Glioblastoma (GBM) is the most common and malignant primary brain cancer in adults with a poor prognosis and high risk of chemotherapy resistance (32). So far, the epidermal growth factor receptor (EGFR) and ATP-binding cassette subfamily G member 2 (ABCG2) have been reported frequently as drug resistance-related genes that are carried by ecDNA in GBM.

EGFR deletions and point mutations are often found in GBM, of which 50% have EGFR gene amplification in ecDNA, but 30%–60% of EGFR genes are mutated, and the most common mutation is EGFRvIII (33). EGFRvIII activates the NF-κB (nuclear factor κB) pathway and increases the aggressiveness of GBM, and cancer cells expressing EGFRvIII are more sensitive to EGFR tyrosine kinase inhibitors (TKIs) (34, 35). Nathanson et al. (18) used erlotinib to treat GBM-loading mice; 80% of the mice had a reduction in cancers, but cancer cells changed from predominantly high EGFRvIII expression to low EGFRvIII expression, accompanied by a decrease in drug sensitivity. The loss of ecDNA with EGFRvIII in erlotinib resistance was specific, as these cells still contained abundant ecDNA that carry other genes, such as murine double minute 2 (MDM2). In addition, they also found that MDM2 gene amplification was also associated with drug resistance during the study through fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). After erlotinib treatment, the copy number of ecDNA with the MDM2 gene was increased and remained elevated, even after drug withdrawal.

The ABCG2 gene is located on chromosome 4, and the protein it encodes can efficiently transport a variety of chemotherapeutic drugs (36). Rao et al. (19) detected DM carrying ABCG2 gene amplification in the SF295 MX50 and MX100 sublines. They generated these sublines by exposing SF295 cells belonging to GBM to mitoxantrone. Interestingly, with the increase in mitoxantrone concentration, fewer DMs were observed, but homogeneously staining regions (HSR) that carried ABCG2 gene amplicons were visible through FISH. Obviously, amplification of ABCG2 occurred initially in the form of DM, followed by chromosomal reintegration of the amplicon at multiple sites and producing stable genotypes associated with drug resistance.

Neuroblastoma is the most common solid extracranial neoplasm in children, showing an appreciable heterogeneity in clinical evolution. Amplification of the MYCN oncogene in this cancer is detected in 20–30% of cases and is associated with non-effective chemotherapy (37, 38). Through FISH and quantitative PCR analyses, Valent et al. (20) found that the MYCN oncogene can be amplified by ecDNA, especially in patients with advanced neuroblastoma who were resistant to chemotherapy. The MRP gene encodes special transmembrane glycoproteins, which can act as plasma membrane drug-efflux pumps, discharge drugs from the cells, eliminate the accumulation of drugs in cells, and make cancer cells acquire tolerance to varieties of drugs (39, 40). In neuroblastoma, the MYCN gene can be amplified with the help of ecDNA, which increased the expression of this gene and then upregulated the expression of multidrug resistance genes, resulting in enhanced resistance. Therefore, reducing the expression of MYCN may avoid the occurrence of chemotherapy resistance. It was reported that hydroxyurea induced the overexpression of MDR1 in cells to reduce the expression of extrachromosomal MYCN (41, 42), which provides a treatment target for the high-risk neuroblastoma in clinical settings.

Cervical carcinoma is the most common female reproductive system cancer in developing countries (43). Chemotherapy is considered as the standard treatment for patients with advanced or recurrent cervical cancer. Resistance to chemotherapy substantially affects the efficacy of cervical cancer treatment. Michael et al. (44) used FISH to analyze cervical cancer cells with methotrexate (MTX) resistance and found that all cells could amplify the dihydrofolate reductase (DHFR) gene via DMs. In addition, they found that chromosome 5 fragmentation events could form HSR with the DHFR gene. HSR could break again and produce fragments because of its instability, then produce DMs. Then, in HeLa cells, it was also confirmed that the defects in homologous recombination (HR) could play a role in the amplification of extrachromosomal DNA elements. HR-deficient cell lines had a significantly higher frequency of gene amplification, and the clone frequency of all MTX-resistant cells was higher than that of HeLa parental cells (21). Recently, Shoshani et al. (7) also performed whole-genome sequencing of clonal isolates developing MTX resistance, and the results further identified chromothripsis as a major driver of DM amplification in DMs and proved the amplification of DHFR genes in DMs, which enabled HeLa cells to rapidly acquire tolerance to altered growth conditions.

