Potential conflict of interest: Nothing to report.
The epithelial-mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis of human cancers. In this study we investigated the expression profiles of the EMT markers, the relationship between EMT markers and patient/tumor/viral factors, and the interplay between major EMT regulators in human hepatocellular carcinoma (HCC). Reduced E-cadherin and nonmembranous β-catenin expression, the hallmarks of EMT, were shown in 60.2% and 51.5% of primary HCC samples, respectively. Overexpression of Snail, Twist, or Slug, the major regulators of EMT, was identified in 56.9%, 43.1%, and 51.4% of primary HCCs, respectively. Statistical analysis determined that Snail and Twist, but not Slug, are major EMT inducers in HCC: overexpression of Snail and/or Twist correlated with down-regulation of E-cadherin, nonmembranous expression of β-catenin, and a worse prognosis. In contrast, there were no such significant differences in samples that overexpressed Slug. Coexpression of Snail and Twist correlated with the worst prognosis of HCC. Hepatitis C-associated HCC was significantly correlated with Twist overexpression. HCC cell lines with increased Snail and Twist expression (e.g., Mahlavu) exhibited a greater capacity for invasiveness/metastasis than cells with low endogenous Twist/Snail expression (e.g., Huh-7). Overexpression of Snail or/and Twist in Huh-7 induced EMT and invasiveness/metastasis, whereas knockdown of Twist or Snail in Mahlavu reversed EMT and inhibited invasiveness/metastasis. Twist and Snail were independently regulated, but exerted an additive inhibitory effect to suppress E-cadherin transcription. Conclusion: Our study provides a comprehensive profile of EMT markers in HCC, and the independent and collaborative effects of Snail and Twist on HCC metastasis were confirmed through different assays. (HEPATOLOGY 2009.)
Hepatocellular carcinoma (HCC) is one of the most common neoplasms in South Africa and Asian countries including Taiwan.1 Most of the HCC in Taiwan stems from the high prevalence of chronic hepatitis B virus (HBV) infection (15%-20%).2 Although successful partial hepatectomy has significantly improved survival, the prognosis of HCC remains poor because of tumor invasiveness, frequent intrahepatic spread, and extrahepatic metastasis.3 Elucidation of the molecular mechanisms underlying HCC invasiveness is of utmost importance for the development of future strategies for treating HCC.
The epithelial-mesenchymal transition (EMT), a developmental process by which epithelial cells reduce intercellular adhesion and acquire fibroblastoid properties, has been shown to be critical for the development of the invasiveness and metastatic potential of human cancers.4 Characteristic changes during EMT include down-regulation of epithelial markers (e.g., E-cadherin and plakoglobin),4, 5 translocation of β-catenin (i.e., dissociation of membranous β-catenin and translocation into the nuclear compartment),6 and up-regulation of mesenchymal markers (e.g., vimentin and N-cadherin).4, 5 The EMT process is initiated by suppression of E-cadherin expression by the major EMT regulators, e.g., Snail, Slug, and Twist.4, 5 The zinc-finger transcriptional repressors Snail (also known as Snail1) and Slug (also known as Snail2) and the basic helix-loop-helix (bHLH) transcription factor Twist (also known as Twist1) were shown to induce EMT through repression of E-cadherin expression.7–9
The clinical significance of EMT has been confirmed in certain types of human cancers,10, 11 and the significance of individual EMT regulators in HCC has been demonstrated.12–15 However, a comprehensive study demonstrating the expression profile of multiple EMT markers in HCC is lacking. In addition, the role of hepatotrophic viruses, such as HBV and hepatitis C virus (HCV), in promoting the EMT/metastasis of HCC is unclear. In this study we investigated the expression profiles of EMT markers, the relationship between EMT markers and patient/tumor/viral factors, and the interplay between major EMT regulators in human HCC.
In all, 123 primary HCC samples with adjacent nontumorous liver tissues were obtained from patients who had undergone curative hepatic resection between 1990 and 2002 at Taipei Veterans General Hospital. Among these 123 cases, 46 of 84 recurrent tumor samples were available for analysis. The study was approved by the Institutional Review Board of Taipei Veterans General Hospital. The clinical characteristics of the patients with HCC are presented in Table 1.
