Up-Regulation of Imp3 Confers In Vivo Tumorigenicity on Murine Osteosarcoma Cells

<div><p>Osteosarcoma is a high-grade malignant bone tumor that manifests ingravescent clinical behavior. The intrinsic events that confer malignant properties on osteosarcoma cells have remained unclear, however. We previously established two lines of mouse osteosarcoma cells: AX cells, which are able to form tumors in syngeneic mice, and AXT cells, which were derived from such tumors and acquired an increased tumorigenic capacity during tumor development. We have now identified Igf2 mRNA-binding protein3 (Imp3) as a key molecule responsible for this increased tumorigenicity of AXT cells in vivo. Imp3 is consistently up-regulated in tumors formed by AX cells, and its expression in these cells was found to confer malignant properties such as anchorage-independent growth, loss of contact inhibition, and escape from anoikis in vitro. The expression level of Imp3 also appeared directly related to tumorigenic ability in vivo which is the critical determination for tumor-initiating cells. The effect of Imp3 on tumorigenicity of osteosarcoma cells did not appear to be mediated through Igf2-dependent mechanism. Our results implicate Imp3 as a key regulator of stem-like tumorigenic characteristics in osteosarcoma cells and as a potential therapeutic target for this malignancy.</p> </div

Tumorigenic activity of osteosarcoma cells correlates with Imp3.

<p>(<b>A</b>) Immunofluorescence analysis of Imp3 expression in AX cells. The boxed region is shown at higher magnification in the lower panel. (<b>B</b>) Real-time PCR analysis of <i>Imp3</i> expression in AX cells and in an AX subclone (designated AX-low) obtained by single-cell cloning. *<i>P</i><0.01. (<b>C</b>) Weight of tumors derived from subcutaneously injected AX-low cells. (<b>D</b>) Real-time PCR analysis of <i>Imp3</i> expression in AX-low cells as well as in the AX-low cell-derived tumors. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. NS, not significant. (<b>E</b>) Immunofluorescence analysis of Imp3 expression in AXT cells. The boxed region is shown at higher magnification in the lower panel. (<b>F</b>) AXT subclones (designated AXT-high and AXT-low) isolated by single-cell cloning were subjected to real-time PCR analysis of <i>Imp3</i> as well as to immunofluorescence analysis of Imp3 protein. (<b>G</b>) Weight of tumors derived from subcutaneously injected AXT-high or AXT-low cells. *<i>P</i><0.001.</p

Knockdown of Imp3 in AXT cells attenuates malignant cellular phenotype.

<p>(<b>A</b>) The efficacy of knockdown of Imp3 in AXT cells was evaluated by real-time PCR analyses and immunoblotting. *<i>P</i><0.01, **<i>P</i><0.001. (<b>B</b>) Cell proliferation assays for AXT-shLUC, AXT-sh1, and AXT-sh2 cells cultured under adherent or nonadherent conditions. *<i>P</i><0.05, **<i>P</i><0.001. NS, not significant. (<b>C</b>) AXT-shLUC, AXT-sh1, and AXT-sh2 cells were cultured under adherent (upper panels) or nonadherent (lower panels) conditions for 24 h and were then stained with annexin V and PI. The percentages of viable (annexin V– and PI-negative) cells are indicated for a representative experiment. The percentage of viable cells was determined as means ± SD for three independent experiments. *<i>P</i><0.01. NS, not significant. (<b>D</b>) AXT-shLUC, AXT-sh1, and AXT-sh2 cells were cultured under normal conditions for 3 days, after which the saturation density was determined by counting total cell number. *<i>P</i><0.01. Representative bright-field images of cells after culture for 3 days are also shown.</p

Imp3 regulates tumorigenic activity independently of Igf2 in AXT cells.

<p>(<b>A</b>) AXT cell lysate was fractionated by centrifugation on 5 to 30% sucrose gradient. The resulting absorbance profile of the gradient was determined at 254 nm for identification of ribosomal subunits, individual ribosomes, and polyribosomes (upper panel). The gradient fractions as well as the original lysate sample (Input) were subjected to immunoblot analysis with antibodies to Imp3, rpS6, and Ago2. (<b>B</b>) Cell proliferation assays for AX and AXT cells and for AXT-shLUC and AXT-sh2 cells performed under nonadherent culture conditions and in complete medium supplemented (or not) with Igf2 (50 ng/ml). *<i>P</i><0.05, **<i>P</i><0.001. (<b>C</b>) AXT cells expressing three different Igf2 shRNAs (AXT-shIgf2-1 to -3) were injected subcutaneously into syngeneic mice. The tumors were weighed and assayed for <i>Igf2</i> expression by real-time PCR analysis. The correlation coefficient (CC) for the two variables was determined.</p

Knockdown of Imp3 in AXT cells suppresses tumorigenic activity in vivo.

<p>(<b>A</b>) Weight of tumors formed in syngeneic mice after subcutaneous injection of AXT-shLUC, AXT-sh1, or AXT-sh2 cells. *<i>P</i><0.001. (<b>B</b>) Kaplan-Meier survival analysis of mice injected intraperitoneally with AXT-shLUC, AXT-sh1, or AXT-sh2 cells. P values for comparison with AXT-shLUC were determined by the log-rank test. (<b>C</b>) Real-time PCR analysis of <i>Imp3</i> expression in lethal osteosarcoma tumors at 49 days after bilateral subcutaneous injection of AXT-sh1 cells (left panel) or at 141 days after intraperitoneal injection of AXT-sh2 cells (right panel). *<i>P</i><0.01, **<i>P</i><0.001.</p

Imp3 overexpression in AX cells promotes cell proliferation and tumorigenic activity.

<p>(<b>A</b>) The expression level of Imp3 was evaluated by real-time PCR analyses, immunofluorescence and immunoblotting. *<i>P</i><0.01. (<b>B</b>) Cell proliferation assays for AX-mock and AX-Imp3 cells cultured under adherent or nonadherent conditions. *<i>P</i><0.01. NS, not significant. (<b>C</b>) Weight of tumors derived from subcutaneously injected AX-mock or AX-Imp3 cells. *<i>P</i><0.001. (<b>D, E</b>) Tumors formed at 1, 2, or 3 weeks after subcutaneous injection of AX-mock or AX-Imp3 cells in mice were subjected to H&E staining and to immunohistochemical staining for GFP in serial sections as well as to real-time PCR analysis of <i>GFP.</i> *<i>P</i><0.01. NS, not significant.</p

Up-regulation of Imp3 expression during tumor formation in vivo.

<p>(<b>A</b>) AX or AXT cells were injected bilaterally and subcutaneously into syngeneic mice, and the weight of the tumors was measured. *<i>P</i><0.0001. (<b>B</b>) Cell proliferation assays for AX or AXT cells cultured under adherent or nonadherent conditions. *<i>P</i><0.001. NS, not significant. Representative bright-field images of cells after culture for 2 days are shown. (<b>C</b>) Immunoblot analysis of Imp3 expression in AX and AXT cells. α-Tubulin was examined as a loading control. Real-time PCR analysis of <i>Imp3</i> expression in AX and AXT cells. *<i>P</i><0.01. (<b>D</b>, <b>E</b>) Real-time PCR analysis of <i>Imp3</i> expression in subcutaneous AX cells at 1 or 2 weeks after injection into mice, and in primary tumors and metastatic lesions formed by AX cells. *<i>P</i><0.05, **<i>P</i><0.01. NS, not significant. (<b>F</b>) Immunohistochemical staining of IMP3 in human osteosarcoma samples. The intensity of staining was scored from 0 to 3. Representative images are shown.</p