Chuan Yin

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.)

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.

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)

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)

Inhibition of extracellular signal-regulated kinase 1 by adenovirus mediated small interfering RNA attenuates hepatic fibrosis in rats†

Extracellular signal‐regulated kinase 1 (ERK1) is a critical part of the mitogen‐activated protein kinase signal transduction pathway, which is involved in hepatic fibrosis. However, the effect of down‐regulation of ERK1 on hepatic fibrosis has not been reported. Here, we induced hepatic fibrosis in rats with dimethylnitrosamine administration or bile duct ligation. An adenovirus carrying small interfering RNA targeting ERK1 (AdshERK1) was constructed to determine its effect on hepatic fibrosis, as evaluated by histological and immunohistochemical examination. Our results demonstrated that AdshERK1 significantly reduced the expression of ERK1 and suppressed proliferation and levels of fibrosis‐related genes in hepatic stellate cells in vitro. More importantly, selective inhibition of ERK1 remarkably attenuated the deposition of the extracellular matrix in fibrotic liver in both fibrosis models. In addition, both hepatocytes and biliary epithelial cells were proven to exert the ability to generate the myofibroblasts depending on the insults of the liver, which were remarkably reduced by AdshERK1. Furthermore, up‐regulation of ERK1 paralleled the increased expression of transforming growth factor β1 (TGF‐β1), vimentin, snail, platelet‐derived growth factor‐BB (PDGF‐BB), bone morphogenetic protein 4 (BMP4), and small mothers against decapentaplegic‐1 (p‐Smad1), and was in reverse correlation with E‐cadherin in the fibrotic liver. Nevertheless, inhibition of ERK1 resulted in the increased level of E‐cadherin in parallel with suppression of TGF‐β1, vimentin, snail, PDGF‐BB, BMP4, and p‐Smad1. Interestingly, AdshERK1 treatment promoted hepatocellular proliferation. Conclusion: Our study provides the first evidence for AdshERK1 suppression of hepatic fibrosis through the reversal of epithelial‐mesenchymal transition of both hepatocytes and biliary epithelial cells without interference of hepatocellular proliferation. This suggests that ERK1 is implicated in hepatic fibrogenesis and selective inhibition of ERK1 by small interfering RNA may present a novel option for hepatic fibrosis treatment. (HEPATOLOGY 2009.)

Hepatic stellate cells modulate the differentiation of bone marrow mesenchymal stem cells into hepatocyte-like cells.

Differentiation of stem cells is tightly regulated by the microenvironment which is mainly composed of nonparenchymal cells. Herein, we investigated effect of hepatic stellate cells (HSCs) in different states on mesenchymal stem cells (MSCs) differentiation. Rat HSCs were isolated and stayed quiescent within 5 days. Primary HSCs were activated by being in vitro cultured for 7 days or cocultured with Kupffer cells for 5 days. MSCs were cocultured with HSCs of different states. Expression of hepatic lineage markers was analyzed by RT‐PCR and immunofluorescence. Glycogen deposition was detected by periodic acid‐schiff staining. MSCs cocultured with HSC‐T6 or Kupffer cell activated HSCs were morphologically transformed into hepatocyte‐like cells. Hepatic‐specific marker albumin was expressed in 78.3% of the differentiated MSCs 2 weeks after initiation of coculture. In addition, the differentiated MSCs also expressed α‐fetoprotein, cytokeratin‐18, glutamine synthetase and phosphoenolpyruvate carboxykinase. Glycogen deposition was detectable in 55.4% of the differentiated MSCs 6 weeks after initiation of coculture. However, the quiescent HSCs or culture activated HSCs did not exert the ability to modulate the differentiation of MSCs. Moreover, Kupffer cell activated HSCs rather than culture activated HSCs expressed hepatocyte growth factor mRNA. We draw the conclusion that fully activated HSCs could modulate MSCs differentiation into hepatocyte‐like cells. J. Cell. Physiol. 217: 138–144, 2008.

