Leslie E. Rogler

Elevated Expression of the miR-17–92 Polycistron and miR-21 in Hepadnavirus-Associated Hepatocellular Carcinoma Contributes to the Malignant Phenotype

Alterations in microRNA (miRNA) expression in both human and animal models have been linked to many forms of cancer. Such miRNAs, which act directly as repressors of gene expression, have been found to frequently reside in fragile sites and genomic regions associated with cancer. This study describes a miRNA signature for human primary hepatitis B virus-positive human hepatocellular carcinoma. Moreover, two known oncomiRs--miRNAs with known roles in cancer--the miR-17-92 polycistron and miR-21, exhibited increased expression in 100% of primary human and woodchuck hepatocellular carcinomas surveyed. To determine the importance of these miRNAs in tumorigenesis, an in vitro antisense oligonucleotide knockdown model was evaluated for its ability to reverse the malignant phenotype. Both in human and woodchuck HCC cell lines, separate treatments with antisense oligonucleotides specific for either the miR-17-92 polycistron (all six members) or miR-21 caused a 50% reduction in both hepatocyte proliferation and anchorage-independent growth. The combination of assays presented here supports a role for these miRNAs in the maintenance of the malignant transformation of hepatocytes.

MicroRNA‐23b cluster microRNAs regulate transforming growth factor‐beta/bone morphogenetic protein signaling and liver stem cell differentiation by targeting Smads

Transforming growth factor‐beta / bone morphogenetic protein (TGFβ/BMP) signaling has a gradient of effects on cell fate choice in the fetal mouse liver. The molecular mechanism to understand why adjacent cells develop into bile ducts or grow actively as hepatocytes in the ubiquitous presence of both TGFβ ligands and receptors has been unknown. We hypothesized that microRNAs (miRNAs) might play a role in cell fate decisions in the liver. miRNA profiling during late fetal development in the mouse identified miR‐23b cluster miRNAs comprising miR‐23b, miR‐27b, and miR‐24‐1 and miR‐10a, miR‐26a, and miR‐30a as up‐regulated. In situ hybridization of fetal liver at embryonic day 17.5 of gestation revealed miR‐23b cluster expression only in fetal hepatocytes. A complementary (c)DNA microarray approach was used to identify genes with a reciprocal expression pattern to that of miR‐23b cluster miRNAs. This approach identified Smads (mothers against decapentaplegic homolog), the key TGFβ signaling molecules, as putative miR‐23b cluster targets. Bioinformatic analysis identified multiple candidate target sites in the 3′ UTRs (untranslated regions) of Smads 3, 4, and 5. Dual luciferase reporter assays confirmed down‐regulation of constructs containing Smad 3, 4, or 5, 3′ UTRs by a mixture of miR‐23b cluster mimics. Knockdown of miR‐23b miRNAs during hepatocytic differentiation of a fetal liver stem cell line, HBC‐3, promoted expression of bile duct genes, in addition to Smads, in these cells. In contrast, ectopic expression of miR‐23b mimics during bile duct differentiation of HBC‐3 cells blocked the process. Conclusion: Our data provide a model in which miR‐23b miRNAs repress bile duct gene expression in fetal hepatocytes while promoting their growth by down‐regulating Smads and consequently TGFβ signaling. Concomitantly, low levels of the miR‐23b miRNAs are needed in cholangiocytes to allow TGFβ signaling and bile duct formation. (HEPATOLOGY 2009.)

Identification of hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver

Hepatocyte function and regeneration are severely compromised in severe liver disease, and a common sequela is cirrhosis. Structural changes caused by cirrhosis create a cellular environment conducive to the formation of ductular reactions (DRs). Ductular reactions are primarily composed of oval cells also known as “intermediate hepatobiliary cells”. We have conducted single, double, and triple staining to study lineages of oval cells present in DRs. Staining with NCAM, CK19, and HepPar1 has revealed a distinctly bipolar structure to DRs that are embedded in cirrhotic tissue. Spatial analysis of cells that are singly HepPar1‐positive, or CK19‐positive, has revealed hepatocytic and biliary poles, respectively, in the DRs. Also, the location of singly NCAM‐positive cells in DRs suggests that they may be bipotent liver stem/progenitor cells. The locations of other intermediate hepatobiliary cells, which have combinations of markers, suggest that CK19+/NCAM+ cells are transitional cells in the biliary lineage and that rare cells that are negative for all three markers are transitional cells in the hepatocytic lineage. A working cell lineage model for DRs is presented. (HEPATOLOGY 2007;45:716–724.)

