Targeted RNAi and pDNA based therapy for gastrointestinal tumors

In this work, we were able to take advantage of a deregulated wnt
signaling pathway – a condition which is found in most
gastrointestinal cancers, in particular in colorectal carcinomas.
In order to restrict reporter gene expression to the desired cell
type, we utilized the β-catenin dependent CTP4-promoter to restrict
the expression of Firefly Luciferase and enhanced green fluorescent
fusion protein (EGFPLuc) to cell lines with deregulated wnt
signaling including SW480, LS174T, HepG2, Coga2 and Coga12. Stable
cell lines containing this CTP4-driven EGFPLuc construct were
established with the help of a lentiviral vector to monitor wnt
activity by transgene expression. With these stably transduced cell
lines, we performed a therapeutic target screen via siRNA-mediated
knock-down of a number of potentially therapeutic targets within
the wnt pathway – osteoprotegerin (OPG), Traf2 and Nck-interacting
kinase (TNIK), SRY-related HMG-box (Sox2), protease-activated
receptor 1 (PAR-1), β-catenin and transcription factor 4 (TCF4).
The in vitro screening system was utilized as a prevalidation tool
for therapeutically relevant targets. The degree of interference of
our novel targets was determined and the search for a suitable
siRNA target in colorectal cancer cells was narrowed down to
β-catenin, PAR-1 and TNIK. As proof of principle the siRNA-mediated
knock down of β-catenin was verified on mRNA and protein level in
LS174T cells. After the initial read-out of various cell lines with
different siRNAs has been established via the reduction of
Luciferase expression levels, the biological effect of these
targets were validated. For this purpose colony formation and cell
motility/invasion assays were conducted for all relevant target
cell lines. Furthermore in the in vitro experiments, the
tumor-selectivtiy of the CTP4-promoter was employed in the delivery
of the cytotoxic protein diphteria toxin A (DTA) in colorectal
cancer target cells. Data evaluation of all in vitro assays pointed
at reduced levels of proliferation, invasive behavior and
aggressiveness, which yielded three candidates (PAR-1, TNIK and
β-catenin) considered as viable for a treatment attempt in vivo. In
the in vivo experiments, systemic delivery of siRNA against
β-catenin, sticky siRNA targeting PAR-1 and plasmid DNA encoding
for CTP4 controlled DTA were evaluated in a disseminated liver
metastasis model of LS174T colorectal cancer. Specific knock-downs
of β-catenin and PAR-1 were achieved which was confirmed via mRNA
analysis. As for CTP4-DTA pDNA delivery the overall tumor load of
the liver was reduced without any significant systemic toxicity,
indicating specific DTA expression in tumor tissue. Also knock down
of PAR1 using sticky siRNA significantly reduced tumor growth. All
in all, the therapeutic effect of PAR-1 and β-catenin knock-down
could be verified in various in vitro assays analyzing invasive
behavior and anchorage independent growth and ultimately also in
vivo. The tumor-specific expression of DTA pDNA could also be
confirmed in vitro and was further investigated in an orthotopic
liver dissemination model in NMRI nude mice.