Gankyrin gene deletion followed by proteomic analysis: insight into the roles of Gankyrin in Tumorigenesis and Metastasis

BMC Medical Genomics, Aug 2012

Background Gankyrin was originally purified and characterized as the p28 component of the 26S proteasome, and later identified as an oncogenic protein in hepatocellular carcinomas (HCC). It has recently been found to be highly expressed in several other malignancies, and compelling evidence show gankyrin plays important roles in tumorigenesis. However, its mechanism of action remains unclear. Methods In order to further clarify the functions of gankyrin and better understand its molecular mechanisms, we generated a gankyrin null cell line, HCT116 gankyrin−/− , by targeted homologous recombination in human colon cancer cells, and then employed two-dimensional electrophoresis (2-DE) based proteomic approaches followed by MS identification to investigate alterations in the proteome due to the gankyrin knockout. Western blot and qRT-PCR assays were also used to examine the protein and mRNA levels of some identified proteins. Results Compared with wild-type control cells, gankyrin null cells were impaired in terms of their proliferation, migration and anchorage-independent growth. A total of 21 altered proteins were identified, which included 18 proteins that had not previously been reported to be related to gankyrin. Notably, eight metastasis-related proteins were identified. Western blot analyses confirmed that the changes in three examined proteins were consistent with 2-DE gel analysis. Conclusions In summary, we have generated a useful cell tool to clarify the functions of gankyrin. Our proteomic data provide novel information to better understand the roles and underlying mechanisms by which gankyrin is involved in tumorigenesis and cancer metastasis.

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Gankyrin gene deletion followed by proteomic analysis: insight into the roles of Gankyrin in Tumorigenesis and Metastasis

Xue Luo 1 2 Liang Chen 1 Jiang Dai 1 Yanfei Gao 1 Hongli Wang 1 Na Wang 1 Yongqiang Zhao 1 Feng Liu 1 Zhihong Sang 1 Jie Wang 1 Weihua Li 1 Kun He 1 Baofeng Jin 1 Jianghong Man 1 Wei Zhang 0 Qing Xia 1 0 Department of Clinical Laboratory, No. 307 Hospital, Academy of Military Medical Sciences , Beijing 100071 , China 1 National Center of Biomedical Analysis , 27 Taiping Road, Beijing 100850 , China 2 Department of Occupational Health, Third Military Medical University , 30 Gaotanyan Road, Chongqing 400038 , China Background: Gankyrin was originally purified and characterized as the p28 component of the 26S proteasome, and later identified as an oncogenic protein in hepatocellular carcinomas (HCC). It has recently been found to be highly expressed in several other malignancies, and compelling evidence show gankyrin plays important roles in tumorigenesis. However, its mechanism of action remains unclear. Methods: In order to further clarify the functions of gankyrin and better understand its molecular mechanisms, we generated a gankyrin null cell line, HCT116 gankyrin/ , by targeted homologous recombination in human colon cancer cells, and then employed two-dimensional electrophoresis (2-DE) based proteomic approaches followed by MS identification to investigate alterations in the proteome due to the gankyrin knockout. Western blot and qRT-PCR assays were also used to examine the protein and mRNA levels of some identified proteins. Results: Compared with wild-type control cells, gankyrin null cells were impaired in terms of their proliferation, migration and anchorage-independent growth. A total of 21 altered proteins were identified, which included 18 proteins that had not previously been reported to be related to gankyrin. Notably, eight metastasis-related proteins were identified. Western blot analyses confirmed that the changes in three examined proteins were consistent with 2-DE gel analysis. Conclusions: In summary, we have generated a useful cell tool to clarify the functions of gankyrin. Our proteomic data provide novel information to better understand the roles and underlying mechanisms by which gankyrin is involved in tumorigenesis and cancer metastasis. - Background Gankyrin was originally purified and characterized as the p28 component of the 19S regulatory subunit of the 26S proteasome. It consists of 226 amino acids that encode a 25-kDa protein with six ankyrin repeats [1]. Fujita et al. identified gankyrin as an oncogenic protein that is overexpressed in hepatocellular carcinomas (HCC) [2]. They and several other groups have demonstrated critical roles of gankyrin in HCC tumorigenesis [2-4]. Gankyrin has recently been found to be highly expressed in several other malignancies, including lung cancer [5], pancreatic cancer [6] and colorectal cancer [7]. Compelling evidences from the literature and our own findings show that gankyrin plays important roles in both tumorigenesis and metastasis. We and other investigators have been devoted to unraveling the mechanism that underlies the oncogenic effects of gankyrin. Hiroaki H et al. [4] found that gankyrin controlled two major tumor suppressors, Rb and p53. Previously, we observed that gankyrin was upregulated in Ras-transformed cells [8], and it played an essential role in Ras-initiated transformation by regulating Akt activation [5]. However, its detailed functions and mechanism of action as a whole remain unclear. In order to further clarify the functions of gankyrin and better understand its molecular mechanisms, we generated a gankyrin null cell line from the human colon cancer cell line, HCT116, by gene targeting. The utilization of HCT116 as the gene targeting cells was borne out of two considerations: Firstly, Tang et al. [7] reported that gankyrin is significantly overexpressed in colorectal cancer tissues and cell lines. We tested the protein expression levels of gankyrin in 50 paired samples of colorectal cancer and adjacent normal tissue, and obtained similar results (unpublished data). Secondly, with a stable chromosomal complement, HCT116 as a diploid cancer cell line has been utilized most often for the generation of knockout and knock-in mutations [9]. Previous studies regarding gankyrin have mainly been based on RNA interference (RNAi) or overexpression experiments. However, RNAi might generate off-target effects that make its results difficult to predict and interpret. On the contrary, gene targeting by homologous recombination is a powerful technique for the analysis of gene function, which produces results that are more straightforward to interpret than overexpression or RNAi studies [10]. Specifically, human somatic cell knockout is a unique model system to study human gene function with conceptual advantages over analogous studies in model organisms. Based on similar strategies, several human knockout cell lines have been established and widely used, such as HCT116 p53/, HCT116 PPAR/ and HCT116 CDC4/ cells, amongst othe (...truncated)


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Xue Luo, Liang Chen, Jiang Dai, Yanfei Gao, Hongli Wang, Na Wang, Yongqiang Zhao, Feng Liu, Zhihong Sang, Jie Wang, Weihua Li, Kun He, Baofeng Jin, Jianghong Man, Wei Zhang, Qing Xia. Gankyrin gene deletion followed by proteomic analysis: insight into the roles of Gankyrin in Tumorigenesis and Metastasis, BMC Medical Genomics, 2012, pp. 36, 5, DOI: 10.1186/1755-8794-5-36