Potential conflict of interest: Nothing to report.
Cyclin G1 deficiency is associated with reduced incidence of carcinogen-induced hepatocellular carcinoma (HCC), but its function in HCC progression remains obscure. We report a critical role of cyclin G1 in HCC metastasis. Elevated expression of cyclin G1 was detected in HCCs (60.6%), and its expression levels were even higher in portal vein tumor thrombus. Clinicopathological analysis revealed a close correlation of cyclin G1 expression with distant metastasis and poor prognosis of HCC. Forced expression of cyclin G1 promoted epithelial-mesenchymal transition (EMT) and metastasis of HCC cells in vitro and in vivo. Cyclin G1 overexpression enhanced Akt activation through interaction with p85 (regulatory subunit of phosphoinositide 3-kinase [PI3K]), which led to subsequent phosphorylation of glycogen synthase kinase-3β (GSK-3β) and stabilization of Snail, a critical EMT mediator. These results suggest that elevated cyclin G1 facilitates HCC metastasis by promoting EMT via PI3K/Akt/GSK-3β/Snail-dependent pathway. Consistently, we have observed a significant correlation between cyclin G1 expression and p-Akt levels in a cohort of HCC patients, and found that combination of these two parameters is a more powerful predictor of poor prognosis. Conclusions: Cyclin G1 plays a pivotal role in HCC metastasis and may serve as a novel prognostic biomarker and therapeutic target. (HEPATOLOGY 2012;55:1787–1798)
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Cyclin G1 was first identified serendipitously in screening for Src kinase family in rat fibroblasts.1 It has been categorized as a cyclin on account of possessing a well-conserved cyclin box, although it lacks a destruction box or PEST (Pro, Glu, Ser and Thr)-rich sequences that are responsible for cyclin degradation.1 Cyclin G1 has been well documented as a p53-responsive gene and could be transcriptionally activated by p53 or p73.2, 3 In fact, p53 is the major factor regulating cyclin G1 expression upon DNA damage.4 Cyclin G1 was reported to interact with the B′ regulatory subunit of protein phosphatase 2A, which in turn dephosphorylated MDM2 followed by p53 degradation.5 A feedback regulation of p53 by cyclin G1 was also observed in cyclin G1-null mouse embryo fibroblasts, which exhibited increased p53 levels.6, 7 Additionally, cyclin G1 was found to interact with certain proteins involved in cell cycle regulation, such as cyclin G–associated kinase and cyclin-dependent kinase 5, but the physiologic significance of these interactions remains elusive.8
Cyclin G1–deficient mice have been reported to be viable without any apparent phenotype. However, these mice exhibited increased mortality after γ-irradiation.4 Cyclin G1 deregulation has been described in colorectal cancer, breast cancer, and leiomyoma.9-11 Moreover, suppression of cyclin G1 in pancreatic cancer cells or osteosarcoma cells resulted in the growth inhibition of xenograft tumor through suppression of proliferation or induction of apoptosis.12, 13 Jensen et al.14 revealed a correlation between increased cyclin G1 expression and G2/M-cell cycle arrest of hepatocytes in response to DNA damage. More importantly, cyclin G1–null mice treated with diethylnitrosamine displayed significantly reduced incidence, tumor size, and malignancy of hepatocellular carcinoma (HCC) compared with control mice.15 Nevertheless, the role of cyclin G1 in HCC invasion and metastasis remains largely unknown.
Epithelial-mesenchymal transition (EMT) is defined as the process wherein epithelial cells lose their epithelial signatures while acquiring the characteristics of mesenchymal cells including morphology, cellular structure, and biological function.16 EMT of tumor cells is well accepted to be closely associated with cancer invasion and metastasis. Characteristic down-regulation of E-cadherin is regarded as the key step of EMT. Zinc-finger transcriptional repressors Snail and Slug, the repressor SIP-1/ZEB-2, ΔEF-1/ZEB-1, as well as the basic helix-loop-helix (bHLH) transcription factors Twist17 and E12/E4718 are the most prominent suppressors of E-cadherin transcription, which bind to E-boxes of the E-cadherin promoter and suppress its transcription in response to upstream signaling.
