Introduction

A genetic basis for human cancer has been recognized for perhaps more than a century and has been supported by data from familial and epidemiological studies and animal studies. However, only during the past three decades has definitive molecular evidence been accumulated to support the view that all cancer types arise from defects in the structure and/or regulation of genes. Studies from many different fields, including tumor virology, chemical carcinogenesis, molecular biology and biochemistry, somatic cell genetics, developmental biology, and genetic epidemiology, have all played critical contributing roles in clarifying the contributions of genetic and epigenetic mechanisms to cancer development. Although environmental and dietary factors have substantial roles in cancer development, it is now well established that the accumulation of multiple genetic and epigenetic alterations in a single cell plays a fundamental role in cancer initiation and progression.

The mutations that arise in cancer cells can be divided into two functionally distinct classes: oncogene and tumor suppressor gene mutations. In addition to the classic alterations of oncogenes and tumor suppressor genes, other genetic alterations occur in genes regulating transcription and translation, as well as in genes responsible for DNA repair (maintaining DNA fidelity during cell division). Although localized mutations are commonly observed, other genetic variations are also important in oncogenesis, such as copy number variation, deletion, and translocations. All of these commonly described genomic alterations in the initiation and progression of cancer can be broadly considered as oncogenic—“gain of function”—or suppressor—“loss of function”—when describing any genetic or epigenetic alterations specific to the cancer phenotype. Some mutations may be present in an individual’s germline and may predispose to particular cancers. Such mutations can also be passed on to future generations. The nature and role of certain germline mutations in cancer development are of great interest to cancer biologists because the mutations provide powerful clues about the identity of genes and pathways that play particularly critical roles in the malignant conversion of cells. Nevertheless, germline mutations in oncogenes or tumor suppressor genes likely have a major contributing role in the development of only a small fraction of all cancers, and the vast majority of mutations in cancer are somatic (i.e., present only in the tumor cells).

A few of the cellular genes that are recurrently affected by inherited and somatic mutations in human cancer will be discussed in more detail later. Brief mention will be made here of some general properties of the mutated genes. Oncogenes act in a positive fashion to promote tumorigenesis. Their normally functioning cellular counterparts, termed proto-oncogenes, have been found to be important regulators of many aspects of cell physiology. The proteins encoded by various proto-oncogenes can be found in virtually all subcellular compartments. The term proto-oncogene does not imply that genes of this class lie dormant in the cell with the purpose of promoting tumorigenesis. Rather, the terminology reflects the fact that mutations in cancer cells alter the normal structure and/or expression pattern of the proto-oncogene, generating oncogenic variants with altered function. In genetic terms, oncogenic alleles have gain-of-function mutations that confer enhanced or novel functions.

In contrast to the activating mutations in oncogenes, tumor suppressor genes harbor loss-of-function defects in cancer cells. Historically, the term antioncogene was sometimes used with respect to the tumor suppressor gene class. The term suggests that the primary function of the genes might be to act in direct opposition to activated oncogenes. Although many of the proteins that are encoded by tumor suppressor genes do in fact bind to and regulate the function of proto-oncogenes or function in pathways that regulate proto-oncogene activity, it is not by necessity a general principle. Hence, genes that contribute to cancer by virtue of their inactivation or loss-of-function mutations in human cancers will be referred to here as tumor suppressor genes. Similarly to the proto-oncogenes, the normal functions of tumor suppressor genes are diverse, and the proteins encoded by these genes are found in essentially all compartments of the cell.

Mutations in genes that regulate the recognition and repair of DNA damage also play critical roles in tumorigenesis. The DNA damage recognition and repair genes could be considered to constitute a distinct class of cancer genes, because at least some of the DNA repair proteins might have a more passive role in cell proliferation, differentiation, and cell survival. Their inactivation in tumor cells might lead predominantly to the acquisition of a “mutator phenotype,” with a resultant increased rate of mutations in other cellular genes with rate-determining roles in the cancer process—that is, oncogenes and tumor suppressor genes. Nevertheless, it is perhaps most straightforward to simply evaluate whether an alteration in any given gene represents a gain-of-function or loss-of-function defect, and DNA damage recognition and repair pathway genes will be considered here based on the nature of the alterations affecting them in cancer.

