Establishment Of Gankyrin Knockout Colon Cancer Cell Model And Function Study

Gankyrin is a newly discovered oncogene. It encodes a 25kDa protein with 226 amino acids which consists of seven ankyrin repeats and is a subunit of the 26S proteasome. Gankyrin interacts with several key molecules of significant signaling pathways such as p53, Rb, MDM2, RhoGDI and etc, and plays critical roles in cell transformation, apoptosis and cell cycle control. It is reported that Gankyrin correlates with hepatocellular carcinomas, lung cancers, pancreatic and esophageal cancers, and the roles of Gankyrin in hepatocellular carcinomas has been well explored, while little is known about how it is involved in colon cancers. Our immunohistochemistry (IHC) results of 100 clinical tissue samples of colon cancers and adjacent normal tissues showed that Gankyrin is obviously overexpressed in colon cancer tissues compared to controls, which imply the important role of Gankyrin in the development of colon cancers.In this study we generated Gankyrin knockout human colon cancer cell line (HCT116 gankyrin^-/-) using gene targeting strategies based on homologous recombination, which provided important cell model for Gankyrin gene function study. Using the cell model we found that the ablilities of HCT116 cell transformation and tumorigenesis were seriously impaired after Gankyrin knockout and the knockout cells growed slowly compared to wild type. Cell cycle synchronization experiments revealed that there were G1-S transition delay and G2/M arrest in HCT116 gankyrin^-/- cells. In addition, knockout of Gankryin inhibited HCT116 cell migration, whereas stable overexpression of it in NIH3T3 cell greatly promoted cell migration. Further to seek for mechanism clues, proteomics analysis of HCT116 and HCT116 gankyrin^-/- cells was done, in which seventeen proteins regulated by Gankyrin were identified, including ubiquitin-proteasome system protein UBE2T, E2 enzyme of protein neddylation Ubc12, cell cycle protein Spc25, p53 related protein PIG3 and cystoskeleton regulator Lasp1, etc.This study, which has perfected the regulation network of Gankyrin, may provide new insight into the role of Gankyrin in the ubiquitin-proteasome system and demonstrate mechanisms of Gankyrin involved in the progression of colon cancers.