Malignant Progression Of Hepatocellular Carcinoma Research Articles

HOXB7 accelerates the malignant progression of hepatocellular carcinoma by promoting stemness and epithelial-mesenchymal transition

BackgroundHomeobox B7 (HOXB7) has been identified associated with poor prognosis of hepatocellular carcinoma (HCC). However, the specific mechanism by which HOXB7 promotes the malignant progression of HCC remains to be determined.MethodsImmunohistochemistry (IHC) was used to detect the expression level of HOXB7 in 77-paired HCC tissue samples, and the correlation between HOXB7 and HCC prognosis was assessed. The location of HOXB7 was confirmed by immunofluorescence. Cell Titer-Blue assay was used to assess the proliferation of hepatoma cells. The stem-like properties of hepatoma cells were analysed by sphere formation and clone formation assays. The effect of HOXB7 on expression of cancer stem cell markers was evaluated. Transwell and wound-healing assays were performed to estimate the invasion and migration abilities of hepatoma cells. A xenograft tumor model was established in nude mice to assess the role of HOXB7 in tumor growth. Bioluminescence imaging was used to survey the effect of HOXB7 on the metastatic ability of hepatoma cells in vivo.ResultsHigher expression of HOXB7 was detected in HCC tissues compared with noncancerous tissues and significantly associated with poor prognosis of HCC. In addition, HOXB7 knockdown suppressed the cell proliferation, clone formation, sphere formation, invasion and migration of hepatoma cells in vitro; conversely, these biological abilities of hepatoma cells were enhanced by HOXB7 overexpression. Moreover, the cancer stem cell markers EPCAM and NANOG were up-regulated by HOXB7. The role of HOXB7 in promoting tumor growth and metastasis was verified in vivo. Further investigation revealed that c-Myc and Slug expression was elevated by HOXB7 and the AKT pathway was activated.ConclusionOverexpression of HOXB7 was significantly correlated with poor prognosis of HCC. HOXB7 up-regulated c-Myc and Slug expression via the AKT pathway to promote the acquisition of stem-like properties and facilitate epithelial-mesenchymal transition of hepatoma cells, accelerating the malignant progression of HCC.

MiR-885-5p suppresses hepatocellular carcinoma metastasis and inhibits Wnt/β-catenin signaling pathway.

MicroRNAs (miRNAs) inhibit or improve the malignant progression of hepatocellular carcinoma (HCC). We previously reported that compared to health controls, patients with liver cirrhosis present the highest levels of circulating miR-885-5p, followed by those with chronic hepatitis B and those with HCC. However, the molecular involvement of miR-885-5p in HCC metastasis is presently unclear. Here, we demonstrated that the expression of miR-885-5p negatively correlated with the invasive and metastatic capabilities of human HCC tissue samples and cell lines. We found that miR-885-5p expression levels correlated with the survival of patients with HCC. Overexpression of miR-885-5p decreased metastasis of HCC cells in vitro and in vivo. Inhibition of miR-885-5p improved proliferation of non-metastatic HCC cells. Furthermore, we disclosed that miR-885-5p targeted gene encoding β-catenin CTNNB1, leading to decreased activity of the Wnt/β-catenin signaling pathway. The present study indicates that miR-885-5p suppresses the metastasis of HCC and inhibits Wnt/β-catenin signaling pathway by its CTNNB1 target, which suggests that miR-885-5p to be a promising negative regulator of HCC progression and as a novel therapeutic agent to treat HCC.

Prognostic significance of eukaryotic initiation factor 4E in hepatocellular carcinoma.

