Chromothripsis events are common across many types of cancer

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Researchers at Harvard Medical School and EMBL-EBI have carried out the largest analysis across cancer types of the newly discovered mutational phenomenon chromothripsis.

This study is the largest of its kind to date, containing whole-genome sequencing (WGS) data from over 2600 tumors spanning 38 different types of cancer.

Chromothripsis, or ‘chromosome shattering’, is a mutational process in which large stretches of a chromosome undergo massive rearrangements in a single catastrophic event.

The chromosomal regions fragment into smaller pieces, rearrange, and rejoin, leading to a new genome configuration.

Fully understanding how these alterations drive cancer genome evolution, and what molecular mechanisms are involved in their generation, are important steps towards understanding cancer genome evolution.

This research was published in Nature Genetics as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) project, a global effort involving the international collaboration of over 1300 scientists.

In this study, the researchers showed that chromothripsis events are much more common across many types of cancer than previously thought.

They could also directly link chromothripsis to common hallmarks of the cancer genome, including oncogene amplification (an increase in the number of copies of a gene that can cause cancer), and the loss of tumor suppressors (genes that regulate cell growth and division).

Chromothripsis prevalence in cancer

“We integrated WGS data from over 2600 tumors spanning more than 30 cancer types,” says Isidro Cortés-Ciriano, group leader at EMBL-EBI and a former postdoctoral researcher at Harvard Medical School.

“From this, we discovered that chromothripsis events and other types of complex genome rearrangements are pervasive across human cancers, with frequencies greater than 50% of tumors in some cancer types.”

Using WGS datasets gave the researchers an enhanced view of chromothripsis events in the cancer genome.

Previous studies looking at the role of chromothripsis in cancer and congenital diseases often used low-resolution array-based technologies.

Here the researchers were able to show that chromothripsis events are much more prevalent in cancer than previously estimated.

They also characterized the patterns of massive genome alterations across cancer types and studied the DNA repair mechanisms involved in their generation.

“This study is yet another demonstration of the power of large-scale whole-genome sequencing,” says Peter Park, Professor of Biomedical Informatics at Harvard Medical School and senior author of the paper.

“It allowed us to probe the bewildering complexity of genome-shattering in cancer genomes and to characterise common features across hundreds of cases.”

Chromothripsis and cancer prognosis

“The discoveries made in this project allow us to better understand how cancer arises and evolves, as well as the patterns of alterations in the DNA of human tumors,” says Cortés-Ciriano.

“Some of these alterations have strong clinical implications and could open new avenues for therapeutic development over the coming years.”

The researchers demonstrate that chromothripsis shapes the tumor genome, leading to the loss of tumor suppressor genes and amplification of oncogenes to drive cancer progression.

Chromothripsis has been associated with poor prognosis for cancer patients, but continuing studies like this help us to understand the impact of chromothripsis and other large-scale genome alterations, and how they may be used for cancer diagnosis in the future.

The Pan-Cancer project

The Pan-Cancer Analysis of Whole Genomes project is a collaboration involving more than 1300 scientists and clinicians from 37 countries.

It involved analysis of more than 2600 genomes of 38 different tumor types, creating a huge resource of primary cancer genomes.

This was the starting point for 16 working groups to study multiple aspects of cancer development, causation, progression, and classification.


Chromothripsis

Chromothripsis is the first of these new catastrophic process (mechanism) described in 2011 [2].

The phenomenon is currently defined as a mutational event driven by multiple double-strand breaks (DSBs) occurring in a single catastrophic event between a limited numbers of chromosomal segments, and followed by the reassembly of the DNA fragments in random order and orientation to form complex derivative chromosomes (Fig. 1).

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The concept of chromothripsis: during a one-step catastrophic event, multiple double-strand breaks occurred, restricted to a simple chromosomal segment or to a few closed chromosome domains, leading to the pulverization of chromosomal fragments. This shattering can produce tens to hundreds DNA fragments. Most of them are stitched back together by non-homologous end-joining (NHEJ), resulting in chaotic derivative chromosome(s), whereas some are lost or combined in small circular extra-chromosomes

Several factors common to all chromothripsis events, such as the generation of numerous clustered chromosomal breakpoints, the low DNA copy number changes and the preservation of heterozygosity in the rearranged segments, allow to distinguish chromothripsis from other complex chromosomal rearrangements and define its molecular signature [34].

Initially described in cancers [1], the phenomenon was rapidly evidenced in patients with congenital abnormalities [57].

Notably, even some translocations and inversions classified as simple balanced rearrangements were identified as more complex than previously appreciated [8].

In the same way, extreme balanced germline chromothripsis were identified in patients with autism spectrum disorders and other developmental abnormalities [910].

Also, chromothripsis was observed in healthy subjects [1112] as well as in prenatal diagnosis [13].

Some studies reported the possible reversibility of chromothripsis [14] and its potential curative effect [15]. Accumulating data on familial chromothripsis validated the notion of the heritability of some chromothripsis rearrangements.

Precise analysis of breakpoint junction sequences have indicated that the re-assembly of DNA fragments was driven by recombination-based mechanism such as classical non-homologous end joining (c-NHEJ) or alternative form of end joining (alt-EJ), operating in all phases of the cell cycles and working independently of micro-homologies but potentially error-prone [1619].

Since the end-joining process mediates the formation of reciprocal translocations and complex three-way translocations, Kloosterman et al. [20] suggested that a similar cascade mechanism could operate in the creation of the derivative complex chromosomes found in constitutional chromothripsis.

