Understand Ted Chiang 57.pdf
Erinn Hickel
Extrachromosomal circular DNA was recently found to be particularly abundant in multiple human cancer cells, although its frequency varies among different tumor types. Elevated levels of extrachromosomal circular DNA have been considered an effective biomarker of cancer pathogenesis. Multiple reports have demonstrated that the amplification of oncogenes and therapeutic resistance genes located on extrachromosomal DNA is a frequent event that drives intratumoral genetic heterogeneity and provides a potential evolutionary advantage. This review highlights the current understanding of the extrachromosomal circular DNA present in the tissues and circulation of patients with advanced cancers and provides a detailed discussion of their substantial roles in tumor regulation. Confirming the presence of cancer-related extrachromosomal circular DNA would provide a putative testing strategy for the precision diagnosis and treatment of human malignancies in clinical practice.
Current understanding of extrachromosomal circular DNA in cancer pathogenesis. Multiple recent studies have demonstrated the increasing importance of extrachromosomal circular DNA on oncogenic features and regulatory capacity in cancer research and treatment
Insights into the t-circle/c-circle formation mechanisms remain limited; to date, several DNA damage-associated proteins, such as XRCC3 [79], NBS1 [64], and Ku70/80 [37], have been strongly implicated in this process [80]. Moreover, because of the lack of the ability to initiate replication, t-circles/c-circles are mainly generated from recombination rather than heritably amplified episomes. The production of t-circles/c-circles using homologous recombination can be obviously inhibited by RAD52 deletion [81, 82]. ATRX loss leads to multiple phenotypic features of ALT in two high-grade glioma cell lines, U-251 and UW479, including c-circle formation [83]. Knockdown of the Ku70/80 heterodimer by shRNAs reduces the levels of t-circles and activates the p53 pathway, ultimately resulting in significantly decreased cell growth in SaOS2 osteosarcoma cells [37]. However, another study showed that while XRCC3 and NBS1 are required for t-circle production, knocking down these two factors does not affect cell proliferation and telomere maintenance [84]. These unexpected findings suggest that in the absence of t-circles/c-circles, there may be some subtypes of cancer cells undergoing additional telomere maintenance pathways, for example, overexpression of telomerase components [17, 85]. Further studies are needed to better understand the mechanistic details of t-circles/c-circles on telomere maintenance and cell growth in ALT cancer cells. In addition to these DNA repair-associated proteins, whether other factors are responsible for t-circle/c-circle formation in different cancers is an interesting question for future investigation. To prove this hypothesis, Touzot et al. [86] demonstrated that the regulator of telomere elongation helicase 1 (RTEL1) is required for telomere stability and thereby prevents rapid telomere deletion and t-circle formation. Moreover, high levels of TERT and telomerase activity can suppress the production of c-circles in ALT-positive cells from brain tumors [87] and promyelocytic leukemia [88]. In addition, deficiency of several telomere-associated factors, such as the CST complex (CTC1-STN1-TEN1) [89], telomeric repeat-containing RNA [90] or homeobox containing protein 1 [91], has been found to significantly increase telomeric DNA damage and diminish the abundance of t-circles/c-circles, thereby impairing cell proliferation in U2OS osteosarcoma cells.
In addition, there is evidence that regions of genomic rearrangement, such as genomic focal amplification sites in human cancers, might be closely related to strong gain-of-function mutations in oncogenes, contributing to the development of drug resistance in cancer therapy [122]. Circular DNA elements act as the cytogenetic hallmarks of genomic focal amplification in cancer cells [123], and the copy number of these elements with oncogenic mutations is altered in response to environmental changes [107]. An example of this is in nervous system neoplasms, including GBM and low-grade glioma, where the receptor tyrosine kinases (RTKs) are frequently mutated and commonly give rise to the driver variant [124]. In particular, the activating mutations of EGFR [125] and PDGF receptor a (PDGFRa) [126], two members of the RTK family, have usually been thought to reside primarily on DM structures. These extrachromosomal mutations are the driving factors that contribute to therapy resistance by increasing tumor heterogeneity. Studies have also indicated that although the constitutively active mutant of EGFR, EGFR variant III (EGFRvIII), plays critical pro-survival roles in GBM pathogenesis, and it also makes cancer cells more sensitive to EGFR inhibitors [127]. Therefore, resistance to EGFR-targeted therapy might develop if extrachromosomal mutations are eliminated. In recent years, several empirical studies have been conducted to address this issue. After treatment with the EGFR inhibitor erlotinib, EGFRvIII-bearing extrachromosomal DNA elements within GBM cells were markedly reduced, resulting in resistance to anti-EGFR therapeutics. However, after withdrawal of the drug, the reemergence of clonal EGFRvIII mutations on DMs effectively resensitized cancer cells to EGFR inhibitors, which could then induce cell death [128]. Unexpectedly, in some cancer cell subclones, resistance to anti-EGFR therapy might not be entirely mediated by EGFRvIII-positive DMs; rather, resistance might occur through other compensatory mechanisms, such as the potential oncogenic roles of extrachromosomal MDM2 amplification [128]. In addition, Vicario et al. [129] analyzed the amplification patterns of HER2 (another member of the RTK family) in response to different anti-HER2 therapies. Although the acquisition of resistance in HER2-positive breast cancer cells is often concomitant with HER2 protein loss, the copy number of HER2-positive DMs did not obviously change after treatment with the HER2 inhibitor trastuzumab. These data indicate that HER2 elimination-mediated resistance might not be due to the loss of HER2-containing circular DNA. Thus, future work is needed to evaluate the compensatory mechanisms that enable cancer cells with or without RTK-positive extrachromosomal DNA to continue to proliferate during anti-RTK treatment. By elucidating these dynamic regulatory mechanisms in detail, we will gain more comprehensive insight into therapy resistance, and this understanding could support the future development of novel therapeutic approaches.
