Polymorphisms,genomic imprinting and cancer susceptibility

Polymorphisms have been identified in proto-oncogenes and tumor suppressor genes that predispose people to cancer. Recent evidence indicates that genomic imprinting, an epigenetic form of gene regulation that results in uniparental gene expression, can also function as a cancer predisposing event. Thus, cancer susceptibility is increased by both Mendelian inherited genetic and non-Mendelian inherited epigenetic events. Consequently, chemical and physical agents cannot only induce cancer through the formation of genetic mutations but also through epigenetic changes that result in the inappropriate expression of imprinted proto-oncogenes and tumor suppressor genes. The role of genomic imprinting in carcinogenesis and cancer susceptibility is examined in this review.     

Mutations and epimutations in the origin of cancer

Cancer is traditionally viewed as a disease of abnormal cell proliferation controlled by a series of mutations. Mutations typically affect oncogenes or tumor suppressor genes thereby conferring growth advantage. Genomic instability facilitates mutation accumulation. Recent findings demonstrate that activation of oncogenes and inactivation of tumor suppressor genes, as well as genomic instability, can be achieved by epigenetic mechanisms as well. Unlike genetic mutations, epimutations do not change the base sequence of DNA and are potentially reversible. Similar to genetic mutations, epimutations are associated with specific patterns of gene expression that are heritable through cell divisions. Knudson's hypothesis postulates that inactivation of tumor suppressor genes requires two hits, with the first hit occurring either in somatic cells (sporadic cancer) or in the germline (hereditary cancer) and the second one always being somatic. Studies on hereditary and sporadic forms of colorectal carcinoma have made it evident that, apart from genetic mutations, epimutations may serve as either hit or both. Furthermore, recent next-generation sequencing studies show that epigenetic genes, such as those encoding histone modifying enzymes and subunits for chromatin remodeling systems, are themselves frequent targets of somatic mutations in cancer and can act like tumor suppressor genes or oncogenes. This review discusses genetic vs. epigenetic origin of cancer, including cancer susceptibility, in light of recent discoveries. Situations in which mutations and epimutations occur to serve analogous purposes are highlighted.     

Co-regulation of histone-modifying enzymes in cancer

Cancer is characterized by aberrant patterns of expression of multiple genes. These major shifts in gene expression are believed to be due to not only genetic but also epigenetic changes. The epigenetic changes are communicated through chemical modifications, including histone modifications. However, it is unclear whether the binding of histone-modifying proteins to genomic regions and the placing of histone modifications efficiently discriminates corresponding genes from the rest of the genes in the human genome. We performed gene expression analysis of histone demethylases (HDMs) and histone methyltransferases (HMTs), their target genes and genes with relevant histone modifications in normal and tumor tissues. Surprisingly, this analysis revealed the existence of correlations in the expression levels of different HDMs and HMTs. The observed HDM/HMT gene expression signature was specific to particular normal and cancer cell types and highly correlated with target gene expression and the expression of genes with histone modifications. Notably, we observed that trimethylation at lysine 4 and lysine 27 separated preferentially expressed and underexpressed genes, which was strikingly different in cancer cells compared to normal cells. We conclude that changes in coordinated regulation of enzymes executing histone modifications may underlie global epigenetic changes occurring in cancer.     

Chromosome instability and tumor lethality suppression in carcinogenesis

The maintenance and survival of each organism depends on its genome integrity. Alterations of essential genes, or aberrant chromosome number and structure lead to cell death. Paradoxically, cancer cells, especially in solid tumors, contain somatic gene mutations and are chromosome instability (CIN), suggesting a mechanism that cancer cells have acquired to suppress the lethal mutations and/or CIN. Herein we will discuss a tumor lethality suppression concept based on the studies of yeast genetic interactions and transgenic mice. During the early stages of the multistep process of tumorigenesis, incipient cancer cells probably have adopted genetic and epigenetic alterations to tolerate the lethal mutations of other genes that ensue, and to a larger extent CIN. In turn, CIN mediated massive gain and loss of genes provides a wider buffer for further genetic reshuffling, resulting in cancer cell heterogeneity, drug resistance and evasion of oncogene addiction, thus CIN may be both the effector and inducer of tumorigenesis. Accordingly, interfering with tumor lethality suppression could lead to cancer cell death or growth defects. Further validation of the tumor lethality suppression concept would help to elucidate the role of CIN in tumorigenesis, the relationship between CIN and somatic gene mutations, and would impact the design of anticancer drug development.     

