studies

1152A AASLD ABSTRACTS HEPATOLOGY, October, 2015
classified according to the integrated molecular profiles using
corresponding and hierarchical clustering analyses, and their
relationship to the clinicopathological backgrounds were examined.
(2) Molecular profiles of 44 HCCs from the patients who
underwent liver transplantation (LT) were determined based
on the mutation, methylation and microsatellite analyses. We
tried to examine the specific molecular features that related to
the extrahepatic recurrence after LT of HCC. Results: (1) We
successfully classified HCCs in to 4 subgroups based on the
genetic and epigenetic alterations. The subgroup A1 (S-A1)
was characterized by high frequencies of extensive TGS methylation,
β-catenin and TERT promoter mutation; however, the
molecular characteristics of the subgroup B2 (S-B2) were the
presence of significant hypomethylation on RSs and p53 mutation
accompanied by CI, which was mutually exclusive to that
of S-A1. Similarly, the subgroup A2 (S-A2) was characterized
by the presence of all the alterations examined. On the other
hand, genetic and epigenetic alterations were rare in HCCs in
the subgroup B1 (S-B1). Characteristics related to tumor progression,
such as high serum AFP (> 200 ng/ml), larger tumor
size (> 3 cm), presence of vascular invasion, and dedifferentiated
tumor were more frequently observed in HCCs belonging
to S-A2 and S-B2 than those in S-A1 and S-B1. (2) Among 44
HCC patients after LT, 6 showed metastatic recurrence of HCC
after LT and 38 had not shown any recurrence for more than 2
years. Five of 6 HCC patients (83%) showing recurrence carried
tumors with S-B2 molecular characteristics, whereas only
11 of 38 HCCs in non-recurrence group (29%) showed S-B2
characteristics. Conclusion: Integrated genetic and epigenetic
profiles are associated with tumor characteristics and predict
mild or aggressive behavior of HCCs.
Disclosures:
Masatoshi Kudo - Advisory Committees or Review Panels: Bayer HealthCare;
Grant/Research Support: Bayer HealthCare
The following authors have nothing to disclose: Naoshi Nishida, Toshimi Kaido
1936
Baicalein targets liver tumor initiating stem cell-like cells
resistant to mTORC1 inhibition
Raymond Wu 1 , Ramachandran Murali 2 , Yasuaki Kabe 3 , Keigo
Machida 1 , Clay Wang 4 , Stan G. Louie 4 , Hidekazu Tsukamoto 1 ;
1 Southern California Research Center for ALPD and Cirrhosis, Keck
School of Medicine, University of Southern California, Los Angeles,
CA; 2 Cedars-Sinai Medical Center, Los Angeles, CA; 3 Keio University
School of Medicine, Tokyo, Japan; 4 Department of Pharmacology
and Pharmaceutical Sciences, School of Pharmacy of the
University of Southern California, Los Angeles, CA
[Introduction] mTORC1 inhibitors are tested for the treatment
of HCC based on hyperactive mTOR in this malignancy. However,
evidence indicates mTORC1 inhibitors may promote
CD133 upregulation and chemoresistance. CD133+ tumor initiating
stem cell-like cells (TIC) isolated from mouse liver tumors
(PNAS 106:1548; JCI 123:2832) are chemoresistant, and
identification of an approach to abrogate this resistance is critical.
[Objective] We searched for a natural compound which
rescinds TIC’s resistance to mTORC1 inhibition and improves
chemotherapy outcome. [Methods and Results] HPLC/MS and
NMR analyses combined with TIC lethality assay, identified
baicalein (BC) as a bioactive compound in the herbal medicine
Yan Gang Wan shown to suppress DEN-induced liver
tumorigenesis in mice. BC dose-dependently inhibits TIC self-renewal
shown by spheroid formation and clonogenic assay
and induces TIC apoptosis at IC50=9.5mM while having no
adverse effects on mouse primary hepatocytes. BC represses
stemness genes (Nanog, Sox2) while upregulating hepatocyte
differentiation genes (Alb, Cyp7a1). Structure-function analysis
reveals –OH groups on C5 and C7 are required for TIC killing.
