Genetic and epigenetic heterogeneity in cancer: A genome‐centric perspective

Genetic and epigenetic heterogeneity (the main form of non‐genetic heterogeneity) are key elements in cancer progression and drug resistance, as they provide needed population diversity, complexity, and robustness. Despite drastically increased evidence of multiple levels of heterogeneity in cancer, the general approach has been to eliminate the “noise” of heterogeneity to establish genetic and epigenetic patterns. In particular, the appreciation of new types of epigenetic regulation like non‐coding RNA, have led to the hope of solving the mystery of cancer that the current genetic theories seem to be unable to achieve. In this mini‐review, we have briefly analyzed a number of mis‐conceptions regarding cancer heterogeneity, followed by the re‐evaluation of cancer heterogeneity within a framework of the genome‐centric concept of evolution. The analysis of the relationship between gene, epigenetic and genome level heterogeneity, and the challenges of measuring heterogeneity among multiple levels have been discussed. Further, we propose that measuring genome level heterogeneity represents an effective strategy in the study of cancer and other types of complex diseases, as emphasis on the pattern of system evolution rather than specific pathways provides a global and synthetic approach. Compared to the degree of heterogeneity, individual molecular pathways will have limited predictability during stochastic cancer evolution where genome dynamics (reflected by karyotypic heterogeneity) will dominate. J. Cell. Physiol. 220: 538–547, 2009. © 2009 Wiley‐Liss, Inc.     

Single cell human genomic analyses: a way to refine the knowledge of cellular heterogeneity origins in individual subject

Single cell genomics performed on individual human subjects' tumors, neural tissues, and sperm samples revealed the existence of genetic heterogeneity arising through either mutations in exomes, deletions, recombinations, and duplications of DNA sequences, as well as aneuploidy. These genetic changes happen during cell cycles followed by cell division. The aim of this review is to strictly focus on single cell human genomics and intends to deliver information that can help to refine fundamental knowledge relating to genetic causes of cellular heterogeneity origins in both healthy and disease states. Allogenic heterogeneity as well as heterogeneity origins of cells possessing the same genome with different gene expression patterns is not the subject of this review. Future research still requires: a) improvement for complete and errorless DNA acquisition and sequencing of not only selected parts of the genome, and b) analyses of more samples that contain millions of cells. These data will deliver a more precise comparative representation of genetic diversity among single cells in an individual human subject. Consequently, we will be able to better distinguish between the role of genetic, versus epigenetic, and stochastic factors in the cellular diversity of over 30 trillion cells present in a human body.     

The dynamics of cancer chromosomes and genomes

A key feature of cancer chromosomes and genomes is their high level of dynamics and the ability to constantly evolve. This unique characteristic forms the basis of genetic heterogeneity necessary for cancer formation, which presents major obstacles to current cancer diagnosis and treatment. It has been difficult to integrate such dynamics into traditional models of cancer progression. In this conceptual piece, we briefly discuss some of the recent exciting progress in the field of cancer genomics and genome research. In particular, a re-evaluation of the previously disregarded non-clonal chromosome aberrations (NCCAs) is reviewed, coupled with the progress of the detection of sub-chromosomal aberrations with array technologies. Clearly, the high level of genetic heterogeneity is directly caused by genome instability that is mediated by stochastic genomic changes, and genome variations defined by chromosome aberrations are the driving force of cancer progression. In addition to listing various types of non-recurrent chromosomal aberrations, we discuss the likely mechanism underlying cancer chromosome dynamics. Finally, we call for further examination of the features of dynamic genome diseases including cancer in the context of systems biology and the need to integrate this new knowledge into basic research and clinical applications. This genome centric concept will have a profound impact on the future of biological and medical research.     

