studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 543A
673
Effects of Targeting Wnt/β-Catenin and RAS/RAF/
MAPK Pathway on Hepatocellular Carcinoma Cell
Growth and Metabolism: Potential for a New Combination
Therapy
Lilia Turcios 1 , Valery Vilchez 1 , David Butterfield 2 , Mihail I. Mitov 3 ,
Francesc Marti 1 , Roberto Gedaly 1 ; 1 Surgery, Univ of Kentucky,
Lexington, KY; 2 Chemistry, University of Kentucky, Lexington, KY;
3 Markey Cancer Center - Core Support, University of Kentucky,
Lexington, KY
Background: Treatment of advanced hepatocellular carcinoma
(HCC) remains a challenge. We aim to evaluate the impact
of a β-catenin inhibitor, FH535, alone or in combination with
a RAS/RAF/MAPK inhibitor, sorafenib, in liver cancer cells.
Methods: Proliferation of liver cancer cell lines (Huh7, PLC,
Hep3B) with or without addition of FH535 (10mM, 5mM,
2.5mM) and sorafenib (2mM, 1 mM) alone or in combination
was assayed by 3 H-thymidine incorporation at different time
points (12h, 24h and 48h). The effect of drug treatment in mitochondrial
function was determined by monitoring changes in
mitochondrial transmembrane potential (ΔΨm) and mitochondrial
respiratory activity. ΔΨm was assessed by tetramethylrhodamine
ethyl ester (TMRE)-labeling and analyzed by flow
cytometry. Mitochondrial respiration (OXPHOS) and glycolysis
were measured by real-time analysis of oxygen consumption
(OCR) and extra-cellular acidification (ECAR) rates, respectively,
in a XF96 Flux Analyzer. Expression of β-catenin and
apoptosis-related proteins were analyzed by Western Blot.
Results: FH535 and sorafenib inhibited HCC cell proliferation
in all three different cell lines tested (Huh7, PLC and Hep3B).
We observed a significant dose-response inhibition with single
treatments, although the effect of combined drugs was
more potent. Our results also indicated the decreased levels of
ΔΨm in cells exposed to the simultaneous exposure to FH535
and sorafenib, which may reflect the efficient mitochondrial
drug-targeting of the combined treatment. Mitochondrial dysfunction
was confirmed by lower oxygen consumption rates
and increased levels of apoptosis in cells treated with FH535
plus sorafenib. Similar ECAR levels in treated and non-treated
cells ruled out a broad cellular metabolic failure and rather
confirmed the predominant mitochondrial effect of the drugs.
Conclusions: The combination of FH535 and sorafenib inhibits
HCC cell proliferation. Mechanistically, our results suggest that
the combined therapeutic targeting of β-catenin and RAS/RAF/
MAPK pathways may alter tumor cell growth by promoting
mitochondrial dysfunction.
Disclosures:
The following authors have nothing to disclose: Lilia Turcios, Valery Vilchez,
David Butterfield, Mihail I. Mitov, Francesc Marti, Roberto Gedaly
674
RA and butyrate induce liver and colon cancer cell
apoptosis through miR-22 silencing of histone deacetylases
Ying Hu, Hui-Xin Liu, Yu-Jui Yvonne Wan; Department of Medical
Pathology and Laboratory Medicine, University of California,
Davis Health Systems, Sacramento, CA
Due to the signicant tumor suppressive role of microRNA-22
(miR-22), it is important to establish the mechanism by which
miR-22 expression is regulated. Our published data showed
that bile acid-activated farnesoid x receptor (FXR) induces miR-
22. In this report, we showed that all-trans retinoic acid (RA)
and butyrate, normally present in digestive tract, also up-regulate
miR-22. RA is a biologically active metabolite of vitamin
A and a natural agonist for tumor suppressor retinoic acid
receptor β (RARβ). Butyrate is a short chain fatty acid and
histone deacetylase inhibitor (HDACi), produced by fiber-fed
Gram-positive bacteria. Both agents can induce apoptosis in
cancer cells. We tested the hypothesis that RA and butyrate
act through miR-22 to inhibit HDACs and induce apoptosis in
liver and colon cancer cells. Our data showed that RA/butyrate
treatment synergistically promoted apoptosis and potently
induced RARβ expression compared to single compound treatment
in liver Huh7 and colon HCT116 cancer cells. miR-22
induction by RA and butyrate was transcriptionally regulated
by RARβ/RXRα and FXR/RXRα direct binding to a DR-5 motif
located at -1568 to -1585 bp upstream of miR-22 based on
transient transfection assays. Using 3’UTR luciferase assay,
histone deacetylase 1 (HDAC1) was uncovered as a novel
miR-22 target. Furthermore, miR-22 mimics also reduced the
protein levels of HDAC4 and SIRT1, which are identified miR-
22 targets in rat cardiomyocytes. Consistently, RA and butyrate
reduced HDAC1, HDAC4, and SIRT1 protein levels in HCT116
cells. In addition, the reduction in protein deacetylases was
accompanied by increased histone acetylation in the RARβ
regulatory region harboring a RA response element (DR-5).
Histone modification of the RARβ gene could account for
enhanced RARβ expression by RA/butyrate treatment. Moreover,
miR-22 inhibitors abolished reduction of protein deacetylase
levels and prevented apoptosis in HCT116 and Huh7 cells
induced by RA/butyrate. Conclusion: RA and butyrate induce
miR-22 to promote apoptosis of gastrointestinal cancer cells.
miR-22 inhibition of HDACs is a novel pathway to explain the
anti-carcinogenic effect of RARβ and FXR.
Disclosures:
The following authors have nothing to disclose: Ying Hu, Hui-Xin Liu, Yu-Jui
Yvonne Wan
675
IRAKM-Mincle axis contributes to alcohol-induced liver
injury via inflammasome activation
Hao Zhou 1 , Laura E. Nagy 2 , Xiaoxia Li 1 ; 1 Immunology, Lerner
Research Institute, Cleveland Clinic Foundation, Cleveland, OH;
2 Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation,
Cleveland, OH
Alcohol-induced liver injury is driven by hepatocyte necrosis
and increased endotoxin translocation into the hepatic portal
system, which together trigger chronic inflammatory liver
damage. In this study, we found that mice deficient in IRAKM,
a proximal Toll-like receptor pathway molecule, are protected
from alcohol-induced liver injury. In response to low doses
of LPS, which better reflect physiologically relevant levels of
endotoxemia, IRAKM mediates the up-regulation of Mincle, a
receptor for danger signals released by damaged cells. Biochemical
analysis revealed that low-dose LPS preferentially
induces the formation of an IRAKM Myddosome, which in turn
promotes MEKK3-dependent NFκB activation. Mincle-deficient
mice are also protected from alcohol-induced liver injury. Ex
vivo studies demonstrated that both IRAKM and Mincle are
required for inflammasome activation by the endogenous Mincle
ligand SAP130, which is a danger signal release by damaged
hepatocytes. Taken together, this study reveals a novel
IRAKM-Mincle axis that critically mediates the pathogenesis of
alcohol-induced liver disease through inflammasome activation.
Disclosures:
The following authors have nothing to disclose: Hao Zhou, Laura E. Nagy, Xiaoxia
Li