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

subgroup of patients with B-Raf mutation will likely benefit, and that
due to the robustness of the healthy cells that have no B-Raf mutation
side effects might be minimal. We believe that analysing robustness of
other signalling pathways in a similar way will be the key to devise efficient
targeted interventions for these, and will unveil which mutations in
the pathway will break robustness and thereby open the door for efficient
intervention.
DO-121
The nuclear localization and transcriptional activation of
β-catenin are independent of each other
S . Ormanns1 , T . Kirchner1 , A . Jung1 1Ludwig-Maximilians-University, Institute of Pathology, München
Aims. Mutations in components of the Wnt signaling pathway are the
drivers of carcinogenesis in the majority of colorectal cancers. They
result in the accumulation of the transcription factor β-catenin which
exerts its function by the induction of the hallmarks of cancer like epithelio-mesenchymal
transition, stemness, chemoresistance, proliferation,
invasion or apoptosis besides others. The transcriptional activity
of β-catenin depends on its nuclear localization and posttranslational
modifications. As it is unknown how both processes are regulated, we
asked if the nuclear accumulation of β-catenin and its activation induced
via the PI3K-AKT signaling pathway were independent of each other.
Methods. To discriminate between the nuclear localization and additional
activation steps of β-catenin an experimental cell culture system
was designed that allowed the forced nuclear translocation of β-catenin
independent of additional activation steps. The subcellular localization
of β-catenin was assessed by immunofluorescence. The transcriptional
activity of β-catenin was determined by TOP-flash luciferase reporter
gene assays. The activity of PI3K-AKT was interfered by the specific inhibitor
LY294.002.
Results. Inhibiting PI3K/AKT lead to a significant dose-dependent reduction
of endogenous β-catenin activity in the cell lines SW480 and
RWP-1 harbouring inactivating mutations in the APC gene. Conversely,
stimulating the Wnt-signaling pathway or adding degradation resistant
β-catenin both resulted in the activation of β-catenin in the cell lines
293T, CHO or HeLa. Here, the nuclear translocation of β-catenin also
resulted in an activation of its transcriptional activity which could be
blocked by inhibiting PI3K. In contrast the nuclear translocation of β-catenin
did not result in transcriptional activity in A431 cells.
Conclusions. We provide experimental evidence that the nuclear transport
and the transcriptional transactivation of β-catenin are independent
processes. Thus, β-catenin signaling depends at least on active PI3K/
AKT signaling. Taken together, the transcriptional activity of β-catenin
is regulated at least by two signals which might open the opportunity
for clinically interfering with the hallmark of CRC, the activation of the
β-catenin pathway.
DO-122
Activation of the EGFR-MAPK signaling pathway is dependent on
FAM125 proteins
S . Müller1 , G . Baretton2 , G . Fitze1 , M . Haase3 1 2 TU Dresden, Pediatric Surgery, Dresden, TU Dresden, Pathology, Dresden,
3TU Dresden, Pediatric Surgery and Pathology, Dresden
Aims. Ionizing radiation leads to complex changes in tissues such as changes
in cell survival, cell differentiation and loss of function. In order to
find proteins that are overexpressed in irradiated tissue, we constructed
a differential cDNA library. Among other proteins, we found FAM125A
(family 125A), a protein component of transport vesicles that has been
reported to play a role in the internalization of epidermal growth factor
receptor (EGFR). The aim of the study was to get further insights into the
function of FAM125 proteins.
Methods. A differential cDNA library was constructed from cDNA obtained
from radiated lung tissue of the rat. mRNA expression analysis
was done by quantitative RT-PCR. Protein expression was quantified
by western blot analysis. Tissue distribution was analyzed by immunohistochemistry
on tissue microarrays (TMAs). Down-regulation of
mRNA/proteins was achieved by stable transfection of sh-RNA vectors
into HELA cells. Protein extracts from these cells were prepared from
the membrane, cytoplasmic and nuclear fractions.
Results. FAM125A protein is expressed in most cell types. A very high
expression is seen in tissues with very active membrane transport processes
such as renal tubule cells and glandular cells. FAM125A mRNA
and protein are overexpressed in irradiated tissue. Down-regulation of
FAM125A and B leads to a decrease of total EGFR and phosphorylated
EGFR (Y1045 and Y1068) in the membrane fraction. In addition, it leads
to an accumulation of phosphorylated Akt (S473) and c-Src (T416)
in the membrane fraction whereas phosphorylation of p42/44 MAPK
(T202-Y204) is decreased.
Conclusions. FAM125 proteins seem to play a role in transport processes
of molecules including signaling proteins. Down-regulation of EGFRactivity
correlates with decreased activity of the p42/44 MAPK pathway
whereas Akt and c-Src activity are not affected. This suggests that the
EGFR-MAPK pathway is dependent on FAM proteins whereas the Akt
and c-Src pathways act independently. Further research should clarify
the association of various signaling molecules to transport vesicles and
should provide an insight into signaling processes in radiation-damaged
cells.
DO-123
DUSP4 expression increases cell proliferation in colorectal cancer
(CRC) cells and is associated with microsatellite instability in CRC
B . Gröschl1 , M . Bettstetter1 , W . Dietmaier1 1University of Regensburg, Institute of Pathology, Regensburg
Aims. DUSP4, a member of the mitogen-activated protein kinase phosphatase
(MKP) family and a potential tumor suppressor, negatively regulates
the MAPKs (Mitogen-activated protein kinases) ERK, p38 and
JNK which play a crucial role in cancer development and progression.
Our aim was to investigate DUSP4 expression in high frequent microsatellite
unstable (MSI-H) and microsatellite stable (MSS) colorectal
cancers (CRC) as well as its influence on potential MAPK downstream
targets and its effect on the proliferation in CRC cells.
Methods. We studied DUSP4 mRNA levels in 19 MSI-H and 19 MSS CRC
compared to matched normal tissue as well as in CRC cell lines by RTqPCR.
Promotor methylation of the DUSP4 gene was analyzed using
Methy-QESD (Quantification of Endonuclease-Resistent DNA) and
coding regions were assessed for mutations through Sanger sequencing.
We overexpressed DUSP4 in CRC cell lines and analyzed expression of
potential downstream target genes as well as cell growth by Real-Time
Cell Analysis (RTCA).
Results. DUSP4 mRNA was elevated in all 19 MSI-H tumors and in
14 MSS tumors. Median expression levels in MSI-H tumors were significantly
higher than in MSS-tumors (p