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

Abstracts
Conclusions. Taken together we analysed mRNA and miRNA quality in
correlation to the time and condition of warm and cold ischemia of the
same tissue samples. The results enabled us to establish a RNA-based
quality assessment procedure of tissue specimen for frozen tissue biobanks.
This study may facilitate and optimise the logistics of biobanking
processes.
DO-117
Quantitative genome-wide methylation profiling of human
breast cancer reveals subtype-specific patterns of epigenetic
instability
U . Lehmann1 , J . Rößler1 , O . Ammerpohl2 , J . Gutwein2 , R . Geffers3 , W . Hofmann4
, F . Länger1 , H . Kreipe1 1 2 Medical School Hannover, Institute of Pathology, Hannover, University
Hospital Schleswig-Holstein, Institute for Human Genetics, Kiel, 3Helm holtz Centre for Infection Research, Genom analysis/Gen Regulation and
Differentation, Braunschweig, 4Medical School Hannover, Institute of Cell
and Molecular Pathology
Aims. This project addresses the question whether a subgroup of human
breast cancer is characterized by widespread epigenetic instability, in
contrast to genetic instability which is typical for e.g., familial breast
cancer.
Methods. High molecular weight DNA was isolated from 28 histologically
examined fresh-frozen human breast cancer specimens and 4 normal
mammary epithelial fractions using standard procedures. DNA methylation
patterns were analyzed using the newly developed 450k methylation
array from Illumina as well as methyl binding domain (MBD)-based
affinity enrichment and subsequent hybridization to a CpG-island
and promotor array from Agilent. For data analysis software provided
by the manufacturers of the arrays were employed. These analyses were
complemented and extended by employing commercially as well as freely
available software packages (Omics Explorer from Qlucore and the R
package IMA). The results for individual loci were validated using conventional
pyrosequencing and independent breast cancer specimens.
Results. In comparison to normal mammary epithelial cell fractions all
breast cancer specimens display several thousand statistically significant
aberrations in DNA methylation (number of CpG sites for pA), and tamoxifen-resistant MCF7 cells (T-
MCF7) were treated with the allosteric mTOR complex 1 (mTORC1)
inhibitor Everolimus and the active-site mTORC1/mTORC2 kinase inhibitor
PP242. In this setting, the effects of insulin receptor signalling on
cell growth, motility and viability were investigated by stimulation with
insulin or IGF1 and in the presence of siRNA inhibition of the insulin
receptor (IR) and insulin like growth factor 1 receptor (IGF-1R).
Results. T-MCF7 showed elevated level of IR/IGFR expression as well
as an activated (phosphorylated) ERK1/2 in contrast to the untreated
MCF7. The addition of insulin resulted in an increased signal transduction
via AKT and ERK1/2. Simultaneous inhibition of mTORC1/2 through
PP242 abolished AKT-phosphorylation and led to a complete cell cycle
arrest in G0/G1 as well as a substantial decrease of cell viability in MCF7
and T-MCF7. However, mTORC1-inhibition alone using Everolimus resulted
only in a partial G0/G1-arrest which could be reversed by addition
of insulin. siRNA inhibition of IR demonstrated an effective reduction of
MAPK-signalling in both MCF7 and T-MCF7 while siRNAs against IR
or IGF1R resulted in an additional decrease of cell viability.
Conclusions. Inhibition of mTOR-signalling reduced cell viability and
proliferation in PIK3CA-mutated breast cancer cells independent of an
acquired Tamoxifen resistance. However, our data indicate that IR and
IGF1R-conferred cell growth may reduce the effects of isolated mTOR
inhibition in Tamoxifen-resistant breast cancer cells and that additional
targeting of the insulin receptor pathway may prove useful in this
setting.
DO-120
Strong negative feedback from Erk to Raf confers robustness to
MAPK signaling
R . Fritsche1 , F . Witzel2 , A . Sieber1 , R . Herr3 , N . Schmidt1 , S . Braun3 , T . Brummer3 ,
C . Sers1 , N . Blüthgen1 1 2 Charité University Hospital Berlin, Pathology, Berlin, Charité University
Hospital Berlin, Pathology, 3ZBSA, Albert Ludwigs-University, Freiburg
Aims. Protein levels within signal transduction pathways vary strongly
from cell to cell. For example, it has been reported that concentrations
of the last kinase within the MAPK signalling module, Erk, varies about
4-fold between clonal cells under the same conditions. In the present study,
we analysed how signalling pathways can still process information
quantitatively despite strong heterogeneity in protein levels.
Methods. Mathematical analysis of isolated de- and phosphorylation
cycles predicts that phosphorylation of a signalling molecule is proportional
to the protein concentration. We combined mathematical modelling
and experimental analysis and systematically perturbed the protein
levels of Erk by siRNA. Our experiments also included the analysis of
Erk phosphorylation under Mek overexpression, measuring transcript
levels of negative feedback regulators, and the application of generic inhibitors.
Results. We found that the steady-state phosphorylation of Erk is very robust
against perturbations of Erk protein level, suggesting that there are
mechanisms that provide robustness to the pathway against protein fluctuations.
Using mathematical modelling, we identified three potential
mechanisms that may provide robustness: 1. kinetic effects, 2. transcriptional
negative feedbacks, 3. negative feedbacks on the post-translational
level. By experimental analysis of the systems we could exclude kinetic
effects and transcriptional negative feedback as mechanisms of robustness.
By analysing a panel of cell lines we found that cells are robust as
long as the signal passes through Raf-1. In contrast, cells where the pathway
is activated by a mutation in B-Raf loose robustness. Therefore,
once the feedback is broken, the system loses robustness and can be readily
modulated by low concentrations of targeted inhibitors. In contrast,
if the feedback is intact, inhibition of the pathway is inefficient.
Conclusions. This finding explains why Mek inhibition has shown little
success in the past in cancer treatment. However, it also shows that a