Multistage carcinogenesis

Carcinogenesis can be divided conceptually into four steps: tumor initiation, tumor promotion, malignant conversion, and tumor progression (Figure 1, Box 1). The distinction between initiation and promotion was recognized through studies involving both viruses and chemical carcinogens.^{13, 14, 57} This distinction was formally defined in a murine skin carcinogenesis model in which mice were treated topically with a single dose of a polycyclic aromatic hydrocarbon (PAH) (i.e., initiator), followed by repeated topical doses of croton oil (i.e., promoter),¹³ and this model has been expanded to a range of other rodent tissues, including bladder, colon, esophagus, liver, lung, mammary gland, stomach, and trachea.⁵⁹ During the last 65 years, the sequence of events comprising chemical carcinogenesis has been systematically dissected and the paradigm increasingly refined, and both similarities and differences between rodent and human carcinogenesis have been identified.⁶³ Carcinogenesis requires the malignant conversion of benign hyperplastic cells to a malignant state, and invasion and metastasis are manifestations of further genetic and epigenetic changes.⁶⁴ The study of this process in humans is necessarily indirect and uses information from lifestyle or occupational exposures to chemical carcinogens. Measures of age-dependent cancer incidence have shown, however, that the rate of tumor development is proportional to the sixth power of time, suggesting that at least four to six independent steps are necessary.⁶⁵ Partial scheduling of specific genetic events in this process has been possible for some cancers. Examples of sequential genetic and epigenetic changes that occur with the highest probability are those found in the development of lung cancer⁶⁶ and colon cancer.⁶⁷ Recent advances in sequencing technology have allowed us to identify the genomic landscape of many tumors. In common solid tumors such as those derived from the colon, breast, brain, or pancreas, an average of 33–66 genes display subtle somatic mutations that can alter their protein products.⁶⁸ Certain tumor types display many more or many fewer mutations than average. Melanomas and lung tumors, for example, contain approximately 200 nonsynonymous mutations per tumor; a reflection of the involvement of potent mutagens [ultraviolet (UV) light and cigarette smoke, respectively] in the pathogenesis of these tumor types.⁶⁸

Epigenetics and chemical carcinogenesis

Epigenetics describes a change in gene activity without a change in the DNA sequence.⁷⁹ Well-described mechanisms involved in epigenetics include DNA methylation and histone modification; each of which alters how genes are expressed without altering the underlying DNA sequence. Another mechanism (described in detail later) also falling under the definition of “epigenetic” is the effects of microRNA (miRNA; Figure 2, Box 2) on carcinogenesis. Both nongenotoxic and genotoxic chemical carcinogens impact these epigenetic processes. At the cellular level, exposure to environmental factors may leave an epigenomic signature that can be exploited in discovering new biomarkers for risk assessment and cancer prevention.^{80, 81}

DNA methylation is catalyzed by a family of DNA methyltransferases (DNMTs). In somatic mammalian cells, DNA methylation occurs at cytosine residues of CpG dinucleotides; in embryonic stem cells, DNA methylation occurs at both CpG and non-CpG sequences. DNMT expression, and therefore indirect control of DNA methylation, is controlled by DNMT3L, lymphoid-specific helicase, miRNAs, and piwi-interacting RNAs.⁸² It is becoming increasingly clear that many chemical carcinogens are involved in DNA methylation. For example, the tobacco-specific carcinogen, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), induces DNMT accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients.⁸³ As well, B(a)P and many others not only genotoxic, but also nongenotoxic chemical carcinogens have been shown to induce aberrant methylation patterns.^{82, 84} Several mechanisms may explain aberrant methylation patterns due to chemical carcinogens. As an example, cigarette smoke may alter DNA methylation by (1) inducing DNA damage, stimulating recruitment of DNMT1; (2) the ability of nicotine to down-regulate DNMT1 mRNA and protein expression; (3) modulating expression and activity of DNA-binding factors, such as Sp1; and (4) inducing hypoxia, which increases HIF1α, leading to the up-regulation of methionine adenosyltransferase 2A⁸⁵ (Figure 2).

Chromatin is the complex of DNA wrapped around histone octamers, consisting of four different histones, H2A, H2B, H3, and H4. Gene expression changes with histone post-transcriptional modification (e.g., acetylation, phosphorylation, and methylation) in the N-terminal tail region. Enzymes that regulate histone modification include histone deacetylases (HDACs), histone acetyltransferases (HATs), and histone methyltransferases (HMTs). Chemical carcinogens can alter the activity of such enzymes. For example, challenging cells with B(a)P induces early enrichment of the transcriptionally active chromatin markers histone H3 trimethylated at lysine 4 (H3K4Me3) and histone H3 acetylated at lysine 9 (H3K9Ac), and reduces association of DNMT1 with the long interspersed nuclear element-1 (L1) promoter. These changes are followed by proteasome-dependent decreases in cellular DNMT1 expression and sustained reduction of cytosine methylation within the L1 promoter CpG island.⁸⁶ Similarly, long-term exposure of immortalized bronchial epithelial cells (HBEC-3KT) to low doses of tobacco-related carcinogens leads to oncogenic transformation, increased HDAC expression, cell-cycle independent increased DNMT1 stability, and DNA hypermethylation.⁸⁷

