Focal chromosomal amplification is an important route to generating cancer through mediating over-expression of oncogenes 1 – 3 or to developing cancer therapy resistance by increasing expression of a gene whose action diminishes efficacy of an anti-cancer drug. Here we used whole-genome sequencing of clonal isolates developing chemotherapeutic resistance to identify chromothripsis as a major driver of extrachromosomal DNA (ecDNA) amplification into circular double minutes (DMs) through PARP- and DNA-PKcs-dependent mechanisms. Longitudinal analyses revealed that DMs undergo continuing structural evolution to promote increased drug tolerance through additional chromothriptic events. In-situ Hi-C sequencing is used to demonstrate that DMs preferentially tether near chromosome ends where they re-integrate when DNA damage is present. Intrachromosomal amplifications formed initially under low-level drug selection undergo continuing breakage-fusion-bridge cycles, generating >100 megabase-long amplicons that we show become trapped within interphase bridges and then shattered, producing micronuclei that mediate DM formation. Similar genome rearrangement profiles linked to localized gene amplification are identified in human cancers with acquired drug resistance or with oncogene amplifications. We propose that chromothripsis is a primary mechanism accelerating genomic DNA amplification and which enables rapid acquisition of tolerance to altered growth conditions.
Cancer genomes are frequently characterized by numerical and structural chromosomal abnormalities. Here we integrated a centromere-specific inactivation approach with selection for a conditionally essential gene, a strategy termed ‘CEN-SELECT’, to systematically interrogate the structural landscape of missegregated chromosomes. We show that single-chromosome missegregation into a micronucleus can directly trigger a broad spectrum of genomic rearrangement types. Cytogenetic profiling revealed that missegregated chromosomes exhibit 120-fold higher susceptibility to developing seven major categories of structural aberrations, including translocations, insertions, deletions, and complex reassembly through chromothripsis coupled to classical non-homologous end joining. Whole-genome sequencing of clonally propagated rearrangements identified random patterns of clustered breakpoints with copy-number alterations resulting in interspersed gene deletions and extrachromosomal DNA amplification events. We conclude that individual chromosome segregation errors during mitotic cell division are sufficient to drive extensive structural variations that recapitulate genomic features commonly associated with human disease.
Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A binding sites are identified within the megabase-long, repetitive α-satellite DNAs at each centromere. CENP-A is shown in early G1 to be assembled into nucleosomes within each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. DNA replication is demonstrated to remove ectopically loaded, non-centromeric CENP-A. In contrast, tethering of centromeric CENP-A to the sites of DNA replication through the constitutive centromere associated network (CCAN) is shown to enable precise reloading of centromere-bound CENP-A onto the same DNA sequences as in its initial pre-replication loading. Thus, DNA replication acts as an error correction mechanism for maintaining centromere identity through its # Correspondence should be addressed to DWC and/or YNA. Author Contributions Y.N-A. and D.W.C. conceived and designed experiments and wrote the manuscript. Y.N-A performed experiments. K.H.M analyzed the sequencing data. M.A.M. and O.S. analyzed data and performed experiments. D.F. suggested experiments and provided key experimental input. A.Y.L and B.R prepared sequencing libraries and provided resources. A.A and J.Y.III performed mass spectrometry experiments and provided resources.
Centrosomes are microtubule-organizing centers that facilitate bipolar mitotic spindle assembly and chromosome segregation. Recognizing that centrosome amplification is a common feature of aneuploid cancer cells, we tested whether supernumerary centrosomes are sufficient to drive tumor development. To do this, we constructed and analyzed mice in which centrosome amplification can be induced by a Cre-recombinase–mediated increase in expression of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts produced supernumerary centrosomes and enhanced the expected mitotic errors, but proliferation continued only after inactivation of the p53 tumor suppressor. Increasing Plk4 levels in mice with functional p53 produced centrosome amplification in liver and skin, but this did not promote spontaneous tumor development in these tissues or enhance the growth of chemically induced skin tumors. In the absence of p53, Plk4 overexpression generated widespread centrosome amplification, but did not drive additional tumors or affect development of the fatal thymic lymphomas that arise in animals lacking p53. We conclude that, independent of p53 status, supernumerary centrosomes are not sufficient to drive tumor formation.
Abnormal numerical and structural chromosome content is frequently found in human cancer. To test the role of aneuploidy in tumor initiation and progression, we generated mice with random aneuploidies by transient induction of polo-like kinase 4 (Plk4), a master regulator of centrosome number. Short-term chromosome instability (CIN) from transient Plk4 induction resulted in formation of aggressive T-cell lymphomas in mice with heterozygous inactivation of one p53 allele and accelerated tumor development in the absence of p53. Transient CIN increased the frequency of lymphoma-initiating cells with a specific karyotype profile, including trisomy of chromosomes 4, 5, 14, and 15 occurring early in tumorigenesis. Tumor development in mice with chronic CIN induced by an independent mechanism (through inactivation of the spindle assembly checkpoint) gradually trended toward a similar karyotypic profile, as determined by single-cell whole-genome DNA sequencing. Overall, we show how transient CIN generates cells with random aneuploidies from which ones that acquire a karyotype with specific chromosome gains are sufficient to drive cancer formation, and that distinct CIN mechanisms can lead to similar karyotypic cancer-causing outcomes.
Chromosome missegregation into a micronucleus can cause complex and localized genomic rearrangements 1,2 known as chromothripsis 3 , but the underlying mechanisms have not been established. Here we developed an inducible Y centromere-selective inactivation strategy by exploiting a CENP-A/histone H3 chimera to directly examine the fate of missegregated chromosomes in otherwise diploid human cells. Using this approach, we identified a temporal cascade of events that are initiated upon centromere inactivation involving chromosome missegregation, fragmentation, and re-ligation that span three consecutive cell cycles. Following centromere inactivation, a micronucleus harboring the Y chromosome is formed in the first cell cycle. Chromosome shattering, producing up to 53 dispersed fragments from a single chromosome, is triggered by premature micronuclear condensation prior to or during mitotic entry of the second cycle. Lastly, canonical non-homologous end joining (NHEJ), but not homology-dependent repair, is shown to facilitate re-ligation of chromosomal fragments in the third cycle. Thus, initial errors in cell division can provoke further genomic instability through fragmentation of micronuclear DNAs coupled to NHEJmediated reassembly in the subsequent interphase.Chromosome segregation errors during mitosis are a well-recognized cause of numerical aneuploidy and have been implicated in the formation of chromosomal translocations 4 , both
Mesenchymal stromal cells (MSC) are used extensively in clinical trials; however, the possibility that MSCs have a potential for malignant transformation was raised. We examined the genomic stability versus the tumorforming capacity of multiple mouse MSCs. Murine MSCs have been shown to be less stable and more prone to malignant transformation than their human counterparts. A large series of independently isolated MSC populations exhibited low tumorigenic potential under syngeneic conditions, which increased in immunocompromised animals. Unexpectedly, higher ploidy correlated with reduced tumor-forming capacity. Furthermore, in both cultured MSCs and primary hepatocytes, polyploidization was associated with a dramatic decrease in the expression of the long noncoding RNA H19. Direct knockdown of H19 expression in diploid cells resulted in acquisition of polyploid cell traits. Moreover, artificial tetraploidization of diploid cancer cells led to a reduction of H19 levels, as well as to an attenuation of the tumorigenic potential. Polyploidy might therefore serve as a protective mechanism aimed at reducing malignant transformation through the involvement of the H19 regulatory long noncoding RNA. Cancer Res; 72(24); 6403-13. Ó2012 AACR.
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