EpCAM inhibits differentiation of human liver progenitor cells into hepatocytes in vitro by activating Notch1 signaling.

In both normal turnover of the hepatic tissue and acute hepatic injury, the liver predominantly activates terminally differentiated hepatocytes to proliferate and repair. However, in chronic and severe chronic injury, this capacity fails, and liver progenitor cells (LPCs) can give rise to hepatocytes to restore both hepatic architecture and liver metabolic function. Although the promotion of LPC-to-hepatocyte differentiation to acquire a considerable number of functional hepatocytes could serve as a potentially new therapeutic option for patients with end-stage liver disease, its development first requires the identification of the molecular mechanisms driving this process. Here, we found that the epithelial cell adhesion molecule (EpCAM), a progenitor cell marker, regulates the differentiation of LPCs into hepatocytes through Notch1 signaling pathway. Western blotting (WB) revealed a consistent expression pattern of EpCAM and Notch1 during LPC-to-hepatocyte differentiation in vitro. Additionally, overexpression of EpCAM blocked LPC-to-hepatocyte differentiation, which was in consistent with the repressive role of Notch signaling during hepatic differentiation. WB and immunofluorescence data also showed that the upregulation of EpCAM expression increased the generation of Notch intracellular domain (N1ICD), indicating the promotion of Notch1 activity. Our results established the EpCAM-Notch1 signaling axis as an inhibitory mechanism preventing LPC-to-hepatocyte differentiation in vitro.

Acetyl-coenzyme A carboxylase alpha promotion of glucose-mediated fatty acid synthesis enhances survival of hepatocellular carcinoma in mice and patients.

UNLABELLED: Solid tumors often suffer from suboptimal oxygen and nutrient supplies. This stress underlies the requirement for metabolic adaptation. Aberrantly activated de novo lipogenesis is critical for development and progression of human hepatocellular carcinoma (HCC). However, whether de novo lipogenesis influences biological behaviors of HCCs under conditions of metabolic stress are still poorly understood. Here, we show that HCCs display distinct levels of glucose-derived de novo lipogenesis, which are positively correlated with their survival responses to glucose limitation. The enhanced lipogenesis in HCCs is characterized by an increased expression of rate-limiting enzyme acetyl-coenzyme A carboxylase alpha (ACCα). ACCα-mediated fatty acid (FA) synthesis determines the intracellular lipid content that is required to maintain energy hemostasis and inhibit cell death by means of FA oxidation (FAO) during metabolic stress. In accord, overexpression of ACCα facilitates tumor growth. ACCα forms a complex with carnitine palmitoyltransferase 1A (CPT1A) and prevents its mitochondria distribution under nutrient-sufficient conditions. During metabolic stress, phosphorylation of ACCα leads to dissociation of the complex and mitochondria localization of CPT1A, thus promoting FAO-mediated cell survival. Therefore, ACCα could provide both the substrate and enzyme storage for FAO during glucose deficiency. Up-regulation of ACCα is also significantly correlated with poorer overall survival and disease recurrence postsurgery. Multivariate Cox's regression analysis identified ACCα as an effective predictor of poor prognosis. CONCLUSION: These results present novel mechanistic insight into a pivotal role of ACCα in maintaining HCC survival under metabolic stress. It could be exploited as a novel diagnostic marker and therapeutic target.

Maintaining viability and characteristics of cholangiocarcinoma tissue by vitrification-based cryopreservation.

Tumor tissue has great clinical and scientific value which relies highly on the proper preservation of primary materials. Conventional tumor tissue cryopreservation using slow-freezing method has yielded limited success, leading to significant cell loss and morphological damage. Here we report a standardized vitrification-based cryopreservation method, by which we have successfully vitrified and warmed 35 intrahepatic cholangiocarcinoma (ICC) tissues with up to 80% viability of the fresh tumor tissues. Cryopreserved ICC tissue could generate patient-derived xenografts (PDXs) with take rates of 68.2% compared to 72.7% using fresh tumor tissues. Histological and genetic analyses showed that no significant alterations in morphology and gene expression were introduced by this cryopreservation method. Our procedure may facilitate collection, long-time storage and propagation of cholangiocarcinoma or other tumor specimens for (pre)clinical studies of novel therapies or for basic research.

CD24+LCN2+ liver progenitor cells in ductular reaction contributed to macrophage inflammatory responses in chronic liver injury.

