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1.  Critical Roles of p53 in Epithelial-Mesenchymal Transition and Metastasis of Hepatocellular Carcinoma Cells 
PLoS ONE  2013;8(9):e72846.
Hepatocellular carcinoma (HCC) is one of the most malignant tumors and the biggest obstacle in curing HCC is its high metastasis potential. Alteration of p53 is the most frequent genetic change found in HCC. Although the biological function of p53 in tumor initiation and progression has been well characterized, whether or not p53 is implicated in metastasis of HCC is largely unknown. In this study, we analyzed the potential functions of p53 in epithelial-mesenchymal transition (EMT) and metastasis of HCC cells. Both insulin- and TGF-β1-induced changes of critical EMT markers were greatly enhanced by p53 knockdown in HCC cells. The insulin- and TGF-β1-stimulated migration of HCC cells were enhanced by p53 knockdown. Furthermore, in vivo metastasis of HCC cells using different mouse models was robustly enhanced by p53 knockdown. In addition, we found that p53 regulation on EMT and metastasis involves β-catenin signaling. The nuclear accumulation and transcriptional activity of β-catenin was modulated by p53. The enhanced EMT phenotype, cell migration and tumor metastasis of HCC cells by p53 knockdown were abrogated by inhibiting β-catenin signal pathway. In conclusion, this study reveals that p53 plays a pivotal role in EMT and metastasis of HCC cells via its regulation on β-catenin signaling.
doi:10.1371/journal.pone.0072846
PMCID: PMC3759437  PMID: 24023784
2.  Toll-Like Receptor 3 Expressing Tumor Parenchyma and Infiltrating Natural Killer Cells in Hepatocellular Carcinoma Patients 
Background
Hepatocellular carcinoma (HCC) is a highly aggressive cancer that is linked to chronically dysregulated liver inflammation. However, appropriate immune responses can control HCC progression. Here we investigated the role and underlying mechanism of toll-like receptor 3 (TLR3) in HCC.
Methods
HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3–9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan–Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided.
Results
TLR3 activation increased cell death in the TLR3+ SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).
Conclusions
TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.
doi:10.1093/jnci/djs436
PMCID: PMC3814220  PMID: 23197495
3.  Alcohol, nutrition and liver cancer: Role of Toll-like receptor signaling 
This article reviews the evidence that ties the development of hepatocellular carcinoma (HCC) to the natural immune pro-inflammatory response to chronic liver disease, with a focus on the role of Toll-like receptor (TLR) signaling as the mechanism of liver stem cell/progenitor transformation to HCC. Two exemplary models of this phenomenon are reviewed in detail. One model applies chronic ethanol/lipopolysaccharide feeding to the activated TLR4 signaling pathway. The other applies chronic feeding of a carcinogenic drug, in which TLR2 and 4 signaling pathways are activated. In the drug-induced model, two major methyl donors, S-adenosylmethionine and betaine, prevent the upregulation of the TLR signaling pathways and abrogate the stem cell/progenitor proliferation response when fed with the carcinogenic drug. This observation supports a nutritional approach to liver cancer prevention and treatment. The observation that upregulation of the TLR signaling pathways leads to liver tumor formation gives evidence to the popular concept that the chronic pro-inflammatory response is an important mechanism of liver oncogenesis. It provides a nutritional approach, which could prevent HCC from developing in many chronic liver diseases.
doi:10.3748/wjg.v16.i11.1344
PMCID: PMC2842526  PMID: 20238401
Toll-like receptor; Hepatocellular carcinoma; Methyl donors, Epigenetic processes; Inflammation; Alcohol; Drug toxicity; Lipopolysaccharides
4.  Differential Roles of Insulin-like Growth Factor Receptor- and Insulin Receptor-mediated Signaling in the Phenotypes of Hepatocellular Carcinoma Cells12 
Neoplasia (New York, N.Y.)  2009;11(9):835-845.
Intrahepatic and extrahepatic metastases are common findings in hepatocellular carcinoma (HCC). Insulin-like growth factor 2 (IGF2) expression is frequently induced in HCC, and serum IGF2 levels correlate with the presence of extrahepatic metastases. Yet, the role of IGF-induced signaling in the dissemination of HCC remains unclear. We have previously observed elevated IGF2 levels in tumors with metastatic potential in an HCC mouse model. Here, we demonstrate that inhibition of IGF2, or its receptor IGF1R, impairs the migration and invasion activities of murine HCC cells. Furthermore, inhibition of IGF1R also impairs the ability of HCC cells to colonize the lungs after introduction into the circulation through the tail vein but does not impair subcutaneous tumor growth. Collectively, these findings suggest that IGF1R-mediated signaling plays a causative role in tumor dissemination but is not required for tumor growth per se. Although previous studies indicate that IGF ligands can signal through IGF1R/insulin receptor (IR) heterodimers, and IR-A homodimers, we demonstrate that the IR is not required for invasion and metastasis by HCC cells. Finally, we identify matrix metalloproteinase 2 as a mediator of the invasive phenotype downstream of IGF1R-induced signaling. Thus, our studies demonstrate the importance of IGF2-induced signaling in the dissemination of HCC cells.
PMCID: PMC2735682  PMID: 19724677
5.  Epithelial-mesenchymal transition expression profiles as a prognostic factor for disease-free survival in hepatocellular carcinoma: Clinical significance of transforming growth factor-β signaling 
Oncology Letters  2012;5(1):149-154.