miRNA’s in chemical carcinogenesis

miRNA expression is regulated by a growing list of factors, including inflammatory cytokines, free radicals, and chemical carcinogens.^88–91 The specific effects of chemical carcinogens on each miRNA in vitro and in vivo have been described in detail elsewhere, so the reader is guided to that source.⁹¹ For the purposes of this chapter, we will provide an overview of miRNAs, examples of key miRNAs affected by chemical carcinogens, and the mechanisms by which this occurs.

miRNAs are nonprotein coding small RNAs of about 22 base pairs and regulate mRNA stability and translation into proteins.⁹² miRNA genes are regulated by transcription factors including by the p53 tumor suppressor protein⁹³ and are involved in carcinogenesis including tumor invasion and metastasis.⁹⁴ miRNAs are produced by processing of a hairpin precursor by an enzyme called dicer, and the mature miRNA incorporates into a protein complex (the RNA-induced silencing complex—RISC), where it acts as a guide to find complementary target RNAs. Binding of RISC to the target RNA causes degradation of the target or blocks translation—in either case, preventing production of the encoded protein. The alteration of miRNA expression by chemical exposure or during inflammation potentially has enormous consequences, because of the large number of genes each miRNA regulates. Each organism encodes hundreds of miRNAs, and these affect many key biological processes, including cell proliferation, differentiation, survival, and metabolism. In the 15 or so years since miRNAs were discovered,^95–97 it has become clear that post-transcriptional control of gene expression by miRNAs provides an important and widely used level of gene regulation. In humans, it is estimated that about 60% of all protein-coding genes are regulated by a miRNA.^{98, 99} Because miRNAs are highly specific, potent suppressors of gene expression; and aberrant regulation of miRNA expression is associated with cancer (and many other diseases), the potential value of these small RNAs as therapeutic agents is now widely recognized.^100–104 Not surprisingly, miRNAs are also clinical biomarkers associated with diagnosis, prognosis, and therapeutic outcome of cancer.^{105, 106} Because miRNA levels change upon exposure to environmental and endogenous chemicals, it is possible that miRNAs can be used for biomonitoring purposes. This is supported by the understanding that miRNAs are released from the target tissues into the blood stream; and therefore, their analysis is feasible via noninvasive sampling (e.g., urine and feces).^107–109

Gene–environment interactions and inter-individual variation

A cornerstone of human chemical carcinogenesis is the concept of gene–environment interactions (Figure 3, Box 3).^{56, 172} Potential inter-individual susceptibility to chemical carcinogenesis may well be defined by genetic variations in the host elements of this compound system. Functional polymorphisms in human proteins that have, or may have, a role in chemical carcinogenesis include enzymes that metabolize (i.e., activate and detoxify) xenobiotic substances, enzymes that repair DNA damage, cell surface receptors that activate the phosphorylation cascade and cell-cycle control genes (i.e., oncogenes and tumor suppressor genes that are elements of the signal transduction cascade).

When chemicals or xenobiotics encounter biologic systems, they become altered by metabolic processes. This is an initial facet of gene–environment interaction. The inter-individual variation in carcinogen metabolism and macromolecular adduct formation arising from such processes was recognized almost 40 years ago.³⁰ The cytochrome P450 (CYP) multigene family is largely responsible for the metabolic activation and detoxification of many different chemical carcinogens in the human environment.¹⁷³ CYP P450s are phase I enzymes that act by adding an atom of oxygen onto the substrate; they are induced by PAHs and chlorinated hydrocarbons.¹⁷³ Phase II enzymes act on oxidized substrates and also contribute to xenobiotic metabolism. Some phase II enzymes are methyltransferases, acetyltransferases, glutathione transferases, uridine 5′-diphosphoglucuronosyl transferases, sulfotransferases, nicotinamide adenine dinucleotide (NAD)- and nicotinamide adenine dinucleotide phosphate (NADP)-dependent alcohol dehydrogenases, aldehyde, steroid dehydrogenases, quinone reductases, NADPH diaphorase, azo reductases, aldoketoreductases, transaminases, esterases, and hydrolases. The pathways of activation and detoxification are often competitive, providing yet further potential for individual differences in propensity for carcinogen metabolism to DNA damaging species. This scenario is further complicated by a second facet of gene–environment interaction that leads to enzyme induction or inhibition. In this case, environmental exposures alter gene expression, and genes responsible for carcinogen metabolism can be upregulated or repressed by certain chemical exposures.

