Daekwan Seo

HEPATOCYTE GROWTH FACTOR/C-MET SIGNALING IS REQUIRED FOR STEM-CELL-MEDIATED LIVER REGENERATION IN MICE

Hepatocyte growth factor (HGF)/c‐Met supports a pleiotrophic signal transduction pathway that controls stem cell homeostasis. Here, we directly addressed the role of c‐Met in stem‐cell–mediated liver regeneration by utilizing mice harboring c‐met floxed alleles and Alb‐Cre or Mx1‐Cre transgenes. To activate oval cells, the hepatic stem cell (HSC) progeny, we used a model of liver injury induced by diet containing the porphyrinogenic agent, 3,5‐diethocarbonyl‐1,4‐dihydrocollidine (DDC). Deletion of c‐met in oval cells was confirmed in both models by polymerase chain reaction analysis of fluorescence‐activated cell‐sorted epithelial cell adhesion molecule (EpCam)‐positive cells. Loss of c‐Met receptor decreased the sphere‐forming capacity of oval cells in vitro as well as reduced oval cell pool, impaired migration, and decreased hepatocytic differentiation in vivo, as demonstrated by double immunofluorescence using oval‐ (A6 and EpCam) and hepatocyte‐specific (i.e. hepatocyte nuclear factor 4‐alpha) antibodies. Furthermore, lack of c‐Met had a profound effect on tissue remodeling and overall composition of HSC niche, which was associated with greatly reduced matrix metalloproteinase (MMP)9 activity and decreased expression of stromal‐cell–derived factor 1. Using a combination of double immunofluorescence of cell‐type–specific markers with MMP9 and gelatin zymography on the isolated cell populations, we identified macrophages as a major source of MMP9 in DDC‐treated livers. The Mx1‐Cre‐driven c‐met deletion caused the greatest phenotypic impact on HSCs response, as compared to the selective inactivation in the epithelial cell lineages achieved in c‐Metfl/fl; Alb‐Cre+/− mice. However, in both models, genetic loss of c‐met triggered a similar cascade of events, leading to the failure of HSC mobilization and death of the mice. Conclusion: These results establish a direct contribution of c‐Met in the regulation of HSC response and support a unique role for HGF/c‐Met as an essential growth‐factor–signaling pathway for regeneration of diseased liver. (HEPATOLOGY 2012)

Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer

BACKGROUND & AIMS Aberrant activation of the RAS pathway is ubiquitous in human hepatocarcinogenesis, but the molecular mechanisms leading to RAS induction in the absence of RAS mutations remain under-investigated. We defined the role of Ras GTPase activating proteins (GAPs) in the constitutive activity of Ras signaling during human hepatocarcinogenesis. METHODS The mutation status of RAS genes and RAS effectors was assessed in a collection of human hepatocellular carcinomas (HCC). Levels of RAS GAPs (RASA1-4, RASAL1, nGAP, SYNGAP1, DAB2IP, and NF1) and the RASAL1 upstream inducer PITX1 were determined by real-time RT-PCR and immunoblotting. The promoter and genomic status of RASAL1, DAB2IP, NF1, and PITX1 were assessed by methylation assays and microsatellite analysis. Effects of RASAL1, DAB2IP, and PITX1 on HCC growth were evaluated by transfection and siRNA analyses of HCC cell lines. RESULTS In the absence of Ras mutations, downregulation of at least one RAS GAP (RASAL1, DAB2IP, or NF1) was found in all HCC samples. Low levels of DAB2IP and PITX1 were detected mostly in a HCC subclass from patients with poor survival, indicating that these proteins control tumor aggressiveness. In HCC cells, reactivation of RASAL1, DAB2IP, and PITX1 inhibited proliferation and induced apoptosis, whereas their silencing increased proliferation and resistance to apoptosis. CONCLUSIONS Selective suppression of RASAL1, DAB2IP, or NF1 RAS GAPs results in unrestrained activation of Ras signaling in the presence of wild-type RAS in HCC.