Epithelial-mesenchymal transition (EMT) plays a pivotal role in cancer invasion and metastasis, and transforming growth factor (TGF)-β signaling is a potent inducer of EMT. However, the clinical significance of the correlation between EMT marker expression and TGF-β signaling in hepatocellular carcinoma (HCC) patients remains unknown. In this study, immunohistochemistry was used to analyze the expression of EMT markers and phospho-Smad2 nuclear positivity, and their association with clinicopathological features in 150 HCC patients. E-cadherinhigh/vimentinlow and E-cadherinlow/vimentinhigh expression profiles were determined in 55 (36.7%) and 21 (14.0%) patients, respectively. The E-cadherinlow/vimentinhigh expression profile was significantly correlated with poor tumor differentiation (P<0.001), vascular invasion (P=0.007) and extrahepatic recurrence following curative surgery (P=0.026). Furthermore, the E-cadherinlow/vimentinhigh expression profile was significantly correlated with shorter disease-free survival compared to E-cadherinhigh/vimentinlow (P=0.002). Forty-one patients (27.3%) were demonstrated to have high phospho-Smad2 nuclear positivity, which was significantly correlated with the E-cadherinlow/vimentinhigh expression profile (P<0.001). In conclusion, this study suggests that EMT expression profiles are useful prognostic markers for disease-free survival in HCC patients, and that the E-cadherinlow/vimentinhigh expression profile is closely associated with high-grade malignant behavior such as tumoral vascular invasion and metastasis in HCC. Additionally, TGF-β-mediated EMT may play an important role in the aggressiveness of HCC.
doi:10.3892/ol.2012.954
PMCID: PMC3525349  PMID: 23255911
TGF-β; epithelial-mesenchymal transition; hepatocellular carcinoma; Smad2
6.  The aPKCι blocking agent ATM negatively regulates EMT and invasion of hepatocellular carcinoma 
Ma, C Q | Yang, Y | Wang, J M | Du, G S | Shen, Q | Liu, Y | Zhang, J | Hu, J L | Zhu, P | Qi, W P | Qian, Y W | Fu, Y
Cell Death & Disease  2014;5(3):e1129-.
Epithelial-to-mesenchymal transition (EMT) has an important role in invasion and metastasis of hepatocellular carcinoma (HCC). To explore the regulatory mechanism of atypical protein kinase C ι (aPKCι) signaling pathways to HCC development, and find an agent for targeted therapy for HCC, immortalized murine hepatocytes were employed to establish an EMT cell model of HCC, MMH-RT cells. Our study showed that EMT took place in MMH-R cells under the effect of transforming growth factor-β1 (TGF-β1) overexpressing aPKCι. Furthermore, we showed that the aPKCι blocking agent aurothiomalate (ATM) inhibited EMT and decreased invasion of hepatocytes. Moreover, ATM selectively inhibited proliferation of mesenchymal cells and HepG2 cells and induced apoptosis. However, ATM increased proliferation of epithelial cells and had little effect on apoptosis and invasion of epithelial cells. In conclusion, our result suggested that aPKCι could be an important bio-marker of tumor EMT, and used as an indicator of invasion and malignancy. ATM might be a promising agent for targeted treatment of HCC.
doi:10.1038/cddis.2014.91
PMCID: PMC3973203  PMID: 24651432
epithelial-to-mesenchymal transition; aPKCι; hepatocellular carcinoma; aurothiomalate; targeted drug
7.  Crosstalk between Beta-Catenin and Snail in the Induction of Epithelial to Mesenchymal Transition in Hepatocarcinoma: Role of the ERK1/2 Pathway 
Epithelial to mesenchymal transition (EMT) is an integral process in the progression of many epithelial tumors. It involves a coordinated series of events, leading to the loss of epithelial features and the acquisition of a mesenchymal phenotype, resulting in invasion and metastasis. The EMT of hepatocellular carcinoma (HCC) cells is thought to be a key event in intrahepatic dissemination and distal metastasis. In this study, we used 12-O-tet-radecanoylphorbol-13-acetate (TPA) to dissect the signaling pathways involved in the EMT of HepG2 hepatocarcinoma cells. The spectacular change in phenotype induced by TPA, leading to a pronounced spindle-shaped fibroblast-like cell morphology, required ERK1/2 activation. This ERK1/2-dependent EMT process was characterized by a loss of E-cadherin function, modification of the cytoskeleton, the acquisition of mesenchymal markers and profound changes to extracellular matrix composition and mobility. Snail was essential for E-cadherin repression, but was not sufficient for full commitment of the TPA-triggered EMT. We found that TPA triggered the formation of a complex between Snail and β-catenin that activated the Wnt pathway. This study thus provides the first evidence for the existence of a complex network governed by the ERK1/2 signaling pathway, converging on the coregulation of Snail and the Wnt/β-catenin pathway and responsible for the onset and the progression of EMT in hepatocellular carcinoma cells.
doi:10.3390/ijms141020768
PMCID: PMC3821642  PMID: 24135872
epithelial-mesenchymal transition; ERK1/2; hepatocellular carcinoma; β-catenin; snail
8.  Krüppel-Like Factor 4, a Tumor Suppressor in Hepatocellular Carcinoma Cells Reverts Epithelial Mesenchymal Transition by Suppressing Slug Expression 
PLoS ONE  2012;7(8):e43593.
Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that plays an important role in differentiation and pathogenesis. KLF4 has been suggested to act as an oncogene or tumor suppressor in different tumor types. However, the role of KLF4 in hepatocellular carcinoma (HCC) remains unclear. Here, we demonstrate that forced expression of Klf4 in murine HCC cell lines reduced anchorage-independent growth in soft agar as well as cell migration and invasion activities in vitro. Ectopic Klf4 expression impaired subcutaneous tumor growth and lung colonization in vivo. By contrast, Klf4 knockdown enhanced HCC cell migration. Interestingly, ectopic expression of Klf4 changed the morphology of murine HCC cells to a more epithelial phenotype. Associated with this, we found that expression of Slug, a critical epithelial mesenchymal transition (EMT)-related transcription factor, was significantly down-regulated in Klf4-expressing cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays showed that Klf4 is able to bind and repress the activity of the Slug promoter. Furthermore, ectopic Slug expression partially reverts the Klf4-mediated phenotypes. Consistent with a role as a tumor suppressor in HCC, analysis of the public microarray databases from Oncomine revealed reduced KLF4 expression in human HCC tissues in comparison with normal liver tissues in 3 out of 4 data sets. By quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we found reduced KLF4 mRNA in 50% of HCC tissues. Importantly, an inverse correlation between the expression of KLF4 and SLUG was found in HCC tissues. Our data suggest that KLF4 acts as a tumor suppressor in HCC cells, in part by suppressing SLUG transcription.
doi:10.1371/journal.pone.0043593
PMCID: PMC3427336  PMID: 22937066
9.  Intratumoral Hepatic Stellate Cells as a Poor Prognostic Marker and a New Treatment Target for Hepatocellular Carcinoma  
PLoS ONE  2013;8(11):e80212.
Hepatic stellate cells (HSCs), a specialized stromal cytotype in the liver, have been demonstrated to actively contribute to hepatocellular carcinoma (HCC) development. However, the previous studies were performed using HSC cell lines, and the prognostic value of intratumoral HSCs (tHSCs) was unclear. Here we isolated tHSCs from fresh human HCC tissues, and analyzed the abilities of tHSCs to promote HCC progression by using in vitro assays for cell viability, migration and invasion as well as epithelial-mesenchymal transition (EMT) phenotype. 252 HCC patients who underwent hepatectomy were enrolled for analysis of tHSCs and E-cadherin expression in tumor tissues, and 55 HCC patients for analysis of tHSCs in tumor tissues and circulating tumor cells (CTCs) in blood. Prognostic factors were then identified. The results showed that coculture of tHSCs with HCC cells had a stronger effect on HCC cell viability, migration and invasion, accompanied with the acquisition of epithelial-mesenchymal transition (EMT) phenotype. In vivo cotransplantation of HCC cells with tHSCs into nude mice more efficiently promoted tumor formation and growth. Icaritin, a known apoptosis inducer of HSCs, was demonstrated to effectively inhibit tHSC proliferation in vitro and tHSC-induced HCC-promoting effects in vivo. Clinical evidence indicated that tHSCs were rich in 45% of the HCC specimens, tHSC-rich subtypes were negatively correlated either with E-cadherin expression in tumor tissues (r = -0.256, p < 0.001) or with preoperative CTCs in blood (r = -0.287, p = 0.033), and were significantly correlated with tumor size (p = 0.027), TNM staging (p = 0.018), and vascular invasion (p = 0.008). Overall and recurrence-free survival rates of tHSC-rich patients were significantly worse than those for tHSC-poor patients. Multivariate analysis revealed tHSC-rich as an independent factor for overall and recurrence-free survival. In conclusion, tHSCs provide a promising prognostic biomarker and a new treatment target for HCC.
doi:10.1371/journal.pone.0080212
PMCID: PMC3835887  PMID: 24278260
10.  A synthetic dsRNA, as a TLR3 pathwaysynergist, combined with sorafenib suppresses HCC in vitro and in vivo 
BMC Cancer  2013;13:527.
Background
Recent studies have demonstrated that synthetic dsRNAs may produce therapeutic effects in a target-independent manner through stimulation of the toll-like receptor-3 (TLR3)/interferon pathway; as a result, angiogenesis and proliferation of tumor cells are inhibited. Thus, this pathway may become a potential target of dsRNA in tumor suppression. In this study, we evaluated the role of synthetic dsRNA as a TLR3 synergist and by combining with sorafenib in anti-hepatocellular carcinoma (HCC) in vitro and in vivo.
Methods
Four dsRNAs were designed and synthesized. One of them that was capable of activating TLR3 most effectively in human HCC cell line (HepG2.2.15) was selected as a TLR3 synergist (called BM-06). Subsequently, the expression of proteins relating to TLR3 signaling pathway, such as NF-κB, caspase 8 survivin, bcl-2 and PCNA affected by BM-06, sorafenib alone or in combination, was compared. The migration, proliferation and apoptosis of HepG2.2.15 cells were evaluated in presence of BM-06, sorafenib alone or in combination of both. The similar treatments were also applied in an SD rat primary HCC model.
Results
qRT-PCR data showed that the expression of TLR3 and NF-κB in HepG2.2.15 cells was enhanced. BM-06 was selected as a TLR3 synergist capable of activating the TLR3/interferon pathway most effective among 4 synthetic dsRNAs. The migration and proliferation were significantly inhibited in treated HepG2.2.15 cells with BM-06 or Sorafenib alone as compared with PBS-sham control (P < 0.01). However, the role of combination BM-06 with Sorafenib was the most prominent. Tumor cell apoptotic rate was increased by BM-06 or combination when compared to PBS or poly(I:C) (P < 0.05). Similarly, in orthotopic HCC SD rats, the effect of the combination was superior to either agent alone on the inhibition of tumor growth and induction of HCC cell apoptosis (P < 0.05).
