Academic Background and Problem Statement

In cancer research, gene amplification is a common form of mutation, particularly in the activation of oncogenes. However, despite the widely recognized importance of gene amplification in cancer, precisely modeling these amplifications in primary cells and model organisms remains a challenge. Specifically, extrachromosomal DNA (ecDNA)-mediated gene amplification is particularly common in cancer, but its specific role in tumor initiation and progression is not yet fully understood. ecDNA is a class of circular DNA molecules that do not rely on chromosomes, often carrying multiple copies of oncogenes. They can rapidly accumulate through random segregation during cell division, thereby promoting tumor heterogeneity and evolution.

To gain a deeper understanding of the role of ecDNA in tumorigenesis, researchers need a method to precisely induce and track ecDNA formation in cell and animal models. However, existing technologies face numerous limitations in modeling ecDNA-mediated gene amplification. Therefore, this study aims to develop a general strategy to spatiotemporally induce large-scale (over 1 Mbp) ecDNA amplification in cells and mice, and to use this strategy to investigate the specific mechanisms of ecDNA in tumorigenesis.

Paper Source and Author Information

This paper was co-authored by Davide Pradella, Minsi Zhang, Rui Gao, and 13 other researchers, primarily from renowned institutions such as Memorial Sloan Kettering Cancer Center, Stanford University, and UC San Diego. The paper was published online on October 31, 2024, in the journal Nature, titled “Engineered extrachromosomal oncogene amplifications promote tumorigenesis.”

Research Process and Experimental Design

1. Development of ecDNA Engineering Strategy

To mimic ecDNA amplification commonly observed in human cancers, the researchers designed a strategy based on the Cre-loxP system. By coupling ecDNA formation with the expression of selectable markers, the researchers were able to track the dynamics of ecDNA under physiological conditions and specific selective pressures. Specifically, the researchers designed two “circularization cassettes” inserted at the ends of the target gene region. When Cre recombinase is expressed, the genomic region between the loxP sites is excised and circularized, forming ecDNA. Simultaneously, fluorescent markers (e.g., H2B-GFP and mScarlet) in the circularization cassettes allowed the researchers to track the presence and dynamics of ecDNA.

2. Validation of ecDNA Formation and Dynamics in Cells

The researchers first validated this strategy in the HCT116 colon cancer cell line. Using CRISPR-Cas9 gene editing, the researchers inserted a 1.5 Mbp region on chromosome 12 containing the MDM2 oncogene. Upon Cre recombinase expression, the researchers successfully induced ecDNA formation and observed its dynamics using flow cytometry and fluorescence microscopy. Additionally, the researchers confirmed the presence and amplification of ecDNA through shallow whole-genome sequencing (SWGS) and quantitative PCR (qPCR).

3. Induction of ecDNA Formation in Mouse Models

To further investigate the role of ecDNA in vivo, the researchers constructed a mouse model carrying loxP sites in the MYC gene region on chromosome 15. Through Cre recombinase induction, the researchers successfully induced the formation of MYC-containing ecDNA in primary mouse cells and observed the spontaneous accumulation of these ecDNA in cells. Furthermore, the researchers constructed a mouse model carrying MDM2-containing ecDNA and found that these ecDNA promoted the proliferation, immortalization, and transformation of primary cells.

4. Role of ecDNA in Tumorigenesis

To validate the specific role of ecDNA in tumorigenesis, the researchers induced the formation of MDM2-containing ecDNA in an autochthonous mouse model of hepatocellular carcinoma. The results showed that mice carrying ecDNA spontaneously developed liver cancer driven by the MYC gene. This result directly demonstrated the important role of ecDNA in tumorigenesis and provided a new animal model for studying ecDNA-driven tumors.

Main Research Findings

1. Formation and Dynamic Tracking of ecDNA: The researchers successfully developed a strategy to precisely induce ecDNA formation in cells and mice and tracked the dynamics of ecDNA using fluorescent markers and genomic sequencing techniques.

2. ecDNA Promotes Cell Proliferation and Immortalization: In primary mouse cells carrying MYC- and MDM2-containing ecDNA, the researchers observed the spontaneous accumulation of ecDNA and found that these ecDNA promoted cell proliferation and immortalization.

3. Role of ecDNA in Tumorigenesis: In an autochthonous mouse model of hepatocellular carcinoma, the researchers found that MDM2-containing ecDNA promoted tumor formation, further validating the important role of ecDNA in tumorigenesis.

Conclusions and Significance

This study developed a general strategy to precisely induce ecDNA-mediated gene amplification in cells and mice and used this strategy to investigate the specific mechanisms of ecDNA in tumorigenesis. The results demonstrated that ecDNA can spontaneously accumulate in primary cells and promote cell proliferation, immortalization, and tumor formation. This discovery not only provides new insights into the role of ecDNA in tumorigenesis but also offers important tools for developing new preclinical immunocompetent mouse models.

Research Highlights

1. Innovative Strategy: This study is the first to develop a strategy to precisely induce ecDNA formation in cells and mice, providing new tools for studying the biological functions of ecDNA.

2. Role of ecDNA in Tumorigenesis: The study directly demonstrated the important role of ecDNA in tumorigenesis, offering a new perspective for understanding tumor heterogeneity and evolution.

3. Application Value: This study provides important tools for developing new preclinical mouse models, which can be used in the future to study immunotherapy and targeted therapy for ecDNA-driven tumors.

Other Valuable Information

This study also revealed the dynamic changes of ecDNA in cells and its relationship with tumorigenesis, providing new directions for future cancer research. Additionally, the researchers found that the accumulation of ecDNA is closely related to specific selective pressures, offering new clues for understanding tumor drug resistance.