Concerning the shattering of chromosome segments, multiple DBSs can arise from various exogenous sources such as ionizing radiation, free radicals, environmental toxins or chemotherapeutic drugs [21].

Even cannabis exposure has been associated with chromothripsis occurrence [22].

Other exogenous causal factors might be certain viral integration such as human papillomavirus (HPV) that can promote genomic instability and multiple DNA breaks [23].

Analysis of the aetiology of chromothripsis has also led to the identification of several cellular mechanisms capable of initiating chromothripsis process.

Tubio and Estivill [24] proposed that chromothripsis might be caused by abortive apoptosis. Whereas apoptosis was considered as an irreversible cascade of extensive chromatin fragmentations leading to cell death, a small subset of cells could undergo a restricted form of apoptosis and thus survive.

The partial DNA fragmentation could be restricted to regions of high chromatin accessibility. The subsequent DNA repair might be accomplished through a fast and incorrect repair process, promoting the emergence of chaotic chromosomal rearrangement [1625].

Since many examples of chromothripsis rearrangements affect chromosome ends, it has been proposed that chromothripsis could also arise via telomere attriction [226].

Indeed, uncapped chromosome-ends are prone to fusion, leading to the formation of dicentric chromosomes [27]. During mitosis, this telomere crisis can yield complex rearrangements through breakage-fusion-bridge (BFB) cycles [28].

Several studies have suggested the association between chromothripsis and the occurrence of BFBs [2629]. By examining the fate of dicentric human chromosomes, Maciejowski et al. [30] evidenced the formation of chromatin bridges connecting daughter cells.

These bridges can undergo nuclear envelope rupture and nucleolytic attack by cytoplasmic TREX1 exonuclease, causing in the restricted area of the bridge, chromothripsis-like rearrangements frequently associated with local hypermutations known as kataegis [3031].

Other proposed models suggest that replication stress and mitotic error could synergize to induce chromosomal instability and chromothripsis occurrence [163233] or that premature chromosome condensation (PCC) induced by the fusion of an interphasic cell with a metaphasic cell could initiate chromothripsis, leading to incomplete replication and subsequent partial pulverization of chromosomes [34].

The emergence of chromothripsis has also been strongly associated with dysregulation or loss of p53 tumour suppressor genes. Known as the guardian of the genome, p53 plays a major role in maintaining genome stability by mediating cell cycle arrest, apoptosis and cell senescence in response to DNA damages [3536].

The potential implication of p53 pathways in chromothripsis occurrence was postulated by Rausch et al. [37] after the discovery of a striking correlation between germline p53 mutations (Li-Fraumeni syndrome) and chromothripsis patterns in patients with Sonic-Hedgehog medulloblastoma brain tumors.

These findings led the authors to propose that germline p53 mutations could either predispose cell to catastrophic DNA rearrangements or facilitate cell survival after these catastrophic events.

An attractive mechanistic explanation to link all these causal processes with the confined nature of damages created during chromothripsis, is that the implicated chromosome(s) can be incorporated into a micronucleus in which chromothripsis-related damages will occur.

Micronuclei are generally considered as passive indicators of chromosomal instability [38]. Crasta et al. [39] provided the first experimental evidence on this mechanism by the generation of micronuclei in several human cell lines and the subsequent observation of extensive genomic rearrangements during the cell cycles following the formation of micronuclei.

Micronuclei display a double-membrane similar to regular nuclei, but micronuclei often undergo defective nuclear envelope assembly and the number of nuclear pore complexes (NPCs) is often inadequate. Recently, Liu et al. [40] showed that only “core” nuclear envelope proteins assemble efficiently around lagging chromosomes whereas “non-core” nuclear envelope proteins, especially NPCs, do not.

This situation leads to a defect in the micronuclear import of essential components for DNA repair and replication, and consequently to reduced functioning in micronuclei. The chromatin sequestrated in micronuclei can undergo defective replication, resulting in the formation of complex rearranged chromosomes [41].

Micronuclei may persist in daughter cells over several cell cycles before being eliminated or reincorporated into the regular nucleus [42]. An additional pathway for the occurrence of DNA damages in micronuclei is the irreversible breakdowns of their membranes during interphase. Zhang et al. [43] proposed that membrane rupture enables enzymes such as endonucleases or topoisomerases to act aberrantly on micronuclear chromosome fragments.

The entry of the cell into mitosis while the micronucleus is still undergoing DNA replication will result in micronuclear DNA pulverization due to premature chromosome compaction, and the subsequent chaotic reassembly of chromosome fragments [3944].

Using an elegant in vitro model to specifically induce mis-segregation of the Y chromosome, Ly et al. [45] observed frequent Y chromosome sequestration into micronuclei, followed by shattering and incorrect reassembly of Y chromosome fragments through 3 consecutive cell cycles. By using inhibitor of DNA repair, the authors demonstrated that NHEJ mechanism was not efficient in the micronucleus, but operated during the subsequent interphase, after the incorporation of Y chromosome fragments in a daughter nucleus.

These micronucleus-based models have the potential to explain many features of chromothripsis, especially how such massive damages can be confined to one or just a few chromosomal segments [46].


More information: Cortes-Ciriano, I., et al. (2020). Comprehensive analysis of chromothripsis in 2,658 human cancers using whole-genome sequencing. Nature Genetics, 5 February 2020; DOI: 10.1038/s41588-019-0576-7

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