Astrocyte elevated gene-1 (AEG-1) functions as an oncogene and regulates angiogenesis

Abstract

Astrocyte-elevated gene-1 (AEG-1) expression is increased in multiple cancers and plays a central role in Ha-ras-mediated oncogenesis through the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Additionally, overexpression of AEG-1 protects primary and transformed human and rat cells from serum starvation-induced apoptosis through activation of PI3K/Akt signaling. These findings suggest, but do not prove, that AEG-1 may function as an oncogene. We now provide definitive evidence that AEG-1 is indeed a transforming oncogene and show that stable expression of AEG-1 in normal immortal cloned rat embryo fibroblast (CREF) cells induces morphological transformation and enhances invasion and anchorage-independent growth in soft agar, two fundamental biological events associated with cellular transformation. Additionally, AEG-1-expressing CREF clones form aggressive tumors in nude mice. Immunohistochemistry analysis of tumor sections demonstrates that AEG-1-expressing tumors have increased microvessel density throughout the entire tumor sections. Overexpression of AEG-1 increases expression of molecular markers of angiogenesis, including angiopoietin-1, matrix metalloprotease-2, and hypoxia-inducible factor 1-alpha. In vitro angiogenesis studies further demonstrate that AEG-1 promotes tube formation in Matrigel and increases invasion of human umbilical vein endothelial cells via the PI3K/Akt signaling pathway. Tube formation induced by AEG-1 correlates with increased expression of angiogenesis markers, including Tie2 and hypoxia-inducible factor-alpha, and blocking AEG-1-induced Tie2 with Tie2 siRNA significantly inhibits AEG-1-induced tube formation in Matrigel. Overall, our findings demonstrate that aberrant AEG-1 expression plays a dominant positive role in regulating oncogenic transformation and angiogenesis. These findings suggest that AEG-1 may provide a viable target for directly suppressing the cancer phenotype.

Fig. 1.

Effects of stable overexpression of AEG-1 in CREF cells on colony formation in soft agar and cell invasion. (A) CREF cells were stably transfected with either the empty pcDNA3.1 vector or the AEG-1 expression vector, respectively. CREF-AEG-1 clones were selected for expression of AEG-1. (Left) Expression of AEG-1 protein by stably transfected CREF-AEG-1 cells is shown by Western blot analysis. EF1α was used as an internal control to ascertain equal loading. (Right) Expression of AEG-1 protein in stably transfected CREF-AEG-1 cells (clone 2 and 30) and normal immortal CREF and a series of normal human cells (PHFA, P69, FM516-SV) and corresponding human tumor cells (U87MG, H4, DU-145, PC-3, HO-1, C8161) by Western blot analysis. EF1α was used as an internal control for protein loading. (B) A total of 1 × 10⁵ cells were seeded in 0.4% agar on 0.8% base agar. Two weeks later, colonies >0.1-mm were counted under a dissection microscope. *, P < 0.05 vs. CREF. (C) Cells (5 × 10⁴) were seeded onto the upper chamber of a Matrigel invasion chamber system in the absence of serum. Twenty-four hours after seeding, the filters were fixed, stained, and photographed. (D) Quantitation of the invasion assay. The data expressed in the graph is the mean ± SE of three independent experiments. *, P < 0.05 vs. CREF.

Fig. 2.

Overexpression of AEG-1 in CREF cells induces tumorigenesis in vivo. (A) 2 × 10⁶ cells of each cell line were subcutaneously injected into the flank of each nude mouse. Tumor volumes were measured at the indicated time points. Results are expressed as means ± SE (n = 5 per group). CREF did not form tumors in nude mice. The average tumor volume in cubic millimeters of five animals ± SD. Unpaired two-tailed Student's t test (P < 0.01). (B) Photograph showing tumor growth in nude mice. (C) Tumor weights were measured after sacrifice of the mice 4 weeks after injection. The data represent mean ± SE in each group (*, P < 0.01 vs. CREF).

Fig. 3.

Histochemical analysis of tumors derived from nude mice injected with AEG-1 stable overexpressing CREF clone 2 and 30. (A) Representative examples of tumors derived from CREF-AEG-1 clone 2 and 30. (B) The sections of CREF-AEG-1 clone 2 and 30 xenografts and human glioma tissue were immunostained for the endothelial cell marker CD31. Immunohistochemical analysis demonstrated robustly increased blood vessel density in CREF-AEG-1 tumor sections (inserts, high magnification view). (C) Serial sections of formalin-fixed, paraffin-embedded tumors were stained with a rabbit polyclonal AEG-1, a rabbit polyclonal MMP-2, a rabbit polyclonal Ang1, and a mouse monoclonal HIF1-α antibodies. Signals were developed with DAB chromogen (brown, AEG-1 and MMP-2) or Vector VIP (purple, Ang1 and HIF1-α) and counterstained with hematoxylin.

