Chemokine ligand 20 enhances progression of hepatocellular carcinoma via epithelial-mesenchymal transition

AIM: To identify the mechanisms of chemokine ligand 20(CCL20)-induced hepatocellular carcinoma(HCC) metastasis and evaluate it as a prognostic marker. METHODS: Expression of CCL20 was evaluated by immunohistochemistry in HCC tissues from 62 patients who underwent curative resection. The relationship between CCL20 expression and clinicopathologic features was analyzed. Univariate and multivariate analyses were performed to evaluate its predictive value for recurrence and survival of HCC patients. The expression levels ofepithelial-mesenchymal transition(EMT)-and signaling pathway-related proteins were evaluated by Western blotting and immunocytochemistry. The effects of CCL20 on HCC cell proliferation and migration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenoltetrazolium bromide(MTT) and Transwell assays. RESULTS: CCL20 immunoreactivity was detected in all 62 patient specimens. CCL20 expression was associated with preoperative alpha-fetoprotein level(P = 0.043), tumor size(P = 0.000), tumor number(P = 0.008), vascular invasion(P = 0.014), and tumor differentiation(P = 0.007). Patients with high CCL20 expression had poorer recurrence-free and overall survivals compared to those with low CCL20 expression(both P 0.001). CCL20 induced EMT-like changes in HCC cells and increased their proliferation and migration ability(P 0.05). Western blotting and immunofluorescence staining showed that CCL20 induced an EMT-like phenotype in HCC cells, and increased expression of phosphorylated AKT, β-catenin and vimentin, and decreased E-cadherin expression(P 0.05). The correlation analysis revealed that high CCL20 expression in HCC tissue specimens was negatively correlated with E-cadherin expression(13.33%, 4/30), and positively correlated with vimentin(90.0%, 27/30), β-catenin(96.67%, 29/30) and p-AKT(76.67%, 23/30) expression.CONCLUSION: CCL20 expression is associated with HCC recurrence and patient survival and promotes HCC cell proliferation and migration by inducing EMT-like changes via PI3K/AKT and Wnt/β-catenin pathways.     

Expression of β-catenin in hepatocellular carcinoma

AIM: The β-catenin has been recognized as a critical member of the Wnt signaling pathway and plays an important role in the generation/differentiation of many tissues. Inappropriate activation of this pathway has been implicated in carcinogenesis. The mechanism underlying the development as well as its prognosis of hepatocellular carcinoma (HCC) has remained unclear. The purpose of this study is to analyze the expression of β-catenin in HCC in relation to histological grades and viral hepatitis backgrounds. METHODS: Thirty-two sections were selected at random from autopsy and surgical cases of HCC. Immuohistologically, the location and positivity of β-catenin expression in HCC was examined. RESULTS: Normal hepatocytes did not express β-catenin. In 78% of HCC β-catenin was expressed at the membrane of the cells, with or without cytoplasmic and/or nuclear expression. The tumor cells with well- and moderately-differentiated grades expressed frequently at the membrane and cytoplasm compared with poorly-differentiated type. Nuclear expression of β-catenin was prone to occur in the tumor cells of poorly-differentiated grade. There were 15% of hepatitis C virus (HCV) backgrounds with nuclear expression. In contrast, there was 38% with nuclear expression in hepatitis B virus (HBV) backgrounds. In nonBnonC hepatitis, no case expressed nuclear β-catenin. CONCLUSION: The β-catenin expression in HCC cells was heterogenous among types of hepatitis viral infection. Wnt signaling pathway might be deeply involved in less-differentiated HCC and HBV background.     