BACKGROUND: CD24+CK19+/CD24+SOX9+ resident liver cells are activated and expanded after chronic liver injury in a ductular reaction. However, the sources and functions of these cells in liver damage remain disputed. RESULTS: The current study combined genetic lineage tracing with in vitro small-molecule-based reprogramming to define liver progenitor cells (LPCs) derived from hepatic parenchymal and non-parenchymal tissues. tdTom+ hepatocytes were isolated from ROSA26tdTomato mice following AAV8-Tbg-Cre-mediated recombination, EpCAM+ biliary epithelial cells (BECs) from wild-type intrahepatic bile ducts and ALB/GFP-EpCAM- cells were isolated from AlbCreERT/R26GFP mice. A cocktail of small molecules was used to convert the isolated cells into LPCs. These in vitro cultured LPCs with CD24 and SOX9 expression regained the ability to proliferate. Transcriptional profiling showed that the in-vitro cultured LPCs derived from the resident LPCs in non-parenchymal tissues expressed Lipocalin-2 (Lcn2) at high levels. Accordingly, endogenous Cd24a+Lcn2+ LPCs were identified by integration of sc-RNA-sequencing and pathological datasets of liver dysfunction which indicates that LPCs produced by ductular reactions might also originate from the resident LPCs. Transplantation of in-vitro cultured Cd24a+Lcn2+ LPCs into CCl4-induced fibrotic livers exacerbated liver damage and dysfunction, possibly due to LCN2-dependent macrophage inflammatory response. CONCLUSIONS: CD24+LCN2+ LPCs constituted the expanding ductular reaction and contributed to macrophage-mediated inflammation in chronic liver damage. The current findings highlight the roles of LPCs from distinct origins and expose the possibility of targeting LPCs in the treatment of chronic hepatic diseases.

Targeting mTORC2/HDAC3 Inhibits Stemness of Liver Cancer Cells Against Glutamine Starvation.

Cancer cells are addicted to glutamine. However, cancer cells often suffer from glutamine starvation, which largely results from the fast growth of cancer cells and the insufficient vascularization in the interior of cancer tissues. Herein, based on clinical samples, patient-derived cells (PDCs), and cell lines, it is found that liver cancer cells display stem-like characteristics upon glutamine shortage due to maintaining the stemness of tumor initiating cells (TICs) and even promoting transformation of non-TICs into stem-like cells by glutamine starvation. Increased expression of glutamine synthetase (GS) is essential for maintaining and promoting stem-like characteristics of liver cancer cells during glutamine starvation. Mechanistically, glutamine starvation activates Rictor/mTORC2 to induce HDAC3-mediated deacetylation and stabilization of GS. Rictor is significantly correlated with the expression of GS and stem marker OCT4 at tumor site, and closely correlates with poor prognosis of hepatocellular carcinomas. Inhibiting components of mTORC2-HDAC3-GS axis decrease TICs and promote xenografts regression upon glutamine-starvation therapy. Collectively, the data provides novel insights into the role of Rictor/mTORC2-HDAC3 in reprogramming glutamine metabolism to sustain stemness of cancer cells. Targeting Rictor/HDAC3 may enhance the efficacy of glutamine-starvation therapy and limit the rapid growth and malignant progression of tumors.

The combined induction of liver progenitor cells and the suppression of stellate cells by small molecules reverts chronic hepatic dysfunction.

Rationale: We developed a cocktail of soluble molecules mimicking the in vivo milieu supporting liver regeneration that could convert mature hepatocytes to expandable liver progenitor-like cells in vitro. This study aimed to induce endogenous liver progenitor cells by the administration of the soluble molecules to provide an alternative approach for the resolution of liver fibrosis. Methods:In vitro cultured hepatocyte-derived liver progenitor-like cells (HepLPCs) were transplanted into CCL4-treated mice to investigate the therapeutic effect against liver fibrosis. Next, we used HGF in combination with a cocktail of small molecules (Y-27632, A-83-01, and CHIR99021 (HACY)) to induce endogenous CD24+ liver progenitor cells and to inhibit the activation of hepatic stellate cells (HSCs) during CCL4-induced hepatic injury. RNA sequencing was performed to further clarify the features of HACY-induced CD24+ cells compared with CCL4-induced CD24+ cells and in vitro derived HepLPCs. Finally, we evaluated the expansion of HACY-induced CD24+ cells in human hepatocyte-spheroids from fibrotic liver tissues. Results: HepLPCs exhibited the capacity to alleviate liver fibrosis after transplantation into CCL4-treated mice. The in vivo administration of HACY not only induced the conversion of mature hepatocytes (MHs) to CD24+ progenitor cells but prevented the activation of HSCs, thus leading to enhanced improvement of liver fibrosis in CCL4-treated mice. Compared to CD24+ cells induced by CCL4 alone, HACY-induced CD24+ cells retained an enhanced level of hepatic function and could promote the restoration of liver function that exhibited comparable gene expression profiles with HepLPCs. CD24+ cells were also observed in human liver fibrotic tissues and were expanded in three-dimensional (3D) hepatic spheroids in the presence of HACY in vitro. Conclusions: Hepatocyte-derived liver progenitor-like cells are crucial for liver regeneration during chronic hepatic injuries. The administration of HACY, which allowed the induction of endogenous CD24+ progenitor cells and the inactivation of HSCs, exerts beneficial effects in the treatment of liver fibrosis by re-establishing a balance favoring liver regeneration while preventing fibrotic responses.

