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Insulin like growth factor binding protein 4 promotes GBM progression and regulates key factors involved in EMT and invasion

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Abstract

Insulin like growth factor binding protein 4 (IGFBP4) regulates growth and development of tissues and organs by negatively regulating IGF signaling. Among most cancers, IGFBP4 has growth inhibitory role and reported as a down-regulated gene, except for renal cell carcinoma, wherein IGFBP4 promotes tumor progression. IGFBP4 expression has been shown to be higher in increasing grades of astrocytoma. However, the functional role of IGFBP4 in gliomas has not been explored. Surgical biopsies of 20 normal brain and 198 astrocytoma samples were analyzed for IGFBP4 expression by qRT-PCR. Highest expression of IGFBP4 mRNA was seen in GBM tumors compared to control brain tissues (median log2 of 2.035, p < 0.0001). Immunohistochemical analysis of 53 tissue samples revealed predominant nuclear staining of IGFBP4, seen maximally in GBMs when compared to DA and AA tumors (median LI = 29.12 ± 16.943, p < 0.001). Over expression of IGFBP4 in U343 glioma cells resulted in up-regulation of molecules involved in tumor growth, EMT and invasion such as pAkt, pErk, Vimentin, and N-cadherin and down-regulation of E-cadherin. Functionally, IGFBP4 over expression in these cells resulted in increased proliferation, migration and invasion as assessed by MTT, transwell migration, and Matrigel invasion assays. These findings were confirmed upon IGFBP4 knockdown in U251 glioma cells. Our data suggest a pro-tumorigenic role for IGFBP4 in glioma.

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Acknowledgments

We gratefully acknowledge the help of Neurosurgeons Drs. B.S.Chandramouli, A.S. Hedge, A. Arivazhagan, and K. Prasanna for the glioma tissues. We thank Profs. Kumaravel Somasundaram and M.R.S. Rao for useful suggestions during the course of the study. We thank Sarwat Naz for help with ICC, Meenakshi for confocal microscopy facility, Sreekanth Reddy, Harish Srinivasan for RT-PCR data analysis, B.S. Shailaja, Vrinda, Lakshmi and Cini Samuel for help with the patient sample collection. Department of Biotechnology (DBT), India. Infrastructure support was from Department of Science and Technology (under FIST program), DBT, and University Grants Commission (DRS) are acknowledged. VRPK received DBT post-doctoral fellowship.

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All the Authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560 012, India

    V. R. Praveen Kumar, Priyanka Sehgal, Shilpa Patil & Paturu Kondaiah

  2. Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore, India

    Balram Thota & Vani Santosh

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  1. V. R. Praveen Kumar
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  2. Priyanka Sehgal
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Correspondence to Paturu Kondaiah.

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Supplementary material 1 Table S1: Primer pairs of genes analyzed by RT-PCR (DOCX 13 kb)

11060_2013_1324_MOESM2_ESM.tif

Supplementary material 2 Fig. S1: IGFBP4 expression in U343 and U251 cells upon IGFBP4 over expression and knockdown respectively. a, U343 and b U251 cells were plated on coverslips and allowed to grow. After transfection cells were fixed, permeabilized, stained for IGFBP4 expression. Expression of IGFBP4 is shown in green. Nucleus was stained using propidium iodide (PI), shown in red. Original magnification was 63X. (TIFF 4438 kb)

11060_2013_1324_MOESM3_ESM.tif

Supplementary material 3 Fig. S2: Gene expression changes upon IGFBP4 over expression in U343 cells (a) and knockdown in U251 cells (b). a, Cells were plated and 24 h later transfected with pCMV6-AC-IGFBP4and/or vector. 48 h post transfection, RNA was extracted and gene expression was analyzed by Semi quantitative RT-PCR analysis. b, Cells were plated and 24 h later transfected with IGFBP4 siRNA and/or control siRNA. 96 h post transfection, RNA was extracted and gene expression was analyzed by Semi quantitative RT-PCR analysis. Representative ethidium bromide gel shows the expression of genes regulated upon forced expression of IGFBP4 in U343 (a) and upon IGFBP4 knockdown in U251 glioma cells (b). RPL35a and HPRT expression served as normalizing genes. (TIFF 6968 kb)

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Praveen Kumar, V.R., Sehgal, P., Thota, B. et al. Insulin like growth factor binding protein 4 promotes GBM progression and regulates key factors involved in EMT and invasion. J Neurooncol 116, 455–464 (2014). https://doi.org/10.1007/s11060-013-1324-y

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  • DOI: https://doi.org/10.1007/s11060-013-1324-y

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