Expression of hepatic Wnt5a and its clinicopathological features in patients with hepatocellular carcinoma

Backgroud: Wingless-type MMTV integration site family member 5 a(Wnt5 a) is involved in carcinogenesis. However, little data are available in Wnt5 a signaling with hepatocellular carcinoma(HCC). In the present study, we investigated the expression of hepatic Wnt5 a in HCC and the role of Wnt5 a in HCC progression and outcome.Methods: Wnt5 a expression and cellular distribution in HCCs and their matched paracancerous tissues from 87 patients were analyzed with tissue microarray and immunohistochemistry and compared with hepatic Wnt3 a signaling. Wnt5 a expression was categorized into low or high based on immunohistochemistry. Overall survival rate of HCC patients was estimated in correlation with the hepatic Wnt5 a level using Kaplan–Meier method; the survival difference between patients with low and those with high Wnt5 a was compared with log-rank test; and prognostic analysis was carried out with Cox regression.Results: Total incidence of Wnt5 a expression in the HCC tissues was 70.1%, which was significantly lower(χ~2= 13.585, P 0.001) than that in their paracancerous tissues(88.5%). Significant difference of Wnt5 a intensity was found between HCC and their paracancerous tissues(Z = 8.463, P 0.001). Wnt5 a intensity was inversely correlated with Wnt3 a signaling(r =-0.402, P 0.001) in HCC tissues. A decrease of Wnt5 a expression in relation to the clinical staging from stage I to IV and low or no staining at advanced HCC were observed. Wnt5 a level was related to periportal embolus(χ~2= 11.069, P 0.001), TNM staging(χ~2= 8.852, P 0.05), 5-year survival(χ~2= 4.961, P 0.05), and confirmed as an independent prognosis factor of HCC patients(hazard ratio: 1.957; 95% confidence interval: 1.109–3.456; P 0.05).Conclusions: The decrease of hepatic Wnt5 a signaling is associated with HCC progression and poor prognosis.     

Prognostic signif icance of HIF-2α/EPAS1 expression in hepatocellular carcinoma

AIM: To evaluate the prognostic signif icance of HIF- 2α/EPAS1 expression in hepatocellular carcinoma (HCC). METHODS: Surgical specimens from 315 patients with HCC as well as 196 adjacent noncancerous lesions and 22 cases of normal liver tissue were investigated by immunohistochemistry (IHC) for HIF-2α/EPAS1 using a standard detection system. Correlations with clinicopathological factors, VEGF, microvessel density (MVD), and prognosis were analyzed. RESULTS: Immunoreactivity of HIF-2α/EPAS1 was positive in 69.5% of HCC, 55.6% of adjacent noncancerous tissue, and 0% of normal liver tissue. And it was significantly correlated with tumor grade, venous invasion, intrahepatic metastasis, necrosis, and capsule infiltration. Correlation analysis of HIF-2α/EPAS1 with angiogenic factor VEGF (P < 0.001), and MVD (P = 0.016) was also noted. HIF-2α/EPAS1 protein was less frequently expressed in low MVD cases, whereas a high rate of expression was noted in cases with both medium and high MVD (P = 0.042). By Kaplan-Meier analysis, strong HIF-2α/EPAS1 staining (> 50% of tumor cells) in HCC correlated with a shortened survival in patients (Cox's regression, P < 0.001, r = 3.699). CONCLUSION: We conclude that HIF-2α/EPAS1 expression may play an important role in tumor progression and prognosis of HCC. Assessment of HIF-2α/EPAS1 expression in HCC may be used as a diagnostic tool and possibly a target in the treatment of HCC.     

Diagnostic value of glypican-3 in serum and liver for primary hepatocellular carcinoma

AIM:To evaluate the diagnostic value of glypican-3(GPC3) in serum and liver for primary hepatocellular carcinoma(HCC).METHODS:Serum levels of GPC3 and α-fetoprotein(AFP) were measured in 75 patients with primary HCC and 32 patients with liver cirrhosis.Expression of GPC3 and AFP in 58 HCC and 12 cirrhotic specimens was detected with immunohistochemical staining.RESULTS:When the cut-off value of serum GPC3 was set at 300 ng/L,its sensitivity and specificity for HCC were 47.0% and 93.5%,respectively.Among the...     

