Repression of the SUMO-conjugating enzyme UBC9 is associated with lowered double minutes and reduced tumor progression.

Double minutes (DMs), extrachromosomal gene fragments found within certain tumors, have been noted to carry onco- and drug resistance genes contributing to tumor pathogenesis and progression. After screening for SUMO-related molecule expression within various tumor sample and cell line databases, we found that SUMO-conjugating enzyme UBC9 has been associated with genome instability and tumor cell DM counts, which was confirmed both in vitro and in vivo. Karyotyping determined DM counts post-UBC9 knockdown or SUMOylation inhibitor 2-D08, while RT-qPCR and Western blot were used to measure DM-carried gene expression in vitro. In vivo, fluorescence in situ hybridization (FISH) identified micronucleus (MN) expulsion. Western blot and immunofluorescence staining were then used to determine DNA damage extent, and a reporter plasmid system was constructed to detect changes in homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Our research has shown that UBC9 inhibition is able to attenuate DM formation and lower DM-carried gene expression, in turn reducing tumor growth and malignant phenotype, via MN efflux of DMs and lowering NHEJ activity to increase DNA damage. These findings thus reveal a relationship between heightened UBC9 activity, increased DM counts, and tumor progression, providing a potential approach for targeted therapies, via UBC9 inhibition.

Gene amplification in neoplasia: A cytogenetic survey of 80 131 cases.

Gene amplification is a crucial process in cancer development, leading to the overexpression of oncogenes. It manifests cytogenetically as extrachromosomal double minutes (dmin), homogeneously staining regions (hsr), or ring chromosomes (r). This study investigates the prevalence and distribution of these amplification markers in a survey of 80 131 neoplasms spanning hematologic disorders, and benign and malignant solid tumors. The study reveals distinct variations in the frequency of dmin, hsr, and r among different tumor types. Rings were the most common (3.4%) sign of amplification, followed by dmin (1.3%), and hsr (0.8%). Rings were particularly frequent in malignant mesenchymal tumors, especially liposarcomas (47.5%) and osteosarcomas (23.4%), dmin were prevalent in neuroblastoma (30.9%) and pancreatic carcinoma (21.9%), and hsr frequencies were highest in head and neck carcinoma (14.0%) and neuroblastoma (9.0%). Combining all three amplification markers (dmin/hsr/r), malignant solid tumors consistently exhibited higher frequencies than hematologic disorders and benign solid tumors. The structural characteristics of these amplification markers and their potential role in tumorigenesis and tumor progression highlight the complex interplay between cancer-initiating gene-level alterations, for example, fusion genes, and subsequent amplification dynamics. Further research integrating cytogenetic and molecular approaches is warranted to better understand the underlying mechanisms of these amplifications, in particular, the enigmatic question of why certain malignancies display certain types of amplification. Comparing the present results with molecular genetic data proved challenging because of the diversity in definitions of amplification across studies. This study underscores the need for standardized definitions in future work.

Genome-wide characterization of extrachromosomal circular DNA in gastric cancer and its potential role in carcinogenesis and cancer progression.

Extrachromosomal circular DNAs (eccDNAs) carrying random genomic segments are broadly found across different cancer types, but their molecular functions and impact in gastric cancer (GC) are rarely known. In this study, we aimed to investigate the potential role of eccDNA in GC. Using the Circle-seq strategy, we observed the eccDNA abundance in gastric cancer tissues (GCT) was aberrantly higher than that of normal adjacent tissues (NAT). The high abundance of eccDNAs carrying oncogene-segments in GCT may represent the DNA damage products of amplified oncogenes. Analysis of GCT over-represented eccDNA carrying enhancer (eccEnhancer) based on data from FANTOM5 project combined with TCGA database suggested the GC over-represented eccEnhancers may contribute to development of GC. GC over-represented eccDNAs carrying pre-miRNA (eccMIR) were enriched to multiple cancer-relevant signal pathways by KEGG analysis. We then synthesized the top six GC over-represented eccMIRs and found four of them enabled high expression of miRNAs and down-regulation of miRNA-target genes in MGC803 cells. Furthermore, we observed the inheritance of GC over-represented eccMIRs benefited host cell proliferation and promoted the aggressive features of host cells. Altogether, this study revealed the GC over-represented eccDNAs carrying functional genomic segments were related to the carcinogenesis of GC and presented the capability to facilitate cancer progression, suggesting the cancerous eccDNAs may serve as a dynamic reservoir for genome plasticity and rapid adaptive evolution of cancer. Therefore, blocking the pathways for eccDNAs generation may provide a novel therapeutic strategy for the treatment of gastric cancer.

