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

Abstracts
The aim was to investigate the association of FMNL2 with Rho signaling
pathway in the invasion of CRC.
Methods. Rho family GTPase activity was tested by Rho pull-down assay.
In vitro invasive ability of cells was detected by Boyden Chamber
assay. And luciferase activities of MAL/SRF were detected using dualluciferase
reporter assay. In addition, Immunofluorescent analyses were
performed to examine F-actin stained by phalloidin and co-localization
of FMNL2 and LARG or p115RhoGEF. Co-immunoprecipitation was
used to determine the direct binding of FMNL2 and LARG. Finally,
GST pull-down assay was used to detect the binding of LARG-CT with
FMNL2 in the absence or presence of active RhoAV14.
Results. In this study, we showed that FMNL2 activated Rho/ROCK pathway,
and required ROCK to promote cell invasion. Moreover, FMNL2
promoted the formation of stress fiber and filopodia, and activated the
SRF transcription factor in the Rho-dependent manner. We also demonstrated
that FMNL2 was necessary for LPA-induced invasion, Rho/
ROCK activation, actin polymerization and SRF activation. FMNL2 is
an essential component of LPA signal transduction toward Rho by directly
interacting with LARG. Finally, we found FMNL2, LARG and
RhoA constituted a positive feedback loop. LARG silencing inhibited
Rho/ROCK pathway and cell invasion.
Conclusions. Our findings provide evidence for the positive feedback
between FMNL2 and RhoA, which promotes actin assembly and cell
invasion of CRC.
SG-P-119
DSC3 expression is regulated by p53, and methylation of DSC3
DNA is a prognostic marker in human colorectal cancer
T . Cui1 , Y . Chen1 , L . Yang1 , T . Knösel1 , K . Zöller1 , O . Huber2 , I . Petersen1 1 2 Institute of Pathology, Jena, Institute of Biochemistry II
Aims. Desmocollin 3 (DSC3), a member of the cadherin superfamily and
integral component of desmosomes, is involved in carcinogenesis. However,
the role of DSC3 in human colorectal cancer (CRC) has not yet
been established. Our aim of the study was to explore the role of DSC3 in
human colorectal cancer.
Methods. DSC3 expression in CRC cell lines was analyzed by RT-PCR
and western blotting. Methylation status of DSC3 was examined by demethylation
tests, methylation-specific PCR, and bisulfite sequencing
(BS). The regulatory role of p53 was investigated by transfection.
Results. DSC3 was downregulated in CRC cells at both mRNA and protein
levels. DSC3 expression was restored in five out of seven cell lines
after 5-aza-2’-deoxycytidine (DAC) treatment. A heterogeneous methylation
pattern was detected by BS in promoter region and exon 1 of
DSC3. Methylation of DSC3 genomic sequences was found in 41% (41 out
of 99) of primary CRC, being associated with poor prognosis (p=0.002).
Transfection of p53 alone or in combination of DAC increased the DSC3
expression. Similarly, treatment with p53 inducer adriamycin alone or in
combination with DAC enhanced DSC3 expression.
Conclusions. DNA methylation contributes to downregulation of DSC3
in CRC cell lines. Methylation status of DSC3 DNA is a prognostic marker
for CRC. P53 appears to play an important role in regulating DSC3
expression.
SG-P-120
ECRG4 is frequently downregulated by promoter 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
90 | Der Pathologe · Supplement 1 · 2012
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-P-121
Clonality analysis of neuroendocrine cells in gastric adenocarcinoma
L . Wang1 , G . Yao1 , Z . Zhao2 , X . Wei1 , R . Xu3 1Institute of Pathology and Forensic Medicine, Zhejiang University,
Hangzhou, China, 2Zhejiang Provincial People’s Hospital, Hangzhou, China,
3Hangzhou First People’s Hospital, Hangzhou, China
Aims. Gastric cancer remains one of the most common cancers and the
leading causes of cancer death worldwide. Neuroendocrine differentiation
(NED) is a common phenomenon in adenocarcinomas, but there
have been only a few studies of NED in gastric adenocarcinoma. However,
it remains unclear whether the glandular and endocrine cells expand
from two distinct precursors, or arise from a single progenitor cell. Therefore,
we studied the clonality of neuroendocrine (NE) cells in gastric
adenocarcinoma.
Methods. In this study, 120 cases of gastric adenocarcinoma and corresponding
non-neoplastic gastric mucosal tissues were obtained, immunohistochemistry
was carried out using the primary antibody against
NE marker (chromogranin A). Frozen section immunohistochemistry
samples were selected with a large quantity of NE cells. Then laser-capture
microdissection (LCM) was performed to obtain NE cells from gastric
adenocarcinoma, ready for DNA extraction and subsequent genetic
analysis. DNA extraction from the captured cells and whole genome
amplification (WGA) were performed using DNA Micro-kit and DNA
Repli-g Midi kit to obtain a large quantity of DNA. Then we chose 26
microsatellite markers with genome-wide scope, and exon 7 and exon 8
of p53, designed primers, and performed PCR. Genome-wide microsatellite
abnormalities (MSI and LOH) and p53 mutation were detected by
PCR-SSCP silver staining and PCR sequencing to identify the clonality
of NE cells. Statistical analyses were performed using SPSS for Windows
version 15.0.
Results. Thirty samples from a total of 120 that contained a large number
of NE cells were chosen for LCM. Through LCM, about 500 NE cells
were precisely captured from each sample. The total incidence rate of
MSI was 27.4%, and LOH rate was 17.9%. The rates for adenocarcinoma
and NE cells were similar. There was no significant relationship between
the incidence rate of MSI or LOH and clinicopathological characteristics.
According to the coincidence of microsatellite changes, cases 2, 3, 5,
6, 11, 12, 18, 24, 27 and 30 had highest the concordance for the two types
of cells. The other samples had similar microsatellite changes, except for
cases 7 and 10. Most p53 mutations were detected in exons 7 and 8. Concordant
mutations were observed in samples 4, 14, 21 and 27, and there
were different mutations in the two types of cells in case 7. In case 17, mutation
was seen only in adenocarcinoma cells. p53 mutation occurred six
times in adenocarcinoma cells (20.0%) and five times in NE cells (16.7%).
Clinicopathological data showed that mutations were present in poorly