RING finger and WD repeat domain 3 regulates proliferation and metastasis through the Wnt/β-catenin signalling pathways in hepatocellular carcinoma

BACKGROUNDHepatocellular carcinoma (HCC) exhibits high invasiveness and mortality rates, and the molecular mechanisms of HCC have gained increasing research interest. The abnormal DNA damage response has long been recognized as one of the important factors for tumor occurrence and development. Recent studies have shown the potential of the protein RING finger and WD repeat domain 3 (RFWD3) that positively regulates p53 stability in response to DNA damage as a therapeutic target in cancers. AIMTo investigate the relationship between HCC and RFWD3 in vitro and in vivo and explored the underlying molecular signalling transduction pathways. METHODS RFWD3 gene expression was analyzed in HCC tissues and adjacent normal tissues. Lentivirus was used to stably knockdown RFWD3 expression in HCC cell lines. After verifying the silencing efficiency, Celigo/cell cycle/apoptosis and MTT assays were used to evaluate cell proliferation and apoptosis. Subsequently, cell migration and invasion were assessed by wound healing and transwell assays. In addition, transduced cells were implanted subcutaneously and injected into the tail vein of nude mice to observe tumor growth and metastasis. Next, we used lentiviral-mediated rescue of RFWD3 shRNA to verify the phenotype. Finally, the microarray, ingenuity pathway analysis, and western blot analysis were used to analyze the regulatory network underlying HCC. RESULTSCompared with adjacent tissues, RFWD3 expression levels were significantly higher in clinical HCC tissues and correlated with tumor size and TNM stage (P < 0.05), which indicated a poor prognosis state. RFWD3 silencing in BEL-7404 and HCC-LM3 cells increased apoptosis, decreased growth, and inhibited the migration in shRNAi cells compared with those in shCtrl cells (P < 0.05). Furthermore, the in vitro results were supported by the findings of the in vivo experiments with the reduction of tumor cell invasion and migration. Moreover, the rescue of RFWD3 shRNAi resulted in the resumption of invasion and metastasis in HCC cell lines. Finally, gene expression profiling and subsequent experimental verification revealed that RFWD3 might influence the proliferation and metastasis of HCC via the Wnt/β-catenin signalling pathway.CONCLUSIONWe provide evidence for the expression and function of RFWD3 in HCC. RFWD3 affects the prognosis, proliferation, invasion, and metastasis of HCC by regulating the Wnt/β-catenin signalling pathway.     

Abnormal beta-catenin gene expression with invasiveness of primary hepatocellular carcinoma in China

AIM: To study the abnormal expression of beta-catenin gene and its relationship ith invasiveness of primary hepatocellular carcinoma among Chinese people. METHODS: Thirty-four hepatocellular carcinoma (HCC) specimens and adjacent para-cancerous tissues, 4 normal liver tissues were immunohistochemically stained to study subcellular distribution of beta-catenin. Semiquantitive analysis of expression of beta-catenin gene exon 3 mRNA was examined by RT-PCR and in situ hybridization. The relationship between expressions of both beta-catenin protein, mRNA and clinicopathological characteristics of HCC was also analyzed. RESULTS: Immuno-histochemistry showed that all normal liver tissues and para-cancerous tissues examined displayed membranous type staining for beta-catenin protein, occasionally with weak expression in the cytoplasm. While 21 cases (61.8%) of HCC examined showed accumulated type in cytoplasms or nuclei. The accumulated type Labling Index (LI) of cancer tissue and para-cancerous tissue was (59.9 +/- 26.3) and (18.3 +/- 9.7) respectively (P<0.01). Higher accumulated type LI was closely related with invasiveness of HCC. Results of RT-PCR showed the beta-catenin gene exon 3 mRNA Expression Index (EI) of 34 HCCs was higher than that of para-cancerous tissue and normal liver tissue. Using in situ hybridization, the signal corresponding to beta-catenin gene exon 3 mRNA was particularly strong in cytoplasm of HCC when compared with those of para-cancerous and normal liver tissues. Over expression of beta-catenin exon 3 was also found to be correlated with high metastatic potential of HCC. CONCLUSION: Abnormal expression of beta-catenin gene may contribute importantly to the invasiveness of HCC among Chinese people.     

