studies

1172A AASLD ABSTRACTS HEPATOLOGY, October, 2015
ulation of over-expressed GPC3. HCC may also have a growth
advantage over normal hepatocytes in HCV infection by missing
CD81, thus being able to undergo clonal expansion, facilitating
HCC early development and growth. Persistent HCV
infection strengthens this expansion process and contributes
carcinogenesis by E2 protein interacting with CD81.
Disclosures:
George K. Michalopoulos - Consulting: Vital Therapies
The following authors have nothing to disclose: Yuhua Xue, Kelly Koral, William
C. Bowen, Anne Orr, Meagan Haynes, Wendy M. Mars
gene that acts as a driving force for the amplification at 3q26
in HCC and that the oncoprotein EVI1 suppresses TGF-β-mediated
growth inhibition of HCC cells.
Disclosures:
Yoshito Itoh - Grant/Research Support: MSD KK, Bristol-Meyers Squibb, Dainippon
Sumitomo Pharm. Co., Ltd., Otsuka Pharmaceutical Co., Chugai Pharm
Co., Ltd, Mitsubish iTanabe Pharm. Co.,Ltd., Daiichi Sankyo Pharm. Co.,Ltd.,
Takeda Pharm. Co.,Ltd., AstraZeneca K.K.:, Eisai Co.,Pharm.Ltd, FUJIFILM Medical
Co.,Ltd., Gelaed Sciences Co., GlaxoSmithKline
The following authors have nothing to disclose: Kohichiroh Yasui, Atsushi
Umemura, Taichiro Nishikawa, Kanji Yamaguchi, Michihisa Moriguchi, Yoshio
Sumida, Hironori Mitsuyoshi, Shinji Tanaka, Shigeki Arii
1980
EVI1 is a target for gene amplification at 3q26 and
suppresses growth inhibition by transforming growth
factor-β in hepatocellular carcinoma
Kohichiroh Yasui 1 , Atsushi Umemura 1 , Taichiro Nishikawa 1 , Kanji
Yamaguchi 1 , Michihisa Moriguchi 1 , Yoshio Sumida 1 , Hironori Mitsuyoshi
1 , Shinji Tanaka 2 , Shigeki Arii 3,4 , Yoshito Itoh 1 ; 1 Department
of Molecular Gastroenterology and Hepatology, Kyoto
Prefectural University of Medicine, Kyoto, Japan; 2 Department
of Molecular Oncology, Tokyo Medical and Dental University,
Tokyo, Japan; 3 Hamamatsu Rosai Hospital, Hamamatsu, Japan;
4 Department of Hepato-Biliary-Pancreatic Surgery, Tokyo Medical
and Dental University, Tokyo, Japan
Background: Amplification of DNA in certain regions of chromosomes
plays a crucial role in the development and progression
of human malignancies, specifically when proto-oncogenic
target genes within those amplicons are overexpressed. A common
criterion for designation of a gene as a putative target
of amplification is that gene amplification leads to its overexpression.
The EVI1 (ecotropic viral integration site 1) gene
codes for a zinc finger transcriptional factor that is involved in
leukemic transformation of hematopoietic cells. EVI1 is one of
the most aggressive oncogenes associated with myeloid leukemia.
EVI1 has been reported to suppress transforming growth
factor (TGF)-β signaling by inhibiting Smad3 in leukemic cells.
