Preliminary Study On The Function And Regulation Machanism Of FAT10in Hepatocellular Carcinoma Occurrence And Progression

Background and objective:Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide.1In China, HCC has become the second highest cause of cancer death. Partial liver resection (LR) or orthotopic liver transplantation (OLT) are regarded as the standard curative treatments for HCC. However, the long-term prognosis following surgery remains unsatisfactory because of the high rate of intrahepatic recurrence and lack of effective adjuvant therapy to prevent recurrence. Thus, it is important to investigate the molecular mechanisms of HCC and identify markers of its prognosis and recurrence.Human leukocyte antigen F-associated transcript (FAT10) was originally discovered through identification of expressed genes from the HLA-F genomic locus. It belongs to the ubiquitin-like protein family (Ubls), which covalently modify target substrates by binding via their C-termini containing conserved Gly-Gly motifs. Recent advances have indicated that Ubls is involved in regulating numerous processes, including cell division, DNA repair, autophagy, signal transduction, and embryonic development. Thus, Ubls members have multiple essential biological effects and it is not surprising that their function (and often dysfunction) plays an important role in various diseases, particularly cancer.Dyregulation of FAT10has been reported in several human cancers. Increased FAT10expression is correlated with the progression and prognosis of colon cancer and gastric cancer, while FAT10overexpression is found in90%of human HCCs.13Studies on the pathogenesis of HCC have indicated that increased expression of FAT10is involved in the induction of HCC by diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridine decarboxylate (DDC) and in the formation of Mallory Denk bodies (MDBs), which are considered to be a precancerous lesion for HCC. Persistent FAT10expression for14months was followed by formation of FAT10-positive tumors and FAT10-positive hepatocytes showed a proliferative advantage over adjacent normal hepatocytes after exposure to DDC. These findings suggest that FAT10plays a role in hepatic oncogenesis. However, there has been little research into the influence of FAT10on the progression and prognosis of HCC. Also, the precise role of FAT10in tumorigenesis and the molecular mechanism involved are unclear.Here, we report that FAT10is overexpressed in human HCC and that its expression is correlated with tumor recurrence and with a poor prognosis. We also report that ectopic expression of FAT10promotes the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of HCC cells via activation of the RhoA/Akt/GSK-3β pathway. These findings may have implications for the role of FAT10in the progression of HCC.Methods:1. Expression of FAT10in HCC and adjacent non-tumoral tissue and its clinical significance in HCC patientsExpression of FAT10was first analyzed by real-time RT-PCR in30HCC tissue specimens and their matched adjacent non-tumoral tissue specimens. It was also assessed by Western blotting in8HCC specimens. We also examined212paraffin-embedded HCC tissues by Immunohistochemical analysis. To verify our observation, we further analyzed the expression of FAT10protein by TMAs including278HCC tissues and80non-tumoral liver tissues.The Mann-Whitney U-test was used to analyse the relationship Between FAT10expression and clinicopathological characteristics. Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Univariate and multivariate analyses were based on the Cox proportional hazards regression model.2. The biological functions of FAT10in HCC cellsThe cDNA encoding FAT10was cloned into pReceiver-M29vector and generated expression vectors of FAT10. FAT10or control vectors were transfected into HepG2and SMMC-7721cells using FuGENE HD Transfection Reagent. G418was added to selected positive colonies. Transfection of the siRNAs in Huh7and MHCC-LM3was performed using LipofectamineTM RNAiMAX. The expression of FAT10was confirmed by Western blot analysis. MTT assay was engineered to detect the effect of FAT10on cell proliferation, bromodeoxyuridine (BrdU) assay was performed to analyze proliferative phase cells, Flow Cytometry was used to examine the cell cycle progression, proliferative and cell cycle related proteins was detected by western blot, Tumor xenografts in nude mice were further performed to conform proliferative active of FAT10in vivo, and proliferative and cell cycle related proteins in tumors of the nude mice with two groups was detected by immunohistochemistry.Confocal microscope was used to analyze morphologic analysis of FAT10- overexpressing cells. Changes in quantity and distribution of EMT related proteins were performed by western blot, immunohistochemistry and Immunofluorescence analysis with FAT10knockdown or overexpression in HCC cells and tumors of the nude mice. We further detected the invasion and migration capabilities of HCC cells with FAT10knockdown or overexpression by transwell invasion assays.3. Exploring the possible molecular mechanism underlying FAT10-mediated effects on the proliferation, invasion, and EMTof HCC cells.The