DECIPHERING NF-YA ISOFORM-SPECIFIC MECHANISMS UNDERLYING PROSTATE CANCER RESISTANCE TO ADT/ARSi THERAPY

NF-Y (Nuclear Transcription Factor Y) is a heterotrimeric transcription factor composed of three subunits – NF-YA, NF-YB and NF-YC – all necessary for the binding to the CCAAT box, a regulatory element located upstream to the transcription start site (TSS) in about 30% of eukaryotic promoters. The subunit NF-YA undergoes alternative splicing of exon 3, generating two isoforms: NF-YA long and NF-YA short. NF-Y regulates the expression of numerous target genes, involved in both physiological and pathological mechanisms such as cell cycle, proliferation, and survival, as well as DNA damage, cancer and apoptosis. Multiple studies identified NF-Y as a key transcriptional regulator in prostate cancer (PCa), involved in both cells’ malignant transformation and in the development of metastases. Notably, we previously demonstrated that NF-YA isoforms have distinct roles in PCa: NF-YA long promotes migration, whereas NF-YA short stimulates proliferation. In this work, we investigated the oncogenic role of NF-Y in prostate cancer, with particular attention to the molecular mechanism through which it can modulate hormone-induced proliferation or androgen-deprivation (ADT) resistance. Based on preliminary bioinformatic analyses showing an enrichment of NF-Y binding sites in DHT (dihydrotestosterone)-activated genes, we conducted ChIP-seq studies to assess whether NF-Y binding is altered under DHT-induced versus hormone-starved conditions in hormone-responsive prostate cancer LNCaP cells. While only a subset of genes is differentially bound by NF-Y under the two different conditions, approximately 3000 genes are associated with - and thus likely regulated by - NF-Y independently of hormone stimulation. Among these, genes involved in cell cycle control and lipid metabolism emerge, two key categories that also contribute to resistance to ADT therapy. Based on this observation, we investigated the response of LNCaP to Enzalutamide, an agent commonly used in ARSi therapy, through ChIP-seq and qRT-PCR. These assays revealed binding of NF-Y to cell cycle genes, associated with H3K4me3 increased expression concurrently to induced levels of the NF-YA long isoform. These results suggested a possible role of the NF-YA long splice variant in ARSi resistance, consistently with its higher expression in AR insensitive cell lines. Cell viability assays in LNCaP over-expressing NF-YA isoforms after treatment with charcoal-stripped FBS (csFBS) and DHT suggested a possible role of NF-YA long in decreasing sensitivity to androgen deprivation and increasing response to low doses of androgens. This differential response was also confirmed in dose-response experiment with Enzalutamide, which showed distinct IC50 values between NF-YA long and NF-YA short overexpressing cells, supporting a major role of NF-YA long in the development of resistance. To further validate this role, we silenced NF-YA long expression with an isoform-specific siRNA in PC3 cells and treated them with csFBS. Preliminary results suggested that NF-YA long silencing can sensitize cells to ADT. Altogether, these results suggest a potential role for the NF-YA long isoform in the response and development of resistance to prostate cancer therapy. Although additional studies will be necessary to confirm these findings, our data open the opportunity for a new NF-YA isoform-specific targeted therapy for ADT and ARSi resistance.