Wei-Fen Xie

Differentiation therapy of hepatocellular carcinoma in mice with recombinant adenovirus carrying hepatocyte nuclear factor-4α gene†

Previous studies have shown that hepatocyte nuclear factor‐4α (HNF4α) is a central regulator of differentiated hepatocyte phenotype and forced expression of HNF4α could promote reversion of tumors toward a less invasive phenotype. However, the effect of HNF4α on cancer stem cells (CSCs) and the treatment of hepatocellular carcinoma (HCC) with HNF4α have not been reported. In this study, an adenovirus‐mediated gene delivery system, which could efficiently transfer and express HNF4α, was generated to determine its effect on hepatoma cells (Hep3B and HepG2) in vitro and investigate the anti‐tumor effect of HNF4α in mice. Our results demonstrated that forced re‐expression of HNF4α induced the differentiation of hepatoma cells into hepatocytes, dramatically decreased “stemness” gene expression and the percentage of CD133+ and CD90+ cells, which are considered as cancer stem cells in HCC. Meanwhile, HNF4α reduced cell viability through inducing apparent apoptosis in Hep3B, while it induced cell cycle arrest and cellular senescence in HepG2. Moreover, infection of hepatoma cells by HNF4α abolished their tumorigenesis in mice. Most interestingly, systemic administration of adenovirus carrying the HNF4α gene protected mice from liver metastatic tumor formation, and intratumoral injection of HNF4α also displayed significant antitumor effects on transplanted tumor models. Conclusion: The striking suppression effect of HNF4α on tumorigenesis and tumor development is attained by inducing the differentiation of hepatoma cells—especially CSCs—into mature hepatocytes, suggesting that differentiation therapy with HNF4α may be an effective treatment for HCC patients. Our study also implies that differentiation therapy may present as one of the best strategies for cancer treatment through the induction of cell differentiation by key transcription factors. (HEPATOLOGY 2008.)

Hepatic transforming growth factor beta gives rise to tumor‐initiating cells and promotes liver cancer development

Liver cirrhosis is a predominant risk factor for hepatocellular carcinoma (HCC). However, the mechanism underlying the progression from cirrhosis to HCC remains unclear. Herein we report the concurrent increase of liver progenitor cells (LPCs) and transforming growth factor‐β (TGF‐β) in diethylnitrosamine (DEN)‐induced rat hepatocarcinogenesis and cirrhotic livers of HCC patients. Using several experimental approaches, including 2‐acetylaminofluorene/partial hepatectomy (2‐AAF/PHx) and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC)‐elicited murine liver regeneration, we found that activation of LPCs in the absence of TGF‐β induction was insufficient to trigger hepatocarcinogenesis. Moreover, a small fraction of LPCs was detected to coexpress tumor initiating cell (T‐IC) markers during rat hepatocarcinogenesis and in human HCCs, and TGF‐β levels were positively correlated with T‐IC marker expression, which indicates a role of TGF‐β in T‐IC generation. Rat pluripotent LPC‐like WB‐F344 cells were exposed to low doses of TGF‐β for 18 weeks imitating the enhanced TGF‐β expression in cirrhotic liver. Interestingly, long‐term treatment of TGF‐β on WB‐F344 cells impaired their LPC potential but granted them T‐IC properties including expression of T‐IC markers, increased self‐renewal capacity, stronger chemoresistance, and tumorigenicity in NOD‐SCID mice. Hyperactivation of Akt but not Notch, signal transducer and activator of transcription 3 (STAT3), or mammalian target of rapamycin (mTOR) was detected in TGF‐β‐treated WB‐F344 cells. Introduction of the dominant‐negative mutant of Akt significantly attenuated T‐IC properties of those transformed WB‐F344 cells, indicating Akt was required in TGF‐β‐mediated‐generation of hepatic T‐ICs. We further demonstrate that TGF‐β‐induced Akt activation and LPC transformation was mediated by microRNA‐216a‐modulated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) suppression. Conclusion: Hepatoma‐initiating cells may derive from hepatic progenitor cells exposed to chronic and constant TGF‐β stimulation in cirrhotic liver, and pharmaceutical inhibition of microRNA‐216a/PTEN/Akt signaling could be a novel strategy for HCC prevention and therapy targeting hepatic T‐ICs. (HEPATOLOGY 2012;56:2255–2267)

