The urinary eccDNA landscape in prostate cancer reveals associations with genome instability and vital roles in cancer progression.

INTRODUCTION: Extrachromosomal circular DNA (eccDNA) plays significant roles in cancer progression and prognosis. However, it remains unclear whether cell-free eccDNA, considered more stable than linear DNA, possesses cancer-specific genomic features. Furthermore, the biogenesis and function of eccDNAs are not yet fully understood. OBJECTIVES: This study aims to characterize the genomic landscape of urinary cell-free eccDNAs in prostate cancer (PCa) and non-cancer (NC) individuals, elucidate their biogenesis and PCa-specific genomic features, and investigate their roles in PCa progression. METHODS: We conducted urine Circle-seq for 21 PCa patients and 16 NC individuals, performed integrated analysis with other omics datasets, and finally validated the function of eccDNA by in vitro transfection and RNA-seq. RESULTS: We pioneered the profiling of urinary cell-free eccDNAs landscape in PCa and uncovered a high association between eccDNA generation and active chromatin status as well as gene transcription. Double strand breaks and R-loops, which preferentially occur in active genomic sites and cause genome instability, can promote eccDNA generation. Genome instability frequently results in genomic mutations, and our study further established a link between eccDNA generation and oncogenic mutations. Additionally, genes specifically exhibiting high eccDNA generation frequency (HFGs) in PCa contributed to PCa progression and were associated with poorer survival outcomes in PCa patients. Finally, we demonstrated that eccDNAs derived from PCa-specific HFGs, in contrast to intergenic eccDNAs, could suppress PCa cell proliferation and migration, which was independent of their host gene expression. CONCLUSION: Our study illustrated the biogenesis of eccDNAs from DSBs in active genes, revealed PCa-specific eccDNA features and suggested new mechanisms underlying eccDNA function.