A third facet of gene–environment interaction occurs when the chemical alters gene structure (environmentally predisposed abnormal DNA). Once a procarcinogen is metabolically activated to an ultimate carcinogenic form, it can bind covalently to cellular macromolecules, including DNA. This DNA damage can be repaired by several mechanisms.¹⁷⁴ Differences in rates and fidelity of DNA repair potentially influence the extent of carcinogen adduct formation (i.e., biologically effective dose) and, consequently, the total amount of genetic damage. The consequences of polymorphisms in genes controlling the cell cycle (serine/threonine kinases, transcription factors, cyclins, cyclin-dependent kinase inhibitors, and cell surface receptors) are much less clear. However, molecular epidemiologic evidence suggests that certain common variants of these types of genes have a role in susceptibility to chemical carcinogenesis.¹⁷⁵ The evaluation of polymorphisms as potential biomarkers of susceptibility in the human population is discussed in Weston and Harris.¹⁷⁶

Carcinogen metabolism

Inter-individual variation in cancer susceptibility, and, consequently, meaningful human cancer risk assessment, involve determination of inherited host factors as well as exposure assessment. Metabolic polymorphisms have historically been determined by the use of indicator drugs (e.g., caffeine, debrisoquine, dextromethorphan, dapsone, and isoniazid), however, these assays are being replaced by direct genetic assays. This approach has allowed the investigation of diverse host factors for which indicator drugs were not available, and it has been applied to a wide variety of cancers.^177–181 Such studies have not only made a key impact in the area of pharmacogenomics,^{182, 183} but genetic indicators of propensity for carcinogen activation and detoxification, DNA-repair capacity, apoptosis, and cell-cycle control are all features of molecular epidemiologic studies that are complementary to adduct studies because of the implications for a biologically effective dose after exposure.

CYP P450 polymorphisms, involved in carcinogen activation, and glutathione-S transferases, uridine diphosphate (UDP) glucuronosyltransferases, sulfotransferases, and N-acyltransferases, involved in both carcinogen activation and detoxification, could explain variations in cancer susceptibility among the human population. Evidence that absent protection of a functionally intact GSTM1 gene correlates with an increased risk of tobacco-related lung cancer.¹⁸⁴ Similarly, the absence of GSTM1 and GSTT1 genes increases the risk of lung cancer because of radon exposure.¹⁸⁵ There is a substantial reduction in risk of lung adenocarcinoma associated with genetic polymorphism in CYP2A13, the most active CYP P450 for the metabolic activation of tobacco-specific carcinogen NNK.¹⁸⁶ The finding that XRCC1, GSTM1, and COMT polymorphisms was strongly associated with lung cancer risk in smoking women is supportive of an interaction of repair, tobacco smoke metabolism, and estrogen metabolism in this disease and continues the controversy of an increased risk of lung cancer in women.¹⁸⁷ Also, UDP glucuronosyltransferases (e.g., UGT1A1, UGT1A9, and UGT2B7) have been implicated in cancers of the head and neck. Persons inheriting reduced activity variants of NAT1 and NAT2 genes, resulting in the slow acetylator phenotype, are at a greater risk of aromatic amine-induced bladder cancer. This may include persons exposed through tobacco smoke inhalation.^{188, 189} Over the past 25 years, there have been advances in the understanding of carcinogen metabolism, inter-individual variations, and cancer risk. A good review of the existing literature can be found elsewhere.¹⁹⁰

PAHs, for example, B(a)P, were the first carcinogens to be chemically isolated.¹⁹¹ They are composed of variable numbers of fused benzene rings that form from incomplete combustion of fossil fuels and vegetable matter, they are common environmental contaminants. PAHs are chemically inert, and require metabolism to exert their biologic effects.¹⁹¹ This multistep process involves the following: initial epoxidation (CYP P450), hydration of the epoxide (epoxide hydrolase), and subsequent epoxidation across the olefinic bond.^{173, 192} The result is the ultimate carcinogenic metabolite; in the case of B(a)P it is r7, t8-dihydroxy-c9, 10 epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (B[a]P-7,8-diol 9,10-epoxide, BPDE).¹⁹³ The biology of CYP P450 (e.g., CYP1A1) metabolism has been elucidated providing a molecular basis for inducibility and inter-individual variation, and variations in CYP levels among humans have been documented.¹⁹⁴ The arene ring of BPDE opens spontaneously at the tenth position, revealing a carbonium ion that can form a covalent addition product (adduct) with cellular macromolecules, including DNA. Several DNA adducts can be formed, the most abundant being at the exocyclic amino group of deoxyguanosine ([7R]-N 2-[10–7β,8a,9a-trihydroxy-7,8,9,10-tetrahydro-benz[a]pyrene}yl]-deoxyguanosine; BPdG). One electron oxidation has been suggested as an alternative pathway of PAH activation, here a radical cation forms at the meso position (L-region). The resulting DNA adducts at the C8 of guanine (B(a)P-6-C8Gua and BP-6-C8dGua), the N7 of guanine and adenine (B(a)P-6-N7Gua and BP-6-N7Ade) likely undergo spontaneous depurination. Firm evidence for the exfoliation of these adducts in urine has been provided.¹⁹⁵

