Function and potential clinical application of circular DNA in cancer

Extrachromosomal circular DNA (eccDNA) has been detected across a broad spectrum of eukaryotes species, from yeast to humans, underscoring its evolutionary conservation, and suggesting its potential fundamental roles in cellular functions. The evolution of cancer cells has been linked to genomic mutations, including eccDNA that carry oncogenes. However, while previous studies have primarily focused on the function of large amplified eccDNA (1 to 3 Mb in size) with oncogenes in cancer, comprehensive understanding of eccDNA across both small and large sizes remains incomplete. In this work, I present eccDNA profiles across three cancer types, describe a method for investigating eccDNA function, and report on a pilot study investigating eccDNA in human urine biopsy of healthy individuals and chronic kidney disease patients. Using these approaches, I investigate the role of circularized genes on eccDNA in cancers and assess the feasibility of developing urinary biomarkers based on eccDNA.

Initially, the introduction section offers an overview of eccDNA detailing both the foundational knowledge and methodologies for newcomers to this field. I will review the diverse types of eccDNA previously identified, highlighting their nomenclature and definitions. Additionally, I focus on the role of eccDNA in facilitating cancer cell adaptation. Moreover, I propose a model suggesting that miRNA gene amplification on eccDNA contributes to both the proliferation and adaptation of cancer cell. Lastly, I introduce the potential application of urinary cell free eccDNA in screening of urinary tract disease including cancer and inflammation.

In Chapter 1, I characterize eccDNA of all sizes, including big ecDNA (1 to 3 Mb in size) with amplified oncogene named ecDNA, and elucidate genome heterogeneity in 80 urothelial bladder carcinomas (UBC). This study uncovers that eccDNA increases oncogene dosage and appears to influence genome-wide gene expression. The genetic changes found in the UBC are associated with hyper-mutation (kataegis), copy number variation, oncogene duplication, structure variation, and poor clinical outcome. Our analy sis supports that eccDNA is associated with cancer genome heterogeneity, progression, and clinical outcomes.

Chapter 2 addresses the function of small size eccDNA in the context of renal cell carcinoma of 122 pairs of cancer and normal biopsies. This study reveals a high abundance of eccDNA across all biopsies, while only one ecDNA (1-3 Mb) was identified in one tumor biopsy. This enabled a focused investigation into the role of smaller eccDNA (1 Mb <) in facilitating cancer progression and cancer-cell evolution mechanisms. Multiple cancer-related miRNA genes on eccDNA were identified across various cancer biopsies, five of them were chosen by integration of their target genes, and I confirmed that they can contribute to cancer-cell phenotypes in in vitro models. This study offers a comprehensive overview of eccDNA’ s prevalence in renal cancer, underscoring the critical role of eccDNA carrying miRNA gene sequences in the adaptive processes of cancer cells.

Chapter 3 presents CAES (Circle-Seq based Artificial EccDNA Synthesis), a novel and robust method for the artificial synthesis of circular DNA. The CAES enables precise synthesis of eccDNA ranging from 300 bp to 2100 bp in size. Additionally, eccDNA synthesized via CAES containing miRNA genes efficiently expresses both miRNA-3’ and 5’ molecules, independent of a canonical promoter, in human cell lines.

Chapter 4 reveals a notably higher abundance of eccDNA in gastric cancer (GC) compared to its adjacent normal tissues. I found that GC over-represented eccDNAs that carry miRNA genes were significantly enriched in multiple cancer-relevant signal pathways. Furthermore, we observed the transfection of GC over-represented eccDNA carrying miRNA gene, benefited host cell proliferation and promoted the aggressive features of host cells.

Lastly, in Chapter 5, I coauthored a study where we acquired and compared the eccDNA profiles in the urine biopsies from healthy individuals and chronic kidney disease (CKD) patients. Here we modified the classical method for purification of eccDNA, Circle-Seq, to perform it in urine biopsies and identified highly abundant eccDNA in the urine of healthy and CKD individuals. This work discovers and provides the first deep characterization of urinary cell-free eccDNA and suggests the application of urinary cell-free eccDNA as a valuable non-invasive biomarker for urogenital disorder diagnosis and monitoring.