The life history of 21 breast cancers.

Cancer evolves dynamically as clonal expansions supersede one another driven by shifting selective pressures, mutational processes, and disrupted cancer genes. These processes mark the genome, such that a cancer's life history is encrypted in the somatic mutations present. We developed algorithms to decipher this narrative and applied them to 21 breast cancers. Mutational processes evolve across a cancer's lifespan, with many emerging late but contributing extensive genetic variation. Subclonal diversification is prominent, and most mutations are found in just a fraction of tumor cells. Every tumor has a dominant subclonal lineage, representing more than 50% of tumor cells. Minimal expansion of these subclones occurs until many hundreds to thousands of mutations have accumulated, implying the existence of long-lived, quiescent cell lineages capable of substantial proliferation upon acquisition of enabling genomic changes. Expansion of the dominant subclone to an appreciable mass may therefore represent the final rate-limiting step in a breast cancer's development, triggering diagnosis.

Mutational processes molding the genomes of 21 breast cancers.

All cancers carry somatic mutations. The patterns of mutation in cancer genomes reflect the DNA damage and repair processes to which cancer cells and their precursors have been exposed. To explore these mechanisms further, we generated catalogs of somatic mutation from 21 breast cancers and applied mathematical methods to extract mutational signatures of the underlying processes. Multiple distinct single- and double-nucleotide substitution signatures were discernible. Cancers with BRCA1 or BRCA2 mutations exhibited a characteristic combination of substitution mutation signatures and a distinctive profile of deletions. Complex relationships between somatic mutation prevalence and transcription were detected. A remarkable phenomenon of localized hypermutation, termed "kataegis," was observed. Regions of kataegis differed between cancers but usually colocalized with somatic rearrangements. Base substitutions in these regions were almost exclusively of cytosine at TpC dinucleotides. The mechanisms underlying most of these mutational signatures are unknown. However, a role for the APOBEC family of cytidine deaminases is proposed.

Author Correction: Landscape of somatic mutations in 560 breast cancer whole-genome sequences.

In the Methods section of this Article, 'greater than' should have been 'less than' in the sentence 'Putative regions of clustered rearrangements were identified as having an average inter-rearrangement distance that was at least 10 times greater than the whole-genome average for the individual sample. '. The Article has not been corrected.

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.

Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

Landscape of somatic mutations in 560 breast cancer whole-genome sequences.

We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.

Identification of the RNA polymerase I-RNA interactome.

Ribosome biogenesis is a complex process orchestrated by a host of ribosome assembly factors. Although it is known that many of the proteins involved in this process have RNA binding activity, the full repertoire of proteins that interact with the precursor ribosomal RNA is currently unknown. To gain a greater understanding of the extent to which RNA-protein interactions have the potential to control ribosome biogenesis, we used RNA affinity isolation coupled with proteomics to measure the changes in RNA-protein interactions that occur when rRNA transcription is blocked. Our analysis identified 211 out of 457 nuclear RNA binding proteins with a >3-fold decrease in RNA-protein interaction after inhibition of RNA polymerase I (RNAPI). We have designated these 211 RNA binding proteins as the RNAPI RNA interactome. As expected, the RNAPI RNA interactome is highly enriched for nucleolar proteins and proteins associated with ribosome biogenesis. Selected proteins from the interactome were shown to be nucleolar in location and to have RNA binding activity that was dependent on RNAPI activity. Furthermore, our data show that two proteins, which are required for rRNA maturation, AATF and NGDN, and which form part of the RNA interactome, both lack canonical RNA binding domains and yet are novel pre-rRNA binding proteins.

Signatures of mutational processes in human cancer.

All cancers are caused by somatic mutations; however, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here we analysed 4,938,362 mutations from 7,042 cancers and extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC family of cytidine deaminases, whereas others are confined to a single cancer class. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, 'kataegis', is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer, with potential implications for understanding of cancer aetiology, prevention and therapy.

Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells.

Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells.

Analysis of the mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer.

BACKGROUND: MAP2K4 is a putative tumor and metastasis suppressor gene frequently found to be deleted in various cancer types. We aimed to conduct a comprehensive analysis of this gene to assess its involvement in ovarian cancer. METHODS: We screened for mutations in MAP2K4 using High Resolution Melt analysis of 149 primary ovarian tumors and methylation at the promoter using Methylation-Specific Single-Stranded Conformation Polymorphism analysis of 39 tumors. We also considered the clinical impact of changes in MAP2K4 using publicly available expression and copy number array data. Finally, we used siRNA to measure the effect of reducing MAP2K4 expression in cell lines. RESULTS: In addition to 4 previously detected homozygous deletions, we identified a homozygous 16 bp truncating deletion and a heterozygous 4 bp deletion, each in one ovarian tumor. No promoter methylation was detected. The frequency of MAP2K4 homozygous inactivation was 5.6% overall, and 9.8% in high-grade serous cases. Hemizygous deletion of MAP2K4 was observed in 38% of samples. There were significant correlations of copy number and expression in three microarray data sets. There was a significant correlation between MAP2K4 expression and overall survival in one expression array data set, but this was not confirmed in an independent set. Treatment of JAM and HOSE6.3 cell lines with MAP2K4 siRNA showed some reduction in proliferation. CONCLUSIONS: MAP2K4 is targeted by genetic inactivation in ovarian cancer and restricted to high grade serous and endometrioid carcinomas in our cohort.