Growing evidence has elucidated that numerous signaling pathways are involved in the regulation of EMT. The cooperation of oncogenic Ras or receptor tyrosine kinases (RTKs) with endogenous transforming growth factor β receptor (TGF-βR) signaling was elucidated to trigger EMT in the context of tumorigenesis. Sustained TGF-βR signaling was required for the maintenance of EMT in epithelial cells and cancer metastasis in mouse models.19, 20 In recent years, several cancer-associated cascades have emerged as important regulatory signaling for EMT, which include phosphoinositide 3-kinase (PI3K)/Akt-, Wnt-, Notch-, Hedgehog- or nuclear factor-κB (NF-κB)-dependent pathways.21 EMT has been considered as a central mechanism responsible for invasiveness and metastasis of various cancers including HCC.22-24
In this report, we explored the expression of cyclin G1 in human HCCs and its clinical significance. The role of cyclin G1 in HCC metastasis and the underlying mechanism were also addressed.
One hundred and seventy HCCs were randomly retrieved from HCC patients who underwent curative resection at Eastern Hepatobiliary Surgery Hospital, Shanghai, China, from September 2001 to July 2007 (see detailed clinicopathological features in Supporting Table 1). All patients were followed up until March 2010, with a median observation time of 40 months. Overall survival (OS) was defined as the interval between the dates of surgery and death. Disease-free survival (DFS) was defined as the interval between the dates of surgery and recurrence; if recurrence was not diagnosed, patients were censored on the date of death or the last follow-up. Matched pairs of primary HCC samples and adjacent liver tissues were used for the construction of a tissue microarray (in collaboration with Shanghai Biochip Company, Shanghai, China). Immunostaining was performed on tissue microarray slides. Assessment of the staining was based on the percentage of positively stained cells and the staining intensity using software Image-Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD). Fifty-eight pairs of human HCC with pericancerous tissues and 38 pairs of HCC with portal vein tumor thrombus samples diagnosed by pathologist were obtained from Eastern Hepatobiliary Surgery Hospital. Patient samples were obtained following informed consent according to an established protocol approved by the Ethic Committee of Eastern Hepatobiliary Surgery Hospital.
In Vitro Cell Behavior Assay.
SMMC-7721/cyclin G1, HepG2/cyclin G1, and their control cells (1 × 103) were cultured in 96-well plates for various time periods. Adenosine triphosphate activity was measured using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) with a Synergy 2 microplate reader to assess the cell proliferation. Hepatoma cells (2.5 × 104) were seeded in 96-well plates coated with 10 μg/mL fibronectin (Calbiochem, La Jolla, CA) and cell adhesion was evaluated. For wound healing assay, monolayers of cells were wounded by scraping with a plastic pipette tip and rinsed several times with medium to remove dislodged cells. Cells that had migrated into the wound area were photographed. For invasion assay, 2 × 105 cells were plated into the upper chamber of a polycarbonate transwell filter chamber coated with Matrigel (BD) and incubated for 60 hours. Cell counts are expressed as the mean number of cells per field of view.
In Vivo Metastasis Assay.
Six-week-old male BALB/c nude mice were randomized into two groups (n = 11) and inoculated with SMMC-7721/cyclin G1 or control cells (2 × 106) in spleen. Four mice were sacrificed 8 weeks after inoculation, and metastatic tumor colonies in the liver were measured. The remaining mice were observed for survival analysis. For the tail vein metastasis model, 22 nude mice were randomized into two groups. SMMC-7721/cyclin G1 or control cells (2 × 106) were injected into the tail vein of nude mice. Four mice were sacrificed 6 weeks after inoculation and consecutive sections of the whole lung were subjected to hematoxylin and eosin staining. All of the metastatic foci in lung were calculated microscopically to evaluate the development of pulmonary metastasis. The remaining mice were monitored for survival analysis.
293T cells were harvested in immunoprecipitation lysis buffer supplemented with a complete protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The cell lysate was immunoprecipitated with anti-p85α or anti-cyclin G1 antibody and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblotting.
PI3K Activity Assay.
PI3K of hepatoma cells overexpressing cyclin G1 was immunoprecipitated with anti-p85α antibody and Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology). PI3K activity in the immunoprecipitates was analyzed by PI3K enzyme-linked immunosorbent assay kit (Echelon Biosciences, Salt Lake City, UT) according to the manufacturer's instructions.
Differences among variables were assessed by χ2 analysis or two-tailed Student t test. Kaplan-Meier and log-rank analysis was used to assess the patient survival between subgroups. Data were presented as the mean ± SEM unless otherwise indicated. P < 0.05 was considered statistically significant.
