studies

1158A AASLD ABSTRACTS HEPATOLOGY, October, 2015
and Ras oncogenes (AKT/Ras), based on the evidence that activation
of AKT/mTOR and Ras/MAPK pathways often occurs in
human HCC and ICC samples. In AKT/Ras mice, either the
Hippo cascade was activated by co-injecting Lats2 or Yap/
TAZ activity was blocked via transfection of dominant negative
TEAD2 (dnTEAD2). Furthermore, Yap or TAZ was silenced via
mir30 based shRNA. qRT-PCR, Western blotting, and immunohistochemistry
(IHC) were applied to detect gene and protein
expression patterns in tissues and cell lines. Results: Both Yap
and TAZ showed nuclear staining in AKT/Ras liver tumor samples.
Co-injecting Lats2 or dnTEAD2 with AKT/Ras strongly
delayed liver tumor development. Similar results were obtained
when the co-expression of AKT/Ras was paralleled by Yap or
TAZ silencing in the mouse liver. Histological analysis showed
decrease in liver tumor number and tumor size as well as the
prevalence of adenomas over carcinomas in the liver of AKT/
Ras mice. Importantly, activation of Hippo or loss of Yap/TAZ
led to the complete elimination of ICC-like lesions in the liver.
The phenotype was highly similar to that observed when AKT/
Ras mice were treated with anti-Notch2 antibodies. Indeed,
further analysis demonstrated the down-regulation of Notch2
in these mouse liver tumor tissues. Silencing of Yap or TAZ in
human HCC cell lines also led to the decreased expression of
CCA-like markers such as Sox9 and CK19. Conclusion: Our
data demonstrate that Hippo pathway negatively regulates
liver tumor formation in mice via regulating Notch2. Both Yap
and TAZ are required for hepatocarcinogenesis.
Disclosures:
The following authors have nothing to disclose: Shanshan Zhang, Junyan Tao,
Xiaolei Li, Diego Calvisi, Xin Chen
1949
Hepatocyte-specific overexpression of B cell leukemia-3
in mice suppresses chemically-induced hepatocarcinogenesis
Nadine Gehrke 1 , Marcus A. Woerns 1 , Yvonne Alt 1 , Nadine Hoevelmeyer
2 , Ari Waisman 2 , Peter R. Galle 1 , Jörn M. Schattenberg 1 ;
1 I. Department of Medicine, University Medical Center of the
Johannes Gutenberg University Mainz, Mainz, Germany; 2 Institute
for Molecular Medicine Mainz, University Medical Center, Mainz,
Germany
Background: B cell leukemia-3 (Bcl-3), which was originally
described as a proto-oncogene in B cell lymphomas, is highly
expressed in the liver. It binds tightly to the NFkB subunits
p50 and p52 homodimers in order to activate or inhibit transcription
from NFkB-dependent promoters regulating especially
inflammation and cell death. Bcl-3, when dysregulated, has be
shown to be widely expressed in several cancer types including
hepatocellular carcinoma (HCC), but a complete understanding
of the function of Bcl-3 in cancer development is still
lacking. Methods: To evaluate the role of hepatic Bcl-3 in hepatocarcinogenesis
we employed the two-step diethylnitrosamine
(DEN)/phenobarbital (PB)-liver cancer mouse model. 7-10
day-old, male mice exhibiting an increased hepatocyte-specific
expression of Bcl-3 (alfp-Cre:bcl-3, Bcl-3 hepar mice) and wild
type (wt) littermates received a single intraperitoneal injection
of 25mg DEN followed, 4 weeks later, by continuous treatment
with 0.5g/l PB dissolved in drinking water. Blood and liver
tissue were harvested at 40 weeks of age for further analysis.
