Identification Of Differentially Expressed Protein Targets In HPV Infected Retinoblastoma Using 2D-DIGE Coupled Mass Spectrometry Approach

Abstract

Purpose: In India, population based cancer registries have reported retinoblastoma (RB) among the top five childhood cancers. There are reports on association of RB with Human Papilloma Virus (HPV) and abrogation of pRB function. But no reports are available on the proteomic profile of HPV positive and negative RB. We hypothesize that HPV may play a role in development of RB with several proteins differentially expressed in patients and controls that may act as potential candidates for therapy.

Methods: Fresh RB tumor and normal retinal tissues (n=42each) were recruited. Screening of 21 different HPV genotypes was done using HPV genoarray kit. Proteomic analysis was performed on four samples each from HPV positive, HPV negative RB and controls. 2D-DIGE coupled MALDI- TOF/TOF mass spectrometry was employed. Dye swapping was done. Image analysis was done using Decyder 2D software. Differentially expressed spots were identified and picked. Using PCA, the two tumor groups could be delineated from normal tissue based on protein expression pattern. mRNA expression of the identified proteins was verified and some selected proteins were validated by western blot as well.

Results: Disease was bilateral in 33% cases. Of the 39 eyes with non- familial RB, 25.6% tested positive for HR HPV16. Among the three groups, 102 protein spots were differentially regulated (p<0.05,±1.5 folds) constituted by 39 unique proteins determined by MALDI-TOF/TOF. 12 proteins were up regulated in HPV positive cases vis-a-vis HPV negative. Patient group exhibited upregulation of 7 proteins compared to controls. Highly deregulated proteins were GFAP, RBP3, CRABP1, SAG and TF. Significant mRNA levels (p<0.05) of RBP3,GFAP,CRABP1,PDIA3,ATP5B,PITPNA were observed in RB compared to normal. Gene ontology revealed majority of proteins to be associated with metabolic processes (26%) and catalytic activity (38%). Pathway analysis identified to be the proteins involved in acute phase signalling in tumor(p=1.42-08). Whereas CTTNB1 and TP53 signalling pathways were more significantly regulated in HPV positive than HPV negative RB.

Conclusions: The study provides a dynamic protein profile of retinoblastoma (HPV positive and negative) and highlights significantly relevant protein targets like GFAP, RBP3 and CRABP1. Their prospects of being used as potential candidates in therapy needs to be further explored.