Live-cell imaging shows uneven segregation of extrachromosomal DNA elements and transcriptionally active extrachromosomal DNA clusters in cancer
Posted on: 28 October 2020
Preprint posted on 21 October 2020
Categories: cancer biology, cell biology, molecular biology, neuroscience
Background1-3
Extrachromosomal DNAs (ecDNAs) are genetic elements present inside the nucleus independent of a linear chromosome that are about 50kb-5Mb in size, harboring a set of genes and regulatory elements. ecDNAs are formed when broken linear DNA ligate end-to-end. ecDNAs are found in many eukaryotic species like yeast, D. melanogaster, C. elegans, and humans. In cancer cells, ecDNAs predominantly accommodate oncogenes or fused oncogenes to facilitate oncogene amplification. However, it is not clear how ecDNA propagate intratumor genetic heterogeneity, a characteristic feature of multidrug-resistant cancer colonies. Unequivocal segregation of ecDNA to daughter cells could impel intratumor genetic heterogeneity. To address this, the authors of the current study used cutting-edge CRISPR based imaging techniques to investigate the dynamics of ecDNA in glioblastoma.
Key findings
1. To gain insights into the dynamics of ecDNA in glioblastoma, the investigators used four glioblastoma samples and a pair of neurospheres derived from the same patients. They evaluated fluorescent in situ hybridization (FISH) of EGFR(an oncogene of glioblastoma) to score ecDNA based on their earlier studies4 and a chromosome 7 (Chr7) loci for linear intact chromosome. They found that EGFR-containing ecDNA numbers vary among samples than Chr7 that was rather evenly distributed. They further report that unequivocal EGFR ecDNA amplification corroborated with heterogeneity in protein expression. In other cell lines, they also found heterogenous gene amplification of ecDNA-associated genes than genes harbored in linear chromosomes. Thus, they report that ecDNA could drive genetic heterogeneity in cancer cells (fig.1).
2. To address how ecDNA heterogeneity is dispersed in cancer cells, the authors implemented a CRISPR-based DNA labeling system (fig.2). For this purpose, they used nuclease dead Cas9, provided a single guide-RNAs with 25 Pumilio/FBF (PUF) RNA-binding sites that would bind to the unique fusion sequences at the ecDNA breakpoints, and recruit fluorescently tagged PUF proteins to identify ecDNA in real-time. They identified four unique ecDNAs – ecEGFRx1, ecEGFR, ecCCAT1, and ecCCDC26– harboring an EGFR exon1, a full-length EGFR, and non-coding genes CCAT and CCDC26(using sequencing, FISH, and PCR-based techniques). They detected all four ecDNA outside chromosomes using breakpoint-specific FISH imaging in metaphase spreads. They were able to demonstrate all four ecDNA using the CRISPR-based system in neurosphere cells but not in prostate cancer cells that acted as a control. Moreover, they found that CRISPR labeling coincided with the breakpoint-specific FISH signal suggesting the reliability of the signal. They also found heterogeneous ecDNA copy numbers than linear chromosome locales (Chr7 and MUC4) in the neurospheres.
3. The authors then tracked the ecDNA live using the same CRISPR-based DNA labeling system and found uneven segregation of the ecDNA to daughter cells even when followed for 48hrs, unlike Chr7and MUC4. They also found that ecDNAs tend to coalesce in at least 50% of neurospheres in the 48hr time frame. Furthermore, they show that these ecDNA clusters co-localized with nuclear bodies5 (Cajal and PML bodies) that act as a macromolecular hub, but there was no significant linear correlation between the number of ecDNAs and the number of nuclear bodies, suggesting that ecDNAs generate its own hub. Intriguingly, they also report ecDNA clusters co-localized with RNA polymerase II in 60% of the cells and the size of ecEGFRfoci indicating its clustering positively correlated with EGFR mRNA levels.
Conclusion
Recent studies revealed intratumor genetic heterogeneity as a critical factor for the perpetuation of multidrug-resistant cancer colonies. Intratumor genetic heterogeneity poses a great challenge to personalized medicine. Here the authors report that intratumor genetic heterogeneity is promoted by ecDNAs through their uneven segregation during cell divisions and clustering to facilitate oncogene expression or amplification (fig.1). The current study raises many interesting hypotheses on how genome instability drives oncogenesis and cancer evolution.
Acknowledgments
I am grateful to all the authors for their support, especially Eunhee Yi and Roel GW Verhaak for being open to discuss the work and replying promptly.
References
- https://doi.org/10.1038/s41568-019-0128-6
- https://doi.org/10.1016/j.molonc.2014.06.005
- https://doi.org/10.1016/j.annonc.2020.03.303
- https://doi.org/10.1038/s41588-018-0105-0
- https://doi.org/10.1016/S0962-8924(99)01606-2
doi: https://doi.org/10.1242/prelights.25485
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