Hepatocyte Nuclear Factor 4α Suppresses the Development of Hepatocellular Carcinoma

Hepatocyte nuclear factor 4α (HNF4α) is a transcription factor that plays a key role in hepatocyte differentiation and the maintenance of hepatic function, but its role in hepatocarcinogenesis has yet to be examined. Here, we report evidence of a suppressor role for HNF4α in liver cancer. HNF4α expression was progressively decreased in the diethylinitrosamine-induced rat model of liver carcinogenesis. In human liver tissues, HNF4α expression was decreased in cirrhotic tissue and further decreased in hepatocarcinoma relative to healthy tissue. Notably, an inverse correlation existed with epithelial-mesenchymal transition (EMT). Enforced expression of HNF4α attenuated hepatocyte EMT during hepatocarcinogenesis, alleviated hepatic fibrosis, and blocked hepatocellular carcinoma (HCC) occurrence. In parallel, stem cell marker gene expression was inhibited along with cancer stem/progenitor cell generation. Further, enforced expression of HNF4α inhibited activation of β-catenin, which is closely associated with EMT and hepatocarcinogenesis. Taken together, our results suggest that the inhibitory effect of HNF4α on HCC development might be attributed to suppression of hepatocyte EMT and cancer stem cell generation through an inhibition of β-catenin signaling pathways. More generally, our findings broaden knowledge on the biological significance of HNF4α in HCC development, and they imply novel strategies for HCC prevention through the manipulation of differentiation-determining transcription factors in various types of carcinomas.

Long-Term Effects of Mid-Dose Ursodeoxycholic Acid in Primary Biliary Cirrhosis: A Meta-analysis of Randomized Controlled Trials

OBJECTIVES:The effect of ursodeoxycholic acid (UDCA) treatment on survival and liver histological progression of primary biliary cirrhosis (PBC) remains uncertain. The aim of this study is to assess the long-term efficacy of mid-dose UDCA treatment for PBC.METHODS:Electronic databases including Medline, Embase, Cochrane controlled trials register, Science Citation Index, and PUBMED (updated to Nov 2005), and manual bibliographical searches were conducted. A meta-analysis of all long-term randomized controlled trials (RCTs) comparing mid-dose UDCA with placebo or no treatment was performed.RESULTS:Seven RCTs and six reports of their extended follow-up including 1,038 patients were assessed. UDCA could significantly improve liver biochemistry, but had no effect on pruritus and fatigue. UDCA could delay the progression of PBC, especially for early-stage patients. Meta-analysis of the seven RCTs including their extended follow-up showed a significant reduction of the incidence of liver transplantation (OR 0.65, p = 0.01), and a marginally significant reduction of the rate of death or liver transplantation (fixed-effect model: OR 0.76, p = 0.05; random-effect model: OR 0.77, p = 0.3) in the UDCA group, except death (OR 1.01, p = 1). In the sensitivity analyses, which included studies administrating placebo as control, long-term studies (≥48 months), or large size studies (total number of patients ≥100), we all found long-term treatment with UDCA could significantly reduce the incidence of liver transplantation, and death or liver transplantation.CONCLUSIONS:Long-term treatment with mid-dose UDCA can improve liver biochemistry and survival free of liver transplantation in patients with PBC. In addition, UDCA therapy can delay the histological progression in the early-stage patients.

Transarterial chemoembolization in combination with percutaneous ablation therapy in unresectable hepatocellular carcinoma: a meta‐analysis

Background: Recent evidence suggests that transcatheter arterial chemoembolization (TACE) combined with radiofrequency ablation (RFA) or a percutaneous ethanol injection (PEI) may have a synergistic effect in treating hepatocellular carcinoma (HCC). The aim of the current meta‐analysis was to identify the survival benefits of TACE combined with percutaneous ablation (PA) therapy (RFA or PEI) for unresectable HCC compared with those of TACE or PA alone.

