Carcinogenesis: Mechanisms and Evaluation

1.1

Prominent Theories of Carcinogenesis

Our understanding of cancer biology is evolving rapidly. Conceptual views of carcinogenesis are formed by the piece-by-piece discovery of key elements of the complex biological puzzle that this disease entails; however, the search for a comprehensive theory of carcinogenesis has not been forthcoming, and investigation into several theories on the origin and pathogenesis of neoplasia still dominates much of the field of cancer research. Three predominant theories on the origin of carcinogenesis include the Somatic Mutation Theory (SMT), the Cancer Stem Cell (CSC) Theory, and the Tissue Organizational Field Theory (TOFT) ( ; ; ).

The SMT, which has dominated the last 60 years of cancer research, is based on the premise that cancer originates when a normal somatic cell successively accumulates multiple mutations in genes controlling cell proliferation and the cell cycle ( ). The transformed cell then passes the mutation to its progeny, generating malignant clones. The longer a population of somatic cells persists, or the higher rate of cell turnover in a given tissue, the higher the likelihood of mutations occurring in that population over time. Then, as the mutated cells proliferate, there is a finite probability that some of them will sustain at least a second mutation. As the process of successive mutation and proliferation continues, cells eventually sustain enough genetic alterations to undergo neoplastic transformation. The accumulation of successive mutations would be expected to increase as a function of age and of the degree of cell proliferation, and finally clones acquire additional mutations until a subclone arises with the ability to invade and metastasize. The SMT has been refined over the years to include alterations in oncogenes, tumor suppressor genes, DNA repair genes, apoptosis genes, and epigenetic mechanisms, and has been the prevalent means to explain the process of neoplastic transformation and progression ( ).

The CSC hypothesis was first proposed over 150 years ago, which stated that dormant embryonic components exist in adult tissues that may be later induced to become tumorigenic ( ). According to the CSC theory of carcinogenesis, a distinct subpopulation of cancer stem cells has the capacity to generate a tumor and sustain its growth. Cancer stem cells perpetuate tumorigenesis through the process of self-renewal, in which a stem cell pool maintains its numbers through symmetric and asymmetric division. In symmetric cell division, the progeny is identical to the initial stem cell; in asymmetric division, one of the two progeny is identical to the initial stem cell, whereas the other cell is a committed progenitor cell, which undergoes cellular differentiation. Through the tightly regulated process of self-renewal, normal stem cells are able to function over the lifespan of the host, and thus their longevity and continued mitotic activity make them a significant target and potential reservoir for the accumulation of the numerous genetic mutations needed for transformation. Through the ability to self-renew, a small population of carcinogenic stem cells within a neoplasm has the ability to maintain malignant clones. Finally, two major factors affecting tumor response to therapy and cancer mortality are chemoresistance and metastasis. The CSC hypothesis considers that a tumor is composed largely of transformed but poorly proliferative tumor cells, and that only a small proportion, the highly carcinogenic cancer stem cell subset, is able to form a new tumor or metastasize, or show enhanced chemoresistance.

The TOFT, a relatively new theory on the origin of carcinogenesis, is based on the premise that cancer is a result of tissue organization, and that proliferation is the default state of a host cell ( ). A tissue-based theory rather than a cellular-based one as is the SMT, the TOFT, proposes that carcinogens cause a disruption in the interactions of cells that maintain normal tissue homeostasis, repair, and tissue organization, and that alteration in the microenvironment relaxes normal forces that maintain tissue order, allowing parenchymal cells to proliferate and migrate. The TOFT states that the normal state of the cell is one of proliferation and migration; that is, the interaction of the physical and biochemical forces of the stromal microenvironment keeps the cell in a resting state, and if this interaction is dysregulated in any way, this leads to release of these forces normally keeping cells in check, and they revert to their natural state of proliferation and migration, thus resulting in uncontrolled growth (tumorigenesis) and migration (metastasis).

These prevailing theories do not necessarily discount one another but may coexist in overlapping space and time. For example, the CSC hypothesis does not discount the SMT, but rather accepts the contribution of genetic instability, mutation, and epigenetic factors that influence the evolution of heterogeneity in a malignancy; however, in this case, the target of the genetic or epigenetic alterations is the stem cell, rather than the somatic cell. In the case of the TOFT, there is increasing support that cancer is also due to abnormalities in the microenvironment as well as tissue organization, in addition to mutations in oncogenes and tumor suppressor genes. Stromal alterations resulting in increased genomic instability and eventual accumulation of genetic mutations in parenchymal cells or overexpression of stromal enzymes such as matrix metalloproteinases inducing reactive oxygen species (ROS) or free radicals inducing DNA damage span the gap between the SMT and the TOFT. In any case, a single path forward to explain the origin of carcinogenesis remains elusive and may likely involve components of multiple predominant theories.