Results: Remarkably, Bcl-3 hepar mice exhibited less tumours compared
to wt mice in response to DEN/PB. Also their relative
liver weight was significantly lower, which could be attributed
to the smaller tumour number and size. Histopathological
tumour grading revealed a comparable presence of dysplastic
foci and nodules in all H&E stained liver sections irrespective
of the genotype. However, more HCC as well as a greater
relative HCC area were detectable in the wt compared to the
Bcl-3 hepar group. In addition, K i
-67 labeling and appropriate
qRT-PCR analyses (K i
-67, p53, cMyc, mTOR) showed significantly
diminished levels of cell proliferation in the HCC region
of Bcl-3 hepar livers compared to wt livers. Moreover, DEN/PB
treatment resulted in a markedly reduced liver injury in Bcl-
3 hepar mice relative to the wt as demonstrated by lower serum
ALT and LDH levels. In agreement, JNK and ERK activation
were more pronounced in tumour and residual tumour tissues of
wt mice, whereas a higher grade of phosphorylated p38 and
NFkB p65 was determined in Bcl-3 hepar livers protecting against
DEN/PB-induced cell death. Compared to the wt, the absolute
number and activation of intrahepatic B cells, that exert a suppressive
function on tumour growth, was reduced in Bcl-3 hepar
mice as measured by FACS analysis and decreased mRNA
expression of CD81 and BLNK. Conclusion: Overexpression of
hepatic Bcl-3 impaired DEN/PB-induced hepatocarcinogenesis
through regulation of cell death and inflammatory processes.
Thus, Bcl-3 could be an important target in the development of
novel therapies.
Disclosures:
Marcus A. Woerns - Advisory Committees or Review Panels: Bayer, Bayer
Peter R. Galle - Advisory Committees or Review Panels: Bayer, BMS, Lilly, Daiichi,
Jennerex; Consulting: Medimmune; Grant/Research Support: Roche, Lilly; Speaking
and Teaching: Bayer, BMS
The following authors have nothing to disclose: Nadine Gehrke, Yvonne Alt,
Nadine Hoevelmeyer, Ari Waisman, Jörn M. Schattenberg
1950
Platelet-Derived Growth Factor D (PDGF-D)-Induced
Secretion of Vascular Endothelial Growth Factor-C by
Cancer-Associated Fibroblasts (CAF) Stimulates Lymphangiogenesis
in Cholangiocarcinoma
Massimiliano Cadamuro 2,1 , Marta Vismara 2 , Simone Brivio 2 ,
Mario Strazzabosco 2,3 , Luca Fabris 1,3 ; 1 Dep. of Molecular Medicine,
University of Padova, Padova, Italy; 2 Dep. of Surgery &
Translational Medicine, University of Milan-Bicocca, Monza, Italy;
3 Section of Digestive Diseases, Yale University, New Haven, CT
Cholangiocarcinoma (CCA) is characterized by a strong
invasiveness and a dismal prognosis. Early metastasisation to
regional lymph nodes often precludes surgery, the only potentially
curative option to treat CCA. Metastatic dissemination
is facilitated by the presence of an abundant reactive stroma,
mainly composed by cancer-associated fibroblasts (CAF), and
by a rich lymphatic vasculature. PDGF-D secreted by CCA cells
plays a major role in CAF recruitment. Mechanisms of lymphangiogenesis
in CCA are unknown. Therefore, we investigated
the possible role of PDGF-D and CAF in this process. Methods/Results.
Human CCA specimens were immunostained with
D2-40 (lymphatic endothelial cells, LEC), CD34 (vascular blood
endothelial cells, VEC), αSMA (CAF) alone or in combination
with antibodies against VEGF-C or its receptor VEGFR-3 (n=6).
This analysis showed that in CCA, lymphatic endothelial cells
(LEC), positive for VEGFR-3, rather than VEC, were closely adjacent
to VEGF-C expressing CAF. Secretion of VEGF-C, VEGF-D,
Ang-1, Ang-2 was then evaluated in human primary cultured
fibroblasts challenged with PDGF-D (n=3). Upon PDGF-D stimulation,
fibroblasts secreted increased levels of the lymphangiogenic
growth factor VEGF-C, whereas VEGF-D and Ang-2
were not expressed. In fibroblasts, PDGF-D-stimulated VEGF-C
expression was significantly inhibited by blocking PDGFRβ
(the cognate receptor of PDGF-D) with imatinib mesylate, or
by inhibiting JNK and ERK/NF-kB signaling with SP600125,
U0126 and BAY11-7082, respectively. LEC recruitment (tran-