Aromatic amines are found in cigarette smoke, diesel exhaust, industrial environments, and certain cooked foods. The compound, 4-aminobiphenyl, is thought to be responsible for bladder cancer among tobacco smokers and rubber industry workers.¹⁹⁶ In addition, nitrated PAHs are environmental contaminants that are related to aromatic amines by nitroreduction. Aromatic amines can be converted to an aromatic amide that is catalyzed by an acetyl coenzyme A-dependent acetylation.¹⁹⁷ The acetylation phenotype varies among the population. Persons with the rapid acetylator phenotype are at a higher risk of colon cancer (especially in smokers),¹⁹⁸ whereas, those who are slow acetylators are at risk of bladder cancer.¹⁹⁸ This latter association may result from the fact that activation of aromatic amines by N-oxidation is a competing pathway for aromatic amine metabolism. The N-hydroxylation products when protonated (e.g., in the urinary bladder) are reactive and can cause DNA damage.

An initial activation step for both aromatic amines and amides is N-oxidation by CYP1A2. The reactions of N-hydroxyarylamines with DNA appear to be acid catalyzed, but they can be further activated by either an acetyl coenzyme A-dependent O-acetylase or a 3′-phosphoadenosine-5′phosphosulfate-dependent O-sulfotransferase. The N-arylhydroxamic acids arise from the acetylation of N-hydroxyarylamines or N-hydroxylation of aromatic amides; they are not electrophilic and require further activation. The predominant pathway here occurs through acetyltransferase-catalyzed rearrangement to a reactive N-acetoxyarylamine. Sulfotransferase catalysis forms N-sulfonyloxy arylamides. This complex pathway results in two major adduct types, amides (acetylated) and amines (nonacetylated).

Heterocyclic amines form in food cooking from pyrolysis (>150 °C) of amino acids, creatinine, and glucose. They have been recognized as food mutagens, shown to form DNA adducts and cause liver tumors in primates.¹⁹⁹ These compounds are activated by CYP1A2 and their metabolites form DNA adducts in humans. The N-hydroxy metabolites of heterocyclic amines like 2-amino-3-methyl-imidazo-[4,5-f]quinoline (IQ) can react directly with DNA. Enzymic O-esterification of N-hydroxy metabolites plays a key role in activating food mutagens, and because the N-hydroxy metabolites are good substrates for transacetylases, these chemicals may be implicated in colorectal cancer.

Aflatoxins (B1, B2, G1, and G2), metabolites of Aspergillus flavus, contaminate cereals, grain, and nuts. A positive correlation exists between dietary aflatoxin exposure and the incidence of liver cancer in developing countries, where grain spoilage is high. Aflatoxins B1 and G1 have an olefinic double bond at the 8,9-position that can be oxidized by several CYP P450.^{173, 192} This implies that the olefinic 8,9-bond is the activation site. Further support for this mechanism comes from studies of DNA adducts and the prevalence of TP53 mutations in liver cancer. In people with liver cancer from parts of China and Africa, where food spoilage caused by molds is high, G : C to T : A transversions in codon 249 are frequent.^{42, 43, 200} This phenomenon is consistent with metabolic activation of aflatoxin B1 and the formation of depurinating carcinogen–deoxyguanosine adducts.

Carcinogenic N-nitrosamines are ubiquitous environmental contaminants and can be found in food, alcoholic beverages, cosmetics, cutting oils, hydraulic fluid, rubber, and tobacco.²⁰¹ Tobacco-specific N-nitrosamines (TSNs), for example, NNK, are not formed by pyrolysis, which accounts for the highly carcinogenic nature of snuff and chewing tobacco.²⁰² TSNs are not symmetric so both small alkyl adducts and large bulky adducts can be formed; for example, NNK metabolism gives rise to either a positively charged pyridyl-oxobutyl ion or a positively charged methyl ion, both of which are able to alkylate DNA.^{70, 203} Endogenous nitrosamines form when an amine reacts with nitrate alone or nitrite in the presence of acid. Thus, nitrite (used to cure meats) and L-cysteine, in the presence of acetaldehyde (from alcohol), form N-nitrosothiazolidine-4-carboxylic acid. N-Nitrosodimethylamine undergoes a-hydroxylation, catalyzed primarily by the alcohol inducible CYP2E1, to form an unstable a-hydroxynitrosamine. The breakdown products are formaldehyde and methyl diazohydroxide. Methyl diazohydroxide and related compounds are powerful alkylating agents that can add a small functional group at multiple sites in DNA.

