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

Abstracts
our study to identify overlapping molecular characteristics in a variety
of histologically defined lymphoma entities. To achieve this goal, comprehensive
expression profiles of non-coding and coding transcripts derived
from a broad spectrum of lymphomas were established. Our data
show that overlapping molecular features can be identified beyond the
current lymphoma classification.
Methods. RNA was extracted from frozen tissue blocks of distinct human
B- and T-cell lymphoma entities as well as tonsils (n=116). The small
(micro) RNA fraction was sequenced by next generation technology
(SOLiD) and total RNA was subjected to Affymetrix Exon 1.0 ST array
hybridisation. In addition immunohistochemical and clinical data was
acquired. Bioinformatic analyses were then applied for subgroup detection.
Results. Unsupervised clustering based on the mature micro RNA expression
derived from deep sequencing demonstrated the presence of
two distinct large clusters within the case collection. One cluster contained
predominantly the so-called indolent lymphoma types, but also a
considerable number of aggressive B-cell lymphoma cases. In the second
cluster the majority of cases were aggressive B-cell lymphoma but interestingly
intermingled with the T-cell lymphoma cases. Differentially
expressed micro RNAs between the two clusters were determined and
logistic regression identified a micro RNA classificator able to distinguish
the two groups. The micro RNA expression data was combined with
the coding RNA data in order to identify the involved pathways.
Conclusions. Micro RNA expression profiles obtained by deep sequencing
were able to identify two groups within a lymphoma collection that
separated the cases beyond the histopathological classification system.
Discriminative micro RNAs and associated pathways were identified.
These findings give new insights into lymphoma biology and point to
alternative treatment options.
*These authors contributed equally to the study .
Poster
Poster: Deutsch-Chinesisches Symposium
SG-P-112
Mitochondrial mortalin (HSPA9) expression in enterocytes is
regulated by ACSL5
N . Gaßler 1 , C . Klaus 1 , E . Kämmerer 2 , M . Adolf 1 , A . Reinartz 1
1 RWTH Aachen University, Institute of Pathology, Aachen, 2 RWTH Aachen
University, Klinik für Kinder- und Jugendmedizin, Aachen
Aims. Mitochondrial acyl-CoA synthetase 5 (ACSL5), a long-chain fatty
acid activating enzyme, is preferentially found in small intestinal enterocytes.
ACSL5 activities are associated with complex changes in the
mitochondrial lipid metabolism and are important for epithelial differentiation
and cell death. The aim of the present study was to identify
and characterize ACSL5 related regulation of mitochondrial proteins.
Methods. Mitochondria isolated from ACSL5 transfected CaCo2 cells
and controls were homogenized and further separated with 2D-gel
electrophoresis. Spots were analyzed using special proteomics software.
Protein spots differentially expressed were further identified with MAL-
DI-TOF. Additional techniques were used for target validation. Laser microdissected
enterocytes from normal human small intestinal mucosa
were investigated to substantiate the in vitro findings.
Results. 14 mitochondrial protein species, probably regulated by ACSL5,
were identified. ACSL5 dependent expression and synthesis of the candidate
molecule mortalin (HSPA9) was confirmed using qRT-PCR,
Western blotting, and siRNA ACSL5 knockdown. Using this approach,
a positive correlation of ACSL5 and mortalin expression was verified.
The positive correlation of ACSL5 and mortalin expression was additio-
88 | Der Pathologe · Supplement 1 · 2012
nally found in human normal small intestinal mucosa. Strong mortalin
expression was seen in ACSL5-rich enterocytes isolated from intestinal
villi, whereas mortalin was diminished in low ACSL5 expressing enterocytes
isolated from intestinal crypts.
Conclusions. In conclusion, ACSL5 activities are associated with changes
in lipid metabolism as well as expression of mitochondrial proteins.
ACSL5-dependent expression and synthesis of mitochondrial mortalin
is probably a phenomenon of stress-response.
SG-P-113
ACSL5 as putative modifier of Wnt activity in intestinal cells
C . Klaus1 , U . Schneider1 , R . Knuechel1 , N . Gaßler1 1RWTH Aachen University, Institute of Pathology, Aachen
Aims. The mitochondrial localized Acyl-CoA synthetase 5 (ACSL5) converts
free long-chain fatty acids into fatty acyl-CoA esters, and thereby
plays a key role in lipid biosynthesis and fatty acid degradation. In particular,
ACSL5 has been recently identified to be involved in apoptotic
cell death of senescent enterocytes along the intestinal crypt-villus axis
and to interact with mitochondrial proteins. The aim of this study was
to investigate ACSL5-dependent effects on intestinal signalling pathways
that coordinate proliferation and/or differentiation of enterocytes, particularly
the Wnt pathway.
Methods. Wnt signalling was analysed in an ACSL5 overexpressing cell
culture model using luciferase assays, immunohistochemistry, qRT-PCR
and Western blot. The findings were substantiated with expression studies
in human colon carcinomas and in a Wnt-associated mouse model.
Results. ACSL5 transgenic intestinal-derived cells displayed a strong
susceptibility to pro-apoptotic stimuli. This phenomenon was accompanied
by caspase-3 activation and significant down-regulation of Wnt
signalling activity. In particular, Wnt pathway associated mitochondrial
localized molecules were identified as interaction partners of ACSL5 activity.
Conclusions. Molecular mechanisms underlying ACSL5-dependent
apoptosis susceptibility of enterocytes are probably bivalent including
pro-apoptotic and anti-proliferative activities.
SG-P-114
MAPK signaling regulates differential WNT activity in colorectal
cancer
D . Horst1 , J . Chen2 , T . Kirchner1 , R . Shivdasani2 1Ludwig-Maximilians-Universität München, Pathologisches Instiut,
München, 2Harvard Medical School, Dana-Farber Cancer Institute, Boston,
United States
Aims. Most colorectal cancers (CRCs) express the WNT effector protein
β-catenin in a heterogeneous pattern. Strong nuclear expression is
often confined to a small fraction of tumor cells at the tumor’s leading
edge. Recent data suggest a role for Mitogen Activated Protein Kinase
(MAPK) signaling in nuclear accumulation of β-catenin. We therefore
investigated if MAPK activity regulates overtly differential WNT activity
in CRC cell subpopulations.
Methods. We used gene expression profiling, and immunohistochemistry
to assess interdependence of MAPK and WNT pathway activity in
CRC. Lentivirus- and drug-based pathway modification in CRC xenograft
tumors of primary human colon cancers and colon cancer cell lines
was used to study the effect of MAPK activation or repression on differential
WNT activity.
Results. CRC cells with high WNT activity showed coincident overexpression
of MAPK target genes and high levels of phospho-ERK, indicating
active MAPK signaling. Forced MAPK activation by lentiviral
expression of constitutively active KRAS enhanced WNT pathway activity
in vivo in CRC xenograft tumors, whereas drug based inhibition of
EGFR signaling attenuated it.