Profiling cellular protein expression in H-Ras mediated oncogenic transformation by functional proteomics.

摘要

H-Ras plays a critical role in the transformation of cells. To further understand the mechanism of H-Ras mediated oncogenic transformation, we utilized an ovarian epithelial transformation model in which H-RasV12 was the primary transforming agent to study systematically the protein changes that occur following transformation. Using 2-D gel proteomic analysis, we identified 32 proteins significantly altered in human ovarian epithelial cells transformed by H-RasV12 (T29H). These proteins are grouped into several functional classes, including antioxidant proteins, metabolic enzymes, proteins involved in DNA/protein methylation, calcium binding/signaling proteins, and apoptotic proteases. The changes in protein expression identified in our proteomic study occurred at both transcriptional and posttranscriptional levels. For example, methionine adenosyl transferase (MAT2A) was up-regulated transcriptionally and may affect H-Ras activity in a positive feedback loop; whereas caspase 4 was altered post-translationally by H-Ras and may affect T29H resistance to apoptosis.; We have also identified several antioxidant enzymes positively regulated by H-Ras transformation. These enzymes make up the second largest group of proteins identified in T29H cells and include selenophosphate synthetase, peroxiredoxin III and IV, and NADH dehydrogenase ubiquinone. Given the number of antioxidant proteins affected, we hypothesized that H-Ras mediated resistance against free radical induced apoptosis to protect cancer cells from higher levels of endogenous oxidative stress. When challenged with H2O 2, we discovered that T29H cells were indeed resistant to H2O 2 mediated apoptosis. This effect was clearly linked to H-Ras signaling, as suppressing H-Ras signaling pathways by small molecule inhibitors and siRNAs eliminated the protective effect. We propose that these enzymes participate in a mechanism whereby H-Ras up-regulate antioxidant enzymes to overcome oxidative stresses mediated by increased metabolic loads in the tumor microenvironment.; We further revealed that the tumor susceptibility protein 101 (TSG101) was up-regulated post-translationally by H-Ras through the Raf/MEK/ERK kinase signaling axis, and this up-regulation was dependent on H-Ras activity. Transient TSG101 gene silencing using siRNA decreased cell proliferation, arrested cells at G2/M, led to cellular apoptosis, and suppressed tumor formation in nude mice. These results demonstrated that TSG101 is an important down-stream effector of Ras and involved in maintenance of tumor growth in human ovarian cancer.