1.2

General Features of Carcinogenesis

Genomic Instability: Initiation, Promotion, and Progression

Cancer develops as the result of the accumulation of numerous mutations over an extended period of time, resulting in progressive genomic instability and increasingly severe genomic alterations ( ). For neoplastic transformation of a normal cell to occur, heritable changes involving multiple, independent genes are required. In early studies, it was first observed that a long latent period could elapse from exposure to carcinogens to the development of cancer. In 1941, skin carcinogenesis experiments by Rous and Kidd showed that if dermal exposure to chemical carcinogens was interrupted, induced tumors would regress, only to reappear if the exposure was readministered ( ). It seemed, therefore, that a reversible process was taking place in those cells that did not attain the complete neoplastic state. These cells had undergone what Rous called “initiation.”

During initiation, a normal cell undergoes an irreversible change that becomes fixed in the genome, making this cell more susceptible to transformation ( ). This change usually involves mutation of a proto-oncogene, tumor suppressor gene, or genes important for DNA repair. Initiation operationally implies that there is alteration to cellular DNA at one or more sites in the genome, which is converted to a stable biological lesion during DNA replication. Thus, if a round of cell replication occurs before the DNA damage is repaired, the lesion in the DNA is regarded as “fixed.” Initiation is additive, and the yield of neoplasms is dose dependent. In other words, increasing the dose of an initiator increases the incidence and multiplicity of neoplasms and shortens the latency to neoplastic transformation. Since initiation requires fixation of a mutation in the genome by a round of cell proliferation, it is also influenced by cell cycle, with more rapidly dividing cells being more susceptible to initiating events. Mutations may either be “driver” mutations, which promote tumorigenesis and contribute to the development of a malignancy when accumulated over time, or “passenger” mutations, which may be numerous, and are generally not essential for transformation, but can still contribute to carcinogenesis through influencing cancer cell fitness, immunologic responses, and altered epigenetic mechanisms ( ; ; ). As a result of genomic instability and defective DNA repair mechanisms, by the time a cell acquires the number of driver mutations necessary for transformation, it may bear hundreds of acquired mutations which ultimately lead to neoplastic transformation over time. Initiated cells may remain functionally and phenotypically normal for a prolonged time before driven toward full conversion to a malignant neoplasm, often months in animals and years in humans.

Mutational events occur frequently in specific regions or key sequences of DNA that are hypermutagenic, i.e., are prone to mutation. Although mutations in genes occur frequently across the genome, there is a significant variation in somatic mutation rates in certain areas of DNA due to regional differences in DNA mismatch repair (MMR) ( ). Certain regions of DNA that are more critical for cellular functions have higher rates of and more efficient repair, whereas those regions that encode for housekeeping or noncritical functions have less efficient repair. Mutations in these regions contribute to genomic instability. Conversely, certain classes of genes cluster in regions with high mutation rates, or “hot spots,” indicate that the region is prone to mutational events. Genes in “hot spots” tend to encode for dynamic processes such as cell signaling and immune responses, which can evolve in function to adapt to various stimuli ( ). In terms of carcinogen exposure, frequencies of single-nucleotide polymorphisms (SNPs), single- and double-strand breaks (SSBs/DSBs), DNA adduct formation, oxidative damage, and DNA–DNA and DNA–protein cross-linking, chromosomal rearrangements, and changes in gene copy number are associated with exposure to various exogenous mutagens ( ; ). Adduct formation is a classic manifestation of chemical carcinogenesis, as many mutagenic chemicals are metabolized to reactive electrophiles that covalently bind and damage DNA ( ). In some cases, patterns of mutation and adduct formation are specific for carcinogen exposure. For example, DNA adducts are observed with various classes of carcinogens including the aromatic amines, polycyclic aromatic hydrocarbons, tobacco-specific nitrosamines, alkylating agents, aldehydes, volatile carcinogens, and oxidative damage ( ). One example of a carcinogen-specific mutation due to adduct formation is that of AFB1 mycotoxin–induced hepatocellular carcinoma. In aflatoxicosis, the intermediate AFB1-8,9-epoxide reacts with guanine bases in DNA, forming adducts resulting in a specific transversion mutation at codon 249 in exon 7 of TP53 . Guanine is susceptible to adduct formation because electrophilic carcinogens often target nucleophilic moieties in DNA, including nitrogen and oxygen in guanine ( ). Another DNA lesion associated with direct damage from a specific carcinogen is the formation of pyrimidine, or thymine, dimers. This lesion is induced directly by ultraviolet light as well as secondary to oxidative damage, in which pyrimidine dimers form (most often) between two thymine bases, interfering with DNA and RNA polymerases and inhibiting replication of the chromosome ( ).

