part 2. mechanisms of

chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks Bernard W. Stewart PART 2 CH A PTER 11

Introduction by describing a tumour The types of biological agents and of as “an abnormal mass of , the now recognized by IARC For many decades, a corollary to growth of which exceeds and is un- as carcinogenic to (Group 1) the contemporary understanding of coordinated with that of the surround- are few compared with the number the nature of and of carcino- ing tissue, and that continues to grow of chemicals in this category (IARC genesis has been the recognition of in the same excessive manner after 2012a, b, c, d, e, f); there is a much causative agents. Since the 1950s, cessation of the stimulus that caused larger number of chemicals for which many agents that contribute to the it”. According to the same textbook, at least some evidence of carcino- development of cancer have been development of tumours of the , genicity is available (see Volumes categorized as initiators or promot- the alimentary canal, or the respira- 1–105 of the IARC Monographs, ers, on the basis of studies of chem- tory tract was to be expected among available from http://publications. ical carcinogenesis in mouse skin individuals exposed “to various iarc.fr). (Berenblum and Shubik, 1947). noxious agents in the environment”. Research has established how Cancer was described with ref- Causation of cancer in humans or many carcinogenic chemicals , erence to causative agents. Thus, by certain chemicals, radia- or are likely to cause, malignant a 1970s text (Cappell tion, and biological agents was rec- transformation, but the biological and Anderson, 1974) introduced ognized by early in the 20th century. processes involved are diverse, and

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 93 Table 11.1. A selection of proposals for the categorization of chemical carcinogensa

Mode of action Exposure context Chemistry relevance Agent type of bioassay data

Genotoxic PAHs DNA binding Atmospheric pollutants

Direct-acting Alcoholic N-nitroso PPARα activation beverages compounds

Pro- Occupation Aromatic amines α2u-Globulin Organic nephropathy

Inorganic carcinogen Halogenated Urinary tract calculi Endocrine disruptors organic compounds

Non-genotoxic Naturally Disinfection occurring by-products compounds

Solid-state carcinogen Pharmaceutical Inorganic Pharmacological drugs compounds steroids

Hormone Exogenous

Immunosuppressant

Promoter

PAHs, polycyclic aromatic ; PPAR, peroxisome proliferator-activated . a Knowledge about chemical is presented from a variety of perspectives apart from that of mechanism of action. The listings indicate those used in particular publications (e.g. Searle, 1984; Tomatis et al., 1990; Vainio et al., 1992; Vainio and Hietanen, 2003; Hsu and Stedeford, 2010) as ways of ordering data, as indicated by chapter headings in many cases, and are not necessarily comprehensive. Categories shown in bold involve or include at least one Volume 100 (Group 1) agent. there is no generally accepted mech- categorization of chemical carcin- “hallmark” or “hallmarks” have been anistic basis for classifying chemi- ogens on the basis of the af- published. These papers typically cal carcinogens (Loeb and Harris, fected (Warshawsky and Landolph, describe path- 2008), beyond categorization ac- 2006). ways and their therapeutic implica- cording to (Weisburger Currently, the most widely rec- tions. Although the characterization and Williams, 1981). There is no ognized description of the nature of by Hanahan and Weinberg (2011) single comprehensive basis for cat- cancer is that presented by Hanahan of did not egorization; chemical carcinogens and Weinberg in two reviews – pub- refer to chemical carcinogens or are sometimes ordered according lished more than a decade apart – causative agents in general, recent- to the context in which information is that identify the “hallmarks” of can- ly the hallmarks have been used to presented, with genotoxicity ordered cer (Hanahan and Weinberg, 2000, characterize chemical carcinogens according to , 2011). These papers have been so (Kleinstreuer et al., 2013). or agents categorized in relation to influential that others refer to “the These considerations give rise to differing classes of receptors. There hallmarks” without further qualifica- two questions: (i) whether previous- have been many proposals for the tion, for example in the title of a re- ly used mechanism-based descrip- categorization of chemical carcin- cent perspective on tumour metabol- tions of chemical carcinogens may ogens according to various crite- ism (Cantor and Sabatini, 2012). be recast in relation to the hallmarks; ria. A selection of these is shown Since 2000, about 200 cancer re- and (ii) whether, and to what extent, in Table 11.1; others include the search papers with a title including the hallmarks provide opportunities

94 to characterize agents apart from Morphological and genetic biological macromolecules had been currently known carcinogens as changes variously demonstrated over dec- contributing to the development of ades, carcinogen adducts in DNA cancer. Both of these matters are Within 20 years of the publica- were crucial. addressed in this chapter. tions cited above, the identification of DNA by N-nitroso of multistage carcinogenesis with compounds was shown by Magee Multistage carcinogenesis particular carcinogens or other exog- and Farber (1962), with tumorigene- enous agents had become irrelevant sis attributable to the pro-mutagenic Exogenous agents to an understanding of cancer de- O6-methylguanine product, which The widely accepted paradigm of velopment. Over the same decades, mispairs with . In rats, acti- carcinogenesis as involving a mul- the context in which carcinogenesis vation of H-Ras in tistage process is generally recog- was best understood changed from tumours induced by N-methyl-N′- nized to have been developed from rodents to humans. Critical to this nitrosourea was correlated with the two-stage model of carcino- transition was the identification of H-Ras at codons 12, 13, genesis in mouse skin (Berenblum multistage carcinogenesis with alter- and 61 (Sukumar et al., 1983). and Shubik, 1947), which typically ations in structure or expres- However, although this insight had involves a polycyclic aromatic hydro- sion rather than with the impact of been gained, it was clear that the eti- exogenous agents. carbon (PAH) and a phorbol ester ology of some types of cancer, such PART 2