Conclusions
dsRNA alone was capable of inhibiting the proliferation of HepG2.2.15 cells and tumor growth of orthotopic HCC SD rats, but the effect of combination of dsRNA with sorafenib was more prominent. These findings implicate the potential role of combined use of a dsRNA, a TLR3 synergist, and sorafenib in inhibition of HCC.
doi:10.1186/1471-2407-13-527
PMCID: PMC3827827  PMID: 24195809
dsRNA; TLR3; NF-κB; sorafenib; HCC
11.  Insufficient radiofrequency ablation promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells through Akt and ERK signaling pathways 
Background
Residual tumor progression after insufficient radiofrequency ablation (RFA) has been recently reported. However, whether epithelial-mesenchymal transition (EMT), which is a key process that drives cancer metastasis, is involved in the tumor progression after insufficient RFA is not well understood.
Methods
Human hepatocellular carcinoma (HCC) cell lines SMMC7721 and Huh7 were used. Insufficient RFA was simulated using a water bath (47°C 5 min, 10 min, 15 min, 20 min and 25 min gradually). MTT assay was used to evaluate the proliferation of HCC cells in vitro. Migration and invasion of HCC cells were determined by transwell assay. The molecular changes in HCC cells after insufficient RFA were evaluated by western blot. LY294002 and PD98059 were used to treat HCC cells. An ectopic nude mice model and a tail vein metastatic assay were used to evaluate the growth and metastatic potential of SMMC7721 cells in vivo after insufficient RFA.
Results
SMMC7721 and Huh7 cells after insufficient RFA (named as SMMC7721-H and Huh7-H respectively) exhibited enhanced proliferation, migration and invasion (6.4% and 23.6%, 33.2% and 66.1%, and 44.1% and 57.4% increase respectively) in vitro. Molecular changes of EMT were observed in SMMC7721-H and Huh7-H cells. LY294002 and PD98059 inhibited the EMT of SMMC7721-H and Huh7-H cells. SMMC7721-H cells also exhibited larger tumor size (1440.8 ± 250.3 mm3 versus 1048.56 ± 227.6 mm3) and more lung metastasis (97.4% increase) than SMMC7721 cells in vivo. Higher expression of PCNA, N-cadherin and MMP-2 and MMP-9, was also observed in SMMC7721-H tumors.
Conclusions
Insufficient RFA could directly promote the invasiveness and metastasis of HCC cells. Insufficient RFA may promote the EMT of HCC cells through Akt and ERK signaling pathways.
doi:10.1186/1479-5876-11-273
PMCID: PMC3842745  PMID: 24168056
Insufficient radiofrequency ablation; Epithelial-mesenchymal transition; Hepatocellular carcinoma; Metastasis
12.  Epithelial–mesenchymal transition markers expressed in circulating tumor cells in hepatocellular carcinoma patients with different stages of disease 
Cell Death & Disease  2013;4(10):e831-.
The presence of circulating tumor cells (CTCs) in peripheral blood is associated with metastasis and prognosis in hepatocellular carcinoma (HCC) patients. The epithelial–mesenchymal transition (EMT) has a pivotal role in tumor invasion and dissemination. To identify more sensitive biomarkers for evaluating metastasis and prognosis, we investigated the expression of EMT markers, including vimentin, twist, ZEB1, ZEB2, snail, slug and E-cadherin in CTCs, primary HCC tumors and adjacent non-tumoral liver tissues. After isolating viable CTCs from the peripheral blood of HCC patients using asialoglycoprotein receptors (ASGPRs), the CTCs were identified with immunofluorescence staining. CTCs were detected in the peripheral blood obtained from 46 of 60 (76.7%) HCC patients. Triple-immunofluorescence staining showed that twist and vimentin expression could be detected in CTCs obtained from 39 (84.8%) and 37 (80.4%) of the 46 patients, respectively. The expression of both twist and vimentin in CTCs was significantly correlated with portal vein tumor thrombus. Coexpression of twist and vimentin in CTCs could be detected in 32 (69.6%) of the 46 patients and was highly correlated with portal vein tumor thrombus, TNM classification and tumor size. Quantitative fluorescence western blot analysis revealed that the expression levels of E-cadherin, vimentin and twist in HCC tumors were significantly associated with the positivity of isolated CTCs (P=0.013, P=0.012, P=0.009, respectively). However, there was no significant difference in ZEB1, ZEB2, snail and slug expression levels in CTCs, primary HCC tumors and adjacent non-tumoral liver tissues across samples with regard to the clinicopathological parameters. Our results demonstrate that the EMT has a role in promoting the blood-borne dissemination of primary HCC cells, and the twist and vimentin expression levels in CTCs could serve as promising biomarkers for evaluating metastasis and prognosis in HCC patients.
doi:10.1038/cddis.2013.347
PMCID: PMC3824657  PMID: 24091674
circulating tumor cells; epithelial–mesenchymal transition; hepatocellular carcinoma; metastasis; biomarkers
13.  Caveolin-1 Is Up-Regulated by GLI1 and Contributes to GLI1-Driven EMT in Hepatocellular Carcinoma 
PLoS ONE  2014;9(1):e84551.