Alpha-fetoprotein expression is a potential prognostic marker in hepatocellular carcinoma

AIM: To characterize the alpha-fetoprotein (AFP) positive and negative hepatocellular carcinoma (HCC) samples. METHODS: Thirty-seven paraffin-embedded human HCC samples were analyzed by immunohistochemistry for the following antigens: AFP, β-catenin, p53, CD44, MSH-2, MLH-1, and HNF-4. The tumors were divided into two groups based on the AFP expression. The immunophenotypic data and important clinical parameters were studied between the two groups. RESULTS: Twenty-one of the thirty-seven examined HCCs were AFP positive. Seven with nuclear p53 staining were AFP positive, while seven tumors with nuclear β-catenin staining were AFP negative. CD44 staining and high histological tumor grade were more frequent among the AFP-positive HCCs. The other immunophenotypical and dinical parameters did not show statistically significant difference in their distribution between the AFP positive and negative samples. CONCLUSION: AFP expression in HCC correlates with unfavorable prognostic factors, while nuclear β-catenin positivity is more common among the AFP-negative liver tumors. This observation supports the microarray data on in vivo human tumors.     

Reduced expression of beta-catenin inhibitor Chibby in colon carcinoma cell lines

AIM: To analyse the Chibby expression and its function in colon carcinoma cell lines and colorectal carcinoma (CRC). METHODS: Chibby expression levels were investigated by quantitative RT-PCR in a panel of seven different colon carcinoma cell lines. By sequencing, we analysed mutational status of Chibby. To test whether Chibby exhibited effects on beta-catenin signalling in colon carcinoma cells, we transfected SW480 cells with Chibby expression plasmid and, subsequently, analysed activity of beta-catenin and tested for alterations in cellular phenotype. In addition, we examined Chibby mRNA levels in samples of colorectal carcinomas and adjacent normal tissues by using quantitative RT-PCR and hybridised gene chips with samples from CRC and normal tissues. RESULTS: Chibby mRNA expression was strongly down-regulated in colon carcinoma cell lines in comparison to normal colon epithelial cells and no mutation in any of the examined colon carcinoma cell lines was found. Further, we could show that Chibby inhibited beta-catenin activity in TOPflash assays when over-expressed in SW480 cells. Proliferation and invasion assays with Chibby transfected SW480 cells did not reveal profound differences compared to control cells. In contrast to these in vitro data, quantitative RT-PCR analyses of Chibby mRNA levels in CRC tumor samples did not show significant differences to specimens in adjacent non-cancerous tissue. Consistent with these findings, gene chips analysing tissue samples of tumors and corresponding normal tissue did not show altered Chibby expression. CONCLUSION: Altered Chibby expression might be observed in vitro in different colon carcinoma cell lines. However, this finding could not be confirmed in vitro in CRC tumors, indicating that Chibby is not likely to promote CRC tumor development or progression. As Chibby is an important inhibitor of beta-catenin signalling, our data implicate that the usability of colon carcinoma cell lines for in vitro studies analysing the Wnt/beta-catenin pathway in colorectal carcinoma needs extensive verification.     

Glypican-3 is a prognostic factor and an immunotherapeutic target in hepatocellular carcinoma

Glypican-3 (GPC3) is a cell surface oncofetal proteoglycan that is anchored by glycosylphosphatidylinositol. Whereas GPC3 is abundant in fetal liver, its expression is hardly detectable in adult liver. Importantly, GPC3 is overexpressed in hepatocellular carcinoma (HCC), and several immunohistochemical studies reported that overexpression predicts a poorer prognosis for HCC patients. Therefore, GPC3 would serve as a useful molecular marker for HCC diagnosis and also as a target for therapeutic intervention in HCC. Indeed, some immunotherapy protocols targeting GPC3 are under investigations; those include humanized anti-GPC3 cytotoxic antibody, peptide vaccine and immunotoxin therapies. When considering the clinical requirements for GPC3-targeting therapy, companion diagnostics to select the appropriate HCC patients are critical, and both immunohistochemical analysis of tissue sections and measurement of serum GPC3 level have been suggested for this purpose. This review summarizes current knowledge regarding the clinical implication of GPC3 detection and targeting in the management of patients with HCC.     

