Extrachromosomal circular DNA (eccDNA): from carcinogenesis to drug resistance

Cancer is a major public health problem that is associated with a high mortality rate and a poor quality-of-life in some cancer patients, thus representing a serious threat to human health [1, 2]. The occurrence of disease is associated with specific causes and mechanisms, and the development of cancer is no exception. Chromatin and its related epigenetic mechanisms in cells of the body can maintain specific gene expression patterns and cellular homeostasis under normal physiological conditions, so as to adapt to changes in various development and survival conditions. However, due to some abnormal genetic, environmental or metabolic stimuli and other factors, there may cause changes in the epigenetic environment of cells and chromatin distortion, thus causing cancer and other diseases [3]. The occurrence and development of cancer is not caused by a single factor; on the contrary, it is caused by an interaction between complex and diverse molecular mechanisms. Abnormal gene expression is the hallmark of cancer [4, 5] and one of the key driving factors of this disease. This includes changes in gene sequence, such as point mutation, chromosomal translocation, deletion and insertion, and changes in the gene copy number, such as gene amplification and other gene activation mechanisms [6, 7]; these events can promote large-scale DNA recombination [8], thus leading to cancer.

Extrachromosomal circular DNA (eccDNA) refers to circular DNA that originates from chromosomes, but is likely to be independent of chromosomal DNA once produced. Human cells have 23 pairs of chromosomes, some of which can be amplified in the DNA outside the chromatin under certain environmental conditions. Therefore, there is a special type of circular DNA molecules that are independent of the chromosome genome in cells, which are collectively referred to as eccDNA. These are separated from normal chromosomes and genomes, and form a single or double stranded closed circular DNA structure [9, 10]. In 1964, Alix Bassel and Yasuo Hoota identified eccDNA in the nucleus of pig sperm for the first time by electron microscopy; researchers named it double minutes (DMs) [11]. Subsequent research revealed that the characteristics and functions of eccDNA are increasingly being exposed to public view, and it is now clear that a wide variety of eccDNA is widely present in eukaryotic cells, with sizes ranging from hundreds of base pairs (bp) to several million bases (Mb). Based on source and size, eccDNA can be divided into mitochondrial DNA (MtDNA), episomes, double minutes (DMs) (100 Kb–3 Mb), telomere rings (T-rings), small polydisperse circular DNA (spcDNA) (100 bp–10 kb), and microDNA (100–400 bp) [12]. With increased research activity, the functions of eccDNA are gradually being reported. In addition to functions such as aging [13‐15] and heterogeneity [16], researchers have found that the length of eccDNA is long enough to feature its own replication starting point, as well as to encode amino acids and form specific proteins to function, which have been detected in tumor tissues. For example, in non-small cell lung cancer, the eccDNA of PLCG2 is upregulated in NSCLC cells, resulting in phosphatidase Cγ2, as a transmembrane signal transduction enzyme, is highly expressed in NSCLC tissues and cells, thereby promoting the progression of NSCLC [17]; the amplification of DHFR gene can also enhance tumor resistance to MTX by increasing the production of DHFR [18]. Therefore, in cancer therapy, eccDNA often features oncogenes or genes associated with drug resistance [19‐22]. These characteristics enable eccDNA to play a positive role in the progression of cancer, effectively promoting cancer progression. In this review, we discuss the biogenesis of eccDNA, its relationship with tumors and its role in tumor development, and provide prospects for its future clinical applications.

In the decades following the discovery of eccDNA, it has been found that the formation of eccDNA in human cells typically involves damage to chromosomal DNA and incorrect behavior through different DNA repair pathways [23]. Studies have shown that heterochromatin in the body is considered the guardian of the genome, which can protect the integrity and stability of the genome [24]. Therefore, it is speculated that one of the reasons for DNA damage may be due to the decreased protective ability of noncoding DNA against the genome [25]. Some studies have also found that transcription can induce the formation of eccDNA during the aging process of yeast cells [15]. Another study has shown that deoxyribonuclease 1 like 3 (DNASE1L3) can digest eccDNA outside the cell, thereby reducing the accumulation of eccDNA [26]. Researchers have performed in-depth studies relating to the mechanisms responsible for eccDNA and found that eccDNA can be produced by many different ways and different mechanisms. Following decades of research, several eccDNA formation models have been proposed, including the break–fusion–bridge (BFB) cycle model, chromosome fragmentation, the episome model, and the translocation–excision–deletion–amplification model [27‐30].

EccDNA is closely related to the occurrence and development of various cancers and affects the progression of cancer through various ways and functions (Table 1) [43]. These findings indicate that eccDNA may represent a new biological target and play an important role in the process of tumor detection and treatment (Fig. 1A).

