Tetraspanin 8 mediates AEG-1-induced invasion and metastasis in hepatocellular carcinoma cells

Abstract

Astrocyte-elevated gene-1 (AEG-1) positively regulates tumor progression and metastasis. Here, we document that AEG-1 upregulates transcription of the membrane protein tetraspanin 8 (TSPAN8). Knocking down TSPAN8 in AEG-1-overexpressing human hepatocellular carcinoma (HCC) cells markedly inhibited invasion and migration without affecting proliferation. TSPAN8 knockdown profoundly abrogated AEG-1-induced primary tumor and intrahepatic metastasis in an orthopic xenograft model in athymic nude mice. Coculture of TSPAN8 knockdown cells with human umbilical vein endothelial cells (HUVEC) markedly inhibited HUVEC tube formation indicating that inhibition of angiogenesis might cause reduction in primary tumor size. TSPAN8 inhibition might be a potential therapeutic strategy for metastatic HCC.

Keywords: angiogenesis; astrocyte-elevated gene-1; invasion; metastasis; tetraspanin 8.

Figure 1

AEG-1 increases transcription of TSPAN8. A. TSPAN8 and GAPDH protein levels were analyzed by Western blotting in Hep-PC-4, Hep-AEG-1-8 and Hep-AEG-1si cells. B. TSPAN8 mRNA expression was analyzed in the indicated cells. C. AEG-1, TSPAN8 and EF1α levels were analyzed by Western blotting in Hc3716-hTERT and QGY-7703 cells. D. TSPAN8-luc reporter plasmid was transfected into the indicated cells and luciferase activity was measured. E. QGY-7703 cells were transfected with TSPAN8-luc plasmid along with control siRNA or AEG-1 siRNA and luciferase activity was measured. F. HEK-293 cells were transfected with TSPAN8-luc plasmid and indicated amounts of empty vector (pcDNA3.1) or AEG-1 expression plasmid and luciferase activity was measured. G-H. Hep-AEG-1-8 (F) and HEK-293 (G) cells were transfected with TSPAN8-luc plasmid and treated with U0126 (10 μM), LY294002 (10 μM) or SB203580 (2 μM) and luciferase activity was measured. For luciferase assays, activity was measured 48 h after transfection and luciferase activity was normalized by protein concentration and by activity of promoter-less pGL4.10[luc2] plasmid. Data represent mean ± SD of at least triplicate experiments. *: p< 0.0002; **: p< 0.0004; #: p<0.05.

Figure 2

Knockdown of TSPAN8 in Hep-AEG-1-8 cells does not affect proliferation.. TSPAN8 mRNA (A) and protein (B) levels were measured in parental Hep-AEG-1-8 cells and stable clones of Hep-AEG-1-8 cells expressing control scrambled shRNA (CON-si) or TSPAN8 shRNA (TS8-si). C. Cell proliferation of the indicated cells were measured by standard MTT assay. D. Colony formation by the indicated cells were measured after 2 weeks. Data represent mean ± SD of at least triplicate experiments. *: p< 0.0002.

Figure 3

Knockdown of TSPAN8 abrogates invasion and migration in Hep-AEG-1-8 cells. A. Matrigel invasion assay was performed in the indicated cells. Top, photomicrograph of the invaded cells. Bottom, graphical quantification of the invaded cells from 10 fields. B. Wound healing (scratch) assay was performed in the indicated cells. Top, photomicrograph of the wound at the time of scratch (0 h) and 72 h later. Bottom, graphical quantification of wound closure. Data represent mean ± SD. *: p< 0.0002. C. F-actin staining at the wound edge in the indicated cells 72 h after the wound. D. Western blot analysis for the indicated proteins in the indicated cells.

Figure 4

TSPAN8 knockdown inhibits intrahepatic metastasis and angiogenesis. A. Photograph of the livers carrying orthotopic xenografts of the indicated cells. B. Liver weight at the end of the study (n = 5 per group). C. Tube formation assay was performed in human umbilical vein endothelial cells (HUVEC) co-cultured with the indicated cells. Left, photomicrograph of the tubes 6 h after co-culture. Right, Graphical quantification of the tubes. Data represent mean ± SD. *: p< 0.0002.