Pediatric oral fluorescein angiography: a retrospective review from a single institution.

PURPOSE: To describe our experience with locally developed evidence-based guidelines for oral fluorescein angiography (FA) for retinal imaging in children. METHODS: The medical records of consecutive pediatric patients (≤18 years of age) at University of Pittsburgh Medical Center Children's Hospital Eye Center who underwent oral FA between November 1, 2018, and April 1, 2022, were reviewed retrospectively. Adherence to or deviation from the guidelines was noted at the time of testing. RESULTS: A total of 55 patients aged 3-18 with 79 examinations were included. No patient was excluded from the retrospective case review because of lack of recorded data. The main indications for oral FA included uveitis, retinal vasculopathy, disk pathology, and retinal lesions. Three children had transient side effects, and 1 had delayed urticaria 4 hours after examination. No child had anaphylaxis. One patient had suboptimal imaging due to nonadherence to the guidelines-recommended fasting protocol. All other examinations (78/79), where guidelines were followed, provided images adequate for clinical decision making. CONCLUSIONS: Based on our experience, we recommend that oral FA be considered, especially in children where intravenous access is less well tolerated while awake. Informed consent that includes the possibility of delayed side effects is advisable.

Thygeson's superficial punctate keratitis in children.

OBJECTIVE: To describe the first paediatric case series of Thygesons' superficial punctate keratitis (TSPK) with management outcomes. METHODS: A retrospective chart review was done for all children either diagnosed at initial presentation or referred with TSPK from 01/2012 to 08/2021 at a tertiary children's hospital. Records were assessed for signs, symptoms, diagnosis, steroid and cyclosporine 0.05% use. The main outcome measures were visual acuity, treatment response and total steroid exposure. RESULTS: Fifteen children (7 females), mean age at presentation 8 ± 4 years were included. All had bilateral disease and a BCVA of >20/40 in the better eye. All patients received topical fluorometholone 0.1%, (FML) initially. 80% had a good response to FML. Corneal scraping was done to exclude infectious causes in four cases due to poor initial response or clinical suspicion. All 4 needed EUA for scraping and anterior segment OCT, after which 2 had molecularly confirmed TGFBI-related stromal dystrophy. For the rest, slow steroid taper was done every 4-6 weeks and recurrences were treated by increasing steroid frequency. Cyclosporine 0.05% was started in nine patients (69%), 8 ± 6 months after initial presentation. The decrease in total steroid exposure per week after starting cyclosporine was statistically significant (p < 0.05). CONCLUSION: Children with TSPK respond quickly to steroids, however, recurrences are common, necessitating a slow taper. Non-response to steroid needs careful reconsideration of the diagnosis and may necessitate the use of an EUA. Using cyclosporine 0.05% reduces the total steroid exposure in TSPK.

A Knock-In Mouse Model of Thymoma With the GTF2I L424H Mutation.

INTRODUCTION: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear. METHODS: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and characterized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1+ thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors. RESULTS: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle-related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial progenitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas. CONCLUSIONS: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.

Spatial meta-transcriptomics reveal associations of intratumor bacteria burden with lung cancer cells showing a distinct oncogenic signature.

BACKGROUND: The lung intratumor microbiome influences lung cancer tumorigenesis and treatment responses, but detailed data on the extent, location, and effects of microbes within lung tumors are missing, information needed for improved prognosis and treatment. METHODS: To address this gap, we developed a novel spatial meta-transcriptomic method simultaneously detecting the expression level of 1,811 host genes and 3 microbe targets (bacteria, fungi, and cytomegalovirus). After rigorous validation, we analyzed the spatial meta-transcriptomic profiles of tumor cells, T cells, macrophages, other immune cells, and stroma in surgically resected tumor samples from 12 patients with early-stage lung cancer. RESULTS: Bacterial burden was significantly higher in tumor cells compared with T cells, macrophages, other immune cells, and stroma. This burden increased from tumor-adjacent normal lung and tertiary lymphoid structures to tumor cells to the airways, suggesting that lung intratumor bacteria derive from the latter route of entry. Expression of oncogenic ß-catenin was strongly correlated with bacterial burden, as were tumor histological subtypes and environmental factors. CONCLUSIONS: Intratumor bacteria were enriched with tumor cells and associated with multiple oncogenic pathways, supporting a rationale for reducing the local intratumor microbiome in lung cancer for patient benefit. TRIAL REGISTRATION NUMBER: NCT00242723, NCT02146170.