A detailed description of the materials and methods can be found in the Supporting Information.
Expression of Cyclin G1 Is Up-regulated in Human HCCs.
To explore the role of cyclin G1 in HCC development, we first evaluated the expression of cyclin G1 in various human HCCs. As shown in Fig. 1A, elevated expression of cyclin G1 was observed in HCC cell lines compared with that in normal liver cell lines. Cyclin G1 transcripts were significantly increased in HCCs relative to paired noncancerous tissues in 58 patients (Fig. 1B), which was further confirmed by western blot assay (Fig. 1C). Immunohistochemical analysis showed that cyclin G1 was up-regulated in 60.6% (103/170) of the HCC patients (Fig. 1D,E). HCC carries a high risk of portal vein invasion. Portal vein tumor thrombus markedly deteriorates hepatic function and serves as a prognostic factor of metastasis.25 Interestingly, cyclin G1 level was significantly increased in portal vein tumor thrombus compared with the matched primary tumors, indicating the potential role of cyclin G1 in HCC metastasis (Fig. 1F and Supporting Fig. 1).
Cyclin G1 Overexpression Predicts Poor Prognosis of HCC.
To investigate the clinical significance of cyclin G1 overexpression in HCC, tissue microarray analysis of HCC tissues from 170 patients underwent liver resection (Supporting Table 1) was performed. The average expression level of cyclin G1 was significantly higher in HCCs than that in peritumoral tissues (Supporting Table 2). More importantly, elevated cyclin G1 expression was associated with larger tumor size or distant metastasis (Fig. 2A,B and Supporting Table 3). Based on the results from immunohistochemistry, all 170 HCC patients were divided into two groups: high cyclin G1 expression (n = 85) and low cyclin G1 expression (n = 85). Patients in the high expression group exhibited shorter DFS (median DFS time, 12 and 41 months, respectively; difference = 29 months; P < 0.05) and worse OS (median OS time, 26 and 42 months, respectively; difference = 16 months; P < 0.05) than those in the low expression group (Fig. 2C,D and Supporting Table 4). Consistently, the 3-year and 5-year OS or DFS rate after surgery was much lower in the cyclin G1-high group than that in the cyclin G1-low group (Supporting Table 5). Additionally, among the HCC patients with similar tumor size (Fig. 2E,F and Supporting Fig. 2A,B) or without distant metastasis (Supporting Fig. 2C,D), the cyclin G1-high group exhibited poor survival rate compared with the cyclin G1-low group. Thus, cyclin G1 overexpression could serve as a valuable predicting factor for recurrence and poor survival of HCC patients.
Cyclin G1 Enhances Metastatic Potential of HCC Cells.
To elucidate the effects of elevated cyclin G1 on hepatoma cell behavior, SMMC-7721 and HepG2 cells were infected by lentiviral-cyclin G1 and stable transfectants were established (Supporting Fig. 3A). Although cyclin G1 is a member of cyclin superfamily, overexpression of cyclin G1 had marginal influence on the growth of SMMC-7721 and HepG2 cells (Supporting Fig. 3B). Scratch wound healing assay showed that hepatoma cells overexpressing cyclin G1 exhibited enhanced mobility (Supporting Fig. 4). Matrigel invasion chamber assays revealed that forced cyclin G1 expression markedly promoted the invasiveness of hepatoma cells (Fig. 3A and Supporting Fig. 5). Adhesion of tumor cells to the extracellular matrix is one of the key steps in metastasis, which allows subsequent invasion and metastasis. Cell adhesion assay demonstrated that forced cyclin G1 expression in SMMC-7721 or HepG2 cells significantly increased the cell adherence to fibronectin (Fig. 3B). As shown in Fig. 3C,D, nude mice inoculated with SMMC-7721/cyclin G1 cells in spleen displayed more and larger xenografts in the liver and reduced median survival period in comparison with control mice (P < 0.05, with 80 days as cutoff). To further verify the metastasis-promoting effect of cyclin G1, SMMC-7721/cyclin G1 cells and HepG2/cyclin G1 cells were injected into lateral tail vein of nude mice. Six weeks later, more and larger micrometastatic lesions were microscopically detected in the lungs of nude mice inoculated with cyclin G1-overexpressing hepatoma cells compared with those inoculated with control cells (Fig. 3F, Supporting Fig. 6). Moreover, nude mice injected with SMMC-7721/cyclin G1 had a shorter survival period than the mice injected with SMMC-7721 expressing green fluorescent protein (P < 0.05, with 65 days as a cutoff).