An extracorporeal bioartificial liver embedded with 3D-layered human liver progenitor-like cells relieves acute liver failure in pigs.

Clinical advancement of the bioartificial liver is hampered by the lack of expandable human hepatocytes and appropriate bioreactors and carriers to encourage hepatic cells to function during extracorporeal circulation. We have recently developed an efficient approach for derivation of expandable liver progenitor-like cells from human primary hepatocytes (HepLPCs). Here, we generated immortalized and functionally enhanced HepLPCs by introducing FOXA3, a hepatocyte nuclear factor that enables potentially complete hepatic function. When cultured on macroporous carriers in an air-liquid interactive bioartificial liver (Ali-BAL) support device, the integrated cells were alternately exposed to aeration and nutrition and grew to form high-density three-dimensional constructs. This led to highly efficient mass transfer and supported liver functions such as albumin biosynthesis and ammonia detoxification via ureagenesis. In a porcine model of drug overdose-induced acute liver failure (ALF), extracorporeal Ali-BAL treatment for 3 hours prevented hepatic encephalopathy and led to markedly improved survival (83%, n = 6) compared to ALF control (17%, n = 6, P = 0.02) and device-only (no-cell) therapy (0%, n = 6, P = 0.003). The blood ammonia concentrations, as well as the biochemical and coagulation indices, were reduced in Ali-BAL-treated pigs. Ali-BAL treatment attenuated liver damage, ameliorated inflammation, and enhanced liver regeneration in the ALF porcine model and could be considered as a potential therapeutic avenue for patients with ALF.

A DMSO-free hepatocyte maturation medium accelerates hepatic differentiation of HepaRG cells in vitro.

The most essential tools for studying drug hepatotoxicity, liver diseases, and bioartificial livers have always been models that can recapitulate liver physiology in vitro. The liver progenitor cell line HepaRG represents an effective surrogate of the primary hepatocyte. However, the differentiation of HepaRG relies on long-term induction using a high concentration of dimethyl sulfoxide (DMSO), which may compromise the research of drug metabolism and restrict the applicability of this hepatic model. Here, we present a novel hepatic maturation medium (HMM) for the differentiation of HepaRG, which is based on a cocktail of soluble molecules that mimick the in vivo environment. We showed that HMM could rapidly (about nine days) induce HepaRG differentiation into polarized hepatocytes with maturely metabolic functions. In addition, under three-dimensional culture conditions, the hepatic spheroids showed multiple liver functions and toxicity profiles close to those of primary human hepatocytes (PHH). Our work demonstrates the utility of HMM as an alternative to the DMSO-dependent differentiation protocol for HepaRG; moreover, these results facilitate the application of HepaRG.

Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens.

The study of pathophysiological mechanisms in human liver disease has been constrained by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. We and others have previously shown that mouse mature hepatocytes can be converted to liver progenitor-like cells in vitro with defined chemical factors. Here we describe a protocol achieving efficient conversion of human primary hepatocytes into liver progenitor-like cells (HepLPCs) through delivery of developmentally relevant cues, including NAD + -dependent deacetylase SIRT1 signaling. These HepLPCs could be expanded significantly during in vitro passage. The expanded cells can readily be converted back into metabolically functional hepatocytes in vitro and upon transplantation in vivo. Under three-dimensional culture conditions, differentiated cells generated from HepLPCs regained the ability to support infection or reactivation of hepatitis B virus (HBV). Our work demonstrates the utility of the conversion between hepatocyte and liver progenitor-like cells for studying HBV biology and antiviral therapies. These findings will facilitate the study of liver diseases and regenerative medicine.