Crystallization and preliminary X-ray crystallographic analysis of zebrafish prototype galectin Drgal1-L2.

Zebrafish (Danio rerio) are an important developmental and embryological model given the optical clarity of the embryos and larvae, which permits real-time viewing of developing pathologies. More recently, a broader scope for these vertebrates to model a range of human diseases, including some cancers, has been indicated. Zebrafish Drgal1-L2 has been identified as an orthologue of mammalian galectin-1, which is is a carbohydrate-binding protein that exhibits ß-galactoside-binding specificity and which is overexpressed by many aggressive human cancers. This study describes the cloning, expression in Escherichia coli, purification and crystallization of recombinant Drgal1-L2 protein in the presence of lactose (ligand). X-ray diffraction data from these novel crystals of zebrafish Drgal1-L2 were collected to a resolution of 1.5 Šusing a synchrotron-radiation source, enabling their characterization.

Are there any more ovarian tumor suppressor genes? A new perspective using ultra high-resolution copy number and loss of heterozygosity analysis.

Ovarian cancer is characterized by complex genetic alterations, including copy number loss and copy number-neutral loss of heterozygosity (LOH). These alterations are assumed to represent the "second hit" of the underlying tumor suppressor gene (TSG), however, relative to the number of LOH hotspots reported, few ovarian TSGs have been identified. We conducted a high-resolution LOH analysis using SNP arrays (500K and SNP6.0) of 106 primary ovarian tumors of various histological subtypes together with matching normal DNA. LOH was detected in at least 35% of samples on chromosomes 17, 19p, 22q, Xp, 13q, 8p, 6q, 4q, 5q, 1p, 16q, and 9q with a median minimal region of overlap of only 300 kb. Subtype-specific differences in LOH frequency were noted, particularly for mucinous cases. We also identified 192 somatic homozygous deletions (HDs). Recurrent HDs targeted known TSGs such as CDKN2A (eight samples), RB1 (five samples), and PTEN (three samples). Additional recurrent HDs targeted 16 candidate TSGs near minimal regions of LOH on chromosomes 17, 13, 8p, 5q, and X. Given the importance of HDs in inactivating known genes, these candidates are highly likely to be ovarian TSGs. Our data suggest that the poor success of previous LOH studies was due to the inability of previous technology to resolve complex genomic alterations and distinguish true LOH from allelic imbalance. This study shows that recurrent regions of LOH and HD frequently align with known TSGs suggesting that LOH analysis remains a valid approach to discovering new candidates.

Biotin proximity tagging favours unfolded proteins and enables the study of intrinsically disordered regions.

Intrinsically Disordered Regions (IDRs) are enriched in disease-linked proteins known to have multiple post-translational modifications, but there is limited in vivo information about how locally unfolded protein regions contribute to biological functions. We reasoned that IDRs should be more accessible to targeted in vivo biotinylation than ordered protein regions, if they retain their flexibility in human cells. Indeed, we observed increased biotinylation density in predicted IDRs in several cellular compartments >20,000 biotin sites from four proximity proteomics studies. We show that in a biotin 'painting' time course experiment, biotinylation events in Escherichia coli ribosomes progress from unfolded and exposed regions at 10 s, to structured and less accessible regions after five minutes. We conclude that biotin proximity tagging favours sites of local disorder in proteins and suggest the possibility of using biotin painting as a method to gain unique insights into in vivo condition-dependent subcellular plasticity of proteins.

Efficient recovery of the RNA-bound proteome and protein-bound transcriptome using phase separation (OOPS).

RNA-protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA-protein adducts in an unbiased way. OOPS avoids molecular tagging or the capture of polyadenylated RNA. Instead, it is based on sampling the interface of a standard TRIzol extraction to enrich RNA-binding proteins (RBPs) and their cognate bound RNA. OOPS specificity is achieved by digesting the enriched interfaces with RNases or proteases to release the RBPs or protein-bound RNA, respectively. Here we present a step-by-step protocol to purify protein-RNA adducts, free protein and free RNA from the same sample. We further describe how OOPS can be applied in human cell lines, Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli and how it can be used to study RBP dynamics.

The cell stress response: extreme times call for post-transcriptional measures.

