studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 1157A
1946
Truncated Hepatitis B virus X protein up-regulates
CD44+ cancer stem like cells through LTBP1
Goki Suda, Seiji Tsunematsu, Masato Nakai, Jun Itoh, Mitsuteru
Natsuizaka, Kenichi Morikawa, Koji Ogawa, Naoya Sakamoto;
hokkaido university, Sapporo, Japan
Background Aims: Hepatitis B virus (HBV) is a major pathogen
of hepatocellular carcinoma (HCC). HBV X protein (HB) is
reported to promote HCC development by activation of various
signals. Recently genome-wide association study about HBV-related
HCC revealed that COOH-truncated HBx was integrated
in host genome at high frequency. Cancer stem like cells (CSCs)
are defined as those cells that can self-renew, drive tumorigenesis.
It is not clear whether COOH-truncated HBx integration
promotes prevalence of CSCs or not. Therefore, we aimed
to analyze the relationship between COOH-truncated HBx
expression and CSCs. Methods: We firstly established stably
HBx (HBx/HepG2) and COOH-truncated HBx (truncated HB/
HepG2). Next we examined the expression levels of stemness
makers in these established cells by Flow cytometry analysis.
Subsequently we analyzed the anti-tumor agent resistance and
tumorigenesis of these cells by MTS assay and soft gel agar
colony formation assay. Finally we investigated the factors that
promote prevalence of CSCs in truncated-HBx /HepG2 cells
by PCR-array. Results: Both HBx/HepG2 and truncated HBx/
HepG2 shows significantly resistant to anti-tumor agent, 5-FU,
compared with control/HepG2 cells. Soft gel agar colony formation
assay shows that truncated-HBx/HepG2 cells make significantly
more colonies than control/HepG2 cells and HBx/
HepG2 cells. Flow cytometry analysis reveals that truncated
HBx/HepG2 cells express high levels of CD44, one of cancer
stemness maker, compared with control/HepG2 and HBx/
HepG2 cells. Next truncated-HBx cells were sorted in CD44
high and low cells and were analyze potential of anti-tumor
agent resistant and tumorigensis. CD44high cells shows resistant
to anti-tumor agent 5-FU compared with CD44low cells,
and soft gel agar colony formation assay shows that CD44high
cells make significantly higher colonies than CD44low cells.
Subsequently we investigated what factor up-regulate CD44 in
truncated HBx/HepG2 cells by Human Stem Cell PCR Array
(Qiagen).This PCR-array results indicated that LTBP1 is significantly
up-regulated in only Truncated-HBx/HepG2 compared
with HBx/HepG2 and con/HepG2 cells. The expression level
of CD44 were significantly down-regulated by siRNA knockdown
of LTBP1. In sorted CD44high cells, LTBP1 were significantly
up-regulated compared with CD44low cells. Conclusion:
COOH-truncated HBx protein up-regulates CD44+ cancer stem
like cells via LTBP1. This finding might contribute development
of efficient strategies for eliminating CSCs
Disclosures:
Naoya Sakamoto - Grant/Research Support: Gilead Sciences, TRSS; Speaking
and Teaching: Bristol Myers Squibb, Janssen Pharm, Chugai co ltd
The following authors have nothing to disclose: Goki Suda, Seiji Tsunematsu,
Masato Nakai, Jun Itoh, Mitsuteru Natsuizaka, Kenichi Morikawa, Koji Ogawa
1947
P300 promotes TGF-β stimulated nuclear translocation
of SMADs and activation of hepatic stellate cells into
liver metastasis promoting myofibroblasts
Luyang Guo 2 , Xiaoyu Xiang 2 , Vijay Shah 1 , Ningling Kang 2 ;
1 Mayo Clinic, Rochester, MN; 2 the Hormel Institute, Austin, MN
Introduction: Liver metastasis is dependent on bidirectional interactions
between cancer cells and the microenvironment of the
liver. TGF-β, released from cancer cells and other cells within
the liver, induces activation of hepatic stellate cells (HSCs) into
myofibroblasts (MFs). In turn, MF/activated HSCs promote liver
metastatic growth. TGF-β induces intracellular signaling events
in HSCs, including activation of receptors, nuclear translocation
of SMADs and transcription of TGF-β target genes, which
present targets to inhibit HSC activation. However, mechanisms
governing these signaling events remain poorly understood.