Life of double minutes: generation, maintenance, and elimination.

Advances in genome sequencing have revealed a type of extrachromosomal DNA, historically named double minutes (also referred to as ecDNA), to be common in a wide range of cancer types, but not in healthy tissues. These cancer-associated circular DNA molecules contain one or a few genes that are amplified when double minutes accumulate. Double minutes harbor oncogenes or drug resistance genes that contribute to tumor aggressiveness through copy number amplification in combination with favorable epigenetic properties. Unequal distribution of double minutes over daughter cells contributes to intratumoral heterogeneity, thereby increasing tumor adaptability. In this review, we discuss various models delineating the mechanism of generation of double minutes. Furthermore, we highlight how double minutes are maintained, how they evolve, and discuss possible mechanisms driving their elimination.

Extrachromosomal circular DNA in cancer: history, current knowledge, and methods.

Extrachromosomal circular DNA (eccDNA) is a closed-circle, nuclear, nonplasmid DNA molecule found in all tested eukaryotes. eccDNA plays important roles in cancer pathogenesis, evolution of tumor heterogeneity, and therapeutic resistance. It is known under many names, including very large cancer-specific circular extrachromosomal DNA (ecDNA), which carries oncogenes and is often amplified in cancer cells. Our understanding of eccDNA has historically been limited and fragmented. To provide better a context of new and previous research on eccDNA, in this review we give an overview of the various names given to eccDNA at different times. We describe the different mechanisms for formation of eccDNA and the methods used to study eccDNA thus far. Finally, we explore the potential clinical value of eccDNA.

Circular DNA in the human germline and its association with recombination.

Extrachromosomal circular DNA (eccDNA) is common in somatic tissue, but its existence and effects in the human germline are unexplored. We used microscopy, long-read DNA sequencing, and new analytic methods to document thousands of eccDNAs from human sperm. EccDNAs derived from all genomic regions and mostly contained a single DNA fragment, although some consisted of multiple fragments. The generation of eccDNA inversely correlates with the meiotic recombination rate, and chromosomes with high coding-gene density and Alu element abundance form the least eccDNA. Analysis of insertions in human genomes further indicates that eccDNA can persist in the human germline when the circular molecules reinsert themselves into the chromosomes. Our results suggest that eccDNA has transient and permanent effects on the germline. They explain how differences in the physical and genetic map might arise and offer an explanation of how Alu elements coevolved with genes to protect genome integrity against deleterious mutations producing eccDNA.

Breaking the vicious circle: Extrachromosomal circular DNA as an emerging player in tumour evolution.

Extrachromosomal circular DNA (ecDNA) or double minutes have gained renewed interest since its discovery more than five decades ago, emerging as potent drivers of tumour evolution. This has largely been motivated by recent discovery that the tumour-exclusive ecDNA are highly prevalent in almost all cancers unlike previously thought. EcDNAs contribute to elevated oncogene expression, intratumoural heterogeneity, tumour adaptation and therapy resistance independently of canonical chromosomal alterations. Importantly, ecDNAs play a critical role in patient survival as ecDNA-based oncogene amplification adversely affects clinical outcome to a significantly greater extent than intrachromosomal amplification. Chromothripsis, a major driver of ecDNA biogenesis and gene amplification, is a mutational process characterised by chromosomal shattering and localised complex genome rearrangement. Chemotherapeutic drugs can lead to chromothriptic rearrangements and therapy resistance. In this review, we examine how ecDNAs mediate oncogene overexpression, facilitate accelerated tumour malignancy and enhance rapid adaptation independently of linear chromosomes. We delve into discoveries pertaining to mechanisms of biogenesis, distinctive features of ecDNA, gene regulation and topological interactions with active chromatin. We also discuss the critical role of chromothripsis in engendering ecDNA amplification and evolution. One envisions that insights into ecDNA biology not only hold importance for the cancer genome and tumour evolutionary dynamics, but could also inform prognostication and clinical intervention, particularly for cancers characterised by high oncogene amplification.

Gene Amplification and the Extrachromosomal Circular DNA.

Oncogene amplification is closely linked to the pathogenesis of a broad spectrum of human malignant tumors. The amplified genes localize either to the extrachromosomal circular DNA, which has been referred to as cytogenetically visible double minutes (DMs), or submicroscopic episome, or to the chromosomal homogeneously staining region (HSR). The extrachromosomal circle from a chromosome arm can initiate gene amplification, resulting in the formation of DMs or HSR, if it had a sequence element required for replication initiation (the replication initiation region/matrix attachment region; the IR/MAR), under a genetic background that permits gene amplification. In this article, the nature, intracellular behavior, generation, and contribution to cancer genome plasticity of such extrachromosomal circles are summarized and discussed by reviewing recent articles on these topics. Such studies are critical in the understanding and treating human cancer, and also for the production of recombinant proteins such as biopharmaceuticals by increasing the recombinant genes in the cells.

