the present study, we investigated the contribution of AEG-1 to glioma-induced neurodegeneration. Pearson correlation coefficient analysis in normal brain tissues and glioma patient samples indicated a strong negative correlation between expression of AEG-1 and a primary glutamate transporter of astrocytes EAAT2. Gain and loss of function studies in normal primary human fetal astrocytes and T98G glioblastoma multiforme cells revealed that AEG-1 repressed EAAT2 expression at a transcriptional level by inducing YY1 activity to inhibit CBP function as a coactivator on the EAAT2 promoter. In addition, AEG-1-mediated EAAT2 repression caused a reduction of glutamate uptake by glial cells, resulting in induction of neuronal cell death. These findings were also confirmed in glioma patient samples demonstrating that AEG-1 expression negatively correlated with NeuN expression. Taken together, our findings suggest that AEG-1 contributes to glioma-induced neurodegeneration, a hallmark of this fatal tumor, through regulation of EAAT2 expression.
AEG-1; glioma; EAAT2; glutamate; glioma-induced neurodegeneration
Astrocyte Elevated Gene-1 (AEG-1) has been proposed as a biomarker for a variety of cancers. This study aimed to investigate the expression of AEG-1 in human astrocytomas and the correlation between AEG-1 expression and clinicopathologic variables of astrocytomas. AEG-1 expression in four pairs of matched astrocytomas tissues and 5 cell lines was detected by immunohistochemical and Western blot analysis. In addition, AEG-1 protein expression was examined by immunohistochemical staining in 204 cases, including 32 normal brain tissues, 80 Low-malignant astrocytomas (LMAs) and 92 High-Malignant astrocytomas (HMAs). AEG-1 expression in 31 LMAs and 29 HMAs samples was detected by RT-PCR and Western blot analysis. We detected AEG-1 expression in normal neurons and glioma cell lines U87, U251 and M059K, but not in normal glial cells. Immunohistochemical analysis showed that 128 of 172 (74.4%) paraffin-embedded archival astrocytomas samples exhibited positive AEG-1 expression. Statistical analysis suggested that higher AEG-1 level was significantly correlated with histological grade of astrocytomas. In addition, AEG-1 mRNA and protein expression was higher in HMAs than in LMAs. AEG-1 expression had no correlation with the gender or age of astrocytoma patients. In summary, our data suggest that AEG-1 may represent a novel prognostic marker for astrocytomas.
Astrocytoma; AEG-1; prognosis
Expression of astrocyte elevated gene-1 (AEG-1) is elevated in multiple human cancers including brain tumors, neuroblastomas, melanomas, breast cancers, non-small cell lung cancers, liver cancers, prostate cancers, and esophageal cancers. This gene plays crucial roles in tumor cell growth, invasion, angiogenesis and progression to metastasis. In addition, over-expression of AEG-1 protects primary and transformed cells from apoptosis-inducing signals by activating PI3K-Akt signaling pathways. These results suggest that AEG-1 is intimately involved in tumorigenesis and may serve as a potential therapeutic target for various human cancers. However, the normal physiological functions of AEG-1 require clarification. We presently analyzed the expression pattern of AEG-1 during mouse development. AEG-1 was expressed in mid-to-hindbrain, fronto-nasal processes, limbs, and pharyngeal arches in the early developmental period from E8.5 to E9.5. In addition, at stages of E12.5-E18.5 AEG-1 was localized in the brain, and olfactory and skeletal systems suggesting a role in neurogenesis, as well as in skin, including hair follicles, and in the liver, which are organ sites in which AEG-1 has been implicated in tumor development and progression. AEG-1 co-localized with Ki-67, indicating a role in cell proliferation, as previously revealed in tumorigenesis. Taken together, these results suggest that AEG-1 may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation, and that temporal regulation of AEG-1 expression may be required during specific stages and in specific tissues during development.
