studies

1162A AASLD ABSTRACTS HEPATOLOGY, October, 2015
1958
Chronic alcohol accelerates early hepatobiliary cancer
by increasing inflammation, stemness, EMT and miR-
122 mediated HIF-1α activation
Aditya Ambade, Abhishek Satishchandran, Gyongyi Szabo; Medicine,
UMass Medical School, Worcester, MA
Background / Aims: Chronic alcohol use is a major risk factor
in the development of hepatocellular carcinoma [HCC].
Inflammation contributes to alcoholic liver disease, particularly
in alcoholic hepatitis, and promotes HCC development. Other
molecular events leading to HCC include proliferation of tumor
stem cells, epithelial-mesenchymal transition (EMT) and altered
microRNA expression in the liver. We hypothesized that the
pro-inflammatory microenvironment in alcoholic hepatitis accelerates
liver tumor development after pre-exposure to a chemical
carcinogen. Methods: Four week old male C57BL/6 mice
were injected intraperitoneally with N, N diethyl nitrosamine
(DEN), 75 mg/kg/week for 3 consecutive weeks followed by
100 mg/kg/week for 3 additional weeks. At age 8 weeks,
mice were divided into alcohol (4% Lieber-DeCarli alcohol
diet) or calorie matched control diet (pair-fed group). After 6
weeks of alcohol feeding mice were sacrificed; blood, liver
tissues were collected for analysis of cytokines, tumor markers
and miR-122. Cyclin D1, p53, vimentin and sonic hedgehog
proteins were evaluated by western blots. HIF-1a activity was
assayed by EMSA. Results: Alcohol-fed DEN-injected mice
had greater liver damage (ALT) and significantly increased
liver-to-body weight ratio compared to pair-fed DEN-injected
mice. Alcohol feeding resulted in steatohepatitis indicated by
increased pro-inflammatory cytokines TNFa, IL-6, MCP-1, IL-17
and fibrotic genes a-SMA, procollagen1a1 and TGFb, that
were significantly higher in alcohol plus DEN mice compared
to other groups. MRI and liver histology of alcohol plus DEN
mice revealed hepatobiliary cysts, early hepatic neoplasia and
increased AFP. No tumors were found in other groups. Proliferation
makers (PCNA, cyclin D1, p53), EMT markers (vimentin,
sonic hedgehog) and cancer stem cell markers (CD133
and nanog) were significantly upregulated in livers of alcohol-fed
DEN-injected mice compared to pair-fed DEN-injected
and alcohol-fed saline-injected mice. Immunostaining for AFP,
PCNA, cytokeratin7 and 19 showed increased number of
stained cells in alcohol-fed DEN-injected mice compared to
all other groups indicating proliferation of hepatobiliary progenitors.
In livers with tumors from alcohol plus DEN mice, we
found significant reduction of miR-122 with upregulation of
miR-122 target gene, HIF-1α. Increased HIF-1a DNA binding
activity and upregulation of its target, VEGFR1, was found in
livers of alcohol plus DEN mice compared to all other groups.
Conclusion: Our findings establish a new model of early HCC
and demonstrate that alcoholic hepatitis accelerates hepatobiliary
tumor development with molecular features of HCC.
Disclosures:
The following authors have nothing to disclose: Aditya Ambade, Abhishek Satishchandran,
Gyongyi Szabo
1959
Variations of the host genome and interaction of hepatitis
B viral X protein associated with hepatocarcinogenesis
Hiroko Nagata 1 , Yasuhiro Itsui 1,2 , Fukiko Kawai-Kitahata 1 , Shun
Kaneko 1 , Miyako Murakawa 1,2 , Sayuri Nitta 1 , Mina Nakagawa 1 ,
Seishin Azuma 1 , Sei Kakinuma 1 , Yasuhiro Asahina 1 ; 1 Department
of Gastroenterology and Hepatology, Tokyo Medical and Dental
University, Tokyo, Japan; 2 Department of Medical Education
Research and Development, Tokyo Medical and Dental University,
Tokyo, Japan
Background and Aims: Association between variation of the
host genome and viral proteins responsible for hepatocarcinogenesis
is unclear. To clarify this, we comprehensively analyzed
variations of the host genome in patients with HCC using
a next-generation sequencer, and investigated the association
between identified host genome variation and HBx protein.
Methods: [1] We performed deep sequenceing in 104 pairs of
HCC and non-tumor samples targeting 54 cancer-related genes
containing 2820 mutational hotspots. [2] To assess the effect of
TERT promoter variations (C228T, C250T), which were identified
as most frequent mutations in our samples, on its transcriptional
activity, reporter assays using TERT promoter-luciferase
plasmids with or without these mutations were performed.
[3] To assess the association between TERT promoter activity
and HBx protein, reporter assays were performed 48 hours
after co-transfection of plasmid expressing HBx protein and
TERT promoter-luciferase plasmids in HepG2 cells and 293T
cells. [4] Moreover, effects of transcriptional factor Sp1 and
c-myc on TERT promoter activity were analyzed by additional
co-transfection using plasmid expressing these transcriptional
factors. Results: [1] We detected somatic mutations in 9 genes
out of 93 samples: TERT, TP53, CTNNB1, PTEN, CDKN2A,
HRAS, PIK3CA, STK11, GNAS and NFE2L2. Among them,
TERT promoter mutations were most frequently found (65% of
the patients) in overall. However, these mutations were significantly
less frequent in HBV-related HCC, while integration of
HBx gene into TERT region was frequently found, which was
mutually exclusive to TERT promoter mutations. [2] The TERT
mutations (C228T, C250T) significantly upregulated the promoter
activity in comparison with wild-type prompter in HepG2
and 293T cell (181±2.2%, 255±5.2%). [3] Expression of HBx
protein resulted in further activation of TERT promoter activity
in both of TERT-mutant and wild type (714±3.1%, 628±2.1%),
and effect of HBx protein on TERT promoter activation was
more remarkable in the TERT-wild promoter than the TERT-mutant
one (560%, 435%). [4] The promoter activity was elevated
by Sp1 or c-myc, which are TERT promoter-binding factors.
Conclusions: TERT promoter mutations were most frequently
detected in non-HBV related HCC, but rare in HBV-related
HCC, in which integration of HBx into TERT region were frequently
and exclusively found. Upregulation of TERT promoter
activity by either TERT promoter mutations or interaction of HBx
protein with TERT promoter via Sp1 and c-myc may associate
with hepatocarcinogenesis or tumor progression.
Disclosures:
Sei Kakinuma - Grant/Research Support: The Japanese Society of Gastroenterology,
MSD Co., Ltd.
The following authors have nothing to disclose: Hiroko Nagata, Yasuhiro Itsui,
Fukiko Kawai-Kitahata, Shun Kaneko, Miyako Murakawa, Sayuri Nitta, Mina
Nakagawa, Seishin Azuma, Yasuhiro Asahina