Fig 1: IMP3 exerts its proproliferative effects through the translational activation of IGF-2 mRNA. A, immunohistochemical of staining for IGF-2 for section from DA (a), 3/9 (33.3%) positive for staining; AA, 2/10 (20%) positive for staining (b); secondary GBM (c) and primary GBM (d), positive for staining (29/50; 58.00%). Including four normal brain samples (all four negative), ANOVA for difference in their IGF-2 positivity was carried out (p = 0.0239). Immunohistochemical staining for AKT expression for a section from DA (e), all cases positive for staining (9/9); AA, all cases positive for staining (10/10) (f); secondary GBM (g), and primary GBM (h), (30/34; 88.20%) positive for staining. Including four normal brain samples (all cases positive) ANOVA for difference in their Akt positivity was carried out (p = 0.4139). Immunohistochemical staining for phospho Akt for section from DA, 1/9 (11.1%) positive for staining (i); AA, 2/10 (20%) cases positive for staining (j); secondary GBM (k) and primary GBM (l), 23/34 (67.60%) positive for staining. Including four normal brain samples (all four negative), ANOVA for difference in their pAkt positivity was carried out. p = 0.0008). B, equal amounts of lysates for the vector and IMP3 overexpressing stable clones of U373 were subjected to Western blotting to detect the levels of IMP3 (anti-IMP3, Sigma), IGF-2 (anti-IGF-2, Santa Cruz Biotechnology), and actin (anti-actin HRP, Sigma). Fold change in the IGF-2 transcript levels obtained from RT-qPCR analysis in the IMP3-overexpressing and vector-stable clones relative to the control cells is depicted below. There is a significant increase in the IGF-2 protein levels upon IMP3 overexpression without a change in the IGF-2 transcript levels (compare lane 2 with 1). C, equal amounts of total protein lysates from cyclophilin (Cyc.) and IMP3 siRNA-transfected U138 cells at 120 h after transfection were subjected to Western blotting to detect levels of IGF-2 (anti-IGF-2, Abcam), IMP3 (anti-IMP3, Sigma), and proliferating cell nuclear antigen (anti-proliferating cell nuclear antigen, oncogene) proteins. The bottom panel shows the fold change in the IGF-2 transcript levels obtained from RT-qPCR analysis of the siRNA transfected U138 cells at 120 h. There is a significant decrease in the IGF-2 protein levels upon IMP3 knockdown without a change in the IGF-2 transcript levels (compare lane 2 with lane 1). D, fold change in the real-time RT-qPCR-based quantitation of the IGF-2 transcript levels in the IMP3 and control antibody immunoprecipitates is plotted. LN229 cells were infected with a control adenovirus and an Ad-IMP3 adenovirus and IMP3 and the control antibody immunoprecipitates were used to analyze for the IGF-2 transcript levels. E, U138 cells were transfected with IMP3 or cyclophilin siRNA, and 4 days after siRNA transfection, the cell lysates were made and subjected to polysomal fractionation. Relative fold change in the IGF-2 transcript levels between cyclophilin and IMP3 siRNA-transfected cells within polysome or non-polysome fractions are shown. F, H1299 vector and IMP3-stable cells were treated with 50 µg/ml cycloheximide (CHX) for 10, 18, and 24 h. Equal amounts of lysates were subjected to Western blotting to detect the levels of IGF-2 and actin.
Fig 2: IMP3 exerts its proproliferative effects through the IGF-2-PI3K and IGF-2-MAPK cascades. A, viability was measured by MTT assay at indicated time points for H1299 vector and IMP3 stable clones treated with a neutralizing antibody for IGF-2 as indicated. The assays were carried out in triplicate, and the mean value for each cell type at each time point was used to generate the graph. A one-way analysis of variance was carried out to test the significance of the observed differences between the groups and a p = 0.05 is represented with an asterisk, p = 0.01 is represented as double asterisks and p = 0.0001 is represented as triple asterisks. B, viability was measured by MTT assay as an indicator of cell proliferation at the indicated time points for either mock, cyclophilin (cyclo.), or IMP3 siRNA-transfected U138 cells. IGF-2 was exogenously supplemented as indicated to monitor its ability to rescue the effects of IMP3 knockdown. A one-way analysis of variance was carried out to test the significance of the observed differences between the groups. A p value = 0.05 is represented as an asterisk, which is significant, and NS refers to nonsignificant difference. C, equal amounts of total protein lysates from cyclophilin and IMP3 siRNA-treated U138 cells were subjected to Western blotting with the indicated antibodies. Note that the intensity of phospho-mTOR, phospho-Akt, phospho-4EBP1, phospho-MEK1/2, and phospho-ERK1/2 bands reduces in lane 2 compared with lane 1, whereas that of total the corresponding total proteins remain unchanged. D, viability was measured by MTT assay on day 9 after plating for the U373 vector clone (#6) and IMP3 stable clones (#5 and 17) treated with dimethyl sulfoxide (vehicle), LY294002 (20 µm), or U0126 (10 µm). Please note that dimethyl sulfoxide (DMSO)-treated IMP3 stable clone (#5 and #17) grow faster than vector stable clone (compare light gray bars). Both LY294002 and U0126 treated IMP3 stable clone (#5 and #17) failed to show faster growth than vector stable clone (compare dark gray and black bars with light gray bars).
