Whole-genome doubling in tissues and tumors.

The overwhelming majority of proliferating somatic human cells are diploid, and this genomic state is typically maintained across successive cell divisions. However, failures in cell division can induce a whole-genome doubling (WGD) event, in which diploid cells transition to a tetraploid state. While some WGDs are developmentally programmed to produce nonproliferative tetraploid cells with specific cellular functions, unscheduled WGDs can be catastrophic: erroneously arising tetraploid cells are ill-equipped to cope with their doubled cellular and chromosomal content and quickly become genomically unstable and tumorigenic. Deciphering the genetics that underlie the genesis, physiology, and evolution of whole-genome doubled (WGD+) cells may therefore reveal therapeutic avenues to selectively eliminate pathological WGD+ cells.

Cancer recurrence and lethality are enabled by enhanced survival and reversible cell cycle arrest of polyaneuploid cells.

We present a unifying theory to explain cancer recurrence, therapeutic resistance, and lethality. The basis of this theory is the formation of simultaneously polyploid and aneuploid cancer cells, polyaneuploid cancer cells (PACCs), that avoid the toxic effects of systemic therapy by entering a state of cell cycle arrest. The theory is independent of which of the classically associated oncogenic mutations have already occurred. PACCs have been generally disregarded as senescent or dying cells. Our theory states that therapeutic resistance is driven by PACC formation that is enabled by accessing a polyploid program that allows an aneuploid cancer cell to double its genomic content, followed by entry into a nondividing cell state to protect DNA integrity and ensure cell survival. Upon removal of stress, e.g., chemotherapy, PACCs undergo depolyploidization and generate resistant progeny that make up the bulk of cancer cells within a tumor.

Cancer cells employ an evolutionarily conserved polyploidization program to resist therapy.

Unusually large cancer cells with abnormal nuclei have been documented in the cancer literature since 1858. For more than 100 years, they have been generally disregarded as irreversibly senescent or dying cells, too morphologically misshapen and chromatin too disorganized to be functional. Cell enlargement, accompanied by whole genome doubling or more, is observed across organisms, often associated with mitigation strategies against environmental change, severe stress, or the lack of nutrients. Our comparison of the mechanisms for polyploidization in other organisms and non-transformed tissues suggest that cancer cells draw from a conserved program for their survival, utilizing whole genome doubling and pausing proliferation to survive stress. These polyaneuploid cancer cells (PACCs) are the source of therapeutic resistance, responsible for cancer recurrence and, ultimately, cancer lethality.

Characteristics of DNA macro-alterations in breast cancer with liver metastasis before treatment.

BACKGROUND: Whole-genome doubling (WGD) has been observed in 30% of cancers, followed by a highly complex rearranged karyotype unfavourable to breast cancer's outcome. However, the macro-alterations that characterise liver metastasis in breast cancer(BC) are poorly understood. Here, we conducted a whole-genome sequencing analysis of liver metastases to explore the status and the time frame model of these macro-alterations in pre-treatment patients with metastatic breast cancer. RESULTS: Whole-genome sequencing was conducted in 11 paired primary tumours, lymph node metastasis, and liver metastasis fresh samples from four patients with late-stage breast cancer. We also chose five postoperative frozen specimens from patients with early-stage breast cancer before any treatment as control. Surprisingly, all four liver metastasis samples were classified as WGD + . However, the previous study reported that WGD happened in 30% of cancers and 2/5 in our early-stage samples. WGD was not observed in the two separate primary tumours and one lymph node metastasis of one patient with metastatic BC, but her liver metastasis showed an early burst of bi-allelic copy number gain. The phylogenetic tree proves her 4 tumour samples were the polyclonal origin and only one WGD + clone metastasis to the liver. Another 3 metastatic BC patients' primary tumour and lymph node metastasis experienced WGD as well as liver metastasis, and they all showed similar molecular time-frame of copy number(CN) gain across locations within the same patient. These patients' tumours were of monoclonal origin, and WGD happened in a founding clone before metastasis, explaining that all samples share the CN-gain time frame. After WGD, the genomes usually face instability to evolve other macro-alterations. For example, a greater quantity and variety of complex structural variations (SVs) were detected in WGD + samples. The breakpoints were enriched in the chr17: 39 Mb-40 Mb tile, which contained the HER2 gene, resulting in the formation of tyfonas, breakage-fusion-bridge cycles, and double minutes. These complex SVs may be involved in the evolutionary mechanisms of the dramatic increase of HER2 copy number. CONCLUSION: Our work revealed that the WGD + clone might be a critical evolution step for liver metastasis and favoured following complex SV of breast cancer.

