Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients

Background: Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile.The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAI. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes. Methods: Data on the expression of NF-YA isoforms were extracted from the TCGA (The Cancer Genome Atlas) database of tumor prostate tissues and validated in prostate cell lines. Lentiviral transduction and CRISPR-Cas9 technology allowed the modulation of the expression of NF-YA splice variants in PCa cells. We characterized 3D cell cultures through in vitro assays and RNA-seq profilings. We used the rank-rank hypergeometric overlap approach to identify concordant/discordant gene expression signatures of NF-YAs/NF-YAI-overexpressing cells and human PCa patients. We performed in vivo studies in SHO-SCID mice to determine pathological and molecular phenotypes of NF-YAs/NFYAI xenograft tumors. Results: NF-YA depletion affects the tumorigenic potential of PCa cells in vitro and in vivo. Elevated NF-YAs levels are associated to aggressive PCa specimens, defined by Gleason Score and TNM classification. NF-YAI overexpression increases cell motility, while NF-YAs enhances cell proliferation in PCa 3D spheroids and xenograft tumors. The transcriptome of NF-YAs-spheroids has an extensive overlap with localized and metastatic human PCa signatures. According to PCa PAM50 classification, NF-YAs transcript levels are higher in LumB, characterized by poor prognosis compared to LumA and basal subtypes. A significant decrease in NF-YAs/NF-YAI ratio distinguishes PCa circulating tumor cells from cancer cells in metastatic sites, consistently with pro-migratory function of NF-YAI. Stratification of patients based on NF-YAs expression is predictive of clinical outcome. Conclusions: Altogether, our results indicate that the modulation of NF-YA isoforms affects prostate pathophysio-logical processes and contributes to cancer-relevant phenotype, in vitro and in vivo. Evaluation of NF-YA splicing may represent a new molecular strategy for risk assessment of PCa patients.