studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 551A
bution of GFP was inversely correlated with alpha-fetoprotein
(AFP) expression, suggesting local insulin signaling mediated
by IRS2 may play a role in tumor heterogeneity and cellular
differentiation. Exogenous expression of IRS2 promoted tumorigenesis
in HepG2 soft agar and clonogenic assays and also
corresponded with decreased AFP immunostaining. In HepaRG
pIRS2-GFP positive cells were observed in discrete sites within
the cultures surrounding “islands” of hepatocyte differentiation.
IRS2 enriched cells had progenitor-like properties and we show
that insulin/IRS2 signaling at a cellular level determines the
differentiation, proliferative expansion and survival of hepatocyte-like
tumor cells within the cultures, whilst expression of
IRS2 is dynamically regulated during the timecourse of hepatocyte
differentiation such that levels diminished as cells matured.
Taken together, our results underscore the heterogeneity of
insulin sensitivity at the cellular level within “homogeneous”
cancer cell lines. We show that IRS2 expression is spatially
patterned both in monolayers in vitro and in tumors in vivo and
serves as a proxy for insulin sensitivity. Further investigation of
how this novel cellular niche reacts to aberrant insulin signaling
in the context of metabolic disease may provide future insights
into the links that underpin the association between type II diabetes
and HCC risk.
Disclosures:
The following authors have nothing to disclose: Fátima Manzano Núñez, Carlos
Acosta Umanzor, Aránzazu Leal Tassias, Deborah J. Burks, Luke A. Noon
692
Sonic Hedgehog-Containing Exosomes Mediate Biliary
Differentiation and Fibronectin Deposition via Epigenetic
Regulation
Nidhi Jalan-Sakrikar, Thiago de Assuncao, Jie Lu, Gwen Lomberk,
Martin E. Fernandez-Zapico, Raul A. Urrutia, Robert C. Huebert;
Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN
Background: Developmental morphogens play an important
role in coordinating the ductular reaction and portal fibrosis
that occur in the setting of cholangiopathies. However, little is
known about how injured cholangiocytes signal to adjacent
progenitor cells to promote repair after biliary injury. Recent
studies show that sonic hedgehog (Shh) signaling is activated
during liver regeneration and repair. In addition, cholangiocytes
release exosomes that contain biologically active Shh and
the secretion of these organelles increases during biliary fibrosis.
Our lab has recently reported a differentiation protocol that
generates induced pluripotent stem cell (iPSC)-derived cholangiocytes
(iDC), a model useful for studying the stem cell to cholangiocyte
transition. The purpose of this study was to dissect
the mechanisms whereby injured cholangiocytes communicate
with the neighboring progenitor cells via Shh-containing exosomes
to initiate both cholangiocyte differentiation and extracellular
matrix (ECM) remodeling. Methods and Results: Next
generation RNA sequencing at each phase of the iPSC to iDC
transition revealed significant upregulation of the Smoothened
(SMO) receptor as well as Gli-1 and Gli-2 transcription factors
during biliary specification and these changes correlated
with a 60-fold increase in fibronectin (FN) levels. Exosomes
isolated from LPS-injured cholangiocytes enhanced progenitor
cell acquisition of cholangiocyte markers (CK7: 2.7±0.3-fold;
CK19: 2.3±0.2-fold) as well as the expression and release
of FN (8.3±2.4 fold). Cholangiocyte differentiation and FN
release were both mediated by Shh as shown using pharmacologic
(cyclopamine) or genetic (SMO shRNA) inhibition of
Shh signaling. Concurrent alterations in epigenetic regulators
(SetD7, KDM5D, EZH2) suggested that a common epigenetic
regulatory program, driven by Shh, may mediate both cholangiocyte
differentiation and ECM deposition. Cholangiocyte
differentiation was associated with temporal alterations in histone
methylation patterns that were Shh-responsive, including
early increases in H3K4me3 (4.6±1.6-fold) and late decreases
in H3K27me3 (-3.8±1.2-fold). In vivo, mice fed the choline-deficient,
ethanolamine supplemented diet had an expansion of
LGR5+ progenitor cells that was associated with Shh activation,
increased FN deposition, and peri-portal fibrosis. Conclusions:
We conclude that Shh-containing exosomes play an
integral role in cholangiocyte differentiation from progenitor
cells following biliary injury and that an epigenetically-driven
cascade of gene regulation may simultaneously promote FN
deposition and biliary fibrosis.
Disclosures:
The following authors have nothing to disclose: Nidhi Jalan-Sakrikar, Thiago de
Assuncao, Jie Lu, Gwen Lomberk, Martin E. Fernandez-Zapico, Raul A. Urrutia,
Robert C. Huebert
693
Interleukin-17 mediates liver progenitor cell transformation
into cancer stem cells in hepatocellular carcinoma.
Imène Gasmi 1,2 , Adrien Guillot 2,3 , Nabila Hamdaoui 1,2 , Arthur
Brouillet 1,2 , Julien Calderaro 1,2 , Benoit Rousseau 1,2 , Jean-Michel
Pawlotsky 1,2 , Fouad Lafdil 1,2 ; 1 INSERM U955 Henri Mondor Hospital,
INSERM / University of Paris Est, Créteil, France; 2 INSERM
/ University of Paris Est, Creteil, France; 3 NIH NIAAA, Bethesda,
MD
Introduction. Hepatocellular carcinoma (HCC) is the third leading
cause of cancer-related death. HCC arises in the setting
of cirrhotic livers in 80 to 90% of cases and progresses in an
inflammatory context. Cancer stem cell biology has recently
sparked the interest of scientists because of their capacity to
initiate and to enhance tumor progression. However the underlying
mechanisms by which expansion of CSCs is initiated are
still unclear. After severe and chronic liver injury, liver progenitor
cell compartment is activated under sustained inflammatory
response. LPCs participate to the regenerative process and are
also observed in HCC. Interestingly, among the large spectrum
of released cytokines in HCC, recent studies identified
IL-17-producing cells as a factor associated with a poor prognosis
of the disease. In this study, we propose to determine
whether IL-17 could be involved in tumorigenesis, in particular,
by promoting the transformation of resident liver progenitor
cells (LPCs) into CSCs. Materiels and methods. Serial sections
from 70 HCC-patients were immuno-stained to identify LPCs
(with anti-CK19) and IL-17-producing cells (with anti-IL-17). In
vitro, a murine LPC line (BMOL) was used for long-term culture
with or without IL-17 for 10, 20, 30 or 40 days. The expression
of cancer stem cell markers were analyzed by quantitative
RT-PCR and flow cytometry. Cell cycle controlling factors
were assessed by western blot. Acquired self-renewal property
of LPCs after long term culture with IL-17 was assessed by
spheroid formation capacity analysis. Results. Identification
and semi-quantitative analysis of CK19+ and IL-17+ cells in
HCC patients showed a positive correlation between the number
of infiltrated IL-17+ cells and the number of LPCs. In vitro,
LPC stimulation with IL-17 led to increased expression of CSC
marker mRNA expression including Klf4, ALDH1A1 or CD133,
EpCAM and Glypican-3. CD133 protein and ALDH1A1 activity
analyzed by flow cytometry, were enhanced in LPC cultured
for 10, 20 or 30 days when compared to none treated LPCs.
Cell cycle analysis showed that IL-17 induces the expression
of the proto-oncogene c-Raf, and cyclin D1. In addition, LPCs
cultured for 10 days in presence of IL-17 were able thereafter
to form spheroids when transferred in low-attachment culture