studies

1166A AASLD ABSTRACTS HEPATOLOGY, October, 2015
The Ki-67 and Tunel staining results show that these inhibitors
can also inhibit proliferation and induce apoptosis of the CCA
xenograft cells. Conclusions: These three TKIs all significantly
inhibited the growth of patient-derived CCA xenografts bearing
an oncogenic FGFR2-CCDC6 fusion gene. Treatment efficacy
from most to least effective was BGJ398 > dovitinib > ponatinib.
Additional studies exploring the potential mechanisms
underlying differences in response to particular FGFR inhibitors
and the effects of combinations of other therapeutic agents with
FGFR inhibitors are underway.
Disclosures:
Joseph M. Gozgit - Employment: ARIAD Pharmaceuticals
Lewis R. Roberts - Grant/Research Support: Bristol Myers Squibb, ARIAD Pharmaceuticals,
BTG, Wako Diagnostics, Inova Diagnostics, Gilead Sciences, Five
Prime Therapeutics
The following authors have nothing to disclose: Yu Wang, Hassan M. Shaleh,
Xiwei Ding, Shaoqing Wang, Roongruedee Chaiteerakij, Kais Zakharia, Loretta
K. Allotey, Essa A. Mohamed, Catherine D. Moser, Steven Alberts, Mitesh J.
Borad
1967
Glypican 3 contributes to HCC invasiveness and tumorigenesis
by up-regulating Angioprotein 2 and initiating
epithelial mesenchymal transition
Thu Le Trinh, William M. Puszyk, Olorunseun Ogunwobi, Quanfeng
Wu, Chen Liu; Department of Pathology, University of Florida,
Gainesville, FL
Background and Rationale: Glypican 3 (GPC3) has been
shown to be up-regulated in HCC and is known to be associated
with HCC advanced stages as well as cancer invasiveness
and recurrence. The aim of this study is to define the
mechanisms of GPC3 in HCC carcinogenesis and metastasis.
Methods: Representations of murine and human HCCs were
created using GPC3 knock-in in 1MEA (1MEA hGPC3 ), a murine
HCC cell line which doesn’t have GPC3 expression; and GPC3
knock-down in Huh7 (Huh7 GPC3 shRNA ), a human HCC cell line
which highly expresses GPC3. Cell proliferation and tumor
development were access in vitro and in vivo using the 1MEA/
Balb/c and Huh7/NSG mice models. The impact of GPC3
knock down on different cancer pathways was approached by
qPCR array analysis on the Huh7 control shRNA and GPC3-knockdown
Huh7 GPC3 shRNA cell lines using Cancer Pathway Finder
array (Qiagen), which consists of 84 different genes that represent
9 different biological pathways involved in carcinogenesis.
The effect of GPC3 repression on cell invasion ability
was also examined by wound-healing assay. Results: We
found that GPC3 didn’t induce a different proliferation rate
in 1MEA hGPC3 cell line, but it promoted tumor development
significantly when 1MEA hGPC3 was implanted into Balb/c mice
compared to 1MEA control. Knock-down of GPC3 resulted in
cell growth inhibition and cell cycle arrest at S phase. Similarly,
tumor proliferation was greatly retarded in Huh7 GPC3
shRNA
xenograft mouse model in comparison to Huh7control shRNA
tumor. In the 84 cancer pathway focused genes, 29 genes
showed at least a 2-fold difference in gene expression between
GPC3-knockdown cells compared to non-silenced cells. These
included genes that maintain telomeres (TINKS, TINKS2, TINF2,
TERF2IP), anti-apoptotic genes (XIAP, NOL3, FASLG), genes
involved in cell cycle regulation (CCND2, CCND3, E2F4),
markers of epithelial to mesenchymal transition (EMT) (CDH2,
DSP, OCLN, SNAI3), and genes that promote angiogenesis
(ANGPT2). Among these, ANGPT2 gene expression was
down-regulated up to 150 fold in Huh7 shRNA GPC3 compared to
Huh7 control shRNA . GPC3 roles in EMT were confirmed by migration
assay and showed significant inhibition of Huh7GPC3 shRNA
migration. Likewise, EMT markers and inducers were upregulated
in 1MEA hGPC3 cell line in comparison to 1MEA control.
