studies

1160A AASLD ABSTRACTS HEPATOLOGY, October, 2015
interacts mainly with Mnt in normal liver but this switches to
c-Maf, MafG and c-Myc in cholestatic livers and CCA. Furthermore,
MATα1 interacts directly with c-Myc, MafG and c-Maf.
Interestingly promoter regions of MAT1A, c-Maf, MafG and
c-Myc have E-box sequences that are bound by MATα1 and
Mnt at baseline that switched to c-Myc, c-Maf and MafG after
BDL and LCA treatment. MATα1 binds to the E-box as a complex
with c-Myc and Max, but not by itself or with c-Myc. While
E-box positively regulates c-Myc, MafG and c-Maf, it negatively
regulates MAT1A. Furthermore, MATα1 binding to the E-box
represses E-box and lowers the promoter activity of c-Myc,
MafG and c-Maf, whereas c-Myc, MafG and c-Maf binding
enhance E-box-driven promoter activity. This results in reciprocal
regulation between MATα1 and c-Myc and Maf proteins.
In CCA cell lines knockdown of MAT1A or overexpression of
MafG or c-Maf enhanced tumorigenesis. Conclusions: we have
uncovered a novel interplay between MATα1, c-Myc and Maf
proteins and their deregulation during chronic cholestasis sets
the stage for CCA oncogenesis.
Disclosures:
The following authors have nothing to disclose: Heping Yang, Ting Liu, Jiaohong
Wang, Tony Li, Jose M. Mato, Shelly C. Lu
suppressed by regorafenib in the sorafenib-resistant clones,
suggesting that sorafenib-resistance might be related to the
acquisition of resistance against ERK signaling pathway inhibition
mediated by sorafenib. Promisingly, regorafenib treatment
suppressed the subcutaneous tumor growth of sorafenib-resistant
Huh7 and Huh1 clones with significantly prolonged overall
survival in vivo compared with sorafenib treatment. Conclusions:Regorafenib
inhibited the growth of sorafenib-resistant
HCC cells, potentially through suppression of the ERK signaling
pathway. The efficacy of regorafenib on the sorafenib-resistant
clones suggests its potential utility in HCC patients with resistance
to sorafenib.
Disclosures:
Hikari Okada - Employment: Kanazawa University
Mariko Yoshida - Grant/Research Support: Bayer
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co.,
Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc,
Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc,
Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer
Japan, Eli lilly Japan
The following authors have nothing to disclose: Tomomi Hashiba, Taro Yamashita,
Kouki Nio, Takehiro Hayashi, Yoshimoto Nomura, Tomoyuki Hayashi, Naoki
Oishi, Masao Honda
1953
Regorafenib inhibits ERK signaling and suppresses the
growth of sorafenib-resistant cells in human hepatocellular
carcinoma
Tomomi Hashiba, Taro Yamashita, Hikari Okada, Kouki Nio,
Takehiro Hayashi, Yoshimoto Nomura, Mariko Yoshida, Tomoyuki
Hayashi, Naoki Oishi, Masao Honda, Shuichi Kaneko; Gastroenterology,
Kanazawa University Graduate School of Medical
Science, Kanazawa, Japan
Background:Regorafenib is a multi-kinase inhibitor targeting
VEGFRs, PDGFRs, and RAF. A recent phase II study suggested
acceptable tolerability and anti-tumor activity of regorafenib in
patients with hepatocellular carcinoma (HCC) that progressed
after initial sorafenib treatment. A confirmatory phase III study
(NCT01774344) is ongoing. However, it remains unclear
how regorafenib inhibits the growth signaling pathways of
sorafenib-resistant HCC. In this study, we evaluated the effects
of regorafenib and sorafenib on HCC growth and signaling
pathways in HCC cell lines and primary HCC cells. Methods:Two
HCC cell lines (Huh1 and Huh7) and three primary
HCC cells obtained from surgically resected specimens were
cultured routinely in DMEM supplemented with 10% fetal bovine
serum and sorafenib tosylate (5-10 μM) for 3 months to generate
sorafenib-resistant clones. Whole exome sequence analysis
was performed to detect mutations in the sorafenib-resistant
clones and their parental cells. Sensitivity against sorafenib
or regorafenib was evaluated using a cell proliferation assay
kit. Gene and protein expression was evaluated by quantitative
RT-PCR and western blotting. A subcutaneous xenotransplantation
model was used to evaluate the efficacy of orally
administered sorafenib (30 mgkg -1 day -1 ) or regorafenib (20
mgkg -1 day -1 ) on sorafenib-resistant clones in vivo. Results:All
established sorafenib-resistant clones showed higher sensitivity
to regorafenib compared with sorafenib in vitro, whereas
parental cells showed similar sensitivity to both sorafenib and
regorafenib. Sorafenib-resistant clones derived from cell lines
showed abundant expression of the cancer stem cell marker
CD44 compared with the parental cells. Although sorafenib-resistant
clones showed frequent missense/nonsense mutations
compared with the parental clones, no common mutations were
detected among all sorafenib-resistant clones. Interestingly,
ERK phosphorylation was not affected by sorafenib, but was
1954
The Dead Box Protein P68 Can Supress The Carcinogenesis
And Α-Fetoprotein (AFP) Gene Expression via Binding
The New Silencer Region Of AFP Gene
Shunsuke Nojiri, Kei Fujiwara, Noboru Shinkai, Etsuko Iio, Takashi
Joh; Department of Gastroenterology and Metabolism, Nagoya
City University Graduate School of Medical Sciences, Nagoya,
Japan
The activity of the α-fetoprotein (AFP)
gene decreases rapidly after birth but is reactivated in hepatocellular
carcinoma (HCC) and also during proliferation of
hepatocyte. In clinical therapy of hepatocellular carcinoma
AFP reduction can be the good marker after anticancer therapies.
We predicted a new silencer lesion that was located
upstream from previous reported areas. The aim of this study
is to identify the precise lesion of the silencer and demonstrate
a new protein which suppresses AFP gene expression via its
new suppresser lesion. A basic vector was constructed
by inserting the 4.9-kb AFP5’-flanking sequence into
PGL2 basic vector. The vectors deleted several sections were
also constructed. The Luciferase activities were expressed in
relation to the activity of the cells that were transfected with
the basic vector and we found the 179 base pair new silencer
lesion. After immunoprecipitation by biotinized PCR products
including the silencer arrangement and the nuclear extracts, the
protein was identified from using an LC-MS/MS assay. Chromatin
immunoprecipitation (ChIP) was performed to confirm the
protein binding to the new silencer. The candidate protein was
suppressed or overexpressed using siRNA duplexes or overexpression
vector in HepG2 cells and AFP expression measured
by Luciferase activities and real time PCR and cell proliferation
were measured by cell proliferation assay. The results
of the LC-MS/MS assay demonstrated the presence of DEAD
box protein p68 (p68). P68 protein could binding to the new
silencer session was confirmed by ChIP. After using RNAi to
p68 the Luciferase activities increased 3.5±1.1 times (p