An Integrated Genomic and Epigenomic Approach Predicts Therapeutic Response to Zebularine in Human Liver Cancer

Drug-induced transcription and epigenetic changes can predict whether liver cancer can be successfully treated with an epigenome-targeting drug. A Crystal Ball for Cancer Therapy If you ask most first-year medical students, they are not likely to list fortune-telling as a required skill for a successful clinician. Yet, predicting how different patients will respond to therapy could save time, money, and lives. Andersen et al. peer into their crystal ball and provide an approach for determining whether patients with liver cancer will respond to treatment or not. Liver cancer causes more than half a million deaths annually. Although early cases may respond to treatment, it is often diagnosed at later stages when symptoms become more severe. Only a subset of patients with advanced-stage liver cancer is eligible for the most successful current therapy, sorafenib, leaving a large number of patients with few options. Epigenetic changes, or changes in gene expression not due to differences in DNA sequence, contribute to the initiation and progression of liver cancer. These changes are reversible, and epigenetic modifier drugs such as zebularine can reactivate tumor suppressor genes and decrease tumor cell growth. Andersen et al. characterized the epigenetic changes induced by zebularine in liver cancer cell lines. They found a distinct signature that identified two groups of cells—those sensitive and those resistant to zebularine. In an animal xenograft model, zebularine treatment of liver tumors with the “sensitive” profile resulted in increased survival and decreased metastasis to the lungs. Moreover, the zebularine-sensitive signature predicted prognosis of patients with liver cancer with an accuracy ranging from 84 to 96%. Therefore, this study not only provides a proof of principle that epigenetic modification may be a viable therapy for liver cancer but also gives us a glimpse into the future, allowing clinicians to predict clinical outcome for liver cancer patients, as well as the success or failure of this emerging treatment regimen. Epigenomic changes such as aberrant hypermethylation and subsequent atypical gene silencing are characteristic features of human cancer. Here, we report a comprehensive characterization of epigenomic modulation caused by zebularine, an effective DNA methylation inhibitor, in human liver cancer. Using transcriptomic and epigenomic profiling, we identified a zebularine response signature that classified liver cancer cell lines into two major subtypes with different drug responses. In drug-sensitive cell lines, zebularine caused inhibition of proliferation coupled with increased apoptosis, whereas drug-resistant cell lines showed up-regulation of oncogenic networks (for example, E2F1, MYC, and TNF) that drive liver cancer growth in vitro and in preclinical mouse models. Assessment of zebularine-based therapy in xenograft mouse models demonstrated potent therapeutic effects against tumors established from zebularine-sensitive but not zebularine-resistant liver cancer cells, leading to increased survival and decreased pulmonary metastasis. Integration of the zebularine gene expression and demethylation response signatures allowed differentiation of patients with hepatocellular carcinoma according to their survival and disease recurrence. This integrated signature identified a subclass of patients within the poor-survivor group that is likely to benefit from therapeutic agents that target the cancer epigenome.

Human hepatic cancer stem cells are characterized by common stemness traits and diverse oncogenic pathways.

Epigenetic mechanisms play critical roles in stem cell biology by maintaining pluripotency of stem cells and promoting differentiation of more mature derivatives. If similar mechanisms are relevant for the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSC isolation and rigorous evaluation. To test this hypothesis, primary human cancer cells and liver cancer cell lines were treated with zebularine (ZEB), a potent DNA methyltransferase‐1 inhibitor, and putative CSCs were isolated using the side population (SP) approach. The CSC properties of ZEB‐treated and untreated subpopulations were tested using standard in vitro and in vivo assays. Whole transcriptome profiling of isolated CSCs was performed to generate CSC signatures. Clinical relevance of the CSC signatures was evaluated in diverse primary human cancers. Epigenetic modulation increased frequency of cells with CSC properties in the SP fraction isolated from human cancer cells as judged by self‐renewal, superior tumor‐initiating capacity in serial transplantations, and direct cell tracking experiments. Integrative transcriptome analysis revealed common traits enriched for stemness‐associated genes, although each individual CSC gene expression signature exhibited activation of different oncogenic pathways (e.g., EGFR, SRC, and MYC). The common CSC signature was associated with malignant progression, which is enriched in poorly differentiated tumors, and was highly predictive of prognosis in liver and other cancers. Conclusion: Epigenetic modulation may provide a tool for prospective isolation and in‐depth analysis of CSC. The liver CSC gene signatures are defined by a pernicious interaction of unique oncogene‐specific and common stemness traits. These data should facilitate the identifications of therapeutic tools targeting both unique and common features of CSCs. (HEPATOLOGY 2011;)

Loss of c-Met Disrupts Gene Expression Program Required for G2/M Progression during Liver Regeneration in Mice

