The NF–Y splicing signature controls hybrid EMT and ECM-related pathways to promote aggressiveness of colon cancer

The NF-Y binding site is one of the most representative regulatory elements in the promoters of eukaryotic genes. NF-YA, the DNA binding subunit of the NF-Y transcription factor, undergoes alternative splicing producing two isoforms, NF-YAs and NF-YAl that differ in the transactivation domain. The expression of NF-YA splicing variants is deregulated in many cancers compared to healthy tissues. Colorectal cancer (CRC) is the third most common type among tumors. Based on comprehensive gene expression profiles, CRC is classified into 4 consensus molecular subtypes (CMSs). In this study, we demonstrated that the lower ratio between NF- YAs/NF-YAl transcripts is associated with the mesenchymal CMS4 subtype, characterized by poor response to therapies and the worst prognosis compared to other CMSs. Moreover, high NF-YAl expression predicts lower patients’ survival. Using in vitro cellular models, we determined the predominant expression of NF-YAs in all CRC cell lines, with NF-YAl level increasing from epithelial to mesenchymal cells. NF-YA isoforms oppositely regulate the transcription of the E-cadherin gene, through direct binding to its promoter. Compared to NF-YAshigh, NF-YAlhigh cells show alterations of genes associated with extracellular matrix and epithelial-mesenchymal transition. 2D and 3D assays identified different migratory and invasive behavior of transduced cells: NF-YAlhigh cells are characterized by single cell ameboid-like migration and form irregular spheroids with low levels of cell-to-cell adhesion, consistently with low E-Cadherin expression. We further confirmed the increased metastatic potential of NF-YAlhigh CRC cells by injection of transduced cells in the zebrafish xenograft model. Altogether, our data highlight the direct role of the NF-YAl in cell dissemination and aggressiveness of CRC. NF-YAl represents a new CRC prognostic factor and splice-switching strategies may reduce metastatic CRC progression.