Aberrant expression of eukaryotic initiation factor 4E (eIF4E) has been observed in human malignancies. However, its role in hepatocellular carcinoma (HCC) remains to be established. The purpose of this study was to detect eIF4E expression and to evaluate its clinical relevance. The eIF4E expression was studied in ninety HCC and randomly selected thirty-one non-tumor tissues from the same patient cohort, as well as in normal hepatic and HCC cell lines. The relation between its expression and clinicopathological parameters was also analyzed. eIF4E expression was higher in HCC samples and cell lines compared with that in non-tumor tissues (P<0.001) and hepatocyte LO2, respectively. eIF4E overexpression was significantly associated with tumor number (P=0.005) and incomplete encapsulation (P=0.001). The 5-year overall survival rate and disease-free survival rate for patients with high eIF4E expression were 32.5 and 31.2%, respectively; and for low eIF4E expression, it was 67.9 and 64.4%, respectively (P<0.001). Furthermore, subgroup analysis showed that high eIF4E level predicted poorer overall survival only for incomplete encapsulation (P=0.001) and cirrhosis (P<0.001), but not for complete encapsulation (P=0.804) and non-cirrhosis (P=0.359). Multivariate analysis revealed that eIF4E overexpression was an independent indicator for both overall survival (hazard ratio, 2.015; P=0.043) and disease-free survival (hazard ratio, 2.666; P=0.006). eIF4E protein might result in the malignant progression of HCC, and its overexpression may be a powerful prognostic biomarker and therapeutic target for HCC patients.

MiR-503 inhibits hepatocellular carcinoma cell growth via inhibition of insulin-like growth factor 1 receptor.

MicroRNAs (miRs) have been demonstrated to play key roles in the development and progression of hepatocellular carcinoma (HCC). However, the regulatory mechanism of miR-503 in HCC has not been fully uncovered. In this study, we found that miR-503 was significantly downregulated in HCC tissues compared to nontumorous liver tissues. Moreover, lower miR-503 levels were associated with the malignant progression of HCC, and the expression of miR-503 was also decreased in several common HCC cell lines compared to normal human liver cell line THLE-3. Overexpression of miR-503 inhibited proliferation but induced apoptosis of LM3 and HepG2 cells. Bioinformatical analysis and luciferase reporter assay further identified insulin-like growth factor 1 receptor (IGF-1R) as a novel target of miR-503 in 293T cells. Moreover, overexpression of miR-503 led to a significant decrease in the protein levels of IGF-1R, while knockdown of miR-503 enhanced its protein levels in LM3 and HepG2 cells. Besides, overexpression of IGF-1R reversed the effects of miR-503-mediated HCC cell proliferation and apoptosis, indicating that IGF-1R acts as a downstream effector of miR-503 in HCC cells. Furthermore, IGF-1R was found to be significantly upregulated in HCC tissues compared to nontumorous liver tissues. In addition, the mRNA levels of IGF-1R were inversely correlated to the miR-503 levels in the HCC tissues. Thus, we demonstrate that miR-503 inhibits the proliferation and induces the apoptosis of HCC cells, partly at least, by directly targeting IGF-1R, and suggest that IGF-1R may serve as a promising target for the treatment of HCC.

Reciprocal activation between MMP-8 and TGF-β1 stimulates EMT and malignant progression of hepatocellular carcinoma

The efficiency of surgery in hepatocellular carcinoma (HCC) is limited due to metastasis and recurrence, but the molecular mechanisms are unclear. Here, we show that MMP-8 and TGF-β1 accumulate in highly invasive HCC cell lines and invasive HCC patient tissues. Upregulation of MMP-8 and TGF-β1 correlated with changes in cellular epithelial–mesenchymal transition (EMT) phenotypes and HCC migration and invasion. The expression of TGF-β1 was markedly restored by MMP-8 overexpression in TGF-β1-depleted HCC cells mainly via the activation of PI3K/Akt/Rac1 pathway. Similarly, the expression of MMP-8 was restored by TGF-β1 treatment in MMP-8-depleted HCC cells mainly through the activation of the same PI3K/Akt/Rac1 pathway. MMP-8 expression was significantly related to TGF-β1 expression in HCC patient tissues, and high expression of MMP-8 or TGF-β1 was significantly associated with TNM stage and HCC metastasis. Specifically, patients with high co-expression of MMP-8 and TGF-β1 had a shorter time-to-recurrence than those with low co-expression. Therefore, the reciprocal positive interplay between MMP-8 and TGF-β1 contributes to HCC invasion and metastasis by inducing EMT mainly through the PI3K/Akt/Rac1 pathway.