Overexpression of miR-21 Promotes an In vitro Metastatic Phenotype by Targeting the Tumor Suppressor RHOB

Metastasis is a multistep process that involves the deregulation of oncogenes and tumor suppressors beyond changes required for primary tumor formation. RHOB is known to have tumor suppressor activity, and its knockdown is associated with more aggressive tumors as well as changes in cell shape, migration, and adhesion. This study shows that oncogenic microRNA, miR-21, represses RHOB expression by directly targeting the 3′ untranslated region. Loss of miR-21 is associated with an elevation of RHOB in hepatocellular carcinoma cell lines Huh-7 and HepG2 and in the metastatic breast cancer cell line MDA-MB-231. Using in vitro models of distinct stages of metastasis, we showed that loss of miR-21 also causes a reduction in migration, invasion, and cell elongation. The reduction in migration and cell elongation can be mimicked by overexpression of RHOB. Furthermore, changes in miR-21 expression lead to alterations in matrix metalloproteinase-9 activity. Therefore, we conclude that miR-21 promotes multiple components of the metastatic phenotype in vitro by regulating several important tumor suppressors, including RHOB. Mol Cancer Res; 8(5); 691–700. ©2010 AACR.

Transcriptional profiling implicates TGFβ/BMP and Notch signaling pathways in ductular differentiation of fetal murine hepatoblasts

Bile duct morphogenesis involves sequential induction of biliary specific gene expression, bilayer generation, cell proliferation, remodeling and apoptosis. HBC-3 cells are a model system to study differentiation of hepatoblasts along the hepatocytic or bile ductular lineage in vitro and in vivo. We used microarray to define molecular pathways during ductular differentiation in response to Matrigel. The temporal pattern of expression of marker genes induced was similar to that observed during bile duct formation in vivo. Notch, HNF1beta, Polycystic kidney disease 2, Bicaudal C 1 and beta-catenin were up regulated during the time course. Functional clustering analysis revealed significant up regulation of clusters of genes involved in extracellular matrix remodeling, ion transport, vacuoles, lytic vacuoles, pro-apoptotic and anti-apoptotic genes, transcription factors and negative regulators of the cell proliferation, while genes involved in the cell cycle were significantly down regulated. Notch signaling pathway was activated by treatment with Matrigel. In addition, TGFbeta/BMP signaling pathway members including the type I TGFbeta receptor and Smads 3, 4 and 5 were significantly up regulated, as were several TGFbeta/BMP responsive genes including Hey 1, a regulator of Notch pathway signaling. SMADS 3, 4 and 5 were present in the nuclear fraction of HBC-3 cells during ductular differentiation in vitro, but not during hepatocyte differentiation. SMAD 5 was preferentially expressed in hepatoblasts undergoing bile duct morphogenesis in the fetal liver, while the TGFbeta/BMP signaling antagonist chordin, was expressed throughout the liver suggesting a mechanism by which TGFbeta/BMP signaling is limited to hepatoblasts that contact portal mesenchyme in vivo.

New cell surface markers for murine fetal hepatic stem cells identified through high density complementary DNA microarrays

Isolation of hepatic stem cells from the adult liver (AL) has not yet been achieved due to the lack of specific cell surface markers. To identify new surface markers for hepatic stem cells, we analyzed differences in the gene expression profile of embryonic day (ED) 13.5 fetal liver stem/progenitor cells (FLSPC) versus AL by complementary DNA (cDNA) microarray technology. Using FLSPC purified to >90% by immunomagnetic selection for E‐cadherin and high density (27k) mouse cDNA microarrays, we identified 474 genes that are more strongly expressed in FLSPC (FLSPC‐up genes) and 818 genes that are more strongly expressed in AL (AL‐up genes). The most highly overrepresented gene ontology (GO) categories for FLSPC‐up genes are nucleus, cellular proliferation, and cell cycle control. AL‐up genes are overrepresented for genes in metabolic pathways for specific hepatic functions. We identified 24 FLSPC‐up gene surface markers and 69 AL‐up gene surface markers. Western blot studies confirmed the expression of the FLSPC‐up gene neighbor of Punc E11 (Nope) in fetal liver, but expression was not detectable in AL. Immunohistochemistry, confocal microscopy, and fluorescence‐activated cell sorting (FACS) analysis of fetal liver demonstrated that Nope is specifically expressed on the surface of FLSPC within the fetal liver. Conclusion: This is the first microarray study to analyze the specific gene expression profile of purified murine FLSPC. Our analysis identified 24 new/potential cell surface markers for murine fetal hepatic stem cells, of which Nope may be particularly useful in future studies to identify, characterize and isolate hepatic stem cells from the AL. (HEPATOLOGY 2007.)

