Abstract
Purpose :
Retinoblastoma (Rb) is the most common primary intraocular malignancy in children. Although highly curable in the United States, there is a high mortality rate in developing countries due to metastatic disease. As a highly angiogenic tumor, anti-VEGF therapies have shown limited success in clinical setting thus illustrating the need for more novel anti-angiogenic therapeutics. We hypothesize that signaling through platelet-derived growth factor-beta (PDGFR-b) underlies angiogenesis and may be a potential adjuvant therapy for Rb.
Methods :
Two Rb cell lines were used: Y79, representing the metastatic form of the disease, and Weri-1-Rb as the non-metastatic control. We investigated the role of the PDGFR- b in the control of angiogenesis and metastasis by culturing cells in the presence of imatinib mesylate –IM- (Sigma-Aldrich, St. Louis, MO), an inhibitor of kinases encoded by the BCR-ABL and PDGFR oncogenes. To address the effect of IM in angiogenesis we measured the expression of angiogenesis-related genes, including VEGFA, PDGFA, PDGFB by qPCR analysis. Additional tests included Western blot, Multiplex-Luminex to assay for protein secretion. Lastly, we performed CytoSelect™ wound-healing assay (Cell Biolabs, Inc., San Diego, CA) to investigate the migration potential of the cells after blockade of the PDGFR-b by IM.
Results :
Initial work showed a dose-dependent effect in proliferation and toxicity in both cell lines, determined by Ki-67 (flow cytometry) and CellTiter 96® AQueous One Solution (Promega, Wisconsin). Western blot analysis demonstrated a post-transcriptional effect on the Rb cells with a significant reduction in the phosphorylation of PDGFR-β (p-PDGFR-β) while mRNA levels of both PDGFB and PDGFRB remained unchanged. Unexpectedly, we observed an increase in PDGFRA mRNA, illustrating a potential compensatory signaling pathway. Protein analyses revealed a reduction in VEGF (untreated versus IM: 120.89±13.82 versus 80.02±30.54, p=0.08), while an increase in PDGF-AA (untreated versus IM: 4.25±1.33 versus 12.54±1.52, p=0.001), in the metastatic line, suggesting a reduction in VEGF-dependent angiogenesis.
Conclusions :
Our results are consistent with our hypothesis that inhibition of PDGFR-β impacts cell signaling and survival. Further experimentation is needed to delineate molecular pathways in both metastatic and non-metastatic cells to determine clinical targets.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.