Correlation of adrenomedullin with the erythropoietin receptor and microvessel density in hepatocellular carcinoma.

IntroductionUncontrolled angiogenesis plays an essential role in the occurrence, metastasis and malignant progression of hepatocellular carcinoma (HCC). This study aimed to investigate the expression of adrenomedullin (ADM) in human HCC and its correlation with the expression of erythropoietin receptor (EPOR), microvessel density (MVD) and the tumor pathological characteristics.Material and methodsFresh tumor tissues were obtained from 30 HCC patients after hepatectomy. Ten cirrhotic and 10 normal liver tissues were included as controls. Expression of ADM and EPOR was determined by real-time PCR. The MVD was determined by counting the number of microvessels.ResultsThe MVD and the mRNA levels of ADM and EPOR in cancer tissues were significantly higher than those in the non-cancer tissues (p < 0.05). Expression of ADM was significantly correlated with the MVD and EPOR (r = 0.68 and 0.74, p < 0.01). Adrenomedullin and EPOR mRNA levels in HCC tissues were correlated with capsule invasion, pathological differentiation and tumor metastasis (p < 0.05).ConclusionsOur findings suggest that ADM and EPOR may serve as new regulatory factors involved in angiogenesis of HCC and represent novel targets for the treatment of HCC.

Common Variants of the Prostaglandin-Endoperoxide Synthase 2 Gene and Hepatocellular Carcinoma Susceptibility.

Hepatocellular carcinoma (HCC) is a heterogeneous disease with substantial genetic constitution. Previous work has evaluated the effect of prostaglandin-endoperoxide synthase 2 (PTGS2) variants (−765G/C, −1195A/G, and +8473T/C) on the development of HCC, but the conclusions are inconsistent. We conducted a meta-analysis in this work. Data from 7 case–control studies were combined to assess the association between PTGS2 variants and HCC. The risk of HCC (OR and 95% CI) was estimated using either the fixed- or the random-effects model according to the Q test. No significant association was identified for −765G/C and +8473T/C. However, we identified a significantly decreased risk in relation to the GG genotype of −1195A/G (OR = 0.70, 95% CI = 0.50–0.98 for GG versus AA). We also observed a similar decrease (OR = 0.47, 95% CI = 0.23–0.95 for GG versus AA) in Caucasian samples. Variant −1195A/G in the promoter PTGS2 may protect against the malignant progression of HCC. This significant association suggests that −1195A/G could be used as a biomarker of HCC.

Contribution of the toxic advanced glycation end-products-receptor axis in nonalcoholic steatohepatitis-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. The main etiologies of HCC are hepatitis B virus and hepatitis C virus (HCV), and non-hepatitis B/non-hepatitis C HCC (NBNC-HCC) has also been identified as an etiological factor. Although the incidence of HCV-related HCC in Japan has decreased slightly in recent years, that of NBNC-HCC has increased. The onset mechanism of NBNC-HCC, which has various etiologies, remains unclear; however, nonalcoholic steatohepatitis (NASH), a severe form of nonalcoholic fatty liver disease, is known to be an important risk factor for NBNC-HCC. Among the different advanced glycation end-products (AGEs) formed by the Maillard reaction, glyceraldehyde-derived AGEs, the predominant components of toxic AGEs (TAGE), have been associated with NASH and NBNC-HCC, including NASH-related HCC. Furthermore, the expression of the receptor for AGEs (RAGE) has been correlated with the malignant progression of HCC. Therefore, TAGE induce oxidative stress by binding with RAGE may, in turn, lead to adverse effects, such as fibrosis and malignant transformation, in hepatic stellate cells and tumor cells during NASH or NASH-related HCC progression. The aim of this review was to examine the contribution of the TAGE-RAGE axis in NASH-related HCC.

