studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 457A
Inherited severe unconjugated hyperbilirubinemia is known as
Crigler-Najjar syndrome (CN). CN is characteristically caused
by deficiency of UDP-glucuronosyl transferase 1A1 (UGT1A1),
encoded by the UGT1A1 gene. This enzyme is responsible
for glucuronidation of bilirubin needed to excrete this toxic
compound via the bile. The severity of inherited unconjugated
hyperbilirubinemia depends on the residual UGT1A1 activity
and can be described as a spectrum from complete absence
(CN type 1) or some activity (CN type 2) to 30% of residual
activity as seen in the benign Gilbert syndrome. In CN
patients with residual UGT1A1 activity a reduction of serum
bilirubin levels can be achieved by phenobarbital administration
that results in transcriptional induction of UGT1A1 via
activation of the Constitutive Androstane Receptor (CAR). We
analyzed the UGT1A1 promoter sequence of a patient with
severe unconjugated hyperbilirubinemia suspected for CN type
2, but unresponsive to phenobarbital treatment and without
clarifying mutation in the UGT1A1 coding region. Besides an
extra TA repeat in the TATA box, we disclosed a homozygous
three nucleotide (3nt) insertion on position -85 to -83 of the
proximal promoter, corresponding with the HNF1α binding
site. We generated UGT1A1 promoters with all different mutations
and cloned them upstream of a luciferase reporter gene
to perform functional studies. The insertion of this 3nt in the
HNF1α binding site resulted in a >95% reduction of promoter
activity, independent of the extra TA-repeat in the TATA-box. In
addition, this insertion rendered the promoter unresponsive to
the induction by phenobarbital via CAR and rifampicin via the
Pregnane X Receptor (PXR), thereby explaining the intermediate
CN phenotype of this patient. In conclusion, disruption of the
HNF1α binding site in the promoter of a liver specific gene
such as UGT1A1 is for the first time demonstrated to have a
major impact on gene expression in man and may result in
severe liver disorder.
Disclosures:
Robert J. de Knegt - Advisory Committees or Review Panels: Roche, Norgine,
Janssen Cilag, AbbVie; Grant/Research Support: Roche, Janssen Cilag, BMS,
AbbVie; Speaking and Teaching: Gilead, Roche, Janssen Cilag, AbbVie
Ulrich Beuers - Consulting: Intercept via University of Amsterdam, Novartis via
University of Amsterdam; Grant/Research Support: Falk, Zambon; Speaking and
Teaching: Falk Foundation, Gilead, Roche, Shire
The following authors have nothing to disclose: Remco van Dijk, Isabel
Mayayo-Peralta, Sem J. Aronson, Anja A. Kattentidt-Mouravieva, Vincent van
der Mark, Nevin Oruç, Piter J. Bosma
493
MicroRNA-122 (miR-122) Regulates Polyploidy in the
Liver
Shu-hao Hsu 1 , Evan Delgado 1 , P. A. Otero 1 , Kolin Meehan 1 , Justin
B. Moroney 1 , Kun-Yu Teng 2 , Kalpana Ghoshal 2 , Andrew W.
Duncan 1 ; 1 Department of Pathology, McGowan Institute for Regenerative
Medicine, University of Pittsburgh, Pittsburgh, PA; 2 Department
of Pathology, Comprehensive Cancer Center, The Ohio State
University, Columbus, OH
A defining feature of the mammalian liver is polyploidy, a
numerical change in the entire complement of chromosomes.
Hepatocytes are either mononucleate or binucleate, and
ploidy is determined by the number of nuclei/cell, as well as
the ploidy of each nucleus. The first step of polyploidization
involves cell division with failed cytokinesis. Although polyploidy
is common, affecting ~90% of hepatocytes in mice and
50% in humans, the specialized role played by polyploid cells
in liver homeostasis and disease remains poorly understood.
The goal of this study was to identify novel signals that regulate
polyploidization, and we focused on microRNAs (miR-
NAs). First, to test whether miRNAs could regulate hepatic
polyploidy we examined livers from Dicer1 knockout mice,
which are devoid of mature miRNAs. Loss of miRNAs resulted
in a 3-fold reduction in binucleate hepatocytes, indicating that
miRNAs could indeed regulate polyploidization. Secondly, we
surveyed age-dependent expression of >500 miRNAs (NanoString)
in wild type mice and identified a subset of miRNAs,
including miR-122, differentially expressed at 2-3 weeks, a
period when extensive polyploidization occurs. Thirdly, we
examined Mir122 knockout mice and observed profound, lifelong
depletion of polyploid hepatocytes, proving that miR-122
is required for complete hepatic polyploidization. Next, we
identified direct targets of miR-122, Cux1, Iqgap1, Mapre1,
Nedd4l and Slc25a34, that regulate cytokinesis. Inhibition of
each target induced cytokinesis failure and promoted hepatic
binucleation. Finally, to validate the relevance of these targets
to liver disease, we examined expression in a subset of human
hepatocellular carcinomas (HCC) with reduced miR-122. Consistent
with the mouse data, target expression was inversely
proportional to miR-122. In summary, our data suggest a novel
regulatory role for miR-122 in liver polyploidization. Moreover,
differential expression of miR-122 targets in HCC provides new
insights into miR-122-mediated tumorigenesis. These studies
will serve as the foundation for future work investigating miR-
122 and polyploidy in lipid metabolism, viral infection, tumorigenesis
and regeneration.
Disclosures:
The following authors have nothing to disclose: Shu-hao Hsu, Evan Delgado, P.
A. Otero, Kolin Meehan, Justin B. Moroney, Kun-Yu Teng, Kalpana Ghoshal,
Andrew W. Duncan
494
Overexpression Screening to Discover Gene-Gene Interactions
that Drive Hepatocellular Carcinoma
Monica Teta-Bissett 1 , Kirk J. Wangensteen 1,2 , Klaus H. Kaestner 1 ;
1 Genetics, University of Pennsylvania, Philadelphia, PA; 2 Gastroenterology,
University of Pennsylvania, Philadelphia, PA
Hepatocellular carcinoma (HCC) is the third leading cause
of cancer death worldwide, and one of the top ten in the
United States according to the Global Cancer Statistics and
the US CDC, respectively. Sorafenib, a small molecule targeting
receptor tyrosine kinases, is the only drug approved for
treatment of advanced HCC in the United States. In order to
determine the relative contribution of HCC candidate genes in
HCC formation and growth, we developed an overexpression
model to screen multiple gene combinations simultaneously
during liver tumor formation. A pooled plasmid library of 34
constructs, each designed to express the FAH gene plus a different
cancer-related gene of interest, was delivered into the
liver of Fah-null mice by hydrodynamic tail vein injection. The
constructs allowed for constitutive expression of FAH, rescuing
the tyrosinemia of Fah-null mice, and creating repopulation
nodules overexpressing random combinations of the genes
within the plasmids. The mouse livers were riddled with tumors
at four and six months following library injection. More than
20 unique tumors were dissected and the tumor phenotypes
were characterized using hematoxylin and eosin staining and
immunohistochemistry for Osteopontin (Opn) on serial sections.
DNA was extracted from unique tumors and, by high-throughput
sequencing of unique 5-nucleotide barcodes attached to
each of the expression plasmids, the profiles of the exogenous
DNA constructs was compiled. We found that c-myc
was linked to all of the tumors that were tested, indicating
that it was a consistent driver of tumor growth in this system.
TGFα, Foxa3, Akt, Met and Nr1h3 were among other factors
detected to drive tumor growth. Interestingly, the combination