Cancer progression by non-clonal chromosome aberrations

The establishment of the correct conceptual framework is vital to any scientific discipline including cancer research. Influenced by hematologic cancer studies, the current cancer concept focuses on the stepwise patterns of progression as defined by specific recurrent genetic aberrations. This concept has faced a tough challenge as the majority of cancer cases follow non-linear patterns and display stochastic progression. In light of the recent discovery that genomic instability is directly linked to stochastic non-clonal chromosome aberrations (NCCAs), and that cancer progression can be characterized as a dynamic relationship between NCCAs and recurrent clonal chromosome aberrations (CCAs), we propose that the dynamics of NCCAs is a key element for karyotypic evolution in solid tumors. To support this viewpoint, we briefly discuss various basic elements responsible for cancer initiation and progression within an evolutionary context. We argue that even though stochastic changes can be detected at various levels of genetic organization, such as at the gene level and epigenetic level, it is primarily detected at the chromosomal or genome level. Thus, NCCA-mediated genomic variation plays a dominant role in cancer progression. To further illustrate the involvement of NCCA/CCA cycles in the pattern of cancer evolution, four cancer evolutionary models have been proposed based on the comparative analysis of karyotype patterns of various types of cancer.     

癌症相关的DNA甲基化连锁区域

DNA甲基化是重要的表观遗传标记之一,在转录调控中起直接作用。DNA甲基化的异常与癌症的发生发展密切相关。高通量测序使得在单碱基分辨率下检测全基因组的DNA甲基化水平成为可能。本文基于临近CpGs位点甲基化水平的相关性挖掘DNA甲基化连锁区域。结果发现DNA甲基化连锁区域的甲基化水平和模式在癌症中存在异常,而且显著富集到分化/发育相关的生物学功能。DNA甲基化连锁区域的挖掘有助于对具有生物学功能的表观遗传标记的进一步理解,有助于对癌症诊断的表观遗传标记的挖掘。     

Stochastic cancer progression driven by non-clonal chromosome aberrations

Cancer research has previously focused on the identification of specific genes and pathways responsible for cancer initiation and progression based on the prevailing viewpoint that cancer is caused by a stepwise accumulation of genetic aberrations. This viewpoint, however, is not consistent with the clinical finding that tumors display high levels of genetic heterogeneity and distinctive karyotypes. We show that chromosomal instability primarily generates stochastic karyotypic changes leading to the random progression of cancer. This was accomplished by tracing karyotypic patterns of individual cells that contained either defective genes responsible for genome integrity or were challenged by onco-proteins or carcinogens that destabilized the genome. Analysis included the tracing of patterns of karyotypic evolution during different stages of cellular immortalization. This study revealed that non-clonal chromosomal aberrations (NCCAs) (both aneuploidy and structural aberrations) and not recurrent clonal chromosomal aberrations (CCAs) are directly linked to genomic instability and karyotypic evolution. Discovery of "transitional CCAs" during in vitro immortalization clearly demonstrates that karyotypic evolution in solid tumors is not a continuous process. NCCAs and their dynamic interplay with CCAs create infinite genomic combinations leading to clonal diversity necessary for cancer cell evolution. The karyotypic chaos observed within the cell crisis stage prior to establishment of the immortalization further supports the ultimate importance of genetic aberrations at the karyotypic or genome level. Therefore, genomic instability generated NCCAs are a key driving force in cancer progression. The dynamic relationship between NCCAs and CCAs provides a mechanism underlying chromosomal based cancer evolution and could have broad clinical applications.     