Reactivation of the maternally imprinted IGF2 allele in TGFα induced hepatocellular carcinomas in mice

The Insulin like growth factor 2 (IGF2) gene is expressed in several types of tumors in humans and mice and has been implicated as an important growth factor in tumor progression. IGF2 expression in the TGFα transgenic mice was analysed in liver and tumors from animals which also contained one or two functional IGF2 alleles. In a two by two mating experiment using transgenic mice containing either a TGFα transgene or a IGF2 gene knockout, we have investigated whether IGF2 imprinting is reversed during hepatocarcinogenesis and the consequences of IGF2 expression for tumor growth. We observed that: (1) 100% of the hepatocellular carcinomas expressed IGF2 (2) the normally imprinted maternal allele is active in the tumors in which the paternal allele is knocked out and (3) all three of the murine IGF2 promoters upstream of the reactivated maternal alleles are transcriptionally active in tumors. We also observed that the total tumor burden of animals with two wild type IGF-2 alleles (paternal and maternal) was the same as the tumor burden in animals which contained only a single reactivated maternal allele. The 100% incidence of reactivation of the imprinted maternal allele suggests that IGF2 expression is selected during murine hepatocarcinogenesis and can substitute for the paternal allele when it is inactivated.

Contributions of hepadnavirus research to our understanding of hepatocarcinogenesis

Publisher Summary This chapter focuses on an experimental model that has provided specific insights into hepatocarcinogenesis in hepadnavirus carriers, which may be of general significance. The new transgenic models that utilize the HBsAg and the hepatitis B virus X (HBx) genes should now be exploited to further understand the molecular genetics of hepatocellular carcinoma (HCC). These models are directly relevant to HCC in humans because both the HBsAg and HBx genes are expressed in precancerous liver and HCCs from humans. These models have at their disposal the vast resources of the human and murine genome projects. The woodchuck hepatis virus (WHV) model provides many opportunities to further analyze molecular genetic changes that occur in a natural host–virus system that is also very relevant to the human disease. So far, work on these models has pointed to the myc family of protooncogenes and the IGF2 gene as important genes in the initiation of transformation and tumor growth and malignant progression, respectively. The effects of HBx on the PKC and Raf 1 signal transduction pathways may also lead to the identification of new genes of importance in hepatocarcinogenesis. When the individual genetic components (e.g., Ras, Raf) of the interactive signal transduction pathways are more fully understood, common mechanisms between HCC and other cancers would undoubtedly become more evident.

Knockdown of miR-23, miR-27 and miR-24 Alters Fetal Liver Development and Blocks Fibrosis in Mice.

MicroRNAs (miRNAs) regulate cell fate selection and cellular differentiation. miRNAs of the miR23b polycistron (miR-23b, miR-27b, and miR-24) target components of the TGF-β signaling pathway and affect murine bile ductular and hepatocyte cell fate selection in vitro. Here we show that miR-23b polycistron miRNAs directly target murine Smad4, which is required for TGF-β signaling. Injection of antagomirs against these miRNAs directly into E16.5 murine fetuses caused increased cytokeratin expression in sinusoids and primitive ductular elements throughout the parenchyma of newborn mice. Similar antagomir injection in newborn mice increased bile ductular differentiation in the liver periphery and reduced hepatocyte proliferation. Antagomir injection in newborn Alb/TGF-β1 transgenic mice that develop fibrosis inhibited the development of fibrosis, and injection of older mice caused the resolution of existing fibrosis. Furthermore, murine stellate cell activation, including ColA1 and ACTA2 expression, is regulated by miR-23b cluster miRNAs. In summary, knockdown of miR-23b cluster miRNAs in fetal and newborn liver promotes bile duct differentiation and can block or revert TGF-β-induced liver fibrosis that is dependent on stellate cell activation. These data may find practical application in the highly needed development of therapies for the treatment of fibrosis.

Clonal, Cultured, Murine Fetal Liver Hepatoblasts Maintain Liver Specification in Chimeric Mice

Recent studies have shown a pluripotential nature of stem cells that were previously thought to be committed to specific lineages. HBC‐3 cells are a clonal fetal murine hepatoblast cell line derived from an e9.5 murine embryo, and these cells can be induced to form hepatocytes and bile ducts in vitro and when transplanted into adult mouse livers. To determine whether HBC‐3 cells can exhibit a pluripotential phenotype, we created chimeric mice by injection of enhanced green fluorescent protein (EGFP)–marked HBC‐3 cells into wild‐type or dipeptidyl dipeptidase IV (DPPIV) knockout blastocysts. Genetically labeled HBC‐3 cells were identified by EGFP polymerase chain reaction (PCR) in all the major organs of many chimeric mice and visualized in chimeras as bright red DPPIV‐positive cells in the DPPIV knockout chimeric mice. Strikingly, the HBC‐3 cells maintained phenotypic and biochemical features of liver specification in every case in which they were identified in nonliver organs, such as brain, mesenchyme, and bone. In adult liver they were present as small foci of hepatocytes and bile ducts in the chimeras. Additional major histocompatibility complex (MHC) marker analysis and X and Y chromosome content analysis further demonstrated that HBC‐3 cells did not acquire the phenotype of the organs in which they resided and that they were not present because of fusion with host cells. Conclusion: In contrast to other stem cell types, these data demonstrate that cultured murine fetal liver stem cells appear to maintain their liver specification in the context of nonliver organs in chimeric mice. (HEPATOLOGY 2007.)