AEG-1; development; mouse embryo; cell proliferation; cancer
Tumor development is initiated by an accumulation of numerous genetic and epigenetic alterations that promote tumor initiation, invasion and metastasis. Astrocyte elevated gene-1 [AEG-1; also known as Metadherin (MTDH) and Lysine-rich CEACAM1 co-isolated (LYRIC)] has emerged in recent years as a potentially crucial mediator of tumor malignancy, and a key converging point of a complex network of oncogenic signaling pathways. AEG-1/MTDH has a multifunctional role in tumor development that has been found to be involved in the following signaling cascades: i) The Ha-Ras and PI3K/Akt pathways; ii) the nuclear factor-κB signaling pathway; iii) the ERK/mitogen-activated protein kinase and Wnt/β-catenin pathways; and iv) the Aurora-A kinase signaling pathway. Studies have established that AEG-1/MTDH is crucial in tumor progression, including transformation, the evasion of apoptosis, invasion, angiogenesis and metastasis. In addition, recent clinical studies have convincingly associated AEG-1/MTDH with tumor progression and poor prognosis in a number of cancer types, including hepatocellular, esophageal squamous cell, gallbladder and renal cell carcinomas, breast, non-small cell lung, prostate, gastric and colorectal cancers, and glioma, melanoma, neuroblastoma and osteosarcoma. AEG-1/MTDH may be used as a biomarker to identify subgroups of patients who require more intensive treatments and who are likely to benefit from AEG-1/MTDH-targeted therapies. The therapeutic targeting of AEG-1/MTDH may simultaneously block metastasis, suppress tumor growth and enhance the efficacy of chemotherapeutic treatments.
astrocyte elevated gene-1; metadherin; neoplasms; metastasis; chemoresistance
Reactive astrogliosis is a ubiquitous but poorly understood hallmark of central nervous system pathologies such as trauma and neurodegenerative diseases. In vitro and in vivo studies have identified proinflammatory cytokines and chemokines as mediators of astrogliosis during injury and disease; however, the molecular mechanism remains unclear. In this study, we identify astrocyte elevated gene-1 (AEG-1), a human immunodeficiency virus 1 or tumor necrosis factor α-inducible oncogene, as a novel modulator of reactive astrogliosis. AEG-1 has engendered tremendous interest in the field of cancer research as a therapeutic target for aggressive tumors. However, little is known of its role in astrocytes and astrocyte-mediated diseases. Based on its oncogenic role in several cancers, here we investigate the AEG-1-mediated regulation of astrocyte migration and proliferation during reactive astrogliosis.
An in vivo brain injury mouse model was utilized to show AEG-1 induction following reactive astrogliosis. In vitro wound healing and cell migration assays following AEG-1 knockdown were performed to analyze the role of AEG-1 in astrocyte migration. AEG-1-mediated regulation of astrocyte proliferation was assayed by quantifying the levels of cell proliferation markers, Ki67 and proliferation cell nuclear antigen, using immunocytochemistry. Confocal microscopy was used to evaluate nucleolar localization of AEG-1 in cultured astrocytes following injury.
The in vivo mouse model for brain injury showed reactive astrocytes with increased glial fibrillary acidic protein and AEG-1 colocalization at the wound site. AEG-1 knockdown in cultured human astrocytes significantly reduced astrocyte migration into the wound site and cell proliferation. Confocal analysis showed colocalization of AEG-1 to the nucleolus of injured cultured human astrocytes.
The present findings report for the first time the novel role of AEG-1 in mediating reactive astrogliosis and in regulating astrocyte responses to injury. We also report the nucleolar localization of AEG-1 in human astrocytes in response to injury. Future studies may be directed towards elucidating the molecular mechanism of AEG-1 action in astrocytes during reactive astrogliosis.
AEG-1; Astrocyte; HIV-1; Reactive astrogliosis
Cancer is the result of the progressive acquisition of multiple malignant traits through the accumulation of genetic or epigenetic alterations. Recent studies have established a functional role of MTDH (Metadherin)/AEG-1 (Astrocyte Elevated Gene 1) in several crucial aspects of tumor progression, including transformation, evasion of apoptosis, invasion, metastasis and chemoresistance. Overexpression of MTDH/AEG-1 is frequently observed in melanoma, glioma, neuroblastoma, and carcinomas of breast, prostate, liver and esophagus and is correlated with poor clinical outcomes. MTDH/AEG-1 functions as a downstream mediator of the transforming activity of oncogenic Ha-Ras and c-Myc. Furthermore, MTDH/AEG-1 overexpression activates the PI3K/Akt, NFκB, and Wnt/β-catenin signaling pathways to stimulate proliferation, invasion, cell survival and chemoresistance. The lung-homing domain of MTDH/AEG-1 also mediates the adhesion of tumor cells to the vasculature of distant organs and promotes metastasis. These findings suggest that therapeutic targeting of MTDH/AEG-1 may simultaneously suppress tumor growth, block metastasis and enhance the efficacy of chemotherapeutic treatments.
