A schematic representation of the metastatic process. (reproduced, by permission of the publisher, from Fidler & Ellis, 1994).

Localization of AEG-1. A. Putative nuclear localization signals (NLS) in AEG-1. B. Subcellular localization of AEG-1 in IM-PHFA. Calreticulin and Mito-Tracker were used for specific ER and mitochondria markers, respectively. Reproduced, by permission of the publishers, from Emdad et al (2006), and Kang et al (2005), respectively.

AEG-1 and NF-κB pathway. A. AEG-1 activates the NF-κB pathway. Upper panel, HeLa cells were infected with Ad.AEG-1 (50 pfu/cell) or treated with TNF-[unk] (10 ng/ml) and 2 days later NF-κB binding activity was analyzed in the nuclear extracts of the cells by EMSA. Supershift analysis was performed with the indicated antibodies. *indicates a supershifted band by anti-p50 antibody; ** indicates a supershifted band by anti-p65 antibody. Lower panel, Ad.AEG-1 infection activates NF-κB by IκB[unk] degradation. HeLa cells were infected with Ad.AEG-1 (50 pfu/cell) or treated with TNF-[unk](10 ng/ml). The expressions of the indicated proteins were analyzed in cytoplasmic extracts by Western blot analysis 2 days later. B. AEG-1 activates NF-κB promoter activity. HeLa cells were infected with Ad.AEG-1 (50 pfu/cell) or treated with TNF-α (10 ng/ml) and 12 hr later were transfected with the indicated plasmids using Lipofectamine 2000. Luciferase assays were performed 48 hr after transfection using a Luciferase Reporter Gene Assay kit according to the manufacturer's protocol. The β-galactosidase activity was determined using the Galacto-Light Plus kit. Luciferase activity was normalized by β-galactosidase activity, and the data presented is the fold-activation relative to pGL3-Basic from triplicate determinations. C. Inhibition of NF-κB blocks AEG-1-induced increased anchorage independent growth. Upper panel, HeLa cells were either uninfected or infected with Ad.AEG-1 or Ad.IκBα–mt32 either alone or in combination at an m.o.i. of 50 pfu/cell for each Ad. Two days later the level of p65 was determined in the nuclear extracts by Western blot analysis. Lower panel, HeLa cells were infected as in the upper panel. Twenty-four hr later, cells (1 × 10⁵) were replated in 0.4% agar on 0.8% base agar. Two weeks later, colonies ≥ 50 cells were counted under a dissection microscope. Data represents mean ± SD of triplicate plates in three independent experiments. D. Inhibition of NF-κB blocks AEG-1-induced enhanced matrigel invasion. Left panel, HeLa cells were infected as in Fig. 4C. Twenty-four hr later, cells (2.5 × 10⁴) were seeded onto the upper chamber of a matrigel invasion chamber system in the absence of serum. Forty-eight hr after seeding the filters were fixed, stained and photographed. Right panel, graphical representation of the invasion assay. Data represents mean ± SD of three independent experiments. Reproduced, by permission of the publisher, from Emdad et al (2006).

Hypothetical model of signal transduction pathways involved in Ha-ras-mediated AEG-1 induction. Ha-ras activates the PI3K signaling cascade resulting in increased binding of Myc-Max to the AEG-1 promoter and augments AEG-1 expression. AEG-1 activates the NF-κB pathway that regulates expression of genes involved in migration and invasion and thus plays a crucial role in Ha-ras-mediated tumor progression.

A schematic representation of the RaSH approach. For details, please refer to Su et al (2003).

AEG-1 in tumor progression and metastasis. A. AEG-1 expression in various cancer cell lines. Northern blots of the indicated RNA samples were probed with AEG-1 and GAPDH probes. FM516-SV: SV40-immortalized normal human melanocyte; WM35: radial growth phase primary human melanoma; WM238, HO-1, FO-1, SKM p53wt, SKM p53mut, C8161, C8161 6.3, MeWo, 3S5: metastatic and variant human melanoma cells; HMEC: early passage human mammary epithelial cells; HBL-100: immortal normal human breast epithelial cell line; T47D, MCF-7, MDA-MB-157, MDA-MB-231, MDA-MB-453: human breast carcinoma cell lines; NC, normal cerebellum cell line; T98G, G18: human malignant glioma cell line. B. Effect on soft agar colony formation of FM516-SV cells following expression of AEG-1 and Ha-ras, alone or in combination. Upper panel, colony forming ability of FM516-SV cells transiently transfected with the indicated expression vectors. Data shown is average ± S.D. of three independent transfections. Middle panel, colony forming ability of FM516-SV cells stably transfected with AEG-1 and Ha-ras. Data shown is average ± S.D. of three independent transfections. Lower panel, expression of AEG-1 and Ha-ras mRNA in stable transfectants determined by Northern blot analysis. C. AEG-1 is required for Ha-ras-mediated colony formation. THV and THR cells were transfected with control or AEG-1 siRNA using the Lipofectamine 2000 transfection reagent according to the manufacturer's instructions. After 2 days, the cells were trypsinized and counted, and 150 cells were plated in 6-cm dishes. Colonies of ≥ 50 cells were scored after 10 days. The data are represented as mean ± SD and represent three independent experiments in triplicate. D. Ad.AEG-1 infection upregulates NF-κB downstream genes. The expression levels of NF-κB signaling pathway genes after Ad.vec and Ad.AEG-1 infection were analyzed in total RNA using the Human NF-κB signaling gene array (SuperArray Bioscience Corporation) according to the manufacturer's protocol. Graphical representation of expression levels of each gene obtained by densitometric analysis. Reproduced, by permission of the publishers, from Kang et al (2005), Lee et al (2006), and Emdad et al (2006), respectively.

Effect of AEG-1 expression on EAAT2 promoter activity. A. A luciferase reporter vector containing the complete EAAT2 or EAAT1 promoter was co-transfected with either pcDNA3.1 or pcDNA3.1-AEG-1-HA. Two days later, cells were harvested and luciferase activity of the protein extracts was measured. Data shown is representative of multiple experiments, which varied by ≤ 15%. Results presented ± S.D. from two independent experiments. B. A luciferase reporter vector containing the complete EAAT2 or EAAT1 promoter was co-transfected with the indicated expression vectors. Samples were obtained and analyzed as above. Data shown is representative of multiple experiments, which varied by ≤ 15%. Results presented ± S.D. from two independent experiments. Reproduced, by permission of the publisher, from Kang et al (2005).