Riassunto analitico
NF-Y is a transcription factor composed by three subunits: NF-YA, NF-YB and NF-YC. The NF-YA subunit recognizes CCAAT sequences, typically located at the promoters of eukaryotic genes, and is directly involved in DNA binding and regulation of transcription. The NF-YA gene encodes for two different isoforms, which originate from alternative splicing : NF-YA long (NF-YAl) and NF-YA short (NF-YAs), the last one lacking 28/29 amino acids within the transactivation domain. NF-YAs is associated with cell proliferation, while NF-YAl is mainly involved in cell differentiation. NF-Y-regulated genes are implicated in many functions, including cellular metabolism. In particular, NF-Y regulates the transcription of genes involved in the biosynthesis of polyamines, purines and glutamine (Gln), controls lipid biosynthetic pathways and activates glycolytic genes. Several studies show how NF-Y is involved in tumor onset and progression, influencing neoplastic cell metabolism in order to meet energy demand. The aim of this thesis project was to investigate how the two NF-YA isoforms can modulate the metabolic behavior of colorectal cancer cells in a hostile microenvironment with restricted nutrient access, in particular low Gln availability. Bioinformatic analyses of transcriptome data deposited in the TCGA database showed that NF-YA is more expressed in tissues from colorectal cancer patients than in healthy donor’s tissues. Moreover, different expression of the two NF-YA isoforms characterizes diverse tumor subtypes of colorectal cancer, allowing the stratification of patients according to tumor aggressiveness. Metabolomic studies carried out in collaboration with Prof. Righi (University of Bologna) have shown that the knock-down of NF-YA induces a decline in metabolites related to Gln metabolism in HCT116 colorectal cancer cells. qRT-PCR analysis of NF-YA transcripts in HCT116 cells cultured under Gln deprivation conditions highlighted a robust transcriptional up-regulation of NF-YAl expression. HCT116 lines stably overexpressing (O/E) NF-YAl or NF-YAs have been created, in order to understand how their modulation may be involved in a different metabolic adaptation. Differently from HCT116 Empty and HCT116 O/E NF-YAs, HCT116 O/E NF-YAl cells have high expression of genes related to Gln metabolism, which further increases under Gln deprivation conditions. The analysis of energy metabolism through the Seahorse XFe24 Analyzer showed higher capacity and dependance for Gln metabolism of HCT116 O/E NF-YAl, compared to the other two cell lines. Next, we performed wound healing assays to evaluate the ability of cells to migrate into an artificially created gap both in basal condition and in Gln deprivation condition. HCT116 O/E NF-YAl cells have a higher migration rate in both cases. Moreover, cell viability studies have shown that only NF-YAl is able to maintain high cell viability even in conditions of Gln deprivation. We further investigated cell migration and viability following the administration of inhibitors of the major players in Gln catabolism and biosynthesis. Treatment with Glutamine Synthetase (GS) inhibitor reduces the survival of HCT116 O/E NF-YAl in Gln-reduced medium at the same level of HCT116 Empty and HCT116 O/E NF-YAs. Moreover, this treatment inhibited the capability of migration of all three cell lines, pointing to a major role of GS in cell migration. Taken together, our data show that NF-YAl can confers aggressive properties to colorectal cancer cells by regulating the expression of Gln metabolism genes, leading to an increase in GS-mediated proliferation and migration.
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