Abstract
Purpose: :
Recent evidence points to the presence of putative cancer stem cell population in Retinoblastoma (Rb) that are characterized by the size and phenotype of cells, gene expression pattern, metastatic pathways and to some extent, response to drugs. In this study, we aim to evaluate the role of Nodal/Activin pathway in Rb, which is known to play a crucial role in development, neural patterning and maintaining the undifferentiated state of human Embryonic Stem Cells (hESCs).
Methods: :
After IRB approval and informed consent, freshly isolated tumor and normal appearing retina (n=2) were harvested from enucleated eyes of patients with Rb (n=11). Total RNA was isolated and processed according to manufacturer’s protocol. Microarray was performed using human whole genome (4x44K) cDNA arrays (Agilent technologies, USA) and data was normalized and analyzed. Real-Time PCR was done (using RNA from tissues) to validate the results of gene expression (for expression of Nodal receptor- Activin receptor 1C (ACVR1C), HMGA2, LIN-28band N-Myc) in tissues compared to the two control retinas. Further, immunolocalization for ACVR1C expression on tissue sections (n=19).
Results: :
In Rb tumors, of the 10,457 genes that were deregulated (p ≤ 0.05 and fold change ≥ 1.5 folds), significant up-regulation of ACVR1C (2.23 fold) and its downstream effector, SMAD3(2.43 fold) was observed. The ligands for this pathway such as NODAL and TGFB were found to be unregulated and the inhibitor, LEFTY, was unregulated in these tumors. ACVR1C showed higher expression in poorly differentiated tumors as compared to well differentiated tumors. Also, HMGA2 (48.6 fold) and its downstream regulator, LIN-28b (2.1 fold), were found to be highly up-regulated in primary tumors.
Conclusions: :
This study highlights the deregulation of genes involved in Nodal signaling such as ACVR1C, SMAD3, LEFTY, etc. This is in concordance with evidence of elevated Nodal pathway in highly aggressive gliomas, ovarian, pancreatic tumors, etc. Further functional studies, specifically in the cancer stem cells population in Rb, would improve the understanding of tumorigenesis and possibly pave way for targeted therapy.
Keywords: retinoblastoma • gene/expression • immunohistochemistry