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

SO-011
Methylation profiling and integrated genomic and transcriptional
analysis reveal new tumor suppressor genes of human
hepatocarcinogenesis
O . Neumann 1 , M . Kesselmeier 2 , B . Radlwimmer 3 , P . Schemmer 4 , P . Lichter 3 ,
P . Schirmacher 1 , J . Lorenzo Bermejo 2 , T . Longerich 1
1 University Hospital Heidelberg/Institute of Pathology, Heidelberg, 2 Institute
of Medical Biometry and Informatics, University Heidelberg, Heidelberg,
3 Division of Molecular Genetics, German Cancer Research Centre,
Heidelberg, 4 Department of General, Visceral and Transplantation Surgery,
University Hospital Heidelberg, Heidelberg
Aims. We aimed at the identification of new tumor suppressor gene candidates
of human hepatocarcinogenesis by vertical integration of genome-wide
array-based CGH, methylation, and expression data from a
cohort of well-characterized human hepatocellular carcinomas (HCC).
Methods. Bisulfite converted DNA from 64 HCCs and 10 normal control
livers was analyzed for the methylation status of about 14,000 genes
using the Illumina Infinium 27k Methylation array. After determining
the differentially methylated genes between HCC and normal liver, we
integrated their genomic alterations as previously determined by array-CGH-data.
The gene set that contained the genes with both hypermethylation
and regional genomic losses was than correlated with gene
expression data to select genes potentially silenced by promoter hypermethylation.
Aberrant methylation of selected candidates was verified
by pyrosequencing and methylation-dependent gene silencing was validated
after treatment of suitable HCC cell lines with 5’-Azacytidine.
Results. Methylation profiling revealed 2239 CpG-sites differentially
methylated between normal control liver and HCCs. 540 CpG-sites of
these were hypermethylated, whereas 1699 showed promoter hypomethylation.
The hypermethylated group was enriched for genes known to
be inactivated by the polycomb repressor complex 2 (PRC2), while the
group of hypomethyated genes was enriched for imprinted genes showing
loss of imprinting in the HCC samples. We identified 18 candidate
genes matching both criteria hypermethylation and regional genomic
loss. After integrating the expression data three candidates finally remained.
Functional characterization of one of these promising candidates
after re-expression in vitro was performed and the data will be presented
during the meeting.
Conclusions. Our data shows that vertical integration of methylation data
with high resolution genomic and transcriptomic data is suitable for the
identification of new tumors suppressor genes in human HCCs.
SO-012
AKT and N-Ras co-activation in the mouse liver promotes rapid
development of hepatocellular carcinomas that are sensitive to
Rapamycin treatment
D .F . Calvisi1 , C . Ho2 , C . Wang2 , S . Mattu1 , G . Destefanis1 , S . Delogu1 ,
J . Armbruster1 , X . Chen2 , F . Dombrowski1 , M . Evert1 1University Medicine Greifswald, Institute for Pathology, Greifswald,
2University of San Francisco, Liver Center, San Francisco, United States
Aims. Activation of AKT and Ras pathways is often implicated in carcinogenesis.
As yet unknown, the aim of this study is to unravel the
mechanisms, underlying the oncogenic cooperation between these two
cascades in relationship to hepatocellular carcinoma (HCC).
Methods. Therefore, we generated a mouse model characterized by combined
overexpression of activated forms of AKT and N-Ras protooncogenes
in the liver via hydrodynamic transfection. Hepatocarcinogenesis
and the anti-tumorigenic effect of Rapamycin treatment was investigated
by morphological and molecular methods and these findings were
verified in vitro, using HCC cell lines.
Results. Co-expression of AKT and N-Ras resulted in a dramatic acceleration
of liver tumor development when compared with mice overexpressing
AKT alone, whereas N-Ras alone did not lead to tumor for-
mation. Accelerated hepatocarcinogenesis driven by AKT and N-Ras
resulted from a strong activation of mammalian target of rapamycin
complex 1 (mTORC1). Furthermore, elevated expression of FOXM1/
SKP2 and c-Myc also contributed to rapid tumor growth in AKT/Ras
mice, yet via mTORC1-independent mechanisms. Of note, treatment of
AKT/Ras mice with the mTORC1 inhibitor Rapamycin was able both to
strongly constrain the growth of AKT/Ras preneoplastic lesions and to
impede malignant transformation. However, liver tumors rapidly emerged
in AKT/Ras mice following Rapamycin withdrawal. This was associated
with induction of the MAPK/ERK cascade. The biological effects
of co-activation of AKT and N-Ras can be recapitulated in vitro using
HCC cell lines which supported the functional significance of mTORC1,
FOXM1/SKP2 and c-Myc signaling cascades in mediating AKT- and N-
Ras-induced liver tumor development.
Conclusions. Thus, our data demonstrate the in vivo crosstalk between
the AKT and Ras pathways in promoting liver tumor development, and
the pivotal role of mTORC1-dependent and independent pathways in
mediating AKT- and Ras-induced hepatocarcinogenesis.
SO-013
Contribution of keratin-18 in SH- and SH-induced HCC development
A .K . Mehta1 , K . Bettermann1 , E . Lederer1 , C . Diwoky2 , A . Thüringer1 ,
C . Stumptner1 , H . Mueller1 , T . Kolbe3 , T .M . Magin4 , C . Lackner1 , H . Denk1 ,
K . Zatloukal1 , J . Haybaeck1 1 2 Medical University of Graz, Graz, Austria, Graz University of Technology,
Austria, 3University of Veterinary Medicine Vienna, Biomodels Austria (Biat),
Vienna, Austria, 4University of Leipzig, Translational Centre for Regenerative
Medicine Leipzig, Leipzig
Aims. Steatohepatitis (SH) is a liver disease morphologically characterized
by steatosis, hepatocyte ballooning and occurrence of cytoplasmic
protein aggregates termed Mallory-Denk bodies (MDBs). SH affects about
20% of alcoholics and up to 50% of obese type II diabetics. MDBs
mainly consist of misfolded keratin (K) 8, 18 and in part p62 and ubiquitin.
Keratin aggregates are known to be essential for MDB formation
and thereby link keratins to SH which is a major precondition for the
development of liver cirrhosis and hepatocellular carcinoma (HCC). In
this study we aimed at elucidating the pathophysiological and molecular
mechanisms of loss of K18 on hepatocarcinogenesis in mice and its functional
implication in human NASH-induced liver cancer.
Methods. 17 month-old krt18-deficient (krt18-/-) mice (129P2/Ola background)
were investigated for the occurrence of SH- and SH-induced
liver tumors by radiology, histology, immunohistochemistry, gene expression
analysis and immunoblotting.
Results. Aged krt18-/- mice developed the entire morphological spectrum
of SH whereas aged wild-type mice displayed simple steatosis.
Aminotransaminase levels were also elevated in aged krt18-/- mice. Interestingly,
91% of male and 46% of 17 months-old krt18-/- female mice
developed liver tumors revealing morphological and genetic features of
HCC. Moreover, 75% of male and 36% of female age-matched krt18+/-
mice developed HCC.
Conclusions. Aged krt18-/- mice represent a new spontaneous SH- and
SH-driven HCC model. Alterations of hepatocellular K18 therefore seem
to determine the susceptibility towards SH and SH-induced HCC.
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