Caveolin-1 (Cav-1) has been recently identified to be over-expressed in hepatocellular carcinoma (HCC) and promote HCC cell motility and invasion ability via inducing epithelial-mesenchymal transition (EMT). However, the mechanism of aberrant overexpression of Cav-1 remains vague. Here, we observed that Cav-1 expression was positively associated with GLI1 expression in HCC tissues. Forced expression of GLI1 up-regulated Cav-1 in Huh7 cells, while knockdown of GLI1 decreased expression of Cav-1 in SNU449 cells. Additionally, silencing Cav-1 abolished GLI1-induced EMT of Huh7 cells. The correlation between GLI1 and Cav-1 was confirmed in tumor specimens from HCC patients and Cav-1 was found to be associated with poor prognosis after hepatic resection. The relationship between protein expression of GLI1 and Cav-1 was also established in HCC xenografts of nude mice. These results suggest that GLI1 may be attributed to Cav-1 up-regulation which plays an important role in GLI1-driven EMT phenotype in HCC.
doi:10.1371/journal.pone.0084551
PMCID: PMC3890273  PMID: 24454730
14.  Hepatocellular carcinoma and macrophage interaction induced tumor immunosuppression via Treg requires TLR4 signaling 
AIM: To investigated the interaction between toll-like receptor 4 (TLR4)-activated hepatoma cells and macrophages in the induction of tumor-immune suppression mediated by CD4+CD25high family of transcription factor P3 (FOXP3) regulatory T cells (Tregs).
METHODS: The proportion of FOXP3+ Tregs was identified in peripheral blood and tumor tissues of 60 hepatocellular carcinoma (HCC) patients. TLR4 expression was examined in tumor tissues and cell lines. The correlation was examined between FOXP3+ Tregs in peripheral blood and TLR4 expression of HCC tissues. Following activation of TLR4 in H22 murine hepatoma cells pre-incubated with lipopolysaccharide (LPS) and co-cultured with macrophage cell line RAW246.7, the synthesis of cytokines tumor necrosis factor-α, CCL22, and interleukin (IL)-10 by the two cell lines was detected and analyzed.
RESULTS: FOXP3+ Tregs were enriched in tumor sites, and circulating FOXP3+ Tregs were increased in HCC patients in correlation with multiple tumor foci and up-regulated TLR4 expression in HCC tissues. Semi-quantitative analysis indicated that TLR4 was over-expressed in HCC compared with the matched normal tissues. Cell cultivation experiments indicated that the mRNAs of IL-10 and CCL22 were significantly up-regulated in the RAW246.7 cell line when co-cultured with LPS pre-incubated H22 cells.
CONCLUSION: In hepatoma cell lines, TLR4 may indirectly facilitate the recruitment of Tregs to the tumor site and promote intrahepatic metastasis through its interaction with macrophages.
doi:10.3748/wjg.v18.i23.2938
PMCID: PMC3380321  PMID: 22736917
CD4+CD25highFOXP3+ regulatory T cell; Toll-like receptor; Tumor immunity; Hepatocellular carcinoma; Macrophage
15.  Proline-Rich Tyrosine Kinase 2 (Pyk2) Promotes Cell Motility of Hepatocellular Carcinoma through Induction of Epithelial to Mesenchymal Transition 
PLoS ONE  2011;6(4):e18878.
Aims
Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase of the focal adhesion kinase (FAK) family, is up-regulated in more than 60% of the tumors of hepatocellular carcinoma (HCC) patients. Forced overexpression of Pyk2 can promote the proliferation and invasion of HCC cells. In this study, we aimed to explore the underlying molecular mechanism of Pyk2-mediated cell migration of HCC cells.
Methodology/Principal Findings
We demonstrated that Pyk2 transformed the epithelial HCC cell line Hep3B into a mesenchymal phenotype via the induction of epithelial to mesenchymal transition (EMT), signified by the up-regulation of membrane ruffle formation, activation of Rac/Rho GTPases, down-regulation of epithelial genes E-cadherin and cytokeratin as well as promotion of cell motility in presence of lysophosphatidic acid (LPA). Suppression of Pyk2 by overexpression of dominant negative PRNK domain in the metastatic HCC cell line MHCC97L transformed its fibroblastoid phenotype to an epithelial phenotype with up-regulation of epithelial genes, down-regulation of mesenchymal genes N-cadherin and STAT5b, and reduction of LPA-induced membrane ruffle formation and cell motility. Moreover, overexpression of Pyk2 in Hep3B cells promoted the phosphorylation and localization of mesenchymal gene Hic-5 onto cell membrane while suppression of Pyk2 in MHCC97L cells attenuated its phosphorylation and localization.
Conclusion
These data provided new evidence of the underlying mechanism of Pyk2 in controlling cell motility of HCC cells through regulation of genes associated with EMT.
doi:10.1371/journal.pone.0018878
PMCID: PMC3080371  PMID: 21533080
16.  Liver Regeneration Associated Protein (ALR) Exhibits Antimetastatic Potential in Hepatocellular Carcinoma 
Molecular Medicine  2010;17(3-4):221-228.
Augmenter of liver regeneration (ALR), which is critically important in liver regeneration and hepatocyte proliferation, is highly expressed in cirrhotic livers and hepatocellular carcinomas (HCC). In the current study, the functional role of ALR in hepatocancerogenesis was analyzed in more detail. HepG2 cells, in which the cytosolic 15 kDa ALR isoform was reexpressed stably, (HepG2-ALR) were used in migration and invasion assays using modified Boyden chambers. Epithelial-mesenchymal transition (EMT) markers were determined in HepG2-ALR cells in vitro and in HepG2-ALR tumors grown in nude mice. ALR protein was quantified in HCC and nontumorous tissues by immunohistochemistry. HepG2-ALR, compared with HepG2 cells, demonstrated reduced cell motility and increased expression of the epithelial cell markers E-cadherin and Zona occludens-1 (ZO-1), whereas SNAIL, a negative regulator of E-cadherin, was diminished. Matrix metalloproteinase MMP1 and MMP3 mRNA expression and activity were reduced. HepG2-ALR cell-derived subcutaneously grown tumors displayed fewer necrotic areas, more epithelial-like cell growth and fewer polymorphisms and atypical mitotic figures than tumors derived from HepG2 cells. Analysis of tumor tissues of 53 patients with HCC demonstrated an inverse correlation of ALR protein with histological angioinvasion and grading. The 15 kDa ALR isoform was found mainly in HCC tissues without histological angioinvasion 0. In summary the present data indicate that cytosolic ALR reduces hepatoma cell migration, augments epithelial growth and, therefore, may act as an antimetastatic and EMT reversing protein.
doi:10.2119/molmed.2010.00117
PMCID: PMC3060984  PMID: 21152698
17.  The Tumor Microenvironment in Hepatocellular Carcinoma: Current Status and Therapeutic Targets 
Seminars in cancer biology  2010;21(1):35-43.