During the process of promotion, certain factors allow initiated cells to clonally expand. Tumor promoters enhance the development of neoplasms from a background of initiated cells by promoting cell growth and proliferation, interfering with normal apoptotic mechanisms, or through inflammatory or stromal mediators within the microenvironment that serve to provide a permissive environment for tumor growth. The temporal sequence of promoter administration is critical to the operational definition of promotion ( Figure 8.1 ). The agent must be administered after initiation and cause enhancement of the neoplastic process to be considered a promoter ( ). In contrast to initiating events, effects of promoters are reversible, and promoters are generally nongenotoxic but influence or alter gene expression through various epigenetic mechanisms (see section below). Some promoters are believed to produce their effect by interaction with receptors in the cell membrane, cytoplasm, or nucleus (e.g., hormones, dioxin, phorbol ester, polychlorinated biphenyls). Alternatively, some hydrophilic and hydrophobic promoting agents exert their effect through their molecular orientation at cellular interfaces. Other promoters are mitogenic, stimulating DNA synthesis and enhanced cell proliferation. This may occur directly or indirectly by targeting cells with a shortened G1 phase, thereby giving them a selective proliferative advantage. Tumor promotion may be modulated by several factors, such as age, sex, diet, and hormone balance. Age- and sex-associated modulations in hormonal levels of estrogens, progesterone (PR), and androgens have been implicated as potential promoters of breast cancer on the basis of epidemiologic studies. Experimental studies have shown repeatedly that these hormones, in addition to pituitary prolactin, serve to promote mammary tumors in rats initiated with mammary carcinogens.

Progression is that part of the multistep neoplastic process associated with the development of an initiated cell into a biologically malignant cell population. Progression is used frequently to signify the stages whereby a benign proliferation becomes malignant one, or where a neoplasm develops from a low grade to a high grade of malignancy. During progression, neoplasms show progressively increased invasive properties, develop the ability to metastasize, and have alterations in biochemical, metabolic, and morphologic characteristics. Tumor cell heterogeneity is an important characteristic of tumor progression. Expression of this heterogeneity includes antigenic and protein product variants, ability to elaborate angiogenesis factors, emergence of chromosomal variants, development of metastatic capability, altered metabolism, and decreased sensitivity to radiation or chemotherapeutics. The development of tumor heterogeneity may come about as a consequence of additional genomic changes, or as a result of altered epigenetic regulatory mechanisms. More than likely, genetic and epigenetic events subsequent to initiation operate in a nonmutually exclusive manner during progression, possibly in an ordered cascade of latter events superimposed on earlier events.

1.3

Cell Growth and Proliferation

The pivotal role of cell proliferation in all phases (e.g., initiation, promotion, progression) of the multistep process of carcinogenesis is inextricably linked to positive and negative cell cycle control mechanisms as influenced by oncogenes, tumor suppressor genes, growth factors and their cognate receptors, hormones and their receptors, and the action of exogenous agents (e.g., chemicals and viruses) on cell cycle control. Uncontrolled cellular proliferation is a hallmark of neoplasia, and many cancer cells demonstrate genetic alterations that regulate their cell cycles directly.

The prevailing model of the cell cycle is that of a series of transitions at which certain criteria must be met before the cell proceeds to the next phase ( Figure 8.2 ). The cell cycle is composed of an S (DNA synthesis) and an M (mitotic) phase, separated by two gap phases (G1 and G2). Progression through the cell cycle is tightly controlled by a group of heterodimeric protein kinases comprising a cyclin as a regulatory element and a catalytic subunit known as a cyclin-dependent kinase (CDK). There are many combinations of cyclin/CDK complexes, and each phase of the cycle is characterized by a specific pattern of expression and activity.

Five major classes of mammalian cyclins (termed A–E) have been described. Cyclins C, D1–3, and E reach their peak of synthesis and activity during the G1 phase and regulate the transition from G1 to S phase. However, cyclins A and Bl–2 achieve their maximal levels later in the cycle, during the S and G2 phases, and are regarded as regulators of the transition to mitosis. Association with cyclins not only activates CDKs but also determines their substrate specificity. Depending on the cyclin partner and therefore the cell cycle stage, different key target molecules are phosphorylated. These events occur in a highly regulated temporal sequence that is maintained through a series of checkpoints.

The importance of DNA damage leading to the DNA damage response (DDR) and consequently cell cycle checkpoint activation is obvious. Replication of a damaged template would certainly result in irreversible chromosomal aberrations and a high mutation rate. Three major checkpoints are thought to be particularly important following DNA damage and have been established at the middle to end of G1 (preceding DNA replication), at G2 (preceding mitosis), and during mitosis (preceding chromosome segregation in anaphase).

Loss of the G1 checkpoint triggers genomic instability at the time of interaction of unrepaired DNA with the DNA replication machinery, leading to deletion-type mutations and aberrant gene amplification. Inactivation of the G1 checkpoint serves as an initiation step that makes the cell susceptible to unregulated growth (initiation), increasing the probability of subsequent genetic alterations and establishing the fully developed neoplastic phenotype. Control at the G1 checkpoint is dependent on cyclin D1 (degraded at the G1/S transition) and cyclin E (degraded in mid-S phase). Overexpression of either cyclin D1 or cyclin E and subsequent activation of the cyclin D1 and cyclin E/CDK complexes result in entry into S phase and decreased G1 time.

