studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 1159A
swell migration) and vascular assembly by LEC were measured
after exposure to conditioned medium (CM) of fibroblasts challenged
with PDGF-D. CM stimulated LEC recruitment and their
tubular assembly (lumen diameter, tube elongation and anastomization),
to an extent comparable to that induced by VEGF-C
alone. These effects of CM on LEC were inhibited by antagonizing
PDGFRβ in fibroblasts and by inhibiting VEGFR-3 in LEC
(n=6). In conclusion, this study provides a working model to
understand the mechanisms by which the interaction between
cancer cells and CAF promotes lymphangiogenesis in CCA.
Our data show that PDGF-D secreted by CCA cells not only
recruits CAF in the tumor reactive stroma, but also stimulates
CAF to release VEGF-C acting through the JNK and ERK/NF-kB
signaling. In turn, VEGF-C promotes tumor lymphangiogenesis
by stimulating VEGFR-3 expressed on LEC. This interplay may
be at the basis of the high rate of lymphatic metastasization
in CCA. Furthermore, the ability to interfere with these mechanisms
may prevent lymphatic metastatization of CCA.
Disclosures:
The following authors have nothing to disclose: Massimiliano Cadamuro, Marta
Vismara, Simone Brivio, Mario Strazzabosco, Luca Fabris
Notch1 up-regulation cells expressed both C-myc and OV6 (A,
white arrow). Representative tumors in liver and lung (B, black
arrow) and their H&E staining. VCAM-1 was activated in Notch1
transfected cells by iTRAQ (C, red frame), immunofluoresecence
(D, white arrow) and western blot analyses (E). VCAM-1 was
overexpressed (F, black arrow) and TAMs were abnormally
recruited in the pulmonary metastasis (G, black arrow).
1951
Notch-1 Up-regulated Rat Oval Cells Generated Poorly
Differentiated Liver Cancer and Developed Lung Metastases
in an Orthotopic Rat Model
Wen-rui Wu, Xiang-de Shi, Rui Zhang, Lei-bo Xu, Mansheng Zhu,
Xian-huan Yu, Hong Zeng, Chao Liu; Sun Yat-sen Memorial Hospital,
Sun Yat-sen University, Guangzhou, China
Background and aims: Oval cells may give rise to liver cancer.
Notch signaling has been reported to play crucial role in hepatocellular
carcinoma. The aim of this study was to investigate
the biological role of Notch1 in rat oval cell line WB-F344.
Methods: Notch1 was stably transfected into WB-F344 cells
by lentivirus. Their proliferation, colony formation, cell cycle
progression and invasion assays in vitro, as well as tumor
formation assay in an orthotopic (liver) rat model were performed.
Results: Gain-of-function analysis showed that Notch1
up-regulation promoted proliferation, colony formation, G1/S
cell cycle transition and invasion of WB-F344 cells in vitro.
Notch1 transfected cells expressed oncogene C-myc and oval
cell marker OV6 simultaneously. These cells showed a pile up
appearance in culture and could generate poorly differentiated
liver cancer orthotopically in isogenic rats (6/6, 100%).
Interestingly, all tumor bearing rats developed lung metastases
(6/6, 100%). In addition, iTRAQ analysis indicated that
vascular cell adhesion molecule 1 (VCAM-1) was activated
in Notch1 transfected cells in vitro, which was confirmed by
immunofluorescence and western blot analysis. Moreover, we
discovered that VCAM-1 was highly expressed in pulmonary
metastasis tumors and tumor-associated macrophages (TAMs)
were abnormally recruited in these tumors by immunohistochemistry.
Conclusions: Notch1 is a crucial regulator of proliferation
and malignant transformation of oval cells. Notch1
may facilitate pulmonary metastasis of primary liver cancer via
VCAM-1 activation and TAMs recruitment.
Disclosures:
The following authors have nothing to disclose: Wen-rui Wu, Xiang-de Shi, Rui
Zhang, Lei-bo Xu, Mansheng Zhu, Xian-huan Yu, Hong Zeng, Chao Liu
1952
Deregulated Interplay Between Methionine Adenosyltransferase
α1, c-Myc and Maf Proteins During Chronic
Cholestasis Facilitate Development of Cholangiocarcinoma
Heping Yang 1,2 , Ting Liu 3 , Jiaohong Wang 1,2 , Tony Li 1,2 , Jose M.
Mato 4 , Shelly C. Lu 1,2 ; 1 Medicine, Cedars-Sinai Medical Center,
Los Angeles, CA; 2 USC Research Center for Liver Diseases, Los
Angeles, CA; 3 Department of Gastroenterology, Xiangya Hospital
Central South University, Changsha, China; 4 Centro de Investigación
Biomédica en Red de Enfermedades Hepáticas y Digestivas
(Ciberehd), CIC bioGUNE, Derio, Spain
Purpose of Study: Cholangiocarcinoma (CCA) develops frequently
in the setting of chronic inflammation and cholestasis.
We reported the importance of c-Myc induction in the
development of cholestatic liver injury and CCA in mice. We
also showed induction of Maf proteins (MafG and c-Maf) contributed
significantly to cholestatic liver injury. The aim of our
current work was to determine whether there is any interplay
between c-Myc and Maf proteins but in the process of our
investigation we uncovered interesting reciprocal regulations
that likely contribute to development of CCA, particularly in
the setting of chronic cholestasis. Methods: Our study used
bile duct ligation (BDL) and lithocholic acid (LCA) treatment
as chronic cholestasis models, diethylnitrosamine followed by
left and median BDL as a murine CCA model, human CCA
cell lines KMCH and Huh-28, and human CCA specimens.
We used immunoprecipitation followed by mass spectrometry
and recombinant proteins to study protein-protein interactions,
chromatin immunoprecipitation (ChIP) and sequential ChIP to
examine co-occupancy of promoter region, overexpression and
siRNA to vary protein expression. Results: We identified novel
interacting proteins with c-Myc, specifically methionine adenosyltransferase
α1 (MATα1, encoded by MAT1A), MafG and
c-Maf. MAT1A expression fell at the mRNA and protein levels
in whole liver and bile duct epithelial cells during BDL and LCA
treatment, and in murine and human CCA samples. The opposite
occurred with c-Myc, MafG and c-Maf expression. MATα1