TIC which are resistant to rapamycin (2mM) and temsirolimus
(~4.5mM), are killed ~90% when co-treated with BC (10mM).
BC also enhances TIC cytotoxicity by sorafenib (3.3mM) and
doxorubicin (1mM), causing ~95% elimination. BC suppresses
autophagosome formation determined by LC3II IB and LC3-
GFP analysis and mitochondrial respiration by Seahorse assay,
and prevents temsirolimus-mediated CD133 induction. Pharmacokinetic
studies reveal orally administered BC is converted to
~50% less potent baicalin (BCi) with glucuronate substituting
C7 -OH in plasma but largely reverted to BC in the mouse
liver. A similar reversion is confirmed in cultured primary
hepatocytes and TIC treated with BCi. BC treatment completely
eliminates temsirolimus chemoresistance of TIC-derived subcutaneous
tumor growth in nude mice. LC/MC analysis of TIC
proteins pulled-down with BCi-conjugated nanobeads, identifies
Rab1 and other Rab family members as potential targets of
BC, and 3D structural analysis and docking studies predicted
BC interaction at the GTP binding site of Rab1 via BC’s –OH
of C5 and C7. Indeed, GTP but not ATP competitively inhibits
BC-Rab1 interaction and Rab1 overexpression rescues BC-induced
inhibition of autophagy, but not TIC killing, suggesting
a role for additional Rab protein target(s) for the latter effect.
[Conclusion] BC promotes killing of chemoresistant TICs, particularly
when combined with mTORC1 inhibitor via mechanisms
which appear to involve interactions with Rab family proteins.
Disclosures:
Hidekazu Tsukamoto - Consulting: Suntory Ltd.; Grant/Research Support: The
Toray Co.
The following authors have nothing to disclose: Raymond Wu, Ramachandran
Murali, Yasuaki Kabe, Keigo Machida, Clay Wang, Stan G. Louie
1937
CD26 (DPP-4) as a molecular target for HCC treatment
Sohji Nishina 1 , Akira Yamauchi 2 , Yuichi Hara 1 , Keisuke Hino 1 ;
1 Hepatology and Pancreatology, Kawasaki medical univercity,
Kurashiki city, Okayama, Japan; 2 Biochemistry, Kawasaki Medical
School, Kurashiki city, Okayama, Japan
Background and Aim: CD 26 is a multifunctional transmembrane
glycoprotein and functions as dipeptidyl peptidase 4
(DPP-4). Although CD26 is expressed in various cancers, the
relationship between hepatocellular carcinoma (HCC) progression
and CD26 expression remains unknown. The aim of this
study was to investigate the potential role of CD26 as a molecular
target for HCC treatment. Methods: CD26 expression was
examined in 41 surgically resected liver specimens from patients
with HCC. In vitro the effect of DPP-4 inhibitor, anagliptin, on
cancer cell growth was investigated using Huh7/HepG2 cells
that expresses CD26. In vivo nude mice (BALBc-nu/nu) were
subcutaneously injected Huh7 cells and then fed control diet,
low-dose anagliptin containing diet or high-dose anagliptin
containing diet for 21 days. Results: CD26 expression was
correlated with cell proliferation, angiogenesis and cell differentiation
in HCC specimens obtained from patients. Anagliptin
did not affect cell proliferation or cell cycle in vitro. However
in nude mice, anagliptin significantly suppressed the growth of
xenograft tumors in dose dependent manner. Anagliptin also
induced NK cells infiltrations more vigorously and reduced
angiogenesis in xenograft tumors, even though body weight
and dietary intake through the feeding period, and glucose tolerance
determined at the 14th day of feeding were not different
among three groups. These results suggested that anagliptin
potentially have antitumor activity against HCC. Furthermore,
we examined whether anagliptin activated chemotaxis of NK