Allele-specific gene expression and epigenetic modifications and their application to understanding inheritance and cancer

Epigenetic information is characterized by its plasticity during development and differentiation as well as its stable transmission during mitotic cell divisions in somatic tissues. This duality contrasts to genetic information, which is essentially static and identical in every cell in an organism with only a few exceptions such as immunoglobulin genes in lymphocytes. Epigenetics is traditionally perceived as a means to regulate gene expression without a change in DNA sequence. This, however, does not exclude a potential role for genetic variations in providing differential backgrounds on which epigenetic modulations and their regulatory consequences are achieved. An effective approach to investigating the interplay between genetic variations and epigenetic variations is through allele-specific analysis of epigenetics and gene expression. Such studies have generated many new insights into functions of genetic variations, mechanisms of gene expression regulation, and the role of mutations and epigenetic alterations in human cancer. This article is part of a Special Issue entitled: Chromatin in time and space.     

The Role of Transposons in Epigenetic Regulation of Ontogenesis

A new insight into the mechanisms underlying implementation of genomic information in the individual development of eukaryotes through interactions of transposons with epigenetic factors dynamically changing during each cell division is described. These mechanisms of stepwise implementation of individual genetic information with characteristic stage- and tissue-specific features in the activities of certain mobile genetic element families are evolutionarily fixed at the species level. In addition, the individual differences caused by their “unscheduled” transpositions can significantly change the regulatory network of the genome altering the phenotype. These changes in individual development can bring about new traits leading to either a disease or better fitness and represent an important component of the variation for natural selection in evolution. A large part of the eukaryotic transposons is altered by mutations and used for formation of the regulatory gene network, changes in the protein-coding genes, and emergence of new nonprotein-coding genes. When inserted into new loci, mobile genetic elements form the basis for microRNA and the domain structures of long noncoding RNA, responding to various types of stress; this is reflected in the specific features of individual development and contributes to variation. The epigenetic factors, including noncoding RNA, DNA methylation, and histone modifications, are tightly associated with mobile genetic elements. The specific features in transposon location in individuals that have emerged owing to spontaneous mutations or those caused by stress impacts can considerably change the interactions in gene networks. This influences the likelihood of survival under changing environmental conditions and reflects a distinct interrelation between the mechanisms of individual development and evolution. There is a parallelism between the mechanisms underlying the rearrangements of genomes caused by transposons in evolution and in individual development. In particular, the responsiveness of transposons to external and internal (microenvironment) factors forms the background for evolutionary construction of transposon-mediated tissue-specific activation patterns of certain transposons during each cell division, which leads to maturation of a reproductive organism. This mechanism is based on tight stage- and tissuespecific interrelation between transposons, epigenetic factors, and protein-coding genes.     

Cancer genomics identifies disrupted epigenetic genes

Latest advances in genome technologies have greatly advanced the discovery of epigenetic genes altered in cancer. The initial single candidate gene approaches have been coupled with newly developed epigenomic platforms to hasten the convergence of scientific discoveries and translational applications. Here, we present an overview of the evolution of cancer epigenomics and an updated catalog of disruptions in epigenetic pathways, whose misregulation can culminate in cancer. The creation of these basic mutational catalogs in cell lines and primary tumors will provide us with enough knowledge to move diagnostics and therapy from the laboratory bench to the bedside.     

Epigenetic drivers and genetic passengers on the road to cancer

Cancer is traditionally viewed as a primarily genetic disorder, however it is now becoming accepted that cancer is also a consequence of abnormal epigenetic events. Genetic changes and aneuploidy are associated with alterations in DNA sequence, and they are a hallmark of the malignant process. Epigenetic alterations are universally present in human cancer and result in heritable changes in gene expression and chromatin structure over many cell generations without changes in DNA sequence, leading to functional consequences equivalent to those induced by genetic alterations. Importantly, intriguing evidence emerged suggesting that epigenetic changes may precede and provoke genetic changes. In this scenario, epigenetic events are primary events while genetic changes (such as mutations) may simply be a consequence of disrupted epigenetic states. This fact may explain why many genetic screens proved to be limited with regard to cancer causality and pathogenesis. Aberrant epigenetic events affect multiple genes and cellular pathways in a non-random fashion and this can predispose to induction and accumulation of genetic changes in the course of tumour initiation and progression. These considerations are critical for a better understanding of tumourigenesis and molecular events underlying the acquisition of drug resistance, as well as development of novel strategies for cancer therapy and prevention.     

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.     