phosphorylation and non-phosphorylation level of Akt/GSK-3β signaling proteins with FAT10knockdown or overexpression were detected by western blot method. We added in LY294002, which was specificlally inhibited PI3K/Akt pathway, to further analyze these above proteins by western blot and to observe the proliferative active by MTT assay.ResultsThe main results and findings are as follows:1. FAT10is overexpressed in HCC specimensExpression of FAT10was first analyzed by real-time RT-PCR in30HCC tissue specimens and their matched adjacent non-tumoral tissue specimens. It was also assessed by Western blotting in8HCC specimens. At both the mRNA and protein levels, FAT10expression was higher in HCC tissues compared with the matched adjacent non-tumorous liver tissues. These findings were confirmed by examining212paraffin-embedded HCC tissues by Immunohistochemical analysis. FAT10protein was positively detected in126of212(59.4%) HCC tissues and specific FAT10was predominantly found to be expressed in carcinoma cells with a strong staining signal in their cytoplasm.To verify this observation, we further analyzed the expression of FAT10protein by TMAs including278HCC tissues and80non-tumoral liver tissues. On immunohistochemical analysis,191(68.7%) of278HCC specimens showed high FAT10expression, whereas only4(13.3%) of30normal liver tissue specimens and11(22%) of50cirrhosis specimens showed high FAT10expression. These findings strongly indicated that FAT10is overexpressed in HCC.2. FAT10overexpression is correlated with poor outcome and recurrence of HCC patientsNext, we analyzed the association between FAT10expression in212HCC specimens and the clinicopathological characteristics of the patients. There was no significant correlation between expression of FAT10and the age, gender, tumor size, pathological grade, HBsAg status, cirrhosis, or AFP level. However, there were significant differences of FAT10expression in relation to metastasis (P=0.039), and tumor recurrence (P<0.001).To assess the relation between FAT10expression and the clinical outcome of HCC patients, Kaplan-Meier analysis and the log-rank test were employed to compare subgroups of patients with high versus low FAT10expression. It was found that patients whose tumors showed high FAT10expression had significantly shorter survival than those whose tumors showed low FAT10expression (n=212, P=0.001). To further evaluate the association between FAT10expression and the prognosis, Cox regression analysis was employed. Univariate analysis showed that age, pathological grade, AFP, metastasis, recurrence, and FAT10expression were significant prognostic factors. A multivariate model including these parameters demonstrated that FAT10expression was an independent prognostic factor (P=0.015).FAT10expression was significantly correlated with tumor recurrence and the FAT10-positive rate was significantly higher in patients with recurrence of HCC (74.4%) than in those without recurrence (P<.001). This result encouraged us to investigate the prognostic value of FAT10expression in patients with tumor recurrence. High expression of FAT10was a poor prognostic factor for patients with recurrent HCC (n=86, P=0.027), whereas, that was no prognostic value for patients with recurrence-free (n=126, P=0.107). AFP is widely regarded as a reliable marker for recurrence of HCC, so we further analyzed FAT10expression in relation to the AFP level and tumor recurrence. Interestingly, high FATIO expression was inversely correlated with the prognosis in patients with recurrence of HCC and high AFP levels (n=67, P=0.003). In contrast, FAT10had no prognostic value for patients with recurrence-free of HCC and low AFP levels (n=42, P=0.820). These findings suggested that FAT10could be a useful marker for predicting tumor recurrence and a poor prognosis and that it played an important role in the progression of HCC.3. FAT10promotes tumor cell growth in vitro and in vivo and induces cell cycle progressionTo further investigate the biological role of FAT10in HCC, we developed stable clones with FAT10overexpression from HepG2and SMMC-7721cells, which exhibit relatively low expression of FAT10among HCC cell lines. We found that FAT10-overexpressing HepG2or SMMC-7721cells showed a significantly higher in vitro proliferation rate than control or vector-transfected cells (P<0.001). FAT10-overexpressing cells also generated larger xenografts in nude mice than control cells, indicating that FAT10stimulated in vivo tumor growth. To verify the specificity of this tumor-promoting effect, we knocked down FAT10in Huh7cells, which normally show relatively high FAT10expression. We found that down-regulation of FAT10strongly inhibited tumor growth (P<0.05). Collectively, these results suggested that FAT10could promote HCC cell proliferation.To understand the mechanism by which FAT10contributes to tumor cell proliferation, we analyzed changes of the cell cycle in FAT10overexpressing or knockdown cells. FAT10overexpression was associated with an increase of cells entering S phase and a corresponding decline of cells in the G1and G2phases on FACS analysis. Consistent with the FACS data, the percentage of BrdU-positive cells was significantly increased by