Cyclin G1 Promotes EMT in HCC Cells.
EMT of tumor cells has been well accepted to closely correlate with cancer metastasis. To explore whether cyclin G1 could promote EMT process, we determined EMT markers in hepatoma cells overexpressing cyclin G1 and the control cells. As shown in Fig. 4A, SMMC-7721 with ectopic expression of cyclin G1 partially displayed the mesenchymal appearance, and enhanced expression of vimentin, a distinct mesenchymal marker, was also observed. Real-time polymerase chain reaction (PCR) assay demonstrated the decreased levels of epithelial markers (E-cadherin, desmoplakin, and ZO-1), and the increased levels of mesenchymal markers (vimentin, twist, and fibronectin) in SMMC-7721/cyclin G1 cells (Fig. 4B). Luciferase reporter assay further indicated that down-regulation of E-cadherin by cyclin G1 was achieved through the suppression of E-cadherin promoter activity (Fig. 4C). Moreover, immunohistochemistry showed that vimentin and Snail were dramatically increased in murine xenografts from cyclin G1-overexpressing hepatoma cells compared with those from control cells (Fig. 4D). Consistently, correlation of cyclin G1 levels and EMT marker expression was observed in human HCC tissues (Fig. 4E,F). These results suggest that the metastasis-promoting effect of cyclin G1 could be attributed to its induction of EMT in HCC cells.
Cyclin G1 Activates PI3K/Akt Signaling via Interaction With p85.
As a highly conserved cellular program, EMT has been documented to involve several important pathways. As shown in Supporting Figs. 7 and 8, activity of NF-κB, activator protein 1 (AP-1), Gli-1, or β-catenin in hepatoma cells was not affected or slightly influenced by cyclin G1 overexpression. Accumulating studies have suggested that PI3K/Akt activation plays a pivotal role in tumor progression via induction of EMT.22, 26-30 Thus, we detected the activity of Akt in cells with forced cyclin G1 expression. As shown in Fig. 5A, phosphorylation level of Akt was significantly increased by enforced cyclin G1 expression and decreased at the presence of small hairpin RNA targeting cyclin G1 (shcyclin G1) (Supporting Fig. 9). Moreover, cyclin G1 robustly intensified Akt activation triggered by mitogen (epidermal growth factor (EGF)) or carcinogen (As2O3) (Supporting Fig. 10). Akt activation is usually up-regulated by PI3K and down-regulated by tumor suppressor phosphatase and tensin homolog (PTEN). Western blotting revealed that PTEN expression was not influenced by cyclin G1 overexpression (Supporting Fig. 11), which excluded the involvement of PTEN in cyclin G1-mediated Akt activation. In order to assess the effect of cyclin G1 on PI3K, we measured PI3K activity using a competitive enzyme-linked immunosorbent assay. The result showed that cyclin G1 significantly enhanced the activity of PI3K in hepatoma cells (Fig. 5B). RTK-mediated activation of PI3K is the predominant regulatory machinery of PI3K activity. WideScreen RTK pTyr Assay was thereby performed to test whether RTKs were required in cyclin G1-midiated PI3K activation. As shown in Supporting Fig. 12, forced cyclin G1 expression did not affect the autophosphorylation of epidermal growth factor receptor, insulin-like growth factor-1 receptor, hepatocyte growth factor receptor, or Tie-2 in hepatoma cells stimulated with a mitogen cocktail, implying that RTKs were not involved in cyclin G1–induced PI3K activation. To test whether cyclin G1 could enhance PI3K activity via a direct mechanism, we performed coimmunoprecipitation assay, and the results revealed that cyclin G1 could associate with p85, the regulatory subunit of PI3K (Fig. 5C). Ectopic expression of Δp85, a dominant negative mutant of p85, significantly attenuated cyclin G1-triggered Akt phosphorylation with or without the stimulation of EGF or As2O3, which further confirmed that PI3K was involved in Akt activation by cyclin G1 (Fig. 5D). Moreover, blockage of Akt by DN-Akt, a dominant negative mutant of Akt, dramatically attenuated cyclin G1–elicited EMT and invasion of hepatoma cells, suggesting that PI3K/Akt signaling is required in cyclin G1–promoted HCC metastasis (Fig. 5E,F).