Since its discovery, nearly one decade of research on astrocyte elevated gene 1 (AEG-1) has witnessed expanding knowledge of this molecule, ranging from its role in cancer biology to molecular mechanisms underlying the biological functions. As a multifunctional oncoprotein, AEG-1 has been shown to overexpress in multiple types of human cancer, and the elevation of AEG-1 in tumor cells leads to enhanced phenotypes characteristic of malignant aggressiveness, including increased abilities to proliferate robustly, to invade surrounding tissues, to migrate, to induce neovascularization, and to enhance chemoresistance. The multifunctional role of AEG-1 in tumor development and progression has been found to be associated with several signaling cascades, namely, 1) activation of NF-kappa B, partially through direct interaction with p65; 2) PI3K/AKT signaling triggered by AEG-1 indirectly; 3) enhancement of the transcriptional activity of beta-catenin by indirect activation of MAPK and induction of LEF1; 4) regulation of mi/siRNA-mediated gene silencing by interacting with SND1; and 5) promotion of protective autophagy; in addition to possibly unknown mechanisms. Elevated AEG-1 expression is seen in nearly all tumor types, and in most cases AEG-1 positively correlates with tumor progression and poorer patient survival. Taken together, AEG-1 might represent a potential prognostic biomarker and therapeutic target.
Astrocyte elevated gene-1 (AEG-1) is a recently discovered oncogene that has been reported to be highly expressed in various types of malignant tumors, including renal cell carcinoma. However, the precise role of AEG-1 in renal cancer cell proliferation and apoptosis has not been clarified. In this study, we transfected the renal cancer cell line Caki-1 with a plasmid expressing AEG-1 short hairpin RNA (shRNA) and obtained cell colonies with stable knockdown of AEG-1. We found that AEG-1 down-regulation inhibited cell proliferation and colony formation and arrested cell cycle progression at the sub-G1 and G0/G1 phase. Western blot analysis indicated that the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E were significantly reduced following AEG-1 down-regulation. In addition, AEG-1 knockdown led to the appearance of apoptotic bodies in renal cancer cells, and the ratio of apoptotic cells significantly increased. Expression of the anti-apoptotic factor Bcl-2 was dramatically reduced, whereas the pro-apoptotic factors Bax, caspase-3 and poly (ADP-ribose) polymerase (PARP) were significantly activated. Finally, AEG-1 knockdown in Caki-1 cells remarkably suppressed cell proliferation and enhanced cell apoptosis in response to 5-fluorouracil (5-FU) treatment, suggesting that AEG-1 inhibition sensitizes Caki-1 cells to 5-FU. Taken together, our data suggest that AEG-1 plays an important role in renal cancer formation and development and may be a potential target for future gene therapy for renal cell carcinoma.
apoptosis; astrocyte elevated gene-1; chemosensitivity; proliferation; renal cancer cells
Astrocyte elevated gene-1 (AEG-1), also known as MTDH and Lyric, is a novel gene that was first cloned by subtraction hybridization in 2002 and has recently been shown to play a role as a crucial oncogene that acts as a promoter of tumor malignancy. Overexpression and inhibition studies both in in vitro and in vivo models have partly shown the oncogenic roles of AEG-1 in a number of crucial aspects of tumor development and progression, including transformation, evasion of apoptosis, proliferation, cell survival, migration, invasion, metastasis, angiogenesis and chemoresistance through the activation of numerous signaling pathways, such as the nuclear factor κB, PI3K/AKT, Wnt/β-catenin and mitogen-activated protein kinase signaling pathways. However the potential roles of AEG-1, particularly in specific organs or tissues, such as breast tissue, require further clarification. Studies have found that in normal human breast tissue, AEG-1 is always expressed at low levels or is absent, while it is widely overexpressed in many breast cancer cell lines and breast tumors. The present review evaluates the current literature with regards to AEG-1 relative to breast cancer development and progression and highlights new perspectives relative to this molecule, indicating its potential to become a new target for the clinical treatment of breast cancer.
astrocyte elevated gene-1; metastasis; angiogenesis; prognosis; breast cancer
Astrocyte-elevated gene-1 (AEG-1/MTDH/LYRIC) is a potent oncogene that regulates key cellular processes underlying disease of the central nervous system (CNS). From its involvement in human immunodeficiency virus (HIV)-1 infection to its role in neurodegenerative disease and malignant brain tumors, AEG-1/MTDH/LYRIC facilitates cellular survival and proliferation through the control of a multitude of molecular signaling cascades. AEG-1/MTDH/LYRIC induction by HIV-1 and TNF highlights its importance in viral infection, and its incorporation into viral vesicles supports its potential role in active viral replication. Overexpression of AEG-1/MTDH/LYRIC in the brains of Huntington’s disease patients suggests its function in neurodegenerative disease, and its association with genetic polymorphisms in large genome-wide association studies of migraine patients suggests a possible role in the pathogenesis of migraine headaches. In the field of cancer, AEG-1/MTDH/LYRIC promotes angiogenesis, migration, invasion, and enhanced tumor metabolism through key oncogenic signaling cascades. In response to external stress cues and cellular mechanisms to inhibit further growth, AEG-1/MTDH/LYRIC activates pathways that bypass cell checkpoints and potentiates signals to enhance survival and tumorigenesis. As an oncogene that promotes aberrant cellular processes within the CNS, AEG-1/MTDH/LYRIC represents an important therapeutic target for the treatment of neurological disease.