Table 1. Univariate and Multivariate Analysis of Factors Associated with Cancer-Free Interval
Median Survival (mo) (95% CI)
ALT, alanine aminotransferase; BCLC, Barcelona Clinic Liver Cancer; CLIP, Cancer of the Liver Italian Program; CI: confidence interval; ICG-15, indocyanine clearance test; PT: Prothrombin time; RR: risk ratio; NA: not applicable; Y: yes, N: no.
Significant in multivariate analysis: positive anti-HCV: RR 2.387, 95% CI: 1.340-4.237, P = 0.003; multinodular tumors: RR 1.941, 95% CI: 1.164-3.237, P = 0.011; AFP > 100 ng/mL: RR 1.936, 95% CI: 1.101-3.392, P = 0.021;E-cadherin downregulation: RR 4.374, 95% CI: 2.289-8.359, P < 0.001; Twist overexpression: RR 2.978, 95% CI : 1.750-5.068, P < 0.001.
The sample processing and IHC procedures were performed as described.16 The antibodies used in IHC and the incubation periods are listed in Supporting Table S1. All IHC staining was independently scored by two experienced specialists. The expression of E-cadherin was scored as preserved, reduced, or absent, as described.17 Reduced or absent expression of E-cadherin was interpreted as down-regulation of E-cadherin.18 Nonmembranous expression of β-catenin (i.e., more than 10% of cells exhibiting cytoplasmic or nuclear staining) was scored as described.19 Nuclear expression of Snail, Slug, and Twist was graded from 0 to 3+ (0, no staining; 1+, 1%-25%; 2+, 26%-50%; 3+, >50% nuclear staining), with only 3+ considered a positive IHC result.20
Plasmids and Cell Lines.
The human hepatocellular carcinoma cell lines Huh7, HepG2, PLC, Hep3B, Sk-Hep1, Mahlavu, and the human embryonic kidney cell line HEK-293T were obtained from the American Type Culture Collection (Rockville, MD). Cell lines HA59T-VGH and HA22T-VGH were gifts from Professor C.P. Hu (Tung-Hai University, Taichung, Taiwan). The plasmids pcDNA3-Snail, pFLAG-Twist, pSUPER-Snail-si, and pSUPER-Twist-si have been described.20 A scrambled sequence with no significant homology to any mammalian gene sequence was cloned into the pSUPER vector (pSUPER-scr-si) for use as a control in short interference RNA (siRNA) experiments. The oligonucleotide sequences used for generating siRNA constructs are listed in Supporting Table S2.
RNA Purification and Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Analysis.
Total RNA extraction, complementary DNA (cDNA) synthesis, and quantitative real-time RT-PCR were performed as described.16 The primer sequences used in real-time PCR are shown in Supporting Table S2.
Western Blot Analysis.
Protein extraction from cultivated cells and western blot analysis were performed as described.16 Antibodies used in western blot experiments are listed in Supporting Table S1.
Detection of E-cadherin Expression by Enzyme-Linked Immunosorbent Assay (ELISA).
The procedures are detailed in the Supporting Methods.
Analysis of the activity of matrix metallopeptidases-2 and -9 (MMP-2 and MMP-9) in the different clones was performed as described.16 The procedures are detailed in the Supporting Methods.
Cell Migration and Invasiveness Assay.
In vitro transwell migration and invasion assays were performed as described.16 The procedures are detailed in the Supporting Methods.
Cloning of Gene Promoter Regions, Generation of Reporter Constructs, Transient Transfections, and Luciferase Assays.
