Role of NF-Y and SMYD3 transcriptional modulators in aggressiveness of hormone-related cancers

Breast and prostate cancers (BRCA and PRCA) are the leading tumors in terms of incidence in theworld and their metastases account for the great majority of cancer-associated deaths. Studies aimedat dissecting novel molecular mechanisms that favor metastasis formation are of primary relevance for the development of novel treatments and diagnostic tools. The project aims at dissecting the activity of the transcriptional modulators NF-Y and SMYD3 and theirfunctional relationship in transcriptional and epigenetic mechanisms controlling the metastaticbehavior of BRCA and PRCA. NF-Y, a pioneer transcription factor composed of the NF-YB/NF-YCdimer and the DNA binding subunit NF-YA, is capable of accessing condensed chromatin at specificDNA elements to recruit chromatin-modifying proteins, among which histone methyltransferases. Aclear correlation exists between the binding of NF-Y and H3K4me3 at gene promoters. Among H3K4methyltransferases, SMYD3 associates with open chromatin and activates the transcription of multipletarget genes through association with RNA polymerase II. Both NF-YA and SMYD3 are overexpressedin BRCA and PRCA and are responsible for the aggressive behavior of cancer cells. The expressionlevels of NF-YA and SMYD3, as well as the splicing signature of the NF-YA gene, which encodes forNF-YAs and NF-YAl transcripts, characterize tumor tissues and specifically highly aggressive andmetastasis-prone BRCA and PRCA subtypes. To understand the functional role of NF-Y and SMYD3 in aggressiveness, we will perform loss andgain of function experiments in BRCA and PRCA cell lines and we will analyze metastatic properties invitro and in vivo. We will determine individual and collective tumor-cell migration strategies andinvasive properties following single and concomitant genetic and pharmacological modulation ofSMYD3 and NF-YAs/l in 2D and 3D cell cultures analyzed by time-lapse microscopy, wound-healing/transwell assays, immunofluorescence, qRT-PCR and western blot. Further, we will usespecific collagen-based scaffold models mimicking bone environment to evaluate the role of NF-YAisoforms and SMYD3 in bone metastasis. To test the importance of SMYD3 and NF-YA in cellmigration and invasion, we will use both zebrafish and mouse models. To dissect the molecularmechanisms underlying NF-Y and SMYD3 interplay in the transcriptional modulation of cancer cells,we will determine NF-Y and SMYD3 genome-wide occupancy and their transcriptional signature inBRCA and PRCA cells through RNA-seq and Cut&Tag approaches. Finally, with the long-term goal touse NF-YA transcripts and SMYD3 as biomarkers to predict cancer aggressiveness or biochemicalrelapse, we will characterize their expression by RNA-FISH in spreading cells of tumor spheroids andin surgical margins of histological samples from BRCA and PRCA patients. This new molecularstrategy associated with routine histological analysis may avoid underestimation of aggressiveness.