Abstract
Extrachromosomal circular DNA (eccDNA) are circular DNA molecules that originate from chromosomal DNA yet function autonomously, existing outside of the chromosomes. A specific subset of eccDNA, known as ecDNA, consists of larger circular DNA molecules commonly found in tumors, often harboring intact oncogenes. Recent studies underscore a significant role of ecDNAs in driving intra-tumoral genetic heterogeneity, tumor progression, and resistance to anti-cancer therapies. These findings suggest adaptive mechanisms in cancer cells, which leverage ecDNA to amplify oncogenes and other genetic elements that direct and enhance tumorigenesis. Despite this progress in our understanding of ecDNA there is still a critical need for cell and animal model systems for investigating the mechanisms by which ecDNA fuels cancer development. Developing robust, reliable models is essential to unraveling the pathways ecDNA influences, potentially leading to more effective cancer therapies and strategies to overcome drug resistance.
Building on findings that ecDNA carrying the oncogene MYC is frequently selected in multiple cancer types, my research work presented below focuses on understanding how MYC-harboring ecDNA contributes to cancer-associated phenotypes. To this end, I developed two model systems in my thesis work: a cell line-based model and a mouse model. Using CRISPR-C technology, I successfully engineered MYCcontaining ecDNA in both cell lines and the mouse liver, precisely replicating the characteristics of naturally occurring tumor ecDNA.
In Chapter 1, based on the cell model, I tracked the dynamics of engineered MYCcontaining ecDNA to investigate its effects on cell proliferation, apoptosis, and migration. My findings indicate that cells harboring MYC ecDNA exhibit enhanced proliferation and migration capabilities, while showing a reduced response to apoptosis. These results suggest that MYC-harboring ecDNA may play a significant role in driving more aggressive cancer phenotypes by promoting growth and migration while evading programmed cell death.
While the cell models provided valuable insights, they were limited in replicating the natural selection processes that ecDNA undergoes during tumor growth and evolution. Specifically, the engineered ecDNA did not undergo replication and accumulation within cells; instead, it gradually diminished over time. This limitation highlights the challenges of mimicking the dynamic, selective pressures present in a tumor environment using cell culture systems alone.
To overcome the limitations of the cell models, in Chapter 2, I further developed a mouse model to enable studies of Myc-harboring ecDNA in liver cancer (hepatocellular carcinoma, HCC) progression. Through DNA and transcriptomic analysis, I discovered that deletion of Pten allows for the persistence and amplification of Myc ecDNA. Additionally, CCL4-induced liver injury further enhanced Myc transcriptional activation, ultimately activating and exacerbating the oncogenic potential of HCC. These findings suggest that the loss of tumor suppressor genes like Pten may create favorable conditions for the stabilization and amplification of oncogene-containing ecDNA and increases tolerance to high Myc expression. Furthermore, under environmental stimuli such as CCL4, this setting leads to the activation of Myc expression, contributing to increased tumor incidence and progression.
Despite significant progress in understanding large amplified ecDNA, particularly its role in oncogene amplification and cancer progression, our knowledge of smaller eccDNA remains limited. These eccDNAs can be more effectively identified by first removing linear chromosomal DNA, followed by sequencing to reveal their potential contributions to cancer biology. To advance our understanding of eccDNA's role, improved detection methods are crucial, as mitochondrial DNA (mtDNA), which is also circular, often diminishes the detection accuracy of eccDNA by diverting reads away from it. Therefore, in Chapter 3, I developed a CRISPR/Cas9-based approach to selectively remove mtDNA, enabling more effective detection and analysis of eccDNA. Applying this method across mouse tissues, human cell lines, and cancer samples, I observed significant improvements in eccDNA yield and identification, laying a strong foundation for further exploration of eccDNA's role in tumorigenesis.
In Chapter 4, I focused on the role of smaller eccDNA (<100,000 base pairs) in colorectal cancer (CRC), with a particular emphasis on the cytokine gene CXCL5. Using the mtDNA removal approach developed in Chapter 3, analysis of 25 CRC tumors showed that eccDNA was significantly more abundant in tumor tissues and that CXCL5 was frequently present on these eccDNAs, resulting in substantial copynumber increases and upregulation. Ectopic expression experiments in cell models further confirmed that CXCL5 eccDNA enhances transcriptional output and immune cell recruitment, suggesting a pivotal role for eccDNA in promoting CRC progression.
