Estrogen receptor beta-mediated nuclear interaction between IRS-1 and Rad51 inhibits homologous recombination directed DNA repair in medulloblastoma

Abstract

In medulloblastomas, which are highly malignant cerebellar tumors of the childhood genotoxic treatments such as cisplatin or gamma-irradiation are frequently associated with DNA damage, which often associates with unfaithful DNA repair, selection of new adaptations and possibly tumor recurrences. Therefore, better understanding of molecular mechanisms which control DNA repair fidelity upon DNA damage is a critical task. Here we demonstrate for the first time that estrogen receptor beta (ERbeta) can contribute to the development of genomic instability in medulloblastomas. Specifically, ERbeta was found highly expressed and active in mouse and human medulloblastoma cell lines. Nuclear ERbeta was also present in human medulloblastoma clinical samples. Expression of ERbeta coincided with nuclear translocation of insulin receptor substrate 1 (IRS-1), which was previously reported to interfere with the faithful component of DNA repair when translocated to the nucleus. We demonstrated that ERbeta and IRS-1 bind each other, and the interaction involves C-terminal domain of IRS-1 (aa 931-1233). Following cisplatin-induced DNA damage, nuclear IRS-1 localized at the sites of damaged DNA, and interacted with Rad51--an enzymatic component of homologous recombination directed DNA repair (HRR). In medulloblastoma cells, engineered to express HRR-DNA reporter plasmid, ER antagonist, ICI 182,780, or IRS mutant (931-1233) significantly increased DNA repair fidelity. These data strongly suggest that both molecular and pharmacological interventions are capable of preventing ERbeta-mediated IRS-1 nuclear translocation, which in turn improves DNA repair fidelity and possibly counteracts accumulation of malignant mutations in actively growing medulloblastomas.

Fig. 1

Immunohistological analysis of human medulloblastoma biopsies. Immunohistochemistry was performed using the avidin–biotin–peroxidase complex system. The sections of normal human cerebellum and two medulloblastoma biopsies were immunolabeled with anti-IRS-1 antibody (rabbit polyclonal, Upstate Biotechnology, Temecula, CA), and anti-ERβ antibodies (rabbit polyclonal, Abcam, Cambridge, MA), and microscopic evaluation was performed under the original magnification of 1,000×.

Fig. 2

Immunohistofluorescent detection of IRS-1 and ERβ. Double immunofluorescent labeling was performed with anti-ERβ (green fluorescence) and anti-IRS-1 (red fluorescence) antibodies using medulloblastoma clinical sample #9 from Table 1. Image marked “Double labeling” represents superimposition of two images in which ERβ and IRS-1 were detected. Note the presence of a large number of medulloblastoma nuclei in which ERβ and IRS-1 are co-expressed in the nucleus (yellow fluorescence).

Fig. 3

Protein levels and subcellular localization of ERs and IRS-1 in medulloblastoma cell lines. Part A: Western blot analysis showing ERβ, ERα, and IRS-1 protein levels in three human (Daoy, D384, D425) and two mouse (Bs-1a and BsB8) medulloblastoma cell lines. Human breast cancer cell line, MCF7, was used as a positive control. Loading conditions are verified by re-probing the same blot with anti-Grb-2 antibody. Part B: Immunocytofluorescent evaluation of ERβ in D384 and BsB8 cells growing in the presence of 10% FBS. D384 cells collected by cytospin on poly-D-lysine coated slides, and BsB8 monolayer cultures were both fixed and immunolabeled with anti-ERβ mouse monoclonal antibody (green fluorescence). The nuclei were counterstained with DAPI (blue fluorescence).

Fig. 4

The activity of estrogen responsive elements (EREs). The ERE transcriptional activity was evaluated in D384, BsB8, and Daoy cells by dual-Firefly/Renilla luciferase reporter assay (Promega). The cells were transfected with 50 ng of pEREluc plasmid (Morelli et al., 2004) using Amaxa nucleoporator according the manufacturer recommendations, and were cultured in the presence of 10% FBS for 24 h. Subsequently the culture medium was changed to SFM and cells were stimulated with 100 mM estradiol (E2), 10 μM ICI 182,780 (ICI), or were left without stimulation (control). The measurements of luciferase activity were taken 6 h after stimulation. Results represent an average of three separate experiments in duplicate (n =6). *Indicates values significantly different from the control (P ≤ 0.05).

Fig. 5

Evaluation of IRS-1–ERβ interaction. Part A: The interaction between ERβ and IRS-1 evaluated by Immunoprecipitation/Western blot analysis (IP/W). Total proteins (500μg/ml) were IP with anti-ERβ antibody, and the resulting nitrocellulose filters were probed with anti-IRS-1 and anti-ERβ antibodies, respectively. In control conditions, 50 μg aliquots of the protein extract were loaded without IP (No IP)—positive control; or IP was carried out without anti-ERβ antibody (No Ab)—negative control. Parts B and C: Double immunolabeling with anti-ERβ (green) and anti-IRS-1(red)antibodies(B), and with anti-IRS-1(green)andanti-histone-γH2AXantibodies(red) (C). The D384 and Daoy cells were cultured in 10% FBS and treated with cisplatin (1 μg/ml for 6 h). Images labeled “Overlap” represents superimposition of three images, which consist of either ERβ andIRS-1(B); or IRS-1 and γH2AX immunolabelings(C), and DAPI labeled DNA(blue). Digital sections (2μm) from D384 and Daoy cells were collected by the inverted fluorescent microscope equipped with motorized Z-axis and the deconvolution software (SlideBook4). Note possible co-localization (yellow fluorescent foci) between IRS-1 and ERβ detected in the nuclei of exponentially growing Daoy and D384 cells, or between IRS-1 and histone γH2AX detected in the nuclei of exponentially growing Daoy cells.

