BackgroundRetinoblastoma is an ocular neoplastic cancer caused primarily due to the mutation/deletion of RB1 gene. Due to the rarity of the disease very limited information is available on molecular changes in primary retinoblastoma. High throughput analysis of retinoblastoma transcriptome is available however the proteomic landscape of retinoblastoma remains unexplored. In the present study we used high resolution mass spectrometry-based quantitative proteomics to identify proteins associated with pathogenesis of retinoblastoma.MethodsWe used five pooled normal retina and five pooled retinoblastoma tissues to prepare tissue lysates. Equivalent amount of proteins from each group was trypsin digested and labeled with iTRAQ tags. The samples were analyzed on Orbitrap Velos mass spectrometer. We further validated few of the differentially expressed proteins by immunohistochemistry on primary tumors.ResultsWe identified and quantified a total of 3587 proteins in retinoblastoma when compared with normal adult retina. In total, we identified 899 proteins that were differentially expressed in retinoblastoma with a fold change of ≥2 of which 402 proteins were upregulated and 497 were down regulated. Insulin growth factor 2 mRNA binding protein 1 (IGF2BP1), chromogranin A, fetuin A (ASHG), Rac GTPase-activating protein 1 and midkine that were found to be overexpressed in retinoblastoma were further confirmed by immunohistochemistry by staining 15 independent retinoblastoma tissue sections. We further verified the effect of IGF2BP1 on cell proliferation and migration capability of a retinoblastoma cell line using knockdown studies.ConclusionsIn the present study mass spectrometry-based quantitative proteomic approach was applied to identify proteins differentially expressed in retinoblastoma tumor. This study identified the mitochondrial dysfunction and lipid metabolism pathways as the major pathways to be deregulated in retinoblastoma. Further knockdown studies of IGF2BP1 in retinoblastoma cell lines revealed it as a prospective therapeutic target for retinoblastoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-016-9128-7) contains supplementary material, which is available to authorized users.
The present study, is the first comprehensive quantitative membrane proteomic atlas of the differentially regulated proteins in RB compared to the retina. LMNB1 and TFRC could be potential biomarkers for this childhood cancer.
In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus–thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3’UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM +ve/let-7bdown-regulated), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM −ve/let-7bup-regulated), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM +ve/let-7bup-regulated) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.
Inhibition of the interaction between p53 and HDM2 is an effective therapeutic strategy in cancers that harbor a wild-type p53 protein such as retinoblastoma (RB). Nanoparticle-based delivery of therapeutic molecules has been shown to be advantageous in localized delivery, including to the eye, by overcoming ocular barriers. In this study, we utilized biocompatible gold nanoparticles (GNPs) to deliver anti-HDM2 peptide to RB cells. Characterization studies suggested that GNP-HDM2 was stable in biologically relevant solvents and had optimal cellular internalization capability, the primary requirement of any therapeutic molecule. GNP-HDM2 treatment in RB cells in vitro suggested that they function by arresting RB cells at the G2M phase of the cell cycle and initiating apoptosis. Analysis of molecular changes in GNP-HDM2-treated cells by qRT-PCR and western blotting revealed that the p53 protein was upregulated; however, transactivation of its downstream targets was minimal, except for the PUMA-BCl2 and Bax axis. Global gene expression and in silico bioinformatic analysis of GNP-HDM2-treated cells suggested that upregulation of p53 might presumptively mediate apoptosis through the induction of p53-inducible miRNAs.
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