Cyclin D1 is overexpressed in many human cancers, including breast and nonsmall cell lung carcinomas, sarcomas, melanomas, B-cell lymphomas, and squamous cell carcinomas of the head and neck. Cyclin D1/CDK4 complexes act to phosphorylate retinoblastoma (RB), the product of the RB susceptibility gene. RB exerts a negative regulatory effect on gene expression through complex formation with DNA-binding proteins, including members of the E2F family. In nondividing or G0 (arrested) cells, hypophosphorylated RB is bound to E2F family members, leading to repression of E2F-mediated transcription. Upon phosphorylation by cyclin/CDK complexes, RB dissociates from E2F proteins, leading to transcription of genes promoting S phase entry. Thus, hypophosphorylated RB maintains cells in G1, whereas phosphorylation inactivates RB and allows exit from G1 ( Figure 8.2 ). In humans, inactivation of RB is observed most commonly in RBs, osteosarcomas, carcinoid tumors, and nonsmall cell lung cancers.

Another tumor suppressor gene, TP53 , is necessary for G1 phase arrest after DNA damage ( Figure 8.2 ). Mutations at the TP53 locus are the most frequent genetic alterations associated with cancer in humans. The majority of TP53 mutations involve several highly conserved regions within the DNA-binding core. Loss of TP53 function allows synthesis of damaged DNA and increases the incidence of selected types of mutations. These increased incidences have been shown after a variety of DNA damage insults, such as ionizing radiation (strand breaks), alkylation by methyl-methane sulfonate, ultraviolet irradiation (photodimers), and a variety of environmental carcinogens. Thus, one of the major roles of TP53 is to ensure that, in response to genotoxic damage, cells arrest in G1 and attempt to repair their DNA before it is replicated.

The wild-type TP53 protein is normally kept at very low steady-state cellular levels by its relatively short half-life. However, it is stabilized and accumulates in the nucleus of cells undergoing or following DNA damage, or in cells responding to certain forms of stress. After DNA damage, TP53 binds a consensus-binding site and activates the transcription of several downstream genes, including P21 . The P21 gene belongs to a family of negative cell cycle regulators, which function as CDK inhibitory molecules. Genes that encode these proteins are designated cyclin-dependent kinase inhibitor genes. These negative regulators form stable complexes with cyclin/CDK units and inactivate them. P21 inactivates cyclin E/CDK2, cyclin A/CDK2, and cyclin D/CDK4/6 complexes, thereby inhibiting RB phosphorylation and preventing progression of the cell cycle during the late stages of G1 and beyond ( Figure 8.2 ).

The TP53 protein also activates the proapoptotic BAX gene, which is involved in the regulation of apoptosis. Apoptosis is a cell suicide mechanism that leads to programmed cell death in response to DNA damage, preventing replication of cells that have sustained a degree of genetic damage beyond repair (see Biochemical and Molecular Basis of Toxicity , Vol 1, Chap 2 and Morphologic Manifestations of Toxic Cell Injury , Vol 1, Chap 6). The TP53 protein regulates its own function through the activation of the MDM2 gene, which acts as a negative regulator, inhibiting wild-type TP53 transcriptional activity through an autoregulatory feedback loop.

An RB-binding site has also been identified at the carboxy-terminal domain of MDM2 that interacts with RB and restrains its function. Thus, overexpression of MDM2 inactivates both TP53 and RB, demonstrating a potential link between these genes in cell cycle regulation, apoptosis, and tumor progression. With the loss of both TP53 and RB, E2F activation stimulates unchecked cellular proliferation, leading to the emergence of neoplastic cell growth. The high rate and mutation pattern of TP53 and RB in primary tumors have rendered them prototype tumor suppressor genes. Furthermore, detection of TP53 and RB mutations and altered expression of their encoded products appear to be of clinical prognostic significance when identified in specific cancers. Additional gene products activated in response to DNA damage include transcription factors, growth factors, growth factor receptors, and enzymes and proteins associated with inflammation and tissue injury and repair.

1.4

Oncogenes and Tumor Suppressor Genes

As a result of progressive DNA damage and subsequent mutations, activating alterations in various oncogenes that drive cell proliferation, and inactivation of tumor suppressor genes that suppress proliferation, swing the balance toward increased proliferation, and ultimately to neoplastic transformation and progression ( ). Oncogenes are dominant-acting structural genes that encode for protein products capable of transforming the phenotype of a cell. Oncogenes were first identified as part of the genetic makeup of retroviruses, which function to transform virally infected cells and produce neoplasia in the host. It was later learned that transforming oncogenes are normal structural genes captured from eukaryotes previously infected by a retrovirus, rather than intrinsic viral genes. While the viral gene is referred to as a viral oncogene, the homologous gene in the host genome is called a cellular oncogene or a proto-oncogene ( ). As the genomes of various mammalian and submammalian species were examined, it was found that homologues to the retroviral oncogenes were present in species as diverse as yeast, fruit flies, amphibians, birds, and mammals. The high degree of evolutionary conservation of these proto-oncogenes suggested that they served important normal functions in the cell. Proto-oncogenes encode for proteins that are important in cell growth, development, and differentiation. An activated proto-oncogene is referred to as an oncogene and may be altered quantitatively or qualitatively. Quantitative alteration results in overexpression of the normal gene, and qualitative alteration results from an activating mutation, both of which result in either inapporopriate expression or overexpression. Activation can occur in several ways, including through acquisition of point mutations, deletions, or gene fusions within the coding sequence of the proto-oncogene, leading to altered levels or schedule of expression of the normal protein product or in expression of an abnormal protein.

