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High levels of NF-YAl drives EMT in Claudinlow tumors

ABSTRACT: NF-Y is a trimeric transcription factor whose binding site -the CCAAT box- is enriched in cancer-promoting genes. The regulatory subunit, the sequence-specificity conferring NF-YA, comes in two major isoforms, NF-YA long (NF-YAl) and short (NF-YAs). Extensive expression analysis in epithelial cancers determined two features: widespread overexpression and changes in NF-YAl/NF-Ys ratios (NF-YAr) in tumors with EMT features. We performed wet and in silico experiments to explore the role of the NF-YA isoforms in breast -BRCA- and gastric -STAD- cancers. We generated clones of two Claudinlow BRCA lines BT549 and SUM159PT ablated of exon-3, thus shifting expression from NF-YAl to NF-YAs. Edited clones show normal growth, but reduced migratory capacities in vitro, and ability to metastatize in vivo. Using TCGA, including upon deconvolution of scRNA-seq data, we formalize the clinical importance of high NF-YAr, associated to EMT genes and cell populations. We derive a novel, 158 genes signature common to BRCA and STAD Claudinlow tumors. Finally, we identify splicing factors associated to high NF-YAr, further validating RBFOX2 as promoting expression of NF-YAl with wet experiments. In conclusion, these data bring three relevant results: (i) concerning Claudinlow tumors, the definition and clinical implications of NF-YAr and the 158 genes signature; (ii) genetic evidence of a role of exon-3 28 aminoacids, empowering NF-YAl with EMT-driving features in BRCA. (iii) The definition of a set of splicing factors fine-tuning the levels of NF-YA isoforms.

ORGANISM(S): Homo sapiens

PROVIDER: GSE208088 | GEO | 2023/01/28

REPOSITORIES: GEO

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Project description:Background: Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patients’ 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 PCa 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. Through lentiviral transduction and CRISPR-Cas9 technology, we modulated the expression of NF-YA splice variants in prostate cancer cells. We investigated cellular and molecular effects on cancer programs through 2D/3D in vitro assays and RNA-seq profilings. The rank-rank hypergeometric overlap approach (RRHO) was used to identify concordant/discordant overlap between gene expression signatures of NF-YAs/NF-YAl 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 prostate cancer cells in vitro and in vivo. Elevated NF-YAs levels are associated to aggressive prostate cancer specimens, defined by Gleason Score and TNM classification. NF-YAl overexpression in prostate cancer cells increases cell motility, while NF-YAs enhances both cell proliferation and invasive properties in 3D cultured spheroids and xenograft tumors. NF-YAs-transcriptome of multi-cellular spheroids has an extensive overlap with both localized and metastatic human PCa signatures. The ratio between NF-YAs and NF-YAl transcripts is higher in LumB PAM50 subtype, characterized by poor prognosis, and 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.

Project description:NF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB (NFYB) and NF-YC (NFYC)) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5,000-15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (non-modified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively co-localizes with FOS in all genomic contexts, and at promoters and enhancers this often occurs in the absence of JUN and the AP-1 motif. NF-Y also co-associates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly-used, proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve co-association with FOS. Scrambled control (shSCM) and NF-YA pLKO.1-shRNAs were designed by Sigma-Aldrich. The puromycin resistance cassette was replaced with an EGFP cassette. Viral production and transduction were carried out as previously described (Benatti et al. 2011). HeLaS3 cells were transduced with shSCM or shNF-YA viral supernatants, in triplicate, and cells collected after 48 hr of incubation. Total RNA was prepared by Trizol extraction and Qiagen RNeasy kit purification, converted to biotinylated aRNA and hybridized to U133 Plus 2.0 GeneChip expression arrays using the 3’ IVT Express Kit (Affymetrix, USA) following the manufacturer’s protocol. Arrays were RMA normalized (Irizarry et al. 2003), gene expression levels calculated, differential expression determined using the following R packages from the Bioconductor project: affy (Gautier et al. 2004), limma (Smyth 2004). HelaS3 cells were transduced with shSCM or shNF-YA in triplicate for 48hrs.

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High levels of NF-YAl drives EMT in Claudinlow tumors

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