Suppression of Cyclin D1 by Hypoxia-Inducible Factor-1 via Direct Mechanism Inhibits the Proliferation and 5-Fluorouracil–Induced Apoptosis of A549 Cells

Hypoxia-inducible factor (HIF) and cyclin D1 are both key mediators of cell growth and proliferation in normal and cancer cells. However, the interrelation between HIF and cyclin D1 remains unclear. In the present study, we observed the inverse correlation between cyclin D1 and HIF-1 in hypoxia condition. Overexpression of the dominant negative mutant of HIF-1alpha (DN-HIF) significantly enhanced cyclin D1 expression upon hypoxia or arsenite exposure, suggesting the negative regulation of cyclin D1 by HIF-1. Furthermore, we found that the impairment of HIF-1 increased cyclin D1 expression in A549 pulmonary cancer cells, which in turn promoted G1-S cell cycle transition and cell proliferation. Cyclin D1 expression was increased in s.c. xenograft of DN-HIF stably transfected A549 cells in nude mice compared with that of control cells. Chromatin immunoprecipitation assay revealed that HIF-1 was able to directly bind to the promoter region of cyclin D1, which indicates that the negative regulation of cyclin D1 by HIF-1 is through a direct mechanism. Inhibition of histone deacetylase (HDAC) by pretreatment of cells with trichostatin A or specific knockdown of HDAC7 by its shRNA antagonized the suppression of cyclin D1 by HIF-1, suggesting that HDAC7 is required for HIF-1-mediated cyclin D1 downregulation. Moreover, we found that 5-fluorouracil-triggered apoptosis of DN-HIF-transfected A549 cells was reduced by sicyclin D1 (cyclin D1-specific interference RNA) introduction, suggesting that clinical observation of HIF-1 overexpression-associated chemoresistance might be, at least partially, due to the negative regulation of cyclin D1.

Cyclin G1–mediated epithelial-mesenchymal transition via phosphoinositide 3-kinase/Akt signaling facilitates liver cancer progression†

Cyclin G1 deficiency is associated with reduced incidence of carcinogen‐induced hepatocellular carcinoma (HCC), but its function in HCC progression remains obscure. We report a critical role of cyclin G1 in HCC metastasis. Elevated expression of cyclin G1 was detected in HCCs (60.6%), and its expression levels were even higher in portal vein tumor thrombus. Clinicopathological analysis revealed a close correlation of cyclin G1 expression with distant metastasis and poor prognosis of HCC. Forced expression of cyclin G1 promoted epithelial‐mesenchymal transition (EMT) and metastasis of HCC cells in vitro and in vivo. Cyclin G1 overexpression enhanced Akt activation through interaction with p85 (regulatory subunit of phosphoinositide 3‐kinase [PI3K]), which led to subsequent phosphorylation of glycogen synthase kinase‐3β (GSK‐3β) and stabilization of Snail, a critical EMT mediator. These results suggest that elevated cyclin G1 facilitates HCC metastasis by promoting EMT via PI3K/Akt/GSK‐3β/Snail‐dependent pathway. Consistently, we have observed a significant correlation between cyclin G1 expression and p‐Akt levels in a cohort of HCC patients, and found that combination of these two parameters is a more powerful predictor of poor prognosis. Conclusions: Cyclin G1 plays a pivotal role in HCC metastasis and may serve as a novel prognostic biomarker and therapeutic target. (HEPATOLOGY 2012;55:1787–1798)

Gemcitabine in the chemoradiotherapy for locally advanced pancreatic cancer: A meta-analysis

AIMS Whether gemcitabine based chemoradiotherapy (GEM-based CRT) is superior to 5-fluorouracil based chemoradiotherapy (5-FU-based CRT) for locally advanced pancreatic cancer (LAPC) remains uncertain. The aim of the present study was to evaluate the effect of GEM-based CRT compared with 5-FU-based CRT. METHODS Electronic database including Medline, Embase, Cochrane controlled trials register, PubMed (update to December 2010) and manual bibliography searches were carried out. A meta-analysis of all randomized clinical trials (RCTs) or other comparative studies comparing GEM-based CRT and 5-FU-based CRT were performed. RESULTS Three RCTs and one retrospective comparative study including 229 patients were assessed. Meta-analysis showed survival advantage of GEM-based CRT compared with 5-FU-based CRT for 12-month (12-mo) survival rates (SRs) (RR=1.54, 95% CI 1.05-2.26, p=0.03). Moreover, there were also trends of benefit for SR after 6-months (RR 1.13, 95% CI 0.98-1.30, p=0.09) and 24-months (24-mo: RR 2.41, 95% CI 0.90-6.48, p=0.08), though the trends did not reach statistical significance. More frequent severe acute hematologic toxicities were found in the GEM-based CRT group. CONCLUSIONS The meta-analysis found that GEM-based CRT was better than 5-FU-based CRT in the treatment of LAPC, especially for 12-mo SRs. However, the acute toxicity should be carefully regarded.