Cyclin G1 Regulates EMT via the Akt/Glycogen Synthase Kinase-3β/Snail Pathway.
Glycogen synthase kinase-3β (GSK-3β) has been documented to be regulated by Akt and is required for the maintenance of epithelial architecture. To test whether GSK-3β is involved in cyclin G1–mediated EMT, we examined the effect of cyclin G1 on GSK-3β activation. As shown in Fig. 6A, phosphorylation of GSK-3β was notably enhanced by cyclin G1 overexpression with or without EGF treatment, and it was dramatically impaired at the presence of shcyclin G1. As expected, expression of Snail, which is a predominant mediator of EMT and tightly regulated by GSK-3β at the protein level, was significantly increased in cells with cyclin G1 overexpression and decreased in cells transfected with shcyclin G1 (Fig. 6B). Suppression of Akt activation by DN-Akt remarkably attenuated cyclin G1–elicited GSK-3β phosphorylation and Snail expression in hepatoma cells (Fig. 6C). Consistently, overexpression of GSK-3βKD, a kinase dead GSK-3β dominant negative mutant, not only enhanced cyclin G1–triggered Snail induction, but also partially restored shcyclin G1–mediated Snail reduction (Fig. 6D). Considering cyclin G1–overexpressing hepatoma cells exhibited enhanced liver and lung metastasis, we detected the cyclin G1–regulated signaling cascade in those metastatic tumors. The results showed that phosphorylation of Akt or GSK-3β and expression of Snail were evidently increased in the metastatic lesions of cyclin G1–overexpressing hepatoma cells (Fig. 6E,F), which further suggests that the Akt/GSK-3β/Snail pathway is critical in cyclin G1–promoted EMT and HCC metastasis.
Combination of Cyclin G1 and p-Akt Exhibits Improved Prognostic Accuracy for HCC.
As shown in Fig. 7A,B, tissue microarray analysis of HCC specimens from 170 patients revealed a close correlation between cyclin G1 expression and p-Akt levels (P < 0.001), which further supports the activation of Akt by cyclin G1 in human HCCs. The achievements of laboratory studies have provided quite a few biological markers to predict the prognosis of HCC patients. Accumulating evidence also indicates that a combination of multiple markers might be more informative than any single one for the prediction of clinical outcome of HCC patients. Elevation of either cyclin G1 or p-Akt in HCC predicts a poor prognosis of patients (Supporting Fig.13). More importantly, those patients with elevated cyclin G1 expression and enhanced p-Akt levels displayed even worse prognosis, indicating that the combination of the two parameters has a better prognostic value in comparison with cyclin G1 or p-Akt alone (Fig. 7C,D). Taken together, these results unravel a novel mechanism that cyclin G1 binds to the p85 subunit of PI3K and activates PI3K/Akt/GSK-3β/Snail signaling, which renders EMT and metastasis of HCC cells (Fig. 7E).
Primary liver cancer is the fifth most common cancer and the third most common cause of cancer mortality worldwide. HCC accounts for 70%-85% of primary liver cancer according to the statistical data of the American Cancer Society in 2007. Great efforts have been made to elucidate the molecular mechanism underlying tumorigenicity, invasion, and metastasis of HCC in order to develop novel treatments and a possible cure in the past several decades. Nevertheless, the detailed mechanism of hepatocarcinogenesis and HCC metastasis remains obscure. Cyclin G1 deregulation is associated with genomic instability, which is frequently induced following DNA damage.4, 14, 31 It has been reported that cyclin G1, together with MDM2, constitutes part of a negative feedback system attenuating p53 activity, and loss of cyclin G1 decreased tumor susceptibility in diethylnitrosamine-induced murine HCC.6, 15 In the current study, we found that cyclin G1 was highly expressed in HCCs and portal vein tumor thrombus, and that cyclin G1 expression was closely associated with the poor prognosis of HCC patients. Therefore, whether cyclin G1 is responsible for HCC metastasis arouses our interest. EMT is an important process during tumor metastasis. By using a variety of HCC cases and mouse HCC metastasis models, we demonstrated that cyclin G1 could promote EMT of hepatoma cells and facilitate HCC metastasis. Furthermore, we clarified that cyclin G1 could interact with PI3K and activate the PI3K/Akt/GSK-3β/Snail pathway, by which E-cadherin expression was down-regulated.