Background Previous work has established that HGF/c-Met signaling plays a pivotal role in regulating the onset of S phase following partial hepatectomy (PH). In this study, we used Metfl/fl;Alb-Cre+/− conditional knockout mice to determine the effects of c-Met dysfunction in hepatocytes on kinetics of liver regeneration. Methodology/Principal Finding The priming events appeared to be intact in Metfl/fl;Alb-Cre+/− livers. Up-regulation of stress response (MAFK, IKBZ, SOCS3) and early growth response (c-Myc, c-Jun, c-Fos, DUSP1 and 6) genes as assessed by RT-qPCR and/or microarray profiling was unchanged. This was consistent with an early induction of MAPK/Erk and STAT3. However, after a successful completion of the first round of DNA replication, c-Met deficient hepatocytes were blocked in early/mid G2 phase as shown by staining with phosphorylated form of histone H3. Furthermore, loss of c-Met in hepatocytes diminished the subsequent G1/S progression and delayed liver recovery after partial hepatectomy. Upstream signaling pathways involved in the blockage of G2/M transition included lack of persistent Erk1/2 activation and inability to up-regulate the levels of Cdk1, Plk1, Aurora A and B, and Mad2 along with a defective histone 3 phosphorylation and lack of chromatin condensation. Continuous supplementation with EGF in vitro increased proliferation of Metfl/fl;Alb-Cre+/− primary hepatocytes and partially restored expression levels of mitotic cell cycle regulators albeit to a lesser degree as compared to control cultures. Conclusion/Significance In conclusion, our results assign a novel non-redundant function for HGF/c-Met signaling in regulation of G2/M gene expression program via maintaining a persistent Erk1/2 activation throughout liver regeneration.

Specific fate decisions in adult hepatic progenitor cells driven by MET and EGFR signaling

The relative contribution of hepatocyte growth factor (HGF)/MET and epidermal growth factor (EGF)/EGF receptor (EGFR), two key signal transduction systems in the normal and diseased liver, to fate decisions of adult hepatic progenitor cells (HPCs) has not been resolved. Here, we developed a robust culture system that permitted expansion and genetic manipulation of cells capable of multilineage differentiation in vitro and in vivo to examine the individual roles of HGF/MET and EGF/EGFR in HPC self-renewal and binary cell fate decision. By employing loss-of-function and rescue experiments in vitro, we showed that both receptors collaborate to increase the self-renewal of HPCs through activation of the extracellular signal-regulated kinase (ERK) pathway. MET was a strong inducer of hepatocyte differentiation by activating AKT and signal transducer and activator of transcription (STAT3). Conversely, EGFR selectively induced NOTCH1 to promote cholangiocyte specification and branching morphogenesis while concomitantly suppressing hepatocyte commitment. Furthermore, unlike the deleterious effects of MET deletion, the liver-specific conditional loss of Egfr facilitated rather than suppressed progenitor-mediated liver regeneration by switching progenitor cell differentiation toward hepatocyte lineage. These data provide new insight into the mechanisms regulating the stemness properties of adult HPCs and reveal a previously unrecognized link between EGFR and NOTCH1 in directing cholangiocyte differentiation.

Contribution of Hepatic Lineage Stage-Specific Donor Memory to the Differential Potential of Induced Mouse Pluripotent Stem Cells

Recent studies suggested that induced pluripotent stem cells (iPSCs) retain a residual donor cell gene expression, which may impact their capacity to differentiate into cell of origin. Here, we addressed a contribution of a lineage stage‐specific donor cell memory in modulating the functional properties of iPSCs. iPSCs were generated from hepatic lineage cells at an early (hepatoblast‐derived, HB‐iPSCs) and end stage (adult hepatocyte, AH‐iPSCs) of hepatocyte differentiation as well as from mouse embryonic fibroblasts (MEFs‐iPSCs) using a lentiviral vector encoding four pluripotency‐inducing factors Oct4, Sox2, Klf4, and c‐Myc. All resulting iPSC lines acquired iPSCs phenotype as judged by the accepted criteria including morphology, expression of pluripotency markers, silencing of transducing factors, capacity of multilineage differentiation in teratoma assay, and normal diploid karyotype. However, HB‐iPSCs were more efficient in directed differentiation toward hepatocytic lineage as compared to AH‐iPSCs, MEF‐iPSCs, or mouse embryonic stem cells (mESCs). Extensive comparative transcriptome analyses of the early passage iPSCs, donor cells, and mESCs revealed that despite global similarities in gene expression patterns between generated iPSCs and mESCs, HB‐iPSCs retained a transcriptional memory (seven upregulated and 17 downregulated genes) typical of the original cells. Continuous passaging of HB‐iPSCs erased most of these differences including a superior capacity for hepatic redifferentiation. These results suggest that retention of lineage stage‐specific donor memory in iPSCs may facilitate differentiation into donor cell type. The identified gene set may help to improve hepatic differentiation for therapeutic applications and contribute to the better understanding of liver development. STEM CELLS 2012;30:997–1007

