Iaboni, Margherita (2013) NEW POSSIBLE APPLICATIONS FOR APTAMERS IN TARGETED ANTI-CANCER THERAPIES. [Tesi di dottorato]

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Abstract

In order of reducing the frequency of unwanted side effects and recurrences, in the last ten years a major challenge has been the development of methods for the specific targeting to cancer cells. Aptamers are single-stranded RNAs able to form different three-dimensional structures, which allows them to specifically recognize their molecular targets. This makes aptamers attractive agents for targeted cancer therapy. Aptamers were first utilized for their ability to bind and inhibit the activity of their target protein, including extracellular ligands and cell surface proteins. More recently, aptamers were are also used as delivery agents. Thanks to their ability to be endocytosed, aptamers may specifically bring a therapeutic cargo inside the cells. In this thesis, I developed several aspects regarding the aptamer-mediated targeting of cancer cells: 1) the selection of aptamers for a cancer stem cells-targeted therapy. In fact, as widely recognized, cancer stem cells are responsible for tumor recurrence, repopulating the tumor after a chemotherapeutic treatment. 2) The formation of an aptamer-microRNA chimera molecule in which the aptamer is the delivery vehicle for a microRNA. The use of microRNAs represent a challenging approach in cancer therapy because they are able to regulate the expression of cellular proteins modulating different pathways. However, to date the absence of reliable means that permit the specific delivery of microRNAs to the appropriate tissue represents an obstacle to the success of this approach. For the first purpose, I performed for the first time a SELEX (Systematic Evolution of Ligands by EXponential enrichment) method on human breast cancer stem cells, using in the counterselection step breast cancer differentiated cells. I selected putative aptamers specific for the stemness phenotype in breast cancer, useful to 1) identify specific cell-surface targets, 2) block molecular pathways and 3) specifically delivery therapeutics. For the second purpose, I selected an internalizing aptamer, GL21, that specifically binds to Axl receptor, overexpressed in many types of cancer cells. I linked this aptamer to miR-212. This microRNA is considered a tumour-suppressor miR because negatively regulates the anti-apoptotic protein PED found overexpressed in many tumors and involved in resistance to therapeutics. In A549 cells, TRAIL- resistant non-small cell lung cancer cells overexpressing Axl, this chimera was able to enter within and carry the microRNA to the processing machinery. MiR-212, subsequently, targeted PED resulting in TRAIL sensitization. In conclusion, in this thesis I studied new possible applications for aptamers aimed at avoiding therapeutic recurrences and at delivering “therapeutic” RNAs, as microRNAs, to the appropriate cells.

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