The evolution and expansion of RWP-RK gene family improve the heat adaptability of elephant grass (Pennisetum purpureum Schum.).

BACKGROUND: Along with global warming, resulting in crop production, exacerbating the global food crisis. Therefore, it is urgent to study the mechanism of plant heat resistance. However, crop resistance genes were lost due to long-term artificial domestication. By analyzing the potential heat tolerance genes and molecular mechanisms in other wild materials, more genetic resources can be provided for improving the heat tolerance of crops. Elephant grass (Pennisetum purpureum Schum.) has strong adaptability to heat stress and contains abundant heat-resistant gene resources. RESULTS: Through sequence structure analysis, a total of 36 RWP-RK members were identified in elephant grass. Functional analysis revealed their close association with heat stress. Four randomly selected RKDs (RKD1.1, RKD4.3, RKD6.6, and RKD8.1) were analyzed for expression, and the results showed upregulation under high temperature conditions, suggesting their active role in response to heat stress. The members of RWP-RK gene family (36 genes) in elephant grass were 2.4 times higher than that of related tropical crops, rice (15 genes) and sorghum (15 genes). The 36 RWPs of elephant grass contain 15 NLPs and 21 RKDs, and 73% of RWPs are related to WGD. Among them, combined with the DAP-seq results, it was found that RWP-RK gene family expansion could improve the heat adaptability of elephant grass by enhancing nitrogen use efficiency and peroxidase gene expression. CONCLUSIONS: RWP-RK gene family expansion in elephant grass is closely related to thermal adaptation evolution and speciation. The RKD subgroup showed a higher responsiveness than the NLP subgroup when exposed to high temperature stress. The promoter region of the RKD subgroup contains a significant number of MeJA and ABA responsive elements, which may contribute to their positive response to heat stress. These results provided a scientific basis for analyzing the heat adaptation mechanism of elephant grass and improving the heat tolerance of other crops.

Genome Doubling Shapes High-Grade Transformation and Novel EWSR1::LARP4 Fusion Shows SOX10 Immunostaining in Hyalinizing Clear Cell Carcinoma of Salivary Gland.

Hyalinizing clear cell carcinoma (HCCC) is a rare indolent malignant tumor of minor salivary gland origin with EWSR1::ATF1 rearrangement. Pathologically, the tumor cells possess a clear cytoplasm in a background of hyalinized stroma. Generally, the tumor cells are positive for p63 and p40 and negative for s100 and α-smooth muscle actin, suggesting that they differentiate into squamous epithelium and not into myoepithelium. In this study, we performed a detailed histopathological and genomic analysis of 6 cases of HCCC, including 2 atypical subtypes-a case of "high-grade transformation" and 1 "possessing a novel partner gene for EWSR1." We performed a sequential analysis of the primary and recurrent tumor by whole-exome sequencing, RNA sequencing, Sanger sequencing, and fluorescence in situ hybridization to investigate the effect of genomic changes on histopathology and clinical prognosis. A fusion gene involving the EWSR1 gene was detected in all cases. Five cases, including the "high-grade transformation," harbored a known EWSR1::ATF1 fusion gene; however, 1 case harbored a novel EWSR1::LARP4 fusion gene. This novel EWSR1::LARP4-fused HCCC has a SOX10-positive staining, which is different from the EWSR1::ATF1-fused HCCC. According to whole-exome sequencing and fluorescence in situ hybridization analysis, the "whole-genome doubling" and focal deletion involving CDKN2A, CDKN2B, and PTEN were detected in HCCC with "high-grade transformation." Conclusively, we identified a novel partner gene for EWSR1, LARP4, in indolent HCCC. Importantly, "high-grade transformation" and poor prognosis were caused by whole-genome doubling and subsequent genomic aberrations.

