CHARACTERIZATION OF PROTEINS INVOLVED IN INTRACELLULAR PATHWAYS OF COLON CANCER STEM CELLS
CORBO, CLAUDIA (2012) CHARACTERIZATION OF PROTEINS INVOLVED IN INTRACELLULAR PATHWAYS OF COLON CANCER STEM CELLS. [Tesi di dottorato] (Inedito)
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Recent findings suggest that malignant neoplasms are derived from a small sub-population of cells that acts as the "root" of tumours. This conclusion comes from the observation that when neoplastic cells of different types were tested for their growth potential both by in vitro and in vivo experiments, only a restricted minority of them displayed extensive proliferation. These cells are called cancer stem cells (CSCs): both anti-cancer drugs and irradiation cause cancer cells to die by apoptosis, however CSCs might survive and regenerate cancer. At present, CSCs theory represents a breakthrough in cancer research. The aim of this project is to characterize the protein expression pattern of CSCs to obtain further insights into the mechanisms of this class of cells. The knowledge of deregulated proteins could be the first step into the accomplishment of novel therapies targeted directly against CSCs. Particularly, we studied colon CSCs by using as experimental model two different colon cancer cell line systems: CaCo-2 and HCT-116. Putative CSCs were separated from non-CSCs by flow cytometry using CD133 as stemness marker. Then, total protein extract of CD133+ cells was compared to protein extract of CD133- cells and differentially expressed proteins were identified by 2D DIGE coupled with tandem mass spectrometry. Forty-nine differentially expressed proteins in CaCo-2 CD133+ vs CD133- cells and thirty-six in HCT-116 CD133+ vs CD133- cells were identified. Bioinformatics analysis of the differentially expressed proteins by using GeneOnthology and Ingenuity Pathway Analysis (IPA) software showed an alteration of energy metabolism, furthermore the examination of this network showed that several proteins were directly or indirectly connected to MCC (mutated in colorectal cancer), a negative regulator of Wnt pathway. Interestingly, among the identified proteins it has been observed a 2-fold change up-regulation of the splicing factor SRp20, newly identified target gene of the Wnt/β-catenin pathway and we demonstrated a direct cause-effect relationship between Wnt pathway activation and the increased level of SRp20 expression. Furthermore, the results of this work show that SRp20 influences cell proliferation thus suggesting a putative function of this protein in tumorigenicity of CD133+ cells. In conclusion, the activation of the Wnt pathway in CD133+ cells and the consequent up-regulation of SRp20, which is implicated in tumorigenesis, raises the possibility of a sequential series of molecular events occurring in connection with this process.
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