Insulin-like growth factor-I receptor (IGF-IR) translocates to nucleus and autoregulates IGF-IR gene expression in breast cancer cells

Abstract

The insulin-like growth factor (IGF) system plays an important role in mammary gland biology as well as in the etiology of breast cancer. The IGF-I receptor (IGF-IR), which mediates the biological actions of IGF-I and IGF-II, has emerged in recent years as a promising therapeutic target. The IGF and estrogen signaling pathways act in a synergistic manner in breast epithelial cells. The present study was aimed at investigating 1) the putative translocation of IGF-IR and the related insulin receptor (IR) to the nucleus in breast cancer cells, 2) the impact of IGF-IR and IR levels on IGF-IR biosynthesis in estrogen receptor (ER)-positive and ER-depleted breast cancer cells, and 3) the potential transcription factor role of IGF-IR in the specific context of IGF-IR gene regulation. We describe here a novel mechanism of autoregulation of IGF-IR gene expression by cellular IGF-IR, which is seemingly dependent on ER status. Regulation of the IGF-IR gene by IGF-IR protein is mediated at the level of transcription, as demonstrated by 1) binding assays (DNA affinity chromatography and ChIP) showing specific IGF-IR binding to IGF-IR promoter DNA and 2) transient transfection assays showing transactivation of the IGF-IR promoter by exogenous IGF-IR. The IR is also capable of translocating to the nucleus and binding the IGF-IR promoter in ER-depleted, but not in ER-positive, cells. However, transcription factors IGF-IR and IR display diametrically opposite activities in the context of IGF-IR gene regulation. Thus, whereas IGF-IR stimulated IGF-IR gene expression, IR inhibited IGF-IR promoter activity. In summary, we have identified a novel mechanism of IGF-IR gene autoregulation in breast cancer cells. The clinical implications of these findings and, in particular, the impact of IGF-IR/IR nuclear localization on targeted therapy require further investigation.

FIGURE 1.

IGF-IR gene expression in ER-positive and ER-depleted breast cancer cells. A, Western blot analysis of IGF-IR levels. MCF7 and C4.12.5 cell lines were lysed in the presence of protease inhibitors, and equal amounts of protein (100 μg) were separated by 10% SDS-PAGE. After electrophoresis, proteins were transferred onto nitrocellulose membranes and blotted with antibodies against total and phosphorylated IGF-IR (pIGF-IR) and ERα, followed by incubation with an HRP-conjugated secondary antibody. Membranes were reprobed with a tubulin antibody. The figure shows the results of a typical experiment, repeated multiple times with similar results. B, quantitative real-time PCR of IGF-IR mRNA levels. Total RNA was prepared from MCF7 and C4.12.5 cells, and IGF-IR mRNA and GAPDH mRNA values were measured using the TaqMan® real-time PCR system. Analysis of the data was performed as described under “Experimental Procedures.” *, p < 0.01 versus MCF7 cells. C and D, IP analysis of phospho-IGF-IR and phospho-IR abundance in MCF7 and C4.12.5 cells. Total cell extracts (650 μg) were immunoprecipitated with antibodies against total IGF-IR or total-IR, electrophoresed, and immunoblotted with antibodies against phospho-IGF-IR/IR, total IGF-IR, or total-IR. Exposure time for the autoradiogram shown in C was 1 min, whereas the autoradiogram shown in D was exposed for 1 h. E–H, mitogenic effects of estradiol and IGF-I in MCF7 and C4.12.5 cells. MCF7 and C4.12.5 cells were starved overnight and then incubated with increasing doses of estradiol (1, 10, and 100 nm) or IGF-I (1, 5, 10, 20, and 50 ng/ml) for 72 h in serum-free medium. Cellular proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. p < 0.05 versus control cells. A value of 100% was given to the number of cells in control (unstimulated) cultures. Error bars, S.E.

FIGURE 2.

Subcellular distribution of IGF-IR in MCF7 and C4.12.5 cells. A, confluent MCF7 and C4.12.5 cells were lysed and fractionated into cytosolic and nuclear fractions, as described under “Experimental Procedures.” Total lysates (T; 80 μg) and cytosolic (C; 20 μg) and nuclear fractions (N; 20 μg) were resolved on 10% SDS-PAGE and blotted with anti-IGF-IR, anti-IR, anti-tubulin, and anti-lamin A/C (as a control for contamination of cytosolic fractions). Quantitative analysis of IGF-IR (B) and IR (C) abundance in MCF7 and C4.12.5 cells was done by scanning densitometry of the corresponding bands. The bars represent the mean ± S.E. of three independent experiments. p < 0.01 versus MCF7 cells.