In addition to the well-established role of oncogene and tumor suppressor gene mutations in cancer, it is now abundantly clear that epigenetic mechanisms play critical roles in altering the patterns and levels of expression of certain proto-oncogenes and tumor suppressor genes in cancer. For instance, in some cancers, altered transcriptional regulatory mechanisms can lead to markedly increased levels of proto-oncogene expression, akin to the level seen in cancer cells with mutational defects that alter the structure or copy number of the proto-oncogene. Conversely, gene-silencing mechanisms can exert dramatic effects on the expression of certain tumor suppressor genes in cancer cells, essentially rendering the genes functionally inactive in the absence of any mutations. It is of great interest that sequence-based analyses of many different cancer types have revealed that many of the mutations in cancer cells directly affect the cancer epigenome. Specifically, the oncogenes and tumor suppressor gene mutations in cancer cells often target transcription factors, chromatin modifying proteins, and other chromatin-associated proteins, leading to potentially quite dramatic global effects on the structure and activity of chromatin, DNA methylation, and patterns of gene expression in cancer cells.

Given the enormous advances during the past two decades in defining oncogene and tumor suppressor gene mutations and gene expression defects in cancer, it will not be possible in this chapter to review in a comprehensive fashion the vast collection of genetic and epigenetic defects that have been catalogued in human cancers. Nor will it be possible to discuss in detail the possible contributions of the many different gene defects to alterations in cell signaling and cell physiology. Rather, the primary aim of this chapter will be to offer a framework for understanding the relationships between genetic and epigenetic defects in cancer cells and the impact of the accumulated defects on the cancer cell phenotype. Although some details on the identity and nature of gene defects in cancer will be offered here, the emphasis will be on concepts with biological and clinical significance.

Cancers Arise From the Accumulation of Multiple Gene Defects

On the basis of a simple consideration of the likely large number of genetic and epigenetic alterations that arise in normal cells during the many years of life, it would seem quite unlikely that cancers arise as the result of any single gene defect. Even in persons who are strongly predisposed to cancer as a result of a germline mutation in a specific oncogene or tumor suppressor gene, the vast majority of cells in a person never develop into cancer or even display definitive morphologic changes akin to those that are seen in benign tumors. In fact, depending on the inherited cancer syndrome, cancer never develops in a significant fraction of persons who carry specific germline mutations in oncogenes or tumor suppressor genes. Therefore any model for cancer must incorporate these data, which suggest that cancers likely arise as the result of the accumulation of multiple gene defects in an affected cell. Another issue to consider is that clinical and histopathological data indicate that the development of nearly all cancers, regardless of the organ site, is often, if not invariably, preceded by precancerous phases or stages in which the neoplastic cells manifest increasing disordered patterns of differentiation and morphology.

Given this background, it would appear that compelling evidence exists that cancers arise from accumulated defects affecting multiple genes and pathways, and precancerous (benign) precursor lesions harbor fewer of the key gene defects. One question, therefore, is how many rate-limiting defects or “hits” are required for cancer development. Although a definitive answer to this question cannot be given at this point, some estimates can be offered. Most common cancers show dramatically increased incidence with increasing age. On the basis of analysis of the age-specific incidence of a number of common cancers and some straightforward assumptions about the rate of mutations and the size of the target cell population, it was argued as early as the mid 1950s that most common epithelial cancers arise as the result of four to seven rate-limiting events.1,2 It was inferred that these rate-limiting events might represent mutational events. Moreover, benign lesions were inferred to arise as the result of fewer gene defects, consistent with the fact that recognizable benign lesions that are often found show an age-incidence distribution that was shifted roughly one to two decades earlier in life than cancers arising in the corresponding organ or tissue sites. Nevertheless, confounding the use of age-incidence data to model the number of rate-limiting mutations were questions about certain key biological assumptions underlying the multihit models. Given the attendant uncertainties with estimates of rate-limiting mutation numbers derived largely or solely from age-incidence data and the practical difficulties in defining the nature and significance of all inherited and somatic gene defects in cancer, a definitive answer to the number of rate-determining defects in a particular cancer type has not yet been obtained. For instance, even one of the early comprehensive molecular analyses of breast and colon cancer indicated that dozens to hundreds of somatic mutations were often present in a given cancer,³ without even considering the even far greater number of epigenetic and gene expression changes present in the cancer cells relative to adjacent normal cells.

