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

Conclusions. Although CRC is characterized by mutational activation of
the WNT pathway, MAPK signaling influences intratumoral β-catenin
heterogeneity, revealing a mechanism for external stimuli to modulate
pathway activity. Because MAPK signaling does not merely coincide
with nuclear β-catenin but also regulates it, this may account for the high
frequency of KRAS mutations in CRC.
SG-P-115
Influence of an anti-TLR2-Intrabody on rheumatoid arthritis
B . Küttner1 , V . Seiffert1 , S . Laggies1 , G . Gross1 , T . Böldicke1 , A . Dellmann2 ,
K . Donhuijsen2 1 2 Helmholtz-Centre, Infection Research, Braunschweig, academical hospital
Braunschweig, department of pathology, Braunschweig
Aims. Rheumatoid arthritis (RA) is a chronic, systemic inflammatory
disease. Recent results showed that toll-like receptors (TLRs) particularly
TLR2 and its signaling pathway is involved in the pathogenesis of
rheumatic arthritis and might be a “key target” in prevention of RA. An
intracellular antibody (intrabody) fused with an endoplasmatic reticulum
retention sequence has been developed that inhibits the function of
TLR2 by preventing the translocation of TLR2 from the ER to the cell
surface.
Methods. To test if the intrabody has the potential to inhibit inflammation
during rheumatoid arthritis two mouse models were established: a
collagen-induced arthritis model and a model based on the injection of
inflammatory synovial fibroblasts into joints. Investigations by immunohistochemistry,
confocal microscopy, flow cytometry and paw swelling
showed the course of the disease. At first we analyzed the efficiency
of the anti-TLR2 intrabody in the collagen arthritis mouse model.
Results. A strong inflammatory response was seen in both rheumatoid
mouse models in the absence of the intrabody. Anti-TLR2 intrabody inhibited
significantly the paw swelling after adenoviral anti-TLR2 intrabody
gene transfer into the joints. Joints with an inflammatory response
showed migration of cells (HE staining) into the lamina synovialis and
the fat-tissue, in fact macrophages (CD11b+) and fibroblasts (CD40+).
Conclusions. Analysis of TLR2 surface representation and intracellular
intrabody expression of cells prepared from joints of rheumatoid mice
treated with intrabody-adenovirus and eGFP adenovirus (control) is
currently under investigation. In the rheumatoid arthritis model using
synovial cells with inflammation, we plan to analyze the influence of
cellular anti-TLR2-intrabody expression on the manifestation of the disease.
SG-P-116
TGF-β and Wnt signaling pathways regulate the expression of
IGFBP7 of fibroblasts in the tumor-stroma interactions
C . Rao1 , J . Xu1 , M . Liu1 , H . Deng1 1Department of Pathology, School of Medicine, Zhejiang, China
Aims. To find out the mechanism of the up-regulation of IGFBP7and the
biological changes in fibroblasts during the interactions with colorectal
cancer cells.
Fibroblasts (HELFs) were cultured in colorectal cancer cells conditioned
media (SW620-CM), treated by TGF-β 1, TGF-β1 receptor antagonist
(SB431542), TGF-β1 specific antibody (AF), Wnt signaling pathway
agonist (LiCl) and inhibitor (DKK-1) respectively. Q-PCR, Western Blot,
ELISA, Immunofluorescence microscopy and flow cytometry were used
to detect the expression of related targeted genes and proteins of TGF-β
and Wnt signaling pathways.
HELFs cultured in SW620-CM were activated with abundant expression
of α-SMA and showed strong proliferation and weak apoptosis and senescence.
The expression of IGFBP7 of HELFs was up-regulated in timedependent
and dose-dependent manners when cultured with SW620-
CM, while TGF-β signaling were activated as Smad2, P-Smad2 and
TGF-βRΠ were up-regulated in HELFs. These effects could be strengthened
by TGF-β1 and inhibited by SB431542 or AF. During the interactions,
the downstream genes of Wnt signaling pathway such as c-myc, cyclinD1
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-P-117
MicroRNA-137, a HMGA1 target, suppresses cell invasion and
metastasis of colorectal carcinoma by directly targeting FMNL2
X . Li1 , X . Zhang1 , W . Zhao1 , J . Wang1 , X .W . Biang2 , L . Liang3 , Y . Ding3 1Department of Pathology, School of Basic Medical Sciences, Guangzhou,
China, 2Institute of Pathology and Southern Cancer Center, Southwest Hospital,
Chongqing, China, 3Guangdong Province Key Laboratory of Molecular
Tumor Pathology, Guangzhou, China
Aims. FMNL2, a member of diaphanous-related formins, has been
strongly associated with tumor progression but the post-transcriptional
regulatory mechanism of FMNL2 remains unknown. The aim of this
study was to investigate whether increased FMNL2 expression is mediated
by microRNAs in colorectal carcinoma (CRC).
Methods. Real-time PCR or Western blot was performed to detect the expression
level of miR-137 or FMNL2 in CRC cells and tissues. The in vitro
and in vivo functional effect of miR-137 was examined further. A luciferase
reporter assay was conducted to confirm the associations between
miR-137 and FMNL2 3’UTR, HMGA1 and miR-137 promoter. CHIP was
used to assess the direct binding of HMGA1 to miR-137 promoter.
Results. We initially chose miR-137 and miR-142-3p as potential miRNAs
targeting FMNL2 which were predicted by bioinformatics. Only miR-137
showed significant inverse correlation with FMNL2 protein level in CRC
cell lines and tissues. We validated that FMNL2 was a target gene of miR-
137, and miR-137 could inhibit cell proliferation, invasion in vitro, hepatic
or intestinal metastasis in vivo by targeting FMNL2. We further show
that HMGA1 enhances miR-137 transcription by binding its promoter,
and then negatively downregulates FMNL2 expression. Finally, miR-137
inhibited the activation of p-MAPK and p-Akt, followed by the suppression
of MMP2, MMP9 and VEGF.
Conclusions. Our findings demonstrate a novel mechanism of post-transcriptional
regulation of FMNL2 expression and identified miR-137,
induced by its upstream transcription factor HMGA1, can suppress its
direct target FMNL2 and lead to tumor invasion and metastasis, at least
in part through inhibiting PI3K/Akt and MAPK/ERK pathways.
SG-P-118
The positive feedback between FMNL2 and RhoA, promotes actin
assembly and cell invasion of colorectal carcinoma
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.
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