The activation of proto-oncogenes in spontaneous and chemically induced neoplasia has received considerable attention over the years. A variety of activated oncogenes have been documented in rodent neoplasms that play a role in the development of human cancers ( Table 8.1 ). From some experimental studies, it appears that certain types of oncogenes are activated by carcinogen treatment and that this activation is sometimes an early event in tumor induction. Other studies with human and rodent neoplasms suggest that oncogene activation is involved later in the carcinogenic process, specifically during tumor progression. One important gene frequently mutated in several types of rodent and human cancer is the proto-oncogene RAS . The encoded protein for RAS is a G-coupled membrane protein and growth regulator that cycles between an inactive GDP-bound and active GTP-bound state. When RAS is mutated, hydrolysis of GTP to GDP is blocked, and RAS is stuck in a constitutively activated state, leading to activation of downstream cellular growth and proliferation pathways, gene transcription, and promotion of the cell cycle ( ). Another proto-oncogene, c- MYC , is commonly dysregulated and overexpressed in a variety of human and rodent cancers due to amplification or rearrangement. The MYC gene mediates fundamental cellular functions including metabolic processes, regulation of growth and proliferation, cell cycle progression, and regulation of differentiation, apoptosis, and stem cell regulation ( ). Dysregulation of MYC is commonly associated with neoplastic development and progression and is a hallmark of most human cancers.

Tumor suppressor genes also play a critical role in carcinogenesis ( Table 8.2 ). These genes are regulatory genes that normally function to inhibit the activity of structural genes responsible for growth. As such, when intact, they have a function opposite to that of oncogenes and might effectively oppose the action of an oncogene. While proto-oncogenes have to be activated to influence carcinogenesis, inactivation of both alleles of suppressor genes must occur for the transformed phenotype to be expressed. Inactivation can be achieved by chromosome loss, gene deletion, recombination, gene conversion, point mutation, or epigenetic silencing (see below). According to Knudson's two-hit hypothesis, loss of both alleles of a tumor suppressor gene either through direct genomic alterations or through epigenetic silencing is required to develop a cancer phenotype ( ). This hypothesis was developed by Alfred Knudson in 1971, through studies of RB in children ( ). These studies resulted in the discovery of tumor suppressor genes and their role in tumorigenesis. Knudson observed that RB was either an inherited or acquired neoplasm that likely developed through two mutational events. In the inherited form, the first “hit” was present already at birth but did not result in disease because the second allele was functional. The second “hit” occurred as an acquired event resulting in loss of heterozygosity (LOH), and subsequently development of RB at an early age. In the acquired form, an individual is born with two normal alleles for the RB1 gene, and in order for disease to occur, both alleles must be lost, which explained a later onset of disease in these individuals. In addition to RB, mutation of RB occurs in most osteosarcomas and small cell lung cancers in humans, and is a component of the INK4A/Cyclin D1/RB/E2F regulatory pathway that is inactivated in most cancers ( ). Exceptions to the two-hit hypothesis for tumor suppressor genes include inactivation of a normal allele by genomic imprinting, an epigenetic process involving histone and DNA methylation which is established, or “imprinted,” in the genome and maintained through mitotic division, or by dominant-negative mechanisms as is proposed for the TP53 tumor suppressor gene, which is the most frequently mutated gene in human cancers ( ). The phosphatase and tensin (PTEN) tumor suppressor gene plays a significant role in the control of cell growth, proliferation, and survival. It is the most commonly mutated gene in sporadic human cancer, and loss of function of PTEN is associated with tumorigenesis in rodents as well as humans; several mouse models have been developed to study the pathogenesis of various human tumors mediated by alterations in this tumor suppressor gene ( ). Another tumor suppressor gene that has been targeted to model human cancer is the adenomatous polyposis coli ( APC ) gene. Genetically engineered mice (GEM) (ApcMin/+) with loss of one allele of the APC gene are commonly used as a model of human colorectal tumorigenesis, often in combination with other mutations in genes associated with intestinal carcinogenesis in humans ( ).

As such, multiple tumor suppressor genes are often altered during neoplastic progression, in line with the understanding that cancer development involves perturbation of several levels of growth control. Taken together, activation of multiple oncogenes in concert with the loss of function of various tumor suppressor genes through genetic or epigenetic modification, influenced by a variety of endogenous and exogenous stimuli, ultimately culminates in the complex process of neoplastic transformation and progression.