Thermodynamic energetics underlying genomic instability and whole-genome doubling in cancer.

Genomic instability contributes to tumorigenesis through the amplification and deletion of cancer driver genes. DNA copy number (CN) profiling of ensembles of tumors allows a thermodynamic analysis of the profile for each tumor. The free energy of the distribution of CNs is found to be a monotonically increasing function of the average chromosomal ploidy. The dependence is universal across several cancer types. Surprisal analysis distinguishes two main known subgroups: tumors with cells that have or have not undergone whole-genome duplication (WGD). The analysis uncovers that CN states having a narrower distribution are energetically more favorable toward the WGD transition. Surprisal analysis also determines the deviations from a fully stable-state distribution. These deviations reflect constraints imposed by tumor fitness selection pressures. The results point to CN changes that are more common in high-ploidy tumors and thus support altered selection pressures upon WGD.

Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs).

BACKGROUND: Genomic instability and chemoresistance can arise in cancer due to a unique form of plasticity: that of polyploid giant cancer cells (PGCCs). These cells form under the stress of chemotherapy and have higher than diploid chromosome content. PGCCs are able to then repopulate tumors through an asymmetric daughter cell budding process. PGCCs have been observed in ovarian cancer histology, including the deadly and common form high-grade serous ovarian carcinoma (HGSC). We previously discovered that drugs which disrupt the cellular recycling process of autophagy are uniquely efficacious in pre-clinical HGSC models. While autophagy induction has been associated with PGCCs, it has never been previously investigated if autophagy modulation interacts with the PGCC life cycle and this form of tumor cell plasticity. METHODS: CAOV3 and OVCAR3 ovarian cancer cell lines were treated with carboplatin or docetaxel to induce PGCC formation. Microscopy was used to characterize and quantify PGCCs formed by chemotherapy. Two clinically available drugs that inhibit autophagy, hydroxychloroquine and nelfinavir, and a clinically available activator of autophagy, rapamycin, were employed to test the effect of these autophagy modulators on PGCC induction and subsequent colony formation from PGCCs. Crystal violet-stained colony formation assays were used to quantify the tumor-repopulating stage of the PGCC life cycle. RESULTS: Autophagy inhibitors did not prevent PGCC formation in OVCAR3 or CAOV3 cells. Rapamycin did not induce PGCC formation on its own nor did it exacerbate PGCC formation by chemotherapy. However, hydroxychloroquine prevented efficient colony formation in CAOV3 PGCCs induced by carboplatin (27% inhibition) or docetaxel (41% inhibition), as well as in OVCAR3 cells (95% and 77%, respectively). Nelfinavir similarly prevented colony formation in CAOV3 PGCCs induced by carboplatin (64% inhibition) or docetaxel (94% inhibition) as well as in OVCAR3 cells (89% and 80%, respectively). Rapamycin surprisingly also prevented PGCC colony outgrowth (52-84% inhibition). CONCLUSIONS: While the autophagy previously observed to correlate with PGCC formation is unlikely necessary for PGCCs to form, autophagy modulating drugs severely impair the ability of HGSC PGCCs to form colonies. Clinical trials which utilize hydroxychloroquine, nelfinavir, and/or rapamycin after chemotherapy may be of future interest.

Inbreeding depression in polyploid species: a meta-analysis.

Whole-genome duplication is a common mutation in eukaryotes with far-reaching phenotypic effects, the resulting morphological and fitness consequences and how they affect the survival of polyploid lineages are intensively studied. Another important factor may also determine the probability of establishment and success of polyploid lineages: inbreeding depression. Inbreeding depression is expected to play an important role in the establishment of neopolyploid lineages, their capacity to colonize new environments, and in the simultaneous evolution of ploidy and other life-history traits such as self-fertilization. Both theoretically and empirically, there is no consensus on the consequences of polyploidy on inbreeding depression. In this meta-analysis, we investigated the effect of polyploidy on the evolution of inbreeding depression, by performing a meta-analysis within angiosperm species. The main results of our study are that the consequences of polyploidy on inbreeding depression are complex and depend on the time since polyploidization. We found that young polyploid lineages have a much lower amount of inbreeding depression than their diploid relatives and their established counterparts. Natural polyploid lineages are intermediate and have a higher amount of inbreeding depression than synthetic neopolyploids, and a smaller amount than diploids, suggesting that the negative effect of polyploidy on inbreeding depression decreases with time since polyploidization.

