Proteomics Analysis of H-RAS-Mediated Oncogenic Transformation in a Genetically Defined Human Ovarian Cancer Model
Total Page：16
File Type：pdf, Size：1020Kb
Oncogene (2005) 24, 6174–6184 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc Proteomics analysis of H-RAS-mediated oncogenic transformation in a genetically defined human ovarian cancer model Travis Young1, Fang Mei1, Jinsong Liu2, Robert C Bast Jr3, Alexander Kurosky4 and Xiaodong Cheng*,1 1Department of Pharmacology and Toxicology, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1031, USA; 2Department of Pathology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA; 3Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA; 4Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1031, USA RAS is a small GTP binding protein mutated in Introduction approximately 30% human cancer. Despite its important role in the initiation and progression of human cancer, the Normal cells in the bodyare programmed bya variety underlying mechanism of RAS-induced human epithelial of distinct signals to grow, divide, and eventuallydie in a transformation remains elusive. In this study, we probe the coordinated fashion. Cancer arises when cells escape cellular and molecular mechanisms of RAS-mediated normal growth control and fail to die. Considerable transformation, by profiling two human ovarian epithelial knowledge of oncogenesis and tumor development has cell lines. One cell line was immortalized with SV40T/t been gained using cellular and animal cancer models. It antigens and the human catalytic subunit of telomerase is clear that oncogenic transformation is a complex (T29), while the second cell line was transformed with an process that involves multiple steps of genetic and additional oncogenic rasV12 allele (T29H). In total, 32 cellular alterations. During this process, cells destined proteins associated with RAS-mediated transformation for oncogenic transformation progressivelyacquire capa- have been identified using peptide mass fingerprinting. bilities of self-sufficiencyin growth signals, insensitivity These protein targets are involved in several cellular to antigrowth signals, limitless replicative potential, pathways, including metabolism, redox balance, calcium evasion of apoptotic signals, tissue invasion and signaling, apoptosis, and cellular methylation. One such metastasis, and sustained angiogenesis (Hanahan and target, the 40kDa procaspase 4 is significantly upregu- Weinberg, 2000). Although identification of various lated at the protein level in RAS-transformed T29H cells, oncogenes and tumor suppressor genes has greatly related directly to signaling through MEK, but not PI3 advanced our understanding of the steps associated kinase. Cellular caspase 4 activity is, however, suppressed with cancer formation, the underlying cellular signaling in the T29H cells, suggesting that the maturation process networks governed byindividual oncogenes or tumor of caspase 4 is abrogated in RAS-transformed T29H cells. suppressor genes remain poorlyunderstood at the Consistent with this notion, transformed T29H cells were molecular level. One important reason for this major less susceptible to the toxic effects of anti-Fas antibody gap is the lack of appropriate human cancer models and than were immortalized, nontransformed T29 cells, subsequently the ability to systematically analyse these associated with less activation of caspase 4. This study model systems. demonstrates that functional proteomic analysis of a Although primaryrodent cells can be readilytrans- genetically defined cancer model provides a powerful formed bytwo cooperating oncogenes (Land et al., approach toward systematically identifying cellular tar- 1983; Ruley, 1983), oncogenic transformation of human gets associated with oncogenic transformation. primarycells was achieved onlyrecentlybyintroducing Oncogene (2005) 24, 6174–6184. doi:10.1038/sj.onc.1208753; a combination of the SV40 T/t oncogenes, the telomer- published online 6 June 2005 ase catalytic subunit gene (hTERT), and an oncogenic allele of H-RASV12 (Hahn et al., 1999). With the advent Keywords: caspase 4; mass spectrometry; ovarian cancer; of this methodology, malignant transformation has been proteomics; ras; transformation demonstrated in a varietyof human cell lines using the same set of defined genetic elements. These systems include human embryonic kidney cells and foreskin fibroblasts (Hahn et al., 1999), primaryairwayepithelial cells (Lundberg et al., 2002), primarymammary epithelial cells (Elenbaas et al., 2001), mesothelial cells *Correspondence: X Cheng; E-mail: [email protected] (Yu et al., 2001), and astrocytes (Rich et al., 2001). Received 18 November 2004; revised 