Characterization of NF-YA alternative splicing in colorectal cancer cells to identify therapeutic vulnerabilities

NF-Y is a heterotrimeric transcription factor composed of the NF-YA, NF-YB and NF-YC subunits. The NF-YA subunit allows sequence-specific recognition of the CCAAT motif and the recruitment of the entire NF-Y trimer to target gene promoters, many of which involved in cell survival and proliferation. The NF-YA gene undergoes alternative splicing, resulting in the production of two transcripts encoding the NF-YA long (NF-YAl) and NF-YA short (NF-YAs) isoforms. In particular, NF-YAl is involved in cell differentiation, while NF-YAs plays a key role in cell proliferation and maintenance of stemness. Colorectal cancer (CRC) is characterized by an altered balance between the expression levels of NF-YAl and NF-YAs. Among the CRC molecular subtypes (CMS), the lower NF-YAs/NF-YAl ratio is observed in the CMS4, which is associated with a mesenchymal phenotype with high migratory and invasive capacity. Building on this evidence, modulation of NF-YA splicing may represent a strategy to reduce CRC aggressiveness of, particularly in the CMS4 subtype. This could be achieved either i) by modulating the RNA-binding proteins (RBPs) that regulate NF-YA alternative splicing or ii) through direct targeting of NF-YA splicing isoforms via isoform-specific silencing or splice-switching approaches. To address this study, we selected the RKO cell line that displays the lowest NF-YAs/NF-YAl ratio among available CRC models and recapitulates the aggressive CMS4 mesenchymal phenotype. To identify the RBPs involved in NF-YA alternative splicing, the NF-YA sequence encompassing exon 3 and its two flanking introns was analyzed using RBPmap and SpliceAid, revealing 52 RBPs with predicted binding sites. To identify RBPs regulating NF-YA splicing in CRC, we employed a funnel-like strategy, applying successive filtering steps to progressively refine the candidate list. Analysis of TCGA data showed that all 52 candidates are expressed in CRC samples. Correlation analysis between RBP expression and a low short/long NF-YA ratio narrowed the candidates to 44 RBPs, for which specific siRNAs were designed. We evaluated basal expression in RKO cells using a qRT-PCR cycle threshold cut-off of 33, bringing the list to 35 RBPs. Subsequently, assessment of siRNA efficacy using a 40% silencing threshold led to the exclusion of 7 additional candidates, reducing the list to 28 RBPs. The final filtering step was based on the modulation of NF-YA isoforms in RKO cells upon RBP siRNA-mediated silencing, retaining only those RBPs whose depletion induced opposite modulation of the two isoforms. This analysis yielded 5 candidate RBPs: HNRNPA2B1, DAZAP1 and RBM23, whose silencing reduced NF-YAs level, suggesting a role in the production of the short isoform; RBFOX2 and SNRPA, whose silencing reduced NF-YAl, indicating involvement in the production of the long isoform. The expression of the 5 RBPs was analyzed across the different CMS subtypes, focusing on their differential expression in CMS4, where NF-YAl is more abundant, compared to the other subtypes. This analysis identified HNRNPA2B1 and DAZAP1 as final candidates associated with NF-YAs, and RBFOX2 as a candidate linked to NF-YAl. As a complementary approach to modulate the NF-YA isoform ratio, the direct targeting was achieved using an exon 3-specific siRNA to selectively downregulate NF-YAl. After validation, we found that this NF-YAl specific silencing reduces the migratory capacity of RKO cells. Finally, considering that CMS4 is the tumor subtype with the poorest response to pharmacological treatments and the most prone to rapid acquisition of resistance, we evaluated the effect of NF-YAl silencing on the response to cetuximab, an EGFR-targeting monoclonal antibody widely used in CRC treatment. NF-YAl downregulation resulted in a decreased GI50, indicating increased cellular sensitivity to treatment.