The sequence-specific transcription factor NF-Y binds the CCAAT box, one of the most representative regulatory element in the promoters of eukaryotic genes involved in cell cycle progression and proliferation. NF-Y consists of the NF-YA, NF-YB and NF-YC subunits, all necessary for chromatin binding. The NF-YA subunit is directly involved in DNA binding through CCAAT sequence recognition. The NF-YA gene undergoes alternative splicing producing two transcripts coding for NF-YAs (short) and NF-YAl (long) isoforms. NF-YAs is mainly associated to stemness and cell proliferation, while NF-YAl is more involved in cell differentiation. Bioinformatic analyses of TCGA (The Cancer Genome Atlas) transcriptomic data from colorectal adenocarcinoma tissues revealed higher expression of total NF-YA in tumor tissues than healthy counterparts. Moreover, different ratio of NF-YAs/NF-YAl transcripts characterizes tumor subtypes. Among consensus molecular subtypes (CMS) of colorectal cancer, CMS4, which is associated to a mesenchymal phenotype with high migratory and invasive capacity, shows the lower level of NF-YAs/NF-YAl ratio.
With the aim to investigate the activity of NF-YA isoforms in controlling the epithelial-mesenchymal transition (EMT) process and aggressiveness of colon cancer cells, firstly we analyzed the signature of NF-YA transcripts in colorectal cell lines. We then transduced HT29 and HCT116 cells to stably overexpress NF-YAs or NF-YAl. We assessed the expression of EMT markers by Western blot and qRT-PCR. The results showed that the overexpression of NF-YAs or NF-YAl was not sufficient to increase the expression of key EMT genes. Differently, we observed a decrease in E-cadherin expression in NF-YAl cells compared to NF-YAs and Empty ones. Immunofluorescence staining of E-cadherin highlighted the presence of amoeboid cells in NF-YAl 2D cultures. Consistently, live imaging analysis of HCT116 cells displayed amoeboid movement of NF-YAl cells, while NF-YAs and Empty cells migrate collectively. We then performed 2D migration assays (Transwell and Wound Healing) in HT29 and HCT116 cell lines: higher migration capacity and speed characterizes NF-YAl cells compared to NF-YAs. Despite this, the addition of extracellular matrix (Matrigel-HC) impairs the invasive potential of NF-YAl cells. We further investigated the effect of the overexpression of NF-YA isoforms on cell growth: NF-YAl cells showed lower ability to proliferate in both adhesion and non-adhesion conditions (clonogenic assays), compared to NF-YAs and Empty cells.
To better evaluate growth and invasion properties of HCT116 overexpressing cells, we set up 3D spheroids cultures using different matrices (Matrigel, Collagen, Vitrogel). NF-YAl spheroids grown in Matrigel and Collagen matrices resulted smaller and less compact compared to NF-YAs and Empty ones. By means of Vitrogel matrix, we explored the capacity of single cells to form colonies in 3D cultures: NF-YAs and Empty cells produced regular and organized structures unlike NF-YAl cells. The analysis of the invasive potential of HCT116 spheroids highlighted the ability of NF-YAl cells to disseminate and spread from the spheroid, differently from NF-YAs and Empty.
Altogether, these results suggest that NF-YAl can enhance the migration capacity of CRC cells, allowing metastatic cells to disseminate but not promoting their growth and invasion abilities. We hypothesize that the two isoforms of NF-YA differently control the migration modes and mechanisms contributing to different CRC cell phenotypes. While high expression of the short isoform is crucial in establishing tumor hallmarks regardless of subtype, high expression of the long isoform confers migratory properties making the tumor more aggressive.