1.5

Apoptosis and DNA Damage Repair

Current data strongly suggest that cell death may be as essential as cell proliferation in carcinogenesis. The ratio between cell birth and counterbalancing cell death determines tumor growth. Two forms of cell death may be seen in cancer development: necrosis and apoptosis. Necrosis typically occurs when a developing cancer outgrows its blood supply. In contrast, apoptosis is an energy-dependent process that involves active gene transcription and translation. In preneoplastic lesions, apoptosis is the predominant form of cell death. Chemicals, nutrient deprivation, certain cytokines, growth factors, and tumor suppressor genes may trigger enhanced apoptosis ( ).

As a result of either endogenous or exogenous DNA damage and genomic instability, a normal cell can either undergo successful DNA repair and return to normal state or if DNA repair is not successful, cells can either undergo apoptosis or survive, resulting in fixed mutations in its DNA structure ( ). This leads to functional alterations in oncogenes and tumor suppressor genes, impairment of apoptosis, and ultimately the formation of a malignant tumor. Apoptosis is a protective response to transformation; when DNA damage occurs that cannot be repaired, the affected cells undergo apoptosis to prevent fixation of DNA damage into the genomic sequence. However, in cancer cells that have defective DNA repair mechanisms, DNA damage not corrected becomes fixed in the genome, and this contributes to further genomic instability ( ). Thus, apoptosis is a barrier that must be surmounted for cancer to develop and progress, and any abnormalities in the function of genes responsible for normal apoptosis pathway function can predispose to neoplastic transformation.

Apoptosis occurs through two primary mechanisms; the intrinsic and extrinsic apoptosis pathways ( ), Biochemical and Molecular Basis of Toxicity , Vol 1, Chap 2 and Morphologic Manifestations of Toxic Cell Injury , Vol 1, Chap 6 . In the intrinsic (mitochondrial) pathway, cells, which sustain irreparable internal injury due to DNA damage, cellular injury, or stress, undergo apoptosis through release of cytochrome C from the inner mitochondrial membrane in a TP53-dependent fashion, triggering activation of downstream initiator (Caspase 9) and effector (Caspase 3) caspases, leading to the morphologic changes typical of apoptosis. Tumor cells may evade the intrinsic apoptosis pathway and escape cell death through dysfunction of the TP53 pathway or upregulation of prosurvival ( BCL2, BCL-XL ) or inhibition of proapoptosis ( BAX, BAK ) mitochondrial genes ( ).

In the extrinsic pathway, external signals such as an immune cell response function by activation of death signaling cascades to mediate cell death due to defects in viability. To evade the extrinsic apoptosis pathway, tumor cells may survive through interference with death receptor (e.g., Fas/FasL) function or binding, resulting in defective Caspase 8 function and inactivation of the apoptosis signaling complex. Interference with the intrinsic or extrinsic pathways leads to inhibition of apoptosis and faulty DNA repair, resulting in fixation of additional mutations in the genome over time, and neoplastic transformation and progression ( ).

Tumor cells can also evade apoptosis through inhibition of senescence. Senescence is the process by which cells exit the cell cycle, stop dividing, and become quiescent. Senescence is prevented by the function of telomeres, which are regions of repetitive nucleotide sequences at the ends of each chromosome ( ). Each time a cell divides, a small amount of each telomere is lost; as telomeres shorten, the cell ages and eventually stops dividing once the telomeres are completely lost. At this point, the cell enters senescence and may die as a normal part of cellular aging. Telomerase is an enzyme that functions to preserve the telomere, adding nucleotides to the ends of chromosomes to preserve the structural integrity of the telomere. Some cancer cells function to escape senescence through induction of telomerase expression, thereby avoiding cell death through telomere-mediated senescence and apoptosis. Additionally, cancer cells may induce expression of embryonic growth pathways, stem cell genes, and developmental mediators that prevent senescence, allowing them to continue to proliferate ( ).

1.6

Angiogenesis, Invasion, and Metastasis

In order for a tumor to survive, it has to be able to grow and proliferate, which requires a sufficient supply of oxygen and nutrients. This requires the acquisition of an independent blood supply. Tumor cells acquire a blood supply through numerous signaling molecules, cytokines, and chemokines, growth factors such as vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factor family members, matrix metalloproteinases, integrins and their receptors, and endothelial cells and endothelial progenitor cells, to induce the formation of new blood vessels ( ), in the form of angiogenesis, neovascularization, or vasculogenic mimicry ( ). These processes are driven by tumor cell signaling within the extracellular matrix (ECM) via a number of angiogenic factors. Angiogenesis is defined as the formation of new blood vessels from preexisting vessels, as quiescent endothelial cells become activated when stromal, tumor, and immune cell–derived proangiogenic factors are secreted into the tumor site, leading to proliferation, migration, degradation of the ECM, and tube formation from existing capillaries. Neovascularization is the formation of new blood vessels from bone marrow progenitor cells, with no involvement of preexisting vasculature; bone marrow–derived endothelial precursor cells are recruited to the tumor site and differentiate into endothelial cells, forming de novo capillaries. Finally, vasculogenic mimicry is the formation of channels within tumors by motile tumor cells (rather than endothelial cells) that act as blood channels to feed the tumor. And so, through various mechanisms, tumors can induce their own blood supply through systemic signaling to the bone marrow (neovascularization), or local proangiogenic signaling to promote vascular ingrowth from preexisting vasculature (angiogenesis), or invasive properties of the tumor itself to form vascular channels through vasculogenic mimicry.