Conclusions: These results indicated that GPC3 played a role
in HCC tumorigenesis via promoting EMT, angiogenesis and
cell migration, and that GPC3 could serve as a key connection
between angiogenesis and metastasis in HCC.
Disclosures:
The following authors have nothing to disclose: Thu Le Trinh, William M. Puszyk,
Olorunseun Ogunwobi, Quanfeng Wu, Chen Liu
1968
Promoted malignant tumor phenotype of hepatitis C
virus transgenic mice fed an atherogenic high-fat diet is
associated with the abnormal expression of the glycolysis-related
gene pyruvate kinase M2.
Riuta Takabatake 1 , Masao Honda 1 , Hikari Okada 1 , Kai Takegoshi
1 , Yoshio Sakai 1 , Taro Yamashita 1 , Naoto Nagata 3 , Toshinari
Takamura 3 , Takuji Tanaka 2 , Shuichi Kaneko 1 ; 1 Gastroenterology,
Kanazawa University Graduate School of Medical Science,
Kanazawa, Japan; 2 Cancer Research and Prevention, The Tohkai
Cytopathology Institute, Gifu, Japan; 3 Department of Disease Control
and Homeostasis, Kanazawa University Graduate School of
Medical Sciences, Kanazawa, Japan
Objective The relationship between hepatitis C virus (HCV)-related
metabolic abnormalities and the progression of hepatic
tumorigenesis remains unclear. The expression of the HCV
open reading frame in the liver of transgenic (Tg) mice induces
steatohepatitis, which is followed by the development of hepatocellular
carcinoma. The atherogenic high-fat diet (Ath HFD)
mouse model develops steatohepatitis similar to human nonalcoholic
steatohepatitis. We investigated the expression of
pyruvate kinase M2 (PKM2), an isoenzyme of the glycolytic
enzyme pyruvate kinase and recently recognized as a transcription
factor promoting cancer cell growth and the progression
of tumorigenesis, in HCV Tg mice fed an Ath HFD
diet. Materials and Methods Four groups of mice (n = 15–20/
group) were fed with a control diet or an Ath HFD diet starting
at 8 weeks after birth for 30 and 60 weeks. The degree of
liver steatosis, liver fibrosis, liver weight, and tumor incidence
were measured. The expression of collagen I and IV, TGF-β,
p-SMAD3, PKM2, Foxo1, p-Akt, Hes1, and Hey1 was evaluated
by immunohistochemical staining, TaqMan PCR, and
western blotting. In vitro analysis was performed by using
PKM2-stable knockdown Huh-7 cells. Results HCV Tg mice fed
an Ath HFD diet showed markedly promoted liver fibrosis at
30 and 60 weeks compared with wild-type (WT) mice fed an
Ath HFD diet. In addition, HCV Tg mice fed an Ath HFD diet
showed markedly increased liver weight (1.5-fold) and tumor
size (1.5-fold), but with no change in tumor frequency compared
to WT mice. HCV Tg mice fed an Ath HFD diet showed
a significant increase in the expression of PKM2 and fibrosis-related
genes, such as collagen I/IV, p-smad3, and TGF-β
together with Notch signal-related genes. HCV core protein
was found to activate the PKM2 promoter through the induction
of CREB. In vitro, PKM2-stable knockdown Huh-7 cells showed
significant repression of EpCAM, AFP, Sox2, Oct4, vimentin,
and KRT19 expression, which are markers of cancer stem cells.
Interestingly, the addition of recombinant Notch/jagged1 to
Huh-7 cells further activated the nuclear translocation of PKM2;
conversely, Notch-stimulated Hes1 and Hey1 expression was
repressed in PKM2-knockdown cells. Conclusion Promoted liver
fibrosis and a malignant tumor phenotype were observed in
HCV Tg mice fed an Ath HFD diet, which were associated with
the abnormal expression of PKM2. PKM2 might be involved in
the expression of pro-fibrotic genes and epithelial-mesenchymal
transition signaling by activation of the Notch signaling