studies

1178A AASLD ABSTRACTS HEPATOLOGY, October, 2015
dense collagen-enriched fibrous stroma. In order to further our
understanding of the biological relationships mediating the desmoplastic
reaction in ICC, we employed a novel rat 3-D culture
model to investigate fibrogenic promoting interactions between
cholangiocarcinoma cells (BDEsp-TDE cc
cells) and α-SMA+CAFs
(BDEsp-TDF sm
cells) when co-cultured in a dilute collagen-type I
gel matrix. Both BDEsp-TDE cc
and BDEsp-TDF sm
cells were established
from a desmoplastic rat cholangiocarcinoma from liver.
Compared with monoculture controls, co-culturing BDEsp-TDE cc
cholangiocarcinoma cells constitutively expressing TGF-β1 with
BDEsp-TDF sm
CAFs provoked an intense desmoplastic-like reaction
within the gel culture matrix, characterized by a prominent
deposition of dense collagen fibers, together with a significant
increase in the numbers of BDEsp-TDF sm
CAFs accumulated
within the gel matrix. This reaction was greatly suppressed by
the TGF-β receptor 1 kinase inhibitor LY2157299. Similarly,
treatment of BDEsp-TDF sm
cells cultured alone within 3-D gel cultures
with TGF-β1 significantly increased the numbers of these
SMA+ CAFs within the gel matrix (Figure), which correlated
with an overproduction of dense collagen fibers, and which
was also inhibited by >70% by LY2157299. These results
strongly suggest that paracrine TGF-β signaling plays a major
role in generating the desmoplastic reaction in ICC and that
targeting this pathway may have important implications for
stromal depletion therapy for desmoplastic ICC. Supported by
NIH R01 CA 083650.
TGF-β1-induced enhancement of BDEsp-TDF sm
CAF cell accumulation
within 3-D gel monoculture compared with vehicle control
cultures (VC).
associated with oxidation of lipids and formation of oxysterols.
Oxysterols are allosteric regulators of Smoothened (Smo), the
receptor of the Hedgehog (Hh) signaling pathway, which is
activated in cholangiocarcinoma. The step wise process of
malignant transformation of biliary epithelial cells is poorly
described. Purpose: We aimed to interrogate malignant transformation
of the biliary epithelium upon stimulation of the Hh
pathway by oxysterols. Methods: The human non-malignant
(H69 and NHC) and primary PSC patient-derived cholangiocyte
cell lines were employed for this study. Upon treatment
with oxysterols [7-keto-27-hydrocholesterol and 20(S)-hydroxycholesterol],
cholangiocytes were analyzed for Hh pathway
stimulation with Smo translocation to the plasma membrane
and cilia by immunofluorescence, migration in modified Boyden
chambers, and colony-formation assay in a soft-agar. A
miRNA array was conducted to identify miRNAs that were
induced in cholangiocytes in an oxysterol- and Smo-dependent
manner. miRNA targets were analyzed in silico. Results: Cholangiocytes
expressed Hh pathway components at a baseline
(Gli2, Smo). Smo translocation to the plasma membrane and
cilia were observed in the cells treated with oxysterols (10mM
for 16 hours) but not in the vehicle treated cells. Stimulation
with oxysterols (10 mM for 7 hours) lead to a 1.3 – 2 (p <
0.01) fold increase in cholangiocyte migration. Oxysterols promoted
anchorage-independent growth evidenced by formation
of colony-like structures by non-malignant cholangiocytes in a
soft-agar over the 6 week period. miR-1246 was upregulated
(logFC = 2.7) in H69 cells upon stimulation with 7-keto-27-hydrocholesterol
and was inhibited (logFC = -2.2) with Vismodegib,
direct-inhibitor of Smo, pre-treatment. The Hh pathway
inhibitory receptor Patched-1, Axin2, and GSK-3β, all of each
are involved in Hh signaling and carcinogenesis, were found
to be a target for miR-1246 in silico. Conclusions: Products of
lipid oxidation, oxysterols, promote malignant transformation
of cholangiocytes in the Hh pathway-dependent manner. The
oxysterol-Smo axis upregulation of miR-1246 might provide a
positive feedback mechanism in this transformation.
Disclosures:
Gregory J. Gores - Advisory Committees or Review Panels: Conatus
The following authors have nothing to disclose: Nataliya Razumilava, Anuradha
Krishnan, Steven P. O’Hara
Disclosures:
The following authors have nothing to disclose: Alphonse E. Sirica, Akihiro Usui,
Deanna J. Campbell
1994
Oxysterols Promote Malignant Transformation of Cholangiocytes
in a Hedgehog Signaling-dependent Manner
Nataliya Razumilava 2 , Anuradha Krishnan 1 , Steven P. O’Hara 1 ,
Gregory J. Gores 1 ; 1 Mayo Clinic, Rochester, MN; 2 Division of
Gastroenterology and Hepatology, University of Michigan, Ann
Arbor, MI
Background: Cholangiocarcinoma is a highly lethal neoplasm.
It is often observed in a milieu of biliary tract inflammation
1995
A Randomized Prospective Open-label Trial Comparing
Peginterferon Plus Adefovir or Tenofovir Combination
Therapy Versus No Treatment in HBeAg-Negative
Chronic Hepatitis B Patients with a Low Viral Load
Annikki de Niet 1 , Louis Jansen 1 , Femke Stelma 1 , Sophie Willemse 1 ,
Sjoerd Kuiken 2 , Sebastiaan Weijer 3 , Carin M. Van Nieuwkerk 4 ,
Hans L. Zaaijer 5,1 , Richard Molenkamp 1 , Bart Takkenberg 1 ,
Maarten Koot 5 , Joanne Verheij 1 , Ulrich Beuers 1 , Hendrik W.
Reesink 1 ; 1 Academic Medical Center, Amsterdam, Netherlands;
2 Sint Lucas Andreas Hospital, Amsterdam, Netherlands; 3 Medical
Center Zuiderzee, Lelystad, Netherlands; 4 VU Medical Center,
Amsterdam, Netherlands; 5 Sanquin, Amsterdam, Netherlands
Background and aims: Chronic hepatitis B (CHB) patients with
a low viral load (LVL) are currently not eligible for antiviral
treatment. However, they comprise the largest group of CHB
patients and are still at increased risk of developing cirrhosis or
hepatocellular carcinoma. Here, we present the final results of a
randomized trial designed to determine HBsAg loss and decline
in CHB patients with LVL receiving combination treatment of
peginterferon alfa-2a (peg-IFN) and a nucleotide analogue or
no treatment. Methods: 134 CHB patients (HBeAg-negative,
HBV-DNA