Hypothesis: We tested if p300, an acetyltransferase
previously known to promote gene transcription, modulates
TGF-β1 induced activation of HSCs into tumor promoting MFs.
Methods: Lentiviruses encoding different p300 shRNAs were
used to knockdown p300 and a small molecule C646 was
used to inhibit p300 acetyltransferase activity. A tumor/HSC
coculture and coimplantation mouse model were used to test
the role of p300 knockdown HSCs for tumor growth. Immunofluorescence
(IF) was used to study subcellular localization of
p300 and SMAD nuclear translocation. Western Blot analysis
(WB) and IF for α-SMA were used to assess HSC activation,
co-immunoprecipitation (coIP) was used to study protein-protein
interactions and microarray analysis was used to study
gene expression profiles of HSCs. Results: p300 knockdown
HSCs were less effective on promoting tumor cell proliferation
than control HSCs in a tumor/HSC coculture and were less
effective on promoting tumor growth in mice in a subcutaneous
tumor/HSC coimplantation model. In vitro, p300 knockdown
inhibited TGF-β1 stimulated activation of HSCs into MFs as
assessed by both WB and IF for α-SMA (p200 cells
per group). IF revealed that p300 resided in both cytoplasm
and nucleus of HSCs and that p300 knockdown or inhibition
by C646 inhibited TGF-β1 stimulated nuclear translocation of
SMADs. Co-IP demonstrated that TGF-β1 stimulation promoted
a p300/Importin7/8/SMADs complex in the cytoplasm that
promotes nuclear transport of SMADs. Furthermore, microarray
analysis identified that p300 knockdown suppressed transcription
of TGF-β1 inducible genes, such as CTGF, TNC, PDGFC,
FGF2 and POSTN encoding either prometastatic niche factors
or growth factors of tumor cells. Conclusions: p300 is required
for TGF-β1 stimulated nuclear translocation of SMADs and transcription
of TGF-β1 target genes encoding numerous tumor
promoting paracrine factors. p300 of HSCs thus presents a
therapeutic target to inhibit HSC activation required for the
development and progression of liver metastasis.
Disclosures:
The following authors have nothing to disclose: Luyang Guo, Xiaoyu Xiang, Vijay
Shah, Ningling Kang
1948
Critical role of Hippo cascade in regulating AKT/Rasdriven
liver cancer development in mice
Shanshan Zhang 2,1 , Junyan Tao 1 , Xiaolei Li 1 , Diego Calvisi 3 ,
Xin Chen 1 ; 1 Department of Bioengineering and Therapeutic Sciences,
University of California San Francisco, San Francisco, CA;
2 Department of Pathology and Pathophysiology, Eastern Hepatobiliary
Surgery Hospital, Second Military Medical University, Shanghai,
China; 3 Institut für Pathologie, Ernst-Moritz-Arndt-Universität,
Greifswald, Germany
Background: Primary liver cancer consists of both hepatocellular
carcinoma (HCC) and intrahepatic cholangiocarcinoma
(ICC). Hippo pathway is a critical regulator in liver cancer
development. However, the precise role of the Hippo cascade
along hepatocarcinogenesis has not been fully explored. Purpose:
In this study, we investigated the functional significance
of Yap and TAZ, the transcriptional activators downstream of
Hippo tumor suppressor kinases, in a murine model of liver
cancer. Methods: We employed a mixed HCC and ICC mouse
model via hydrodynamic transfection of activated forms of AKT