Study of the genetic alterations of pediatric cancers that occur much earlier and are perhaps less complex in terms of their genetic signature may offer insights into key cancer target genes (see Chapter 95 on pediatric solid tumors).

Clonal Selection and Evolution in Cancer

As previously noted, most if not all cancers arise from preexisting precancerous populations of cells, and multiple genetic and epigenetic defects are likely needed for conversion of a normal cell to a cancerous cell. Molecular studies of cancers of various types and their corresponding associated precancerous lesions have yielded some fundamental insights into the processes likely to be critical in the emergence of the cancer. First, although normal tissues and tissues from noncancerous disease states display polyclonal (balanced) cell populations, the neoplastic component that is present in benign lesions and cancers displays a clonally related cell population, consistent with the notion that neoplastic transformation of one or at most a few cells within a tissue give rise to all daughter cells that are present in the tumor. Second, in tumors in which it has been possible to analyze both cancer cells and associated precancerous cell populations, a subset of the somatic gene defects present in the cancer are clonally represented in the precancerous cell population. Other somatic gene defects appear to be acquired during progression from the precancer subclones to the dominant subclone in the cancer.

These molecular findings in benign and malignant tumors are essentially consistent with a model initially proposed by Foulds⁴ and subsequently advanced by Nowell⁵ (Fig. 13-1). In brief, the clonal evolution model predicts that cancers arise as the result of successive expansions of clonally related cell populations. The successive expansions are driven by the gradual or punctuated acquisition of mutations and gene expression changes that endow a particular cell and its progeny with a selective advantage over cells that do not harbor the gene defects. In essence, clonal selection is an evolutionary process that allows the outgrowth of precancerous and cancerous cells that carry mutations and gene expression changes that confer the most potent proliferative and survival properties on the cancer cells. It is important to note that the specific constellation of genetic and epigenetic changes that are present in precancerous and cancerous cells is context-dependent and certainly varies considerably from one cancer type to another and even most certainly varies to a significant degree among patients whose cancers display quite similar clinical and histopathological features. The basis for the context-dependent relationship of the changes that confer a selective growth advantage in a particular cancer may reflect physiological differences in organ site and the tissue microenvironment within the organ site, the identities of the preceding somatic gene defects in the precancerous or cancerous clone, and even the constitutional sequence variations and particular gene expression patterns that are present in nonneoplastic tissues of a given patient. This issue of context-dependent effects of gene defects that promote clonal selection in neoplastic cells will be addressed further in the following sections.

The clonal evolution model has biological and clinical ramifications, just a few of which will be mentioned here. First, the clonal gene defects that are present in a cancer can be traced in precancerous lesions from the same organ site with a goal of attempting to clarify the preferred order in which gene defects arise in the natural history of a particular cancer type. The particular order in which defects accumulate during the initiation and subsequent progression of one cancer type often differs from that in another cancer type. As a result, a genetic or epigenetic change that is critical in tumor initiation in one cancer type might contribute to tumor progression in another tumor type and vice versa. Second, defects that arise at “early” stages of tumorigenesis might play a vital role not only in tumor initiation but also in the aggressive behavior of advanced stage cancers. Third, the model predicts that the acquisition of further genetic heterogeneity will be a common and important factor in primary cancer lesions and metastases. Genetic heterogeneity likely plays a significant role in resistance to chemotherapy and the emergence of aggressive cell populations in patients with advanced cancer.