Poorly differentiated chordoma with whole-genome doubling evolving from a SMARCB1-deficient conventional chordoma: A case report.

Evolution of poorly differentiated chordoma from conventional chordoma has not been previously reported. We encountered a case of a poorly differentiated chordoma with evidence of whole-genome doubling arising from a SMARCB1-deficient conventional chordoma. The tumor presented as a destructive sacral mass in a 43-year-old man and was comprised of a highly cellular poorly differentiated chordoma with small, morphologically distinct nodules of conventional chordoma accounting for <5% of the total tumor volume. Immunohistochemistry (IHC) revealed both components were strongly reactive for brachyury and lacked normal staining for INI1. Single nucleotide polymorphism (SNP) array analysis identified multiple genomic imbalances in the conventional component, including deletions of 1p, 3p, and 22q (involving SMARCB1) and loss of chromosomes 5 and 15, while the poorly differentiated component exhibited the same aberrations at a more profound level with additional loss of chromosome 4, low level focal deletion of 17p (involving TP53), and tetraploidy. Homozygous deletion of SMARCB1 was present in both components. Fluorescence in situ hybridization (FISH) analysis confirmed the relevant deletions in both components as well as genome doubling in the poorly differentiated tumor. This case suggests that SMARCB1 loss is an early event in rare conventional chordomas that could potentially evolve into poorly differentiated chordoma through additional genomic aberrations such as genome doubling. Further studies with additional patients will be needed to determine if genome doubling is a consistent pathway for evolution of poorly differentiated chordoma.

H3K27me3 expression and methylation status in histological variants of malignant peripheral nerve sheath tumours.

Diagnosing MPNST can be challenging, but genetic alterations recently identified in polycomb repressive complex 2 (PRC2) core component genes, EED and SUZ12, resulting in global loss of the histone 3 lysine 27 trimethylation (H3K27me3) epigenetic mark, represent drivers of malignancy and a valuable diagnostic tool. However, the reported loss of H3K27me3 expression ranges from 35% to 84%. We show that advances in molecular pathology now allow many MPNST mimics to be classified confidently. We confirm that MPNSTs harbouring mutations in PRC2 core components are associated with loss of H3K27me3 expression; whole-genome doubling was detected in 68%, and SSTR2 was amplified in 32% of MPNSTs. We demonstrate that loss of H3K27me3 expression occurs overall in 38% of MPNSTs, but is lost in 76% of histologically classical cases, whereas loss was detected in only 23% cases with heterologous elements and 14% where the diagnosis could not be provided on morphology alone. H3K27me3 loss is rarely seen in other high-grade sarcomas and was not found to be associated with an inferior outcome in MPNST. We show that DNA methylation profiling distinguishes MPNST from its histological mimics, was unrelated to anatomical site, and formed two main clusters, MeGroups 4 and 5. MeGroup 4 represents classical MPNSTs lacking H3K27me3 expression in the majority of cases, whereas MeGroup 5 comprises MPNSTs exhibiting non-classical histology and expressing H3K27me3 and cluster with undifferentiated sarcomas. The two MeGroups are distinguished by differentially methylated PRC2-associated genes, the majority of which are hypermethylated in the promoter regions in MeGroup 4, indicating that the PRC2 target genes are not expressed in these tumours. The methylation profiles of MPNSTs with retention of H3K27me3 in MeGroups 4 and 5 are independent of mutations in PRC2 core components and the driver(s) in these groups remain to be identified. Our results open new avenues of investigation. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

A primary thymic adenocarcinoma with two components that traced distinct evolutionary trajectories.