The genomic regions flanking the E-cadherin, Snail, and Twist gene promoter regions (Fig. 6A,C,E) were generated by PCR amplification of human genomic DNA and inserted into the HindIII/BglII sites in the pXP2 vector to generate the pXP2-Ecadherin, pXP2-Twist, and pXP2-Snail reporter constructs. Transfections and reporter assays were performed as described, and the bacterial β-galactosidase gene (pCMV-βgal) was used as a control for transfection efficiency.20
All procedures involving animals were performed in accordance with the institutional animal welfare guidelines of Taipei Veterans General Hospital. Cells were injected into the splenic vein or the tail vein of 8-week-old nude mice (BALB/c strain) at 1 × 106 cells/injection site. The mice were sacrificed after 6 weeks and the number and volume of metastatic tumors were assessed. Tumor size was measured by use of a caliper and volume was calculated as length × height × width × 0.5236 with reference to a previous report.21
The Kaplan-Meier estimate was used for survival analysis and the log-rank test was selected to compare the difference. A Cox proportional hazards model was applied to multivariate survival analysis for testing independent prognostic factors. Statistical significance was accepted when P < 0.05 for all tests.
Down-regulation of E-cadherin in HCC.
IHC was performed to investigate the significance of down-regulated E-cadherin expression, the hallmark of EMT,4, 5 in HCC patients. Among primary HCCs, 39.8% of the samples showed preserved expression of E-cadherin (Case 1 of Fig. 1A; Supporting Fig. S1A, left panel). Decreased (Case 2 of Fig. 1A; Supporting Fig. S1A, middle panel) or absent E-cadherin expression (Supporting Fig. S1A, right panel) was identified in 52.8% and 7.4% of the samples, respectively. Among the samples with recurrent HCC, preserved, decreased, or absent expression of E-cadherin was shown in 23.9%, 63.1%, and 13% cases, respectively. An increased proportion of down-regulated E-cadherin expression was shown in recurrent compared with primary samples (Fig. 1B). The results of IHC analysis for E-cadherin were validated by ELISA and real-time RT-PCR in representative HCC samples (Supporting Fig. S1B,C). Survival analysis demonstrated a significant decrease in cancer-free interval (CFI) and overall survival (OS) in patients with down-regulated expression of E-cadherin (Table 1, Supporting Table S11, Fig. 1D). Down-regulated expression of E-cadherin was also associated with factors indicating clinical aggressiveness (e.g., large tumor size; Supporting Table S3), and a trend toward multinodular tumors was observed (Supporting Table S3).
Nonmembranous Expression of β-Catenin in HCC.
Translocation of β-catenin is an important marker of EMT.6 IHC analysis for β-catenin was performed in the available 103 primary and 33 recurrent HCC samples. A higher incidence of nonmembranous expression of β-catenin (Case 2 of Fig. 1A) was shown in recurrent versus primary HCCs (66.7% versus 51.5%; Fig. 1C). Nonmembranous expression of β-catenin was associated with down-regulated E-cadherin (Supporting Table S4), a shorter CFI, and a worse OS (Table 1, Supporting Table S11, Fig. 1E) and a trend toward multinodular tumors and microscopic venous invasion was observed (Supporting Table S5).
Expression Profile and Clinical Significance of the Major EMT Regulators (Snail, Slug, and Twist) in HCC.
The expression profiles of Snail, Slug, and Twist in HCC were evaluated. An increased incidence of Snail (Supporting Fig. S2A) or Twist overexpression (Supporting Fig. S3A) was shown in the recurrent versus primary HCCs (Supporting Figs. S2B, S3B). However, the percentages of Slug overexpression (samples available in 105 primary and 33 recurrent cases; Supporting Fig. S4A) were similar in the recurrent versus primary samples (Supporting Fig. S4B). Expression of Snail or Twist, but not Slug, was associated with down-regulation of E-cadherin and β-catenin translocation (Supporting Table S6). Overexpression of Snail or Twist was associated with a shorter CFI and OS (Table 1, Supporting Table S11, Supporting Figs. S2C, S3C) and aggressive tumor behavior (e.g., large tumor size and multinodular tumors; Supporting Tables S7, S8). Surprisingly, increased Twist expression was significantly correlated with serum anti-HCV positivity and HBsAg negativity (Supporting Table S8). Although increased Slug expression was associated with aggressive clinical factors (e.g., large tumor size; Supporting Table S9), no prognostic significance of Slug on OS and CFI was demonstrated (Table 1, Supporting Table S11, Supporting Fig. S4).