FIGURE 3.

Coimmunoprecipitation analysis of IGF-IR and IR SUMOylation. Nuclear extracts of MCF7 and C4.12.5 (35 μg) were immunoprecipitated (IP) with anti-IGF-IR or anti-IR, electrophoresed through 10% SDS-polyacrylamide gels, and immunoblotted (WB) with anti-SUMO-1 (A and C) as described under “Experimental Procedures.” Membranes were reprobed with IGF-IR (B) or IR (D) antibodies.

FIGURE 4.

Confocal microscopy analysis of IGF-IR nuclear localization. A, fluorescence microscope imaging of IGF-IR and IR-expressing MCF7 cells by confocal immunofluorescence microscopy. B, amplified fluorescence imaging of IGF-IR-expressing MCF7 cells. C, fluorescence microscope imaging of IGF-IR- and IR-expressing C4.12.5 cells. Fixed cells were stained for DNA with PI (red) and for IGF-IR and IR with fluorescent goat anti-rabbit IgG (FITC) (green). Merging of pictures (FITC + PI) gives a yellow color with yellow grains in the nucleus. NRS was used as a negative control (A and C).

FIGURE 5.

Binding of IGF-IR and IR to IGF-IR promoter DNA. A, DNA affinity chromatography. Nuclear extracts of MCF7 and C4.12.5 cells were incubated with a PCR-amplified, biotin-labeled IGF-IR proximal promoter DNA probe extending from nt −458 to +53, after which DNA-protein complexes were adsorbed to streptavidin beads. Bound proteins were eluted with a high salt buffer, electrophoresed through 10% SDS-PAGE, and blotted with antibodies against IGF-IR or IR β-subunits. The left lanes in each gel correspond to nuclear extracts, and the right lanes represent the DNA affinity chromatography eluates. B and C, chromatin immunoprecipitation. MCF7 and C4.12.5 cells were cross-linked with formaldehyde, lysed, sonicated, and immunoprecipitated with IGF-IR (B), IR (C), or ERα (B) antibodies, followed by PCR amplification of precipitated chromatin using primers encompassing the IGF-IR promoter. The position of the 510-bp amplified fragments is indicated. The input bands represent the amplified PCR product in the absence of antibodies. Immunoprecipitated (IP) IGF-IR, IR, and ER were detected by Western blots (WB) using specific antibodies (insets).

FIGURE 6.

Effect of IGF-IR and IR levels on IGF-IR promoter activity. A, MCF7 and C4.12.5 cells were cotransfected with the p(−188/+640)LUC IGF-IR reporter construct, along with a full-length IGF-IR cDNA expression vector (GFP-IGF-IR) (or empty pcDNA3) and a β-galactosidase vector. Forty-eight hours after transfection, cells were harvested and luciferase and β-galactosidase activities were measured. Promoter activities are expressed as luciferase values normalized to β-gal values. A value of 100% was given to the promoter activity generated by the reporter plasmid in empty vector-transfected MCF7 or C4.12.5 cells. Bars, mean ± S.E. (error bars) of three independent experiments in duplicate wells. *, p < 0.05 versus control cells; **, p < 0.05 versus MCF7 cells transfected with GFP-IGF-IR. The inset shows a Western blot (WB) of C4.12.5 cells transfected with GFP-IGF-IR or GFP-pcDNA in comparison with endogenous IGF-IR expression. B, MCF7 and C4.12.5 cells were infected with an IR-containing adenoviral vector (or empty virus). After 24 h, the medium was changed, and cells were transfected with the p(−188/+640)LUC IGF-IR reporter construct for 48 h. Bars, mean ± S.E. of three independent experiments in duplicate wells. *, p < 0.01 versus control cells. The inset shows a Western blot using a specific IR antibody of MCF7 cells infected with the IR viral vector or empty vector in comparison with endogenous IR expression. C and D, MCF7 cells were transfected with the p(−188/+640)LUC IGF-IR promoter plasmid along with the full-length IGF-IR (C) or IR (D) vectors described above, in serum-containing medium. After 24 h, medium was replaced with serum-free medium, and cells were incubated for an additional 24 h in the presence of 50 ng/ml of IGF-I (C) or insulin (D) (or left untreated). After an additional 24 h, cells were harvested, and luciferase and β-galactosidase activities were measured as described above. A value of 100% was given to the promoter activities of expression vector-transfected cells in the absence of exogenous ligand treatments; *, p < 0.05 versus control cells.