EUS elastography for the differentiation of benign and malignant lymph nodes: a meta-analysis.

BACKGROUND EUS elastography is a new technique for differentiating benign and malignant lymph nodes (LNs) by describing the mechanical property of the target tissue. OBJECTIVE To assess the accuracy of EUS elastography by pooling data of existing trials. DESIGN Seven studies involving 368 patients with 431 LNs were included. Meta-analysis was performed. Pooling was conducted in a fixed-effect model or a random-effect model. PATIENTS This study involved 368 patients. INTERVENTION EUS elastography. MAIN OUTCOME MEASUREMENTS Meta-analysis and meta-regression analysis. RESULTS The pooled sensitivity of EUS elastography for the differential diagnosis of benign and malignant LNs was 88% (95% confidence interval [CI] 0.83-0.92), and the specificity was 85% (95% CI, 0.79-0.89). The area under the curve under summary receiver operating characteristic (SROC) was 0.9456. The pooled positive likelihood ratio was 5.68 (95% CI, 2.86-11.28), and the negative likelihood ratio was 0.15 (95% CI, 0.10-0.21). The subgroup analysis by excluding the outliers provided a sensitivity of 85% (95% CI, 0.79-0.90) and a specificity of 91% (95% CI, 0.85-0.95) for the differential diagnosis of benign and malignant LNs. The area under the curve under SROC was 0.9421. LIMITATIONS A small number of studies met inclusion criteria. CONCLUSION EUS elastography is a promising, noninvasive method for differential diagnosis of malignant LNs and may prove to be a valuable supplemental method to EUS-guided FNA.

Recombinant Adenovirus Carrying the Hepatocyte Nuclear Factor-1alpha Gene Inhibits Hepatocellular Carcinoma Xenograft Growth in Mice

Hepatocyte nuclear factor‐1alpha (HNF1α) is one of the key transcription factors of the HNF family, which plays a critical role in hepatocyte differentiation. Substantial evidence has suggested that down‐regulation of HNF1α may contribute to the development of hepatocellular carcinoma (HCC). Herein, human cancer cells and tumor‐associated fibroblasts (TAFs) were isolated from human HCC tissues, respectively. A recombinant adenovirus carrying the HNF1α gene (AdHNF1α) was constructed to determine its effect on HCC in vitro and in vivo. Our results demonstrated that HCC cells and HCC tissues revealed reduced expression of HNF1α. Forced reexpression of HNF1α significantly suppressed the proliferation of HCC cells and TAFs and inhibited the clonogenic growth of hepatoma cells in vitro. In parallel, HNF1α overexpression reestablished the expression of certain liver‐specific genes and microRNA 192 and 194 levels, with a resultant increase in p21 levels and induction of G2/M arrest. Additionally, AdHNF1α inhibited the expression of cluster of differentiation 133 and epithelial cell adhesion molecule and the signal pathways of the mammalian target of rapamycin and transforming growth factor beta/Smads. Furthermore, HNF1α abolished the tumorigenicity of hepatoma cells in vivo. Most interestingly, intratumoral injection of AdHNF1α significantly inhibited the growth of subcutaneous HCC xenografts in nude mice. Systemic delivery of AdHNF1α could eradicate the orthotopic liver HCC nodules in nonobese diabetic/severe combined immunodeficiency mice. Conclusion: These results suggest that the potent inhibitive effect of HNF1α on HCC is attained by inducing the differentiation of hepatoma cells into mature hepatocytes and G2/M arrest. HNF1α might represent a novel, promising therapeutic agent for human HCC treatment. Our findings also encourage the evaluation of differentiation therapy for tumors of organs other than liver using their corresponding differentiation‐determining transcription factor. (HEPATOLOGY 2011)