Prostate epithelial cell transformation: the role of NF-Y transcription factor and its isoforms in lipid metabolism

Luca Bianchini - PROSTATE EPITHELIAL CELL TRANSFORMATION: THE ROLE OF NF-Y TRANSCRIPTION FACTOR AND ITS ISOFORMS IN LIPID METABOLISM. Nuclear Transcription Factor Y (NF-Y) is a heterotrimeric sequence-specific transcription factor composed of the NF-YA, NF-YB and NF-YC subunits. NF-YB and NF-YC form a heterodimer that recruits NF-YA, which recognizes the CCAAT box, a frequent eukaryotic regulatory promoter sequence. NF-YA exists as a long or short isoform, resulting from alternative splicing of the NF-YA gene. NF-Y regulates the expression of genes involved in cell cycle, proliferation, differentiation, and various metabolic processes. In prostate cancer, NF-Y contributes to the progression from benign prostatic epithelium to a localized and metastatic tumour. In particular, NF-YA long and short isoforms promote cell dissemination and proliferation respectively. NF-Y plays a key role in modulating target genes by interacting with other transcription regulators, among which cholesterol regulatory element-binding proteins (SREBPs) involved in lipid metabolism, increasingly recognized as a key factor in the development and progression of prostate cancer. With this work, we investigated the possible oncogenic role of NF-YA isoforms in promoting tumoral transformation and modulating lipid metabolism in epithelial prostate cells. First, we induced PrEC primary cells to differentiate by treatment with KGF and R1881 to study the role of NF-YA long and NF-YA short in differentiation of prostate basal epithelial cells. RT-qPCR highlighted an increase in the NF-YA short isoform that may be implicated in luminal differentiation, as supported by bioinformatic analysis of previously published RNA-seq studies. Immortalized RWPE1 basal cell lines overexpressing NF-YA long or NF-YA short were used as a model to study if changes in the splicing signature affect cell differentiation or confer tumour-relevant-properties. RT-qPCR analysis revealed that NF-YA short overexpression is not sufficient to induce a luminal cytokeratins profile in RWPE1 cells. NF-YA long overexpression associates with an intermediate cytokeratins profile that is seemingly precursor to tumorigenic transformation. Even though NF-YA isoforms did not significantly influence proliferation, RWPE1 cells overexpressing NF-YA short exhibited a higher 2D clonogenic capacity, whereas NF-YA long led to the formation of fewer but larger colonies. Preliminary results from transwell assays showed that NF-YA long enhances the migratory ability of RWPE1 cells. RNA-sequencing underlined the differentially expressed genes of NF-YA long and NF-YA short RWPE1 cells. The bioinformatic analysis carried out with the enrichment analysis web-based tool Enrichr revealed that NF-YA long primarily associates with lipid metabolism signature. This finding was further explored by RT-qPCR, which showed that NF-YA long significantly correlates with higher expression of the lipid metabolism genes SREBP1 and ACLY, the latter generating acetyl-CoA from citrate for fatty acid and cholesterol biosynthesis. Furthermore, we observed increased histone H3 acetylation levels by western blot, consistent with the role of acetyl-CoA in protein acetylation. Although lipid metabolism appears to be transcriptionally altered only in association with NF-YA long, both isoforms increased RWPE1 resistance to Fatostatin, a SREBPs inhibitor. Altogether, these results suggest that NF-YA short may be involved in luminal differentiation, while NF-YA long promotes tumoral transformation via modulation of acetyl-CoA, which is reported to modulate lipid metabolism and chromatin acetylation. However, further studies will be necessary to validate these results, to clarify whether acetyl-CoA levels are indeed altered, and determine whether ACLY and NF-Y transcriptional activity are directly involved and could serve as viable therapeutic targets.