Integrated Genomic Comparison of Mouse Models Reveals Their Clinical Resemblance to Human Liver Cancer.

Hepatocellular carcinoma (HCC) is a heterogeneous disease. Mouse models are commonly used as preclinical models to study hepatocarcinogenesis, but how well these models recapitulate molecular subtypes of human HCC is unclear. Here, integration of genomic signatures from molecularly and clinically defined human HCC (n = 11) and mouse models of HCC (n = 9) identified the mouse models that best resembled subtypes of human HCC and determined the clinical relevance of each model. Mst1/2 knockout (KO), Sav1 KO, and SV40 T antigen mouse models effectively recapitulated subtypes of human HCC with a poor prognosis, whereas the Myc transgenic model best resembled human HCCs with a more favorable prognosis. The Myc model was also associated with activation of ß-catenin. E2f1, E2f1/Myc, E2f1/Tgfa, and diethylnitrosamine (DEN)-induced models were heterogeneous and were unequally split into poor and favorable prognoses. Mst1/2 KO and Sav1 KO models best resemble human HCC with hepatic stem cell characteristics. Applying a genomic predictor for immunotherapy, the six-gene IFNγ score, the Mst1/2 KO, Sav1 KO, SV40, and DEN models were predicted to be the least responsive to immunotherapy. Further analysis showed that elevated expression of immune-inhibitory genes (Cd276 and Nectin2/Pvrl2) in Mst1/2 KO, Sav1 KO, and SV40 models and decreased expression of immune stimulatory gene (Cd86) in the DEN model might be accountable for the lack of predictive response to immunotherapy.Implication: The current genomic approach identified the most relevant mouse models to human liver cancer and suggests immunotherapeutic potential for the treatment of specific subtypes. Mol Cancer Res; 16(11); 1713-23. ©2018 AACR.

Loss of c-Met signaling sensitizes hepatocytes to lipotoxicity and induces cholestatic liver damage by aggravating oxidative stress.

Recent studies confirmed a critical importance of c-Met signaling for liver regeneration by modulating redox balance. Here we used liver-specific conditional knockout mice (MetKO) and a nutritional model of hepatic steatosis to address the role of c-Met in cholesterol-mediated liver toxicity. Liver injury was assessed by histopathology and plasma enzymes levels. Global transcriptomic changes were examined by gene expression microarray, and key molecules involved in liver damage and lipid homeostasis were evaluated by Western blotting. Loss of c-Met signaling amplified the extent of liver injury in MetKO mice fed with high-cholesterol diet for 30days as evidenced by upregulation of liver enzymes and increased synthesis of total bile acids, aggravated inflammatory response and enhanced intrahepatic lipid deposition. Global transcriptomic changes confirmed the enrichment of networks involved in steatosis and cholestasis. In addition, signaling pathways related to glutathione and lipid metabolism, oxidative stress and mitochondria dysfunction were significantly affected by the loss of c-Met function. Mechanistically, exacerbation of oxidative stress in MetKO livers was corroborated by increased lipid and protein oxidation. Western blot analysis further revealed suppression of Erk, NF-kB and Nrf2 survival pathways and downstream target genes (e.g. cyclin D1, SOD1, gamma-GCS), as well as up-regulation of proapoptotic signaling (e.g. p53, caspase 3). Consistent with the observed steatotic and cholestatic phenotype, nuclear receptors RAR, RXR showed increased activation while expression levels of CAR, FXR and PPAR-alpha were decreased in MetKO. Collectively, our data provide evidence for the critical involvement of c-Met signaling in cholesterol and bile acids toxicity.

DNMT1 is a required genomic regulator for murine liver histogenesis and regeneration.

