Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 Influences Sensitivity to Anti-IGF System Agents Through the Translational Regulation of IGF1R

Abstract

Insulin-like growth factor 2 (IGF2) mRNA-binding protein 3 (IGF2BP3) is an oncofetal protein that binds RNA, thereby influencing the fate of target transcripts. IGF2BP3 is synthesized de novo in cancer, where it promotes proliferation, drug resistance, and metastasis via both IGF2-dependent and IGF2-independent mechanisms. Ewing sarcoma (ES) is a rare bone and soft tissue tumor in which the IGF system plays a pivotal role. This study aimed to investigate the effect of IGF2BP3 on the regulation of the IGF system in ES. Among the components of the IGF axis, a direct significant correlation was identified between IGF2BP3 and IGF1R at mRNA and protein levels in two independent series of clinical specimens from patients with localized ES. After the formal demonstration of a direct association between IGF2BP3 and IGF1R mRNA using ribo-immunoprecipitation assay, we performed in vitro studies using A673 and TC-71 ES cell lines to demonstrate that IGF2BP3 loss promotes the downregulation of IGF1R and a decreased biological response to IGF1, represented by reduced migration and cell growth. Additionally, the compensatory activation of insulin receptor (IR) and its mitogenic ligand IGF2 is triggered in some but not all cell lines in response to IGF2BP3-mediated IGF1R loss. These findings have therapeutic implications because cells with a decreased expression of IGF2BP3/IGF1R axis but an increased expression of the IR/IGF2 loop display higher sensitivity to the dual inhibitor OSI-906 than do control cells. Therefore, studies on IGF2BP3, which was confirmed as a post-transcriptional regulator of IGF1R, provide a step forward in the identification of new mechanisms regulating the IGF system. In addition, our results demonstrate that the detection of IGF2BP3 expression should be combined with the assessment of the IGF1R/IR ratio to predict cell responses to anti-IGF1R/IR agents.

Keywords: Ewing sarcoma; IGF1R; anti-IGF agents; insulin-like growth factor 2 mRNA-binding protein 3; personalized treatment.

Figure 1

Insulin-like growth factor 2 (IGF2) mRNA-binding protein 3 (IGF2BP3) expression is directly correlated with IGF1R expression in Ewing sarcoma (ES) cases. Scatter plot displaying the correlation between IGF2BP3 and IGF2 (A) or IGF1R (B) mRNA levels in 89 ES cases. Correlation coefficient (r) and p-value were calculated using Spearman’s correlation test. Human mesenchymal stem cells were used as calibrators (2^−ΔΔCt = 1).

Figure 2

Assessment of IGF1R as an insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) mRNA target in Ewing sarcoma (ES) cells. (A) Quantitative real-time PCR analysis of IGF2BP3-associated mRNAs isolated from the cytoplasmic extracts of A673 cells by immunoprecipitation using an anti-IGF2BP3 antibody. Non-immune goat IgG was used as a negative control. ABCG2 was used as positive control. Columns represent the mean values of two independent experiments, in which samples were run in triplicates, and the bars represent the SE. *p < 0.05, **p < 0.01, Student’s t-test. (B) mRNA expression levels of IGF2BP3 and IGF1R in IGF2BP3-depleted or empty vector-transfected (shCTR) A673 ES cells. GAPDH was used as a housekeeping gene. Columns represent the mean values of at least two independent experiments, in which samples were run in triplicates, and the bars represent the SE. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with respect to shCTR. (C) Western blotting showing IGF2BP3 and IGF1R expression in IGF2BP3-depleted or empty vector-transfected (shCTR) A673 (left) or TC-71 (right) ES cells. GAPDH was used for normalization. Data from one experiment representative of three.

Figure 3

Evaluation of the oncogenic potential of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3)/IGF1R in Ewing sarcoma (ES) cell lines. (A) Migration of IGF2BP3-depleted or empty vector-transfected (shCTR) A673 or TC-71 ES cells in response to IGF1 (50 ng/ml) stimulation. Columns represent the mean values of two independent experiments, in which samples were run in triplicate, and the bars represent the SE. (B) Growth capacity of IGF2BP3-depleted or empty vector-transfected (shCTR) A673 or TC-71 ES cells after 24 h of IGF1 (50 ng/ml) stimulation. Trypan blue staining was used for cell counting. Columns represent the mean values of two independent experiments, in which samples were run in duplicates, and the bars represent the SE. (A,B) *p < 0.05, **p < 0.01, one-way ANOVA.

Figure 4

Insulin receptor (IR) signaling in response to insulin-like growth factor 2 (IGF2) mRNA-binding protein 3 (IGF2BP3)-mediated IGF1R regulation. (A,B) Western blotting depicting the expression of IR and phosphorylation of Akt and ERK 1/2 in IGF2BP3-depleted or empty vector-transfected (shCTR) (A) A673 or (B) TC-71 Ewing sarcoma (ES) cells. GAPDH, Lamin B, and total proteins were used for normalization purposes. Data from one experiment representative of three. (C,D) mRNA level of IGF1 and IGF2, expressed as 2^−ΔΔCt, in IGF2BP3-depleted or empty vector-transfected (shCTR) (C) A673 or (D) TC-71 cells. Columns represent the mean values of at least two independent experiments, in which samples were run in triplicates, and the bars represent the SE. *p < 0.05, **p < 0.01, one-way ANOVA compared with shCTR.

Figure 5

Efficacy of agents blocking the IGF system in insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3)-depleted models. Cell growth was assessed using the MTT assay after 72 h of exposure to OSI-906 at indicated doses in IGF2BP3-depleted or empty vector-transfected (shCTR) A673 (A) and TC-71 (B) Ewing sarcoma cells. Results are displayed as the percentage of growth inhibition relative to that in untreated control cells. Histograms represent the mean of three independent experiments, in which each sample was in triplicate, and bars represent the SE. *p < 0.05, **p < 0.01, one-way ANOVA compared with shCTR.