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. 2007 Dec 26;104(52):20926-31.
doi: 10.1073/pnas.0710359105. Epub 2007 Dec 17.

Enhanced Sensitivity to IGF-II Signaling Links Loss of Imprinting of IGF2 to Increased Cell Proliferation and Tumor Risk

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Free PMC article

Enhanced Sensitivity to IGF-II Signaling Links Loss of Imprinting of IGF2 to Increased Cell Proliferation and Tumor Risk

Atsushi Kaneda et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to abnormal activation of the normally silent maternal allele, is a common human epigenetic population variant associated with a 5-fold increased frequency of colorectal neoplasia. Here, we show first that LOI leads specifically to increased expression of proliferation-related genes in mouse intestinal crypts. Surprisingly, LOI(+) mice also have enhanced sensitivity to IGF-II signaling, not simply increased IGF-II levels, because in vivo blockade with NVP-AEW541, a specific inhibitor of the IGF-II signaling receptor, showed reduction of proliferation-related gene expression to levels half that seen in LOI(-) mice. Signal transduction assays in microfluidic chips confirmed this enhanced sensitivity with marked augmentation of Akt/PKB signaling in LOI(+) cells at low doses of IGF-II, which was reduced in the presence of the inhibitor to levels below those found in LOI(-) cells, and was associated with increased expression of the IGF1 and insulin receptor genes. We exploited this increased IGF-II sensitivity to develop an in vivo chemopreventive strategy using the azoxymethane (AOM) mutagenesis model. LOI(+) mice treated with AOM showed a 60% increase in premalignant aberrant crypt foci (ACF) formation over LOI(-) mice. In vivo IGF-II blockade with NVP-AEW541 abrogated this effect, reducing ACF to a level 30% lower even than found in exposed LOI(-) mice. Thus, LOI increases cancer risk in a counterintuitive way, by increasing the sensitivity of the IGF-II signaling pathway itself, providing a previously undescribed epigenetic chemoprevention strategy in which cells with LOI are "IGF-II addicted" and undergo reduced tumorigenesis in the colon upon IGF-II pathway blockade.

Conflict of interest statement

Conflict of interest statement: R.A. and M.A.P. (Novartis, Inc.) have a proprietary interest in NVP-AEW541. The Johns Hopkins University and the National Institutes of Health have filed a patent on data presented in this manuscript.

