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GPC3 activates ERK phosphorylation. (A) ERK phosphorylation in GPC3-expressing NIH3T3 stable lines. Cells were serum starved for 24 h. Forty micrograms of cell extracts were analyzed by western blot with either anti-phospho-ERK or anti-ERK antibody. Whole-cell extracts from 12-O-tetradecanoylphorbol 13-acetate-treated HEK293 cells (lane 2) were used as a positive control. (B) ERK phosphorylation in GPC3-knocked-down HuH-7 cells. shRNA was transiently transfected into HuH-7 cells. After serum starvation, 35 μg cell extracts were subjected for western blot analysis with anti-phospho-ERK, anti-ERK, 1G12 or anti-actin. ERK phosphorylation decreased after GPC3 knockdown. (C) ERK phosphorylation in PLC-PRF-5 cells. Cells were stably transfected with p gpc3-GFP (GPC3) or control vector (vector) and serum starved for 24 h. Cell extracts were analyzed with either anti-phospho-ERK or anti-ERK. (D) ERK phosphorylation in HA22T/VGH cells. Cells were stably transfected with pcDNA-gpc3 [wild-type GPC3 (WT-GPC3)], RR → AA, P25-29A or control vector (vector) and serum starved at 0.5% fetal calf serum for 24 h. Cell extracts were immunobloted with either anti-phospho-ERK, anti-ERK or 1G12. (E) IGF-II knockdown by siRNA (siIGF-II). Chemically synthesized, double-stranded siRNAs for IGF-II and control were transfected into HEK293 cells. Total RNA was extracted and analyzed by reverse transcription–polymerase chain reaction. Specific primers for IGF-II were used in polymerase chain reaction and actin was served as the RNA loading control. (F) ERK phosphorylation was decreased by IGF-II knockdown. HuH-7 cells were transfected with either control or IGF-II siRNA and serum starved. Cell extracts were analyzed with either anti-phospho-ERK or anti-ERK. (G) Growth rate of HuH-7 cells after IGF-II knockdown. Cells were seeded in 12-well plates in triplicate, transfected with either control or IGF-II siRNA and were serum starved. Cells were harvested at 48 h intervals.
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fig5: GPC3 activates ERK phosphorylation. (A) ERK phosphorylation in GPC3-expressing NIH3T3 stable lines. Cells were serum starved for 24 h. Forty micrograms of cell extracts were analyzed by western blot with either anti-phospho-ERK or anti-ERK antibody. Whole-cell extracts from 12-O-tetradecanoylphorbol 13-acetate-treated HEK293 cells (lane 2) were used as a positive control. (B) ERK phosphorylation in GPC3-knocked-down HuH-7 cells. shRNA was transiently transfected into HuH-7 cells. After serum starvation, 35 μg cell extracts were subjected for western blot analysis with anti-phospho-ERK, anti-ERK, 1G12 or anti-actin. ERK phosphorylation decreased after GPC3 knockdown. (C) ERK phosphorylation in PLC-PRF-5 cells. Cells were stably transfected with p gpc3-GFP (GPC3) or control vector (vector) and serum starved for 24 h. Cell extracts were analyzed with either anti-phospho-ERK or anti-ERK. (D) ERK phosphorylation in HA22T/VGH cells. Cells were stably transfected with pcDNA-gpc3 [wild-type GPC3 (WT-GPC3)], RR → AA, P25-29A or control vector (vector) and serum starved at 0.5% fetal calf serum for 24 h. Cell extracts were immunobloted with either anti-phospho-ERK, anti-ERK or 1G12. (E) IGF-II knockdown by siRNA (siIGF-II). Chemically synthesized, double-stranded siRNAs for IGF-II and control were transfected into HEK293 cells. Total RNA was extracted and analyzed by reverse transcription–polymerase chain reaction. Specific primers for IGF-II were used in polymerase chain reaction and actin was served as the RNA loading control. (F) ERK phosphorylation was decreased by IGF-II knockdown. HuH-7 cells were transfected with either control or IGF-II siRNA and serum starved. Cell extracts were analyzed with either anti-phospho-ERK or anti-ERK. (G) Growth rate of HuH-7 cells after IGF-II knockdown. Cells were seeded in 12-well plates in triplicate, transfected with either control or IGF-II siRNA and were serum starved. Cells were harvested at 48 h intervals.

Mentions: Lastly, we checked if GPC3 could activate the downstream mitogenic pathway. In western blot analysis, the levels of total ERK in the parental NIH3T3 cells, 12-O-tetradecanoylphorbol 13-acetate treatment HEK293 cells or GPC3 expression NIH3T3 cells were equal (Figure 5A). 12-O-tetradecanoylphorbol 13-acetate induced prominent ERK phosphorylation in HEK293 cells (Figure 5A, lane 2). Phospho-ERK was barely visible in the parental NIH3T3 cells, but was abundant in GPC3-expressing cell lines GPC3-60 and GPC3-65 (Figure 5A, lanes 3 and 4). In the GPC3-high-expressing HuH-7 cells, ERK phosphorylation was high, but phospho-ERK decreased when GPC3 was knocked down by gpc3 shRNA (Figure 5B). In the GPC3-low-expressing PLC-PRF-5 and HA22T/VGH cells, stable expression of GPC3 caused the elevation of phospho-ERK, but mutants RR → AA or P25-29A had much less effects (Figure 5C and D). Serum starvation for the HA22T/VGH cells was done in 0.5% serum because in 0% serum no ERK phosphorylation could be seen. This could be due to the low IGF-II expression in HA22T/VGH cells (Figure 3A).

Glypican-3-mediated oncogenesis involves the Insulin-like growth factor-signaling pathway

Cheng W, Tseng CJ, Lin TT, Cheng I, Pan HW, Hsu HC, Lee YM - Carcinogenesis (2008)

Bottom Line: Also, GPC3 knockdown in HCC cells decreased the phosphorylation of both IGF-1R and ERK.Therefore, GPC3 confers oncogenecity through the interaction between IGF-II and its receptor, and the subsequent activation of the IGF-signaling pathway.This data are novel to the current understanding of the role of GPC3 in HCC and will be important in future developments of cancer therapy.

Affiliation: Graduate Institute of Pathology, College of Medicine, National Taiwan University, Taipei 100, Taiwan.

ABSTRACT
Glypican-3 (gpc3) is the gene responsible for Simpson-Golabi-Behmel overgrowth syndrome. Previously, we have shown that GPC3 is overexpressed in hepatocellular carcinoma (HCC). In this study, we demonstrated the mechanisms for GPC3-mediated oncogenesis. Firstly, GPC3 overexpression in NIH3T3 cells gave to cancer cell phenotypes including growing in serum-free medium and forming colonies in soft agar, or on the other way, GPC3 knockdown in HuH-7 cells decreased oncogenecity. We further demonstrated that GPC3 bound specifically through its N-terminal proline-rich region to both Insulin-like growth factor (IGF)-II and IGF-1R. GPC3 stimulated the phosphorylation of IGF-1R and the downstream signaling molecule extracellular signal-regulated kinase (ERK) in an IGF-II-dependent way. Also, GPC3 knockdown in HCC cells decreased the phosphorylation of both IGF-1R and ERK. Therefore, GPC3 confers oncogenecity through the interaction between IGF-II and its receptor, and the subsequent activation of the IGF-signaling pathway. This data are novel to the current understanding of the role of GPC3 in HCC and will be important in future developments of cancer therapy.

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