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

Abstracts
were up-regulated and the proteins of Wnt signaling pathway such as
β-catenin, Dvl3, Dvl2 and Naked2 were obviously up-regulated which
suggested the canonical Wnt signaling pathway was also activated.
TGF-β and Wnt signaling pathways were activated during colorectal
cancer cells-fibroblasts interactions. Upregulating of IGFBP7 in HELFs
was mainly through TGF-β1/ALK5/ Smad-2 signaling pathway. Wnt signaling
pathway may also play an important role in this process.
SG-134
FMNL2 is regulated by MIR-137 and promotes actin assembly and
cell invasion
Y . Zeng1 , Y . Qiao1 , W . Zhao1 , X . Zhu1 , J . Wang2 , X . Zhang2 , X . Bian3 , Y . Ding2 ,
L . Liang2 1Department of Pathology, School of Basic Medical Sciences, Guangzhou,
China, 2Guangdong Province Key Laboratory of Molecular Tumor Pathology,
Guangzhou, China, 3Institute of Pathology and Southern Cancer Center,
Southwest hospital, Chongqing, China
Aims. FMNL2 is a member of diaphanous-related formins which act as
effectors of Rho family GTPases and control the actin-dependent processes
such as cell motility or invasion. We previously validated FMNL2
as a positive regulator of cell motility and metastasis in colorectal carcinoma
(CRC) but the mechanisms of FMNL2 in CRC remain unclear.
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-135
The p53 target gene desmocolin 3 acts as a novel tumor suppressor
through inhibiting AKT signaling pathoway in human colon
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.
84 | Der Pathologe · Supplement 1 · 2012
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-136
Connexin 43 reverses malignant phenotypes of glioma stem cells
by modulating E-cadherin
X .-W . Bian1 , S .-c . Yu1 , H .-l . Xiao1 , X .-f . Jiang1 , Q .-l . Wang1 , Y . Li1 , X .-j . Yang1 ,
Y .-f . Ping1 , J .-j . Duan1 , X . Zhang1 1Third Military Medical University, Institute of Pathology and Southwest
Cancer Center, Chongqing, China
Aims. The purpose of this study was to investigate the role of gap junction
related proteins such as connexins for sustaining the malignant behavior
of cancer stem cells (CSCs) in glioma.
Methods. Tumorspheres from U87 cells and primary specimens were
isolated and characterized as previously described. CD133-positive cells
were sorted by flow sorting cytometry and termed as glioma stem cells
(GSCs). Electron microscopy was used for observation of ultrastructure
of GSCs. Laser confocal microscopy was used for fluorescence recovery
after photobleaching (FRAP) on GSCs. Cells and tissues were immunocyto(histo)chemically
stained by using stemness and differentiation
markers. Real time RT-PCR and western blotting were used for detection
of mRNA and protein levels of the stem cell markers. Cx43 or E-cadherin
pulldown assays were performed with anti-Cx43 antibody or anti-E-cadherin
antibody using the Co-immunoprecipitation. In addition, analyses
for promoter methylation of GJA1 gene, overexpression and knock-down
of Cx43, gene expression array and gene set enrichment analysis (GSEA)
were performed. Proliferation assay Matrigel invasion assay were carried
out in vitro and in vivo experiments were performed in nude mice.
Results. We obtained tumorspheres formed by glioma stem cells (GSCs)
and adherent GSCs, and then examined their GJIC. All GSCs showed
reduced GJIC, and differentiated glioma cells had more gap junction-like
structures than GSCs. GSCs expressed very low level of connexins, Cx43
in particular, which are key components of gap junction. We observed
hypermethylation in the promoter of gap junction protein ***1 (GJA1),
which encodes Cx43 in GSCs. Reconstitution of Cx43 in GSCs inhibited
their capacity of self-renewal, invasiveness and tumorigenicity via influencing
E-cadherin and its coding protein, which leads to changes of the
expression of Wnt/***-catenin targeting genes.
Conclusions. In this study, we provide evidence for the first time that
dysfunction of GJIC is an important feature of GSCs. Reconstitution of
Cx43, the key component to maintain the functional GJIC, does not alter
functional status of GJIC in GSCs but ablates their self-renewal, invasiveness
and tumorigenicity though the interaction with E-cadherin and
its coding protein to downregulate the express of those Wnt/β-catenin
targeting genes. Our results identify a potential role of Cx43 in maintaining
the malignant phenotype of GSCs.