Molecularly targeted therapy on a new preclinical mouse model for gastric neuroendocrine tumors

Neuroendocrine tumors are a heterogeneous group of malignancies
with an increasing prevalence. Since there is not much progress in
therapy, model systems are urgently needed. We have a CEA424-SV40
TAg transgenic mouse model which develops spontaneous tumors in the
antral region of the stomach. In addition, several cell lines
derived from the tumor were established. Gene expression analysis
of the tumor tissue as well as cell lines revealed neuroendocrine
markers. Therefore we further characterized this model with special
emphasis on the cells of origin and used it for testing new
targeted treatment protocols. To analyze CEA424-SV40 TAg mouse
model in more detail, tumor tissue as well as the cell lines
derived from the primary tumor were investigated by
immunohistochemistry, immunofluorescence, western blot, and ELISA.
Antibodies used were directed at SV40 TAg, Ki-67, chromogranin A,
chromogranin B, secretin, H+-K+-ATPase, glucagon, and transcription
factors NeuroD1 and Nkx2.2. Plasma hormone levels of serotonin and
secretin were measured by ELISA. Immunostainings of SV40 TAg and
Ki-67 revealed highly proliferative tumors cells. The tumors
stained intensively for the neuroendocrine markers chromogranin A,
chromogranin B, secretin and glucagon. The tumor tissue as well as
the cell lines expressed transcription factors NeuroD and Nkx2.2,
which are involved in the differentiation of the neuroendocrine
lineage. Hormone levels of serotonin and secretin in the plasma of
the transgenic mice were dramatically elevated when compared with
normal littermates, thus supporting the neuroendocrine phenotype.
As the neuroendocrine phenotype of CEA424-SV40 TAg transgenic mouse
was confirmed, molecularly targeted therapies were tested in this
model system both in vitro and in vivo. Cell lines were tested for
drug sensitivity with mTOR inhibitors (RAD001, NVP-BEZ235),
paclitaxel, E2F inhibitor, HSP90 inhibitor, and p53 stabilizer
Nutlin-3a. All the drugs tested in vitro could efficiently inhibit
cell proliferation in a dose dependent manner. From these drugs the
mTOR inhibitor RAD001 was chosen for the in vivo experiment. Daily
feeding of 10 mg/kg RAD001 inhibited the tumor development and
prolonged the survival time of the CEA424-SV40 TAg transgenic mice
dramatically. The effects of the RAD001 treatment on tumor cells
were achieved mainly through inactivating mTOR-p70S6K and
mTOR-4EBP1 signaling as proven by western blot and
immunohistochemistry. Still, some cells must develop escape
mechanisms, since the tumor tend to grow. To gain a better
understanding of the T antigen transforming mechanisms as well as
the possible escape mechanisms, some efforts were made on the tumor
originating cells in the CEA424-SV40 Tag transgenic mouse model.
Possible candidates for these tumor originating cells in the
stomach are the newly described epithelial as well as mesenchymal
stem cells. In a first attempt, the expression feature of
epithelial and mesenchymal stem cell markers were analyzed.
Established cell lines as well as tumor tissue from the tumor
bearing mice were investigated by reverse transcription PCR
(RT-PCR), immunohistochemistry, immunofluorescence, western blot,
and microarray analysis. From several markers analyzed, the tumor
cell lines showed a high expression level of the potential
epithelial stem cell marker Bmi1 in RT-PCR and cDNA expression
array. This could be further substantiated by western-blotting and
immunostaining. Consequently, Bmi1 message could also be found in
the growing tumors both in mRNA and protein levels. Experiments
using siRNA to knock down the SV40-TAg expression showed that the
Bmi1 expression went down in the cell lines thus showing the
interrelationship. On the other hand, the mesenchymal stem cell
marker Etv1 was also found to be expressed in the tumor tissue and
cell lines derived from the tumor. More interestingly, Etv1
expression level was up-regulated over the time course of the tumor
development. From these, an Etv1 positive mesenchymal cell could be
a possible candidate for transformation. Since the CEA-promoter
used for the generation of the T-antigen transgenic animals
contains Etv1 binding sites, it is tempting to speculate, that this
may drive the transcription of the T antigen. In conclusion, our
data provide convincing evidence that CEA424-SV40 TAg mice are a
clinically relevant model for neuroendocrine tumor. Testing of
molecularly targeted therapies both in vitro and in vivo offered
promising candidates for further clinical evaluation. Thus, this
new model system could be of great value not only for studies on
the mechanisms of how SV40 TAg induces neuroendocrine tumors but
also for exploring novel targeted therapy in a preclinical setting.