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

Abstracts
the pLenti6.3 to generate pLenti6.3-RC-U. Lentiviruses were packaged in
HEK 293T cells. The KRas mRNA and protein expression after transfecting
the pRC-U expression plasmid into human pancreatic cancer
cells or HEK293T cells were detected by real-time reverse transcription
polymerase chain reaction (real-time RT-PCR), Western blotting and
immunofluorescence. After pRC-U transfection, with mg-132 or cycloheximide
treatments, the KRas protein expression was tested by Western
blotting. The interaction between RC-U and KRas, whether KRas could
be ubiquitinated by RC-U or not were both investigated by immunoprecipitation.
The expression of HRas, NRas, phosphorylated extracellular
signal-regulated protein kinases (pERK) and extracellular signal-regulated
protein kinases (ERK) in pancreatic cancer cells was also examined
by Western blotting after pRC-U transfection. The effects of RC-U
on pancreatic cancer cell growth in vitro were detected by CCK-8 and
soft agar colony formation assays after lentivirus infection. In vivo, with
RC-U treatment, the volumes of the subcutaneous xenografts in nude
mice were measured.
Results. RC-U dramatically decreased the protein level of KRas compared
to the controls. No significant change of KRas mRNA expression
within different groups was observed. After pRC-U transfection, the
stability of KRas was significantly increased in the presence of specific
proteasome inhibitor mg-132. HEK293T cells were transfected by mutant
KRas construct together with either pRC-U or empty vector and then
incubated with cycloheximide to inhibit novel protein synthesis. The
exogeneous mutant KRas oncoprotein in pRC-U transfected cells was
degraded more quickly than that in controls. RC-U can bind with KRas.
KRas can be ubiquitinated by RC-U. It was shown that RC-U also degradate
Kras as well as HRas and NRas. The protein expression of pERK was
also decreased, but there was no significant change in total ERK expression.
When the pancreatic cancer cells infected with lentivirus expressing
RC-U, the ability of the cell growth in vitro was decreased. In vivo,
the reduced volumes of the subcutaneous xenografts in nude mice with
RC-U treatment group versus the control group were observed.
Conclusions. Our findings for the first time, implicate the chimeric ubiquitin
ligase RC-U can decrease KRas protein expression. Destruction
of KRas by RC-U through a ubiquitin-dependent, proteasome-mediated
degradation pathway. RC-U degradates HRas and NRas simultaneously.
RC-U inhibited pancreatic cancer cell growth in vitro and in vivo. This
may represent an effective alternative strategy for the targeted therapy of
KRas in pancreatic cancers.
SG-P-126
Neuropilin-2 in progression and therapy resistance of pancreatic
cancer
M . Muders1 , M .J . Stanton2 , S . Dutta2 , P . Hönscheid1 , D . Aust1 , K . Datta2 ,
G .B . Baretton1 1Institute of Pathology, University Hospital “Carl-Gustav-Carus”, Dresden,
2Department of Biochemistry, University of Nebraska Medical Center,
Omaha, United States
Aims. Exocrine pancreatic adenocarcinoma is a deadly disease with
5-year survival rates of 25 to 30 percent in patients without and 10 percent
in patients with lymph node metastasis. Surgical resection is the only
curative treatment option. Unfortunately only 15 to 20 percent of pancreatic
cancer patients are eligible for curative surgical therapy due to
advanced disease at presentation. Therefore, new treatment options are
urgently needed.
Methods. To investigate the role of Neuropilin-2 in therapy resistance
of pancreatic cancer we knocked down Neuropilin-2 and its ligand
VEGF-C by RNA interference in standard pancreatic cancer cells lines
and treated these cell lines with Gemcitabine. After treatment the cell
death was measured using a YO-PRO/PI apoptosis assay. The role of autophagy
in the treatment resistance was tested by a standard autophagic
flux assay.
92 | Der Pathologe · Supplement 1 · 2012
Results. Neuropilin-2 and its ligand VEGF-C are important molecules
of chemotherapy resistance in pancreatic cancer. Furthermore, we have
found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy,
which maintains cancer cell survival following treatment.
Conclusions. Together, these data suggest a link between the VEGF-C/
NRP-2 axis in pancreatic cancer cell progression and therapy resistance.
Effective targeting of this pathway may lead to the development of new
cancer therapies.
SG-P-127
Salinomycin induces autophagic and apoptotic cell death in pancreatic
carcinoma cell lines
M . Vogt1 , B . Verdoodt1 , S .-T . Liffers1 , A . Tannapfel1 , A . Mirmohammadsadegh1 1Ruhr-University of Bochum, Institute of Pathology, Bochum
Aims. Salinomycin a polyether antibiotic, is produced by a strain of
streptomyces albus and has anti-microbial and anti-coccodial activities.
Recently, a number of studies described anti-tumor properties of salinomycin,
in particular its effect on chemoresistant tumor initiating cells.
In the present study, we investigated the impact of salinomycin mediated
activation of MEK/ERK signalling pathway on autophagy and apoptotic
cell death in pancreatic cancer cell lines
Methods. Two pancreatic cell lines MIAPaCa-2 and PaTu8902 were used
to analyze the effect of salinomycin on cell viability, autophagy and
apoptosis. The effect of salinomycin on autophgay was investigated by
transmission electron microscopy (TEM) for detection of autophagic
vesicles and processing of LC3B, microtubule-associated protein 1 light
chain 3 isoform B (LC3B). Towards investigating the role of salinomycin
on apoptotic cell death we used caspase3/7, FACS, Western blot analysis
and immunofluorescence staining.
Results. Salinomycin treatment inhibits cell viability and colony forming
in MIA PaCa-2 and PaTu8902 in a time and concentration depending
manner. In both cell lines salinomycin was able to induce autophagic cell
death, detected by LC3 processing and formation of autophagic vacoules.
Salinomycin was able to induce autophagic and apoptotic cell death
in MIAPaCa-2 cell lines. In MIAPaCa-2 cells the salinomycin induced
autophagic cell death was dependent on ERK1/2 pathway. In contrast,
salinomycin induced autophagic cell death in PaTu8902 was independent
of ERK1/2.
Conclusions. Salinomycin, a novel anti-tumor drug is able to induce autophagic
and apoptotic cell death depending on cellular background.
SG-P-128
Up-regulation of Kindlin-2 promotes progression of human
breast cancer cells by increasing their proliferation, drug resistance,
genomic instability, and tumorigenesis
W . Fang1 , T . Zhao1 , H .-q . Zhang2 1Department of Pathology, Key Laboratory of Carcinogenesis and Translational
Research, Beijing, China, 2Peking University Health Science Center,
Beijing, China
Aims. Kindlin-2 has been confirmed as an essential element of bidirectional
integrin signaling. In recent years, the relationship between Kindlin-2
expression and cancers has been a focus of interest. Our previous
studies have shown that Kindlin-2 expression was up-regulated in several
types of human cancers, and a strong correlation between Kindlin-2
expression and clinical outcome of breast cancer patients was found.
However, the functional role of Kindlin-2 in breast cancer has not been
studied. This study was designed to investigate the role of Kindlin-2 in
the progression of human breast cancer cells.
Methods. Firstly, Kindlin-2 expression at protein level was detected by
Western blot in several breast cancer cell lines. Two luminal-like breast
cancer cell lines, MCF-7 and T47D, expressed low level of Kindlin-2.
Two basal-like breast cancer cell lines, MDA-MB-231 and HS578T, ex-