Proteomics analysis of H-RAS-mediated oncogenic transformation in a genetically defined human ovarian cancer model

Abstract

RAS is a small GTP binding protein mutated in approximately 30% human cancer. Despite its important role in the initiation and progression of human cancer, the underlying mechanism of RAS-induced human epithelial transformation remains elusive. In this study, we probe the cellular and molecular mechanisms of RAS-mediated transformation, by profiling two human ovarian epithelial cell lines. One cell line was immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase (T29), while the second cell line was transformed with an additional oncogenic ras V12 allele (T29H). In total, 32 proteins associated with RAS-mediated transformation have been identified using peptide mass fingerprinting. These protein targets are involved in several cellular pathways, including metabolism, redox balance, calcium signaling, apoptosis, and cellular methylation. One such target, the 40 kDa procaspase 4 is significantly upregulated at the protein level in RAS-transformed T29H cells, related directly to signaling through MEK, but not PI3 kinase. Cellular caspase 4 activity is, however, suppressed in the T29H cells, suggesting that the maturation process of caspase 4 is abrogated in RAS-transformed T29H cells. Consistent with this notion, transformed T29H cells were less susceptible to the toxic effects of anti-Fas antibody than were immortalized, nontransformed T29 cells, associated with less activation of caspase 4. This study demonstrates that functional proteomic analysis of a genetically defined cancer model provides a powerful approach toward systematically identifying cellular targets associated with oncogenic transformation.

Up-regulation of Tumor Susceptibility Gene 101 Conveys Poor Prognosis through Suppression of p21 Expression in Ovarian Cancer

“…Although originally identified as a potential tumor suppressor gene in NIH3T3 cells (12), the precise roles of TSG101 in tumorigenesis remain undefined. Previously, we identified TSG101 as a downstream target of oncogenic RAS up-regulated during in vitro transformation of human ovarian epithelial cells (27,36). This increased expression of TSG101 is also observed in ovarian cancer patients because more than 70% of ovarian carcinomas express elevated levels of TSG101.…”

“…We previously identified TSG101 as a downstream target of the RAS oncogene in a genetically defined human ovarian cancer model (27). To investigate the clinical implication of RASmediated TSG101 up-regulation in human ovarian surface epithelial cells, we probed the expression levels of TSG101 in EOC using human ovarian cancer tissue arrays.…”

Up-regulation of Tumor Susceptibility Gene 101 Protein in Ovarian Carcinomas Revealed by Proteomics Analyses

“…2-DE Analysis-2-DE proteomics analysis was performed as described previously (34,35). Briefly cells were trypsinized, washed in PBS, and lysed in buffer containing the following: 7 M urea, 2 M thiourea, 4% CHAPS, 1 mM EDTA, 1 mM EGTA, 60 mM DTT, 1 mM PMSF, 25 g/ml leupeptin, 10 g/ml aprotinin, 1 mM benzamidine, 1 mM sodium orthovanadate, and 1 mM microcystin.…”

Ras-induced Epigenetic Inactivation of the RRAD (Ras-related Associated with Diabetes) Gene Promotes Glucose Uptake in a Human Ovarian Cancer Model

“…The immortalized but non-oncogenic T29 cells were further transformed by introducing oncogenic HRas V12 to generate the T29H cell line, which resembles natural ovarian cancer in several aspects (10,12). Functional proteomics analysis of Ras-mediated transformation in these cell lines enabled the identification of proteins targeted by Ras that mediate cellular metabolism, apoptosis, and the methylation pathways (10).…”

“…The immortalized but non-oncogenic T29 cells were further transformed by introducing oncogenic HRas V12 to generate the T29H cell line, which resembles natural ovarian cancer in several aspects (10,12). Functional proteomics analysis of Ras-mediated transformation in these cell lines enabled the identification of proteins targeted by Ras that mediate cellular metabolism, apoptosis, and the methylation pathways (10). Taking advantage of the Ras-T29H ovarian epithelial cell model, Cheng's group also found that the OPCML (opioid-binding protein/cell adhesion molecule-like) gene is epigenetically regulated by Ras in oncogenic transformation (11).…”