In summary, by developing effective detection methods and establishing robust cell and animal models, this work provides important tools for investigating eccDNA's influence on tumor behavior. Future research by our team will aim to clarify the mechanisms by which eccDNA drives cancer development. A deeper understanding of these pathways could reveal novel targets for therapeutic intervention, ultimately contributing to more precise and effective cancer treatments
Building on findings that ecDNA carrying the oncogene MYC is frequently selected in multiple cancer types, my research work presented below focuses on understanding how MYC-harboring ecDNA contributes to cancer-associated phenotypes. To this end, I developed two model systems in my thesis work: a cell line-based model and a mouse model. Using CRISPR-C technology, I successfully engineered MYCcontaining ecDNA in both cell lines and the mouse liver, precisely replicating the characteristics of naturally occurring tumor ecDNA.
In Chapter 1, based on the cell model, I tracked the dynamics of engineered MYCcontaining ecDNA to investigate its effects on cell proliferation, apoptosis, and migration. My findings indicate that cells harboring MYC ecDNA exhibit enhanced proliferation and migration capabilities, while showing a reduced response to apoptosis. These results suggest that MYC-harboring ecDNA may play a significant role in driving more aggressive cancer phenotypes by promoting growth and migration while evading programmed cell death.
While the cell models provided valuable insights, they were limited in replicating the natural selection processes that ecDNA undergoes during tumor growth and evolution. Specifically, the engineered ecDNA did not undergo replication and accumulation within cells; instead, it gradually diminished over time. This limitation highlights the challenges of mimicking the dynamic, selective pressures present in a tumor environment using cell culture systems alone.
To overcome the limitations of the cell models, in Chapter 2, I further developed a mouse model to enable studies of Myc-harboring ecDNA in liver cancer (hepatocellular carcinoma, HCC) progression. Through DNA and transcriptomic analysis, I discovered that deletion of Pten allows for the persistence and amplification of Myc ecDNA. Additionally, CCL4-induced liver injury further enhanced Myc transcriptional activation, ultimately activating and exacerbating the oncogenic potential of HCC. These findings suggest that the loss of tumor suppressor genes like Pten may create favorable conditions for the stabilization and amplification of oncogene-containing ecDNA and increases tolerance to high Myc expression. Furthermore, under environmental stimuli such as CCL4, this setting leads to the activation of Myc expression, contributing to increased tumor incidence and progression.
Despite significant progress in understanding large amplified ecDNA, particularly its role in oncogene amplification and cancer progression, our knowledge of smaller eccDNA remains limited. These eccDNAs can be more effectively identified by first removing linear chromosomal DNA, followed by sequencing to reveal their potential contributions to cancer biology. To advance our understanding of eccDNA's role, improved detection methods are crucial, as mitochondrial DNA (mtDNA), which is also circular, often diminishes the detection accuracy of eccDNA by diverting reads away from it. Therefore, in Chapter 3, I developed a CRISPR/Cas9-based approach to selectively remove mtDNA, enabling more effective detection and analysis of eccDNA. Applying this method across mouse tissues, human cell lines, and cancer samples, I observed significant improvements in eccDNA yield and identification, laying a strong foundation for further exploration of eccDNA's role in tumorigenesis.
In Chapter 4, I focused on the role of smaller eccDNA (<100,000 base pairs) in colorectal cancer (CRC), with a particular emphasis on the cytokine gene CXCL5. Using the mtDNA removal approach developed in Chapter 3, analysis of 25 CRC tumors showed that eccDNA was significantly more abundant in tumor tissues and that CXCL5 was frequently present on these eccDNAs, resulting in substantial copynumber increases and upregulation. Ectopic expression experiments in cell models further confirmed that CXCL5 eccDNA enhances transcriptional output and immune cell recruitment, suggesting a pivotal role for eccDNA in promoting CRC progression.
In summary, by developing effective detection methods and establishing robust cell and animal models, this work provides important tools for investigating eccDNA's influence on tumor behavior. Future research by our team will aim to clarify the mechanisms by which eccDNA drives cancer development. A deeper understanding of these pathways could reveal novel targets for therapeutic intervention, ultimately contributing to more precise and effective cancer treatments
| Original language | English |
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| Publisher | Department of Biology, Faculty of Science, University of Copenhagen |
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| Number of pages | 176 |
| Publication status | Published - 2025 |