The role of YBX1 in m5C-mediated control of cutaneous squamous cell carcinoma invasion and metastasis

Cutaneous squamous cell carcinoma (cSCC) is a highly prevalent skin malignancy and, although most cases are managed successfully by surgical excision, a significant subset develops invasive or metastatic disease associated with high morbidity and limited therapeutic options. Posttranscriptional mechanisms increasingly emerge as key regulators of tumor progression and cellular plasticity. Among these, RNA modifications and RNA binding proteins (RBPs) constitute a dynamic layer of control that influences mRNA stability, translation, and fate. However, the contribution of these pathways to cSCC biology remains poorly understood. In this thesis, I investigate the role of the RBP YBX1 as a post-transcriptional regulator of program associated with cSCC invasion and metastasis. The first part of this thesis focused on profiling YBX1-RNA interactions through enhanced crosslinking immunoprecipitation (eCLIP) and RNA nanopore sequencing. These analyses reveal that YBX1 engages distinct sets of transcripts in tumor cells compared with non-transformed epidermis and demonstrate a preferential enrichment of m⁵C modifications at YBX1 binding sites in cSCC, suggesting that RNA methylation serves as a determinant of target specificity. This modification-guided interaction is consistent with elevated NSUN2 expression in tumor cells, implicating NSUN2 as the writer that shapes the m⁵C landscape required for YBX1 target engagement. The second part explores the functional consequences of YBX1 and NSUN2 depletion in primary cSCC cell lines. Both factors support proliferation meanwhile suppressing cSCC migration. YBX1 loss induces a marked increase in invasion and metastatic dissemination across in vitro, 3D spheroid, and in vivo models. These data uncover a dual role for YBX1, in which proliferative support is accompanied by an anti-invasive function that appears progressively lost in metastatic lesions. The final part of the thesis aimed to identify and characterize m⁵C-modified transcripts bound by YBX1 that contribute to invasive behavior in cSCC. Loss of YBX1 reduces transcript stability and protein expression of these targets, suggesting a mechanistic model in which m⁵C deposition by NSUN2 enhances YBX1 binding, thereby promoting transcript stabilization and translation of anti-invasive targets. This establishes an epitranscriptional mechanism in which coupling between a methylation writer and an RBP regulates gene expression programs relevant to tumor progression. Collectively, this work uncovers a previously unexplored regulatory mechanism in cSCC, demonstrating that NSUN2-dependent m⁵C modifications direct YBX1 activity to modulate the stability and expression of anti-invasive transcripts, thereby sustaining local proliferation while inhibiting dissemination. These findings provide mechanistic insight into cSCC progression and individuate the YBX1–m⁵C–NSUN2 regulatory axis as a potential target for therapeutic intervention.