96. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V ...

HCV-positive formalin-fixed and paraffin-embedded biopsies, sections
were prepared and HCC were macrodissected. Subsequently, miR-125b
was analyzed by real-time PCR. Putative miR-125b binding sites were fused
to the luciferase reporter and reporter assays were carried out with
miR-125b treated hepatoma cells.
Results. During development of mouse HCC, the expression of miR-125b
progressively decreased. In agreement miR-125b was reduced in human
hepatoma cells in comparison to normal liver. Furthermore, miR-125b
decrease depending on the progression of hepatocarcinogenesis was
confirmed in human samples, showing significant lower levels in HCC
high grades than in dysplastic foci or cirrhosis. Overexpression of miR-
125b in Hep3B and Pop10 cells resulted in a pronounced reduction of cell
growth. Screening of putative miR-125b target transcripts by various
algorithm calculations identified various pathways involved in proliferation
and apoptosis. Reporter assays of 3’-UTR-regions of the putative
targets identified miR-125b binding sites in lin-28 mRNA. Since lin28 is
known to effect synthesis of the mitogen IGF-II, the miR-125b/lin28 axis
is suggested to be involved in HCC pathogenesis by IGF-II mediated regulation
of cell growth.
Conclusions. Expression of miR-125b is down-regulated during progression
of hepatocarcinogenesis leading to up-regulation of lin-28 that in
turn triggers enhanced cell growth and proliferation.
DO-005
The PI3K-AKT-mTOR axis contributes to the functional inactivation
of p53 through stabilization of MDM4 in human hepatocellular
carcinoma
R . Pellegrino1 , O . Neumann1 , P . Schirmacher1 , T . Longerich1 1University Hospital Heidelberg/Institute of Pathology, Heidelberg
Aims. Mutational inactivation of p53 gene is rare in Western hepatocellular
carcinoma (HCC). MDM4, one of the main p53-regulating factors,
is frequently upregulated in human HCC. This overexpression can be
in part explained by chromosomal gains at 1q34.1. Here we investigated
the role of the PI3K-AKT axis in the stabilization of the MDM4 protein.
Methods. All experiments were performed in human HCC cell lines
with different p53 gene status. PI3K and mTOR were specifically inhibited
using chemical compounds in vitro; specific siRNAs were transiently
transfected to target AKT1 and ATK2 and to validate the results from
drug treatment. Realtime RT-PCR analysis was used to check for restored
p53 transcriptional activity. In addition, combined cycloheximide
and siRNAs treatment was performed to study the protein stability of
MDM4 under these experimental conditions.
Results. Using a specific PI3K inhibitor or siRNAs targeting AKT1/2 in
HCC cell lines, we observed a strong decrease of MDM4 protein levels,
which resulted in the activation of p53 target genes (e.g. PUMA, BAX and
p21) indicating restored p53 gene function in these lines. Cycloheximide
treatment combined with siRNA-mediated AKT inhibition indicated
that MDM4 is phosphorylated by AKT2 and that this phosphorylation
is responsible for the stabilization of the protein via the protection from
proteasomal degradation. This effect was independent from both MDM2
and p53 gene status. Furthermore, we showed that the Eukaryotic translational
Elongation Factor 1 alpha 2 (EEF1A2), which we reported upregulated
in human HCC, is involved in the activation of the PI3K-AKT
axis. Specific siRNA inhibition of EEF1A2 resulted in decreased pAKT
and MDM4 protein levels in HCC cell lines. Moreover, treatment with
the mTOR inhibitors, Rapamycin and PI-103, decreased MDM4 protein
levels indicating that the PI3K-AKT-mTOR axis is involved in the
MDM4 regulation in vitro.
Conclusions. Our data demonstrate that the EEF1A2-PI3K-AKT-mTOR
axis is involved in maintaining protumorigenic MDM4 levels in human
HCC cell lines, which in turn promotes functional inactivation of
p53. Moreover, we showed that the AKT-mediated phosphorylation of
MDM4 is the crucial mechanism to prevent its proteasomal degradation.
