studies

534A AASLD ABSTRACTS HEPATOLOGY, October, 2015
653
Lysophosphatidic acid receptor 3 (LPAR3) is expressed
by cancer progenitor cells in human HCC tissue at the
tumor:non-tumor margin and regulates cell migration.
Valentina Zuckerman 1 , Eugene Sokolov 1 , Jacob H. Swet 1 , Victor
C. Showalter 1 , William A. Ahrens 2 , David A. Iannitti 1 , Iain H.
McKillop 1 ; 1 Department of Surgery, Carolinas Medical Center,
Charlotte, NC; 2 Department of Pathology, Carolinas Medical Center,
Charlotte, NC
Background The poor prognosis for hepatocellular carcinoma
(HCC) patients can be attributed, in part, to late detection,
rapid tumor expansion, and metastases. LPAR3 is detected
at low levels in the liver/ hepatocytes, and previous studies
report LPAR3 expression increases in HCC and diseased/cirrhotic
non-tumor liver (NTL) from HCC patients compared to
healthy normal liver (NL). A growing interest in stem cells in
tumor biology, and the role of LPAR3 in other cancers, led us
to hypothesize that changes in LPAR3 expression in HCC may
occur in a subset of hepatic progenitor cells involved in tumor
progression/expansion. Methods Sections from human HCC-
NTL, and lysates from SKHep1 cells (liver cancer progenitor
cells) and NL were analyzed for LPAR3, CD44 (mesenchymal
stem cell marker), EpCAM (cancer progenitor cell marker),
and Hepar1 (hepatocyte marker) expression-localization. The
effect of LPA on cell function was determined in SKHep1 using
2D and 3D cell migration analysis. Cell signaling (AKT/MEK
activity) and migration following LPA (10μM) treatment was
analyzed in conjunction with inhibition of Gi-protein dependent
signaling (PI3K inhibitor (LY294002 [LY], 40μM) and MEK
inhibitor (U0126, 5μM)). In parallel shRNA was used to inhibit
endogenous LPAR3 expression. Results Extensive co-localization
of LPAR3 with CD44 and EpCAM was detected in the
HCC-NTL margin, but not the NTL or tumor mass, data mirrored
in SKHep1 cells (LPAR3, CD44 and EpCAM detected). Conversely,
Hepar1 was readily detected in NL and the HCC mass,
but not SKHep1 cells, or cells in the HCC margin that stained
positive for LPAR3. In vitro analysis demonstrated LPA-dependent
PI3K and ERK activation in SKHep1 cells resulting in
significant cell migration. Pretreatment with LY and U0126
abrogated LPA-dependent PI3K and MEK activation. However,
inhibition of PI3K (LY) failed to significantly alter LPA-dependent
migration whereas ERK inhibition (U0126) abolished LPA-dependent
migration. The effects of pharmacological inhibition
were mirrored by inhibiting LPAR3 expression (shRNA), in
which exogenous LPA failed to stimulate ERK activation and
cell migration. Summary These data identify a unique subset
of LPAR3 positive cancer progenitor cells at the tumor–NTL
margin in HCC patients. Using an in vitro model of hepatic
tumor progenitor (SKHep1) cells, with similar LPAR profiles to
in vivo tissue, we report LPA regulates tumor cell migration via
Gi-MEK-ERK dependent signaling. These data suggest targeting
LPA-LPAR3 signaling within this progenitor cell subset in the
liver may slow tumor expansion and/or metastasis and limit
disease progression.
Disclosures:
David A. Iannitti - Consulting: Davol, Covidien, Ethicon Endosurgery, Davol,
Covidien, Ethicon Endosurgery, Davol, Covidien, Ethicon Endosurgery, Davol,
Covidien, Ethicon Endosurgery; Speaking and Teaching: Davol, Covidien,
Ethicon Endosurgery, Davol, Covidien, Ethicon Endosurgery, Davol, Covidien,
Ethicon Endosurgery, Davol, Covidien, Ethicon Endosurgery
The following authors have nothing to disclose: Valentina Zuckerman, Eugene
Sokolov, Jacob H. Swet, Victor C. Showalter, William A. Ahrens, Iain H. McKillop
654
Hepatitis B virus X protein promotes HCC metastasis
through activation of CD44v6 splicing form switching by
regulation of splicing factor ESRP1/ESRP2
Ya-Ju Chang 1 , Li-Chen Wu 2 , Ja-an Annie Ho 1 ; 1 Department of
Biochemical Science and Technology, National Taiwan University,
Taipei, Taiwan; 2 Department of Applied Chemistry, National Chi
Nan University, Puli, Taiwan
Purpose Hepatitis B virus x (HBx) has been implicated in the
development of hepatocellular carcinoma (HCC) through the
promotion of malignant transformation, upregulation of the
properties of liver cancer stem cells (CSC), and manipulation
of cell cycle progression, but it is not clear how HBx promotes
HCC metastasis. Among CSC markers, CD44 is associated
with migration, proliferation, and invasion, especially CD44v6,
which is critical for metastasis. Herein the effects of HBx on
CD44v6-dependant HCC metastasis was investigated. Methods
Stable tet-on inducible HBx overexpression cells were obtained
by transfection of pLV-CMVTO-GFP-HBx plasmids to HepG2
for studying HCC metastasis. The expression of CD44v6 was
induced by doxycycline (Doxy.) and was determined by immunoblotting,
quantitative real-time PCR (Q-PCR), and flow cytometry.
The metastatic ability of HBx-induced HCC cell lines was
investigated by real-time cell analysis. The effect of HBx/snail
complex on the expression of CD44 splicing factor ESRP1 and
ESRP2 was analyzed. The inhibitory effects of siESRP1 and
siESRP2 were also studied. Xenograft tumor model was used for
in vivo study for the investifation of the association of CD44v6
in metastasis and the splicing factor on HBx expressed tumors.
Results HBx significantly induced the expression of CD44v6
as identified by immunoblotting, Q-PCR, and flow cytometry.
Through interaction with snail, HBx promoted the expression
of downstream signaling CD44-related splicing factor ESRP2,
and restricted ESRP1. Inhibition of snail and ESRP2 but not
ESRP1 markedly reduced the mobility of HBx-expressed cells.
Addtionally, overexpressed N-cadherin induced by HBx but not
E-cadherin was detected, indicating the undergoing of EMT.
MMP2 and 9 were also evaluated for the potential of metastasis.
HBx enhanced the expression of these two metalloproteinases.
Futher metastasis was confirmed by enhanced RhoA-GTP,
a ROCK family protein participating in cell migration/invasion,
determined by IP/IB in HBx expressed HepG2, and cell metastatic
ability as determined by real-time cell monitoring. Based
on these findings, HBx appears to significantly promote HCC
invasion through snail modulated ESRP1/ESRP2, which might
be a driven force to enhance CD44v6-dependent metastatic
ability of HCC. Conclusion HBx was able to promote metastatic
ability in HCC cell line through epigenetic regulation. The metastatic
modification of CD44 variant was through the ESPR1
and ESRP2, that function as specific modulators in CD44 splicing
to maneuver metastatic ability. Inhibition of CD44 splicing
seems to be a potential approach to arrest the progression of
HBV-related HCC metastasis.
Disclosures:
The following authors have nothing to disclose: Ya-Ju Chang, Li-Chen Wu, Ja-an
Annie Ho