AEG-1/MTDH/LYRIC; CNS; HIV; Cancer; Glioblastoma
Hepatocellular carcinoma (HCC) is a highly aggressive vascular cancer characterized by diverse etiology, activation of multiple signal transduction pathways, and various gene mutations. Here, we have determined a specific role for astrocyte elevated gene-1 (AEG1) in HCC pathogenesis. Expression of AEG1 was extremely low in human hepatocytes, but its levels were significantly increased in human HCC. Stable overexpression of AEG1 converted nontumorigenic human HCC cells into highly aggressive vascular tumors, and inhibition of AEG1 abrogated tumorigenesis by aggressive HCC cells in a xenograft model of nude mice. In human HCC, AEG1 overexpression was associated with elevated copy numbers. Microarray analysis revealed that AEG1 modulated the expression of genes associated with invasion, metastasis, chemoresistance, angiogenesis, and senescence. AEG1 also was found to activate Wnt/β-catenin signaling via ERK42/44 activation and upregulated lymphoid-enhancing factor 1/T cell factor 1 (LEF1/TCF1), the ultimate executor of the Wnt pathway, important for HCC progression. Inhibition studies further demonstrated that activation of Wnt signaling played a key role in mediating AEG1 function. AEG1 also activated the NF-κB pathway, which may play a role in the chronic inflammatory changes preceding HCC development. These data indicate that AEG1 plays a central role in regulating diverse aspects of HCC pathogenesis. Targeted inhibition of AEG1 might lead to the shutdown of key elemental characteristics of HCC and could lead to an effective therapeutic strategy for HCC.
Recent studies have linked chemotherapy resistance to the altered expression of microRNAs (miRNAs). Thus, miRNA-based approaches to modulating sensitivity to temozolomide (TMZ) may overcome chemoresistance. The aim of the present study was to investigate whether miR-136 could modulates glioma cell sensitivity to TMZ.
The proliferation of glioma U251 cell line was evaluated by MTT assay. The expression of astrocyte elevated gene-1 (AEG-1)was detected by real‑time polymerase chain reaction (RT-PCR)and Western blot. The luciferase reporter gene was used to test whether AEG-1 was the target of the miR-136.
The MTT assay showed that U251 cells with miR-136 overexpression were significantly more sensitive to the therapy of TMZ than control cells. Luciferase assays revealed that miR-136 directly targeted the 3’UTR of AEG-1. qRT–PCR and western blotting analysis found that AEG-1 expression at the mRNA and protein levels decreased in the miR-136 mimic-treatment group relative to control group. Downregulation of AEG-1 expression by siRNAs, U251 cells became more sensitive to the therapy of TMZ. In addition, the enhanced growth-inhibitory effect by the miR-136 mimics transfection was enhanced after the addition of AEG-1 siRNA.
The present study provides the first evidence that miR-136 plays a key role in TMZ resistance by targeting the AEG-1 protein in glioma cell line, suggesting that miR-136 can be used to predict a patient’s response to TMZ therapy as well as serve as a novel potential maker for glioma therapy.
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_173
Glioma, miR-136; Sensitivity; Temozolomide; AEG-1
There is virtually no effective treatment for advanced hepatocellular carcinoma (HCC) and novel targets need to be identified to develop effective treatment. We recently documented that the oncogene Astrocyte elevated gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis. Employing yeast two-hybrid assay and co-immunoprecipitation followed by mass spectrometry we identified Staphylococcal nuclease domain containing 1 (SND1), a nuclease in the RNA-induced silencing complex (RISC) facilitating RNAi-mediated gene silencing, as an AEG-1 interacting protein. Co-immunoprecipitation and co-localization studies confirmed that AEG-1 is also a component of RISC and both AEG-1 and SND1 are required for optimum RISC activity facilitating siRNA and miRNA-mediated silencing of luciferase reporter gene. In 109 human HCC samples SND1 was overexpressed in ∼74% cases compared to normal liver. Correspondingly, significantly higher RISC activity was observed in human HCC cells compared to immortal normal hepatocytes. Increased RISC activity, conferred by AEG-1 or SND1, resulted in increased degradation of tumor suppressor mRNAs that are target of oncomiRs. Inhibition of enzymatic activity of SND1 significantly inhibited proliferation of human HCC cells. As a corollary, stable overexpression of SND1 augmented and siRNA-mediated inhibition of SND1 abrogated growth of human HCC cells in vitro and in vivo thus revealing a potential role of SND1 in hepatocarcinogenesis.