Fig. 6

Nuclear interaction between IRS-1 and Rad51. Part A: Double immunolabeling with anti-Rad51 (green) and anti-IRS-1 (red) antibodies was applied to exponentially growing Daoy cells at 6 h after the treatment with cisplatin (1 μg/ml). The image marked “Overlap” represents superimposition of three images in which IRS-1, Rad51, and DAPI labeling were applied. A digital section from a single Daoy cell was obtained by inverted fluorescent microscope equipped with motorized Z-axis and the deconvolution software (SlideBook4). Part B: Quantification of the co-localization between IRS-1 and Rad51 was calculated from the entire volume of the nucleus by utilizing Slide Book 4 software (Intelligent Imaging Innovations, Denver CO), according to the manufacturer instructions for mask operations. Data represent average values obtained from the evaluation of 10 randomly selected cells in three independent experiments (n =30). Part C: Western blot analysis with proteins isolated from nuclear (N) and cytosolic (C) fractions of exponentially growing Daoy cells in the presence (FBS +ICI) or absence (FBS) of ERαβ/antagonist, ICI 182,780. The blot was probed first with anti-IRS-1 antibody and was re-probed with anti-ERβ antibody (predominant nuclear localization), and with anti-Grb-2 antibody (cytosolic marker) to evaluate potential contamination of the nuclear fraction with cytosolic fraction. Note that cells treated with ICI 182,780 are characterized by a much lower level of nuclear IRS-1. Part D: The interaction between Rad51 and IRS-1 evaluated by IP/Western. Total proteins (500 μg/ml) were IP with anti-Rad51 antibody, and resulting nitrocellulose filters were probed with anti-IRS-1 and anti-Rad51 antibodies. In control conditions, 50 μg aliquots of the protein extract were loaded without IP (Western)—positive control; or IP was carried out without anti-Rad51 antibody (No Rad51 Ab)—negative control. Note that there is significantly less IRS-1, which co-precipitates with anti-Rad51 antibody in Daoy cells pretreated with ICI 182,780 (n =3). * Indicates value significantly different from FBS. ** Indicate value significantly different from E2 (P ≤ 0.05).

Fig. 7

ERβ–IRS-1 interaction affects HRR. Part A: GST-pull down assay was performed to determine which portion of the IRS-1 molecule binds ERβ. A collection of GST–IRS-1 over lapping truncation mutants (Lassak et al., 2002) and nuclear extracts from Daoy cells were utilized to pull down ERβ. Following pull down reaction one of the five IRS-1 truncation mutants, GST-IRS-1 (931–1233), bound ERβ. All other mutants and GST alone were negative. Part B: Schematic illustration of the pcDNA3/IRS-1-931–1233 —the expression vector in which the 931–1233 C-terminal fragment of IRS-1 was cloned in frame with Myc tag under the control of CMV promoter. Part C: Western blot analysis with protein extracts from Daoy cells expressing the 931–1233/IRS-1 mutant. The cells were transfected in transient (Amaxa nucloporator) with the pcDNA3/IRS-1-931–1233 or with pcDNA3 empty vector (EV). Proteins were isolated at 24, 48, and 72 h after transfection and the resulting blots were probed with anti-IRS-1 rabbit polyclonal antibody, which recognizes C-terminal domain of IRS-1 (UBI, Lake Placid, NY). The cells transfected with 931–1233/IRS-1 mutant, express endogenous IRS-1 (160 kDa band) and expected 30 kDa band (931–1233 IRS-1). Two lower bands were detected as well. These additional bands may represent products of restricted proteolysis from the N-terminal end of this fusion protein, since the same blot striped and re-probed with anti myc-tag antibody, demonstrated the same pattern and intensity of the bands. Note that myc-tag was cloned at the C-terminus of 931–1233 IRS-1. Part D: Homologous recombination directed DNA repair (HRR) was evaluated by the DRGFP reporter system. Daoy/DRGFP cells growing in 10%FBS (control) were additionally treated with17β-estradiol (E2)(100mM), ICI 182,780(10 μM)or were transfected withthepcDNA3-IRS-1-931–1233 expression vector. To evaluate HRR, the cells were transfected in transient (Amaxa) with I-SceI and mito-red expression vectors, and the reconstitution of GFP function was analyzed under fluorescent microscope and quantified by flow-cytometry. The results were collected from three separate experiments, induplicate (n =6). Note a significant increase in HRR in Daoy cells treated either with ICI182, 780 or transfected with the 931–1233-IRS-1 mutant. * Indicates values significantly different from control. ** Indicate values significantly different from EV (P ≤ 0.05). The inset in Figure 6D illustrates an example of HRR-mediated reconstitution of GFP function in Daoy/DRGFP cells treated with ICI 182,780.