studies

458A AASLD ABSTRACTS HEPATOLOGY, October, 2015
of four genes, c-myc, AKT, MET and Nr1h3, correlated with a
subset of Opn-positive HCC tumors. This innovative paradigm
allows for prospective testing of millions of combinations of
overexpressed factors to determine the interaction of known
and suspected liver oncogenes and tumor suppressors in a
systematic fashion.
Disclosures:
The following authors have nothing to disclose: Monica Teta-Bissett, Kirk J. Wangensteen,
Klaus H. Kaestner
495
Peretinoin, an Acyclic Retinoid, Inhibits Hepatocarcinogenesis
through Suppression of Sphingosine Kinase 1
Expression In vitro and In vivo
Masaya Funaki 1 , Tetsuro Shimakami 1 , Tsuguhito Ota 2 , Masao
Honda 1 , Takayoshi Shirasaki 1 , Shuichi Kaneko 1 ; 1 Disease Control
and Homeostasis, Kanazawa university, Kanazawa, Japan;
2 Brain/Liver Interface Medicine Research Center, Kanazawa University,
Kanazawa, Japan
[Objective] A sphingolipid, sphingosine-1-phospate (S1P) is
a potent bioactive lipid which can regulate carcinogenesis
and cancer progression. Both sphingosine kinase 1 (SPHK1)
and SPHK2 are the essential kinases that produce S1P. Therefore,
SPHK can be a therapeutic target by crucially regulating
sphingolipid metabolism in several kinds of cancer. Peretinoin
(NIK-333), an acyclic retinoid, was reported to inhibit the post
therapeutic recurrence of hepatocellular carcinoma (HCC) in
patients with chronic hepatitis C. However, the mechanism of
its inhibitory effects against recurrent HCC remains unclear.
We hypothesized that peretinoin could prevent hepatocarcinogenesis
by modifying a SPHK-S1P axis. In the present study,
we assessed the effect of peretinoin on SPHK activation and
development of liver cancer in vivo and in vitro. [Method] We
examined the effect of peretinoin on the expression and the
enzymatic activity of SPHK1 in a human hepatoma cell line,
Huh-7 cells. Next, using a liver cancer mice model with atherogenic
high fat (AHF) diet-induced NASH we administrated
an AHF diet with and without peretinoin (0.03%) for 48 weeks
and examined the effect of peretinoin on hepatocarcinogenesis
and expression of SPHK1. Finally, we clarified the effect of
SPHK1 on hepatocarcinogenesis induced by Diethylnitrosoamine
(DEN) using SPHK1 knockout mice. [Results] [In vitro] The
treatment with peretinoin (10 to 40 μM) reduced the mRNA
and protein expression of SPHK1 in Huh-7 cells in a time- and
dose- dependent manner. However, peretinoin did not change
the expression of SPHK2 in Huh-7 cells. Furthermore, peretinoin
markedly reduced the enzymatic activity of SPHK1 assessed by
in vitro 32P labeled SPHK activity assay. Next, we performed
reporter gene assays by using constructs containing the SPHK1
promoter region. Peretinoin reduced the promoter activity of
SPHK1, and overexpression of SP1 restored promoter activity.
In addition, three deletion constructs with and without SP1
binding sites were created. The deletion constructs without SP1
binding sites abolished the promoter activity. Interestingly, we
previously reported that peretinoin suppresses the expression of
SP1. Collectively, peretinoin reduced the expression of SPHK1
mRNA via SP1. [In vivo] At week 48 week of treatment, peretinoin
down-regulated SPHK1 mRNA expression and prevented
AHF diet-induced hepatocarcinogenesis. The SPHK1 knockout
mice developed significantly less numbers of HCC induced by
DEN than did wild-type mice. [Conclusion] Our data indicate
that peretinoin prevents hepatocarcinogenesis at least partly
through reduction of expression and activation of SPHK1.