Recent comprehensive sequence-based analyses of cancer cell populations from individual patients in which the tumor cells under study were distinct from one another in location and/or time has begun to reveal that quite dramatic intratumoral genetic heterogeneity may be a “rule” in cancer, rather than an exception.⁶ Certain, perhaps key, initiating genetic lesions might be shared among all neoplastic clones, but geographically distinct regions of large primary tumors may have very distinct mutation profiles from those in other portions of the primary tumor, and the metastatic cell populations may have considerable mutational divergence from the nonmetastatic cells. As such, it seems that quite extensively branched evolutionary growth may be an important feature in both primary tumors and metastatic lesions, with multiple competing clonal populations evolving through divergent and convergent mutational mechanisms.⁶ This more recent view of the potentially quite extensive intratumoral genetic heterogeneity in any given cancer and the contributions of intratumoral heterogeneity to tumor progression contrasts with some earlier views. Prior to the recent studies, it was suspected that the cell populations in many primary cancers might be more homogeneous, where somatic mutations were accumulated in a more stepwise fashion as a result of multiple sequential clonal sweeps of each variant cell populations in the primary cancer, with metastases perhaps most often arising from the clonally dominant cell population in the primary tumor.

It is important to note that clonal somatic mutations are often presumed to have a causal role in promoting further tumor outgrowth or progression because somatic mutations can become clonal (i.e., present in all neoplastic cells) by only a limited number of mechanisms. For instance, the genetic alteration itself could have been selected for because it provided the neoplastic cell with a growth advantage, allowing it to become the predominant cell type in the tumor (clonal expansion). Genes with critical roles in promoting clonal outgrowth in a given cancer have been termed drivers.⁷ Alternatively, a somatic mutation, when detected, might have arisen essentially coincident with another, perhaps undetected, alteration that was the crucial change underlying clonal outgrowth. Somatic mutations of this latter type have been termed passengers.⁷ Genes that are mutant in a significant fraction of cancers and for which other lines of evidence link them to the cancer process can more readily be classified as drivers. However, on the basis of early data from some large-scale sequencing analyses that reveal large numbers of distinct genes that are each mutated in only a minority of cancers of a given type,^3,8–10 sorting out drivers and passengers might not be entirely straightforward, based solely on sequencing data, but will likely require a significant body of functional studies and data. Statistical arguments may fall short, at least before large volumes of sequencing are obtained for the alterations that are private or rare, yet still biologically important when present in a given patient’s cancer.

Given this discussion about the potential uncertainties associated with linking specific somatic mutations to cancer development, it is perhaps apparent why it is even more challenging to assign causal significance to any given gene expression change in precancerous and cancerous cells. In particular, the ambiguities in assigning a causal role to gene expression and epigenetic changes in the absence of mutational or functional data that might inform the situation are in large part due to the difficulties in determining whether a gene expression or epigenetic change merely reflects or is causally involved in the cancer process. Nonetheless, if a specific gene or epigenetic alteration can be shown to promote tumorigenesis or neoplastic transformation in a variety of in vitro tumor models and animal model experiments or if the same gene or chromosomal region is recurrently altered by particular epigenetic changes in tumors, then it might be reasonable to infer that the particular defect might indeed have a causal (driver) role in tumorigenesis.

Contribution of Gene Defects to the Signature Traits of Cancer Cells

Cancer represents a highly heterogeneous collection of diseases. Each cancer type has distinct biological and clinical features and a variable prognosis. Even cancers that arise at a single organ site, such as the ovary, kidney, or lung, represent a hodgepodge of different diseases at the molecular level. Morphologic features often allow the particular cancer types to be distinguished to some degree from one another. Yet even for patients whose cancers have essentially identical gross and microscopic appearances and very similar clinical manifestations, there may be vast differences in outcome. In spite of this complexity, the development of all cancers, regardless of type, is likely to be critically dependent on the acquisition of certain phenotypic features that allow the cancer cells not only to grow in an unchecked fashion in their tissue of origin but also to gain the ability to disseminate into surrounding tissues and organs, lymphatics, and the bloodstream and ultimately to grow as metastatic lesions in distant sites in the body.¹¹ As is indicated in Figure 13-2, among the signature traits that are likely to be expressed in the majority, if not all, of cancers are the following: (1) an increased tendency to manifest a stem cell or progenitor-like phenotype; (2) an enhanced response to growth-promoting signals; (3) a relative resistance to growth-inhibiting cues; (4) an increased mutation rate to allow for the rapid generation of new variant daughter cells; (5) the ability to attract and support a new blood supply (angiogenesis); (6) the capacity to minimize an immune response and/or evade destruction by immune effector cells; (7) the capacity for essentially limitless cell division; (8) a failure to respect tissue boundaries, allowing for invasion into adjacent tissues and organs as well as blood vessels and lymphatics; and (9) the ability to grow in organ sites with microenvironments markedly different from the one where the cancer cells arose. The development of some traits is likely to be associated with certain stages of tumorigenesis (see Fig. 13-2), but acquisition of signature traits in cancers is more likely to show a preferred order than an invariant order. Furthermore, many of the signature traits of cancer cells represent complex biological capabilities (e.g., angiogenic activity, immune evasion/resistance, and metastatic competence). Therefore it is likely that substantial changes in many signaling pathways are needed for the cancer cell to manifest these traits.