[Amplification of Extrachromosomal Oncogene and Tumorigenesis and Development].

Extrachromosomal DNA (ecDNA) is a small segment of circular DNA located outside the chromosome, which has the function of self-replication. Recently, amplification of oncogenes on ecDNA has been proved to be a common phenomenon in tumor cells, and has some characteristics worth studying, such as correlation with patients' poor prognosis. Multiple chromosomal events are involved in the formation of ecDNA, and its amplification can directly increase the number of DNA copies of extra-chromosomal oncogenes and accelerate the generation and development of tumors. Moreover, the segregation pattern of unequal transmission of parental ecDNA cells to offspring not only increases tumor heterogeneity, but also enhances tumor adaptation to environment and response to therapy. This article reviews the current status and potential significance of ecDNA in tumor cells.
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Molecular structure and evolution mechanism of two populations of double minutes in human colorectal cancer cells.

Gene amplification chiefly manifests as homogeneously stained regions (HSRs) or double minutes (DMs) in cytogenetically and extrachromosomal DNA (ecDNA) in molecular genetics. Evidence suggests that gene amplification is becoming a hotspot for cancer research, which may be a new treatment strategy for cancer. DMs usually carry oncogenes or chemoresistant genes that are associated with cancer progression, occurrence and prognosis. Defining the molecular structure of DMs will facilitate understanding of the molecular mechanism of tumorigenesis. In this study, we re-identified the origin and integral sequence of DMs in human colorectal adenocarcinoma cell line NCI-H716 by genetic mapping and sequencing strategy, employing high-resolution array-based comparative genomic hybridization, high-throughput sequencing, multiplex-fluorescence in situ hybridization and chromosome walking techniques. We identified two distinct populations of DMs in NCI-H716, confirming their heterogeneity in cancer cells, and managed to construct their molecular structure, which were not investigated before. Research evidence of amplicons distribution in two different populations of DMs suggested that a multi-step evolutionary model could fit the module of DM genesis better in NCI-H716 cell line. In conclusion, our data implicated that DMs play a very important role in cancer progression and further investigation is necessary to uncover the role of the DMs.

Mechanisms driving acentric chromosome transmission.

The kinetochore-microtubule association is a core, conserved event that drives chromosome transmission during mitosis. Failure to establish this association on even a single chromosome results in aneuploidy leading to cell death or the development of cancer. However, although many chromosomes lacking centromeres, termed acentrics, fail to segregate, studies in a number of systems reveal robust alternative mechanisms that can drive segregation and successful poleward transport of acentrics. In contrast to the canonical mechanism that relies on end-on microtubule attachments to kinetochores, mechanisms of acentric transmission largely fall into three categories: direct attachments to other chromosomes, kinetochore-independent lateral attachments to microtubules, and long-range tether-based attachments. Here, we review these "non-canonical" methods of acentric chromosome transmission. Just as the discovery and exploration of cell cycle checkpoints provided insight into both the origins of cancer and new therapies, identifying mechanisms and structures specifically involved in acentric segregation may have a significant impact on basic and applied cancer research.

Extrachromosomal DNA-relieving heredity constraints, accelerating tumour evolution.

Oncogene amplification on extrachromosomal DNA (ecDNA) provides a mechanism by which cancer cells can rapidly adapt to changes in the tumour microenvironment. These circular structures contain oncogenes and their regulatory elements, and, lacking centromeres, they are subject to unequal segregation during mitosis. This non-Mendelian mechanism of inheritance results in increased tumour heterogeneity with daughter cells that can contain increasingly amplified oncogene copy number. These structures also contain favourable epigenetic modifications including transcriptionally active chromatin, further fuelling positive selection. ecDNA drives aggressive tumour behaviour, is related to poorer survival outcomes and provides mechanisms of drug resistance. Recent evidence suggests one in four solid tumours contain cells with ecDNA structures. The concept of tumour evolution is one in which cancer cells compete to survive in a diverse tumour microenvironment under the Darwinian principles of variation and fitness heritability. Unconstrained by conventional segregation constraints, ecDNA can accelerate intratumoral heterogeneity and cellular fitness. In this review, we highlight some of the recent discoveries underpinning this process.