Nongenotoxic carcinogens may function at the level of the microenvironment by dysregulation of hormones and growth factors, or indirectly inducing DNA damage and mutations through the action of free radicals. These chemicals are none or poorly reactive and are resistant to activation through metabolism. They are also characterized by their persistence in biological systems and consequently tend to accumulate in the food chain. However, they can stimulate oxyradical formation by at least three mechanisms: organochlorine species interact with the Ah receptor, which can lead to CYP P450 induction and associated oxyradical formation; interaction with other receptors, like interferon (IFN)-γ, can stimulate elements of the primary immune response and again generate oxyradicals; and agents like asbestos can promote oxyradical formation through interaction with ferrous metal. The resulting oxyradicals can then damage DNA. Some of the so-called “nongenotoxic” carcinogens might more appropriately be considered to be “oxyradical triggers.” Indeed, chronic inflammatory states, which involve oxyradical formation, can also be cancer risk factors.^204–208

DNA damage and repair

Another facet of inter-individual variation in risk for cancer from chemical carcinogens is inter-individual differences in the ability to repair damage from chemical carcinogens. DNA damage initiates a complex network of signaling cascades.²⁰⁹ The chemical structure of DNA can be altered by a carcinogen in several ways: the formation of large bulky aromatic adducts, small alkyl adducts, oxidation, dimerization, and deamination. In addition, double- and single-strand breaks can occur. Chemical carcinogens can cause epigenetic changes, such as altering the DNA methylation status that leads to the silencing of specific gene expression.⁶⁹ A complex pattern of carcinogen–DNA adducts likely results from a variety of environmental exposures, because of the mixture of different chemical carcinogens present.

Many DNA-repair genes have been described recently, and a growing number of polymorphisms have been identified for which molecular epidemiologic studies have provided evidence that genetic variation in these attributes can be a human cancer risk factor; as well as be used to tailor cancer chemotherapy.^{210, 211} Typically, these types of molecular epidemiological studies initially focus on high-exposure groups such as workers, patients taking therapeutic drugs, and tobacco smokers. Several polymorphisms in DNA-repair genes have now been implicated in tobacco-related neoplasms.²¹² An interesting theory developed recently is a so-called, “hide-then-hit” hypothesis. This describes the importance of DNA-repair variants in escaping checkpoint surveillance. Only cells with subtle defects in repair capacity arising from low-penetrance variants of DNA-repair genes would have the opportunity to grow and to accumulate the genetic changes needed for cancer formation, without triggering cell-cycle checkpoint surveillance.²¹³

BPDE reacts with the exocyclic (N2) amino group of deoxyguanosine and resides within the minor groove of the double helix, it is typical of PAHs. This adduct, BPdG, is probably the most common, persistent adduct of B(a)P in mammalian systems, but others are possible. Adducts like BPdG are thought to induce ras gene mutations, which are common in tobacco-related lung cancers.^{214, 215} Aromatic amine adducts are more complex, because they have both acetylated and nonacetylated metabolic intermediates, and they form covalent bonds at the C8, N2, and sometimes O6 positions of deoxyguanosine as well as deoxyadenosine. The major adducts, however, are C8-deoxyguanosine adducts, which reside predominantly in the major groove of the DNA double helix.²¹⁶

Aflatoxin B1 and G1 activation through hydroxylation of the olefinic 8,9-position results in adduct formation at the N7 position of deoxyguanosine. These are relatively unstable with a half-life of approximately 50 h at neutral pH; depurination products have been detected in urine.²¹⁷ The aflatoxin B1-N7-deoxyguanosine adduct also can undergo ring opening to yield two pyrimidine adducts; alternately, aflatoxin B1-8,9-dihydrodiol could result, restoring the DNA molecular structure if hydrolysis of the original adduct occurs.²¹⁸

DNA alkylation can occur at many sites either following the metabolic activation of certain N-nitrosamines, or directly by the action of the N-alkyl ureas (N-methyl-N-nitrosourea) or the N-nitrosoguanidines. The protonated alkyl-functional groups that become available to form lesions in DNA generally attack the following nucleophilic centers: adenine (N1, N3, and N7), cytosine (N3), guanine (N2, O6, and N7), and thymine (O2, N3, and O4). Some of these lesions are known to be repaired (O6-methyldeoxyguanosine), while others are not (N7-methyldeoxyguanosine), which explains why O6-methyldeoxyguanosine is a promutagenic lesion and N7-methyldeoxyguanosine is not.^{219, 220}

Another potentially mutagenic cause of DNA damage is the deamination of DNA-methylated cytosine residues. 5-Methylcytosine comprises approximately 3% of deoxynucleotides. In this case, deamination at a CpG dinucleotide gives rise to a TpG mismatch. Repair of this lesion most often restores the CpG; however, repair may cause a mutation (TpA).²²¹ Deamination of cytosine also can generate a C to T transition if uracil glycosylation and G-T mismatch repair are inefficient.