In order to develop an invasive and metastatic phenotype, a tumor cell must acquire a number of important features, including epithelial–mesenchymal transition (EMT), ability to escape anoikis (anchorage-dependent apoptosis) and survive in the circulation, and evasion of the immune system. EMT is the process by which an epithelial tumor cell develops a mesenchymal phenotype necessary for migration, especially during local invasion and metastasis. During this process, cells gradually lose expression of epithelial-type proteins (e.g., cytokeratins, E-cadherin, claudins, desmoplakin) that anchor them to the basement membrane and acquire mesenchymal-type proteins (e.g., vimentin, fibronectin, collagen I/III, α-smooth muscle actin) that allow them to reorganize their cytoskeleton, degrade ECM, and migrate through tissue ( ). Having obtained an EMT phenotype, tumor cells subsequently need mechanisms to survive outside of the supporting basement membrane, within the circulation, and at a distant site. Normal epithelial cells that lose integrity of tight junctions or detach from the supporting basement membrane undergo anoikis or anchorage-dependent apoptosis. Obviously, for neoplastic epithelial cells to migrate, this cannot happen. Therefore, tumor cells develop mechanisms to escape anoikis, through increasing genetic instability, intratumoral hypoxia, EMT, and activation of stem cell pathways, in order to survive in the ECM and circulation ( ). Finally, to survive migration through the stroma, into and out of the vasculature, within the circulation, and for survival and establishment at the distant site, metastatic tumor cells require the ability to evade the immune system. Evasion of the immune system is accomplished by tumor cells through three primary mechanisms: (1) downregulation or shedding of strong tumor associated antigens that allow the immune system to recognize them, (2) upregulation of antiapoptotic (e.g., BCL2 ) or growth factor (e.g., human epidermal growth factor receptor-2 genes, and (3) induction of an immunosuppressive tumor microenvironment through tumoral expression of antiinflammatory cytokines (e.g., transforming growth factor-beta, interleukin-10) and other molecules that interfere with adaptive immunity. Through these complex and intricate mechanisms, cancer cells are able to survive and migrate in the ECM and systemic circulation, evade host antitumor mechanisms, and undergo growth and proliferation at distant sites to establish metastatic disease.

2.1

Genotoxic Carcinogens

Genotoxic carcinogens are DNA reactive, leading to damage and ensuing mutation of the genome. Numerous genotoxic carcinogens have been identified with established mechanisms of action including urethane, benzo[ a ]pyrene (BaP), N -ethyl- N -nitrosourea (ENU), 2-acetylaminofluorene, and diaminobenzamide. Genotoxic carcinogens can mutate the genome either with metabolic activation (i.e., parent compound needing metabolism to engage the DNA duplex) or without this occurring (e.g., compounds that bind the DNA duplex without a need for metabolism).

2.2

Direct Acting Carcinogens

Compounds that do not require metabolic activation to induce mutagenesis associated with cancer risk are known as direct acting carcinogens. Direct acting carcinogens are highly electrophilic and bind DNA with high affinity due to the nature of their chemical structure and composition. Additional factors that promote DNA reactivity of agents include nucleophilicity properties, accessibility of the DNA, and van der Waals forces that regulate electrostatic interactions between an agent and DNA ( ). These properties render such mutagenic compounds carcinogenic as tumors can potentially form in exposed tissues. The potential of mutagenic compounds to cause cancer depends not only on the replicative capacity of the exposed tissue but also membrane permeability of the exposed cell, the rate of interaction between carcinogen and the DNA duplex, intracellular free radicals triggered by the mutagen, and stability of the chemical itself. Direct acting carcinogens include nanoparticles ( ; ), epoxides ( ), chemotherapeutics ( ; ), alkyl halides, and esters ( ). The Ames bacterial reverse mutagenicity test ( ) assesses mutagenesis in the bacterial strains Salmonella typhimurium and/or Escherichia coli and has been used successfully for decades. It is able to identify the mutagenic capacity of most direct acting electrophilic carcinogens that do not require additional metabolic activation (for example, using S9 fraction of rat liver homogenate) ( ; ). Even so, false positives and false negatives are known to have occurred due to the artificially high doses of the chemical and factors involving the mitogenic responses and mutagenicity for a chemical/xenobiotic established using the Ames test and that may not necessarily translate to carcinogenic potential ( ).