We unravel a novel mechanism that overexpression of AEG-1 and SND1 leading to increased RISC activity might contribute to hepatocarcinogenesis. Targeted inhibition of SND1 enzymatic activity might be developed as an effective therapy for HCC.
AEG-1; SND1; protein-protein interaction; RNAi; hepatocarcinogenesis
Astrocyte elevated gene-1 (AEG-1) is associated with tumor genesis and progression in a variety of human cancers. This study aimed to explore the significance of AEG-1 in glioma and investigate whether it correlated with radioresistance of glioma cells. Immunohistochemical staining showed that the intensity of AEG-1, CD133 and PPP6c protein expression in glioma tissues increased significantly, mainly in the cytoplasm. The expression rate of AEG-1, CD133 and PPP6c were 85.9% (67/78), 60.3% (47/78) and 65.8% (51/78), respectively. AEG-1 expression was correlated with age (r = 0.227, P = 0.045), clinical stage (r = 0.491, P<0.001) and clinical grade (r = 0.450, P<0.001). No correlation was found between AEG-1 expression and other clinicopathologic parameters (P>0.05). The expression of AEG-1 was positively correlated with the expression of CD133 (r = 0.240, P = 0.035) and PPP6c (r = 0.250, P = 0.027). In addition, retrieved data on TCGA implied co-occurrence of genomic alterations of AEG-1 and PPP6c in glioblastoma. Our findings indicate that AEG-1 is positively correlated with CD133 and AEG-1 expression. It may play an important role in the progression of glioma and may serve as potential novel marker of chemoresistance and radioresistance.
glioma; AEG-1; immunohistochemistry; radioresistance
Malignant tumors are the leading cause of mortality worldwide. The search for new biomarkers for the early diagnosis of the onset of cancer to reduce high mortality is crucial. The potential of minimal invasive testing using serum from patients renders auto-antibodies promising biomarkers for cancer diagnosis. In this study, a 181 amino acid peptide of extracellular astrocyte elevated gene-1 (AEG-1) was expressed and purified, and the peptide was used in an ELISA assay to detect anti-AEG-1 auto-antibodies (AEG-1-Abs) in 483 serum samples from different cancer patients and 230 serum samples from normal blood donors. The results showed that AEG-1-Abs at titers ≥1:50 were detected in 238 of 483 (49%) cancer patients, and the positive antibody responses in different cancer patients were as follows: 44 of 98 (45%) in breast cancer patients, 48 of 96 (50%) in hepatic carcinoma patients, 43 of 88 (49%) in rectal cancer patients, 51 of 113 (45%) in lung cancer patients, and 52 of 88 (59%) in gastric cancer patients. These results were compared with 0 of 230 (0%) in normal individuals. Moreover, AEG-1-Abs at titers ≥1:50 were also detected in 24 of 94 (26%) cancer patients in TNM stages I and II, and the positive rates of AEG-1-Abs decreased with age. These results suggest that the AEG-1-Ab response acts as a diagnostic biomarker for cancer patients with AEG-1-positive expression, and may also prove to be a possible inducer, with substantial immunity against AEG-1 by immunization boosting with AEG-1 vaccines.
astrocyte elevated gene-1; anti-AEG-1 auto-antibody; serum; tumor biomarker; cancer
Astrocyte elevated gene-1 (AEG-1), also known as metadherin (MTDH) and lysine-rich CEACAM1 coisolated (LYRIC), was initially cloned in 2002. AEG-1/MTDH/LYRIC has emerged as an important oncogene that is overexpressed in multiple types of human cancer. Expanded research on AEG-1/MTDH/LYRIC has established a functional role of this molecule in several crucial aspects of tumor progression, including transformation, proliferation, cell survival, evasion of apoptosis, migration and invasion, metastasis, angiogenesis, and chemoresistance. The multifunctional role of AEG-1/MTDH/LYRIC in tumor development and progression is associated with a number of signaling cascades, and recent studies identified several important interacting partners of AEG-1/MTDH/LYRIC in regulating cancer promotion and other biological functions. This review evaluates the current literature on AEG-1/MTDH/LYRIC function relative to signaling changes, interacting partners, and angiogenesis and highlights new perspectives of this molecule, indicating its potential as a significant target for the clinical treatment of various cancers and other diseases.