Genetic and Epigenetic Regulation of TOX3 Expression in Breast Cancer

Genome wide association studies (GWAS) have identified low penetrance and high frequency single nucleotide polymorphisms (SNPs) that contribute to genetic susceptibility of breast cancer. The SNPs at 16q12, close to the TOX3 and CASC16 genes, represent one of the susceptibility loci identified by GWAS, showing strong evidence for breast cancer association across various populations. To examine molecular mechanisms of TOX3 regulation in breast cancer, we investigated both genetic and epigenetic factors using cell lines and datasets derived from primary breast tumors available through The Cancer Genome Atlas (TCGA). TOX3 expression is highly up-regulated in luminal subtype tumors compared to normal breast tissues or basal-like tumors. Expression quantitative trait loci (eQTL) analyses revealed significant associations of rs3803662 and rs4784227 genotypes with TOX3 expression in breast tumors. Bisulfite sequencing of four CpG islands in the TOX3 promoter showed a clear difference between luminal and basal-like cancer cell lines. 5-Aza-2’-deoxycytidine treatment of a basal-like cancer cell line increased expression of TOX3. TCGA dataset verified significantly lower levels of methylation of the promoter in luminal breast tumors with an inverse correlation between methylation and expression of TOX3. Methylation QTL (mQTL) analyses showed a weak or no correlation of rs3803662 or rs4784227 with TOX3 promoter methylation in breast tumors, indicating an independent relationship between the genetic and epigenetic events. These data suggest a complex system of TOX3 regulation in breast tumors, driven by germline variants and somatic epigenetic modifications in a subtype specific manner.     

Genetic Determinants of Epigenetic Patterns: Providing Insight into Disease

The field of epigenetics and our understanding of the mechanisms that regulate the establishment, maintenance and heritability of epigenetic patterns continue to grow at a remarkable rate. This information is providing increased understanding of the role of epigenetic changes in disease, insight into the underlying causes of these epigenetic changes and revealing new avenues for therapeutic intervention. Epigenetic modifiers are increasingly being pursued as therapeutic targets in a range of diseases, with a number of agents targeting epigenetic modifications already proving effective in diseases such as cancer. Although it is well established that DNA mutations and aberrant expression of epigenetic modifiers play a key role in disease, attention is now turning to the interplay between genetic and epigenetic factors in complex disease etiology. The role of genetic variability in determining epigenetic profiles, which can then be modified by environmental and stochastic factors, is becoming more apparent. Understanding the interplay between genetic and epigenetic factors is likely to aid in identifying individuals most likely to benefit from epigenetic therapies. This goal is coming closer to realization because of continual advances in laboratory and statistical tools enabling improvements in the integration of genomic, epigenomic and phenotypic data.     

Epigenetic modulations rendering cell-to-cell variability and phenotypic metastability

Tumor cells display phenotypic plasticity and heterogeneity due to genetic and epigenetic variations which limit the predictability of therapeutic interventions.Chromatin modifications can arise stochastically but can also be a consequence of environmental influences such as the microenvironment of cancer cells.A better understanding of the impact and dynamics of epigenetic modulation at defined chromosomal sites is required to get access to the underlying mechanisms.We investigated the epigenetic modulations leading to cell-to-cell heterogeneity in a tumor cell line model.To this end,we analyzed expression variance in 80 genetically uniform cell populations having a single-copy reporter randomly integrated in the genome.Single-cell analysis showed high intraclonal heterogeneity.Epigenetic characterization revealed that expression heterogeneity was accompanied by differential histone marks whereas contribution of DNA methylation could be excluded.Strikingly,some clones revealed a highly dynamic,stochastically altered chromatin state of the transgene cassette which was accompanied with a metastable expression pattern.In contrast,other clones represented a robust chromatin state of the transgene cassette with a stable expression pattern.Together,these results elucidate locus-specific epigenetic modulation in gene expression that contributes to phenotypic heterogeneity of cells and might account for cellular plasticity.     

Inter- and intramouse heterogeneity of radiation response for a growing paired organ