(identified as the active agent in the A key development was the as cancer in humans, did not CH A PTER 11 irritant croton oil). Because tumor- correlation by Vogelstein et al. (1988) primarily involve alkylating agents. igenesis in animals is amenable to of morphological change during the Thus, in human cancer RAS activa- histological examination at all stag- development of colon cancer in hu- tion is a relevant genetic change in es, morphological criteria can be mans with particular genetic change. tumour tissue, without reference to used to characterize the process. The concept was applicable to all exogenous agents (Bos et al., 1987). With the production of malignant tu- tumour types. Thus, in a diagram il- Although the concept of multi- mours as the end-point, two-stage or lustrating multistage carcinogenesis stage carcinogenesis was estab- multistage carcinogenesis was read- with respect to human cancer, lished through the use of exogenous ily described in various organ sites in Harris (1992) made no reference to agents that target particular organ animals, including the and the any particular exogenous agents as sites in animals, by 1990 multistage bladder (Slaga et al., 1978). mediating specific stages in tumori- carcinogenesis was primarily iden- Thus, in relation to hepatocarcino- genesis, and showed the transitions tified with altered structure or ex- genesis, agents such as phenobarbi- between stages as being mediated pression of associated with tal, dichlorodiphenyltrichloroethane, by alterations in the structure or ex- proliferation, specifically as de- polychlorinated biphenyls, butylated pression of and tumour scribed in human tumours. However, hydroxytoluene, and ben- suppressor genes. the focus of that research has not zoate were identified as promoters Oncogenes and tumour suppres- involved the specification of genetic (Dohi et al., 1996). The relevant sor genes mediate altered prolifera- change over time in a manner that experimental observations, in addi- tive activity in a positive and nega- might account for the emergence of a tion to indicating the possible risk to tive sense, respectively. Classically, metastatic cell population from with- humans presented by the relevant increased proliferative activity due in normal tissue. Rather, the relevant chemicals, also led to the contempo- to expression accounted research has involved the identifica- rary understanding of the nature of for the transformation of NIH 3T3 tion of disordered signal transduction malignancy itself. That understand- cells by DNA isolated from tumours pathways, with a view to developing ing was based on the identification of and not by DNA from normal tissue targeted . The archetype particular abnormal cell populations, (Shih et al., 1981). Oncogene ac- of such research is that establish- specifically including chemically in- tivation (e.g. mutation of Ras) has ing the transforming role of the ty- duced hyperplastic nodules in rat liv- shown that although binding of many rosine BCR-ABL in chronic er (Farber, 1973). chemical carcinogens to diverse myeloid leukaemia, and its inhibition

