Abstract
Purpose :
Tumor-associated adhesion G-protein coupled receptors (a-GPCRs), such as EMR2, are known to mediate migration, invasion and metastasis. Many of these cellular processes take place in retinoblastoma tumor cells as they have the ability to detach from the primary tumor mass and metastasize to the brain. Our study seeks to gain insight on different a-GPCRs, EMR2, GPR125 and GPR97, and their role in regulating the invasive properties of this intraocular tumor. We hypothesize that changes in the expression and localization of a-GPCRs within retinoblastoma cells contribute to their invasive phenotype.
Methods :
Gene expression levels of a-GPCRs in retinoblastoma tumors were elucidated by conducting a Robust Microarray Analysis (RMA). Fetal retinas were used as controls. Western Blot Assays using specific antibodies against a-GPCRs was used to assess relative protein expression levels in the most invasive, Y79, and the least invasive, Weri retinoblastoma cells (N=4). Localization of endogenous a-GPCRs in both cell types was addressed by immunolabeling (N=3). Images were taken using total internal reflection fluorescence or confocal microscopy. Finally, transwell assays were used to determine the role of a-GPCRs in retinoblastoma cell migration.
Results :
RMA gene expression analysis showed that GPR125 is upregulated in retinoblastoma tumors when compared to EMR2 and GPR97. Western blot results show that while GPR125 is elevated in the most invasive Y79 retinoblastoma cells (p<0.05), EMR2 is higher, but not significantly different, in the less invasive, Weri cells. GPR97 levels are also high in Weri cells (N=4). Immunocytochemistry results show that GPR125 differentially distributes in Y79 versus Weri cells; such that it localizes in presumptive leading edges of the Y79, whereas it is more diffuse within Weri cells (N=3). On the other hand, EMR2 and GPR97 are highly enriched in presumptive leading edges of both cell types (p<0.01).
Conclusions :
Our results show that GPR125 high expression and polarized distribution in Y79 may contribute to their invasive phenotype, whereas EMR2 and GPR97 high expression and polarized distribution in Weri cells may contribute to their adhesive phenotype. Should these receptors show a role in retinoblastoma cell adhesion and migration, regulation of their expression and localization will provide novel drug targets to prevent optic nerve invasion and hence, brain metastasis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.