Proteomics Analysis Reveals The Role Of Gankyrin In H-Ras Mediated Transformation

The small GTPase RAS has long been known to play a significant role in tumor formation and development. RAS mutations are found in 30% of all human cancers. Overexpression of activated Ras leads to tumorigenesis in transgenicmice and in naturally occurring human neoplasia¹. RAS associated cell signaling is involved in many important cellular processes, such as cell growth, differentiation and survival under physiological conditions². The recent report suggests that Ras may mediate its transfotming activity through the activation of multiple downstream effector-mediated pathways. Several well-known intracellular signaling cascades including the Raf/MEK/ERK pathway, the PI3 kinase pathway, and the Ral-GDS pathway, have been identified as mediators of RAS downstream effects³. Although multiple mediators of Ras signaling have been discovered, the precise molecular mechanism of Ras-mediated oncogenic transformation is not clear. It is important to resolve what these pathways are and to establish their contributions to tumorigenicity.Gankyrin is a new oncoprotein with potent cell cycle and apoptotic properties that is overexpressed early in hepatocarcinogenesis and in hepatocellular carcinomas'. Gankyrin was initially purified and characterized as the p28 component of the regulatory subunit of the 26S proteasome^5-7. Gankyrin controls the functions of two major tumour suppressors, when overexpressed. Gankyrin can bind to CDK4 and the tumor suppressor RB, and accelerate phosphorylation and proteasomal degradation of RB, enhance E2F1-mediated transcription, and cell cycle progression. Gankyrin also binds to MDM2, a major E3 ubiquitin ligase for p53, and increases ubiquitylation and degradation of p53. And then, gankyrin has an anti-apoptotic activity in cells exposed to DNA-damaging agents. Furthermore, inhibition of gankyrin induces apoptosis in cancer cells^8-11. Gankyrin induced anchorage-independent growth and tumorigenicity in NIH3T3 cells¹².In our previously study, we found that gankyrin was up-regulated at both mRNA and protein levels in response to Ras-mediated transformation. This was the first evidence to show that gankyrin pathway was implicated in Ras-activated transformation¹³. With regards to the importance of gankyrin in the regulation of oncogenic transformation and that its overexpression contributes to tumorigenesis, to explore the relationship between gankyrin and H-Ras-mediated transformation in NIH3T3 cells and to investigate whether gankyrin play a significant role in tumor formation of Ras-mediated, we identified the protein targets associated with H-Ras and gankyrin-mediated oncogennic transformation by profiling two transformation cell lines. In our study, using two-dimensional gel electrophoresis (2-DE) followed by identifications with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) and electrospray ionization MS/MS (ESI-MS/MS). We have identified numerous proteins associated with Ras-mediated and gankyrin-mediated transformation, respectively. The ratio of proteins based on themselves functions in two transformation cell lines is similar. 14 proteins similarly changed in H-Ras and gankyrin-transformation NIH3T3 cells compared with control cells. The proteins similarly changed accounted for 32% and 44% of the proteins altered significantly in their expression in two transformation NIH3T3 cells, respectively. Moreover, the similarly changed proteins related to cells transform and tumor formation had rate of 46% and 60% in altered proteins related to H-Ras and gankyrin-transformation NIH3T3 cells, respectively. In order to further investigate the role of endogenous gankyrin in the Ras-mediated transformation, the Ras-transformed NIH3T3 cells were stably transfected with gankyrin shRNA or a control shRNA. Knocking down gankyrin caused a significant decrease of BTF3 in Ras-transformed NIH3T3 cells, confirming that gankyrin is indeed directly responsible for the maintaining Ras-mediated transformation and critically importantly for Ras signaling pathway.