RESUMO
Aberrant splicing events are associated with colorectal cancer (CRC) and provide new opportunities for tumor diagnosis and treatment. The expression of the splice variants of NF-YA, the DNA binding subunit of the transcription factor NF-Y, is deregulated in multiple cancer types compared to healthy tissues. NF-YAs and NF-YAl isoforms differ in the transactivation domain, which may result in distinct transcriptional programs. In this study, we demonstrated that the NF-YAl transcript is higher in aggressive mesenchymal CRCs and predicts shorter patients' survival. In 2D and 3D conditions, CRC cells overexpressing NF-YAl (NF-YAlhigh) exhibit reduced cell proliferation, rapid single cell amoeboid-like migration, and form irregular spheroids with poor cell-to-cell adhesion. Compared to NF-YAshigh, NF-YAlhigh cells show changes in the transcription of genes involved in epithelial-mesenchymal transition, extracellular matrix and cell adhesion. NF-YAl and NF-YAs bind similarly to the promoter of the E-cadherin gene, but oppositely regulate its transcription. The increased metastatic potential of NF-YAlhigh cells in vivo was confirmed in zebrafish xenografts. These results suggest that the NF-YAl splice variant could be a new CRC prognostic factor and that splice-switching strategies may reduce metastatic CRC progression.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Animais , Humanos , Peixe-Zebra/genética , Fatores de Transcrição , Neoplasias do Colo/genética , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular , Sorbitol , Movimento Celular/genética , Neoplasias Colorretais/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
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-YAs ratios (NF-YAr) in tumours with EMT features. We performed wet and in silico experiments to explore the role of the isoforms in breast -BRCA- and gastric -STAD- cancers. We generated clones of two Claudinlow BRCA lines SUM159PT and BT549 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, prognostic 158 genes signature common to BRCA and STAD Claudinlow tumours. Finally, we identify splicing factors associated to high NF-YAr, validating RBFOX2 as promoting expression of NF-YAl. These data bring three relevant results: (i) the definition and clinical implications of NF-YAr and the 158 genes signature in Claudinlow tumours; (ii) genetic evidence of 28 amino acids in NF-YAl with EMT-promoting capacity; (iii) the definition of selected splicing factors associated to NF-YA isoforms.
Assuntos
Fator de Ligação a CCAAT , Neoplasias , Humanos , Fator de Ligação a CCAAT/genética , Neoplasias/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Processamento de RNA/metabolismo , Sorbitol , Fatores de Transcrição/metabolismo , Transição Epitelial-MesenquimalRESUMO
NF-Y is a pioneer transcription factor-TF-formed by the Histone-like NF-YB/NF-YC subunits and the regulatory NF-YA. It binds to the CCAAT box, an element enriched in promoters of genes overexpressed in many types of cancer. NF-YA is present in two major isoforms-NF-YAs and NF-YAl-due to alternative splicing, overexpressed in epithelial tumors. Here we analyzed NF-Y expression in stomach adenocarcinomas (STAD). We completed the partitioning of all TCGA tumor samples (450) according to molecular subtypes proposed by TCGA and ACRG, using the deep learning tool DeepCC. We analyzed differentially expressed genes-DEG-for enriched pathways and TFs binding sites in promoters. CCAAT is the predominant element only in the core group of genes upregulated in all subtypes, with cell-cycle gene signatures. NF-Y subunits are overexpressed, particularly NF-YA. NF-YAs is predominant in CIN, MSI and EBV TCGA subtypes, NF-YAl is higher in GS and in the ACRG EMT subtypes. Moreover, NF-YAlhigh tumors correlate with a discrete Claudinlow cohort. Elevated NF-YB levels are protective in MSS;TP53+ patients, whereas high NF-YAl/NF-YAs ratios correlate with worse prognosis. We conclude that NF-Y isoforms are associated to clinically relevant features of gastric cancer.
Assuntos
Fator de Ligação a CCAAT/genética , Regulação Neoplásica da Expressão Gênica , Subunidades Proteicas/genética , Neoplasias Gástricas/genética , Fator de Ligação a CCAAT/química , Fator de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Humanos , Prognóstico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Isoformas de Proteínas/genética , Subunidades Proteicas/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , TranscriptomaRESUMO
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.
Assuntos
Processamento Alternativo/genética , Fator de Ligação a CCAAT/metabolismo , Edição de Genes/métodos , Neoplasias da Próstata/genética , Animais , Humanos , Masculino , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
NF-Y is the CCAAT-binding trimer formed by the histone fold domain (HFD), NF-YB/NF-YC and NF-YA. The CCAAT box is generally prevalent in promoters of "cancer" genes. We reported the overexpression of NF-YA in BRCA, LUAD and LUSC, and of all subunits in HCC. Altered splicing of NF-YA was found in breast and lung cancer. We analyzed RNA-seq datasets of TCGA and cell lines of head and neck squamous cell carcinomas (HNSCC). We partitioned all TCGA data into four subtypes, deconvoluted single-cell RNA-seq of tumors and derived survival curves. The CCAAT box was enriched in the promoters of overexpressed genes. The "short" NF-YAs was overexpressed in all subtypes and the "long" NF-YAl in Mesenchymal. The HFD subunits are overexpressed, except Basal (NF-YB) and Atypical (NF-YC); NF-YAl is increased in p53 mutated tumors. In HPV-positive tumors, high levels of NF-YAs, p16 and ΔNp63 correlate with better prognosis. Deconvolution of single cell RNA-seq (scRNA-seq) found a correlation of NF-YAl with Cancer Associated Fibroblasts (CAFs) and p-EMT cells, a population endowed with metastatic potential. We conclude that overexpression of HFD subunits and NF-YAs is protective in HPV-positive tumors; expression of NF-YAl is largely confined to mutp53 tumors and malignant p-EMT cells.