Fig. 4.

AEG-1 promotes angiogenesis. (A) AEG-1 induces tube formation on Matrigel through the PI3K/Akt signaling pathway. HUVECs were infected with Ad.vec or Ad.AEG-1 (25 PFU/cell) in combination with Ad.DN.Akt (25 PFU/cell). One day after infection, cells (5 ×10⁴), which were labeled with a fluorescent dye, calcein AM, were seeded onto Matrigel and tube formation was assayed after 16 h by fluorescence microscopy. Upper, cells that were infected with Ad.AEG-1 clearly showed an increase in tube formation on Matrigel (arrows), whereas the Ad.vec-treated cells were a poor inducer of tube-like structures (arrowheads). (Lower) Graphical presentation of tube formation assay, data expressed in the graph is the mean ± SE of three independent experiments. *, P < 0.05 vs. Ad.vec-infected cells; #, P < 0.05 vs. Ad.AEG-1-infected cells. (B) HUVECs were treated with either control siRNA or AEG-1 siRNA, plated on Matrigel and stimulated with VEGF (10 ng/mL). Tube formation was assayed after 16 h by fluorescence microscopy. *, P < 0.05 vs. control siRNA treated cells. (C) Inhibition of AEG-1 by siRNA inhibits angiogenesis in in vivo CAM assays. CAMs of 9-day-old chicken embryos were injected with either control siRNA or AEG-1 siRNA-transfected H4 glioma cells. Angiogenesis in yolk sac was monitored 1 week after inoculation, and representative fields were photographed. (D) HUVECs were infected with Ad.vec or Ad.AEG-1 (25 PFU/cell) in combination with Ad. DN.Akt (25 PFU/cell). One day after infection, cells (5 × 10⁴) were seeded onto the upper chamber of a Matrigel invasion chamber system in the absence of serum. Twenty-four hours after seeding, the filters were fixed, stained, and photographed. (Right panel) Graphical representation of the invasion assay. The data expressed in the graph is the mean ± SE of three independent experiments. *, P < 0.05 vs. Ad.vec-infected cells, #P < 0.05 vs. Ad.AEG-1-infected cells.

Fig. 5.

AEG-1 enhances expression of angiogenesis-associated genes and promotes VEGF promoter activity. (A) HUVECs, U87 and H4 glioma cells were infected with the indicated virus as in Fig. 4. Forty-eight hours after infection, total cellular extracts were prepared in RIPA buffer. Equal amounts of proteins were separated on 8–12% SDS-PAGE, transferred onto nitrocellulose membrane and probed with a chicken polyclonal AEG-1, a rabbit polyclonal Tie2 and Ang1, and a mouse monoclonal HIF1-α antibodies. EF1α was used as a control to confirm equal protein loading. (B) HUVECs were transfected with either control siRNA or Tie2 siRNA and then infected with Ad.vec or Ad.AEG-1 (25 PFU/cell). One day after infection, cells (5 × 10⁴) were seeded onto Matrigel, and tube formation was assayed after 16 h. The data expressed in the graph is the mean ± SE of three independent experiments. *, P < 0.05 vs. Ad.vec-infected cells; #, P < 0.05 vs. Ad.AEG-1-infected cells. (C) H4 cells were infected with the indicated virus as in Fig. 4. The next day cells were transfected with pGL3/VEGF promoter and pSV-β-gal vectors, and 48 h later, luciferase and β-gal activity were determined as described in Materials and Methods. The data expressed in the graph is the mean ± SE of three independent experiments. *, P < 0.05 vs. Ad.vec-infected cells; #, P < 0.05 vs. Ad.AEG-1-infected cells. (D) One day after infection with the indicated virus, H4 cells were transfected with pGL3/VEGF promoter and pSV-β-gal and either control or Tie2 siRNA. The data in the graph is the mean ± SE of three independent experiments. *, P < 0.05 vs. Ad.vec-infected cells; #, P < 0.05 vs. Ad.AEG-1-infected cells.

Fig. 6.

A hypothetical model of the signal transduction pathways involved in AEG-1-mediated oncogenic transformation and angiogenesis. PI3K is activated by AEG-1, and AEG-1 induces VEGF and Tie-2/Ang. PI3K is also activated by growth factors and angiogenesis inducers such as VEGF and angiopoietins. Akt is an essential downstream target of PI3K for mediating angiogenic signals. Akt activation increases HIF1 expression, which in turn increases VEGF transcriptional expression. Additionally AEG-1 activates NF-κB, which is involved in increased invasion and migration and thus indirectly facilitates tumor angiogenesis. VEGF and VEGFR can form an autocrine loop to regulate tumor angiogenesis.