Mammalian cells acquire epigenetic hallmarks of human cancer during immortalization

Progression to malignancy requires that cells overcome senescence and switch to an immortal phenotype. Thus, exploring the genetic and epigenetic changes that occur during senescence/immortalization may help elucidate crucial events that lead to cell transformation. In the present study, we have globally profiled DNA methylation in relation to gene expression in primary, senescent and immortalized mouse embryonic fibroblasts. Using a high-resolution genome-wide mapping technique, followed by extensive locus-specific validation assays, we have identified 24 CpG islands that display significantly higher levels of CpG methylation in immortalized cell lines as compared to primary murine fibroblasts. Several of these hypermethylated CpG islands are associated with genes involved in the MEK–ERK pathway, one of the most frequently disrupted pathways in cancer. Approximately half of the hypermethylated targets are developmental regulators, and bind to the repressive Polycomb group (PcG) proteins, often in the context of bivalent chromatin in mouse embryonic stem cells. Because PcG-associated aberrant DNA methylation is a hallmark of several human malignancies, our methylation data suggest that epigenetic reprogramming of pluripotency genes may initiate cell immortalization. Consistent with methylome alterations, global gene expression analysis reveals that the vast majority of genes dysregulated during cell immortalization belongs to gene families that converge into the MEK–ERK pathway. Additionally, several dysregulated members of the MAP kinase network show concomitant hypermethylation of CpG islands. Unlocking alternative epigenetic routes for cell immortalization will be paramount for understanding crucial events leading to cell transformation. Unlike genetic alterations, epigenetic changes are reversible events, and as such, can be amenable to pharmacological interventions, which makes them appealing targets for cancer therapy when genetic approaches prove inadequate.     

A unified theory for the development of cancer

It is postulated that cancer is the result of genetic and epigenetic changes that occur mainly in stem (precursor) cells of various cell types. I propose that there are three classes of genes which are involved in the development of cancer. These are: Class I, II and III oncogenes. The classification is based on the way the oncogene acts at the cellular level to further the development of cancer. Genetic changes, that is point mutations, deletions, inversions, amplifications and chromosome translocations, gains or losses in the genes themselves or epigenetic changes in the genes (e.g. DNA hypomethylation) or in the gene products (RNA or protein) are responsible for the development of cancer. Changes of oncogene activity have a genetic or epigenetic origin or both and result in quantitative or qualitative differences in the oncogene products. These are involved in changing normal cells into the cells demonstrating a cancer phenotype (usually a form of dedifferentiated cell) in a multistep process. There are several pathways to cancer and the intermediate steps are not necessarily defined in an orderly fashion. Activation of a particular Class I or II oncogene and inactivation of a Class III oncogene could occur at any step during the development of cancer. Most benign or malignant tumors consist of a heterogeneous mixture of dedifferentiated cells arising from a single cell.     

Genetic and epigenetic alterations in pancreatic carcinogenesis

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Despite significant progresses in the last decades, the origin of this cancer remains unclear and no efficient therapy exists. PDAC does not arise de novo: three remarkable different types of pancreatic lesions can evolve towards pancreatic cancer. These precursor lesions include: Pancreatic intraepithelial neoplasia (PanIN) that are microscopic lesions of the pancreas, Intraductal Papillary Mucinous Neoplasms (IPMN) and Mucinous Cystic Neoplasms (MCN) that are both macroscopic lesions. However, the cellular origin of these lesions is still a matter of debate. Classically, neoplasm initiation or progression is driven by several genetic and epigenetic alterations. The aim of this review is to assemble the current information on genetic mutations and epigenetic disorders that affect genes during pancreatic carcinogenesis. We will further discuss the interest of the genetic and epigenetic alterations for the diagnosis and prognosis of PDAC. Large genetic alterations (chromosomal deletion/amplification) and single point mutations are well described for carcinogenesis inducers. Mutations classically occur within key regions of the genome. Consequences are various and include activation of mitogenic pathways or silencing of apoptotic processes. Alterations of K-RAS, P16 and DPC4 genes are frequently observed in PDAC samples and have been described to arise gradually during carcinogenesis. DNA methylation is an epigenetic process involved in imprinting and X chromosome inactivation. Alteration of DNA methylation patterns leads to deregulation of gene expression, in the absence of mutation. Both genetic and epigenetic events influence genes and non-coding RNA expression, with dramatic effects on proliferation, survival and invasion. Besides improvement in our fundamental understanding of PDAC development, highlighting the molecular alterations that occur in pancreatic carcinogenesis could provide new clinical tools for early diagnosis of PDAC and the molecular basis for the development of new effective therapies.     

Stem Cell Chromatin Patterns: An Instructive Mechanism for DNA Hypermethylation?

Epigenetic gene silencing, and associated promoter CpG island DNA hypermethylation, is an alternative mechanism to mutations by which tumor suppressor genes may be inactivated within a cancer cell 1-4,5-7. These epigenetic changes are prevalent in all types of cancer, and their appearance may precede genetic changes in pre-malignant cells and foster the accumulation of additional genetic and epigenetic hits8. These epigenetically modified genes constitute important categories of tumor suppressor genes including cell cycle regulators, pro-differentiation factors, and anti-apoptotic genes3, and many of these genes are known to play a role in normal development 9-11. While the silencing of these genes may play an essential role in tumor initiation or progression, the mechanisms underlying the specific targeting of these genes for DNA hypermethylation remains to be determined. The large numbers of epigenetically silenced genes that may be present in any given tumor, and the clustering of silenced genes within single cell pathways12, begs the question of whether gene silencing is a series of random events resulting in an enhanced survival of a pre-malignant clone, or whether silencing is the result of a directed, instructive program for silencing initiation reflective of the cells of origin for tumors. In this regard, the current review stresses the latter hypothesis and the important possibility that the program is linked, at least for silencing of some cancer genes, to the epigenetic control of stem/precursor cell gene expression patterns.     