RESUMO
NF-Y is a pioneer trimeric transcription factor formed by the Histone Fold Domain (HFD) NF-YB/NF-YC subunits and NF-YA. Three subunits are required for DNA binding. CCAAT-specificity resides in NF-YA and transactivation resides in Q-rich domains of NF-YA and NF-YC. They are involved in alternative splicing (AS). We recently showed that NF-YA is overexpressed in breast and lung carcinomas. We report here on the overexpression of all subunits in the liver hepatocellular carcinoma (HCC) TCGA database, specifically the short NF-YAs and NF-YC2 (37 kDa) isoforms. This is observed at all tumor stages, in viral-infected samples and independently from the inflammatory status. Up-regulation of NF-YAs and NF-YC, but not NF-YB, is associated to tumors with mutant p53. We used a deep-learning-based method (DeepCC) to extend the partitioning of the three molecular clusters to all HCC TCGA tumors. In iCluster3, CCAAT is a primary matrix found in promoters of up-regulated genes, and cell-cycle pathways are enriched. Finally, clinical data indicate that, globally, only NF-YAs, but not HFD subunits, correlate with the worst prognosis; in iCluster1 patients, however, all subunits correlate. The data show a difference with other epithelial cancers, in that global overexpression of the three subunits is reported and clinically relevant in a subset of patients; yet, they further reinstate the regulatory role of the sequence-specific subunit.
Assuntos
Fator de Ligação a CCAAT/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Biomarcadores Tumorais , Fator de Ligação a CCAAT/química , Fator de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Isoformas de Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
The trimeric transcription factor (TF) NF-Y regulates the CCAAT box, a DNA element enriched in promoters of genes overexpressed in many types of cancer. The regulatory NF-YA is present in two major isoforms, NF-YAl ("long") and NF-YAs ("short"). There is growing indication that NF-YA levels are increased in tumors. Here, we report interrogation of RNA-Seq TCGA (The Cancer Genome Atlas)-all 576 samples-and GEO (Gene Expression Ominibus) datasets of lung adenocarcinoma (LUAD). NF-YAs is overexpressed in the three subtypes, proliferative, inflammatory, and TRU (terminal respiratory unit). CCAAT is enriched in promoters of tumor differently expressed genes (DEG) and in the proliferative/inflammatory intersection, matching with KEGG (Kyoto Encyclopedia of Genes and Genomes) terms cell-cycle and signaling. Increasing levels of NF-YAs are observed from low to high CpG island methylator phenotypes (CIMP). We identified 166 genes overexpressed in LUAD cell lines with low NF-YAs/NF-YAl ratios: applying this centroid to TCGA samples faithfully predicted tumors' isoform ratio. This signature lacks CCAAT in promoters. Finally, progression-free intervals and hazard ratios concurred with the worst prognosis of patients with either a low or high NF-YAs/NF-YAl ratio. In conclusion, global overexpression of NF-YAs is documented in LUAD and is associated with aggressive tumor behavior; however, a similar prognosis is recorded in tumors with high levels of NF-YAl and overexpressed CCAAT-less genes.
Assuntos
Adenocarcinoma de Pulmão/classificação , Fator de Ligação a CCAAT/genética , Ilhas de CpG , Perfilação da Expressão Gênica/métodos , Neoplasias Pulmonares/classificação , Regulação para Cima , Adenocarcinoma de Pulmão/genética , Linhagem Celular Tumoral , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Pulmonares/genética , Prognóstico , Regiões Promotoras Genéticas , Análise de Sequência de RNARESUMO
The CCAAT box is recognized by the trimeric transcription factor NF-Y, whose NF-YA subunit is present in two major splicing isoforms, NF-YAl ("long") and NF-YAs ("short"). Little is known about the expression levels of NF-Y subunits in tumors, and nothing in lung cancer. By interrogating RNA-seq TCGA and GEO datasets, we found that, unlike NF-YB/NF-YC, NF-YAs is overexpressed in lung squamous cell carcinomas (LUSC). The ratio of the two isoforms changes from normal to cancer cells, with NF-YAs becoming predominant in the latter. NF-YA increased expression correlates with common proliferation markers. We partitioned all 501 TCGA LUSC tumors in the four molecular cohorts and verified that NF-YAs is similarly overexpressed. We analyzed global and subtype-specific RNA-seq data and found that CCAAT is the most abundant DNA matrix in promoters of genes overexpressed in all subtypes. Enriched Gene Ontology terms are cell-cycle and signaling. Survival curves indicate a worse clinical outcome for patients with increasing global amounts of NF-YA; same with hazard ratios with very high and, surprisingly, very low NF-YAs/NF-YAl ratios. We then analyzed gene expression in this latter cohort and identified a different, pro-migration signature devoid of CCAAT. We conclude that overexpression of the NF-Y regulatory subunit in LUSC has the scope of increasing CCAAT-dependent, proliferative (NF-YAshigh) or CCAAT-less, pro-migration (NF-YAlhigh) genes. The data further reinstate the importance of analysis of single isoforms of TFs involved in tumor development.