UNLABELLED: DNA methyltransferase 1 (DNMT1) is an essential regulator maintaining both epigenetic reprogramming during DNA replication and genome stability. We investigated the role of DNMT1 in the regulation of postnatal liver histogenesis under homeostasis and stress conditions. We generated Dnmt1 conditional knockout mice (Dnmt1(Δalb) ) by crossing Dnmt1(fl/fl) with albumin-cyclization recombination transgenic mice. Serum, liver tissues, and primary hepatocytes were collected from 1-week-old to 20-week old mice. The Dnmt1(Δalb) phenotype was assessed by histology, confocal and electron microscopy, biochemistry, as well as transcriptome and methylation profiling. Regenerative growth was induced by partial hepatectomy and exposure to carbon tetrachloride. The impact of Dnmt1 knockdown was also analyzed in hepatic progenitor cell lines; proliferation, apoptosis, DNA damage, and sphere formation were assessed. Dnmt1 loss in postnatal hepatocytes caused global hypomethylation, enhanced DNA damage response, and initiated a senescence state causing a progressive inability to maintain tissue homeostasis and proliferate in response to injury. The liver regenerated through activation and repopulation from progenitors due to lineage-dependent differences in albumin-cyclization recombination expression, providing a basis for selection of less mature and therefore less damaged hepatic progenitor cell progeny. Consistently, efficient knockdown of Dnmt1 in cultured hepatic progenitor cells caused severe DNA damage, cell cycle arrest, senescence, and cell death. Mx1-cyclization recombination-driven deletion of Dnmt1 in adult quiescent hepatocytes did not affect liver homeostasis. CONCLUSION: These results establish the indispensable role of DNMT1-mediated epigenetic regulation in postnatal liver growth and regeneration; Dnmt1(Δalb) mice provide a unique experimental model to study the role of senescence and the contribution of progenitor cells to physiological and regenerative liver growth. (Hepatology 2016;64:582-598).

Oncogenic driver genes and the inflammatory microenvironment dictate liver tumor phenotype.

UNLABELLED: The majority of hepatocellular carcinoma develops in the background of chronic liver inflammation caused by viral hepatitis and alcoholic or nonalcoholic steatohepatitis. However, the impact of different types of chronic inflammatory microenvironments on the phenotypes of tumors generated by distinct oncogenes is largely unresolved. To address this issue, we generated murine liver tumors by constitutively active AKT-1 (AKT) and ß-catenin (CAT), followed by induction of chronic liver inflammation by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and carbon tetrachloride. Also, the impact of DDC-induced chronic liver inflammation was compared between two liver tumor models using a combination of AKT-CAT or AKT-NRAS(G12V) . Treatment with DDC and carbon tetrachloride significantly facilitated the adenoma-to-carcinoma conversion and accelerated the growth of AKT-CAT tumors. Furthermore, DDC treatment altered the morphology of AKT-CAT tumors and caused loss of lipid droplets. Transcriptome analysis of AKT-CAT tumors revealed that cellular growth and proliferation were mainly affected by chronic inflammation and caused up-regulation of Cxcl16, Galectin-3, and Nedd9, among others. Integration with transcriptome profiles from human hepatocellular carcinomas further demonstrated that AKT-CAT tumors generated in the context of chronic liver inflammation showed enrichment of poor prognosis gene sets or decrease of good prognosis gene sets. In contrast, DDC had a more subtle effect on AKT-NRAS(G12V) tumors and primarily enhanced already existent tumor characteristics as supported by transcriptome analysis. However, it also reduced lipid droplets in AKT-NRAS(G12V) tumors. CONCLUSION: Our study suggests that liver tumor phenotype is defined by a combination of driving oncogenes but also the nature of chronic liver inflammation. (Hepatology 2016;63:1888-1899).

Curcumin effectively inhibits oncogenic NF-κB signaling and restrains stemness features in liver cancer.

