Although gankyrin has been acknowledged as an important oncogene in HCC and several other malignancies, its oncogenic effects and mechanisms of action remain unclear. In the present study, we generated a human colon cancer cell line, HCT116 gankyrin−/−, by gene targeting, which enabled us to rigorously evaluate the role of this oncogene in tumorigenesis and cancer metastasis. By comparing isogenic cell lines that differed only in the presence or absence of the gankyrin gene, we have unambiguously shown that this protein can directly affect the growth and migration of colorectal cancer cells.
By 2DE/MS based approaches, we systematically analyzed the alterations in the cellular proteome due to the gankyrin knockout, which enabled the identification of numerous changes in protein expression and post-translational processing. Overall, 21 differentially expressed proteins were identified, including gankyrin and another two proteosome subunits, proteasome subunit beta type 7 (PSMB7) and proteosome beta-subunit 2 (PSME2).
The 26S proteasome is the core machinery of the ubiquitin proteasome system (UPS), which executes the degradation of most unwanted proteins in the cytosol and nucleus. Protein degradation in the proteosome plays an important role in cell proliferation, differentiation, signal transduction and the stress response, amongst others. Defects in the UPS have been linked to many human diseases, including cancer. By affinity purification and tandem mass spectrometry, [Wang et al. 17
] characterized the human 26S proteasome complex, and identified gankyrin, PSMB7 and PSME2. These gankyrin–regulated proteins have been reported to play roles in several important biological processes, such as the DNA damage response, cell cycle control, metabolism and cell motility. The dysregulation of these proteins is closely related to tumorigenesis and cancer metastasis.
DNA damage activates a complex signaling network that mediates DNA repair and activates cell-cycle checkpoints. Defects in the ability to properly respond to and repair DNA damage can result in genomic instability and lead to the transformation of normal cells into cancer cells. Three DNA damage response proteins, PIG3, PPIA, CAMK2G, were identified in the present study. PIG3 has been reported to be a p53 downstream target gene, and plays central roles in DNA damage response as a “genome guard”. The observation that PIG3 was upregulated due to the deletion of gankyrin expression was consistent with former reports that gankyrin negatively regulates p53 by increasing its ubiquitynation and degradation. [Hiroaki et al. 18
] also reported that, when treated with Adriamycin, a DNA damaging agent, the induction of PIG3 by p53 was decreased by the overexpression of gankyrin. As the RT-PCR results showed that the mRNA level of PIG3 was upregulated in gankyrin knockout cells, we concluded that this resulted from the increasing transcriptional activity of p53.
The dysregulation of proliferation is one of the most fundamental traits of cancer cells. Three cell cycle-related proteins, PCNA, CYP1A1 and Ubc12, were found to be regulated by gankyrin, which sheds light on the molecular mechanisms that underlie the oncogenic activity of gankyrin. PCNA forms a ring around DNA to facilitate and control DNA replication, which plays a role in many other essential cellular processes, such as the maintenance of chromatin structure, chromosome segregation and other cell-cycle progression stages. In this study, PCNA was observed to be dramatically downregulated after the deletion of gankyrin. Yun et al. obtained similar results when gankyrin was knocked down in pancreatic cancer cells.
Ubc12, an E2 NEDD8-conjugation enzyme, is a key molecule in the neddylation cascade. One of its main substrate, cullin, which is part of the SCF ubiquitin E3 ligase complex, plays critical roles in cell cycle progression. This finding suggests that gankyrin could promote cell cycle progression through regulating the neddylation of important cell cycle molecules.
The analysis of clinical samples has shown that gankyrin is overexpressed in colorectal cancer tissues and cell lines compared to controls, and its expression level was correlated with the tumor, node, and metastasis (TNM) staging system [7
]. Our wound healing assays demonstrated that the gankyrin knockout significantly decreased cell motility, which provides in vitro evidence of the role of gankyrin in cancer metastasis. Notably, in the present proteomic analysis, 40% of the proteins identified to be involved in cancer metastasis processes, including cofilin 1, annexin A2 isoform 2 (ANXA2), protein kinase C inhibitor protein 1 (YWHAZ), S100A9, chain K acetyl-cypa:cyclosporine complex (PPIA), phosphatidylethanolamine binding protein (PEBP1) and p27BBP. Cofilin is a small ubiquitous protein that can bind both monomeric and filamentous actin [19
]. It has been reported to be a key player in regulating the dynamics of the actin cytoskeleton of migrating cells and important for the motility of mammary cancer cells [20
]. Cofilin and its regulatory proteins are involved in the initiation of the early steps in the motility cycle, and evidence has emerged that the expression of certain genes of the cofilin pathway are altered in invasive tumor cells. The activity of the cofilin pathway (Rho-Rock-LIMK-Cofilin) is believed as one of the major determinants of metastasis [21
]. The inhibition of cofilin in several cell types has been found to alter cell protrusion and motility. [22
] In the present study, cofilin was downregulated after the deletion of gankyrin, which was consistent with the phenotype of impaired cell migration. Our previous study revealed that gankyrin is a key mediator of Ras–induced transformation by regulating the RhoA/ROCK/PTEN pathway [5
]. Whether gankyrin mediates tumor metastasis by affecting the cofilin pathway needs to be further clarified.
Cyclophilin A (CypA) has peptidylprolyl cis-trans isomerase activity, which plays important roles in protein folding, trafficking, assembly, immune-modulator and cell signaling. The upregulation of CypA in several cancers has been reported, including small cell lung cancer, pancreatic cancer, breast cancer, colorectal cancer and hepatocellular carcinoma (HCC). Zhang et al. reported that CypA promotes HCC cell metastasis through the upregulation of MMP3 and MMP9. Our results suggests that CypA is positively regulated by gankyrin, and CypA-MMP3/MMP9 may underlie the role of gankyrin in cancer metastasis.
ANXA2 is one of the well studied receptors for plasminogen, as it converts plasminogen to plasmin after binding. Plasmin is a serine protease that plays a key role in the activation of metalloproteinases and the degradation of extracellular matrix components that are essential for metastatic progression. Accumulating evidence suggests that ANXA2 and its receptor axis plays an important role in the tumor microenvironment and metastasis, and it has been recognized as an attractive target for the development of anti-cancer/anti-metastatic agents [23
]. Our 2D/MS result showed that ANXA2 was downregulated due to the gankyrin knockout, which is the first evidence that ANXA2 could be upregulated by gankyrin.
The YWHAZ gene encodes 14-3-3 protein zeta (ζ), a member of the 14-3-3 family, whose members mediate signal transduction by binding to phosphoserine-containing proteins. The upregulated expression of 14-3-3ζ is associated with histological grade, lymph node metastasis and poor clinical outcomes in some cancer types [25
]. Recent studies have shown that 14-3-3ζ interacts with many key cellular proteins that are involved in tumor development and progression. [Gohla et al. 26
] reported that the 14-3-3ζ protein could regulate cellular actin structures through the maintenance of phosphorylated-cofilin levels. Moreover, 14-3-3ζ forms the regulatory complex with Slingshot-1
L), a selective cofilin-regulatory phosphatase. The identification of YWHAZ and other metastasis-related proteins presents novel mechanistic insights into the critical role of gankyrin in cancer metastasis.