Travis W. Young

Activation of Antioxidant Pathways in Ras-Mediated Oncogenic Transformation of Human Surface Ovarian Epithelial Cells Revealed by Functional Proteomics and Mass Spectrometry

Cellular transformation is a complex process involving genetic alterations associated with multiple signaling pathways. Development of a transformation model using defined genetic elements has provided an opportunity to elucidate the role of oncogenes and tumor suppressor genes in the initiation and development of ovarian cancer. To study the cellular and molecular mechanisms of Ras-mediated oncogenic transformation of ovarian epithelial cells, we used a proteomic approach involving two-dimensional electrophoresis and mass spectrometry to profile two ovarian epithelial cell lines, one immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase and the other transformed with an additional oncogenic rasV12 allele. Of ∼2200 observed protein spots, we have identified >30 protein targets that showed significant changes between the immortalized and transformed cell lines using peptide mass fingerprinting. Among these identified targets, one most notable group of proteins altered significantly consists of enzymes involved in cellular redox balance. Detailed analysis of these protein targets suggests that activation of Ras-signaling pathways increases the threshold of reactive oxidative species (ROS) tolerance by up-regulating the overall antioxidant capacity of cells, especially in mitochondria. This enhanced antioxidant capacity protects the transformed cells from high levels of ROS associated with the uncontrolled growth potential of tumor cells. It is conceivable that an enhanced antioxidation capability may constitute a common mechanism for tumor cells to evade apoptosis induced by oxidative stresses at high ROS levels.

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

Small GTPase RAS plays a critical role in cellular signaling and oncogenic transformation. Proteomics analysis of genetically defined human ovarian cancer models identified the tumor susceptibility gene 101 (TSG101) as a downstream target of RAS oncogene. Mechanistic studies revealed a novel post-translational regulation of TSG101 through the RAS/RAF/MEK/MAPK signaling pathway and downstream molecules p14ARF/HDM2. Immunoanalysis using ovarian cancer samples and microtissue array revealed elevated TSG101 levels in human ovarian carcinomas. Silencing of TSG101 by short interfering RNA in ovarian cancer cells led to growth inhibition and cell death. Concurrent with the apparent growth-inhibitory effect, the levels of the CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) and hypoxia-inducible factor 1α (HIF-1α), as well as its cellular activity, were markedly reduced after TSG101 knockdown. These results demonstrate that TSG101 is important for CITED2- and HIF-1α-mediated cellular regulation in ovarian carcinomas.

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

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 rasV12 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

Purpose: The function of tumor susceptibility gene 101 (TSG101) in ovarian carcinogenesis is largely unexplored. The aim of this study is to investigate the role of TSG101 in human ovarian cancer development, to examine the expression levels of TSG101 in ovarian carcinomas, and to correlate the results with clinicopathologic variables and survival. Experimental Design: Human ovarian cancer tissue arrays that contain duplicates of 422 cases of primary ovarian carcinoma were used to probe the expression levels of TSG101 and p21 in epithelial ovarian cancer. In vitro studies in ovarian cancer cells using TSG101-specific small interfering RNA (siRNA) were done to further elucidate the mechanism of TSG101-mediated p21 regulation. Results: We show that TSG101 is increasingly overexpressed in borderline tumors and low-grade and high-grade carcinomas. Patients with low expression of TSG101 survive longer than those with high expression. Suppressing TSG101 by siRNA in ovarian cancer cells led to growth inhibition, cell cycle arrest, and apoptosis with concurrent increases in p21 mRNA and protein. Consistent with this negative association between TSG101 and p21, expression levels of these two markers are inversely correlated in ovarian cancer. Conclusions: TSG101 negatively regulates p21 levels, and up-regulation of TSG101 is associated with poor prognosis in ovarian cancer.

Priming of Eosinophils by GM-CSF Is Mediated by Protein Kinase CβII-Phosphorylated L-Plastin

The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF, but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein that we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. Additionally, coimmunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with protein kinase CβII (PKCβII), GM-CSF receptor α-chain, and two actin-associated proteins, paxilin and cofilin. Inhibition of PKCβII with 4,5-bis(4-fluoroanilino)phtalimide or PKCβII-specific small interfering RNA blocked GM-CSF–induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of αMβ2 integrin, which was sensitive to PKCβII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin, which was abrogated by the above-mentioned PKCβII inhibitors. Similarly, inhibition of PKCβII blocked GM-CSF induced priming for degranulation as assessed by release of eosinophil cationic protein and eosinophil peroxidase in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2–19) phosphorylated on Ser5 upregulated αMβ2 integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCβII and L-plastin in linking GM-CSF–induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCβII-mediated L-plastin phosphorylation.

Toward the proteome of the human peripheral blood eosinophil

Eosinophils (EOSs) are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood EOSs from normal human donors primarily employing 2‐DE with protein spot identification by MALDI‐MS. Protein subfractionation methods employed included IEF (Zoom® Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3141 proteins, which had Mascot expectation scores of 10−3 or less. Of these 426 were unique and non‐redundant of which 231 were novel proteins not previously reported to occur in EOSs. Ingenuity Pathway Analysis showed that some 70% of the non‐redundant proteins could be subdivided into categories that are clearly related to currently known EOS biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the EOS. This data set of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.

Proteomics analyses of ovarian caner using genetically defined human ovarian cancer models

Using a genetically defined human ovarian cancer model, we have analyzed the protein expression profile of human ovarian cancer cells established by oncogenic RAS transformation. Cells were immortalized by retroviral transfection of SV40 t/T antigens and the catalytic subunit of telomerase (hTERT). Careful analyses of protein targets associated with oncogenic transformation have enabled us to identify several novel signaling pathways that play important roles in oncogenic transformation of human ovarian epithelial cells.