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Issue Date: 2-May-2011
Authors: Fabrizi, Eros
Title: Identification of novel therapeutic targets for colon adenocarcinoma
Abstract: Colorectal cancer (CRC) is the third most common form of cancer in the Western world. Despite the emergence of new targeted agents and the use of various therapeutic combinations, none of the treatment options available is curative in patients with advanced cancer. A growing body of evidence is increasingly supporting the idea that malignancies originate from a small fraction of cancer cells, called Cancer Stem Cells (CSC), that show self-renewal and pluripotency and are capable of initiating and sustaining tumor growth. Several studies have shown that, with respect to the bulk of tumor cells, CSC posses a higher degree of resistance to chemotherapy and radiotherapy that could explain the inefficacy of current therapies. The ability to isolate and study these tumor cells provided a powerful tool for the investigation of drug-and radio-resistance mechanisms thus paving the way for the development of novel targeted therapies aimed at the tumor complete eradication. The aim of this PhD thesis was to use CSC lines, derived from CRC specimens, to individuate new potential molecular targets for the development of novel therapies. To this end four colon-CSC lines were subjected to phosphoproteomic analysis by RPPA (Reverse Phase Protein Array) technology. Through this analysis phosphorylation levels of various protein kinases and their substrates were evaluated in order to create an activation map of the main colon-CSC proliferation and cell survival pathways. In parallel colon-CSC lines have been screened in vitro to the action of 80 commercially available protein-kinase inhibitors. This screening has revealed a partial correlation between in vitro sensitivity and phosphoproteomic analysis, but in this study, was not possible to identify predictive factors to infer colon-CSC sensitivity to specific kinase inhibitors. Colon-CSC was sensitive to the inhibition of protein kinase C (PKC), known regulator of cell proliferation and survival. Among PKC inhibitors, the most interesting was the UCN-01, a staurosporine derivative that can also inhibit PDK1 and Chk1. This compound has been already used in clinical trials as antineoplastic agent in combination with conventional chemotherapy. The in vitro treatment of colon-CSC with UCN-01 has demonstrated its ability to enhance the irinotecan cytotoxicity by increasing the apoptotic response. The combined action of UCN-01 and irinotecan caused a marked reduction in the levels of antiapoptotic proteins such as Bcl-XL and Mcl-1 and the activation of caspase 3. The in vivo administration of UCN-01/irinotecan combination, in a mouse model of subcutaneous xenograft, confirmed the observations obtained in vitro, leading to a significant reduction in tumor growth compared to the single treatments. UCN-01 has also shown efficacy in the inhibition of Chk1, as demonstrated by the reduction of the phosphorylation of its target protein cdc25. Inhibition of Chk1, an important regulator of cell cycle, in combination with chemotherapy, could help in reducing the viability of colon-CSC, thus preventing cell cycle arrest and repair DNA damage induced by irinotecan. Although UCN-01 exerts its effect by inhibiting the activity of various protein kinases, this reduced selectivity could be the basis of its effectiveness. The present study demonstrated that it is possible to identify, among the commercially available compounds, those that interfere with processes that regulate colon-CSC survival or proliferation and therefore are potentially able to interfere with tumor growth. The use of newly developed inhibitors, combined with the analysis of genetic alterations or phosphoproteomic, will identify factors predictive of response to therapy and lead to the possibility of developing individualized therapeutic strategies, increasing the likelihood of success of targeted therapy.
Appears in Collections:Area 05 - Scienze biologiche

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