HDAC2 Provides a Critical Support to Malignant Progression of Hepatocellular Carcinoma through Feedback Control of mTORC1 and AKT

Aberrant regulation of histone deacetylase 2 (HDAC2) contributes to malignant progression in various cancers, but the underlying mechanism leading to the activation of oncogenic HDAC2 remains unknown. In this study, we show that HDAC2 expression is upregulated in a large cohort of patients with human hepatocellular carcinoma, and that high expression of HDAC2 was significantly associated with poor prognosis of patients with hepatocellular carcinoma. We found that mTORC1/NF-κBp50 signaling is necessary for the growth factor-induced HDAC2 and is sustained in hepatocellular carcinoma, but not in normal hepatic cells. Growth factor-induced mTORC1 activates the nuclear translocation of NF-κBp50, where it binds to the intragenic sequences of the HDAC2 gene and promotes its transcription. Hepatocellular carcinoma tissues derived from chemical-induced mouse and rat liver cancer models validated that mTORC1 activation and NF-κBp50 nuclear translocation are essential for the transcriptional activation of oncogenic HDAC2 in hepatocellular carcinoma. In addition, we demonstrate that HDAC2 is required to maintain mTORC1 activity by stabilizing the mTOR/RAPTOR complex. Elevated expression of HDAC2 triggers a positive feedback loop that activates AKT phosphorylation via the transcriptional modulation of phosphoinositide signaling molecules. Bioinformatics analysis of HDAC2 signature and immunoblot analysis of mesenchymal genes also evidenced that HDAC2 plays a role in the malignant behavior of tumor cells by Snail induction and simultaneously E-cadherin suppression in hepatocellular carcinoma cells. These findings establish a molecular mechanism responsible for the activation of oncogenic HDAC2, which explains how growth factor-induced HDAC2 maintains mitogenic signaling and function during hepatocellular malignant progression and provide a novel strategy for therapeutic intervention in liver cancer. Cancer Res; 74(6); 1728-38. ©2014 AACR.

Role of TGF-β1 and its receptors in malignant progression of hepatocellular carcinoma

Role of TGF-β1 and its receptors in malignant progression of hepatocellular carcinoma

Perinodular CK19 loss in hepatocarcinogenesis

Cirrhotic nodules containing hepatocytes are surrounded by perinodular stroma, that consists of an expanded fibrous matrix and epithelial cells with ductular phenotype, the "ductular-reaction". Stromal invasion is a key histopathologic feature used to differentiate premalignant dysplastic nodules from malignant hepatocellular carcinoma (HCC). K19 immunoreactivity in the stromal compartment in cirrhotic explants was examined. Quantitative differences were manifested in three distinct histologically identifiable patterns: "complex" around cirrhotic nodules, "attenuated" around dysplastic nodules, and "absent" around HCC. These findings suggest marked alterations in cellular identity as an underlying mechanism for the reproducible extralesional K19 pattern that parallels progressive stages of intranodular hepatocarcinogenesis. Paracrine signalling is proposed as a link that emphasizes the importance of the epithelial-stromal compartment in malignant progression of HCC in cirrhosis.