Following cell stress, a wide range of molecular pathways are initiated to orchestrate the stress response and enable adaptation to an environmental or intracellular perturbation. The post-transcriptional regulation strategies adopted during the stress response result in a substantial reorganization of gene expression, designed to prepare the cell for either acclimatization or programmed death, depending on the nature and intensity of the stress. Fundamental to the stress response is a rapid repression of global protein synthesis, commonly mediated by phosphorylation of translation initiation factor eIF2α. Recent structural and biochemical information have added unprecedented detail to our understanding of the molecular mechanisms underlying this regulation. During protein synthesis inhibition, the translation of stress-specific mRNAs is nonetheless enhanced, often through the interaction between RNA-binding proteins and specific RNA regulatory elements. Recent studies investigating the unfolded protein response (UPR) provide some important insights into how posttranscriptional events are spatially and temporally fine-tuned in order to elicit the most appropriate response and to coordinate the transition from an early, acute stage into the chronic state of adaptation. Importantly, cancer cells are known to hi-jack adaptive stress response pathways, particularly the UPR, to survive and proliferate in the unfavorable tumor environment. In this review, we consider the implications of recent research into stress-dependent post-transcriptional regulation and make the case for the exploration of the stress response as a strategy to identify novel targets in the development of cancer therapies. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution Translation > Translation Mechanisms > Translation Regulation.

Author Correction: Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS).

Existing high-throughput methods to identify RNA-binding proteins (RBPs) are based on capture of polyadenylated RNAs and cannot recover proteins that interact with nonadenylated RNAs, including long noncoding RNA, pre-mRNAs and bacterial RNAs. We present orthogonal organic phase separation (OOPS), which does not require molecular tagging or capture of polyadenylated RNA, and apply it to recover cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. We validated OOPS in HEK293, U2OS and MCF10A human cell lines, and show that 96% of proteins recovered were bound to RNA. We show that all long RNAs can be cross-linked to proteins, and recovered 1,838 RBPs, including 926 putative novel RBPs. OOPS is approximately 100-fold more efficient than existing methods and can enable analyses of dynamic RNA-protein interactions. We also characterize dynamic changes in RNA-protein interactions in mammalian cells following nocodazole arrest, and present a bacterial RNA-interactome for Escherichia coli. OOPS is compatible with downstream proteomics and RNA sequencing, and can be applied in any organism.

Partially methylated domains are hypervariable in breast cancer and fuel widespread CpG island hypermethylation.

Global loss of DNA methylation and CpG island (CGI) hypermethylation are key epigenomic aberrations in cancer. Global loss manifests itself in partially methylated domains (PMDs) which extend up to megabases. However, the distribution of PMDs within and between tumor types, and their effects on key functional genomic elements including CGIs are poorly defined. We comprehensively show that loss of methylation in PMDs occurs in a large fraction of the genome and represents the prime source of DNA methylation variation. PMDs are hypervariable in methylation level, size and distribution, and display elevated mutation rates. They impose intermediate DNA methylation levels incognizant of functional genomic elements including CGIs, underpinning a CGI methylator phenotype (CIMP). Repression effects on tumor suppressor genes are negligible as they are generally excluded from PMDs. The genomic distribution of PMDs reports tissue-of-origin and may represent tissue-specific silent regions which tolerate instability at the epigenetic, transcriptomic and genetic level.

Trans-acting translational regulatory RNA binding proteins.

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms.

HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures.

Approximately 1-5% of breast cancers are attributed to inherited mutations in BRCA1 or BRCA2 and are selectively sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. In other cancer types, germline and/or somatic mutations in BRCA1 and/or BRCA2 (BRCA1/BRCA2) also confer selective sensitivity to PARP inhibitors. Thus, assays to detect BRCA1/BRCA2-deficient tumors have been sought. Recently, somatic substitution, insertion/deletion and rearrangement patterns, or 'mutational signatures', were associated with BRCA1/BRCA2 dysfunction. Herein we used a lasso logistic regression model to identify six distinguishing mutational signatures predictive of BRCA1/BRCA2 deficiency. A weighted model called HRDetect was developed to accurately detect BRCA1/BRCA2-deficient samples. HRDetect identifies BRCA1/BRCA2-deficient tumors with 98.7% sensitivity (area under the curve (AUC) = 0.98). Application of this model in a cohort of 560 individuals with breast cancer, of whom 22 were known to carry a germline BRCA1 or BRCA2 mutation, allowed us to identify an additional 22 tumors with somatic loss of BRCA1 or BRCA2 and 47 tumors with functional BRCA1/BRCA2 deficiency where no mutation was detected. We validated HRDetect on independent cohorts of breast, ovarian and pancreatic cancers and demonstrated its efficacy in alternative sequencing strategies. Integrating all of the classes of mutational signatures thus reveals a larger proportion of individuals with breast cancer harboring BRCA1/BRCA2 deficiency (up to 22%) than hitherto appreciated (∼1-5%) who could have selective therapeutic sensitivity to PARP inhibition.