RNAi-based anti-cancer strategies - targeting the proto-oncogene PIM1 and oncogenic miRNAs

Cancer is a chronic disease caused by a degeneration of somatic cells due to mutations. In general cells grow erratic and under certain conditions this leads to an infiltration of even healthy tissues. To date cancer is the second most common cause of death in the Western world and belongs to one of the most important epidemiological diseases of our society. Besides traditional methods in cancer treatment such as resection of tumor tissue, chemo or immunotherapy, the development of novel therapeutic strategies is of the highest importance. One therapeutic option would include the use of small non coding regulatory RNAs such as miRNAs. MiRNAs are involved in the regulation of crucial biological processes such as development, differentiation or proliferation due to the control of the cell’s gene expression via RNA interference. Since the discovery of miRNAs, evidence has emerged that they can exert either tumor suppressive or oncogenic functions. Thus miRNAs are not only applied as diagnostic or prognostic biomarkers of cancerous tissues, but can also function as therapeutics. In miRNA replacement therapy a loss or downregulation of a tumor suppressive miRNA is adjusted with addition of this single miRNA. On the contrary an antisense therapy approach mediates the inhibition of oncogenic miRNAs by antisense oligonucleotides. Safe and efficient delivery of the oligonucleotides has to be guaranteed for both approaches. Currently the development of such efficient delivery systems is the critical and limiting factor upon miRNA based therapy designs. In this thesis the proto oncogene PIM1 was considered regarding a putative RNA based anti cancer strategy. Overexpression of PIM1 kinase usually is associated with severe forms of cancers with bad prognosis in several solid tumors or malign degenerations of the hematopoietic system. In vitro studies revealed a specific reduction of PIM1 protein levels in human leukemia and colon carcinoma cell lines due to a miRNA with tumor suppressive potential, namely miR 33a. Additionally this decrease in PIM1 resulted in an inhibition of proliferation. Successful delivery with nanoparticle complexes composed of miR 33a oligonucleotides and a non toxic branched polyethylenimine (PEI F25 LMW) showed an anti tumor effect in mouse colon carcinoma models that was obtained through a reduction in tumor size. These in vitro and in vivo studies provide evidence for the first time that the proto oncogene PIM1 is a promising target for miRNA replacement therapy. The second project dealt with the development of novel LNA based miRNA inhibitors, termed LNA antiseeds, composed of single stranded RNA oligonucleotides with locked nucleic acid (LNA) modifications and a natural phosphodiester backbone. In general an LNA design enhances the resistance of oligonucleotides against cellular degradation by nucleases. Furthermore affinity of the molecule to the miRNA of interest is increased. Cellular functions of the targeted oncogenic miRNAs are silenced due to complementary binding to the inhibitor. A use of minor amounts of LNA antiseeds already revealed an almost complete inhibition of miRNAs of the miR 106b family in human cancer cell lines which was connected to an endogenous derepression of the tumor suppressor P21. Successful complex formation of the novel LNA antiseeds with the branched polyethylenimine PEI F25 LMW has been proven. Furthermore in vitro experiments confirmed a functional delivery of the resulting nanoparticles into human cancer cell lines. Application of PEI F25 LMW complexed LNA antiseed miRNA inhibitors could now be tested as a promising antisense therapy approach in xenograft tumor mouse models. The third topic addressed the transcriptional regulation of the oncogenic miRNA cluster miR 17 92. Overexpression of this miRNA cluster is associated with enhanced proliferation, sustained angiogenesis and reduced apoptosis in several hematopoietic malignancies and solid tumors. Transcription of human miR 17 92 is significantly regulated by the oncogene MYC. In vitro experiments in human cancer cell lines confirmed that miR 17 92 belongs to one of those 20 % of MYC regulated genes which transcription is controlled by a synergism of MYC and PIM1. Further investigations regarding transcriptional control of miRNA promoters could provide profound evidences for a better understanding of a connection between miRNA expression levels and the development of cancer