The adaptive potential of circular DNA accumulation in ageing cells.

Carefully maintained and precisely inherited chromosomal DNA provides long-term genetic stability, but eukaryotic cells facing environmental challenges can benefit from the accumulation of less stable DNA species. Circular DNA molecules lacking centromeres segregate randomly or asymmetrically during cell division, following non-Mendelian inheritance patterns that result in high copy number instability and massive heterogeneity across populations. Such circular DNA species, variously known as extrachromosomal circular DNA (eccDNA), microDNA, double minutes or extrachromosomal DNA (ecDNA), are becoming recognised as a major source of the genetic variation exploited by cancer cells and pathogenic eukaryotes to acquire drug resistance. In budding yeast, circular DNA molecules derived from the ribosomal DNA (ERCs) have been long known to accumulate with age, but it is now clear that aged yeast also accumulate other high-copy protein-coding circular DNAs acquired through both random and environmentally-stimulated recombination processes. Here, we argue that accumulation of circular DNA provides a reservoir of heterogeneous genetic material that can allow rapid adaptation of aged cells to environmental insults, but avoids the negative fitness impacts on normal growth of unsolicited gene amplification in the young population.

Circle-Seq: Isolation and Sequencing of Chromosome-Derived Circular DNA Elements in Cells.

Chromosome-derived extrachromosomal circular DNA elements (eccDNAs) are detected in all eukaryotes examined so far. Here I describe the Circle-Seq protocol, applicable for physical enrichment of eccDNAs of a broad size range, combined with sequence confirmation of circular structures.Briefly, by concise alkaline treatment and gentle gravity flow-through an ion-exchange column, eccDNAs are enriched in the eluate fraction. EccDNAs are enzymatically isolated by extensive Plasmid-Safe DNase digestion of linear chromosomes and further enriched by φ29 rolling circle amplification. By means of high throughput sequencing of amplified eccDNA and custom eccDNA mapping software, around ten-thousand unique eccDNA types could be detected at nucleotide resolution in a million human muscle nuclei by this method.

New insights of extrachromosomal DNA in tumorigenesis and therapeutic resistance of cancer.

In the past few decades, the studies of extrachromosomal DNA (ecDNA), which existed independently of chromosomes, were tepid. However, recent studies on ecDNA rekindled the enthusiasm of oncologists for further studying ecDNA. In this review, we summarized the recent advances of ecDNA in oncogenesis and oncotherapy. ecDNA consists of highly open chromatin, and its circular structure enables ultra-long-range chromatin contacts. ecDNA is not inherited in accordance with Mendel's laws. Furthermore, ecDNA is widely existed in cancer cells, but almost never found in normal cells. It has been found that ecDNA played important roles in tumorigenesis and tumor progression, including oncogene amplification, tumor heterogeneity, enhancer hijacking and genomic rearrangement. More importantly, ecDNA is closely related to cancer treatment resistance. In hence, further understanding of ecDNA would contribute to developing innovative targeting ecDNA therapies.

Double-strand breakage in the extrachromosomal double minutes triggers their aggregation in the nucleus, micronucleation, and morphological transformation.

Gene amplification plays a pivotal role in malignant transformation. Amplified genes often reside on extrachromosomal double minutes (DMs). Low-dose hydroxyurea induces DM aggregation in the nucleus which, in turn, generates micronuclei composed of DMs. Low-dose hydroxyurea also induces random double-strand breakage throughout the nucleus. In the present study, we found that double-strand breakage in DMs is sufficient for induction of DM aggregation. Here, we used CRISPR/Cas9 to introduce specific breakages in both natural and artificially tagged DMs of human colorectal carcinoma COLO 320DM cells. Aggregation occurred in the S phase but not in the G1 phase within 4 hours after breakage, which suggested the possible involvement of homologous recombination in the aggregation of numerous DMs. Simultaneous detection of DMs and the phosphorylated histone H2AX revealed that the aggregation persisted after breakage repair. Thus, the aggregate generated cytoplasmic micronuclei at the next interphase. Our data also suggested that micronuclear entrapment eliminated the DMs or morphologically transformed them into giant DMs or homogeneously staining regions (HSRs). In this study, we obtained a model explaining the consequences of DMs after double-strand breakage in cancer cells. Because double-strand breakage is frequently involved in cancer therapy, the model suggests how it affects gene amplification.