Genomic sequencing of key genes in mouse pancreatic cancer cells

Pancreatic cancer is a multiple genetic disorder with many mutations identified during the progression. Two mouse pancreatic cancer cell lines were established which showed different phenotype in vivo: a non-metastatic cell line, Panc02, and a highly metastatic cell line, Panc02-H7, a derivative of Panc02. In order to investigate whether the genetic mutations of key genes in pancreatic cancer such as KRAS, TP53 (p53), CDKN2A (p16), SMAD4, ZIP4, and PDX-1 contribute to the phenotypic difference of these two mouse pancreatic cancer cells, we sequenced the exonic regions of these key genes in both cell lines and in the normal syngeneic mouse pancreas and compared them with the reference mouse genome sequence. The exons of KRAS, SMAD4, CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes were amplified and the genotype of these genes was determined by Sanger sequencing. The sequences were analyzed with Sequencher software. A mutation in SMAD4 was identified in both cell lines. This homozygote G to T mutation in the first position of codon 174 (GAA) generated a stop codon resulting in the translation of a truncated protein. Further functional analysis indicates that different TGF-β/SMAD signaling pathways were involved in those two mouse cell lines, which may explain the phonotypic difference between the two cells. A single nucleotide polymorphism (SNP) in KRAS gene (TAT to TAC at codon 32) was also identified in the normal pancreas DNA of the syngenic mouse and in both derived tumoral Panc02 and Panc02-H7 cells. No mutation or SNP was found in CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes in these two cell lines. The absence of mutations in genes such as KRAS, TP53, and CDKN2A, which are considered as key genes in the development of human pancreatic cancer suggests that SMAD4 might play a central and decisive role in mouse pancreatic cancer. These results also suggest that other mechanisms are involved in the substantial phenotypic difference between these two mouse pancreatic cancer cell lines. Further studies are warranted to elucidate the molecular pathways that lead to the aggressive metastatic potential of Panc02-H7.     

Cause and Consequences of Genetic and Epigenetic Alterations in Human Cancer

Both genetic and epigenetic changes contribute to development of human cancer. Oncogenomics has primarily focused on understanding the genetic basis of neoplasia, with less emphasis being placed on the role of epigenetics in tumourigenesis. Genomic alterations in cancer vary between the different types and stages, tissues and individuals. Moreover, genomic change ranges from single nucleotide mutations to gross chromosomal aneuploidy; which may or may not be associated with underlying genomic instability. Collectively, genomic alterations result in widespread deregulation of gene expression profiles and the disruption of signalling networks that control proliferation and cellular functions. In addition to changes in DNA and chromosomes, it has become evident that oncogenomic processes can be profoundly influenced by epigenetic mechanisms. DNA methylation is one of the key epigenetic factors involved in regulation of gene expression and genomic stability, and is biologically necessary for the maintenance of many cellular functions. While there has been considerable progress in understanding the impact of genetic and epigenetic mechanisms in tumourigenesis, there has been little consideration of the importance of the interplay between these two processes. In this review we summarize current understanding of the role of genetic and epigenetic alterations in human cancer. In addition we consider the associated interactions of genetic and epigenetic processes in tumour onset and progression. Furthermore, we provide a model of tumourigenesis that addresses the combined impact of both epigenetic and genetic alterations in cancer cells.     

Cancer genome sequencing: the challenges ahead

A major challenge for The Cancer Genome Atlas (TCGA) Project is solving the high level of genetic and epigenetic heterogeneity of cancer. For the majority of solid tumors, evolution patterns are stochastic and the end products are unpredictable, in contrast to the relatively predictable stepwise patterns classically described in many hematological cancers. Further, it is genome aberrations, rather than gene mutations, that are the dominant factor in generating abnormal levels of system heterogeneity in cancers. These features of cancer could significantly reduce the impact of the sequencing approach, as it is only when mutated genes are the main cause of cancer that directly sequencing them is justified. Many biological factors (genetic and epigenetic variations, metabolic processes) and environmental influences can increase the probability of cancer formation, depending on the given circumstances. The common link between these factors is the stochastic genome variations that provide the driving force behind the cancer evolutionary process within multiple levels of a biological system. This analysis suggests that cancer is a disease of probability and the most-challenging issue to the TCGA project, as well as the development of general strategies for fighting cancer, lie at the conceptual level.