Perturbation of MicroRNA-370/Lin-28 homolog A/nuclear factor kappa B regulatory circuit contributes to the development of hepatocellular carcinoma

MicroRNA 370 (miR‐370) is located within the DLK1/DIO3 imprinting region on human chromosome 14, which has been identified as a cancer‐associated genomic region. However, the role of miR‐370 in malignances remains controversial. Here, we report that miR‐370 was repressed in human hepatocellular carcinoma (HCC) tissues and hepatoma cell lines. Using gain‐of‐function and loss‐of‐function experiments, we demonstrated that miR‐370 inhibited the malignant phenotype of HCC cells in vitro. Overexpression of miR‐370 inhibited growth and metastasis of HCC cells in vivo. Moreover, the RNA‐binding protein, LIN28A, was identified as a direct functional target of miR‐370, which, in turn, blocked the biogenesis of miR‐370 by binding to its precursor. LIN28A also mediated the suppressive effects of miR‐370 on migration and invasion of HCC cells by post‐transcriptionally regulating RelA/p65, which is an important effector of the canonical nuclear factor kappa B (NF‐κB) pathway. Interleukin‐6 (IL‐6), a well‐known NF‐κB downstream inflammatory molecule, reduced miR‐370 but increased LIN28A levels in HCC. Furthermore, miR‐370 levels were inversely correlated with LIN28A and IL‐6 messenger RNA (mRNA) levels, whereas LIN28A mRNA expression was positively correlated with IL‐6 expression in human HCC samples. Interestingly, reduction of miR‐370 expression was associated with the development of HCC in rats, as well as with aggressive tumor behavior and short survival in HCC patients. Conclusions: These data demonstrate the involvement of a novel regulatory circuit consisting of miR‐370, LIN28A, RelA/p65 and IL‐6 in HCC progression. Manipulating this feedback loop may have beneficial effect in HCC treatment. (Hepatology 2013; 58:1977–1991)

Apoptosis signal-regulating kinase 1 mediates the inhibitory effect of hepatocyte nuclear factor-4α on hepatocellular carcinoma

Previous studies provided substantial evidence of a striking suppressive effect of hepatocyte nuclear factor 4α (HNF4α) on hepatocellular carcinoma (HCC). Apoptosis signal-regulating kinase 1 (ASK1) is involved in death receptor-mediated apoptosis and may acts as a tumor suppressor in hepatocarcinogenesis. However, the status and function of ASK1 during HCC progression are unclear. In this study, we found that HNF4α increased ASK1 expression by directly binding to its promoter. ASK1 expression was dramatically suppressed and correlated with HNF4α levels in HCC tissues. Reduced ASK1 expression was associated with aggressive tumors and poor prognosis for human HCC. Moreover, ASK1 inhibited the malignant phenotype of HCC cells in vitro. Intratumoral ASK1 injection significantly suppressed the growth of subcutaneous HCC xenografts in nude mice. More interestingly, systemic ASK1 delivery strikingly inhibited the growth of orthotopic HCC nodules in NOD/SCID mice. In addition, inhibition of endogenous ASK1 partially reversed the suppressive effects of HNF4α on HCC. Collectively, this study highlights the suppressive effect of ASK1 on HCC and its biological significance in HCC development. These outcomes broaden the knowledge of ASK1 function in HCC progression, and provide a novel potential prognostic biomarker and therapeutic target for advanced HCC.

Inhibition of plasminogen activator inhibitor-1 expression by siRNA in rat hepatic stellate cells

Background and Aim:  The plasminogen activator/plasmin system is known to regulate the extracellular matrix turnover. The aim of this study was to detect the role of plasminogen activator inhibitor‐1 (PAI‐1) during liver fibrogenesis and investigate the functional effects of PAI‐1 gene silencing in rat hepatic stellate cells (HSCs) using small interfering RNA (siRNA).