Morales et al. (45) studied the resistance of colon cancer HT29 cells to MTX and the dynamic process of DHFR amplification. They characterized the DHFR genome region at the cytogenetic and molecular levels in HT29 cells treated with increasing doses of MTX. HSRs were the main form of DHFR amplification in the process of increasing the dose of MTX. DMs with the DHFR gene appear in large numbers only after the cells have been exposed to higher doses of drugs for 3 months, and cancer cell resistance to MTX also increases. HT29 cells’ resistance was reduced after withdrawal of the drug, and the sensitivity of these cells to MTX was restored. At the same time, the DM carrying the DHFR gene also disappeared. Some studies showed that the homologous recombination activity of MTX-resistant cells containing DM was increased compared with MTX-sensitive cells. With the silence of the key player BRCA1 in the HR pathway, the attenuation of HR activity decreased the number of DMs and DM-form-amplified gene copies (such as DHFR, ZFYVE16, and MSH3) and increased the exclusion of micronuclei and nuclear buds that contained DM-form amplification, which were accompanied by increased MTX sensitivity (46). Similar studies had also reported that non-homologous end joining (NHEJ) decreased MTX resistance and cell proliferation in MTX-resistant colon cancer cells, which was related to blocking of the generation of DM and the exclusion of DHFR. Therefore, the DNA repair pathway might represent a novel target to reverse drug resistance and improve therapeutic outcome by eliminating extrachromosomal amplification in cancer (22).

In KB cells from human epidermoid carcinoma, the amplified multidrug resistance (MDR) genes were contained in DM molecules; cells were then treated with colchicine to analyze MDR amplification events. As the concentration of colchicine increased, circular DNAs (890 kb) harboring MDR dimerized to large DM structures (1,780 kb) by intramolecular homologous recombination, which then dimerized to form the larger DMs (3,560 kb). Their studies revealed that the dimerization of circular amplicons was the crucial mechanism for DM generation and MDR gene amplification. Colchicine exposure also induced the mutation of the MDR1 gene. The mutated MDR gene residing on an extrachromosomal DNA element underwent random segregation at mitosis and then enhanced drug resistance to cancer cells in KB cells (24). Joseph et al. (23) also revealed that DM molecules contained the amplified MDR1 genes. Although there were few DMs in each cell, there was a >100-fold amplification of the MDR1 gene. MDR1 overexpression results in cross resistance to a variety of lipophilic compounds, including anthracene-clines (e.g., doxorubicin) and vinca alkaloids (e.g., vinblastine). In addition, Sanchez et al. (47) emphasized that fractionated ionizing radiation obviously reduced the extrachromosomal copy number of MDR1 in KB cancer cells, and this decrease was accompanied by a reduction in multidrug resistance and in P-glycoprotein levels, which might help to improve the efficacy of anticancer therapies.

Hypopharyngeal squamous cell carcinoma (HSCC) was an aggressive form of head and neck squamous cell carcinoma (HNSCC) that had a poor prognosis and was rapidly rising in incidence (48). Cisplatin (DDP)-based chemotherapy was an important factor impairing the effectiveness of chemotherapy for HSCC (49, 50). Lin et al. (25) recently identified more than 10,000 eccDNA in DDP-resistant FaDu cell samples from HSCC and amplified encoding genes (such as RAB3B and RAD54L) from eccDNA (chr1^{circle 46219–52682 kb}) that carried different gene fragments. Furthermore, research found that RAB3B could promote DDP resistance in hypopharyngeal squamous cell carcinoma by inducing autophagy. However, loss of MDR1-carrying ecDNA induced by hydroxyurea increased the sensitivity of vinblastine in oral squamous cell carcinoma (OSCC), and the specific mechanism by which hydroxyurea induced to accelerate loss of extrachromosomal amplified genes is still unclear, one of which may involve the formation of micronuclei. Hydroxyurea did not deeply affect the synthesis of cell DNA and preferentially inhibited the replication of DNA outside chromosomes (26). These studies suggest that eccDNA might play a significant role in cancer drug resistance by amplifying related functional genes, and we need to explore further the novel mechanisms of eccDNA in drug resistance.

In cancer cells from a patient with small-cell lung carcinoma (SCLC) who received MTX treatment, a large number of DMs were discovered, and the DHFR gene was amplified and overexpressed. During serial passages of this cell line in drug-free medium, the number of DMs and the expression level of DHFR declined. The results showed that cancer cells were more sensitive to MTX and the prevalence of DMs in metaphase cells correlated with the concentrations of resistant MTX (27). Amplification of the DHFR gene on DMs led to an increase in mRNA and protein and provided the MTX resistance of human choriocarcinoma cells (28). A similar phenomenon was also observed in mouse EMT-6 cells from breast cancer. The cells that had been irradiated and subjected to stepwise increases in MTX concentration were detected as having numerous DMs. These studies showed that ecDNA-mediated gene amplification played an important role in the MTX resistance of cancer cells. The DHFR genes on DMs were also amplified in a dose-dependent manner (29). Recent studies used CRISPR-C, which is a technology that uses Clustered Regularly Interspaced Short Palindromic Repeats to generate extrachromosomal circular DNA (30), to generate ecDNA containing the dihydrofolate reductase (DHFR) gene in the HAP1 cell line of chronic myelogenous leukemia in humans. In the absence of methotrexate, the cells maintain their initial ecDNA copies. Then, the ecDNA copy number of DHFR ecDNA rose in a strong, dose-dependent pattern in response to MTX treatment (31).