Cancer is one of the main causes of death in humans. For cancer, in addition to surgical treatment, chemotherapy is also one of the main treatment methods for cancer patients [65, 66]. However, cancer patients often experience drug resistance during chemotherapy. It is because cancer cells develop tolerance and resistance to chemotherapy drugs, leading to a decrease in the inhibitory effect of drugs on cancer cells during the treatment process [67]. Thus, drug resistance is one of the main reasons for the failure of clinical chemotherapy. Drug resistance in cancer can be divided into endogenous drug resistance and acquired drug resistance [68]. Endogenous drug resistance occurs prior to chemotherapy and refers to the ability of cancer cells without chemotherapy to survive the initial treatment due to preexisting genetic changes or cell states, while acquired drug resistance develops through the acquisition of new mutations [69]. Most cancer cell resistance is acquired, that is, it develops through new mutations during chemotherapy. Therefore, there are many cancer patients who, even with corresponding chemotherapy drugs available for treatment, have poor chemotherapy effects, and even patients in advanced stages of cancer do not have available chemotherapy drugs. Therefore, the in-depth study of tumor resistance to chemotherapy drugs has become an urgent problem to be solved. Over recent years, an increasing number of studies have shown that eccDNA is involved in the process of drug resistance in cancer treatment and may also promote the generation of resistance to tumor drugs by carrying oncogenes and increasing oncogene copy number [27, 70].

In the process of studying eccDNA, researchers have been constantly trying various experimental methods in order to explore the true face of eccDNA. In the past few decades of studying eccDNA, researchers have used research methods such as electron microscopy [22], Karyotype [20], 2D gel electrophoresis [74, 75], Southern blotting [74], Inverse PCR [76, 77], fluorescence in site hybridization (FISH) [20‐22], density gradient centralization [78], etc., to explore the secrets of eccDNA. These technologies can locate, identify, and quantify eccDNA, but there are also limitations. Some shortcomings, such as the inability of electron microscopy, Karyotype, 2D gel electrophoresis and other technologies to provide sequence information of eccDNA; Technologies such as Southern blotting, reverse PCR, and fluorescence in situ hybridization (FISH) can only provide a small amount of sequence information; however, density gradient centralization is very time-consuming and labor-intensive. A high-throughput sequencing technology [79] has recently been developed, which has extremely high resolution and sensitivity, but the high cost makes this technology unable to be considered as the primary method by researchers. Therefore, in the subsequent research process, finding a new experimental technique that can effectively avoid these shortcomings is a challenge that researchers have to face.

EccDNA has only been discovered for decades, but its existence and importance to the organism are undisputed. This discovery has provided a new direction for cancer research; that is, to study the specific relationship between eccDNA and cancer. In this article, we review existing research relating to eccDNA and its relationship with cancer, propose some challenges and future opportunities, and provide some ideas for future research on eccDNA. For example, although scientists have proposed four models for the biogenesis of eccDNA (the break–fusion–bridge (BFB) cycle model, chromosome fragmentation, the episome model, and the translocation–excision–deletion–amplification model), its biogenesis and cycle mechanisms have yet to be fully elucidated. There are also significant differences between eccDNA and chromosome DNA in key aspects such as location, topology, and biology; therefore, researchers urgently need to develop new research tools and experimental methods with which to explore the specific role of eccDNA in the pathogenesis of cancer [80]. Furthermore, due to differences in the production and expression of eccDNA in cancer cells, eccDNA can be investigated as a potential biomarker for detecting cancer in the future. Furthermore, eccDNA has been shown to be related to drug resistance in some diseases. Therefore, there is an urgent need to improve our understanding of eccDNA by performing new research studies. Providing further evidence to support the potential use of eccDNA as a new biological target to inhibit resistance to tumor drugs is a crucial and significant research focus. Similarly, it is important that we combine therapy with traditional chemoradiotherapy or classical anticancer drugs to improve the efficacy and synergy of tumor therapy; this is an important direction of future research for eccDNA.

Since researchers first discovered the existence of eccDNA in 1964, with the joint efforts of researchers around the world, the mysterious veil of eccDNA has been gradually revealed, allowing people to gradually see its true face. In these 60 years, the mechanisms responsible for the biogenesis of eccDNA has been studied, and four models have been proposed, namely the break–fusion–bridge (BFB) cycle model, chromosome fragmentation, the episome model, and the translocation-excision-deletion-amplification model. Researchers have also reported abnormal oncogene amplification in eccDNA; this is prevalent in various human cancers and can lead to a high copy number and expression levels of oncogenes, thus promoting tumorigenesis and adaptive evolution. Furthermore, eccDNA is also an important driving force for resistance to cancer drugs and has a close relationship with the poor prognosis of cancer and the development of genetic heterogeneity. Therefore, a deep understanding of the structure, mechanism of action, and function of eccDNA can contribute to our research on cancer progression, providing new detection targets and treatment plans for cancer detection and treatment, which is of great significance for the treatment and prognosis of cancer patients. In future research, more in-depth exploration of eccDNA is needed to reveal the regularity and characteristics of eccDNA, in order to be applied for detecting and treating cancer.