Amplification of Extrachromosomal Oncogene and Tumorigenesis and Development / 中国肺癌杂志

Extrachromosomal DNA (ecDNA) is a small segment of circular DNA located outside the chromosome, which has the function of self-replication. Recently, amplification of oncogenes on ecDNA has been proved to be a common phenomenon in tumor cells, and has some characteristics worth studying, such as correlation with patients' poor prognosis. Multiple chromosomal events are involved in the formation of ecDNA, and its amplification can directly increase the number of DNA copies of extra-chromosomal oncogenes and accelerate the generation and development of tumors. Moreover, the segregation pattern of unequal transmission of parental ecDNA cells to offspring not only increases tumor heterogeneity, but also enhances tumor adaptation to environment and response to therapy. This article reviews the current status and potential significance of ecDNA in tumor cells.
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Cytogenomic characterization of double minute heterogeneity in therapy related acute myeloid leukemia.

Breast cancer patients treated with adjuvant chemotherapy regimens containing alkylating agents and anthracyclines are at an increased risk for secondary myeloid malignancies, either acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Complex genomic changes (karyotypes and/or gene amplification) accompany the development of the secondary neoplasms. Here we present a unique case of a breast cancer patient who developed secondary AML within 18 months of treatment with trastuzumab, pertuzumab, docetaxel, carboplatin (TCHP) and radiation. Leukemia cells had catastrophic alterations in chromosomes 8, 11, and 17. Genetic abnormalities in the leukemia cells included amplification of MYC and KMT2A as double minutes, and deletion and mutational inactivation of TP53 Concurrent amplification of different genes at different levels and on different double minutes, we have named "double minute heterogeneity." Clinically, this case highlights the need to identify genes amplified in secondary myeloid malignancies by cytogenomic microarray (CMA) analysis since these may have therapeutic implications.

Generation and maintenance of acentric stable double minutes from chromosome arms in inter-species hybrid cells.

BACKGROUND: Extrachromosomal acentric double minutes (DMs) contribute to human malignancy by carrying amplified oncogenes. Recent cancer genomics revealed that the pulverization of defined chromosome arms (chromothripsis) may generate DMs, however, nobody had actually generated DMs from chromosome arm in culture. Human chromosomes are lost in human-rodent hybrid cells. RESULTS: We found that human acentric DMs with amplified c-myc were stable in human-rodent hybrid cells, although the degree of stability depended on the specific rodent cell type. Based on this finding, stable human-rodent hybrids were efficiently generated by tagging human DMs with a plasmid with drug-resistance gene. After cell fusion, human chromosomes were specifically pulverised and lost. Consistent with chromothripsis, pulverization of human chromosome arms was accompanied by the incorporation into micronuclei. Such micronucleus showed different replication timing from the main nucleus. Surprisingly, we found that the hybrid cells retained not only the original DMs, but also new DMs without plasmid-tag and c-myc, but with human Alu. These DMs were devoid of telomeres and centromeres, and were stable in culture for more than 3 months. Microarray analysis showed that the new DMs were generated from several human chromosomal regions containing genes advantageous for cellular growth. Such regions were completely different from the original DMs. CONCLUSIONS: The inter-species hybrid mimics the chromothripsis in culture. This is the first report that experimentally demonstrates the generation of multiple stable acentric DMs from the chromosome arm.

Structure and evolution of double minutes in diagnosis and relapse brain tumors.

Double minute chromosomes are extrachromosomal circular DNA fragments frequently found in brain tumors. To understand their evolution, we characterized the double minutes in paired diagnosis and relapse tumors from a pediatric high-grade glioma and four adult glioblastoma patients. We determined the full structures of the major double minutes using a novel approach combining multiple types of supporting genomic evidence. Among the double minutes identified in the pediatric patient, only one carrying EGFR was maintained at high abundance in both samples, whereas two others were present in only trace amounts at diagnosis but abundant at relapse, and the rest were found either in the relapse sample only or in the diagnosis sample only. For the EGFR-carrying double minutes, we found a secondary somatic deletion in all copies at relapse, after erlotinib treatment. However, the somatic mutation was present at very low frequency at diagnosis, suggesting potential resistance to the EGFR inhibitor. This mutation caused an in-frame RNA transcript to skip exon 16, a novel transcript isoform absent in EST database, as well as about 700 RNA-seq of normal brains that we reviewed. We observed similar patterns involving longitudinal copy number shift of double minutes in another four pairs (diagnosis/relapse) of adult glioblastoma. Overall, in three of five paired tumor samples, we found that although the same oncogenes were amplified at diagnosis and relapse, they were amplified on different double minutes. Our results suggest that double minutes readily evolve, increasing tumor heterogeneity rapidly. Understanding patterns of double minute evolution can shed light on future therapeutic solutions to brain tumors carrying such variants.