Altered expression of astrocyte elevated gene-1 (AEG-1) is associated with tumorigenesis and progression. The present study aimed to investigate the clinical and prognostic significance of AEG-1 expression in pancreatic ductal adenocarcinoma (PDAC).
Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot analyses were employed to assess AEG-1 expression in three pancreatic cancer cell lines and normal pancreatic duct epithelial cells. qRT-PCR and immunohistochemical analyses were performed to detect AEG-1 expression in ten pairs of PDAC and normal pancreas tissues. Immunohistochemistry was then used to examine AEG-1 expression in paraffin-embedded tissues obtained from 105 patients, and its association with clinicopathological parameters including cancer classification was examined. Kaplan-Meier analysis was performed to study the survival rates of patients.
Expression of AEG-1 mRNA and protein was markedly higher in pancreatic cancer cell lines than that in the normal pancreatic duct epithelial cells. AEG-1 expression was evidently upregulated in PDAC tissues compared to that of the matched distant normal pancreas tissues. qRT-PCR data revealed that the tumor/non-tumor ratio of AEG-1 expression was >1.5-fold (up to 6.5-fold). Immunohistochemical data showed that AEG-1 protein was detected in 98.09% (103/105) of PDAC tissues; and they were found to be associated with tumor size (P = 0.025), advanced clinical stage (P = 0.004), T classification (P = 0.006), N classification (P = 0.003), and M classification (P = 0.007). Furthermore, Kaplan-Meier analysis showed that patients with high AEG-1-expressed PDAC had shorter overall survival. A multivariate Cox regression analysis revealed that clinical stage, T classification, and AEG-1 expression were the independent prognostic predictors for PDAC.
This study suggests that AEG-1 protein was highly expressed in PDAC and associated with poor prognosis of the patients.
AEG-1; Biomarker; Prognosis; Pancreatic ductal adenocarcinoma
Astrocyte elevated gene-1 (AEG-1) is a key contributor to hepatocellular carcinoma (HCC) development and progression. To enhance our understanding of the role of AEG-1 in hepatocarcinogenesis, a transgenic mouse with hepatocyte-specific expression of AEG-1 (Alb/AEG1) was developed. Treating Alb/AEG-1, but not Wild type (WT) mice, with N-nitrosodiethylamine (DEN), resulted in multinodular HCC with steatotic features and associated modulation of expression of genes regulating invasion, metastasis, angiogenesis and fatty acid synthesis. Hepatocytes isolated from Alb/AEG-1 mice displayed profound resistance to chemotherapeutics and growth factor deprivation with activation of pro-survival signaling pathways. Alb/AEG-1 hepatocytes also exhibited marked resistance towards senescence, which correlated with abrogation of activation of a DNA damage response. Conditioned media (CM) from Alb/AEG-1 hepatocytes induced marked angiogenesis with elevation in several coagulation factors. Among these factors, AEG-1 facilitated association of Factor XII (FXII) mRNA with polysomes resulting in increased translation. siRNA-mediated knockdown of FXII resulted in profound inhibition of AEG-1-induced angiogenesis.
We uncover novel aspects of AEG-1 functions, including induction of steatosis, inhibition of senescence and activation of coagulation pathway to augment aggressive hepatocarcinogenesis. The Alb/AEG-1 mouse provides an appropriate model to scrutinize the molecular mechanism of hepatocarcinogenesis and to evaluate the efficacy of novel therapeutic strategies targeting HCC.
Astrocyte elevated gene-1 (AEG-1); transgenic; hepatocellular carcinoma (HCC); senescence; angiogenesis
Hepatocellular carcinoma (HCC) is a highly virulent malignancy with no effective treatment thus requiring innovative and effective targeted therapies. The oncogene Astrocyte elevated gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis and profoundly downregulates insulin-like growth factor binding protein-7 (IGFBP7). The present study focuses on analyzing potential tumor suppressor functions of IGFBP7 in HCC and the relevance of IGFBP7 downregulation in mediating AEG-1 function.
