Bei-Fang Ning

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.

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)

MiR-21 Simultaneously Regulates ERK1 Signaling in HSC Activation and Hepatocyte EMT in Hepatic Fibrosis

Background MicroRNA-21 (miR-21) plays an important role in the pathogenesis and progression of liver fibrosis. Here, we determined the serum and hepatic content of miR-21 in patients with liver cirrhosis and rats with dimethylnitrosamine-induced hepatic cirrhosis and examined the effects of miR-21 on SPRY2 and HNF4α in modulating ERK1 signaling in hepatic stellate cells (HSCs) and epithelial-mesenchymal transition (EMT) of hepatocytes. Methods Quantitative RT-PCR was used to determine miR-21 and the expression of SPRY2, HNF4α and other genes. Immunoblotting assay was carried out to examine the expression of relevant proteins. Luciferase reporter assay was performed to assess the effects of miR-21 on its predicted target genes SPRY2 and HNF4α. Primary HSCs and hepatocytes were treated with miR-21 mimics/inhibitors or appropriate adenoviral vectors to examine the relation between miR-21 and SPRY2 or HNF4α. Results The serum and hepatic content of miR-21 was significantly higher in cirrhotic patients and rats. SPRY2 and HNF4α mRNA levels were markedly lower in the cirrhotic liver. MiR-21 overexpression was associated with enhanced ERK1 signaling and EMT in liver fibrosis. Luciferase assay revealed suppressed SPRY2 and HNF4α expression by miR-21. Ectopic miR-21 stimulated ERK1 signaling in HSCs and induced hepatocyte EMT by targeting SPRY2 or HNF4α. Downregulating miR-21 suppressed ERK1 signaling, inhibited HSC activation, and blocked EMT in TGFβ1-treated hepatocytes. Conclusions MiR-21 modulates ERK1 signaling and EMT in liver fibrosis by regulating SPRY2 and HNF4α expression. MiR-21 may serve as a potentially biomarker as well as intervention target for hepatic cirrhosis.

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)

Long non-coding RNA DILC regulates liver cancer stem cells via IL-6/STAT3 axis

BACKGROUND & AIMS Emerging evidence has demonstrated the aberrant expression of long non-coding RNAs (lncRNAs) in various malignancies including HCC. However, the knowledge of cancer stem cell-related lncRNAs remains limited. METHODS lnc-DILC (lncRNA downregulated in liver cancer stem cells (LCSCs)) was identified by microarray and validated by real-time PCR. The role of lnc-DILC in LCSCs was assessed both in vitro and in vivo. Pull down assay and oligoribonucleotides or oligodeoxynucleotides treatment were conducted to evaluate the interaction between lnc-DILC and interleukin-6 (IL-6) promoter. RESULTS Depletion of lnc-DILC markedly enhanced LCSC expansion and facilitated HCC initiation and progression, whereas ectopic expression of lnc-DILC dramatically inhibited LCSC expansion. Mechanistically, lnc-DILC inhibited the autocrine IL-6/STAT3 signaling. The putative binding locus of lnc-DILC within IL-6 promoter was confirmed by pull down assay. Consistently, the oligoribonucleotide mimics and an oligodeoxynucleotide decoy of lnc-DILC abrogated the effects on IL-6 transcription, STAT3 activation and LCSC expansion triggered by lnc-DILC depletion and lnc-DILC overexpression. Moreover, our data suggested that lnc-DILC mediated the crosstalk between TNF-α/NF-κB signaling and IL-6/STAT3 cascade. Clinical investigation demonstrated the reduction of lnc-DILC in patient HCCs, and suggested the correlation between lnc-DILC levels and IL-6, EpCAM or CD24 expression. Decreased lnc-DILC expression in HCCs predicts early recurrence and short survival of patients, highlighting its prognostic value. CONCLUSIONS lnc-DILC mediates the crosstalk between TNF-α/NF-κB signaling and autocrine IL-6/STAT3 cascade and connects hepatic inflammation with LCSC expansion, suggesting that lnc-DILC could be not only a potential prognostic biomarker, but also a possible therapeutic target against LCSCs.

Hepatocyte nuclear factor-4α reverses malignancy of hepatocellular carcinoma through regulating miR-134 in the DLK1-DIO3 region

