Akt2 siRNA-nanoparticulate system as a new tool to restoration of e-cadherin and eradicate tumor metastic phenotype

______________________________________________________________________________ _______ vii Abstract Cancer has currently a major impact in the worldwide health, with breast and colon carcinomas presenting the second and third highest incidence rates, respectively. Despite the important advances in understanding the mechanism of carcinogenesis, disease progression, and development of new and more effective therapies, advanced cancer is still generally an incurable disease. The currently used treatments have improved both the survival and cure rates of patients; however there are some serious drawbacks, mostly concerning high toxicity and serious side-effects, which may cause even the death of a small percentage of patients. Furthermore, many patients suffer from tumors that are resistant to the conventional treatments, thus not taking profit from any therapeutic benefit. The resistance seen in the aggressive forms of the disease remains to be the biggest challenge of current treatments. More than half of the treated patients suffer from a disease relapse, most of them with distant metastases. Cancer maintenance, resistance to therapy, and metastatic disease seem to be sustained by the presence of cancer stem cells (CSC) within the tumors. These cells retain the capacity of repopulating the tumor after the treatment, while being insensitive to conventional anticancer therapies, antimitotic agents or radiation. It is still not entirely clear if CSC originate from the epithelial stem cells which are essential to maintain proliferative homeostasis or result by accumulation of mutations from differentiated cells. However it is known that only a few number of CSC are necessary and sufficient for tumor regeneration. In this scenario, new cancer therapies under development should fulfil two conditions: (i) reduce the toxicity displayed in current therapies and most importantly, (ii) specifically target and eliminate CSC to overcome drug resistance and tumor recurrence. Both criteria could be accomplished using innovative targeted nanomedicines for drug and/or gene delivery, which is widely expected to bring promise and create novel therapeutics in advanced cancer therapy. Nanoparticles have shown, in both preclinical and clinical practice, to improve the therapeutic window of the therapeutic agents by specifically delivering higher concentrations to the tumor lesions, while reducing the systemic toxicity. This behavior explains the increased interest of research groups and pharmaceutical companies in nanomedicine-based systems to treat cancer, resulting in an increase in the approved and clinically tested products in the last decades. The advances observed in molecular biology and biotechnology have been contributing for the identification and understanding of the complex biological pathways associated with cancer development, and consequently for the emergence of gene therapy as a promising AKT2 siRNA-Nanoparticulate System as a New Tool To Restoration of E-Cadherin and Eradicate Tumor Metastic Phenotype ______________________________________________________________________________ _______ viii therapeutic alternative in diseases with a strong genetic component. New biomarkers and therapeutic targets have been identified. AKT2 is one of the three Protein (serine/threonine) Kinase B (PKB or AKT) isoforms that is amplified in solid tumors. This oncogene is increased in response to apoptosis and its activation is also associated with phosphoinositide 3-kinase (PI3K) related effects as well as TWIST-promoted metastatic process by enhancing cell migration and invasion. Activation of TWIST/AKT2 signaling pathway is involved in E-cadherin silencing in the population of CSC, conferring them high tumorigenic potential. Thus, a therapeutic RNAi-mediated strategy using AKT2 as a downstream target represents an enormous step forward CSC eradication and anti-cancer therapy. The effects of AKT2 silencing in different breast and colon cancer cells, expressing and non-expressing TWIST are discussed in the present work. The obtained results demonstrated that the AKT2 inhibition is effective in terms of reducing cells migration, invasion, and transformation via different mechanisms and do not depend on the presence of TWIST within the tumor. We were able to observe that for cell lines expressing TWIST, AKT2 acts via Epithelial-Mesenchymal Transition (EMT) reversion, while for cells not expressing TWIST, AKT2 acts via mTOR pathway promoting the reduction of stemness markers and alteration in the expression of apoptotic genes such as the Bcl-2 and p53. In summary, the aim of this work was to silence the AKT2 expression using an small interfering RNA (siRNA) against the AKT2 (siAKT2) in different cancer cells lines in order to prove that, not only in bulk cancer cells but also in the sub-population of CSC, the AKT2 knockdown is able to impair their increased tumorigenic ability, reverting their mesenchymal phenotype, reducing cell invasion, inhibiting colony formation or inducing apoptosis. Due to the effect of AKT2 in both bulk cells and CSC, the therapy with siAKT2 could be used as two different strategies: i) to revert the EMT and maintain the tumors in its primary stage for easier chirurgical removal, and ii) prevent the tumor recurrence through the inhibition of CSC tumorigenic and metastatic potential. Despite the enormous outbreak of gene therapy in the last years, its clinical use is still limited mainly due to genetic material-associated delivery difficulties, such as fast degradation, insufficient transfection efficiency or dose limiting vectors toxicity. In order to overcome these problems, we pursued the development and validation of a nanocarrier composed by Pluronic F127 micelles associated with polyethylenimine (PEI)-based polyplexes to deliver a siAKT2. The proposed system for the delivery of siAKT2 seems to gather the requirements for an efficient and safe transport of siRNA in terms of their physicochemical characteristics, internalization capacity, biological efficacy and toxicity profile. These results make us believe that this new formulation will constitute a Abstract _____________________________________________________________________________________________________________________________________________________________ _______ ix technological platform for the development of systems to encapsulate different siRNAs, as well as other types of genetic material. We are putting efforts in the improvement of these formulation in order to achieve a potential gene or multifunctional delivery system, approaching the so increasingly required personalized and combined therapies and bringing new hope into the field of cancer therapy.