Disclosures:
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co.,
Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc,
Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc,
Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer
Japan, Eli lilly Japan
The following authors have nothing to disclose: Masaya Funaki, Tetsuro Shimakami,
Tsuguhito Ota, Masao Honda, Takayoshi Shirasaki
496
Therapeutic overexpression of miR-122 protects mice
from chronic alcoholic liver injury through regulation of
hypoxia-inducible factor-1α
Abhishek Satishchandran 1 , Aditya Ambade 2 , Banishree Saha 2 ,
Benedek Gyongyosi 2 , Patrick Lowe 2 , Nicita Mehta 2 , James V. Zatsiorsky
2 , Arvin Iracheta-Vellve 2 , Jia Li 3,4 , Donna Catalano 2 , Karen
Kodys 2 , Li Zhong 3,4 , Jun Xie 3,5 , Shashi Bala 2 , Guangping Gao 3,5 ,
Gyongyi Szabo 2 ; 1 University of Massachusetts Medical School,
Worcester, MA; 2 Medicine, UMass Medical School, Worcester,
MA; 3 Gene Therapy Center, UMass Medical School, Worcester,
MA; 4 Pediatrics, UMass Medical School, Worcester, MA; 5 Microbiology
and Physiology Systems, UMass Medical School, Worcester,
MA
Introduction: miR-122 regulates essential pathways in alcoholic
liver disease (ALD). We have observed that chronic alcohol
administration reduces miR-122 levels in murine hepatocytes.
Given its essential role in hepatic homeostasis, we hypothesized
that loss of miR-122 contributes to ALD and can be a therapeutic
target. In this study, our goals were to asses: first, the
role of miR-122 in the pathogenesis of ALD and the therapeutic
potential of miR-122 restoration; second, the role of Hypoxia
Inducible Factor 1-α (HIF-1α), a putative miR-122 target; and
third, the mechanisms by which alcohol downregulates miR-
122. Methods: Wild-type 8-week-old, C57Bl/6 or hepatocyte
specific HIF-1α knockout (hepHIFKO) mice were injected intravenously
with hepatocyte-tropic AAV (AAV8) containing antimiR-122
TuD (TuD), or scrambled (Scr) vector. After 14 days,
mice were started on a Lieber-DeCarli (LDC) alcohol diet (Et) or
calorie-matched control diet (PF) for 4 weeks. Some WT mice
were treated with AAV8 pri-miR-122 (OX) on day 7 of the
LDC diet. Results: Anti-miR-122 TuD treatment alone resulted in
significant increases in liver injury (ALT), steatosis, inflammation
(TNFα, IL1-β, MCP-1), and fibrosis (Acta2, pro-col1α) in PF
mice compared to Scr-treated controls. The co-administration of
miR-122 TuD and alcohol resulted in a synergistic effect, further
increasing liver injury, inflammation and fibrosis. Restoration of
miR-122 in hepatocytes of alcohol-fed mice by treatment with
scAAV8 OX resulted in a significant improvement in serum ALT,
inflammation and fibrosis suggesting a protective role for miR-
122 in ALD. The hepatic expression and DNA-binding activity
of HIF-1α, a miR-122 target, was increased in TuD+PF mice
equivalent to that of alcohol feeding alone. HIF1-α activity was
the highest in TuD+Et mice compared to all other groups. Hep-
HIF deficiency protected mice from liver damage, inflammation
and fibrosis induced either by alcohol or miR-122 knockdown.
We discovered that alcohol specifically inhibits expression
of the precursor miR-122 transcript (pri-miR-122) at the transcriptional
level. Furthermore, grainyhead-like 2 (GRHL2), an
inhibitor of miR-122 transcription, was significantly increased
in hepatocytes of alcohol fed mice compared to controls. Conclusions:
Our findings dissect a novel mechanistic regulatory
axis of miR-122 and demonstrate the therapeutic potential of
miR-122 restoration in ALD. miR-122 inhibition alone mimics
and together with alcohol augments the steatosis, inflammation
and early fibrosis seen in ALD through its downstream target,