Oxyradical damage can form thymine glycol or 8-hydroxydeoxyguanosine adducts. Exposure to organic peroxides (catechol, hydroquinone, and 4-nitroquinoline-N-oxide) leads to oxyradical damage; however, oxyradicals and hydrogen peroxide can be generated in lipid peroxidation and the catalytic cycling of some enzymes, as well as environmental sources (e.g., tobacco smoke).^{204, 222} Certain drugs and plasticizers can stimulate cells to produce peroxisomes, and oxyradical formation is mediated through protein kinase C when inflammatory cells are exposed to tumor promoters like phorbol esters.²²³ Oxyradicals can contribute to deamination through induction of NO synthase.²²⁴

Maintenance of genome integrity requires mitigation of DNA damage. Thus, diminished DNA repair capacity is associated with carcinogenesis, birth defects, premature aging, and foreshortened life span. DNA-repair enzymes act at DNA damage sites caused by chemical carcinogens, and six major mechanisms are known: direct DNA repair, nucleotide excision repair, base excision (BER) repair, nonhomologous end joining (double-strand break repair), mismatch repair, and homologous recombination (HR) (postreplication repair).^{225, 226}

In the presence of nonlethal DNA damage, cell-cycle progression is postponed for repair mechanisms. This highly coordinated process involves multiple genes. A DNA-damage recognition sensor triggers a signal transduction cascade and downstream factors direct G1 and G2 arrest in concert with the proteins operationally responsible for the repair process. Although there are at least six discrete repair mechanisms, within five of them, there are numerous multiprotein complexes comprising all the machinery necessary to accomplish the step-by-step repair function.

Generically, DNA repair requires damage recognition, damage removal or excision, resynthesis or patch synthesis, and ligation. Recent advances have led to the cloning of more than 130 human genes involved in five of these DNA-repair pathways. A list of these genes and their specific functions was published elsewhere.²²⁷ These genes are responsible for the fidelity of DNA repair, and when they are defective, the mutation rate increases. This is the mutator phenotype.^{29, 60, 61} Mutations in at least 30 DNA-repair associated genes have been linked to increased cancer susceptibility or premature aging (Table 3).²²⁷ Moreover, the role of common polymorphisms in some of these genes is associated with increased susceptibility in a gene–environment interaction scenario (this is discussed in Weston and Harris¹⁷⁶). Indeed, molecular epidemiologic evidence suggests that tobacco-smoking-related lung cancer is associated with a polymorphism in the nucleotide excision repair gene, XPC (ERCC2).²²⁸

Repair mechanisms: BER, base excision; DSB, double-strand break; HR, homologous recombination; MMR, mismatch; NER, nucleotide excision; PRR, postreplication; SB, strand break.

Diseases: HNPCC, hereditary nonpolyposis colon cancer; NHL, non-Hodgkin lymphoma.

Other abbreviations: SCE, sister chromatid exchange; SCID, severe combined immunodeficiency.

Direct DNA repair is affected by DNA alkyltransferases. These enzymes catalyze translocation of the alkyl moiety from an alkylated base (e.g., O6-methyldeoxyguanosine) to a cysteine residue at their active site in the absence of DNA strand scission. Thus, one molecule of the enzyme is capable of repairing one DNA alkyl lesion, in a suicide mechanism. The inactivation of this mechanism by promoter hypermethylation is associated with Kras G to A mutations in colon cancer.²²⁹

In DNA nucleotide excision repair, lesion recognition, preincision, incision, gap filling, and ligation are required, and the so-called excinuclease complex comprises 16 or more different proteins. Large distortions caused by bulky DNA adducts (e.g., BPDE-dG and 4ABP-dC) are recognized [xeroderma pigmentosum (XPA)] and removed by endonucleases (XPF, XPG, and FEN). A patch is then constructed (pol and pol e) and the free ends are ligated.

Base excision repair also removes a DNA segment containing an adduct; however, small adducts (e.g., 3-methyladenine) are generally the target so that there is overlap with direct repair. The adduct is removed by a glycosylase (hOgg1 and UDG), an apurinic endonuclease (APE1 or HAP1) degrades a few bases on the damaged strand, and a patch is synthesized (pol β) and ligated (DNA ligases: I, II, IIIa, IIIβ, and IV).

DNA mismatches occasionally occur, because excision repair processes incorporate unmodified or conventional, but noncomplementary, Watson–Crick bases opposite each other in the DNA helix. Transition mispairs (G-T or A-C) are repaired by the mismatch repair process more efficiently than transversion mispairs (G-G, A-A, G-A, C-C, C-T, and T-T). The mechanism for correcting mispairings is similar to that for nucleotide excision repair and resynthesis described earlier, but it generally involves the excision of large pieces of the DNA containing mispairings. Because the mismatch recognition protein is required to bind simultaneously to the mismatch and an unmethylated adenine in a GATC recognition sequence, it removes the whole intervening DNA sequence. The parental template strand is then used by the polymerase to fill the gap.

Double-strand DNA breaks can occur from exposure to ionizing radiation and oxidation. Consequences of double-strand DNA breaks are the inhibition of replication and transcription, and loss of heterozygosity. Double-strand DNA break repair occurs through HR, where the joining of the free ends is mediated by a DNA-protein kinase in a process that also protects the ends from nucleolytic attack. The free ends of the DNA then undergo ligation by DNA ligase IV. Genes known to code for DNA-repair enzymes that participate in this process include XRCC4, XRCC5, XRCC6, XRCC7, HRAD51B, HRAD52, RPA, and ATM.