However, little is known about its relevance for hepatocellular
carcinoma (HCC). Aim: To identify a novel gene that acts as a
driving force for gene amplification in HCC and is involved in
the development and progression of HCC. Methods: We investigated
DNA copy number aberrations in 20 HCC cell lines
using a high-density oligonucleotide microarray (GeneChip
Mapping 100K and 250K arrays, Affymetrix). DNA copy
number and expression of EVI1 were determined by using fluorescence
in situ hybridization (FISH) and quantitative PCR in
HCC cell lines and primary HCCs. Knockdown experiments of
EVI1 were performed. Results: We found that a novel amplification
at the chromosomal region 3q26 occurs in the HCC cell
line, JHH-1, and that MDS1 (myelodysplastic syndrome 1) and
EVI1 complex locus (MECOM ), which lies within the 3q26
region, was amplified. Quantitative PCR analysis of the three
transcripts transcribed from MECOM indicated that only EVI1,
but not the fusion transcript MDS1-EVI1 or MDS1, was overexpressed
in JHH-1 cells and was significantly up-regulated in 22
(61%) of 36 primary HCC tumors when compared with their
non-tumorous counterparts. A copy number gain of EVI1 was
observed in 24 (36%) of 66 primary HCC tumors. High EVI1
expression was significantly associated with larger tumor size
and higher level of des-γ-carboxy prothrombin (DCP), a tumor
marker for HCC. Knockdown of EVI1 resulted in increased
induction of the cyclin-dependent kinase inhibitor p15 INK4B by
TGF-β and decreased expression of c-Myc, cyclin D1, and
phosphorylated Rb in TGF-β-treated cells. Consequently, knockdown
of EVI1 led to reduced DNA synthesis and cell viability.
Conclusions: Our results suggest that EVI1 is a probable target
1981
Hyperinsulinemia, not hyperglycemia accelerates the
progression of hepatocellular carcinoma in neonatal
streptozotocin induced mouse model
Koichi Tsuneyama, Ryosuke Bessho, Masahiro Miki, Hayato Baba,
Tetsuyuki Takahashi, Hirohisa Ogawa, Hisanori Uehara; Department
of Pathology and Laboratory Medicine, Institute of Biomedical
Sciences, Tokushima University Graduate School, Tokushima,
Japan
Purpose: Diabetes mellitus (DM) is a well-known risk factor
for hepatocellular carcinoma (HCC); however, the underlying
mechanisms are not well understood. We have previously
reported that neonatal streptozotocin (STZ) treatment in DIAR
mice (DIAR-nSTZ) causes type 1 diabetes and subsequent HCC
without special diet. In the following study, to examine the
relation between hyperglycemia and HCC, we normalized the
blood glucose level by applying excess insulin in DIAR-nSTZ
mice. Although diabetic features were completely improved,
frequency of HCC did not improve in 12 weeks of age. We
hypothesized that hyperinsulinemia, not hyperglycemia have
a close association to carcinogenesis in this model. To clarify
this hypothesis, we examined neonatal STZ treatment in IV CS
mice, which showed quick normalization of blood glucose level
after STZ treatment by rapid regeneration of islet cells. Methods:
Newborn male IV-CS mice were prepared at institute of
animal reproduction (Ibaraki, Japan). STZ (60 mg/g) was subcutaneously
injected into the IV CS-STZ treated group (IV-CSnSTZ
mice, N13), whereas physiologic solution was injected
into the control group (IV-CS-control mice, N=13) at 1.5 days
after birth. All mice were fed a normal diet, and physiologically
and histopathologically assessed at 8 (N=2), 12 (N=3) and
16 (N-8) weeks of age. Blood was taken once in month and
measured glucose and insulin level in all mice. Results: Blood
glucose level of IV CS-nSTZ mice showed slightly increase till
5 week of age, but it was normalized after 6 weeks of age. In
contrast, blood insulin level showed higher titer after 6 weeks
of age. Even at 8 weeks of age, one out of two mice showed
hepatic nodule in a diameter of 2mm. Histopathologically, it
showed mild structural and nuclear atypia showing dysplastic
nodule. At 12 weeks of age, one out of three mice contained
2 hepatic nodules of dysplastic nodule. At 16 weeks of age,
six out of eight mice showed one or more hepatic nodules.
Histologically, well differentiated hepatocellular carcinoma
as well as dysplastic nodules were observed. In contrast, no
hepatic nodules were recognized in IV CS-control mice. Conclusions:
IV CS mice showed hepatocarcinogenesis after neonatal
STZ treatment. Differently from other strains including ddY
and DIAR, blood glucose level did not show marked increase
after STZ treatment, and it was normalized after 6 weeks of
age. However, frequency of HCC showed similar tendency
of DIAR-nSTZ mice. In addition to our previous data of insulin
treatment of DIAR-nSTZ mice, we strongly hypothesized that