2.3

Indirect Acting Carcinogens

Metabolic activation and transformation of a parent compound (procarcinogen) by liver enzymes is a key determinant of the carcinogenic potential of many xenobiotics. Cytosol and microsome fractions (e.g., S9) obtained from rat liver homogenates are most often used together with a compound in the standard in vitro genetic toxicology battery comprising the Ames test (determines whether there has been mutagenicity), micronucleus test (assesses aneugenicity), the COMET assay (detects DNA breakage and damage), or chromosomal aberration (clastogenicity) tests ( Table 8.4 ). Pivotal work done by Miller et al. involving indirect genotoxic nonelectrophilic carcinogens p-Dimethylaminoazobenzene and BaP showed a requirement for metabolism of the parent compound associated with its carcinogenic potential ( , ; ). Xenobiotics may also promote mutagenicity and/or cancer risk following persistent signal transduction pathway activation/inhibition, oxidative damage, disequilibrium in ROS within the cell, errors in machinery required for replication/repair of the DNA duplex, or alterations in the mammalian epigenome or chromatin structure/function ( ; ; ; ; ). Several signal transduction pathways and those that function via various upstream kinases, including the phosphatidylinositol 3-kinase delta (PI3Kδ), p38 kinases, mitogen-activated protein kinases (MAPK), c-Jun N-terminal kinases, and extracellular signal-regulated kinases, can be influenced by either agonistic or antagonistic effects of xenobiotics ( ; ; ; ). An imbalance of such pathways can be directly linked with perturbed downstream mechanisms that may promote mutagenicity of the DNA duplex. Recent reports examining the mutagenic capability of PI3Kδ inhibition by xenobiotics and endogenous aldehydes provide direct evidence for xenobiotics traditionally classified as “nonmutagenic” that can promote mutagenesis and increase genome instability by pathway activation in normal mammalian genomes ( ; ). Persistent PI3Kδ inhibition results in triggering of the activation-induced deaminase (AID), leading to increased off-target AID-dependent mutagenicity ( ). Furthermore, activation or inhibition of signal transduction pathways by an imbalance in ROS can lead to perturbation in expression of genes whose function is required for cellular processes including DNA duplication, mitochondrial function, apoptosis/programmed cell death, or differentiation ( ; ; ; ; ).

2.4

Mechanisms of High-fidelity/Nonmutagenic or Low-fidelity/Mutagenic DNA Repair

Direct acting carcinogens are strong electrophiles that can readily bind genomic DNA and form DNA adducts, induce DNA cross-links, or induce other forms of DNA damage including SSBs or DSBs. Cells are proficient in the repair of DNA lesions ( Table 8.5 ); however, the capacity to repair these lesions in an error-free manner can be dependent on the persistence of the damage at the impacted region of the genome or underlining inherent repair capacity impacted by heritable mutations in certain genes (e.g., BRCA1/2 ) and their involvement in high-fidelity DNA repair mechanisms ( ; ).

High-fidelity DNA repair pathways including error-free homologous recombination (HR) ( ), the Fanconi Anemia (FA) pathway ( ), MMR ( ), base excision repair (BER) ( ), and classical non-homologous end joining (NHEJ) ( ) suppress error-prone mutagenic repair of DNA lesions and promote genomic stability. However, persistence of the lesion(s) as a result of sustained carcinogen exposure can lead to an exhaustive state that activates error-prone DNA repair pathways that serve to promote cell viability albeit at a cost of increased mutagenicity and genomic instability ( ). Similarly, genome editing therapeutics including CRISPR–Cas9 and TALENs can promote off-target mutations or structural changes in the genome following the erroneous repair of induced double-strand break replication intermediates ( ; ). This balance of DNA repair capacity is further impacted when there is an underlying genetic defect or mutation in genes that function as tumor suppressors ( ). For example, individuals having inherited mutations in the BRCA1 or BRCA2 tumor suppressor genes are at an increased risk of developing breast and/or ovarian cancers ( ). Therefore, exposure of individuals having these underlying genetic defects could predispose them further to cancers at a faster rate than their wild-type counterparts following carcinogen-induced DNA damage because of salvage/backup error-prone DNA repair pathway activation. Salvage DNA repair pathways including alternative non-HR ( ; ), single-strand annealing ( ), microhomology-mediated break-induced repair (MMBIR) ( ), or mitotic DNA synthesis (MiDAS) ( ) are activated under conditions of persistent drug-induced replication stress. The activation of these carcinogen-induced error-prone mechanisms can lead to mutations at tumor suppressor genes and increased fitness of preneoplastic cells to clonally expand and form tumors over time ( ) as evidenced by the effects of known carcinogens including urethane, ENU, and BaP that are well established to induce tumors ( ; ; ). Additionally, blocking of PI3Kδ by the small molecule inhibitors idelalisib or duvelisib, compounds that are not directly mutagenic, has recently been shown to increase expression of the AID in mice which leads to increased AID-induced random mutagenesis and ultimately gives rise to plasma cell tumors ( ).

2.5

DNA Replication and Repair Mechanisms

Cells are proficient for the repair of DNA damage resulting from exogenous or endogenous stressors. However, persistent DNA damage and stress can inundate the capacity to carry out high-fidelity DNA repair and activate mutagenic DNA repair pathways that serve to maintain cell viability, albeit at a cost of increased genome instability that can give rise to human diseases/disorders including cancers, premature aging, neurological abnormalities, and immunodeficiency syndromes ( ; ; ).