Ribozym- und RNAi-vermittelter Knockdowndes Wachstumsfaktors Pleiotrophin (PTN)und des PTN-Rezeptors ALK zur funktionellen Analyseund zur therapeutischen Anwendung im Maus-Tumormodell

Pleiotrophin (PTN) is a member of the protein family of secreted, heparin-binding, matrix-associated growth factors, and functionally resembles fibroblast growth factors. It plays a physiological role during ontogenesis and is relevant in different tumor types. The protooncogenic impact of PTN results mainly from the stimulation of proliferation and angiogenesis. The receptor anaplastic lymphoma kinase (ALK) was initially discovered in connection with the large cell anaplastic T-Cell lymphoma as constitutively active fusion protein resulting from chromosomal aberrations. In later studies, ALK was identified as a PTN-receptor. ALK is a typical tyrosine kinase from the insulin receptor family, and triggers the ligand-specific induction of several mitogene-activated cascades responsible for its oncogenic potential via further protein adapters. In this study, the influence of PTN and ALK on proliferation, colony-forming in soft agar, tumor growth, apoptosis and angiogenesis in glioblastoma, teratocarcinoma and coloncarcinoma cell lines was investigated. For this purpose, cell lines that were PTN- and ALK-positive in screening experiments were stably transfected with ribozyme-bearing vectors. The cells with reduced PTN or ALK expression displayed a decrease in contact-dependent and contact-independent proliferation, and formed smaller tumor nodes after subcutaneous injection in mice. The effects of the PTN knockdown could be reverted in vitro by applying recombinant PTN. For further investigation, truncated mutants of ALK were generated and overexpressed in tumor cell lines. These tyrosine kinase deficient ALK mutants had a negative impact on the proliferation of the tumor cells. The ribozyme mediated double targeting of PTN and ALK showed additive antitumoral effects both in cell culture studies and in in vivo models with glioblastoma and colon carcinoma cell lines. To develop novel therapeutic strategies based on these results, we used RNA interference (RNAi) instead of ribozyme targeting. Using the transfection reagent polyethyleneimine (PEI), which is suitable for the delivery of RNA, we succeeded in knocking down PTN by means of PEI/siRNA complexes in established U87MG glioblastoma tumor xenotransplants, with a subsequent considerable reduction of tumor growth. This treatment worked both very efficiently and specifically with intraperitoneal, as well as with subcutaneous injections. The systemic administration of PEI/siRNA complexes in order to knock down PTN and / or ALK thus represents a promising treatment method for different solid tumors.