Table 1.
Clinical, histopathological, and molecular features of adrenal adenoma, IGF2-low carcinoma, and IGF2-high carcinoma.
Figure 1.
IGF2 mRNA levels and prognoses of IGF2-high and IGF2-low ACC.
A: Boxplot showing IGF2 mRNA levels in normal adrenals (n = 4), ACA (n = 87), IGF2-low ACC (n = 10), and IGF2-high ACC (n = 43). IGF2-low ACC have similar levels of IGF2 to normal adrenals and ACAs. There is a minimal 6.5 fold difference between IGF2-high and IGF2-low ACC and there is a 200-fold difference between their median expressions. B: Kaplan-Meier survival curves showing overall (left) and disease-free (right) survival of patients with IGF2-low ACC (n = 10) or IGF2-high ACC (n = 43). P-values from a Cox regression analysis between IGF2-high and IGF2-low ACC are indicated, showing that there is no difference between the two groups.
Figure 2.
Quantitative expression of IGF signaling pathway members.
Boxplots showing the mRNA (quantitative RT-PCR) and/or protein (western blot) levels of several IGF2 pathway members in IGF2-high (n = 23 for RT-PCR and n = 10 for western blot) and IGF2-low (n = 10) ACC. Y-axis for mRNA: result of the ΔΔCT method (2-ΔΔCT value), with RNA18S5 as a reference gene. Y-axis for protein: results of the quantification of the western blot bands, normalized to actin. Wilcoxon test results (*p<0.05; **p<0.01; ***p<0.00; NS = not significant) are indicated for each mRNA or protein studied. A: IGF2 and its receptors. B: Erk and Akt. C: other IGF2 pathway members with significantly different expression between IGF2-high and IGF2-low ACC.
Figure 3.
IGF signaling pathway activation in IGF2-high and IGF2-low ACC.
The activation of Erk (A), Akt (B), and IGF1R/INSR receptors (C) was analyzed by western blotting with antibodies directed against the phosphorylated form of these proteins, in IGF2-high (n = 10) and IGF2-low (n = 10) ACC. Boxplots show the quantification of the results of western blots. Y-axis: results of the quantification of the western blot bands normalized to total Erk, Akt or actin. Wilcoxon test results (*p<0.05; NS = not significant) are indicated. The phosphorylation of the receptors is higher in IGF2-high than in IGF2-low ACC, whereas the activation of Erk and Akt downstream pathways is similar.
Figure 4.
Consequences of IGF2 knock-down on cellular growth, apoptosis, and the cell cycle in H295R cells.
A, B: Consequences of long-term knock-down of IGF2 on H295R cell growth as assessed by a MTT proliferation test. A: Doxycycline treatment leads to IGF2 knock-down and to a significant impairment of cellular proliferation in a clone with integrated IGF2 shRNA. Results are presented for clone 4, which showed the most significant impairment of cell growth B: Doxycycline (black square) does not inhibit proliferation in a control clone. C, D: Effect of IGF2 knock-down on the cell cycle. After 7 days of doxycycline treatment, the cell cycle was analyzed by PI staining and flow cytometry. Cells expressing IGF2 shRNA were largely arrested in the G1 phase of the cell-cycle and few cells were in S phase (C). Results are presented for clone 1, which showed the most significant G1 cell cycle arrest. Doxycycline treatment did not affect the cell cycle of a control clone (D). E-H: Apoptosis studied by flow cytometry after PI (Propidium iodide) and Annexin V staining. E: Example of FACS results for clone 4, showing the apoptotic status of cells according to Annexin V (X axis) and PI (Y axis) staining. F, G: Study of non-induced apoptosis after prolonged IGF2 knock-down. Ten days after shRNA induction by doxycycline treatment, cells were stained and analyzed by flow cytometry. Doxycycline treatment did not affect apoptosis of the control clone without integrated shRNA (G). Both early and late apoptosis were significantly higher in cells expressing IGF2 shRNA than in control cells (F). Results are presented for clone 4, which showed the most significant difference in apoptosis. H: Study of TNF-alpha-induced apoptosis after the transient knock-down of IGF2 in H295R cells. Cells were treated with TNF-alpha 48 h after siRNA transfection, and were analyzed by flow cytometry 48 h later. The number of viable cells was significantly lower in cells transfected with siRNA against IGF2 (black bars), and the percentage of cells in late apoptosis was significantly higher than in cells transfected with a control siRNA (white bars). Results were analyzed with the Wilcoxon test. *: p<0.05. ***: p<0.001. All the results presented in this figure are representative of at least three independent experiments.
Figure 5.
Structure and methylation of the 11p15 locus.
A. Schematic representation of the 11p15 locus. The 11p15 locus is represented with the 2 differentially imprinted regions (ICR1 & ICR2). In the maternal allele H19 is expressed but not IGF2 as the consequence of CTCF binding to a sequence located between the 2 genes and acting as an insulator. Therefore the enhancer (E) can only activate the transcription of the most proximal gene. The methylation (CH3) of this sequence (paternal allele) prevents the binding of CTCF, allows the expression of IGF2 and represses that of H19. The methylation (maternal allele) or not (paternal allele) of ICR2 results in the opposite expression of CDKN1C, KCNQ1 and KCNQ10T. Genes with activation or repression of their expression are indicated in green or red respectively. The most frequent patterns observed in IGF2-high (left) and IGF2-low (right) ACC are indicated in the lower part of the figure. B: Methylation of the 11p15 imprinting center regions in IGF2-high (n = 15) and IGF2-low (n = 9) ACC. The percentage of methylation (y-axis) is shown for ICR1 (blue circles) and ICR2 (red squares). Wilcoxon test results (**p<0.01; NS = not significant) are indicated. Dotted lines connect data from the same tumor, and indicate paternal uniparental disomy (pUPD) when ICR1 is highly methylated and ICR2 is weakly methylated. This pUPD pattern is present in almost all IGF2-high ACC, whereas it is present in only three out of nine IGF2-low ACC.
Figure 6.
Expression of the genes in the 11p15 imprinted region.
A: Snapshot heat map of the 11p15 chromosomal region generated from a previous transcriptomic study (21). Expression levels of imprinted genes IGF2, IGF2AS, H19, CDKN1C, KCNQ1, and KCNQ1OT1 are shown for 47 ACA, six IGF2-low ACC and 23 IGF2-high ACC. Red: high expression. Green: low expression. B: Boxplots showing expression data for the same genes. Wilcoxon test results (*p<0.05; **p<0.01; ***p<0.001; NS = not significant) are indicated. Except for IGF2 and IGF2AS, there are no significant differences between IGF2-high and IGF2-low ACC.