IGFBP7 expression was detected by immunohistochemistry in HCC tissue microarray and real-time PCR and ELISA in human HCC cell lines. Dual Fluorescence in situ hybridization was performed to detect loss of heterozygosity at IGFBP7 locus. Stable IGFBP7-overexpressing clones were established in the background of AEG-1-overexpressing human HCC cells and were analyzed for in vitro proliferation and senescence and in vivo tumorigenesis and angiogenesis.
IGFBP7 expression is significantly downregulated in human HCC samples and cell lines compared to normal liver and hepatocytes, respectively, and inversely correlates with the stages and grades of HCC. Genomic deletion of IGFBP7 was identified in 26% of HCC patients. Forced overexpression of IGFBP7 in AEG-1 overexpressing HCC cells inhibited in vitro growth and induced senescence, and profoundly suppressed in vivo growth in nude mice that might be an end result of inhibition of angiogenesis by IGFBP7.
The present findings provide evidence that IGFBP7 functions as a novel putative tumor suppressor for HCC and establish the corollary that IGFBP7 downregulation can effectively modify AEG-1 function. Accordingly, targeted overexpression of IGFBP7 might be a potential novel therapy for HCC.
Insulin-like growth factor binding protein-7 (IGFBP7); Astrocyte elevated gene-1; gene deletion; senescence; angiogenesis
Astrocyte elevated gene-1 (AEG-1) was originally characterized as a HIV-1-inducible gene in primary human fetal astrocyte. Recent studies highlight a potential role of AEG-1 in promoting tumor progression and metastasis. The aim of this study was to investigate if AEG-1 serves as a potential therapeutic target of human neuroblastoma.
We employed RNA interference to reduce AEG-1 expression in human neuroblastoma cell lines and analyzed their phenotypic changes.
We found that the knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibited cell proliferation and apoptosis. The specific downregulation induced cell arrest in the G0/G1 phase of cell cycle. In the present study, we also observed a significant enhancement of chemo-sensitivity to cisplatin and doxorubicin by knockdown of AEG-1.
Our study suggests that overexpressed AEG-1 enhance the tumorogenic properties of neuroblastoma cells. The inhibition of AEG-1 expression could be a new adjuvant therapy for neuroblastoma.
Glioblastomas continue to carry poor prognoses for patients despite advances in surgical, chemotherapeutic and radiation regimens. One feature of glioblastoma associated with poor prognosis is the degree of hypoxia and expression levels of hypoxia-inducible factor-1α (HIF-1α). HIF-1α expression allows metabolic adaptation to low oxygen availability, partly through upregulation of VEGF and increased tumor angiogenesis. Here, we demonstrate an induced level of astrocyte-elevated gene-1 (AEG-1) by hypoxia in glioblastoma cells. AEG-1 has the capacity to promote anchorage-independent growth and cooperates with Ha-ras in malignant transformation. In addition, AEG-1 was recently demonstrated to serve as an oncogene and can induce angiogenesis in glioblastoma. Results from in vitro studies show that hypoxic induction of AEG-1 is dependent on HIF-1α stabilization during hypoxia and that PI3K inhibition abrogates AEG-1 induction during hypoxia through loss of HIF-1α stability. Furthermore, we show that AEG-1 is induced by glucose deprivation and that prevention of intracellular reactive oxygen species (ROS) production prevents this induction. Additionally, AEG-1 knockdown results in increased ROS production and increased glucose deprivation-induced cytotoxicity. On the other hand, AEG-1 overexpression prevents ROS production and decreases glucose deprivation-induced cytotoxicity, indicating that AEG-1 induction is necessary for cells to survive this type of cell stress. These observations link AEG-1 overexpression in glioblastoma with hypoxia and glucose deprivation and targeting these physiological pathways may lead to therapeutic advances in the treatment of glioblastoma in the future.
AEG-1; glioblastoma; hypoxia; glucose deprivation; necrosis
The survival rate of head and neck squamous cell carcinoma (HNSCC) at advanced stage is poor, despite contemporary advances in treatment modalities. Recent studies have indicated that astrocyte elevated gene-1 (AEG-1), a single transmembrane protein without any known functional domains, is overexpressed in various malignancies and is implicated in both distant metastasis and poor survival.
High expression of AEG-1 in HNSCC was positively correlated with regional lymph node metastasis and a poor 5-year survival rate. Knockdown of AEG-1 in HNSCC cell lines reduced their capacity for colony formation, migration and invasion. Furthermore, decreased tumor volume and metastatic foci were observed after knockdown of AEG-1 in subcutaneous xenografts and pulmonary metastasis assays in vivo, respectively. We also demonstrated that AEG-1 increased phosphorylation of the p65 subunit of NF-κB, and regulated the expression of MMP1 in HNSCC cells. Moreover, compromised phosphorylation of the p65 (RelA) subunit of NF-κB at serine 536 was observed upon silencing of AEG-1 in both HNSCC cell lines and clinical specimens.
