CTCF Binding Site Mutations: Linking Topologically Associated Domains Dysregulation to Cutaneous Squamous Cell Carcinoma Progression

Background Cutaneous squamous cell carcinoma (cSCC) is the most common lethal malignancy with metastatic potential. The high mutational burden in cSCC has made it difficult to understand the significance of variants in the noncoding and regulatory genome. This study presents the first investigation of mutations at CCCTC-binding factor binding sites (CTCFbs) of topologically associating domains (TADs) across defined stages of disease progression —primary tumours that have not metastasized, primary tumours that have metastasized, and lymph node metastasis. Results By integrating matched whole-genome sequencing and RNA sequencing data from the same tumors, we reveal that CTCFbs are mutation hotspots (~1,100 mutations/Mb) in cSCC, with mutation densities far exceeding genome-wide averages (170–250 mutations/Mb). This study is also the first to prove genome-wide association of CTCFbs mutations with gene expression changes in cSCC. We report the novel finding that CTCFbs that overlap with other regulatory elements such as promoters and untranslated regions exhibit even higher mutational densities than overall CTCFbs. A pattern of mutually exclusive TAD loop CTCFbs mutations was observed between non-metastasizing and metastasizing primary tumors. TAD loops with mutated CTCFbs, are associated with significant transcriptional changes in the genes within those TADs, implicating genes including HHIPL2 , LINC02870 , and GDA in cSCC progression. Conclusions Our findings highlight the functional relevance of noncoding mutations at CTCFbs in cSCC and suggest their potential influence as drivers of tumor progression and metastasis. This integrative genomic analysis with detailed examination of TADs provides a foundation for future studies into 3D genome dysregulation in skin cancers.