Even though it is a rare subtype, identifying the genetic features of thymic adenocarcinoma is valuable for a multifaceted understanding of thymic epithelial tumors. We experienced a female patient with thymic adenocarcinoma associated with thymic cysts. The tumor consisted of a solid whitish lesion (lesion-1) and a large cystic lesion with small papillary nodules (lesion-2). Microscopically, lesion-1 exhibited poorly differentiated adenocarcinoma accompanying numerous inflammatory cell infiltrates, and lesion-2 (the nodules within the cystic lesion) exhibited enteric-type adenocarcinoma. Consistent with the histological difference, whole-exome sequencing revealed that these two components exhibited distinct genetic features, except for only a few shared mutations, including CDKN2A truncation. Lesion-1 exhibited microsatellite instability-high signature with high mutation burden, for which immune checkpoint inhibitors might apply; and lesion-2 exhibited whole-genome doubling with KRAS hotspot mutation. Our case presents novel genetic features of thymic adenocarcinoma and demonstrates that distinct mutational processes can be operative within a single tumor.

The effects of hybridization and genome doubling in plant evolution via allopolyploidy.

Polyploidy is a pervasive and recurring phenomenon across the tree of life, which occurred at variable time scales, ecological amplitudes and cell types, and is especially prominent in the evolutionary histories of plants. Importantly, many of the world's most important crops and noxious invasive weeds are recent polyploids. Polyploidy includes two major types, autopolyploidy, referring to doubling of a single species genome, and allopolyploidy referring to doubling of two or more merged genomes via biological hybridization of distinct but related species. The prevalence of both types of polyploidy implies that both genome doubling alone and doubling coupled with hybridization confer selective advantages over their diploid progenitors under specific circumstances. In cases of allopolyploidy, the two events, genome doubling and hybridization, have both advantages and disadvantages. Accumulated studies have established that, in allopolyploidy, some advantage(s) of doubling may compensate for the disadvantage(s) of hybridity and vice versa, although further study is required to validate generality of this trend. Some studies have also revealed a variety of non-Mendelian genetic and genomic consequences induced by doubling and hybridization separately or concertedly in nascent allopolyploidy; however, the significance of which to the immediate establishment and longer-term evolutionary success of allopolyploid species remain to be empirically demonstrated and ecologically investigated. This review aims to summarize recent advances in our understanding of the roles of hybridization and genome doubling, in separation and combination, in the evolution of allopolyploid genomes, as well as fruitful future research directions that are emerging from these studies.

Distinguishing successive ancient polyploidy levels based on genome-internal syntenic alignment.

BACKGROUND: A basic tool for studying the polyploidization history of a genome, especially in plants, is the distribution of duplicate gene similarities in syntenically aligned regions of a genome. This distribution can usually be decomposed into two or more components identifiable by peaks, or local maxima, each representing a different polyploidization event. The distributions may be generated by means of a discrete time branching process, followed by a sequence divergence model. The branching process, as well as the inference of fractionation rates based on it, requires knowledge of the ploidy level of each event, which cannot be directly inferred from the pair similarity distribution. RESULTS: For a sequence of two events of unknown ploidy, either tetraploid, giving rise to whole genome doubling (WGD), or hexaploid, giving rise to whole genome tripling (WGT), we base our analysis on triples of similar genes. We calculate the probability of the four triplet types with origins in one or the other event, or both, and impose a mutational model so that the distribution resembles the original data. Using a ML transition point in the similarities between the two events as a discriminator for the hypothesized origin of each similarity, we calculate the predicted number of triplets of each type for each model combining WGT and/or WGD. This yields a predicted profile of triplet types for each model. We compare the observed and predicted triplet profiles for each model to confirm the polyploidization history of durian, poplar and cabbage. CONCLUSIONS: We have developed a way of inferring the ploidy of up to three successive WGD and/or WGT events by estimating the time of origin of each of the similarities in triples of genes. This may be generalized to a larger number of events and to higher ploidies.

Maternal doubled haploid production in interploidy hybridization between Brassica napus and Brassica allooctaploids.

