studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 1171A
expression of keratin 19(K19), epithelial cell adhesion molecule(EpCAM),
matrix metalloproteinase 2/9(MMP2/9) and
CXC chemokine receptor 4(CXCR4) were also down-regulated
in Bel 7402 cells; Migration and invasion, expression of K19,
EpCAM, MMP2/9 and CXCR4 were significant enhanced
when HLE cells(non-AFP-producing) were transfected with AFP
expressed vector. Conclusions: AFP plays a critical role in promoting
metastasis of HCC; AFP is significantly promote HCC
cell invasion and metastasis via up-regulated expression of
metastasis-related proteins. Thus, AFP may be used as a novel
therapeutic target for treating HCC patients.
Disclosures:
The following authors have nothing to disclose: Mingyue Zhu, Junli Guo, Wei
Li, Mengsen Li
1978
Osteopontin-c isoform promotes a mesenchymal phenotype
in human cholangiocarcinoma cells
Marco A. Briones-Orta 1 , Jason D. Coombes 1 , Massimiliano Mellone
7 , Rossella Rispoli 5 , Paul P. Manka 1,3 , Rasha Younis 1 , Naoto
Kitamura 1 , Shannon S. Glaser 6,8 , Gianfranco Alpini 6,8 , Alberto
Quaglia 9 , Roger Williams 1 , Salvatore Papa 4 , Ali Canbay 3 , Wing-
Kin Syn 1,2 ; 1 Regeneration and Repair, The Institute of Hepatology,
London, United Kingdom; 2 Liver Unit, Barts Health NHS Trust, London,
United Kingdom; 3 Gastroenterology and Hepatology, University
Hospital Essen, Essen, Germany; 4 Cell Signaling, The Institute
of Hepatology, London, United Kingdom; 5 Weatherall Institute of
Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom;
6 Research, Central Texas Veterans Health Care Systems,
Temple, TX; 7 Experimental Pathology Group, Cancer Sciences
Unit, Southampton General Hospital, Southampton, United Kingdom;
8 Scott & White Digestive Disease Research Center, Texas
A&M Health Science Center College of Medicine, Temple, TX;
9 Liver Pathology, Kings College London, London, United Kingdom
Introduction: Osteopontin (OPN) is upregulated in tissue fibrosis
and cancer. The OPN gene generates 3 isoforms (a, b and
c) by alternative splicing, which can induce differing cellular
responses in tumorigenesis. Although these isoforms have been
investigated in breast, prostate, and ovarian cancers, studies in
cholangiocarcinoma (CCA) have been limited to OPN-a (total
OPN). We hypothesized that OPN isoforms are overexpressed
in CCA, and the pattern of isoform overexpression is associated
with either a more or less aggressive phenotype. Crosstalk
between OPN and TGF-b signaling was evaluated as both
are key modulators of cancer progression. Methods: HuCCT1,
SG231 and CCLP1, human intrahepatic cholangiocarcinoma
cell lines were used. Plasmids for each OPN isoform were used
for overexpression. Expression of OPN-a, b, c, and epithelial-mesenchymal
transition (EMT) markers (vimentin, aSMA,
E-cadherin) were evaluated by qRT-PCR. Global gene expression
changes were examined by microarray using SG231.
OPN isoform overexpression and their effects on the components
of TGF-b pathway were evaluated by immunoblots. To
evaluate the in vivo significance of the individual isoforms, a
xenograft model was used. FFPE human cholangiocarcinoma
and healthy liver sections were stained for total OPN. Results:
Total OPN was upregulated in cholangiocarcinoma. CCLP1
cells expressed the highest levels of total OPN and exhibited
the most mesenchymal phenotype (high vimentin and low
E-cadherin). In contrast, HuCCT1 expressed the lowest amount
of OPN and exhibited an epithelial phenotype (high E-cadherin
and low vimentin). In all 3 cell lines, OPN-a, and b mRNA
were more abundant than OPN-c (~10 fold). Overexpression
of OPN-c led to the greatest increase in mesenchymal genes
and proteins (vimentin and aSMA), and was associated with
the highest level of TGF-β signalling as shown by Smad2/3
phosphorylation. Microarray confirmed that overexpression
of OPN-c induced the largest alterations in gene expression
profile, and an enrichment analysis revealed that OPN modulated
genes which regulate phosphoprotein and acetylation. In
the xenograft model, overexpression of all OPN isoforms was
associated with greater rates of tumour growth than control,
but OPN-c had the greatest rate. Conclusions: OPN expression
correlates with the degree of EMT (marker of aggressiveness)
in human cholangiocarcinoma. OPN-c is associated with the
most mesenchymal phenotype and TGF-β pathway activation.