Usefulness of serum des-γ-carboxy prothrombin in detection of hepatocellular carcinoma

AIM: Des-gamma-carboxy prothrombin (DCP) has been reported to be more sensitive and specific in diagnosing hepatocellular carcinoma (HCC) when compared with alpha-fetoprotein (AFP). However, its ability to identify small HCC still remains unclear. Thus, we conducted a cross-sectional case control study to evaluate whether DCP is better than AFP for differentiating HCC from nonmalignant liver disease and further evaluate the usefulness of DCP in early diagnosis of small HCC. METHODS: Serum DCP and AFP levels were determined in 127 patients. Among these patients, 32 were with non-cirrhotic chronic hepatitis, 34 were with compensated cirrhosis, and 61 were with HCC. The cut-off value for the DCP and AFP were set as 40 mAU/mL and 20 ng/mL, respectively. To compare the diagnostic value of DCP and AFP in distinguishing HCC from nonmalignant chronic liver disease, receiver operating characteristic (ROC) curves were constructed for each assay. RESULTS: The accuracy, sensitivity and specificity of DCP were higher than AFP in detecting HCC (81.9%, 77% and 86.4% vs 68.5%, 59% and 77.3%, respectively). The area under the ROC (AUROC) curves revealed that DCP had a better accuracy than AFP in diagnosis of HCC (0.85 [95%CI, 0.78-0.91] vs 0.73 [95%CI, 0.65-0.81], P = 0.013). In 39 patients with solitary HCC, the positive rates of DCP were 100% in patients with tumor size larger than 3 cm, 66.7% in patients with tumor size 2-3 cm and 50% in patients with tumor size less than 2 cm. The positive rates of AFP in patients with tumor size larger than 3 cm, 2-3 cm and less than 2 cm were 55.6%, 50%, and 33.3%, respectively. The median level of DCP in HCC patients with tumor size larger than 3 cm was significantly higher than those with tumor size 2-3 cm and those with the size of less than 2 cm. CONCLUSION: Our study indicates that DCP has a better diagnostic value than AFP in differentiating HCC from nonmalignant chronic liver disease. DCP has not only a stronger correlation with HCC than AFP in tumor size but also more effectiveness than AFP in detecting small size of HCC.     

Biomarkers for the early diagnosis of hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the fifth most common cancer and the second leading cause of cancer-related deaths worldwide. Although the prognosis of patients with HCC is generally poor, the5-year survival rate is 70% if patients are diagnosed at an early stage. However, early diagnosis of HCC is complicated by the coexistence of inflammation and cirrhosis. Thus, novel biomarkers for the early diagnosis of HCC are required. Currently, the diagnosis of HCC without pathological correlation is achieved by analyzing serum α.fetoprotein levels combined with imaging techniques. Advances in genomics and proteomics platforms and biomarker assay techniques over the last decade have resulted in the identification of numerous novel biomarkers and have improved the diagnosis of HCC. The most promising biomarkers,such as glypican-3, osteopontin, Golgi protein-73 and nucleic acids including microRNAs, are most likely to become clinically validated in the near future. These biomarkers are not only useful for early diagnosis of HCC, but also provide insight into the mechanisms driving oncogenesis. In addition, such molecular insight creates the basis for the development of potentially more effective treatment strategies. In this article,we provide an overview of the biomarkers that are currently used for the early diagnosis of HCC.     