Postreplication repair is a damage-tolerance mechanism and it occurs in response to DNA replication on a damaged template. The DNA polymerase stops at the replication fork when DNA damage is detected on the parental strand. Alternately, the polymerase proceeds past the lesion, leaving a gap in the newly synthesized strand. The gap is filled in one of two ways: either by recombination of the homologous parent strand with the daughter strand in a process that is mediated by a helical nucleoprotein (RAD51); or when a single nucleotide gap remains, mammalian DNA polymerases insert an adenine residue. Consequently, this mechanism may lead to recombinational events as well as base-mispairing.

Persistent nonrepaired DNA damage blocks the replication machinery. Cells have evolved translesion synthesis (TLS) DNA polymerases to bypass these blocks.²³⁰ Most of these TLS polymerases belong to the recently discovered Y family, have much lower stringency than replicative polymerases, and thus are error prone. An increased mutation frequency is an evolutionary trade-off for cellular survival.

Mutator phenotype

Cancer cells contain substantial numbers of genetic abnormalities when compared to normal cells. These abnormalities range from gross changes such as nondiploid number of chromosomes, that is, aneuploidy, and translocations or rearrangements of chromosomes, to much smaller changes in the DNA sequence including deletions, insertions, and single nucleotide substitutions. Therefore, carcinogenesis involves errors in (1) chromosomal segregation, (2) repair of DNA damage induced by either endogenous free radicals or environmental carcinogens, and (3) DNA replication. Loeb originally formulated the concept of the mutator phenotype in 1974^{29, 60} to account for the high numbers of mutations in cancer cells when compared to the rarity of mutations in normal cells. Recent advances in the molecular analysis of carcinogenesis in human cells and animal models have refined the mutator phenotype^{61, 231} concept that is also linked to the clonal selection theory proposed by Nowell (Figure 4).²³² Generally, the mutator phenotype hypothesis proposes that mutation rates in normal cells are insufficient to account for the large number of mutations found in cancer cells. Consequently, human tumors exhibit an elevated mutation rate that increases the likelihood of a tumor acquiring advantageous mutations. The hypothesis predicts that tumors are composed of cells harboring hundreds of thousands of mutations, as opposed to a small number of specific driver mutations, and that malignant cells within a tumor therefore constitute a highly heterogeneous population.²³¹

Racial, gender, and socioeconomic disparities in chemical carcinogenesis

Genetic polymorphisms may also partially explain the increased risk of cancer-associated chemical carcinogens amongst different genders, race, and ethnicity. One of the biggest controversies surrounding this issue is the risk of lung cancer in women smokers. The initial studies finding a higher risk of lung cancer in smoking women versus smoking men²³³ have been explained biologically in a number of ways. Several sources of evidence, though inconsistent, suggest that estrogens may play a role in lung cancer. Variation in risk factors and tumor characteristics between men and women has been reported in studies showing that women are more likely to have adenocarcinomas of the lung, a higher risk in never smokers, higher levels of PAH–DNA adducts, higher levels of expression of the gene encoding CYP P450 (CYP) 1A1, more frequent G : C to T : A transversions in p53, and more frequent epidermal growth factor receptor (EGFR) mutations than men.¹⁸⁷ Inherited genetic polymorphisms affecting activating and detoxifying enzymes could also explain a different susceptibility between the sexes to tobacco carcinogens.²³⁴ In addition, several lifestyle and behavioral factors related to smoking habits or environmental and occupational exposures could account for some sex differences.²³⁵ However, in recent years, differences in the susceptibility to lung cancer among smoking men and women have been called into question.^{236, 237}

A consistent theme is that nonsmoking women are at increased risk for lung cancer compared to nonsmoking men^{237, 238}; and nonsmoking women are at increased risk for lung cancer due to environmental tobacco smoke (ETS) compared to nonsmoking women not exposed to ETS.^239–244 This risk may be associated with polymorphisms. For example, in female populations, it appears that a common genetic polymorphism divides the population of never smokers into two groups of approximately equal size, one (homozygous carriers of the GSTM1 null allele) that has a statistically significant greater risk of lung cancer from ETS than the other (heterozygous or homozygous carriers of the wild-type GSTM1 allele).²⁴⁰ In a similar context, genetic variants at 13q31.3 alter the expression of the glypican-5 (GPC5) gene, and down-regulation of GPC5 might contribute to the development of lung cancer in never smokers.^{245, 246} Other genes that have been found in never smokers and associated with lung cancer risk are LEM domain-containing protein 3 (LEMD3), transmembrane Bax inhibitor containing motif (TMBIM), ataxin 7-like 2 (ATXN7L2), Src homology 2 domain containing E (SHE), inter-α-trypsin inhibitor heavy chain H2 (ITIH2), Nudix (nucleoside diphosphate linked moiety X)-type motif 5 (NUDT5), EGFR, and echinoderm microtubule-associated protein like 4–anaplastic lymphoma kinase (EML4–ALK) fusions.^{243, 247, 248} Similarly, α(1)ATD carriers are at a 70–100% increased risk of lung cancer.²⁴⁹ Finally, as is the case for p53, the mutational profile of k-ras seems to differ greatly according to smoking status: G to T transversions are more common in ever smokers, whereas G to A transitions are seen more frequently in never smokers. K-ras and EGFR mutations are generally mutually exclusive, underlining their ordered participation in an intracellular signaling pathway, and suggesting different oncogenic mechanisms in lung cancer as a function of smoking status: tobacco carcinogens seem to be directly responsible for k-ras mutations, whereas the cause of mutations in EGFR, found particularly in never smokers, is currently not clear.

