Batoul Y. Abdallah

Chromosomal instability (CIN): what it is and why it is crucial to cancer evolution

Results of various cancer genome sequencing projects have “unexpectedly” challenged the framework of the current somatic gene mutation theory of cancer. The prevalence of diverse genetic heterogeneity observed in cancer questions the strategy of focusing on contributions of individual gene mutations. Much of the genetic heterogeneity in tumors is due to chromosomal instability (CIN), a predominant hallmark of cancer. Multiple molecular mechanisms have been attributed to CIN but unifying these often conflicting mechanisms into one general mechanism has been challenging. In this review, we discuss multiple aspects of CIN including its definitions, methods of measuring, and some common misconceptions. We then apply the genome-based evolutionary theory to propose a general mechanism for CIN to unify the diverse molecular causes. In this new evolutionary framework, CIN represents a system behavior of a stress response with adaptive advantages but also serves as a new potential cause of further destabilization of the genome. Following a brief review about the newly realized functions of chromosomes that defines system inheritance and creates new genomes, we discuss the ultimate importance of CIN in cancer evolution. Finally, a number of confusing issues regarding CIN are explained in light of the evolutionary function of CIN.

Evolutionary mechanisms and diversity in cancer.

The recently introduced genome theory of cancer evolution provides a new framework for evolutionary studies on cancer. In particular, the established relationship between the large number of individual molecular mechanisms and the general evolutionary mechanism of cancer calls upon a change in our strategies that have been based on the characterization of common cancer gene mutations and their defined pathways. To further explain the significance of the genome theory of cancer evolution, a brief review will be presented describing the various attempts to illustrate the evolutionary mechanism of cancer, followed by further analysis of some key components of somatic cell evolution, including the diversity of biological systems, the multiple levels of information systems and control systems, the two phases (the punctuated or discontinuous phase and gradual Darwinian stepwise phase) and dynamic patterns of somatic cell evolution where genome replacement is the driving force. By linking various individual molecular mechanisms to the level of genome population diversity and tumorigenicity, the general mechanism of cancer has been identified as the evolutionary mechanism of cancer, which can be summarized by the following three steps including stress-induced genome instability, population diversity or heterogeneity, and genome-mediated macroevolution. Interestingly, the evolutionary mechanism is equal to the collective aggregate of all individual molecular mechanisms. This relationship explains why most of the known molecular mechanisms can contribute to cancer yet there is no single dominant mechanism for the majority of clinical cases. Despite the fact that each molecular mechanism can serve as a system stress and initiate the evolutionary process, to achieve cancer, multiple cycles of genome-mediated macroevolution are required and are a stochastically determined event. Finally, the potential clinical implications of the evolutionary mechanism of cancer are briefly reviewed.

Decoding the genome beyond sequencing: The new phase of genomic research

While our understanding of gene-based biology has greatly improved, it is clear that the function of the genome and most diseases cannot be fully explained by genes and other regulatory elements. Genes and the genome represent distinct levels of genetic organization with their own coding systems; Genes code parts like protein and RNA, but the genome codes the structure of genetic networks, which are defined by the whole set of genes, chromosomes and their topological interactions within a cell. Accordingly, the genetic code of DNA offers limited understanding of genome functions. In this perspective, we introduce the genome theory which calls for the departure of gene-centric genomic research. To make this transition for the next phase of genomic research, it is essential to acknowledge the importance of new genome-based biological concepts and to establish new technology platforms to decode the genome beyond sequencing.

Diverse system stresses: common mechanisms of chromosome fragmentation

Chromosome fragmentation (C-Frag) is a newly identified MCD (mitotic cell death), distinct from apoptosis and MC (mitotic catastrophe). As different molecular mechanisms can induce C-Frag, we hypothesize that the general mechanism of its induction is a system response to cellular stress. A clear link between C-Frag and diverse system stresses generated from an array of molecular mechanisms is shown. Centrosome amplification, which is also linked to diverse mechanisms of stress, is shown to occur in association with C-Frag. This led to a new model showing that diverse stresses induce common, MCD. Specifically, different cellular stresses target the integral chromosomal machinery, leading to system instability and triggering of MCD by C-Frag. This model of stress-induced cell death is also applicable to other types of cell death. The current study solves the previously confusing relationship between the diverse molecular mechanisms of chromosome pulverization, suggesting that incomplete C-Frag could serve as the initial event responsible for forms of genome chaos including chromothripsis. In addition, multiple cell death types are shown to coexist with C-Frag and it is more dominant than apoptosis at lower drug concentrations. Together, this study suggests that cell death is a diverse group of highly heterogeneous events that are linked to stress-induced system instability and evolutionary potential.