MAIN CONCLUSION: We found a new in vivo route to produce maternal doubled haploid of Brassica napus . The pollen donor, an allooctaploid rapeseed, acts as a DH inducer. Inbred line has a powerful advantage in cultivar breeding and genetic analysis. Compared to the traditional breeding methods, doubled haploid production can save years off the breeding process. Though genotype-dependent tissue culture methods are widely used in the Brassica crops, seed-based in vivo doubled haploid developing systems are rare in nature and in the laboratory. As interspecific cross and interploid hybridization play an important role in genome evolution and plant speciation, we created a new Brassica artificial hybrid, a Brassica allooctaploid (AAAACCCC, 2n = 8× = 76), by interspecific crossing and genome doubling. A homozygous line was observed at the third self-generation of a synthesized Brassica allohexaploid (AAAACC, 2n = 6× = 58). Crosses between B. napus as female and Brassica allooctaploid as pollen donor were conducted, which yielded maternal doubled haploid B. napus that were identified based on phenotype, ploidy, and molecular analysis. The Brassica octaploid acted as a maternal doubled haploid inducer and had a relatively high induction rate. Our research provides a new insight for generation of homozygous lines in vivo using a single-step approach, as well as promotes the understanding in breeding programs and genetic studies involving the Brassicas.

Novel technologies in doubled haploid line development.

haploid inducer line can be transferred (DH) technology can not only shorten the breeding process but also increase genetic gain. Haploid induction and subsequent genome doubling are the two main steps required for DH technology. Haploids have been generated through the culture of immature male and female gametophytes, and through inter- and intraspecific via chromosome elimination. Here, we focus on haploidization via chromosome elimination, especially the recent advances in centromere-mediated haploidization. Once haploids have been induced, genome doubling is needed to produce DH lines. This study has proposed a new strategy to improve haploid genome doubling by combing haploids and minichromosome technology. With the progress in haploid induction and genome doubling methods, DH technology can facilitate reverse breeding, cytoplasmic male sterile (CMS) line production, gene stacking and a variety of other genetic analysis.

Niche divergence between diploid and autotetraploid Tolmiea.

PREMISE OF STUDY: Polyploidy is common in eukaryotes and is of major evolutionary importance over both short and long time-scales. Compared to allopolyploids, autopolyploids remain understudied; they are often morphologically cryptic and frequently remain taxonomically unrecognized, although there is increasing recognition of the high frequency of autopolyploidy in angiosperms. While autopolyploidy can serve as an instant speciation mechanism, little is known about the ecological consequences of this process. We describe the ecological divergence of a diploid-autotetraploid species pair in Tolmiea. METHODS: We investigated whether abiotic niche divergence has shaped the current allopatric distribution of diploid T. diplomenziesii and its autotetraploid derivative, T. menziesii, in the Pacific Northwest of North America. We employed field measures of light availability, as well as niche modeling and a principal component analysis of environmental space. Within a common garden, we also investigated physiological responses to changes in soil moisture. KEY RESULTS: Diploid and autotetraploid Tolmiea inhabit significantly different climatic niche spaces. The climatic niche divergence between these two species is best explained by a shift in precipitation availability, and we found evidence of differing physiological response to water availability between these species. CONCLUSIONS: We found that spatial segregation of T. diplomenziesii and T. menziesii was accompanied by adaptation to changes in climatic regime. Tolmiea menziesii is not a nascent autotetraploid, having persisted long enough to be established throughout the Pacific Northwest, and therefore both polyploidization and subsequent evolution have contributed to the observed differences between T. menziesii and T. diplomenziesii.

The polyploidy revolution then and now: Stebbins revisited.

Polyploidy has long been considered a major force in plant evolution. G. Ledyard Stebbins, Jr., an architect of the Modern Synthesis, elegantly addressed a broad range of topics, from genes to chromosomes to deep phylogeny, but some of his most lasting insights came in the study of polyploidy. Here, we review the immense impact of his work on polyploidy over more than 60 years, from his entrance into this fledgling field in the 1920s until the end of his career. Stebbins and his contemporaries developed a model of polyploid evolution that persisted for nearly half a century. As new perspectives emerged in the 1980s and new genetic tools for addressing key aspects of polyploidy have become available, a new paradigm of polyploidy has replaced much of the Stebbinsian framework. We review that paradigm shift and emphasize those areas in which the ideas of Stebbins continue to propel the field forward, as well as those areas in which the field was held back; we also note new directions that plant geneticists and evolutionists are now exploring in polyploidy research. Perhaps the most important conclusion from recent and ongoing studies of polyploidy is that, following Levin and others, polyploidy may propel a population into a new adaptive sphere given the myriad changes that accompany genome doubling.