OPN-c also induces greater changes in the transcriptome and
supports greater rates of tumor growth than OPN-a, and -b.
High OPN-c expression may be a predictor of worse clinical
outcomes.
Disclosures:
The following authors have nothing to disclose: Marco A. Briones-Orta, Jason D.
Coombes, Massimiliano Mellone, Rossella Rispoli, Paul P. Manka, Rasha Younis,
Naoto Kitamura, Shannon S. Glaser, Gianfranco Alpini, Alberto Quaglia, Roger
Williams, Salvatore Papa, Ali Canbay, Wing-Kin Syn
1979
Glypican 3 (GPC3)-CD81 axis in regulation Hippo pathway
in hepatocytes and hepatocellular carcinoma (HCC)
Yuhua Xue, Kelly Koral, William C. Bowen, Anne Orr, Meagan
Haynes, Wendy M. Mars, George K. Michalopoulos; Pathology,
University of Pittsburgh, Pittsburgh, PA
GPC3 is over-expressed in HCC. Mutations of GPC3 in humans
are the cause of Simpson-Golabi-Behmel syndrome, associated
to organomegaly. Previous studies have shown that GPC3 is
associated with inhibition of hepatocyte growth and GPC3
transgenic mice with targeted expression in hepatocytes have
decreased liver regeneration. We have also shown that GPC3
binds to the membrane tetraspanin CD81, which is one of the
portals of entry of hepatitis C virus (HCV). Studies elsewhere
have shown that activation of CD81 is associated with phosphorylation
of Ezrin. The latter has been shown to regulate
the Hippo pathway, responsible for regulating nuclear levels
of the protein known as Yes-associated protein (Yap). Expression
of Yap in the nucleus relates to activation of hepatocyte
growth. Mice over-expressing GPC3 have decreased levels of
nuclear Yap. Since we have demonstrated that GPC3 binds
to CD81, the purpose of this study is to explore the role of
GPC3 in regulation of Yap via CD81 and Hippo pathway and
to investigate the possible mechanisms. We found that treatment
of primary rat hepatocytes with CD81 agonist antibody
led to higher levels of phosphorylated Ezrin and lower Hippo
activity. Partial hepatectomy (PHx) exhibited that loss of GPC3
antagonistic modulation during early liver regeneration, CD81
activated Ezrin and down regulated Hippo activity in vivo. Rat
hepatoma cell lines JM1 and JM2 had dramatically down regulated
CD81 expression and Hippo activity, and up regulated
Ezrin activity in vitro. Re-gain of CD81 expression in JM2 rat
hepatoma cells up regulated Hippo activity while down regulated
Ezrin activity in vitro. Human HCC tissue array revealed
absence of CD81 in most HCCs but present in all the normal
tissue controls, while GPC3 was present in most HCCs and all
the normal tissue controls. Treatment with HCV envelope protein
E2 led to more Hippo activity and decrease in nuclear Yap
in HepaRG cells and primary human hepatocytes. Conclusions:
The CD81-phosphoEzrin signaling enhances nuclear Yap levels.
This pathway in normal hepatocytes is inhibited by GPC3.
In human hepatocytes, GPC3 and the E2 protein of HCV also
inhibit the CD81-phosphoEzrin effect on Hippo pathway and
cause a decrease in nuclear Yap. Since most HCC lack expression
of CD81, they are likely to escape from antagonistic mod-