For race, it appears that compared to Caucasians, African-Americans are diagnosed earlier, have more aggressive, and consequently, have lower survival rates from breast cancers.^{250, 251} Racial disparities also exist in colorectal cancer.²⁵² Although these differences can be explained by socioeconomic differences, screening behaviors, and other behavioral differences, biological influences can affect risk. For example, polymorphisms in nucleotide excision repair genes may modify the relationship between breast cancer and smoking in African-American women.²⁵³ As well, the chemical carcinogen detoxifying gene, GSTM1, has a polymorphism that plays a role in the development of lung cancer and modifies the risk for smoking-related lung cancer in African-Americans.²⁵⁴ Interesting recent findings highlighting gene–environment interactions indicated household income is associated with the p53 mutational frequency in patients who develop breast cancer (especially ER-negative). Furthermore, high-income patients may acquire fewer p53 mutations than other patients, suggesting that lifetime exposures associated with socio-economic status may impact breast cancer biology.^{255, 256}

Chronic inflammation and cancer

More than a century ago, the German pathologist, Virchow proposed that inflammation was associated with cancer.⁸ Infection and inflammation significantly contribute to more than 25% of cancer cases worldwide (Table 4).²⁰⁴ Free radicals, endogenous chemicals, are released during the inflammatory response. These ROS and reactive nitrogen species are generated as a physiological protective response to pathogenic microorganisms and toxic agents. During chronic inflammation (e.g., chronic viral hepatitis) and oxyradical overload conditions (e.g., hemochromatosis and inflammatory bowel diseases), these free radicals can induce genetic and epigenetic changes including somatic mutations in cancer-related genes and post-translational modifications in proteins involved in DNA repair, apoptosis, and arachidonic acid cascade (Figure 5).⁸ Epigenetic transcriptional silencing of cancer-related genes including p16, Runt-related transcription factor 3 (RUNX3), and MutL homolog 1 (MLH1), by DNA methylation of their promoter regions has been associated with chronic inflammation in ulcerative colitis and Barretts esophagus.^{257, 258} Inflammation-mediated genetic and epigenetic alterations can also drive cancer development in the neighboring epithelium upon stromal abrogation of transforming growth factor-β signaling.²⁵⁹ Finally, polymorphisms in genes involved in the production of [e.g., COX2, NOS’s (NOS1, NOS2; but mostly NOS3)] or protection from (e.g., GpX, catalase, and MnSOD) free radicals also exist; can modify the effects of drugs; and increase or decrease cancer risk associated with exogenous chemical carcinogens.^260–268

“18% of human cancers, that is, 1.6 million per year, are related to infection.”—B. Stewart and P. Kleihues, World Cancer, Report, IARC Press, Lyon 2003, p. 57.

Rheumatoid arthritis is an example of a chronic inflammatory disease without a marked increased cancer risk, for example, joint sarcoma.

Oncogenic human papilloma viruses are examples of cancer-prone chronic infections without inflammation.

Oncogenes and tumor suppressor genes

Chronic exposures to carcinogens, accumulation of mutations, development of the mutator phenotype, and clonal selection during several decades result in cancer. Although the phenotypic traits of individual cancers are highly variable, commonly acquired capabilities include limitless replicative potential, self-sufficiency in growth signals, insensitivity to antigrowth signals, evading apoptosis, tissue invasion, sustained angiogenesis, and metastasis.⁶⁴ These phenotypic traits reflect a complex molecular circuitry of biochemical pathways and protein machines within cancer cells.²⁶⁹

The genes encoding the proteins within the cancer-associated molecular circuitry are of many functional classes and, historically, have been conceptually divided into oncogenes and tumor suppressor genes^{269, 270} (Box 4). Detailed descriptions of oncogenes and tumor suppressor genes are found in Chapters 4–7. The ras oncogene and the TP53 tumor suppressor gene will be used as examples of molecular targets of chemical carcinogens.

Box 4 Mutational hotspots in cancer genes

Oncogenes and suppressor genes are targets of chemical carcinogens. Many genes, including K-ras and p53, have specific sequences targeted by chemical carcinogens resulting in mutational “hotspots.” These mutations cause a change in expression and activity of the RNA and protein they code for.

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