Identification of differentially expressed proteins in retinoblastoma tumors using mass spectrometry-based comparative proteomic approach

Abstract

In India, retinoblastoma is among the top five childhood cancers. Children mostly present with extraocular extension and high risk features that results in unsatisfactory treatment and low survival rate. In addition, lack of potential therapeutic and prognostic targets is another challenge in the management of retinoblastoma. We studied comparative proteome of retinoblastoma patients (HPV positive and negative (n=4 each) and controls (n=4), in order to identify potential retinoblastoma-specific protein targets. 2D-DIGE coupled MALDI-TOF/TOF mass spectrometry identified 39 unique proteins. Highly deregulated proteins were GFAP,RBP3,APOA1,CRYAA,CRABP1,SAG and TF. Gene ontology (Panther 7.0) revealed majority of proteins to be associated with metabolic processes (26%) and catalytic activity (38%). 8 proteins were significantly upregulated in HPV positive vis-a-vis HPV negative cases. Patient group exhibited 12 upregulated and 18 downregulated proteins compared to controls. Pathway and network analysis (IPA software) revealed CTNNB1 as most significantly regulated signalling pathway in HPV positive than HPV negative retinoblastoma. The trends in transcriptional change of 9 genes were consistent with those at proteomic level. The Western blot analysis confirmed the expression pattern of RBP3,GFAP and CRABP1. We suggest GFAP,RBP3,CRABP1,CRYAAA,APOA1 and SAG as prospective targets that could further be explored as potential candidates in therapy and may further assist in studying the disease mechanism.

Significance: In this study we evaluated tumor tissue specimens from retinoblastoma patients and identified 39 differentially regulated proteins compared to healthy retina. From these, we propose RBP3, CRABP1, GFAP, CRYAA, APOA1 and SAG as promising proteomic signatures that could further be explored as efficient prognostic and therapeutic targets in retinoblastoma. The present study is not only a contribution to the ongoing endeavour for the discovery of proteomic signatures in retinoblastoma, but, may also act as a starting point for future studies aimed at uncovering novel targets for further therapeutic interventions and improving patient outcomes.

Keywords: 2D-DIGE; Biomarkers; Clinical proteomics; MALDI-TOF/TOF; Mass spectrometry; Retinoblastoma.