Snail and Twist were frequently coexpressed (30.1% of primary and 60.9% of recurrent HCCs; Fig. 2A). Coexpression of Snail and Twist was associated with down-regulated expression of E-cadherin, nonmembranous expression of β-catenin (Supporting Table S6), and the worst CFI and OS (Table 1, Supporting Table S11, Fig. 2B). Coexpression of both markers was also associated with aggressive factors (e.g., a trend toward multinodular tumors; Supporting Table S10).
Analyses of Factors Associated with CFI and OS in HCC.
Survival analysis was performed to determine the independent factors associated with CFI or OS. An impact on CFI was shown for many factors in the univariate analysis (Table 1); however, only anti-HCV positivity, elevated levels of α-fetoprotein (AFP), multinodular tumors, down-regulation of E-cadherin, and overexpression of Twist were independent predictors of CFI (Table 1). A number of factors were also associated with OS in the univariate analysis (Supporting Table S11), but multivariate analysis limited this list to patient age, anti-HCV positivity, prolonged prothrombin time, multinodular tumors, and coexpression of Snail and Twist (Supporting Table S11).
Expression of Snail and Twist Is Associated with a Mesenchymal Phenotype and Invasiveness/Metastasis of HCC Cells.
Because Snail and Twist are the major inducers of EMT in HCC, we investigated the expression of Snail/Twist and its association with EMT markers/phenotypic changes in eight HCC cell lines. The results indicated that cells with a higher migratory ability and invasiveness showed associated mesenchymal changes, higher levels of Snail or Twist expression, and higher MMPs activity, whereas cells with lower invasiveness/migration exhibited epithelial markers, lower levels of Snail or Twist, and lower MMPs activity (Fig. 3). An in vivo tail-vein metastasis assay was performed to compare the metastatic capacity of Mahlavu, the most invasive cell line, with Huh7, the least invasive one. A significant increase in pulmonary metastasis was noted in mice receiving injections of Mahlavu versus Huh7 (Fig. 4A -C). A similar result was shown in the splenic-vein model (Fig. 4D-F). These results indicated that HCC cells with a mesenchymal phenotype (e.g., Mahlavu cells) were more invasive and exhibited greater in vivo metastatic ability, whereas HCC cells expressing epithelial markers (e.g., Huh7 cells) were associated with a lower tendency to migrate, invade, and metastasize.
Twist and Snail Are Regulated Independently and Act Additively in Promoting EMT.
To investigate the independent and nonredundant roles of Snail and Twist in HCC metastasis, we used Huh7 cells to generate clones with stable expression of Snail, Twist, or both proteins. Overexpression of Snail and/or Twist resulted in a shift in EMT marker expression (Fig. 5A). Expression of either Snail or Twist alone did not influence the expression levels of the other (Fig. 5A). Overexpression of Snail or Twist increased migration/invasion/metastasis, and cells that coexpressed both proteins demonstrated the highest ability for migration/invasion/metastasis (Fig. 5C, D). Interestingly, overexpression of Snail resulted in higher activities of MMP-2 and MMP-9 than overexpression of Twist (Fig. 5B), but expression of Twist tended to result in slightly greater migration/metastasis than did expression of Snail (Fig. 5C,D).
Promoter activity assays were performed to determine whether Twist and Snail act collaboratively or independently. Overexpression of Snail or Twist resulted in a significant decrease in E-cadherin promoter activity, as expected; and combined overexpression of Snail and Twist seemed to exert an additive inhibitory effect on the E-cadherin promoter compared to either agent alone (Fig. 6A,B). Snail expression could not activate the reporter driven by the Twist promoter. Likewise, Twist expression did not influence the activity of the Snail promoter (Fig. 6C-F). These results confirmed the independent expression and regulation of Twist and Snail.
Twist and Snail Are Both Critical for HCC Cell EMT/Migration/Metastasis.