High expression of AEG-1 is associated with lymph node metastasis and its potentially associated mechanism is investigated.
Astrocyte elevated gene-1 (AEG-1); Head and neck squamous cell carcinoma (HNSCC); Metastasis; Matrix metalloproteinase 1 (MMP1); p65
Astrocyte elevated gene 1 (AEG-1), an important oncogene, has been shown to be overexpressed in several types of cancers. In colorectal cancer (CRC), the protein level of AEG-1 is up-regulated in tumour tissue compared to normal mucosa, showing prognostic significance. Since little is known about the transcriptional level of AEG-1 expression and its biological pathway in CRC the aim of the present study was to examine the relationship of AEG-1 mRNA expression, the protein level and clinicopathological variables as well as its biology pathway in CRC.
Material and methods
The mRNA expression of AEG-1 was analysed by qPCR in fresh frozen patient samples including 156 primary tumours, along with the corresponding normal mucosa, and in five colon cancer cell lines, SW480, SW620, KM12C, KM12SM and KM12L4a. AEG-1 protein expression was investigated by immunohistochemistry in paraffin-embedded materials from 74 distant normal mucosa, 107 adjacent mucosa, 158 primary tumour, 35 lymph node metastasis and 9 liver metastasis samples. In addition, the AEG-1 protein expression was elucidated in the cell lines by Western blot.
The lymph node metastatic cell line SW620 had a significantly higher AEG-1 mRNA (0.27 ± 0.02) expression compared to the primary tumour cell line SW480 (0.17 ± 0.04, p = 0.026). AEG-1 expression at the mRNA level and/or the protein level was significantly up-regulated gradually from normal mucosa to primary CRC, and then to lymph node metastasis and finally to liver metastasis (p < 0.05). There were significant associations of AEG-1 mRNA expression with tumour location (p = 0.047), as well as mRNA and protein expression with the tumour stage (p < 0.03). Furthermore AEG-1 protein expression was positively related to biological variables including NF-κB, p73, Rad50 and apoptosis (p < 0.05).
AEG-1 is up-regulated, at the mRNA and the protein level, during CRC development and aggressiveness, and is related to tumour location and stage. It may play its role in CRC through the NF-κB signaling pathway.
Astrocyte elevated gene-1 (AEG-1) and endothelin-1 (ET-1)/endothelin A receptor (ETAR) signaling have been demonstrated to be important in osteosarcoma (OS) progression. In the present study, we explored the interaction between AEG-1 and ET-1/ETAR signaling in OS cells, and investigated the mechanism(s) through which the functional interaction may impact OS cell invasion and chemoresistance. Overexpression and knockdown of AEG-1 were performed in Saos-2 and MG-63 OS cells, respectively. Overexpression of AEG-1 in Saos-2 cells significantly increased ET-1 expression (at both the mRNA and protein levels), cell invasion, MMP-2 expression and cell survival against cisplatin. These effects were eradicated using a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, or a selective ETAR inhibitor, BQ123. Knockdown of AEG-1 in MG-63 cells significantly decreased ET-1 expression (at both the mRNA and protein levels), cell invasion, MMP-2 expression and cell survival against cisplatin. Exogenous ET-1 restored cell invasion and MMP-2 expression levels in MG-63 cells, in which AEG-1 had been knocked down, in the presence of LY294002, but not in the presence of BQ123. However, exogenous ET-1 only partially rescued cell survival against cisplatin-induced apoptosis in the presence of LY294002, in cells in which AEG-1 had been knocked down. In conclusion, we have demonstrated that AEG-1 regulates ET-1 expression at the transcriptional level in a PI3K-dependent manner in OS cells. Downstream of PI3K, ET-1/ETAR signaling primarily mediates the promoting effect of AEG-1 on OS cell invasion, likely through the upregulation of MMP-2 expression, thus, ET-1/ETAR signaling partially, but significantly, mediates the AEG-1-induced chemoresistance in OS cells. To the best of our knowledge, this study has provided the first evidence of a functional association between AEG-1 and ET-1/ETAR signaling in OS cells, which adds novel insights into the molecular mechanism of OS metastasis and chemoresistance.
astrocyte elevated gene-1; endothelin-1; endothelin A cell invasion; chemoresistance; osteosarcoma; phosphatidylinositol 3-kinase; receptor