Inhibition of dipeptidyl peptidase IV prevents high fat diet-induced liver cancer angiogenesis by downregulating chemokine ligand 2.

Obesity is a major risk factor for hepatocellular carcinoma (HCC) and is typically accompanied by higher levels of serum dipeptidyl peptidase 4 (DPP4). However, the role of DPP4 in obesity-promoted HCC is unclear. Here, we found that consumption of a high-fat diet (HFD) promoted HCC cell proliferation and metastasis and led to poor survival in a carcinogen-induced model of HCC in rats. Notably, genetic ablation of DPP4 or treatment with a DPP4 inhibitor (vildagliptin) prevented HFD-induced HCC. Moreover, HFD-induced DPP4 activity facilitated angiogenesis and cancer cell metastasis in vitro and in vivo, and vildagliptin prevented tumor progression by mediating the pro-angiogenic role of chemokine ligand 2 (CCL2). Loss of DPP4 effectively reversed HFD-induced CCL2 production and angiogenesis, indicating that the DPP4/CCL2/angiogenesis cascade had key roles in HFD-associated HCC progression. Furthermore, concomitant changes in serum DPP4 and CCL2 were observed in 210 patients with HCC, and high serum DPP4 activity was associated with poor clinical prognosis. These results revealed a link between obesity-related high serum DPP4 activity and HCC progression. Inhibition of DPP4 may represent a novel therapeutic intervention for patients with HCC.

Blocking preferential glucose uptake sensitizes liver tumor-initiating cells to glucose restriction and sorafenib treatment.

Cancer cells display altered metabolic phenotypes characterized by a high level of glycolysis, even under normoxic conditions. Because of a high rate of glycolytic flux and inadequate vascularization, tumor cells often suffer from nutrient deficiency and require metabolic adaptations to address such stresses. Although tumor-initiating cells (T-ICs) have been identified in various malignancies, the cells' metabolic phenotypes remain elusive. In this study, we observed that liver T-ICs preferentially survived under restricted glucose treatment. These cell populations compete successfully for glucose uptake by preferentially expressing glucose transporters (GLUT1 and GLUT3), whereas inhibition of GLUT1 or GLUT3 abolished the survival advantage and suppressed the tumorigenic potential of liver T-ICs. Among signaling pathways related to T-ICs, IL-6/STAT3 was identified to be responsible for the elevation of glucose uptake in liver T-ICs under glucose limitation. Further investigation revealed that IL-6 stimulation upregulated GLUT1 and GLUT3 expressions in CD133+ cells, particularly during glucose deprivation. More importantly, inhibition of glucose uptake sensitized liver T-ICs to sorafenib treatment and enhanced the therapeutic efficacy in vivo. Our findings suggest that blocking IL-6/STAT3-mediated preferential glucose uptake might be exploited for novel therapeutic targets during hepatocellular carcinoma (HCC) progression.

HBx regulates fatty acid oxidation to promote hepatocellular carcinoma survival during metabolic stress.

Due to a high rate of nutrient consumption and inadequate vascularization, hepatocellular carcinoma (HCC) cells constantly undergo metabolic stress during tumor development. Hepatitis B virus (HBV) X protein (HBx) has been implicated in the pathogenesis of HBV-induced HCC. In this study, we investigated the functional roles of HBx in HCC adaptation to metabolic stress. Up-regulation of HBx increased the intracellular ATP and NADPH generation, and induced the resistance to glucose deprivation, whereas depletion of HBx via siRNA abolished these effects and conferred HCC cells sensitive to glucose restriction. Though HBx did not affect the glycolysis and oxidative phosphorylation capacity of HCC cells under normal culture conditions, it facilitated fatty acid oxidation (FAO) in the absence of glucose, which maintained NADPH and ATP levels. Further investigation showed that HBx expression, under glucose deprivation, stimulated phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) via a calcium/CaMKK-dependent pathway, which was required for the activation of FAO. Conversely, inhibition of FAO by etomoxir (ETO) restored the sensitivity of HBx-expressing cells to glucose deficiency in vitro and retarded xenograft tumor formation in vivo. Finally, HBx-induced activation of the AMPK and FAO pathways were also observed in xenograft tumors and HBV-associated HCC specimens. Our data suggest that HBx plays a key role in the maintenance of redox and energy homeostasis by activating FAO, which is critical for HCC cell survival under conditions of metabolic stress and might be exploited for therapeutic benefit.