While circular extrachromosomal DNA has been able to be determined for some time, higher-resolution imaging and cytogenetic methods have only recently been developed to evaluate these molecules. Advances in sequencing technologies have allowed for the genome-scale screening and mapping of circular DNA from human cancer cells [153]. Based on short-read sequencing and connecting amplified DNA segments, some computational analysis tools have been developed to explore the profiles of circular DNA signals in tumor samples in an unsupervised manner [154], such as AmpliconArchitect [155]. Defining eccDNAs using AmpliconArchitect showed that oncogenes mainly amplify eccDNA elements and are surprisingly preserved during cancer progression, which has been shown in previous reports [11]. However, these short-read maps could not unambiguously align the long-range duplications crossing the circular structure. As a consequence, the characterization and functional profiles of circular DNA elements might have been drastically underestimated in cancer patients. To effectively solve these problems, the detection systems are expected to be improved in subsequent studies. Recently, several improved tools with high precision, such as AmpliconReconstructor [131] and Circle-Map [156], were developed to better map the physical structure of long contiguous reads. Based on the BioNano technology platform, AmpliconReconstructor was used to provide more conclusive evidence that oncogenes carrying ecDNAs produced substantially more transcripts in multiple cancer cells and tissue types. Today, other higher-resolution technologies are being developed to improve the resolution of circular DNA from high-throughput DNA sequencing data. For example, a higher-resolution image analysis algorithm, named ECdetect, has been developed and is used to effectively quantify ecDNAs from DAPI-stained metaphases in an unbiased and highly accurate fashion [11, 55]. Taken together, these effective tools will permit a deeper understanding of the characteristics of extrachromosomal DNA particles in cancer pathology and their association with clinical features.
It is also essential to expand the cytogenetic toolbox to explore the function of key components with respect to extrachromosomal DNA particles. For a long time, fluorescence in situ hybridization (FISH)-based approaches have been used to resolve amplified oncogenes linked with cancer development. However, FISH probes have been unable to discriminate the location of specific genes on chromosomal and extrachromosomal DNA molecules [157]. A more advanced cell-imaging technology, ecSeg [158], has been introduced to accurately quantify oncogene amplification from ecDNA at the single-cell level. Moreover, the application of genome editing techniques, such as CRISPR-Cas [159, 160], could be leveraged to advance our understanding of the basic biologic functions of these molecules, especially in circular DNA-driven oncogene amplification and drug resistance. Moreover, as delineated above, such methods could help researchers discover agents that target extrachromosomal circular DNA elements.
As mentioned above, cancer-associated genes can be amplified in circular extrachromosomal DNA elements. In particular, because of the unequal segregation into daughter cells, the amplification of eccDNA/ecDNA elements could be a primary mechanism by which oncogenes rapidly reach higher transcript levels and copy numbers than in intrachromosomal amplification. There is growing evidence supporting the functional importance of circular DNA in promoting genetic heterogeneity and accelerating the progression of human cancer pathologies. Moreover, the amplification of circular DNA elements containing oncogenes (such as EGFR and c-Myc) could help cancer cells adapt more effectively to variable environmental stress by acquiring fate-enhancing advantages. Thus, some subclones would express higher levels of oncogenes, leading to cancers that become more aggressive, have poorer prognoses, and are more difficult to treat clinically over time. Accordingly, understanding the underlying molecular mechanisms of tumor heterogeneity, including oncogene amplification from circular extrachromosomal DNA, might help to develop remarkable potential therapeutic strategies that either prevent cancer progression or overcome therapy resistance. Such important scientific topics are currently being addressed by pioneering novel work on these issues.
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