NF-YA ISOFORMS ROLES IN CLAUDIN LOW CANCERS

NF-Y is a pioneer transcription factor that regulates the expression of genes involved in cell cycle progression and metabolism. It is composed of: NF-YA, NF-YB and NF- YC subunits. NF-YA recognizes and binds to the CCAAT box, a DNA element found enriched in the promoters of cancer-specific genes in several tumor gene expression profiles analyzed, including those of breast, colon, thyroid, prostate, and leukemia cancers. NF-YA is present in two major isoforms called NF-YAl (long), and NF-YAs (short), which alternative splice exon 3. NF-YAl/NF-YAs ratio changes in different tissues, cell lines and differentiational stages. A robust increase in mRNA levels of NF- YA has been observed in different tumors of epithelial origin, among which BRCA and STAD. In human breast and gastric cancers and cell lines, NF-YAs isoform is highly expressed in all subtypes except in Claudinlow in which NF-YAl isoform is predominant. Recently it has been identified a 158 genes signature defining Claudinlow tumor subclass common to BRCA and STAD, correlating with high NF-YAl/NF-YAs ratio. For the first part of my PhD project, I worked with two different BRCA Claudinlow cell lines that mainly express NF-YAl. Through CRISPR/Cas9n genome-editing I obtained homozygous exon 3 deleted clones (NF-YAl-KO). NF-YAl-KO clones had an impaired ability to form compact aggregates, their migration, extravasation, invasion, and metastasis formation abilities were significantly impaired both in vitro and in vivo compared to negative controls. RNA-seq data underlined pathways downregulated in all NF-YAl-KO clones, among which EMT and angiogenesis, corroborating in vitro and in vivo data. In parallel, I have investigated the role of different RNA-binding proteins in driving NF-YA alternative splicing, among which RBFOX2, QKI and CELF2 both in BRCA and in STAD cell lines. Through transient overexpression, I validated RBFOX2 as splicing factor promoting the maturation of NF-YAl. Finally, through a frameshift mutation in NF-YA gene, I have investigated the effect of NF-YA alternative translation initiation sites (TISs) usage in HeLa cells and its impact at cellular and molecular levels.