Astrocyte elevated gene-1 regulates hepatocellular carcinoma development and progression

Abstract

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/beta-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-kappaB 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.

Figure 1. AEG1 is overexpressed in HCC cells and HCC tumor samples, and AEG1 inhibition blocks HCC tumorigenesis.

(A) Expression of AEG1 was analyzed in the indicated cell lines. Hepatocytes represent primary rat hepatocytes. Expression of β-tubulin was used as loading control. (B–I) Analysis of AEG1 expression in tissue microarray: (B) normal human liver; (C) stage IV, poorly differentiated; (D) stage I, well differentiated; (E) stage I, poorly differentiated; (F) stage II, well differentiated; (G) stage II, poorly differentiated; (H) stage III, well differentiated; and (I) stage III, poorly differentiated. Original magnification, ×400. (J) Analysis of AEG1 expression in HCC samples by gene expression microarray (Human Affymetrix 133 plus 2.0). Fold changes in gene expression in different stages of human HCC. LGDN, low-grade dysplastic nodule; HGDN, high-grade dysplastic nodule. Asterisk indicates significant difference. (K) Analysis of correlation between AEG1 copy number and AEG1 expression. (L) Inhibition of growth of QGY-7703 xenografts in athymic nude mice by AEG1 siRNA. Treatment protocol is described in Methods. Data represent mean ± SEM with 15 animals in each group.

Figure 2. Characterization of HepG3 cells stably overexpressing AEG1.

(A) The expression of the indicated proteins was analyzed by Western blot in Hep-pc-4 (pc-4) and 5 clones selected for AEG1 overexpression. (B) Localization of AEG1 protein in Hep-pc-4 and Hep-AEG1-14 (AEG1-14) clones. Immunofluorescence studies were performed as described in Methods. Hep-AEG1-8, AEG1-8. (C) Cell viability studies performed in the cell lines at the indicated time points by standard MTT assay. (D) Anchorage-independent growth in soft agar using the indicated clones. Colonies were scored after 2 weeks. (E) Matrigel invasion assay using the indicated clones. All experiments were performed at least 3 times. Left panel represents graphical representation of results. Data represent mean ± SEM. Right panel shows photomicrography of invading cells. Original magnification, ×400 (B); ×100 (E).

Figure 4. AEG1 activates multiple signal transduction pathways.

(A) Expression of the indicated proteins was analyzed by Western blot in Hep-pc-4 and Hep-AEG14 (AEG1-14) clones. (B) Analysis of NF-κB–luciferase activity in Hep-pc-4 and Hep-AEG1-14 clones. Cells were treated with TNF-α for 12 hours at a dose of 10 ng/ml. (C) Analysis of viability of Hep-pc-4 and Hep-AEG14 clones upon treatment with PD98059 and SB203580 by standard MTT assay. Data represent mean ± SEM. (D) Matrigel invasion assay using the indicated clones upon treatment with PD98059 and SB203580. (E) Anchorage-independent growth in soft agar using the indicated clones upon treatment with PD98059 and SB203580. Colonies were scored after 2 weeks. All experiments were performed at least 3 times. Data represent mean ± SEM.

Figure 3. Analysis of the tumors generated by Hep-AEG1-14 cells in athymic nude mice.

Measurement of tumor volume (A) and tumor weight (B) at the end of the study at 3 weeks. Data represent mean ± SEM. (C) Hep-AEG1-14–induced tumor showing high vascularity. (D) H&E-stained section of Hep-AEG1-14–induced tumor showing nodular pattern. Arrows indicate the margins of the nodule. (E) Immunofluorescence analysis of AEG1 and CD31 in section of Hep-AEG1-14–induced tumor. Original magnification, ×100 (D); ×400 (E). (F) Photomicrograph of the lungs of nude mice after tail vein metastasis assay. Notice the knobby appearance of the lungs in Hep-AEG1-14 clones. (G) Human angiogenesis array. Molecules shown in red are upregulated in Hep-AEG1-14 clones compared with Hep-pc-4. Pos, positive control; Neg, negative control. Each item in the grid is represented in duplicate in the arrays.

Figure 5. LEF1 plays a role in mediating AEG1 function.

(A) Analysis of LEF1 mRNA expression by TaqMan Real-Time PCR. (B) Analysis of LEF1 and Myc proteins by Western blot analysis in Hep-pc-4, Hep-AEG14 (AEG1-14), and Hep-AEG1-8 (AEG1-8) clones. (C) LEF1 expression analysis by immunofluorescence. (D) Immunohistochemical analysis of AEG1, LEF1, TFCP2, DPYD, and IGFBP7 expression in normal liver and matched HCC from the same patient. Figure represents data from 1 patient. Similar findings were observed in 13 out of 18 HCC patients. (E) LEF1-responsive luciferase reporter (TOPflash) assay. Transfection procedure of the indicated plasmid is described in Methods. Firefly luciferase activity was normalized by renilla luciferase activity, and the activity of the empty pGL3-basic vector was considered as 1. Data represent mean ± SEM. (F) Effect of LEF1 siRNA (siLEF1) and AEG1 siRNA (siAEG1) on downregulation of LEF1 and AEG1 proteins, respectively, was analyzed by Western blot analysis. siCon, control scrambled siRNA. (G) Matrigel invasion assay using Hep-AEG1-14 clones upon treatment with the indicated siRNA. Data represent mean ± SEM. (H) Matrigel invasion assay in QGY-7703 cells upon treatment with indicated siRNA. Graphical representation of the results is shown in the upper panel. Data represent mean ± SEM. Lower panel shows photomicrograph of the invading cells. Original magnification, ×400 (C); ×100 (D and H).

Figure 6. Activation of β-catenin is mediated by AEG1–induced activation of ERK42/44.

(A) Localization of β-catenin by immunofluorescence analysis. Original magnification, ×400. (B) Expression of indicated proteins was analyzed in the indicated clones upon treatment with PD98059 by Western blot analysis. (C) Schematic representation of the molecular mechanism of activation of Wnt/β-catenin signaling by AEG1.