An intensive search for predictive markers of individual radiation response of apparently normal tissues in cancer patients is in progress at the genetic and epigenetic levels. However, the relative impact of variability at these levels is not clear. Experimental results obtained in inbred rodents, which have significantly reduced genetic heterogeneity relative to a population of human patients, may help to clarify this issue. We investigated a paired-organ mouse system in a strain of inbred mice to evaluate the intermouse variability of normal tissue radiation response, singled out from measurement errors and stochastic effects. The legs of 5-day-old C3H mice were homogeneously gamma-irradiated with a range of single doses. The lengths of the right and left tibiae were measured in 30 kVp X-ray images taken at the time of irradiation and at 84 days postirradiation. The dose-effect curves were smooth and well defined, with bone growth retardation evident at approximately 14 Gy and higher, and were marginally gender-dependent. The intramouse (left compared to right) variability of the tibia length on day 89, which characterized stochastic effects, was not distinguishable from the measurement error for doses less than 16-18 Gy and slightly exceeded measurement errors only at the largest doses of 20-22 Gy. The corresponding intermouse variability was greater than the measurement error and stochastic effects at all doses used. Interestingly, the total variability, judged by the gamma(50) values of approximately 7 we obtained, was similar to that reported for severe late reactions in human normal tissue. If the variations of response determined by epigenetic events in human patients free of known factors associated with altered radiation sensitivity are comparable to those observed in this mouse model, our results imply a relatively low power of genetic approaches alone to predict individual side effects in radiotherapy.     

Transposable Elements Cross Kingdom Boundaries and Contribute to Inflammation and Ageing

The de-repression of transposable elements (TEs) in mammalian genomes is thought to contribute to genome instability, inflammation, and ageing, yet is viewed as a cell-autonomous event. In contrast to mammalian cells, prokaryotes constantly exchange genetic material through TEs, crossing both cell and species barriers, contributing to rapid microbial evolution and diversity in complex communities such as the mammalian gut. Here, it is proposed that TEs released from prokaryotes in the microbiome or from pathogenic infections regularly cross the kingdom barrier to the somatic cells of their eukaryotic hosts. It is proposed this horizontal transfer of TEs from microbe to host is a stochastic, ongoing catalyst of genome destabilization, resulting in structural and epigenetic variations, and activation of well-evolved host defense mechanisms contributing to inflammation, senescence, and biological ageing. It is proposed that innate immunity pathways defend against the horizontal acquisition of microbial TEs, and that activation of this pathway during horizontal transposon transfer promotes chronic inflammation during ageing. Finally, it is suggested that horizontal acquisition of prokaryotic TEs into mammalian genomes has been masked and subsequently under-reported due to flaws in current sequencing pipelines, and new strategies to uncover these events are proposed.     

The epigenetic progenitor origin of human cancer

Cancer is widely perceived as a heterogeneous group of disorders with markedly different biological properties, which are caused by a series of clonally selected genetic changes in key tumour-suppressor genes and oncogenes. However, recent data suggest that cancer has a fundamentally common basis that is grounded in a polyclonal epigenetic disruption of stem/progenitor cells, mediated by 'tumour-progenitor genes'. Furthermore, tumour cell heterogeneity is due in part to epigenetic variation in progenitor cells, and epigenetic plasticity together with genetic lesions drives tumour progression. This crucial early role for epigenetic alterations in cancer is in addition to epigenetic alterations that can substitute for genetic variation later in tumour progression. Therefore, non-neoplastic but epigenetically disrupted stem/progenitor cells might be a crucial target for cancer risk assessment and chemoprevention.     

Targeted deep resequencing of the human cancer genome using next-generation technologies

Next-generation sequencing technologies have revolutionized our ability to identify genetic variants, either germline or somatic point mutations, that occur in cancer. Parallelization and miniaturization of DNA sequencing enables massive data throughput and for the first time, large-scale, nucleotide resolution views of cancer genomes can be achieved. Systematic, large-scale sequencing surveys have revealed that the genetic spectrum of mutations in cancers appears to be highly complex with numerous low frequency bystander somatic variations, and a limited number of common, frequently mutated genes. Large sample sizes and deeper resequencing are much needed in resolving clinical and biological relevance of the mutations as well as in detecting somatic variants in heterogeneous samples and cancer cell sub-populations. However, even with the next-generation sequencing technologies, the overwhelming size of the human genome and need for very high fold coverage represents a major challenge for up-scaling cancer genome sequencing projects. Assays to target, capture, enrich or partition disease-specific regions of the genome offer immediate solutions for reducing the complexity of the sequencing libraries. Integration of targeted DNA capture assays and next-generation deep resequencing improves the ability to identify clinically and biologically relevant mutations.     