DO-006
HSF1 is a downstream effector of Ras and AKT protooncogenes
and contributes to hepatocellular carcinoma development and
progression
D .F . Calvisi1 , S . Mattu1 , S . Delogu1 , V . De Murtas1 , G . Gasparetti1 , G . Destefanis1 ,
X . Chen2 , F . Dombrowski1 , M . Evert1 1University Medicine Greifswald, Institute for Pathology, Greifswald,
2University of San Francisco, Liver Center, San Francisco, United States
Aims. Recent evidence suggests an oncogenic role of heat shock transcription
factor 1 (HSF1) in cancer, but its functional relevance in hepatocellular
carcinoma (HCC) remains poorly delineated.
Methods. We have investigated HSF1 function both via in vitro and in
vivo approaches as well as in a collection of human HCC.
Results. In human liver specimens, we found that HSF1 was progressively
induced from non-tumorous surrounding livers to HCC, reaching the
highest levels in tumors with a poorer outcome (as defined by the length
of patient’s survival). In HCC cell lines, overexpression of HSF1 resulted
in increased activity of MAPK and AKT/mTOR pathways and suppression
of JNK cascade, leading to augmented proliferation and angiogenesis
and reduced apoptosis in vitro. Conversely, suppression of HSF1 in
HCC cell lines decreased MAPK and AKT/mTOR activity, and induced
JNK-dependent apoptosis. Forced overexpression of either Ras or AKT
protooncogenes triggered upregulation of HSF1 in HCC cell lines via the
small RalA GTPase. Of note, HSF1-overexpressing cells were specifically
sensitive to growth inhibition and induction of apoptosis following the
treatment with either AMPK activators or hexokinase inhibitors. Finally,
overexpression of a HSF1 dominant negative form by hydrodynamic
gene delivery strongly reduced the oncogenic potential of activated Ras
and AKT in a mouse model of aggressive liver cancer.
Conclusions. Altogether, the present data indicate that activation of HSF1
plays a major role in hepatocarcinogenesis by enhancing the activity of
Ras and AKT, and might represent a valuable candidate for innovative
targeted therapies against human HCC.
DO-007
Perturbation of hepatocytes metabolism by AKT contributes to
growth in insulin-induced hepatocarcinogenesis and is reverted
by the PI3K/mTOR dual inhibitor NVP-BEZ235
M . Evert1 , D .F . Calvisi1 , K . Evert1 , V . De Murtas1 , G . Gasparetti1 , S . Mattu1 ,
G . Destefanis1 , S . Thiel1 , A . Thiele1 , S . Ribback1 , F . Dombrowski1 1University Medicine Greifswald, Institute for Pathology, Greifswald
Aims. Mounting evidence supports a role of insulin signaling deregulation
and diabetes mellitus in human hepatocarcinogenesis. To study the
oncogenic effect of chronically elevated secretion of insulin on hepatocytes
in the presence of mild hyperglycemia, we developed a model of
pancreatic islet transplantation into the liver.
Methods. In this model, islets of a donor rat are transplanted into the
liver of a recipient diabetic rat, with resulting local hyperinsulinism that
leads to the development of preneoplastic lesions and hepatocellular carcinoma
(HCC). Here, we investigated the metabolic and growth properties
of the AKT pathway in this model of insulin-induced hepatocarcinogenesis.
These findings were recapitulated in HCC cell lines in vitro.
Results. We found that activation of insulin signaling triggers a strong
induction of the AKT cascade that is paralleled by increased synthesis
of fatty acids, cholesterol, and triglycerides, induction of glycolysis and
decrease of fatty acid oxidation and gluconeogenesis in rat preneoplastic
and neoplastic liver lesions when compared with normal liver. AKT-dependent
metabolic effects of insulin on hepatocytes were recapitulated in
vitro using human HCC cell lines. In these cells, suppression of lipogenesis,
glycolysis, and the pentose phosphate pathway triggered a strong
growth restraint despite insulin administration. Of note, metabolic abnormalities
and proliferation driven by insulin were effectively reverted
Der Pathologe · Supplement 1 · 2012 |
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