Keratin 19 Epithelial Patterns in Cirrhotic Stroma Parallel Hepatocarcinogenesis

Cirrhotic septa harbor vessels and inflammatory, fibrogenic, and ductular epithelial cells, collectively referred to as the ductular reaction (DR). Lack of the DR in the stromal compartment around hepatocellular carcinoma (HCC) has been documented; however, the relationship of epithelial keratin 19 (K19) structures to progression of intralesional carcinogenesis has not been explored. K19 immunoreactivity in the stromal compartment around 176 nodules in cirrhotic explants was examined. Quantitative differences (P < 0.0001) were manifested in three distinct histologically identifiable patterns: "complex" around cirrhotic nodules (CN), "attenuated" around dysplastic nodules (DN), and "absent" around HCC. Markers of necrosis or apoptosis could not explain the perinodular K19 epithelial loss; however, multicolor immunolabeling for K19, vimentin, E-Cadherin, SNAIL, and fibroblast-specific protein 1 (FSP-1) demonstrated discrepancies in immunophenotype and cytomorphologic features. Variability of cellular features was accompanied by an overall decrease in epithelial markers and significantly increased fractions of SNAIL- and FSP-1-positive cells in the DR around DN when compared with CN (P < 0.0001). Immunolabeling of transforming growth factor-β signaling components (TGFβR1, SMAD3, and pSMAD2/3) demonstrated increased percentages of pSMAD2/3 around DN when compared with CN (P < 0.0001). These findings collectively suggest marked alterations in cellular identity as an underlying mechanism for the reproducible extralesional K19 pattern that parallels progressive stages of intranodular hepatocarcinogenesis. Paracrine signaling is proposed as a link that emphasizes the importance of the epithelial-stromal compartment in malignant progression of HCC in cirrhosis.

Expression and clinicalsignificance of candidate tumor suppressor gene RASSFIA,BIU and SEMA3B at 3p21,3 in livercancer

：Objective To investigatethe expression of candidate tumor suppressor gene RASSF1A,BIU and SEMA3B being located atthe commonly deleted region 3p21.3 in hepatocellular carcinoma(HCC).Methods RT-PCR wasused to detect RASSF1A,BIU and SEMA3B expression in HCC and its adjacent liver tissues in79 patients.Results(1)The lossing rates of RASSF1A,B1U and SEMA3B mRNAs were significantlyhigher in HCC than in the paracancerous tissue(P＜0.05).(2)The lossing rates ofthree genes' mRNAs were significantly higher in the patients with hepatic cirrhosis orHBsAg(+)than in those without hepatic cirrhosis or HBsAg(+)(P＜0.05).(3)The expression ofthree genes was reduced in poorly differentiated HCC compared with moderatelydifferentiated and well-differentiated HCC,but the difference was not significant forSEMA3B gene(P＞0.05).(4)There was no significant association of expression of the3 genes with age,sex,Child-Puch classification before operation or serum AFP level of thepatients(P＞0.05).ConclusionLoss or abnormal down-regulation of three genes' mRNAs is a frequent event in HCC,whichmay play an important role in the malignant progression of HCC. Key words: Carcinoma hepatocellular; 3p21.3; Tumor suppressorgene; Reverse transcriptase polymerase chain reaction

The overexpression of polycomb group proteins Bmi1 and EZH2 is associated with the progression and aggressive biological behavior of hepatocellular carcinoma

Polycomb-group proteins Bmi1 and EZH2 are involved in the malignant transformation and biological aggressiveness of several human carcinomas. We herein examined the significance of the Bmi1 and EZH2 expression in hepatocellular carcinoma (HCC) and its preneoplastic lesions, dysplastic nodules. The expression of Bmi1 and EZH2 were examined immunohistochemically in HCC (n=27) and dysplastic nodules (n=14), and combined hepatocellular and cholangiocarcinoma (HC-CC) (n=14). The effect of Bmi1 and EZH2 knockdown was examined in cultured HCC cells (HuH7 and HepG2) using siRNA. It was determined that Bmi1 was constantly expressed in cholangiocytes, but not in hepatocytes, and EZH2 was detected in neither cholangiocytes nor hepatocytes. Bmi1 and EZH2 were overexpressed in HCC and more extensively in HC–CC (P<0.01). Interestingly, Bmi1 and EZH2 were not overexpressed in the dysplastic nodules. The expression of Bmi1 and EZH2 was heterogeneous and associated with vascular infiltration, the histological grades, and the cell proliferation activity in HCC and HC–CC. In cultured carcinoma cells overexpressing Bmi1 and EZH2, knockdown of Bmi1 and EZH2 resulted in decreased cell proliferation activities. Therefore, the overexpression of polycomb-group proteins Bmi1 and EZH2 is associated with the malignant progression of HCC, thereby reflecting the aggressive biological behavior in HCC and HC–CC.