Figures

Fig. 1.
Fig. 1.
Gene expression levels in microdissected intestinal crypts. Quantitative real-time PCR was performed on laser capture microdissected intestinal crypts from 12 LOI(+) and 9 LOI(−) mice. Expression was normalized to β-actin, and the expression level in LOI(+) samples (gray) relative to LOI(−) samples (black) is shown. The bars indicate standard error. (A) Among up-regulated genes in the top ranking GO-annotation categories (DNA replication/cell cycle genes listed in SI Table 3), six genes were validated, and all of the genes showed a statistically significant difference in LOI(+) compared with LOI(−) crypts: Cdc6, 1.55-fold (P = 0.003); Mcm5, 1.47-fold (P = 0.007); Mcm3, 1.49-fold (P = 0.002); Chaf1a, 1.61-fold (P = 0.009); Lig1, 1.54-fold (P = 0.008); and Ccne1, 1.38-fold (P = 0.04). In addition, Igf2 was up-regulated 2.54-fold (P = 0.002) in LOI(+) LCM-dissected crypts. (B) Receptor inhibition by NVP-AEW541 had a differential effect on proliferation-related gene expression in LOI(+) crypts. Analysis was by quantitative real-time PCR of laser capture microdissected intestinal crypts from four LOI(+) and four LOI(−) mice treated with NVP-AEW541 for 3 weeks. Reduction in gene expression in microdissected crypts of LOI(+) mice (gray bars) relative to LOI(−) mice (black bars, normalized to 1.0): Cdc6, 0.49-fold (P = 0.048); Mcm5, 0.48-fold (P = 0.007); Mcm3, 0.65-fold (P = 0.1); Chaf1a, 0.42-fold (P = 0.010); Lig1, 0.42-fold (P = 0.029); and Ccne1, 0.57-fold (P = 0.030). Asterisks indicate significant difference between LOI(+) and LOI(−).
Fig. 2.
Fig. 2.
Single cell analysis of Akt activation by IGF2 in LOI(+) and LOI(−) cells. (A) Akt activation by IGF2. Akt/PKB activation was assayed by single cell immunocytochemistry with an antibody to phosphorylated Akt (Ser-473), in a monolithic two-layer PDMS chip sealed with a glass coverslip, with defined media delivery controlled by a multiplexed system of valves. Live LOI(+) and LOI(−) MEF cells were stimulated within the microfluidic chips with varying doses of IGF2, with measurements at multiple time points at each IGF2 concentration. The y axis shows the ratio of nuclear to background fluorescence. For each cell type, IGF2 concentration, and time point, at least 200 individual cellular measurements were obtained by digital imaging and analysis. LOI(+) but not LOI(−) showed a sustained signaling response to low dose IGF2. Standard error bars are not visible behind symbols at this scale. (B) Inhibition of Akt activation by NVP-AEW541. The cells were assayed as in A at 60 min after coincubation with 400 ng/ml IGF2 and 3 μM NVP-AEW541 and compared with the unstimulated control. Each bar is based on measurements of >400 cells. Whereas NVP-AEW541 inhibited Akt activation to the baseline levels in LOI(−) cells, the inhibitor inhibited Akt activation significantly below the baseline in LOI(+) cells. Error bars show SDs. Asterisk indicates statistical difference vs. LOI(+) control (t test, P < 0.001). (C) Single cell analysis of Erk activation by IGF2 in LOI(+) and LOI(−) MEF cells. Erk2 activation was assayed by single cell immunocytochemistry within microfluidic chips using an antibody to phosphorylated Erk2 from Upstate (Charlottesville, VA). LOI(+) cells (red) and LOI(−) cells (black) were exposed to 400 ng/ml IGF2 for indicated times. The y axis shows the ratio of nuclear to background fluorescence normalized to the maximum level achieved in the LOI(+) cells. Error bars represent SD. Standard error bars are completely subsumed by the symbols at this scale. (D) Gene expression levels in mouse embryonic fibroblasts. Quantitative real-time PCR was performed on LOI(+) and LOI(−) MEF cells, with expression normalized to transferrin receptor expression. The expression level in LOI(+) samples (black) relative to LOI(−) samples (white) is shown. The bars indicate standard error. Both Igf1R and Insr show significant differences in expression: Igf1R, 1.93-fold (P = 0.05); Igf2R 0.75-fold (P = 0.33); and Insr, 2.19-fold (P = 0.05).
Fig. 3.
Fig. 3.
Inhibition of azoxymethane (AOM)-induced aberrant crypt foci (ACF) by NVP-AEW541. (A) ACF formation in the colon was induced by AOM i.p. injection, and treatment with NVP-AEW541 was by gastric gavage. Each ACF was formed of one to four aberrant crypts, and the number of ACF (# of ACF), the number of total aberrant crypts (# of AC), and the average number of aberrant crypts per ACF were measured. LOI(+) mice (gray bars) formed 1.60-fold more ACF than LOI(−) mice (black bars) (P = 0.002). Similarly, LOI(+) mice formed 1.65-fold more AC than LOI(−) mice (P = 0.01). LOI(−) mice treated with AOM and NVP-AEW541 (pink bars), showed no significant reduction of ACF over LOI(−) mice treated with AOM injection alone (P = 0.5). However, LOI(+) mice treated with AOM and NVP-AEW541 (azure bars) showed a 61% decrease in ACF compared with LOI(+) mice treated with AOM alone (P = 0.0002), and a 37% decrease compared with LOI(−) mice treated with AOM alone (P = 0.007). Similarly, LOI(+) mice treated with AOM and NVP-AEW541 showed a 64% decrease in AC from LOI(+) mice treated with AOM alone (P = 0.0002), and a 40% reduction compared with LOI(−) mice treated with AOM alone (P = 0.003). (B) The number of ACF and the number of total aberrant crypts (AC) were corrected by colon surface area (cm2). LOI(+) mice (gray bars) formed 1.59-fold more ACF (P = 0.004) and 1.63-fold more AC than LOI(−) mice (black bars) (P = 0.002). LOI(−) mice treated with AOM and NVP-AEW541 (pink bars), showed no reduction of ACF over LOI(−) mice treated with AOM injection alone (P = 0.5). However, LOI(+) mice treated with AOM and NVP-AEW541 (azure bars) showed a 56% decrease in ACF compared with LOI(+) mice treated with AOM alone (P = 0.0008), and a 30% decrease compared with LOI(−) mice treated with AOM alone (P = 0.05). Similarly, LOI(+) mice treated with AOM and NVP-AEW541 showed a 60% decrease in AC from LOI(+) mice treated with AOM alone (P = 0.03), and a 33% reduction compared with LOI(−) mice treated with AOM alone (P = 0.02). Asterisks indicate significant difference between LOI(+) and LOI(−), and between NVP-AEW541 treatment and no treatment for a given epigenotype.

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