A growing body of literature highlights the cross-talk between tumor cells and the surrounding peri-tumoral stroma as a key modulator of the processes of hepatocarcinogenesis, epithelial mesenchymal transition (EMT), tumor invasion and metastasis. The tumor microenvironment can be broadly classified into cellular and non-cellular components. The major cellular components include hepatic stellate cells, fibroblasts, immune, and endothelial cells. These cell types produce the non-cellular components of the tumor stroma, including extracellular matrix (ECM) proteins, proteolytic enzymes, growth factors and inflammatory cytokines. The non-cellular component of the tumor stroma modulates hepatocellular carcinoma (HCC) biology by effects on cancer signaling pathways in tumor cells and on tumor invasion and metastasis. Global gene expression profiling of HCC has revealed that the tumor microenvironment is an important component in the biologic and prognostic classification of HCC. There are substantial efforts underway to develop novel drugs targeting tumor–stromal interactions.
In this review, we discuss the current knowledge about the role of the tumor microenvironment in pathogenesis of HCC, the role of the tumor microenvironment in the classification of HCC and efforts to develop treatments targeting the tumor microenvironment.
doi:10.1016/j.semcancer.2010.10.007
PMCID: PMC3050428  PMID: 20946957
Hepatocellular carcinoma; tumor microenvironment; hepatic stellate cells; cancer-associated fibroblast; Kupffer cell; T cell; TGF-β1; matrix metalloproteinase; tissue inhibitor of metalloproteinase; inflammatory cytokine; gene signature; treatment
18.  Antioxidant N-Acetylcysteine Attenuates Hepatocarcinogenesis by Inhibiting ROS/ER Stress in TLR2 Deficient Mouse 
PLoS ONE  2013;8(10):e74130.
Hepatocellular carcinoma (HCC) remains one of the most deadly solid tumor malignancies worldwide. We recently find that the loss of toll-like receptor 2 (TLR2) activities promotes the diethylnitrosamine (DEN) induced hepatocellular carcinogenesis and tumor progression, which associates with an abundant accumulation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. This finding suggests that the ROS/ER stress plays a role in TLR2 modulated carcinogenesis of HCC. To investigate the mechanism of TLR2 activity defending against hepatocarcinogenesis, the TLR2-deficient mice were treated with or without antioxidant N-acetylcysteine (NAC) before DEN administration. We found that pretreatment of these animals with NAC attenuated carcinogenesis and progression of HCC in the TLR2-deficient mice, declined ROS/ER stress, and alleviated the unfold protein response and inflammatory response in TLR2-deficient liver tissue. Moreover, the NAC treatment significantly reduced the enhanced aggregation of p62 and Mallory-Denk bodies in the DEN-induced HCC liver tissue, suggesting that NAC treatment improves the suppressive autophagic flux in the TLR2-deficient liver. These findings indicate that TLR2 activity defends against hepatocarcinogenesis through diminishing the accumulation of ROS and alleviating ER stress and unfold protein response mediated inflammatory response in the liver.
doi:10.1371/journal.pone.0074130
PMCID: PMC3788783  PMID: 24098333
19.  Hypoxia induces epithelial-mesenchymal transition via activation of SNAI1 by hypoxia-inducible factor -1α in hepatocellular carcinoma 
BMC Cancer  2013;13:108.
Background
High invasion and metastasis are the primary factors causing poor prognosis of patients with hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying these biological behaviors have not been completely elucidated. In this study, we investigate the molecular mechanism by which hypoxia promotes HCC invasion and metastasis through inducing epithelial-mesenchymal transition (EMT).
Methods
The expression of EMT markers was analyzed by immunohistochemistry. Effect of hypoxia on induction of EMT and ability of cell migration and invasion were performed. Luciferase reporter system was used for evaluation of Snail regulation by hypoxia-inducible factor -1α (HIF-1α).
Results
We found that overexpression of HIF-1α was observed in HCC liver tissues and was related to poor prognosis of HCC patients. HIF-1α expression profile was correlated with the expression levels of SNAI1, E-cadherin, N-cadherin and Vimentin. Hypoxia was able to induce EMT and enhance ability of invasion and migration in HCC cells. The same phenomena were also observed in CoCl2-treated cells. The shRNA-mediated HIF-1α suppression abrogated CoCl2-induced EMT and reduced ability of migration and invasion in HCC cells. Luciferase assay showed that HIF-1α transcriptional regulated the expression of SNAI1 based on two hypoxia response elements (HREs) in SNAI1 promoter.