Genetically Distinct and Clinically Relevant Classification of Hepatocellular Carcinoma: Putative Therapeutic Targets

The biological aggressiveness of hepatocellular carcinoma (HCC) and the lack of optimal therapeutic strategies have rendered the disease a major challenge. Highly heterogeneous genetic alteration profiles of HCC have made it difficult to identify effective tailor-made molecular therapeutic targets. Therefore, classification of HCC into genetically homogeneous subclasses would be of great worth to develop novel therapeutic strategies. We clarified genome-scale chromosomal copy number alteration profiles and mutational statuses of p53 and beta-catenin in 87 HCC tumors. We investigated the possibility that HCC might be classifiable into a number of homogeneous subclasses based solely on their genetic alteration profiles. We also explored putative molecular therapeutic targets specific for each HCC subgroup. Unsupervised hierarchical cluster analysis based on chromosomal alteration profiles suggested that HCCs with heterogeneous genetic backgrounds are divisible into homogeneous subclasses that are highly associated with a range of clinicopathologic features of the tumors and moreover with clinical outcomes of the patients (P < .05). These genetically homogeneous subclasses could be characterized distinctively by pathognomonic chromosomal amplifications (eg, c-Myc-induced HCC, 6p/1q-amplified HCC, and 17q-amplified HCC). An in vitro experiment raised a possibility that Rapamycin would significantly inhibit the proliferative activities of HCCs with 17q amplification. HCC is composed of several genetically homogeneous subclasses, each of which harbors characteristic genetic alterations that can be putative tailor-made molecular therapeutic targets for HCCs with specific genetic backgrounds. Our results offer an opportunity for developing novel individualized therapeutic modalities for distinctive genome types of HCC.

