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Issue Date: 14-Feb-2014
Authors: Maugeri, Marco
Title: Analysis of the involvement of exosomal miRNAs and proteins in the response of CRC cells to Cetuximab
Abstract: It has been demonstrated that intercellular communication via cell- released vesicles is very important both for normal and tumor cells, and specifically to determine tumor development and progression as well as invasion and angiogenesis. Cell communication could involve the exosomes, small vescicles of endocytic origin, which are released from different kinds of donor cells; they can transfer molecular signals as proteins and RNAs through the extracellular environment to specific recipient cells in a autocrine, paracrine or endocrine way. Exosomes can strongly influence the recipient cells phenotype by transferring oncogenes that could influence the response of cells to drugs or immune reactions. Recently, it has been demonstrated the presence of miRNAs inside the exosomes and their potential involvement in cancer development. Considering the important role of miRNAs in colorectal cancer, one of the most diffused and studied tumor, it was considered interesting to investigate the role of exosomal miRNAs and associated proteins in the response of CRC cells to Cetuximab (an anti-EGFR therapeutic antibody). The EGFR signaling pathway is very importantly in relationship both to CRC and miRNA biogenesis and expression, and recently also to the exosomal communication system. Therefore, one of the major aims of this thesis was to analyze the possible involvement of exosomes in the response to Cetuximab of two CRC cell lines (wild-type KRAS Caco-2 cells and KRAS mutated HCT-116 cells) through the transfer of specific miRNAs and proteins to recipient cells. To carry out this analysis, we performed cellular and exosomal miRNA profiling after Cetuximab treatment, for 745 miRNAs by using Real-Time PCR. The results of the analysis showed that exosomal miRNA profiles globally reflect those of whole cells at steady-state, but there exists an important quantitative asymmetrical distribution. After Cetuximab treatment, Caco-2 sensitive cells showed several exosomal differentially expressed (DE) miRNAs in comparison to HCT-116 cells. Many DE miRNAs are involved in cancer and immunity. These data could be explained by considering that the EGFR pathway can regulate miRNA biogenesis via the MAPK/ERK cascade. Exosomal proteins analysis was performed for 741 cancer-related proteins through a specific antibody microarrays platform. Also the profile of exosomal proteins from Caco-2 cells showed important alterations after Cetuximab treatment. Globally, several DE miRNAs and proteins from Caco-2 exosomes were related to cancer, stimulation of immunity and inflammation. Interestingly, exosomes transfection experiments between Caco-2 and HCT-116 cell lines (performed to investigate their effect on cell viability) showed that the transfection of steady state Caco-2 exosomes in the HCT-116 cell line determined a decrease of cell viability of recipient cells, while Cetuximab-treated Caco-2 cells exosomes, transfected in HCT-116 cells, increased their viability. These data could be explained considering that exosomes from Cetuximab-treated cells are enriched in oncogenic- and immune stimulation-related miRNAs. Finally, DE proteins were searched to find potential RNA-binding proteins. Globally, the results of this thesis could be useful to: (1) verify the existence of horizontal transfer of genetic informations in eukaryotes; (2) search for potential miRNAs and proteins biomarkers of Cetuximab response in CRC in vivo. Eventually, it will be interesting to perform the characterization of exosomal miRNAs and proteins expression profiles of plasma from CRC patients after Cetuximab treatment. Moreover, the characterization of the asymmetrical distribution of miRNAs between cells and exosomes could be important to further investigate the potential and specific mechanism of miRNA sorting within exosomes.
Appears in Collections:Area 06 - Scienze mediche

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