BACKGROUND & AIMS: The cancer stem cells (CSCs) have important therapeutic implications for multi-resistant cancers including hepatocellular carcinoma (HCC). Among the key pathways frequently activated in liver CSCs is NF-κB signaling. METHODS: We evaluated the CSCs-depleting potential of NF-κB inhibition in liver cancer achieved by the IKK inhibitor curcumin, RNAi and specific peptide SN50. The effects on CSCs were assessed by analysis of side population (SP), sphere formation and tumorigenicity. Molecular changes were determined by RT-qPCR, global gene expression microarray, EMSA, and Western blotting. RESULTS: HCC cell lines exposed to curcumin exhibited differential responses to curcumin and were classified as sensitive and resistant. In sensitive lines, curcumin-mediated induction of cell death was directly related to the extent of NF-κB inhibition. The treatment also led to a selective CSC-depletion as evidenced by a reduced SP size, decreased sphere formation, down-regulation of CSC markers and suppressed tumorigenicity. Similarly, NF-κB inhibition by SN50 and siRNA against p65 suppressed tumor cell growth. In contrast, curcumin-resistant cells displayed a paradoxical increase in proliferation and expression of CSC markers. Mechanistically, an important component of the CSC-depleting activity of curcumin could be attributed to a NF-κB-mediated HDAC inhibition. Co-administration of the class I/II HDAC inhibitor trichostatine sensitized resistant cells to curcumin. Further, integration of a predictive signature of curcumin sensitivity with human HCC database indicated that HCCs with poor prognosis and progenitor features are most likely to benefit from NF-κB inhibition. CONCLUSIONS: These results demonstrate that blocking NF-κB can specifically target CSC populations and suggest a potential for combined inhibition of NF-κB and HDAC signaling for treatment of liver cancer patients with poor prognosis.

The transcription factor GTF2IRD1 regulates the topology and function of photoreceptors by modulating photoreceptor gene expression across the retina.

The mechanisms that specify photoreceptor cell-fate determination, especially as regards to short-wave-sensitive (S) versus medium-wave-sensitive (M) cone identity, and maintain their nature and function, are not fully understood. Here we report the importance of general transcription factor II-I repeat domain-containing protein 1 (GTF2IRD1) in maintaining M cone cell identity and function as well as rod function. In the mouse, GTF2IRD1 is expressed in cell-fate determined photoreceptors at postnatal day 10. GTF2IRD1 binds to enhancer and promoter regions in the mouse rhodopsin, M- and S-opsin genes, but regulates their expression differentially. Through interaction with the transcription factors CRX and thyroid hormone receptor ß 2, it enhances M-opsin expression, whereas it suppresses S-opsin expression; and with CRX and NRL, it enhances rhodopsin expression. In an apparent paradox, although GTF2IRD1 is widely expressed in multiple cell types across the retina, knock-out of GTF2IRD1 alters the retinal expression of only a limited number of annotated genes. Interestingly, however, the null mutation leads to altered topology of cone opsin expression in the retina, with aberrant S-opsin overexpression and M-opsin underexpression in M cones. Gtf2ird1-null mice also demonstrate abnormal M cone and rod electrophysiological responses. These findings suggest an important role for GTF2IRD1 in regulating the level and topology of rod and cone gene expression, and in maintaining normal retinal function.

MYC activates stem-like cell potential in hepatocarcinoma by a p53-dependent mechanism.

Activation of c-MYC is an oncogenic hallmark of many cancers, including liver cancer, and is associated with a variety of adverse prognostic characteristics. Despite a causative role during malignant transformation and progression in hepatocarcinogenesis, consequences of c-MYC activation for the biology of hepatic cancer stem cells (CSC) are undefined. Here, distinct levels of c-MYC overexpression were established by using two dose-dependent tetracycline-inducible systems in four hepatoma cell lines with different p53 mutational status. The CSCs were evaluated using side population (SP) approach as well as standard in vitro and in vivo assays. Functional repression of p53 was achieved by lentiviral shRNA transduction. The results show that c-MYC expression levels have a differential impact on liver CSC characteristics. At low levels, c-MYC activation led to increased proliferation and enhanced CSC properties including activation of reprogramming transcription factors and CSC marker expression (e.g., NANOG, OCT4, and EpCAM), expansion of SP, and acceleration of tumor growth upon subcutaneous transplantation into immunocompromised mice. However, when exceeding a threshold level, c-MYC induced a proapoptotic program and loss of CSC potential both in vitro and in vivo. Mechanistically, c-MYC-induced self-renewal capacity of liver cancer cells was exerted in a p53-dependent manner. Low c-MYC activation increased spheroid formation in p53-deficient tumor cells, whereas p53-dependent effects were blunted in the absence of c-MYC overexpression. Together, our results confirm the role of c-MYC as a master regulator during hepatocarcinogenesis and establish a new gatekeeper role for p53 in repressing c-MYC-induced CSC phenotype in liver cancer cells.

