IGF2 is required for HCC cell migration and invasion. (A) Migration (gray bars) and invasion (white bars) activities of MM189 HCC cells after inhibition of IGF2 using neutralizing antibodies. Error shown is SEM. The panel on the right displays the invasion index (see Materials and Methods). Data are from a representative experiment. (B) Immunoblot of whole-cell lysates confirming knockdown of IGF2 in MM189 HCC cells by a targeting shRNA. β-Actin serves as a loading control. (C) Immunoblot of conditioned serum-free medium collected from IGF2 knockdown cells and controls to determine levels of secreted IGF2 and IGF1. (D) Migration (gray bars) and invasion (white bars) activities of MM189 HCC cells after inhibition of IGF2 by shRNA. Error shown is SEM. The panel on the right displays the invasion index. Data are from a representative experiment. (E) Migration and invasion activities of MM189 cells with IGF2 knockdown or controls in the presence of serum-free medium (first two samples) or in the presence of serum-free conditioned medium collected from either control MM189 cells or MM189 cells expressing IGF2 shRNA (last two samples). Error shown is SEM. The panel on the right displays the invasion index. Data are from a representative experiment. (F) Migration (gray bars) and invasion (white bars) activities of MM189 HCC cells stimulated with recombinant IGF2. Error shown is SEM. The panel on the right displays the invasion index. IGF2 concentrations are shown on the x-axis. Data are from a representative experiment.

IGF1R signaling is required for lung colonization. (A) Representative lung fields of nude mice after the delivery of MM189 cells with IGF1R knockdown or vector controls through tail vein injection. The boxed area in the upper panel is shown at a higher magnification in the lower panel. Quantification of the number of lung lesions (B) and total tumor area (C) in the lungs of mice injected with MM189 cells with IGF1R knockdown or vector controls. Error shown is SEM. *P < .01 by Student's t test. **P < .05 by Student's t test. (D) Representative lung fields of nude mice after the delivery of MM189 cells with IR knockdown or vector controls through tail vein injection. The boxed area in the upper panel is shown at a higher magnification in the lower panel. Quantification of the number of lung lesions (E) and total tumor area (F) in the lungs of mice injected with MM189 cells with IR knockdown or vector controls. Error shown is SEM. P, not significant.

IGF2-induced signaling is not required for SC tumor formation. Effect of RNAi-mediated depletion of IGF1R (A), IGF2 (B), or IR (C) on SC tumor growth of MM189 HCC cells. pLKO- or pLKO-GFP-infected cells are used as vector controls.

MMP2 mediates IGF1R-induced cell invasion. (A) qRT-PCR data demonstrating the relative mRNA levels for the indicated genes in IGF1R knockdown cells and vector controls. (B) qRT-PCR measurement of MMP2 mRNA levels in cells with either IGF2 or IR knockdown. (C) Gelatinase zymogaphy demonstrating the activity of secreted MMP2 and MMP9 in IGF1R knockdown cells and vector controls. (D) Migration (gray bars) and invasion activities (white bars) of MM189 cells treated with an MMP2 inhibitor. Error shown is SEM. The panel on the right displays the invasion index. Data are from a representative experiment. (E) Measurement of MMP2 mRNA levels in MM189 HCC cells treated with inhibitors targeting the IGF1R, MAP kinase signaling pathway, or the PI3 kinase signaling pathway.

IGF1R-mediated, but not IR-mediated, signaling is required for HCC cell migration and invasion. (A) Immunoblot confirming knockdown of IGF1R in MM189 HCC cells by independent targeting shRNA. β-Actin serves as a loading control. (B) Migration (gray bars) and invasion (white bars) activities of vector controls and MM189 cells with IGF1R knockdown. Error shown is SEM. The panel on the right displays the invasion index. Data are from a representative experiment. (C) Immunoblot confirming knockdown of IGF1R in BL322 HCC cells by independent targeting shRNA. β-Actin serves as a loading control. (D) Migration (gray bars) and invasion (white bars) activities of vector controls and BL322 cells with IGF1R knockdown. Error shown is SEM. The panel on the right displays the invasion index. Data are from a representative experiment. (E) Immunoblot confirming knockdown of IR by targeting shRNA. (F) Migration (gray bars) and invasion (white bars) activities of vector controls and MM189 cells with IR knockdown. The panel on the right displays the invasion index. Data are from a representative experiment.

Inhibition of IGF2 does not block lung colonization. (A) Representative lung fields of nude mice after the delivery of MM189 cells with IGF2 knockdown or vector controls through tail vein injection. The boxed area in the upper panel is shown at higher magnification in the lower panel. Quantification of the number of lung lesions (B) and total tumor area (C) in the lungs of mice injected with MM189 cells with IGF2 knockdown or vector controls. Error shown is SEM. P, not significant.

IGF1R-mediated HCC cell invasion involves IRS2. (A) Immunoblot confirming specific knockdown of either IRS1 or IRS2 using targeting shRNA. β-Actin serves as a loading control. Invasion activity of MM189 HCC cells with knockdown of either IRS2 (B) or IRS1 (C). Migration (gray bars) and invasion (white bars) activities of vector controls and cells with indicated knockdown. Error shown is SEM. The panel on the right displays the invasion index. Data are from representative experiments.