In this issue

The mutator phenotype and genomic instability are key steps in the origin of many human cancers. In hereditary nonpolyposis colorectal carcinoma, defects in the DNA mismatch repair proteins are associated with microsatellite instability. In this issue, Catto and colleagues (pages 484–490) examine the expression levels of two DNA mismatch repair proteins and the presence of microsatellite instability in 111 cases of transitional cell carcinoma of the bladder (TCC). They report that a significant proportion of TCCs exhibit reduced expression of hMLH1 or hMSH2, two DNA mismatch repair proteins. This occurred in younger patients and was related to both tumor stage and grade. The least differentiated and most invasive tumors were more likely to exhibit reduced DNA mismatch repair protein expression. Unexpectedly, reduced expression of either protein appeared to predict subsequent outcome, with fewer recurrences and relapses in all tumors and in superficial tumors; however, 5-year survival rates did not reach statistical significance with respect to protein expression. Microsatellite instability was observed in less than 10% of all tumors but, unexpectedly, was not correlated with the loss of DNA mismatch repair proteins. These observations suggest that mismatch repair proteins expression levels could serve as prognostic indicators for TCC. Despite improved diagnostic and treatment options, colorectal cancer remains one of the most common and fatal cancers. Nakayama and colleagues previously reported that p16 promoter methylation is present in the tumors of colorectal cancer patients and can be detected in the serum of these patients using methylation-specific PCR. In this article (pages 491–493) the same group has tested a group of 45 colorectal cancer patients for the presence of p16 promoter methylation in serum. They report that methylated tumor DNA was detected in the serum samples of 31 out of 45 (69%) patients with recurrent colorectal cancer. In sharp contrast, none of the 50 patients whose tumor DNA had no methylation in p16 promoter were positive. These observations suggest that analysis of the state of methylation of the p16 promoter in serum DNA could become a sensitive and selective method to detect recurrent colorectal cancer. DNA methylation controls gene expression and is often dysregulated in cancer cells. Aberrant DNA methylation not only can silence gene transcription when it occurs near promoter regions, it can also lead to enhanced gene mutations as 5-methylcytosine is deaminated to thymidine or can promote carcinogenesis through enhanced allelic loss. As a measure of DNA methyltransferase activity in certain tumors, levels of DNA methyltransferase I (DNMT1) mRNA have been studied in the past. DNMT1 is the best characterized of the three methyltransferase enzymes known so far. In this issue, Saito and colleagues focus on the measurement of DNMT1 protein levels in hepatocellular carcinoma (pages 527–532). They generated a specific antibody directed against human DNMT1 and measured protein levels by immunohistochemistry. Normal liver tissue or precancerous conditions such as cirrhosis or chronic hepatitis did not stain positive. However, nearly half of the samples obtained from individuals with hepatocellular carcinoma were positive for DNMT1. DNMT1-positive tumors were poorly differentiated and showed portal vein involvement and, most importantly, correlated significantly with low survival. These results indicate that increased DNMT1 protein expression might play a role in the malignant progression of hepatocellular carcinoma. Thymomas develop in the thymic epithelium, which normally directs T-lymphocyte maturation. These tumors manifest asymptomatically as mediastinal masses, cause local chest pain or present concurrently with neurological symptoms of myasthenia gravis. This issue of IJC features the first population-based study describing the demographic pattern of thymoma incidence in the United States. Engels and colleagues (pages 546–551) focused exclusively on malignant thymomas, which differ from the more common benign thymomas based on evidence for capsular invasion. Their data confirmed that malignant thymoma is an extremely rare disease. Thymoma incidence was higher in men than in women and peaked late in adulthood (72 years). Interestingly, incidence rates were nearly doubled in individuals from Hawaii, indicating a genetic influence of the disease. To examine whether malignant thymomas arise as a component of an inherited cancer syndrome, special attention was placed on subsequent malignancies. Interestingly, the risk of developing a second malignancy was not broadly elevated although the risk for B-cell non-Hodgkin lymphoma was enhanced in surviving thymoma patients. Whether non-Hodgkin lymphomas were caused by abnormal B-cell proliferation due to dysfunctional T cells or presented as a consequence of radiotherapy remains to be determined. The authors further pointed out that 5 patients developed 2 subsequent malignancies, and 4 of the 5 patients developed lung tumors. They will further examine this novel association in future studies. Kaplan-Meier survival curves for individuals with hepatocellular carcinoma grouped by their DNMT1 status. (A) Recurrence-free survival is shown, while overall survival is plotted in (B).

Beta-catenin accumulation and mutation of exon 3 of the beta-catenin gene in hepatocellular carcinoma.

study was conducted to clarify the contribution of β‐catenin accumulation and mutation of the β‐catenin gene to hepatocarcinogenesis. β‐Catenin accumulation was examined immunohistochemically in 38 paired samples of hepatocellular carcinoma (HCC) and corresponding non‐cancerous liver tissue. Gene mutation was analyzed by polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) and direct sequencing using intronic primers encompassing exon 3. Neither accumulation nor mutation was detected in non‐cancerous liver tissues that showed no remarkable histological features, chronic hepatitis or liver cirrhosis. Accumulation of β‐catenin was seen in the nucleus, cytoplasm or cell membrane in 15 of 38 (39%) HCC samples, and gene mutation was seen in 9 of 38 (24%) HCC samples. Although there was a significant correlation between accumulation and mutation (P<0.01), six HCCs without mutation also showed accumulation. Samples of early HCC showed neither accumulation nor mutation, and accumulation and mutation were each correlated significantly with portal vein tumor involvement (P<0.05). The present results indicate that (1) mutation of exon 3 of the β‐catenin gene can lead to β‐catenin accumulation, although other mechanisms of accumulation may also operate in HCC, and (2) β‐catenin accumulation and mutation of the β‐catenin gene are not early events in hepatocarcinogenesis, and may be associated with the malignant progression of HCC.