Antitumor effects in hepatocarcinoma of isoform-selective inhibition of HDAC2.

Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis, and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit HCC progression. siRNA-mediated silencing of HDAC inhibited HCC cell growth by blocking cell-cycle progression and inducing apoptosis. These effects were associated with deregulation of HDAC-regulated genes that control cell cycle, apoptosis, and lipid metabolism, specifically, by upregulation of p27 and acetylated p53 and by downregulation of CDK6 and BCL2. We found that HDAC2 silencing in HCC cells also strongly inhibited PPARγ signaling and other regulators of glycolysis (ChREBPα and GLUT4) and lipogenesis (SREBP1C and FAS), eliciting a marked decrease in fat accumulation. Notably, systemic delivery of HDAC2 siRNA encapsulated in lipid nanoparticles was sufficient to blunt the growth of human HCC in a murine xenograft model. Our findings offer preclinical proof-of-concept for HDAC2 blockade as a systemic therapy for liver cancer.

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 TGFß, 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.

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.

mTOR inhibitors synergize on regression, reversal of gene expression, and autophagy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) affects more than half a million people worldwide and is the third most common cause of cancer deaths. Because mammalian target of rapamycin (mTOR) signaling is up-regulated in 50% of HCCs, we compared the effects of the U.S. Food and Drug Administration-approved mTOR-allosteric inhibitor, RAD001, with a new-generation phosphatidylinositol 3-kinase/mTOR adenosine triphosphate-site competitive inhibitor, BEZ235. Unexpectedly, the two drugs acted synergistically in inhibiting the proliferation of cultured HCC cells. The synergistic effect closely paralleled eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) dephosphorylation, which is implicated in the suppression of tumor cell proliferation. In a mouse model approximating human HCC, the drugs in combination, but not singly, induced a marked regression in tumor burden. However, in the tumor, BEZ235 alone was as effective as the combination in inhibiting 4E-BP1 phosphorylation, which suggests that additional target(s) may also be involved. Microarray analyses revealed a large number of genes that reverted to normal liver tissue expression in mice treated with both drugs, but not either drug alone. These analyses also revealed the down-regulation of autophagy genes in tumors compared to normal liver. Moreover, in HCC patients, altered expression of autophagy genes was associated with poor prognosis. Consistent with these findings, the drug combination had a profound effect on UNC51-like kinase 1 (ULK1) dephosphorylation and autophagy in culture, independent of 4E-BP1, and in parallel induced tumor mitophagy, a tumor suppressor process in liver. These observations have led to an investigator-initiated phase 1B-2 dose escalation trial with RAD001 combined with BEZ235 in patients with HCC and other advanced solid tumors.

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.

Loss of c-Met accelerates development of liver fibrosis in response to CCl(4) exposure through deregulation of multiple molecular pathways.

HGF/c-Met signaling plays a pivotal role in hepatocyte survival and tissue remodeling during liver regeneration. HGF treatment accelerates resolution of fibrosis in experimental animal models. Here, we utilized Met(fl/fl);Alb-Cre(+/-) conditional knockout mice and a carbon tetrachloride(CCl(4))-induced liver fibrosis model to formally address the role of c-Met signaling in hepatocytes in the context of chronic tissue injury. Histological changes during injury (4weeks) and healing phase (4weeks) were monitored by immunohistochemistry; expression levels of selected key fibrotic molecules were evaluated by western blotting, and time-dependent global transcriptomic changes were examined using a microarray platform. Loss of hepatocyte c-Met signaling altered hepatic microenvironment and aggravated hepatic fibrogenesis. Greater liver damage was associated with decreased hepatocyte proliferation, excessive stellate cell activation and rapid dystrophic calcification of necrotic areas. Global transcriptome analysis revealed a broad impact of c-Met on critical signaling pathways associated with fibrosis. Loss of hepatocyte c-Met caused a strong deregulation of chemotactic and inflammatory signaling (MCP-1, RANTES, Cxcl10) in addition to modulation of genes involved in reorganization of the cytoskeletal network (Actb, Tuba1a, Tuba8), intercellular communications and adhesion (Adam8, Icam1, Itgb2), control of cell proliferation (Ccng2, Csnk2a, Cdc6, cdk10), DNA damage and stress response (Rad9, Rad52, Ercc4, Gsta1 and 2, Jun). Our study demonstrates that deletion of c-Met receptor in hepatocytes results in pronounced changes in hepatic metabolism and microenvironment, and establishes an essential role for c-Met in maintaining the structural integrity and adaptive plasticity of the liver under adverse conditions.