An exhaustive cataloging of the observations associating specific gene defects to the altered phenotypic traits of cancer cells will not be offered here, in part because of space limitations and in part because of uncertainties about the significance of many of the linkages between single gene defects and cancer phenotypic traits. Nonetheless, some general concepts regarding the relationships among gene defects and cancer cell phenotype have emerged, and two of these concepts are offered here. First, although some of the specific mutations and major gene expression defects that are seen in cancer cells may contribute predominantly to a few or perhaps even only one of the signature traits of cancer cells previously listed, it seems likely that many of the gene defects and expression changes have been selected for in large part because they exert pleiotropic effects on the cancer cell phenotype. Second, for certain tumor types, it has been possible to gain some insights into the apparent order in which genetic and epigenetic changes might arise and contribute to cancer pathogenesis. The data suggest that defects in certain genes and signaling pathways might be strongly selected for at a certain point in cancer development and progression, perhaps in large part because the alterations allow the precancerous or cancerous cells to acquire certain critical phenotypic features. Gene defects that might be nearly uniformly present in early-stage lesions of one tumor type might preferentially arise in later-stage tumors in another organ site. The data suggest that cellular and tissue context have critical, albeit poorly understood, modifying effects on the specific genetic defects that give rise to neoplastic transformation, clonal outgrowth, and tumor progression.

Two arguably more speculative points concerning genetic and epigenetic defects and the altered phenotypic traits manifest in cancer will be briefly discussed here. One issue is that the oncogene and tumor suppressor gene mutations present in cancer cells are likely to be far less “plastic” and reversible than some of the epigenetic alterations (e.g., DNA methylation and/or chromatin modification changes) that might achieve the same net effect on a particular gene or signaling pathway. Indeed, the reversibility of most epigenetic changes might allow cancer cells to alter signaling pathways, gene expression patterns, and cell phenotypes in a much more malleable fashion than mutational events. This characteristic perhaps allows the cancer cells to elaborate a particular collection of signature phenotypic traits in a specific tissue or physiological context and to extinguish, enhance, and/or elaborate other signature traits in a different tissue context (e.g., a distant metastatic site) or in response to therapeutic agents. A second issue is that, given that considerable genetic and epigenetic heterogeneity may exist within a primary cancer and among or even within individual grossly visible metastatic lesions, it remains uncertain whether each individual cancer cell in a large primary tumor mass need manifest many or all of the signature phenotypic traits or whether the cancer cells can essentially complement one another, such that the cancer population as a whole manifests all key traits, but few if any individual’s cells within a large tumor mass need manifest all traits. Furthermore, the evolving of different functions of individual precancerous cells may speak to the possibility of cooperation between different cell types in the codevelopment of the cancer and later in the process of tumor progression/metastasis.