Conclusions
We demonstrated that hypoxia-stabilized HIF1α promoted EMT through increasing SNAI1 transcription in HCC cells. This data provided a potential therapeutic target for HCC treatment.
doi:10.1186/1471-2407-13-108
PMCID: PMC3614870  PMID: 23496980
Epithelial-mesenchymal transition; Hypoxia; Hypoxia-inducible factor-1α; SNAI1; Hepatocellular carcinoma
20.  Slug promoted vasculogenic mimicry in hepatocellular carcinoma 
Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumour cells to mimic the pattern of embryonic vasculogenic networks. Epithelial–mesenchymal transition (EMT) regulator slug have been implicated in the tumour invasion and metastasis of human hepatocellular carcinoma (HCC). However, the relationship between slug and VM formation is not clear. In the study, we demonstrated that slug expression was associated with EMT and cancer stem cell (CSCs) phenotype in HCC patients. Importantly, slug showed statistically correlation with VM formation. We consistently demonstrated that an overexpression of slug in HCC cells significantly increased CSCs subpopulation that was obvious by the increased clone forming efficiency in soft agar and by flowcytometry analysis. Meantime, the VM formation and VM mediator overexpression were also induced by slug induction. Finally, slug overexpression lead to the maintenance of CSCs phenotype and VM formation was demonstrated in vivo. Therefore, the results of this study indicate that slug induced the increase and maintenance of CSCs subpopulation and contributed to VM formation eventually. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.
doi:10.1111/jcmm.12087
PMCID: PMC3780534  PMID: 23815612
Slug; cancer stem cells; vasculogenic mimicry; hepatocellular carcinoma
21.  Adiponectin Modulates C-Jun N-Terminal Kinase and Mammalian Target of Rapamycin and inhibits hepatocellular carcinoma 
Gastroenterology  2010;139(5):1762-1773.e5.
Background & Aims
Epidemiological studies have shown that obesity is a risk factor for hepatocellular carcinoma (HCC). Lower adiponectin levels are associated with poor prognosis in obese HCC patients hence it is plausible that adiponectin acts as a negative regulator of HCC. Here, we investigated the effects of adiponectin on HCC development and elucidated the underlying molecular mechanisms.
Methods
We utilized various in vitro assays using Huh7 and HepG2 HCC cells to examine the signal transduction pathways involved in protective function of adiponectin in HCC. These studies were followed by in vivo approaches using HCC xenografts and tumor analysis. Results from in vitro and in vivo findings were corroborated using human HCC tissue micro-array (TMA) and analysis of clinicopathological characteristics.
Results
Adiponectin treatment resulted in increased apoptosis of HCC cells via activation of caspase-3. Adiponectin increased phosphorylation of c-Jun-N-terminal kinase (JNK) and inhibition of JNK-phosphorylation inhibited adiponectin-induced apoptosis and caspase-3 activation. Adiponectin increased phosphorylation of AMP-activated protein kinase (AMPK) and tumor suppressor TSC2 and inhibited mammalian target of rapamycin (mTOR) phosphorylation. Inhibition of AMPK phosphorylation not only inhibited adiponectin-induced JNK phosphorylation but also effectively blocked biological effects of adiponectin. In vivo study showed that adiponectin treatment substantially reduced liver tumorigenesis in nude mice. Importantly, analysis of adiponectin expression levels in TMA of human HCC patients revealed an inverse correlation of adiponectin expression with tumor size.
Conclusions
These novel findings show protective role of adiponectin in liver tumorigenesis and could help explain poor prognosis of obese HCC patients who typically have low adiponectin levels.
doi:10.1053/j.gastro.2010.07.001
PMCID: PMC2967590  PMID: 20637208
Hepatocellular carcinoma; Adiponectin; TSC2; mTOR
22.  Antiangiogenic effects of pazopanib in xenograft hepatocellular carcinoma models: evaluation by quantitative contrast-enhanced ultrasonography 
BMC Cancer  2011;11:28.
Background
Antiangiogenesis is a promising therapy for advanced hepatocellular carcinoma (HCC), but the effects are difficult to be evaluated. Pazopanib (GW786034B) is a pan-vascular endothelial growth factor receptor inhibitor, the antitumor effects or antiangiogenic effects haven't been investigated in HCC.
Methods
In vitro direct effects of pazopanib on human HCC cell lines and endothelial cells were evaluated. In vivo antitumor effects were evaluated in three xenograft nude mice models. In the subcutaneous HCCLM3 model, intratumoral blood perfusion was detected by contrast-enhanced ultrasonography (CEUS), and serial quantitative parameters were profiled from the time-intensity curves of ultrasonograms.
Results
In vitro proliferation of various HCC cell lines were not inhibited by pazopanib. Pazopanib inhibited migration and invasion and induced apoptosis significantly in two HCC cell lines, HCCLM3 and PLC/PRF/5. Proliferation, migration, and tubule formation of human umbilical vein endothelial cells were inhibited by pazopanib in a dose-dependent manner. In vivo tumor growth was significantly inhibited by pazopanib in HCCLM3, HepG2, and PLC/PRF/5 xenograft models. Various intratumoral perfusion parameters changed over time, and the signal intensity was significantly impaired in the treated tumors before the treatment efficacy on tumor size could be observed. Mean transit time of the contrast media in hotspot areas of the tumors was reversely correlated with intratumoral microvessel density.
Conclusions
Antitumor effects of pazopanib in HCC xenografts may owe to its antiangiogenic effects, and the in vivo antiangiogenic effects could be evaluated by quantitative CEUS.
doi:10.1186/1471-2407-11-28
PMCID: PMC3033852  PMID: 21251271
23.  Hepatic tumor–stroma crosstalk guides epithelial to mesenchymal transition at the tumor edge 
Oncogene  2009;28(45):4022-4033.