Both glypican-3/Wnt/β-catenin signaling pathway and autophagy contributed to the inhibitory effect of curcumin on hepatocellular carcinoma

Aim

The aim of this study is to investigate the role of glypican-3(GPC3)/wnt/β-catenin signaling pathway and autophagy in the regulation of hepatocellular carcinoma (HCC) growth mediated by curcumin.

Model based on γ-glutamyltransferase and alkaline phosphatase for hepatocellular carcinoma prognosis

AIM: To determine the prognostic value of alkaline phosphatase (ALP) and γ-glutamyltransferase (GGT) for hepatocellular carcinoma (HCC) .METHODS: We analyzed the outcome of 172 HCC patients who underwent liver resection. Receiver operating characteristic (ROC) curve analysis was performed to determine the cut-off value of ALP and GGT. Then, preoperative risk factors for survival were evaluated by multivariate analysis. Based on the significant factors, a prognostic score model was established.RESULTS: By ROC curve analysis, ALP > 120 U/L and GGT > 115 U/L were considered elevated. Overall survival (OS) and tumor-free survival (TFS) for patients with elevated ALP and GGT were significantly worse than for patients with ALP and GGT within the normal range. Multivariate analysis showed that the elevated levels of ALP, GGT and tumor size were independent prognostic factors. Giving each positive factor as a score of 1, we established a preoperative prognostic score model. The 5-year OS for patients with a score of 0, 1, 2 and 3 were 84.0%, 45.9%, 44.1% and 0%, respectively, while the TFS was 80.6%, 40.0%, 38.8% and 0%, respectively. When combining patients with scores of 1 and 2 into the middle risk group, and patients with scores of 0 and 3 into the low-risk and high-risk groups, respectively, different outcomes would be significantly distinguished by the risk groups.CONCLUSION: Elevated ALP and GGT levels were risk predictors in HCC patients. Our prognostic model might vary the outcomes of patients from different risk groups.     

Wnt Signaling: Role in Regulation of Haematopoiesis

Hematopoietic stem cells (HSCs) are a unique population of bone marrow cells which are responsible for the generation of various blood cell lineages. One of the significant characteristics of these HSCs is to self-renew, while producing differentiating cells for normal hematopoiesis. Deregulation of self-renewal and differentiation leads to the hematological malignancies. Several pathways are known to be involved in the maintenance of HSC fate among which Wnt signaling is a crucial pathway which controls development and cell fate determination. Wnt signaling also plays a major role in differentiation, self-renewal and maintenance of HSCs. Wnt ligands activate three major pathways including planar cell polarity, Wnt/β-catenin and Wnt/Ca²⁺. It has been shown that Wnt/β-catenin or canonical pathway regulates cell proliferation, survival and differentiation in HSCs, deregulation of this pathway leads to hematological malignancies. Wnt non-canonical pathway regulates calcium signaling and planar cell polarity. In this review, we discuss various signaling pathways induced by Wnt ligands and their potential role in hematopoiesis.     

Targeting Wnt/β-catenin pathway in hepatocellular carcinoma treatment

Hepatocellular carcinoma(HCC) is one of the most common causes of cancer-related death worldwide. Liver cancer is generally related to hepatitis B or Cinfection and cirrhosis. Usually, patients with HCC are asymptomatic and are diagnosed at late stages when surgical treatment is no longer suitable. Limited treatment options for patients with advanced HCC are a major concern. Therefore, there is an urge for finding novel therapies to treat HCC. Liver cancer is highly heterogeneous and involved deregulation of several signaling pathways. Wnt/β-catenin pathway is frequently upregulated in HCC and it is implicated in maintenance of tumor initiating cells, drug resistance, tumor progression, and metastasis. A great effort in developing selective drugs to target components of the β-catenin pathway with anticancer activity is underway but only a few of them have reached phase Ⅰ clinical trials. We aim to review the role of β-catenin pathway on hepatocarcinogenesis and liver cancer stem cell maintenance. We also evaluated the use of small molecules targeting the Wnt/β-catenin pathway with potential application for treatment of HCC.