Hepatocyte nuclear factor‐4α (HNF4α) is a dominant transcriptional regulator of hepatocyte differentiation and hepatocellular carcinogenesis. There is striking suppression of hepatocellular carcinoma (HCC) by HNF4α, although the mechanisms by which HNF4α reverses HCC malignancy are largely unknown. Herein, we demonstrate that HNF4α administration to HCC cells resulted in elevated levels of 28 mature microRNAs (miRNAs) from the miR‐379‐656 cluster, which is located in the delta‐like 1 homolog (DLK1) ‐iodothyronine deiodinase 3 (DIO3) locus on human chromosome 14q32. Consistent with the reduction of HNF4α, these miRNAs were down‐regulated in human HCC tissue. HNF4α regulated the transcription of the miR‐379‐656 cluster by directly binding to its response element in the DLK1‐DIO3 region. Interestingly, several miRNAs in this cluster inhibited proliferation and metastasis of HCC cells in vitro. As a representative miRNA in this cluster, miR‐134 exerted a dramatically suppressive effect on HCC malignancy by down‐regulating the oncoprotein, KRAS. Moreover, miR‐134 markedly diminished HCC tumorigenicity and displayed a significant antitumor effect in vivo. In addition, inhibition of endogenous miR‐134 partially reversed the suppressive effects of HNF4α on KRAS expression and HCC malignancy. Furthermore, a positive correlation between HNF4α and miR‐134 levels was observed during hepatocarcinogenesis in rats, and decreases in miR‐134 levels were significantly associated with the aggressive behavior of human HCCs. Conclusion: Our data highlight the importance of the miR‐379‐656 cluster in the inhibitory effect of HNF4α on HCC, and suggest that regulation of the HNF4α‐miRNA cascade may have beneficial effects in the treatment of HCC. (Hepatology 2013; 58:1964–1976)

Hepatocyte nuclear factor 4α-nuclear factor-κB feedback circuit modulates liver cancer progression.

Hepatocyte nuclear factor 4α (HNF4α) is a liver enriched transcription factor and is indispensable for liver development. However, the role of HNF4α in hepatocellular carcinoma (HCC) progression remains to be elucidated. We report that reduced HNF4α expression correlated well with the aggressive clinicopathological characteristics of HCC and predicted poor prognosis of patients. HNF4α levels were even lower in metastatic HCCs, and ectopic HNF4α expression suppressed the metastasis of hepatoma cells both in vitro and in vivo. Forced HNF4α expression attenuated the expression and nuclear translocation of RelA (p65) and impaired NF‐κB activation through an IKK‐independent mechanism. Blockage of RelA robustly attenuated the suppressive effect of HNF4α on hepatoma cell metastasis. MicroRNA (miR)‐7 and miR‐124 were transcriptionally up‐regulated by HNF4α, which repressed RelA expression by way of interaction with RelA‐3′ untranslated region (UTR). In addition, nuclear factor kappa B (NF‐κB) up‐regulated the expression of miR‐21 in hepatoma cells, resulting in decreased HNF4α levels through down‐regulating HNF4α‐3′UTR activity. Conclusions: Collectively, an HNF4α‐NF‐κB feedback circuit including miR‐124, miR‐7, and miR‐21 was identified in HCC, and the combination of HNF4α and NF‐κB exhibited more powerful predictive efficiency of patient prognosis. These findings broaden the knowledge of hepatic inflammation and cancer initiation/progression, and also provide novel prognostic biomarkers and therapeutic targets for HCC. (Hepatology 2014;60:1607‐1619)

Gankyrin‐mediated dedifferentiation facilitates the tumorigenicity of rat hepatocytes and hepatoma cells

Gankyrin is a critical oncoprotein overexpressed in human hepatocellular carcinoma (HCC). However, the mechanism underlying gankyrin‐mediated hepatocarcinogenesis remains elusive. Herein, we provide evidence that gankyrin expression was progressively elevated in liver fibrosis, cirrhosis, and HCC. Levels of gankyrin expression were closely associated with the dedifferentiation status of hepatoma in patients. Decrease of hepatocyte characteristic markers and increase of cholangiocyte‐specific markers were observed in rat primary hepatocytes with enforced gankyrin expression and diethylnitrosamine (DEN)‐triggered rat hepatocarcinogenesis. Overexpression of gankyrin also attenuated the hepatic function of primary hepatocytes, which further suggests that gankyrin promotes the dedifferentiation of hepatocytes. Moreover, elevated expression of gankyrin closely correlated with the expression of HCC stem/progenitor cell markers in DEN‐triggered hepatocarcinogenesis and human HCCs. Hepatoma cells derived from suspension‐cultured spheroids exhibited a higher gankyrin level, and enforced gankyrin expression in hepatoma cells remarkably enhanced cluster of differentiation (CD)133, CD90, and epithelial cellular adhesion molecule expression, indicating a role of gankyrin in hepatoma cell dedifferentiation and the generation of hepatoma stem/progenitor cells. In contrast, down‐regulation of gankyrin in hepatoma cells by lentivirus‐mediated microRNA delivery significantly improved their differentiation status and attenuated malignancy. Interference of gankyrin expression in hepatoma cells also diminished the proportion of cancer stem/progenitor cells and their self‐renewal capacity. Furthermore, gankyrin was found to bind hepatocyte nuclear factor 4α (HNF4α), which determines hepatocyte differentiation status and enhances proteasome‐dependent HNF4α degradation in hepatoma cells. The inverse correlation of gankyrin and HNF4α was further confirmed in primary hepatocytes, DEN‐induced hepatocarcinogenesis, and human HCCs. Conclusion: Gankyrin‐mediated dedifferentiation of hepatocytes and hepatoma cells via, at least partially, down‐regulation of HNF4α facilitates HCC development, and interference of gankyrin expression could be a novel strategy for HCC prevention and differentiation therapy. (HEPATOLOGY 2011;54:1259–1272)