The tumor–stroma crosstalk is a dynamic process fundamental in tumor development. In hepatocellular carcinoma (HCC), the progression of malignant hepatocytes frequently depends on transforming growth factor (TGF)-β provided by stromal cells. TGF-β induces an epithelial to mesenchymal transition (EMT) of oncogenic Ras-transformed hepatocytes and an upregulation of platelet-derived growth factor (PDGF) signaling. To analyse the influence of the hepatic tumor–stroma crosstalk onto tumor growth and progression, we co-injected malignant hepatocytes and myofibroblasts (MFBs). For this, we either used in vitro-activated p19ARF MFBs or in vivo-activated MFBs derived from physiologically inflamed livers of Mdr2/p19ARF double-null mice. We show that co-transplantation of MFBs with Ras-transformed hepatocytes strongly enhances tumor growth. Genetic interference with the PDGF signaling decreases tumor cell growth and maintains plasma membrane-located E-cadherin and β-catenin at the tumor–host border, indicating a blockade of hepatocellular EMT. We further generated a collagen gel-based three dimensional HCC model in vitro to monitor the MFB-induced invasion of micro-organoid HCC spheroids. This invasion was diminished after inhibition of TGF-β or PDGF signaling. These data suggest that the TGF-β/PDGF axis is crucial during hepatic tumor–stroma crosstalk, regulating both tumor growth and cancer progression.
doi:10.1038/onc.2009.253
PMCID: PMC2900602  PMID: 19718050
TGF-β; PDGF; tumor–stroma interaction; epithelial to mesenchymal transition; spheroid
24.  FOXC1 Contributes To Microvascular Invasion In Primary Hepatocellular Carcinoma Via Regulating Epithelial-Mesenchymal Transition 
The existence of microvascular invasion (MVI) formation is one of the most important risk factors predicting poor outcome in hepatocellular carcinoma (HCC) and its mechanism remains largely unknown. Epithelial-Mesenchymal Transition (EMT) has been suggested to be involved in many steps of the invasion-metastasis cascade. To elucidate the possible contribution of EMT to MVI, we initially evaluated the expression of 8 EMT-related transcription factors (TFs) in HCC patients with or without MVI and found that FOXC1 expression was significantly higher in patients with MVI than those without MVI (P < 0.05). Knockdown of FOXC1 expression in HCC cells resulted in a partial conversion of their EMT progresses, mainly regulating the mesenchymal component. Ectopic expression of snail, twist or TGF-β1 could induce expression of FOXC1, but none of the expression of snail, twist, slug or TGF-β was consistently down-regulated in response to FOXC1 silencing, suggesting FOXC1 might operate the downstream of other EMT regulators. In addition, knockdown of FOXC1 expression led to cytoskeleton modification accompanied by decreased ability of cell proliferation, migration, and invasion. Meanwhile, some matrix metalloproteinases (MMPs) and VEGF-A were also simultaneously down-regulated. Together, our findings demonstrate that FOXC1 is one of candidate predictive markers of MVI, and that inhibition of FOXC1 expression can partially reverse EMT program, offering a potential molecular therapeutic target for reducing tumor metastasis in HCC patients.
doi:10.7150/ijbs.4769
PMCID: PMC3445051  PMID: 22991501
Hepatocellular carcinoma; Microvascular invasion; Epithelial-Mesenchymal Transition; FOXC1.
25.  Expression and prognostic significance of Golgiglycoprotein73 (GP73) with Epithelial-mesenchymal transition (EMT) related molecules in Hepatocellular Carcinoma (HCC) 
Diagnostic Pathology  2013;8:197.
Background
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third cause of cancer-related deaths, worldwide. It is essential to develop an effective prognostic biomarker and determine the mechanisms underlying HCC invasion and metastasis.
Aims
This study aimed to investigate the expression of Golgi glycoprotein73 (GP73) and Epithelial-mesenchymal transition (EMT) molecules such as E-cadherin and Vimentin in HCC. We also evaluated the prognostic value of GP73 in HCC.
Methods
Immunohistochemistry (IHC) was used to determine the expression of GP73 and EMT molecules in 75 HCC specimens and the corresponding paracarcinomatous liver (PCL) tissues. Spearman’s correlation test was used to analyze the correlation of GP73 and EMT molecules. Clinicopathological features of the HCC patients were also analyzed. Univariate survival analysis was performed by the Kaplan–Meier method and differences among the groups were analyzed by the Log-rank test.
Results
GP73 expression in HCC was higher compared with PCL tissues (χ 2  = 73.60, P < 0.05). EMT molecules were also detected in HCC and PCL tissues. GP73 was negatively correlated with E-cadherin (r = − 0.49, P < 0.05), but positively correlated with Vimentin (r = 0.46, P < 0.05) in HCC. GP73 was correlated with the clinicopathological features including Edmondson grade, vascular invasion and TNM stage (P < 0.05), which was also associated with overall survival (OS) (P < 0.05).
Conclusions
GP73 was negatively with E-cadherin and positively correlated with Vimentin. It might be associated with aggressive behavior of HCC and had influence on patients’ OS. Further research is needed to determine the potential of GP73.
Virtual slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/29 vs/1504046946108618; http://med.motic.com/MoticGallery/Slide?id=3b6a037e-f60e-4c68-9106-41e790de9431&user=2C69F0D6-A478-4A2B-ABF0-BB36763E8025; http://med.motic.com/MoticGallery/Slide?id=a25b5b32-b613-47b0-9f8b-e1e67a95d1bf&user=2C69F0D6-A478-4A2B-ABF0-BB36763E8025.
doi:10.1186/1746-1596-8-197
PMCID: PMC3924912  PMID: 24313979

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