Fig 3: IGF2 activated downstream signaling mainly through IGF1R(A) IGF1R inhibitor NVP-AEW541 blocked the proliferations of G401 and BT16 cell lines. G401 and BT16 cells were cultured in a/Ham medium supplemented with 10% FBS containing DMSO or NVP-AEW541 at indicated concentrations (0.5, 1, 2.5, 5 µM). Triplicate dishes were counted in a haemocytometer at Day 3. Each point is represented as the mean ± SD. (B) Activities of downstream signaling in G401 and BT16 cells upon the treatment of NVP-AEW541 in absence or presence of IGF2. Cells were starved for 12 h, and medium was changed again 2 hours before stimulation. Vehicle control DMSO or IGF1R inhibitor NVP-AEW541 (0.5, 1, 2.5, 5 µM) was added 1 hour before rhIGF2 stimulation. Then G401 and BT16 cell lines were treated with 200 ng/ml rhIGF-2 or 10% FBS under serum-free conditions for 15mins. Activation of AKT and ERK1/2 were detected by western blot analysis. ß-actin served as a loading control. A representative example from 3 independent experiments is shown.
Fig 4: The serum-free growth of MRT cell lines is accompanied by the activation of IGF2 axis(A) Growth curve of HEK293T, G401 and BT16 in a/Ham medium containing 10% FBS (-10% FBS) or serum-free a/Ham medium (-SFM). Cells were plated out in 60 mm dishes as described in Methods. After attached overnight, all plates were washed and medium replaced with serum-free a/Ham medium. 24 hours later medium was changed again in the presence or absence of serum. Medium was changed daily for the indicated days. On each day triplicate dishes were counted in a haemocytometer. Data represent the mean ± SD. (B) Serum deprivation induced IGF2 axis activation. G401, BT16, HEK293T were cultured with a/Ham medium containing 10% FBS or with serum-free a/Ham medium for 72 hours. The IGF2, IGF1R, INSR, IGF2R mRNA levels, determined by reverse transcription quantitative real-time PCR in three independent experiments, are shown as fold change on the graph. Data represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. (C) The protein levels of SNF5, IGF1R, INSR and IGF2R in control and starved G401, BT16, HEK293T cells. Cells were seeded into 60 mm dishes. After attachment, cells were cultured for 72 hours with a/Ham medium containing 10% FBS or with serum-free a/Ham medium. The expression levels of SNF5, IGF1R, INSR and IGF2R were investigated by western blot analysis. ß-actin served as a loading control. A representative example from 3 independent experiments is shown. (D) The expression levels of IGF1R and INSR following serum-free treatment for the indicated days. G401 were cultured with a/Ham medium containing 10% FBS (lane 1) or with serum-free a/Ham medium (lane 2-5) for 1-4 days. Upregulation of IGF1R and INSR were analyzed by immunoblotting. ß-actin served as a loading control. A representative example from 3 independent experiments is shown.
Fig 5: Schematic illustration of IGF2 axis, downstream kinase pathways, potential therapeutic targets in MRT cellsIn the SNF5/INI1/SMARCB1/BAF47 deficient MRT cells, IGF2 was upregulated when cells suffer from microenvironmental stress including serum deprivation and chemotherapeutic agent exposure. The autocrine IGF2 in turn activated intracellular PI3K/AKT and RAS/ERK pathways through IGF1R and INSR. These two pathways further contribute to survival, proliferation and anti-apoptosis of the tumor cells. Inhibiting this pathway, including IGF2 neutralizing antibody, IGF1R inhibitor NVP-AEW541 and AKT inhibitor MK-2206 2HCl, effectively abolished the ability of the tumor cells to grow in vitro.
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