猕猴桃倍性混合居群基因组遗传和表观遗传变异

植物倍性混合居群的形成和维系常伴随着明显的基因组遗传及表观遗传变异。利用AFLP和MSAP两种分子标记探讨了中华猕猴桃复合体(Actinidia chinensis)倍性混合居群的遗传变异和结构及其基因组甲基化变异方式。结果表明, 该倍性混合居群具有较高的遗传和表观遗传多样性, 但两者之间没有明显的相关性。种群的遗传多样性与海拔呈显著的负相关(P<0.05), 但表观遗传多样性与海拔不具显著相关性。AMOVA分析显示, 主要的遗传和表观遗传分化出现在倍性小种内部(97.65% vs 99.84%, P<0.05); 同时, AFLP邻接聚类分析显示二者存在一定程度的倍性相关性, MSAP分析则未显示有明显的倍性相关性。进一步研究发现, 中华猕猴桃居群的总甲基化程度为24.86%, 且多倍体具有更多的甲基化位点变异。该研究结果为深入探讨猕猴桃倍性混合居群的形成和维系机制奠定了基础。     

DREAMing: a simple and ultrasensitive method for assessing intratumor epigenetic heterogeneity directly from liquid biopsies

Many cancers comprise heterogeneous populations of cells at primary and metastatic sites throughout the body. The presence or emergence of distinct subclones with drug-resistant genetic and epigenetic phenotypes within these populations can greatly complicate therapeutic intervention. Liquid biopsies of peripheral blood from cancer patients have been suggested as an ideal means of sampling intratumor genetic and epigenetic heterogeneity for diagnostics, monitoring and therapeutic guidance. However, current molecular diagnostic and sequencing methods are not well suited to the routine assessment of epigenetic heterogeneity in difficult samples such as liquid biopsies that contain intrinsically low fractional concentrations of circulating tumor DNA (ctDNA) and rare epigenetic subclonal populations. Here we report an alternative approach, deemed DREAMing (Discrimination of Rare EpiAlleles by Melt), which uses semi-limiting dilution and precise melt curve analysis to distinguish and enumerate individual copies of epiallelic species at single-CpG-site resolution in fractions as low as 0.005%, providing facile and inexpensive ultrasensitive assessment of locus-specific epigenetic heterogeneity directly from liquid biopsies. The technique is demonstrated here for the evaluation of epigenetic heterogeneity at p14^ARF and BRCA1 gene-promoter loci in liquid biopsies obtained from patients in association with non-small cell lung cancer (NSCLC) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), respectively.     

A twin approach to unraveling epigenetics

The regulation of gene expression plays a pivotal role in complex phenotypes, and epigenetic mechanisms such as DNA methylation are essential to this process. The availability of next-generation sequencing technologies allows us to study epigenetic variation at an unprecedented level of resolution. Even so, our understanding of the underlying sources of epigenetic variability remains limited. Twin studies have played an essential role in estimating phenotypic heritability, and these now offer an opportunity to study epigenetic variation as a dynamic quantitative trait. High monozygotic twin discordance rates for common diseases suggest that unexplained environmental or epigenetic factors could be involved. Recent genome-wide epigenetic studies in disease-discordant monozygotic twins emphasize the power of this design to successfully identify epigenetic changes associated with complex traits. We describe how large-scale epigenetic studies of twins can improve our understanding of how genetic, environmental and stochastic factors impact upon epigenetics, and how such studies can provide a comprehensive understanding of how epigenetic variation affects complex traits.     

Microbial cell individuality and the underlying sources of heterogeneity

Single cells in genetically homogeneous microbial cultures exhibit marked phenotypic individuality, a biological phenomenon that is considered to bolster the fitness of populations. Major phenotypes that are characterized by heterogeneity span the breadth of microbiology, in fields ranging from pathogenicity to ecology. The cell cycle, cell ageing and epigenetic regulation are proven drivers of heterogeneity in several of the best-known phenotypic examples. However, the full contribution of factors such as stochastic gene expression is yet to be realized.