6 April 2005; accepted 12 April Recently, a genetically defined model for human ovarian 2005; published online 6 June 2005 cancer has also been established using normal human Proteomic analysis of ras-mediated oncogenic transformation T Young et al 6175 ovarian surface epithelial (HOSE) cells (Liu et al., 2004). nude mice. Extracts from each cell line were analysed The observation that a few discrete genetic alterations in triplicate by2-DE. From preliminarystudies using a can transform human cells in vitro supports the notion 3–10 pH gradient, we concluded that the majorityof 2- that there maybe common molecular pathways DE detectable proteins fell within a pH range of 5–7 on associated with the seeminglycomplex and diverse the gel. Therefore, a pH gradient of 4–7 was used in the phenotypes of cancer. Equally important, these model first dimension to obtain maximal resolution of protein systems provide an opportunity to study the complex spots. We also found that the use of 10% tricine–SDS processes of tumorigenesis, especiallythe dissection of polyacrylamide gels, instead of glycine–SDS gradient the functional roles of individual tumor promoter and gels, achieved a better dynamic separation range and suppressor genes in oncogenic transformation of human reproducibility. For each cell line, at least two indepen- cells from different organ sites (Watnick et al., 2003; dent cell lysates were obtained from cultures at different Young et al., 2004). earlypassages and four or more well-resolved gels from The small GTPase RAS has long been known to play six different runs were analysed. Figure 1 shows a significant role in tumor formation and development. representative 2-DE images of T29 and T29H; approxi- RAS mutations are found in 30% of all human cancers mately2200 distinct protein spots were resolved within (Bos, 1989). Although multiple mediators of RAS each gel. signaling, including Raf, PI3 kinase, AF-6, and Ral- The intensityof each protein spot was determined, GDS, have been discovered, the exact role that normalized to the sum of intensities of all spots on the oncogenic RAS plays in the multistep process of gel, and quantified as a percentage volume in each gel oncogenesis is not completelyunderstood and many using Phoretix 2-D analysis software (Nonlinear). Each downstream functions of RAS signaling in cellular individual protein spot was then matched with the transformation remain a mystery. In the current study, identical protein spot from each replicate gel. Data for we applied a proteomics approach to analyse a these matched spots were then averaged over replicate transformation model in which normal HOSE cells are gels for each cell line. The average normalized volume of immortalized bySV40 T/t oncogenes and hTERT, and each spot in the transformed T29H cells was then further transformed through the addition of the compared to that of the matched spot in immortalized constitutivelyactive H-RAS V12. Proteomics provides a T29 cells. In order to determine what would constitute powerful tool for the systematic analysis of alterations significant changes between transformed and untrans- in protein expression and post-translational modifi- formed cells, the intrinsic variance of each protein spot cations, and has been successfullyapplied recentlyfor was determined for each cell line. The average variance identifying tumor markers in bladder squamous cell for individual spots in the replicate gels for the T29 carcinomas (Ostergaard et al., 1997), hepatoma (Yu immortalized cell line was 34%. The average variance in et al., 2000), and melanoma (Bernard et al., 2003). By the transformed T29H cell line was similar at 29% profiling the protein expression differences between two (Figure 2). These intrinsic variances determined the geneticallydefined cell lines from an ovarian cancer basal noise levels of our 2-DE analysis. Based on these model using two-dimensional gel electrophoresis observed values, we selected spots whose average (2-DE), we have identified numerous proteins and normalized volume increased or decreased byat least several specific cellular pathways associated with RAS- 50% between the T29 and T29H cell lines as signifi- mediated oncogenic transformation. Manyof these cantlychanged candidates. We further narrowed the targets are novel downstream molecules that have not field of potential differentiallychanged protein by previouslybeen linked to RAS signaling pathwaysor eliminating spots with greater than 50% variance. transformation. Results from our studynot only Finally, two independent observers examined the spots provide further insights into our understanding of the visuallyto confirm that changes from automated RAS-mediated tumorigenic