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

Abstracts
SG-140
Sialinomycin induces autophagic and apoptotic cell death in
pancreatic carcinoma cell lines
M . Vogt 1 , B . Verdoodt 1 , S .-T . Liffers 1 , A . Tannapfel 1 , A . Mirmohammadsadegh 1
1 Ruhr-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.
Mammary Cancer
SG-141
Kindlin2 up-regulation promotes tumor progression
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, expressed
moderate levels of the protein. Then, Kindlin-2 gene was overexpressed
by transfected into MCF-7 cells. In comparison, short hairpin
RNA (ShRNA)-mediated knockdown of Kindlin-2 was performed in
HS578T cells. Vector controls were also done in the same cell lines. Ki67
Li, FCM cell cycle, anchorage-independent colony formation (in vitro
tumorigenesis), and in vivo tumorigenesis in NOD/SCID mice were observed.
Apoptotic cells were labeled by fluorescent annexin V assay and
86 | Der Pathologe · Supplement 1 · 2012
quantified by FACS. Array CGH analysis and spectral karyotyping were
performed to detect the genomic instability of these cells.
Results. The growth rate of Kindlin-2-transfected MCF-7 cells was much
quicker than that of the controls. The proportion of G2-M phase cells,
clone formation and tumorigenicity were significantly higher than these
of the controls. The change of Kindlin-2-ShRNA transfected cells was
just the reverse. Moreover, Up-regulation of Kindlin-2 can also reduce
the rate of apoptosis induced by the chemotherapy drugs, and these cells
showed much more genomic instability compared with the controls.
Conclusions. These findings suggested that up-regulation of Kindlin-2
promotes the progression of human breast cancer cells by increasing
their proliferation, drug resistance, genomic instability, and tumorigenesis.
SG-142
ECRG4 is frequently downregulated by CpG island hypermethylation
in human breast cancer
W . Zhang1 1Zhejiang University, Institute of Pathology, Hangzhou, China
Aims. Esophageal cancer related gene4 (ECRG4) is a recently reported
candidate tumor suppressor gene frequently hypermethylated in several
human tumor types, including esophageal squamous cell carcinoma,
colorectal carcinoma, glioma and prostatic carcinoma. This study is to
investigate if ECRG4 is transcriptionally silenced by promoter CpG island
methylation in breast cancer.
Methods. We analyzed several breast cell lines and 12 pairs of fresh samples
from breast cancer patients for ECRG4 promoter CpG island methylation
by COBRA and bisulfite sequencing. ECRG4 mRNA and protein
expression analysis was carried out by semi-quantitative RT-PCR, realtime
PCR, Western Blot and immunohistochemistry.
Results. We found that all of three immortalized normal breast cell lines
and three normal breast tissues expressed ECRG4 proteins, whereas
ECRG4 expression were reduced or absent in most breast cancer cell lines
and primary breast cancer samples. We also identified that ECRG4
promoter was frequently methylated in breast cancer samples. An inverse
correlation between mRNA expression and methylation status of
the ECRG4 promoter CpG island was observed in primary breast cancer
samples.
Conclusions. The expression of ECRG4 is frequently decreased due to
promoter CpG island hypermethylation in breast cancer.
SG-143
Novel biomarkers in breast carcinoma: DKK3, ITIH5, SFRP-1
V . Kloten1 , B . Becker1 , M .G . Schrauder 2 , P .A . Fasching 2 , A . Hartmann3 ,
J . Veeck1 , R . Knüchel1 , E . Dahl1 1University Hospital of the RWTH Aachen, Institute of Pathology, Aachen,
2University Hospital Erlangen, University Breast Center, Erlangen,
3University Hospital Erlangen, Erlangen
Aims. For early detection of breast cancer the development of robust
blood-based biomarkers that accurately reflect the host tumor is mandatory
and thus a growing field of research. The most common alterations
in human cancers including breast cancer are changes in the status of
DNA methylation, which are therefore quickly emerging as a new pool
of potential biomarkers. Thus, we investigated the feasibility of detecting
aberrant tumor suppressor gene methylation in cancer cell-derived free
circulating DNA in the bloodstream of patients.
Methods. Using qualitative MSP, we examined the methylation status of
six biologically significant putative tumor suppressor genes, i.e. ITIH5,
DKK3, WIF1, SFRP1, SFRP2 and SFRP5 in DNA extracted from serum.
Clinical performance was determined in a large training study on 150
serum samples (120 breast cancers, 30 healthy controls). 20 benign di-