Discovery and delivery of anti-angiogenic miRNAs for cancer treatment

Abstract

Cancer belongs to the top three causes of death in high-income countries. Several hallmarks of cancer have been identified that contribute to tumor growth. One of these hallmarks is angiogenesis, which occurs when the need for oxygen and nutrients to sustain the tumor cannot be maintained through passive means. At that point, tumor cells secrete growth factors, inducing blood vessel formation from preexisting blood vessels. Since the recognition of angiogenesis as a prominent factor contributing to solid tumor development, angiogenesis has become an attractive therapeutic target for cancer treatment. Several pharmaceuticals have been successfully developed to inhibit tumor angiogenesis and are currently approved for use in treatment of various types of cancer. Unfortunately, the therapeutic benefit of these therapeutics remains modest. At the same time, their high cost coupled to severe adverse reactions in some cases, create a need for better, more efficacious and safer strategies for anti-angiogenic therapy. One of these strategies exploits naturally occurring small non-coding RNAs, known as microRNAs (miRNAs). miRNAs have a key role in regulation of genes at a posttranscriptional level, including angiogenesis. Because miRNA expression levels in endothelial cells (EC) shift after angiogenic stimulation towards pro-angiogenic miRNA species that inhibit anti-angiogenic genes, miRNAs that inhibit pro-angiogenic genes might be an interesting therapeutic strategy to inhibit cancer angiogenesis. miRNAs have an important advantage over currently applied anti-angiogenic strategies because they regulate multiple genes at the same time, potentially leading to profound alteration of cell behavior compared to single pathway inhibition achieved by conventional anti-angiogenic agents. This thesis describes the discovery of anti-angiogenic miRNAs through functional screening with a lentiviral miRNA library to identify miRNAs that inhibit the viability of primary and immortalized EC. Number of anti-angiogenic miRNAs were identified and explored in depth in several in vitro and in vivo models. These miRNAs did not only inhibit neovascularization in a chicken chorioallantoic membrane assay, but also in two different mouse tumor models, which are known for their ability to form vascularized tumors. The potential of miRNAs as therapeutic applications were tested locally and systemically. To permit the usage of these miRNAs systemically, a novel delivery system was used, which we characterized in depth. In addition to their therapeutic potential, additional possibilities of using miRNAs as a tool to discover and identify novel target genes in drug development were explored in a comparative study between the miRNAs with a small molecule acting against the same target genes was investigated. Therefore, miRNAs offer a promising strategy for the exploration as well as development of new therapeutic anti-angiogenic applications.