EMT usually occurs in the critical phases of embryonic development. However, this important developmental program also has a sinister role in tumor metastasis. There is solid evidence indicating that EMT gives rise to the dissemination of single carcinoma cell from the site of the primary tumor.32 EMT is also reported to be involved in the progression of HCC and correlates with the prognosis of patients.24 Although numerous factors have been identified to participate in EMT, whether cyclin G1 promotes EMT and cancer metastasis remains unclear. To investigate the precise function of cyclin G1 in HCC progression, we established stable cell line overexpressing cyclin G1 by recombinant lentivirus. The morphological changes of the tumor cells led us to link the biological function of cyclin G1 with EMT induction. As anticipated, mesenchymal markers were significantly up-regulated in the cyclin G1 stable transfectants, whereas the epithelial markers were remarkably decreased. These results were further confirmed by immunocytochemical analysis of cultured cells and immunohistochemical staining of murine metastatic tumors. Down-regulation of E-cadherin is considered the initial and critical step of the EMT process. In this study, we found that cyclin G1 delivery by adenovirus led to significant repression of E-cadherin promoter activity in hepatoma cells. Biological functional studies demonstrated that cyclin G1 enhanced the metastatic potential of HCC cells and induced more metastatic lesions in the livers and lungs of nude mice. Therefore, these data indicated that cyclin G1 might promote HCC metastasis via, at least partially, induction of EMT in hepatoma cells.
To date, multiple oncogenic pathways have been reported to participate in EMT regulation, such as MAPKs, NF-κB, Hedgehog signaling, and so forth. To determine whether cyclin G1 affects these pathways, we analyzed the activity of AP-1, NF-κB, and Gli-1 by luciferase reporter assay in cells with cyclin G1 overexpression. The results excluded the involvement of these cascades in cyclin G1 function. Increasing evidence has demonstrated that the activated PI3K/Akt pathway plays a central role in the EMT process and correlates with an aggressive phenotype in several kinds of malignancies.22, 26-30 It is therefore of interest to investigate whether Akt participates in cyclin G1–meditated EMT. We found that overexpression of cyclin G1 remarkably intensified the phosphorylation of Akt, which was further enhanced upon the stimulation of mitogen or carcinogen. Meanwhile, we also found that enhanced cyclin G1 expression intensified the kinase activity of PI3K. These results suggest that cyclin G1 may activate Akt by modulating PI3K activity. Activation of PI3K is usually triggered by the ligand-activated receptor tyrosine kinase. However, cyclin G1 overexpression did not show significant influence on the autophosphorylation of several critical RTKs. Considering PI3K activity is principally regulated by the p85 subunit, we hypothesized that cyclin G1 might interact with p85 and eventually increase the catalytic activity of PI3K. As expected, cyclin G1 and p85α were coprecipitated in an immunoprecipitation assay, indicating that cyclin G1–induced Akt activation may depend on the interaction of cyclin G1 and p85. This notion was further supported by the result that overexpression of Δp85 remarkably abolished cyclin G1–induced Akt activation.
Snail acts as the most important transcription factor in the negative regulation of E-cadherin expression and EMT process of epithelial cells.33, 34 Phosphorylation and subsequent degradation of Snail is controlled by GSK-3β, which is predominantly regulated by PI3K/Akt.35 Through the activation of PI3K/Akt signaling, cyclin G1 suppressed GSK-3β activity, and the level of Snail was correspondingly increased, which led to the down-regulation of E-cadherin and the subsequent EMT of hepatoma cells.
In conclusion, cyclin G1 could play an important role in HCC metastasis by EMT induction via, at least partially, the PI3K/Akt/GSK-3β/Snail-dependent pathway, and may serve as a valuable prognostic biomarker and potential therapeutic target. Uncovering a novel function and molecular mechanism for cyclin G1 in HCC will shed new light on the understanding of tumor progression and metastasis.
We thank Dong-Ping Hu, Shan-Na Huang, Dan-Dan Huang, Shan-Hua Tang, Lin-Na Guo, and Dan Cao for technical assistance. We also thank Mu-Sheng Zeng (Sun Yat-sen University School of Medicine, Guangzhou, China) for providing E-box-luciferase reporter plasmid.