FAT10overexpression compared with the control. After knockdown of FAT10by specific siRNA, both FACS analysis and the BrdU incorporation assay showed a significant decrease of cells in S phase, further suggesting that FAT10has a role in regulating cell proliferation and the cell cycle. This conclusion was further confirmed by examining proliferation-and cell cycle-related proteins. Expression of cyclin D1, c-myc, CDK4, and CDK6was significantly upregulated in FAT10-overexpressing cells, whereas expression of these proteins was downregulated in FAT10knockdown cells. Moreover, FAT10-overexpressing tumors grown in nude mice showed strong staining for PCNA, c-myc, and cyclin D1. These results suggested that FAT10promotes cell proliferation by facilitating cell cycle progression.4. Ectopic expression of FAT10induces the epithelial-mesenchymal transition (EMT) and enhances tumor cell invasion in vitroSurprisingly, we found that FATIO itself was sufficient to induce the EMT in HCC cells. After we developed stable clones overexpressing FAT10from HepG2and SMMC-7721cells, we found a dramatic morphological change of these FAT10-overexpressing cells, with the polygonal cells seen under normal culture conditions becoming spindle-like fibroblastic cells, which is one of the main characteristics of the EMT. At the molecular level, there was a corresponding decrease in expression of the epithelial marker E-cadherin and an increase in expression of the mesenchymal marker vimentin. After silencing of FAT10expression in Huh7and MHCC-LM3cells, their morphology was not changed, but E-cadherin expression increased and vimentin expression decreased.Previous studies have revealed that the EMT is transcriptionally regulated by a family of transcription factors, including snail1and smad2/3, which repress E-cadherin in various cells. We found that snail1and smad2/3were significantly elevated in FAT10-overexpressing HepG2and SMMC-7721cells, but were significantly reduced in FAT10knockdown Huh7and MHCC-LM3cells. Similar changes of expression were also found in tumor xenografts by immunohistochemistry, with decreased expression of E-cadherin and increased expression of vimentin and snail1being observed in FAT10-overexpressing tumors from nude mice. These findings suggested that FAT10enhances the EMT in vivo.It has been reported that β-catenin is overexpressed in HCC and its accumulation in tumor cells might contribute to a decrease of E-cadherin expression and induction of the EMT. The present study showed that changes of FAT10expression led to subcellular redistribution of β-catenin. Immunofluorescence staining demonstrated that β-catenin mainly accumulated in the nuclei of FAT10-overexpressing cells stimulated with EGF, whereas it mainly displayed a cytoplasmic distribution in FAT10knockdown cells.Since the EMT could confer invasive capacity on tumor cells, we also investigated the effect of FAT10expression on cell invasion. In the cell invasion assay, FAT10-overexpressing cells exhibited a significant increase of invasiveness (P<0.001), whereas FAT10knockdown showed significantly less invasiveness (P<0.001). Overall, these findings demonstrate that FAT10overexpression promotes the EMT and cell invasion.5. FAT10activates the Akt/GSK-3β pathway in HCCTo investigate the molecular basis of FAT10-mediated promotion of tumor cell proliferation, tumor cell invasion, and the EMT, we first assessed the activation of several signaling pathways. Among the molecules we screened, phosphorylation of Akt was increased in FAT10-overexpressing cells compared with control cells. The PI3K/Akt pathway was reported to have a role in various cellular functions, including proliferation, invasion, and the EMT. We found that phosphorylated Akt was significantly elevated in FAT10-overexpressing HepG2cells and SMMC-7721cells, whereas it was reduced when FAT10was knocked down in Huh7and MHCC-LM3cells. Serine/threonine protein kinase GSK-3β, a downstream target of PI3K/Akt, was inactivated by phosphorylation at serine9, whereas the total GSK-3β level was unchanged. As GSK-3β regulates snail and β-catenin, these results were consistent with our finding that inactivation of GSK-3β upregulated both snail and β-catenin, potentially inducing cell migration, cell proliferation, and the EMT. Blocking the PI3K/Akt pathway with LY294002led to marked reduction in the expression of phosphorylated Akt, phosphorylated GSK-3β, and P-catenin in FAT10-overexpressing HepG2cells and SMMC-7721cells. Furthermore, blocking of the PI3K/Akt pathway significantly reduced the proliferative capacity of FAT10-overexpressing cells, demonstrating that this pathway is crucial for FAT10-mediated signaling. Taken together, we disclosed a novel FAT10/Akt/GSK3β pathway that contributed to the effects of FAT10in HCC cells.Conclusion1. FAT10is overexpression in HCC tissues and could be serve as a useful marker for predicting tumor recurrence and a poor prognosis.2. FAT10promotes tumor cell prolifrative active in vitro and in vivo by inducing cell cycle progression.3. FAT10induces the epithelial-mesenchymal transition (EMT) and enhances HCC cells invasion in vitro.4. FAT10regulates the biological roles of HCC cells via the Akt/GSK-3β pathway.