Genomic and genetic characterization of cholangiocarcinoma identifies therapeutic targets for tyrosine kinase inhibitors.

BACKGROUND & AIMS: Cholangiocarcinoma is a heterogeneous disease with a poor outcome that accounts for 5%-10% of primary liver cancers. We characterized its genomic and genetic features and associated these with patient responses to therapy. METHODS: We profiled the transcriptomes from 104 surgically resected cholangiocarcinoma samples collected from patients in Australia, Europe, and the United States; epithelial and stromal compartments from 23 tumors were laser capture microdissected. We analyzed mutations in KRAS, epidermal growth factor receptor (EGFR), and BRAF in samples from 69 tumors. Changes in gene expression were validated by immunoblotting and immunohistochemistry; integrative genomics combined data from the patients with data from 7 human cholangiocarcinoma cell lines, which were then exposed to trastuzumab and lapatinib. RESULTS: Patients were classified into 2 subclasses, based on 5-year survival rate (72% vs 30%; χ(2) = 11.61; P < .0007), time to recurrence (13.7 vs 22.7 months; P < .001), and the absence or presence of KRAS mutations (24.6%), respectively. Class comparison identified 4 survival subgroups (SGI-IV; χ(2) = 8.34; P < .03); SGIII was characterized by genes associated with proteasomal activity and the worst prognosis. The tumor epithelium was defined by deregulation of the HER2 network and frequent overexpression of EGFR, the hepatocyte growth factor receptor (MET), pRPS6, and Ki67, whereas stroma was enriched in inflammatory cytokines. Lapatinib, an inhibitor of HER2 and EGFR, was more effective in inhibiting growth of cholangiocarcinoma cell lines than trastuzumab. CONCLUSIONS: We provide insight into the pathogenesis of cholangiocarcinoma and identify previously unrecognized subclasses of patients, based on KRAS mutations and increased levels of EGFR and HER2 signaling, who might benefit from dual-target tyrosine kinase inhibitors. The group of patients with the worst prognosis was characterized by transcriptional enrichment of genes that regulate proteasome activity, indicating new therapeutic targets.

Hepatocyte growth factor/c-met signaling is required for stem-cell-mediated liver regeneration in mice.

UNLABELLED: 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-Met(fl/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.

Genomic modeling of tumor onset and progression in a mouse model of aggressive human liver cancer.

A comprehensive understanding of molecular mechanisms driving cancer onset and progression should provide a basis for improving early diagnosis, biomarker discovery and treatment options. A key value of genetically engineered mice for modeling human cancer is the possibility to analyze the entire process of tumor development. Here, we applied functional genomics approach to study step-by-step development of hepatocellular carcinoma (HCC) in the c-Myc/Tgfα transgenic mouse model of aggressive human liver cancer. We report that coexpression of c-Myc and Tgfα induces progressive and cumulative transcriptional alterations in the course of liver oncogenesis. Functional analysis of deregulated genes at the early stage of HCC disease supports a model of active hepatocyte proliferation on the background of chronic oxidative stress generated by a general metabolic disorder. In addition, early and persistent deregulation of numerous immune-related genes suggested that disruption of immune microenvironment may contribute to oncogenic process in this model of accelerated liver carcinogenesis. In particularly, by flow cytometry analysis, we found loss of the major histocompatibility complex class I expression in dysplastic hepatocytes followed by upregulation of numerous activating ligands for natural killer (NK) cells concomitant with a drastic decrease in hepatic NK cell frequency. In conclusion, our study provides a comprehensive characterization of sequential molecular changes during a stepwise progression of preneoplastic lesions toward HCC and highlights a critical role of metabolic disorders and innate immunity at the early stages of liver cancer.