Alternative splicing of NF-YA shapes tumour-macrophages crosstalk in colorectal cancer

Martina Sansone, “Alternative splicing of NF-YA shapes tumour-macrophages crosstalk in colorectal cancer” - ABSTRACT Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related deaths. A key factor in its progression and treatment resistance is the tumour microenvironment (TME), a complex network comprising cancer cells, stromal cells, extracellular matrix (ECM) and immune cells, including macrophages, which account for 50% of the immune population. Tumour associated macrophages (TAMs) can adopt pro-inflammatory M1 or anti-inflammatory M2 phenotypes depending on local cues. In many cancers, TAMs skew toward an M2-like state, promoting angiogenesis, immunological suppression, and ECM remodelling. NF-Y is a transcription factor composed of the subunits NF-YB and NF-YC, that feature a histone-fold domain, and the regulatory subunit NF-YA, which recognizes the CCAAT box, a DNA motif highly enriched in tumour overexpressed genes. NF-YA undergoes alternative splicing to generate two isoforms: the long form (NF-YAl), which includes exon-3 and is associated with cell differentiation, and the short form (NF-YAs), which lacks exon 3 and is linked to stemness. In CRC, the expression of the two NF-YA isoforms aligns with the four molecular consensus subtypes (CMS1-4), which have prognostic significance. NF-YAs is up-regulated in all CMSs, while NF-YAl is down-regulated in all subtypes except for the aggressive and metastatic CMS4 group. CMS4 tumours comprise 23% of all CRC cases and are marked by poor prognosis and treatment resistance compared to other subtypes. They feature a high number of mesenchymal stromal cells, including cancer-associated fibroblasts, and an inflammatory profile, with increased M2 macrophages, creating a pro-tumoral microenvironment. NF-YA alternative splicing events play a key role in tumour progression, but the way they influence colorectal cancer - immune cells interactions is not clear. This thesis project aims to unravel the role of NF-YA splicing isoforms modulation in tumour cells as a driver of interactions within the TME, particularly with macrophages. To explore this, we developed 2D and 3D in vitro models using HCT116 colon cancer cells (CMS4 subtype) and monocytes/macrophages. Cell culture medium conditioned by HCT116 overexpressing NF-YAs (NF-YAs high) increases the levels of M1 markers in monocytes and M0 macrophages, in contrast to NF-YAl high conditioned medium that enhances M2 markers. These results were upheld by indirect co-culture experiments, where NF-YAl high cells induce M2 polarization. In turn, M2 macrophages promote epithelial to mesenchymal transition (EMT) in HCT116 cells by upregulating EMT-related genes. 3D multicellular tumour spheroids confirmed the functional interplay between CRC cells and macrophages. Live confocal imaging showed macrophages clustering, typically observed following M1 polarization, only around NF-YAs high spheroids. In opposition, dispersed macrophages infiltrated the tumour mass composed by NF-YAl cells, being potentially ineffective. Overall, our results are consistent with a different role of NF-YA splicing variants in CRC immune modulation.