Comparison of mitotic cell death by chromosome fragmentation to premature chromosome condensation

Mitotic cell death is an important form of cell death, particularly in cancer. Chromosome fragmentation is a major form of mitotic cell death which is identifiable during common cytogenetic analysis by its unique phenotype of progressively degraded chromosomes. This morphology however, can appear similar to the morphology of premature chromosome condensation (PCC) and thus, PCC has been at times confused with chromosome fragmentation. In this analysis the phenomena of chromosome fragmentation and PCC are reviewed and their similarities and differences are discussed in order to facilitate differentiation of the similar morphologies. Furthermore, chromosome pulverization, which has been used almost synonymously with PCC, is re-examined. Interestingly, many past reports of chromosome pulverization are identified here as chromosome fragmentation and not PCC. These reports describe broad ranging mechanisms of pulverization induction and agree with recent evidence showing chromosome fragmentation is a cellular response to stress. Finally, biological aspects of chromosome fragmentation are discussed, including its application as one form of non-clonal chromosome aberration (NCCA), the driving force of cancer evolution.

Why imatinib remains an exception of cancer research.

The archetype driving the drug targeting approach to cancer therapy is the success of imatinib against chronic phase chronic myeloid leukemia (CML‐CP). Molecular targeting success of this magnitude has yet to be repeated for most solid tumors. To answer why imatinib remains an exception of cancer research, we summarize key features and patterns of evolution that contrast CML‐CP from prostate cancer, an example of a solid tumor that also shares a signature fusion gene. Distinctive properties of CML‐CP include: a large cell population size that is not geographically constrained, a highly penetrant dominant oncogene that sweeps the entire cell population, subsequent progressive and ordered clonal genetic changes, and the effectiveness of molecular targeting within the chronic phase, which is comparable to the benign phase of solid tumors. CML‐CP progression resembles a clonal, stepwise model of evolution, whereas the pattern of solid tumor evolution is highly dynamic and stochastic. The distinguishing features and evolutionary pattern of CML‐CP support why the success of imatinib does not carry over to most solid tumors. Changing the focus of cancer research from a gene‐based view to a genome‐based theory will provide insight into solid tumor evolutionary dynamics. J. Cell. Physiol. 228: 665–670, 2013.

Karyotype Heterogeneity and Unclassified Chromosomal Abnormalities

In a departure from traditional gene-centric thinking with regard to cytogenetics and cytogenomics, the recently introduced genome theory calls upon a re-focusing of our attention on karyotype analyses of disease conditions. Karyotype heterogeneity has been demonstrated to be directly involved in the somatic cell evolution process which is the basis of many common and complex diseases such as cancer. To correctly use karyotype heterogeneity and apply it to monitor system instability, we need to include many seemingly unimportant non-specific chromosomal aberrations into our analysis. Traditionally, cytogenetic analysis has been focused on identifying recurrent types of abnormalities, particularly those that have been linked to specific diseases. In this perspective, drawing on the new framework of 4D-genomics, we will briefly review the importance of studying karyotype heterogeneity. We have also listed a number of overlooked chromosomal aberrations including defective mitotic figures, chromosome fragmentation as well as genome chaos. Finally, we call for the systematic discovery/characterization and classification of karyotype abnormalities in human diseases, as karyotype heterogeneity is the common factor that is essential for somatic cell evolution.