As discussed previously, in genetic terms, oncogenic alleles have gain-of-function mutations. Oncogenic variant alleles that are present in cancer are generated from the normal counterpart proto-oncogenes by various mutational mechanisms, including point or localized mutations, gross chromosomal rearrangements, or gene amplification. Some representative oncogene mutations in human cancer are summarized in Table 13-1. From a brief review of the data in Table 13-1, some generalizations can be offered. First, the mutations affect proteins functioning in various compartments of the cell, including growth factor receptors, cytoplasmic signal transducers, and nuclear proteins, such as transcription factors. Second, although some oncogene mutations may be unique to cancers of a particular type, such as the specific chromosomal translocations and resultant fusion proteins that are seen in cancers of hematopoietic origin (e.g., the BCR-ABL translocation that is seen in chronic myelogenous leukemia and a subset of acute lymphoid leukemias and the PML-RARα translocation that is seen in acute promyelocytic leukemia), other mutations, such as those affecting the KRAS, β-catenin, and c-MYC genes, are found in a broad spectrum of different cancer types. Third, oncogene mutations in cancer are nearly always somatic, because only a very limited number of germline mutations in proto-oncogenes have been linked to cancer predisposition thus far. Fourth, some proto-oncogenes, such as KRAS or BCL2, are somatically altered in cancer by a single mutational mechanism, namely, point mutations in the KRAS gene and chromosomal translocations affecting the BCL2 gene. In contrast, other proto-oncogenes, such as c-MYC, may be activated by more than one mechanism in cancer, including chromosomal translocation and gene amplification. Both mutational mechanisms lead to increased levels of c-MYC transcripts and protein. In fact, specific missense mutations at threonine 58 of the c-MYC gene in some lymphomas may further enhance c-MYC protein levels by abrogating a phosphorylation-ubiquitination mechanism targeting c-MYC for proteosomal degradation.¹² Of course, enhanced c-MYC protein levels can result from alterations in c-MYC–specific microRNAs, methylation, and other noncoding regulatory elements functioning to regulate c-MYC transcription and translation.

Table 13-1

Selected Oncogene Mutations in Cancer

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Jun 13, 2016 | Posted by admin in ONCOLOGY | Comments Off on Genetic and Epigenetic Alterations in Cancer

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Gene	Activation Mechanism	Protein Properties	Tumor Types
KRAS	Point mutation	Signal transducer	Pancreatic, colorectal, lung (adeno), endometrial, other carcinomas
NRAS	Point mutation	Signal transducer	Myeloid leukemia, colorectal cancer
HRAS	Point mutation	Signal transducer	Bladder carcinoma
EGFR (ERBB)	Amplification, mutation	Growth factor (EGF) receptor	Gliomas, lung (non–small cell) carcinoma
NEU (HER2/ERBB2)	Amplification	Growth factor receptor	Breast, ovarian, gastric, other carcinomas
c-MYC	Chromosome translocation	Transcription factor	Burkitt lymphomas
	Amplification		Small cell lung carcinoma (SCLC); other carcinomas; glioblastoma
MYCN	Amplification	Transcription factor	Neuroblastoma, SCLC; glioblastoma
MYCL1	Amplification	Transcription factor	SCLC, ovarian carcinoma
BCL2	Chromosome translocation	Antiapoptosis protein	B-cell lymphoma (follicular type)
CCND1	Amplification	Cyclin D, cell cycle control	Breast and other carcinomas
	Chromosome translocation		B-cell lymphoma, parathyroid adenoma
BCR-ABL	Chromosome translocation	Chimeric nonreceptor tyrosine kinase	CML, ALL (T cell)
RET	Chromosome translocation	GDNF receptor tyrosine kinase	Thyroid cancer (papillary type)
	Point mutation		Thyroid cancer (medullary type: germline mutations)
CDK4	Amplification
	Point mutation	Cyclin-dependent kinase	Sarcoma, glioblastoma
MET	Point mutation	Hepatocyte growth factor (HGF) receptor	Renal carcinoma (papillary type: germline mutations)
SMO	Point mutations	Transmembrane signaling molecule in sonic hedgehog pathway	Basal cell skin cancer
CTNNB1 (β-CAT)	Point mutation, in-frame deletion	Transcriptional coactivator, links E-cadherin to cytoskeleton	Melanoma; colorectal, endometrial, ovarian, hepatocellular, and other carcinomas, hepatoblastoma, Wilms tumor
FGF4	Amplification	Growth factor (FGF-like)	Gastric carcinoma
PML-RARA	Chromosome translocation	Chimeric transcription factor	APL
TCF3-PBX1	Chromosome translocation	Chimeric transcription factor	Pre-B ALL
MDM2	Amplification	p53 binding protein	Sarcoma
GLI1	Amplification	Transcription factor	Sarcoma, glioma
TTG2