Molecular targeting of CSN5 in human hepatocellular carcinoma: a mechanism of therapeutic response

Development of targeted therapy for hepatocellular carcinoma (HCC) remains a major challenge. We have recently identified an elevated expression of the fifth subunit of COP9 signalosome (CSN5) in early HCC as compared with dysplastic stage. In the present study, we explored the possibility of CSN5 being a potential therapeutic target for HCC. Our results show that CSN5 knockdown by small-interfering (si) RNA caused a strong induction of apoptosis and inhibition of cell-cycle progression in HCC cells in vitro. The down-regulation of CSN5 was sufficient to interfere with CSN function as evidenced by the accumulation of neddylated Cullin 1 and changes in the protein levels of CSN-controlled substrates SKP2, p53, p27 and nuclear factor-κB, albeit to a different degree depending on the HCC cell line, which could account for the CSN5 knockdown phenotype. The transcriptomic analysis of CSN5 knockdown signature showed that the anti-proliferative effect was driven by a common subset of molecular alterations including down-regulation of cyclin-dependent kinase 6 (CDK6) and integrin β1 (ITGB1), which were functionally interconnected with key oncogenic regulators MYC and TGFβ1 involved in the control of proliferation, apoptotic cell death and HCC progression. Consistent with microarray analysis, western blotting revealed that CSN5 depletion increased phosphorylation of Smad 2/3, key mediators of TGFβ1 signaling, decreased the protein levels of ITGB1, CDK6 and cyclin D1 and caused reduced expression of anti-apoptotic Bcl-2, while elevating the levels of pro-apoptotic Bak. A chemically modified variant of CSN5 siRNA was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of the CSN5 3/8 variant by stable-nucleic-acid-lipid particles significantly suppressed the tumor growth in Huh7-luc+ orthotopic xenograft model. Taken together, these results indicate that CSN5 has a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy.

Definition of Ubiquitination Modulator COP1 as a Novel Therapeutic Target in Human Hepatocellular Carcinoma

The development of targeted therapeutics for hepatocellular carcinoma (HCC) remains a major challenge. The ubiquitination modulator COP1 regulates p53 activity by ubiquitination and it is frequently overexpressed in human HCC. In this study, we tested the hypothesis that COP1 blockade by short interfering RNA (siRNA)-mediated inhibition could affect the course of HCC progression. The COP1 isoform COP1-1 was selected as the most effective target for siRNAs in terms of growth inhibition and apoptotic induction in several HCC cell lines. Growth inhibition occurred in HCC cells that retained wild-type p53 or expressed mutant p53 (Y220C or R249S), whereas p53-null Hep3B cells were resistant. Microarray expression analysis revealed that the antiproliferative effects of COP1 blockade were driven by a common subset of molecular alterations including a p53-associated functional network. In an orthotopic mouse xenograft model of HCC, systemic delivery of a modified COP1 siRNA by stable nucleic acid-lipid particles suppressed neoplastic growth in liver without unwanted immune responses. Our findings offer a first proof of principle that COP1 can be a promising target for systemic therapy of HCC.

Sequential transcriptome analysis of human liver cancer indicates late stage acquisition of malignant traits

BACKGROUND & AIMS Human hepatocarcinogenesis is as a multi-step process starting from dysplastic lesions to early carcinomas (eHCC) that ultimately progress to HCC (pHCC). However, the sequential molecular alterations driving malignant transformation of the pre-neoplastic lesions are not clearly defined. This lack of information represents a major challenge in the clinical management of patients at risk. METHODS We applied next-generation transcriptome sequencing to tumor-free surrounding liver (n = 7), low- (n = 4) and high-grade (n = 9) dysplastic lesions, eHCC (n = 5) and pHCC (n = 3) from 8 HCC patients with hepatitis B infection. Integrative analyses of genetic and transcriptomic changes were performed to characterize the genomic alterations during hepatocarcinogenesis. RESULTS We report that changes in transcriptomes of early lesions including eHCC were modest and surprisingly homogenous. Extensive genetic alterations and subsequent activation of prognostic adverse signaling pathways occurred only late during hepatocarcinogenesis and were centered on TGFβ, WNT, NOTCH, and EMT-related genes highlighting the molecular diversity of pHCC. We further identify IGFALS as a key genetic determinant preferentially down-regulated in pHCC. CONCLUSIONS Our results define new hallmarks in molecular stratification and therapy options for patients at risk for HCC, and merit larger prospective investigations to develop a modified clinical-decision making algorithm based on the individualized next-generation sequencing analyses.