Chromosomal instability and transcriptome dynamics in cancer

Whole transcriptome profiling has long been proposed as a method of identifying cancer-specific gene expression profiles. Indeed, a multitude of these studies have generated vast amounts of expression data for many types of cancer, and most have identified specific gene signatures associated with a given cancer. These studies however, often contradict with each other, and gene lists only rarely overlap, challenging clinical application of cancer gene signatures. To understand this issue, the biological basis of transcriptome dynamics needs to be addressed. Chromosome instability (CIN) is the main contributor to genome heterogeneity and system dynamics, therefore the relationship between CIN, genome heterogeneity, and transcriptome dynamics has important implications for cancer research. In this review, we discuss CIN and its effects on the transcriptome during cancer progression, specifically how stochastic chromosome change results in transcriptome dynamics. This discussion is further applied to metastasis and drug resistance both of which have been linked to multiple diverse molecular mechanisms but are in fact driven by CIN. The diverse molecular mechanisms that drive each process are linked to karyotypic heterogeneity through the evolutionary mechanism of cancer. Karyotypic change and the resultant transcriptome change alter network function within cells increasing the evolutionary potential of the tumor. Future studies must embrace this instability-induced heterogeneity in order to devise new research and treatment modalities that focus on the evolutionary process of cancer rather than the individual genes that are uniquely changed in each tumor. Care is also needed in evaluating results from experimental systems which measure average values of a population.

Single cell heterogeneity: Why unstable genomes are incompatible with average profiles

Multi-level heterogeneity is a fundamental but underappreciated feature of cancer. Most technical and analytical methods either completely ignore heterogeneity or do not fully account for it, as heterogeneity has been considered noise that needs to be eliminated. We have used single-cell and population-based assays to describe an instability-mediated mechanism where genome heterogeneity drastically affects cell growth and cannot be accurately measured using conventional averages. First, we show that most unstable cancer cell populations exhibit high levels of karyotype heterogeneity, where it is difficult, if not impossible, to karyotypically clone cells. Second, by comparing stable and unstable cell populations, we show that instability-mediated karyotype heterogeneity leads to growth heterogeneity, where outliers dominantly contribute to population growth and exhibit shorter cell cycles. Predictability of population growth is more difficult for heterogeneous cell populations than for homogenous cell populations. Since “outliers” play an important role in cancer evolution, where genome instability is the key feature, averaging methods used to characterize cell populations are misleading. Variances quantify heterogeneity; means (averages) smooth heterogeneity, invariably hiding it. Cell populations of pathological conditions with high genome instability, like cancer, behave differently than karyotypically homogeneous cell populations. Single-cell analysis is thus needed when cells are not genomically identical. Despite increased attention given to single-cell variation mediated heterogeneity of cancer cells, continued use of average-based methods is not only inaccurate but deceptive, as the “average” cancer cell clearly does not exist. Genome-level heterogeneity also may explain population heterogeneity, drug resistance, and cancer evolution.

Unstable genomes elevate transcriptome dynamics

The challenge of identifying common expression signatures in cancer is well known, however the reason behind this is largely unclear. Traditionally variation in expression signatures has been attributed to technological problems, however recent evidence suggests that chromosome instability (CIN) and resultant karyotypic heterogeneity may be a large contributing factor. Using a well‐defined model of immortalization, we systematically compared the pattern of genome alteration and expression dynamics during somatic evolution. Co‐measurement of global gene expression and karyotypic alteration throughout the immortalization process reveals that karyotype changes influence gene expression as major structural and numerical karyotypic alterations result in large gene expression deviation. Replicate samples from stages with stable genomes are more similar to each other than are replicate samples with karyotypic heterogeneity. Karyotypic and gene expression change during immortalization is dynamic as each stage of progression has a unique expression pattern. This was further verified by comparing global expression in two replicates grown in one flask with known karyotypes. Replicates with higher karyotypic instability were found to be less similar than replicates with stable karyotypes. This data illustrates the karyotype, transcriptome, and transcriptome determined pathways are in constant flux during somatic cellular evolution (particularly during the macroevolutionary phase) and this flux is an inextricable feature of CIN and essential for cancer formation. The findings presented here underscore the importance of understanding the evolutionary process of cancer in order to design improved treatment modalities.