Abstract 1110: Molecular and functional alterations associated with repression of tumor suppressor p16INK4a during immortalization of human endothelial cells
Authors: Wen Victoria W. , Kan Flora , Pasquier Eddy , Chang Melissa , Kavallaris Maria , Mackenzie Karen L. 
Published: February 2011

The immortalization of endothelial cells is central to the development of endothelial cell-derived malignancies, such as hemangioblastoma and angiosarcoma. Through our previous studies, we have developed a panel of immortal bone marrow-derived endothelial cells (BMECs) that model molecular aspects of these malignancies. Among BMECs transduced with telomerase reverse transcriptase, ∼40% of the cell lines silenced p16INK4a during immortalization, while inactivation of p53 was a much rarer event, occurring in only 1 out of 12 clones. Loss of p16INK4a expression was associated with the development of complex karyotypes that included recurring abnormalities in chromosomal regions that are frequently altered in angiosarcoma, including +20, -Y and genetic loss at 13p11, 17p13 and 19q13. In the present study, we have investigated whether loss of p16INK4a expression conferred immortal BMECs with malignant properties. Comparison of immortal clones revealed that p16INK4a-negative cell lines had a more transformed phenotype, which featured a higher rate of proliferation, reduced growth factor requirements and impaired angiogenic capacity in an in vitro assay. siRNA-mediated suppression of p16INK4a expression also impaired the angiogenic capacity of normal BMECs and a BMEC cell line that retained p16INK4a during immortalization, thus confirming the link between p16INK4a and endothelial cell morphogenesis. To further define molecular alterations that occurred during immortalization and/or identify molecular events that may have contributed to functional alterations in p16INK4a negative clones, array-based comparative genomic hybridization, and fine tiling of recurring chromosome break points were performed. These analyses refined regions such as 17p13, where 22 genes were found to be deleted in the p16INK4a-negative cells. The deleted genes in this region included the putative tumor suppressor TUSC5 and the gene encoding the adapter protein 14-3-3ε. A four-way comparison of the proteomes of a p16INK4a-positive and a p16INK4a-negative cell line at early and late passage time points using two-dimensional differential in gel electrophoresis (2D DIGE) complemented the genomic analyses. Cytoskeletal proteins were predominant on the list of proteins that were differentially expressed in p16INK4a-negative versus p16INK4a-positive immortal BMECs. Overall our investigations describe previously unreported molecular and functional changes that occur in association with repression of p16INK4a during immortalization of endothelial cells. The loss of p16INK4a expression was associated with the development of a more transformed phenotype. Novel molecular alterations that have been identified in this study warrant further investigation as candidates that may have contributed to the phenotypic changes observed in p16INK4a-immortal BMECs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1110.