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

Abstract

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-YAl. 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-YAl-overexpressing cells and human PCa patients. We performed in vivo studies in SHO-SCID mice to determine pathological and molecular phenotypes of NF-YAs/NF-YAl 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-YAl 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-YAl ratio distinguishes PCa circulating tumor cells from cancer cells in metastatic sites, consistently with pro-migratory function of NF-YAl. 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 pathophysiological 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.

NF-YAl drives EMT in Claudinlow tumours

“…This view changed when it was realised that NF-YA is absent in some post-mitotic cells, it is associated to cell proliferation in vivo and that the targeted genes are enriched in growth-promoting categories, namely cell-cycle progression and metabolism. Not surprisingly, functional removal of NF-YA -by siRNA or shRNA- leads to impairment -or lack- of cell growth [ 41 ]; conversely, overexpression promotes different aspects of cell proliferation and tumorigenesis [ 24 , 42 – 45 ]. Overexpression of TF genes can have widespread direct -and indirect- transcriptional consequences, and this is most likely true for NF-YA, whose protein expression levels are tightly controlled [ 46 ].…”

Curcumin-Based β-Diketo Ligands for Ga3+: Thermodynamic Investigation of Potential Metal-Based Drugs

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“…The CCAAT box represents the binding site specifically recognized by the transcription factor NF-Y, whose overexpression has been associated with cancer progression and aggressiveness. [38,39] Assessment of the developed genosensor was performed by evaluating its ability to hybridize and selectively detect its reverse complementary sequence and to form double-stranded DNA (dsDNA). By exposing the biosensor to fully complementary DNA, and by performing control experiments with mismatch-bearing and noncomplementary strands, we demonstrate the potential use of the device not only for the detection of oligonucleotides at low concentrations (theoretical LOD ≈100 × 10 −15 m) but also for investigating the specificity of biorecognition and fundamental aspects of the hybridization processes.…”