Cancers are not static entities: they start from a genetically normal cell and end with billions of malignant cells that have accumulated a large number of mutations in the process. Most of those occur during chromosome replication in mitosis (51). Due to the accumulation of these mutations, tumor heterogeneity is promoted, which is characteristic of malignancies (52). The existence of eccDNA is an important factor driving genetic heterogeneity in cancer. It can further enhance the heterogeneity of cancer cells, depending on its unique genetic mechanism.

In cytogenetics, the inheritance of eccDNA is not the same as that of chromosomes, which does not follow Mendel’s law of inheritance (Figure 1). It is randomly distributed to daughter cells during mitosis, making the number of eccDNA in daughter cells unequal (17). Consequently, one of these daughter cells may have multiple oncogene copies of eccDNA during each division, thereby gaining a proliferation advantage. This approach enhances genomic diversity and promotes tumor heterogeneity, which helps cancer adapt to different environmental changes. It enables different cell subsets to have different sensitivities to therapeutic agents.

Early studies have proved that eccDNA is an important form of oncogene amplification, and its contribution to oncogene expression is mainly caused by the increase in gene copy number (10). eccDNA has the same complete domain as chromatin, although it lacks the higher-order compression state of chromosomes (53). Therefore, genes on eccDNA are more easily transcribed than those on chromosomes. The enhancers carried by eccDNA molecules can drive the transcription of their own genes and promote the transcription of other eccDNA molecules and even genomic genes ( Figure 2 ) (54). We can speculate that both the increase in copy number and the high transcriptional activity of eccDNA itself can enable the overexpression of oncogenes. On the premise that ecDNA may carry a variety of genes, including oncogenes and drug resistance genes, ecDNA can make cancer cells resistant through gene amplification. Andrew et al. detected copy number alterations in 4,577 human cancer samples representing nine different solid cancers and discovered that cell-derived enhancers were co-amplified with oncogenes in multiple solid tumors, including MYCN, which bore a compact relationship with drug resistance in medulloblastoma (55). Another significant study on ecDNA in GBM revealed the molecules’ function as mobile transcriptional enhancers, which were features of widespread intra-ecDNA and genome-wide chromosomal interactions. It was co-located in the same chromatin structure region to regulate the transcriptional activity of specific genes. These genes also included MYC and EGFR, which were closely related to drug resistance, and there was mutual regulation between ecDNA molecules derived from these genes (56). Therefore, ecDNA can make cancer cells acquire drug resistance through high expression of drug resistance-related genes.

The amplification of oncogenes or drug-resistant genes, which plays a pivotal role in human cell malignant transformation, confers a growth advantage to the cells through the overproduction of the amplified gene product. In cytogenetic research, the amplified gene is located in ecDNA or HSR (57).

HSR was initially detected in Chinese hamster cells resistant to MTX in 1976. Moreover, HSR are longer segments of chromosome than any single band in the karyotype. The staining intensity of HSRs in G-band staining was medium, rather than the normal pattern of alternating dark and light bands in the rest of the chromosome (58). ecDNA and HSR could be converted to each other and are homologous sequences (59). The structure of ecDNA is unstable. ecDNA can be integrated into the chromosome arm, where it efficiently initiated the breakage-fusion-bridge cycle (BFB) that generated HSR (10). As described above, the ABCG2 gene carried by ecDNA in GBM (19) and the DHFR gene carried by ecDNA in HeLa cells resistant to MTX (44) were integrated into a certain site of the chromosome to form HSR and produce a stable condition, so that the drug resistance of cancer cells was more stable. There is evidence that, compared with oncogenes or drug resistance genes amplified on ecDNA, HSR-amplified oncogenes or drug resistance genes located in chromosomes are not easy to eliminate from cells (60).

Previously, it was reported that EGFR derived from adult GBM was often mutated to produce a constitutively active oncogenic variant, EGFRvIII (18, 61). EGFRvIII amplification on ecDNA can provide growth advantages for cancer cells and make cancer cells more sensitive to TKI treatment (34, 62). After stopping TKI treatment, the amount of ecDNA carrying EGFRvIII may increase again, suggesting that ecDNA deletion of EGFRvIII leads to TKI resistance, allowing cancer to adapt to its growth environment and evade treatment against oncogenes maintained on ecDNA (18). This suggests a highly specific dynamic mechanism, in contrast to cancer cells amplifying resistance genes through ecDNA to improve drug resistance, highlighting the diversity and complexity of ecDNA-promoting resistance mechanisms.