To determine whether Twist and Snail are critical mediators of EMT in HCC, Twist or Snail expression in the highly invasive Mahlavu cells was suppressed by siRNA. Repression of either Snail or Twist resulted in down-regulation of vimentin and up-regulation of E-cadherin expression (Fig. 7A), and suppression of in vitro migration/invasion (Fig. 7C) and in vivo metastasis (Fig. 7D). However, knockdown of Snail caused a more significant decrease in MMPs activity than Twist (Fig. 7B). These results further confirmed the critical but different roles of Snail and Twist in mediating EMT and metastasis of HCC cells.
Induction of EMT is distinguished by the repression of E-cadherin transcription by zinc-finger proteins (e.g., Snail, Slug)7, 8 or bHLH family transcriptional factors (e.g., Twist)9 through binding to three E-boxes located in the proximal promoter of E-cadherin. Although EMT has been considered the critical mechanism involved in cancer metastasis, in comparison with other types of human cancers only a few sporadic studies have focused on the significance of EMT in HCC.12–15 The present study is the first to provide a comprehensive profile of multiple EMT markers and to demonstrate that Snail and Twist, but not Slug, are the major inducers of EMT in HCC. These results not only pinpoint the major signal pathways in the induction of EMT in HCC, but also demonstrate its clinical relevance through comprehensive analysis.
Transforming growth factor β (TGF-β) is a master regulator of EMT through activation of the Smad2/3 complex.22 TGF-β1 plays a key role in triggering EMT in HCC through cooperation with laminin-5,23 and inhibition of TGF-β1 attenuates migration/invasion of HCC cells.24 One recent report showed that in HCC the expression of HCV-derived core protein switches the cellular response to TGF-β exposure from inhibition of growth to induction of EMT,25 providing a possible linkage between HCV-induced HCC and EMT. Although HCV-related HCC has been shown to be associated with a higher recurrence rate after surgery,26 the underlying mechanism is unclear, and the correlation between HCV and EMT has never been explored. We report the original finding that overexpression of Twist is correlated with HCV-related HCC. This interesting finding may partially explain the highly invasive behavior and poor prognosis of HCV-related HCC. The mechanism underlying increased Twist expression in HCV-related HCC deserves further study.
The interplay between Snail and Twist in the promotion of EMT in HCC has not been reported. In the present study we provide evidence that Twist and Snail are regulated independently and act collaboratively to promote EMT: coexpression of Snail and Twist indicates the worst prognosis for HCC; HCC cell lines harboring both Snail and Twist showed a higher migratory/metastatic ability; coexpression of Snail and Twist in Huh7 promoted the highest invasiveness and metastasis; and Twist and Snail did not influence the expression/promoter activity of each other. This is the first report to demonstrate the independent regulation of Snail and Twist and their additive effects on EMT promotion.
Differences in the roles of Snail and Twist in promoting HCC metastasis were disclosed in our study: although Snail was more potent in activating MMPs, HCC cells overexpressing Twist tended to be more invasive than cells overexpressing Snail, and this observation was supported by analysis of clinical samples. These results indicate that Twist may be more critical in HCC metastasis, possibly acting through an MMP-independent mechanism. Previous studies disclosed that some critical molecules (e.g., N-cadherin, Akt2, and YB-1) in cancer metastasis are direct targets of Twist.27–29 Overexpression of Twist induces angiogenesis.30 Collectively, our results indicate that Snail and Twist act additively to promote HCC metastasis through different mechanisms.
In conclusion, our study demonstrates the prognostic significance of EMT markers in HCC and also establishes different roles for Snail and Twist in the promotion of EMT in HCC through independent regulation. A correlation between HCV-induced HCC and Twist expression is also reported. These results elaborate on the major mechanisms involved in HCC metastasis and provide essential information for prediction of prognosis and identification of new treatment targets for future HCC management.
We thank Ms. Hsiao-Jung Wang for excellent technical assistance. The authors thank the Sequencing Core Facility of the National Yang-Ming University Genome Research Center (YMGC) for technical support. The Sequencing Core Facility is supported by the National Research Program for Genome Medicine (NRPGM), Taiwan.