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 95 – to the great benefit of patients – by emerge early, whereas others – sus- • enabling replicative ; the low-molecular-weight inhibitor tained , and tissue inva- and STI-571 () (Bilanges and sion and – are seen later. • activating and metastasis. Stokoe, 2007; Rosa et al., 2008). Although hallmarks such as sus- It is notable that, in almost every tained angiogenesis and metasta- instance, the hallmark is not the Molecular changes sis involve morphological change, name of a but refers to Among a series of reviews marking all of the hallmarks were identified a dynamic process. Consistent with the publication of the 100th volume with reference to changes in gene this perception, the authors wrote, of the journal Cell, Hanahan and expression and not by reference to, “The hallmarks of cancer comprise Weinberg (2000) delineated the very or necessarily in correlation with, six biological capabilities acquired wide (even then) spectrum of stud- a change in morphology. Diversity during the multistep development ies addressing the of can- between tumour types and within a of human tumours. The hallmarks cer by reference to phenotype. Six given tumour type was noted, and no constitute an organizing principle for characteristics of how cancer cells reference was made to any particu- rationalizing the complexities of neo- behave could be identified in rela- lar type of for illustrative .” tion to particular genes or classes of purposes. In addition, a decade of progress genes. The phenotypic characteris- In such a description of the mani- had enabled the specification of two tics were: uncontrolled proliferative festation of essential alterations that “emerging hallmarks”: activity (Hall, 1984), tumour growth collectively characterize malignant attributable to familial risk (Hussain • deregulating cellular energetics; growth, there is no requirement to and Harris, 1998), survival of can- and identify exogenous agents as act- cer cells (Vaux et al., 1988), immor- • evading immune destruction. ing on normal or premalignant cells talization of cancer cells (Sedivy, The enabling characteristic iden- to cause the change. The focus is 1998), growth of vessels in tified in 2000 as “genomic instabili- tumours (angiogenesis) (Cavallaro on the nature of tumours and how ty” was described in 2011 as “geno­ and Christofori, 2000), and metastat- they may be distinguished from rele- mic instability and mutation”, and a ic growth (Webb and Vande Woude, vant normal tissue. Finally, Hanahan second enabling characteristic was 2000). Accordingly, the hallmarks and Weinberg (2000) identified an identified as “tumour-promoting of cancer were initially identified as en­abling characteristic: genomic ”. Superficially, such follows: instability, which is equated with in- reference to mutation and to pro- • self-sufficiency in growth signals; creased mutability evident during motion might be seen as implying, if • insensitivity to anti-growth signals; the process of tumour progression not specifying, the roles that DNA- (Loeb, 1994). • evasion of ; damaging and proliferation-induc- ing agents have in carcinogenesis. • sustained angiogenesis; A decade on: “the next However, this is not the case. generation” • limitless replicative potential; and In this context, “mutation” refers • tissue invasion and metastasis. to an acceleration of the accumu- In 2011, Hanahan and Weinberg The 2000 “hallmarks” review was lation of , due to, among provided a new assessment of the concerned primarily with the charac- other things, defects in the DNA hallmarks (Hanahan and Weinberg, terization of the genes and associat- maintenance machinery (Kinzler 2011). They commented, “The past ed signal transduction pathways that and Vogelstein, 1997). As a result, decade has witnessed remarkable mediate these respective activities mutation occurs more readily, ir- in malignant cells and tumours. In progress towards understanding the respective of whether it is mediat- that paper, hypothetical patterns of mechanistic underpinnings of each ed by exogenous or endogenous multistage carcinogenesis were illus- hallmark.” One indication of progress agents. Accordingly, DNA adducts, trated by a linear arrangement of the is that the original hallmarks were strand breakage, and related phe- pictograms for the hallmarks, without rebadged as follows: nomena are not to be identified with reference to any morphological crite- • sustaining proliferative signalling; this enabling characteristic and do ria. From that diagram, it can be in- • evading growth suppressors; not account for, or are not proper- ferred that some hallmarks – such as • resisting cell ; ly identified with, a particular hall- self-sufficiency in growth signals – • inducing angiogenesis; mark. Mutation, in the context of

96 carcinogenesis, identifies a mecha- “genotoxic” indicated, among other include bacterial, mammalian, and nism whereby a chemical carcinogen things, that the covalent binding of other cells, with weight being given may cause the emergence of any of a carcinogen adduct to DNA, when to the extent to which the test system the hallmarks, and almost certainly evident, might account for carcino- has been “validated”, as summa- of several of them, or perhaps of all genesis. Thus, Weisburger and rized by sensitivity and specificity in of them. The enabling characteristic Williams (1981) categorized carcin- relation to known carcinogens and “genomic instability and mutation” ogens primarily on the basis of ge- non-carcinogens. In vivo indicators renders such outcomes more likely notoxicity. Research over the sub- of genotoxicity include, among oth- (Wang et al., 2012), rather than refer- sequent 30 years did not alter that ers, (i) of a chemical to ring to the mechanism through which approach (Hsu and Stedeford, 2010). produce reactive, typically electro- the change occurs. The multiplicity of agents and the philic, intermediates, which are the The identification of “tumour-pro- relatively limited understanding of source of adducts bound to DNA and moting inflammation” as the second their respective mechanisms of ac- other macromolecules, and (ii) ev- enabling characteristic recognizes tion have precluded the adoption of a idence of subsequent DNA repair that inflammation causes the emer- scheme for categorizing carcinogens and/or mutation. gence of several of the hallmarks, beyond the consideration of geno- This description of indicators of including sustaining proliferative sig- . Arguably, until the present genotoxicity also summarizes the nalling and inducing angiogenesis. In IARC Scientific Publication, the most

relevant mechanism of chemical PART 2 their discussion of this enabling char- authoritative assessment on how

carcinogenesis as currently under- CH A PTER 11 acteristic, Hanahan and Weinberg carcinogens act was the 35-page stood (Cohen and Arnold, 2011). (2011) were concerned primarily with consensus report in the publica- Thus, carcinogen metabolism and cellular infiltration by cells of both tion Mechanisms of Carcinogenesis DNA repair processes have been the innate and the adaptive arms of in Risk Identification (Vainio et al., used to identify candidate genes the immune response. They made 1992); this was the agreed position for susceptibility stud- scant, if any, reference to exogenous of a Working Group of more than 40 ies (Yokota et al., 2010). Compared agents provoking an inflammatory scientists in 1991. The consensus re- response. with the relatively modest number of did not centre on a scheme for From a broad perspective, refer- classifying carcinogens according to genes that account for the absorp- ence to the multistep development their mechanism of action. tion, metabolism, and elimination of a of human tumours provides a way Across decades, commentaries carcinogen, together with the repair to consider the particular impact of on chemical carcinogens (Van of corresponding DNA adducts, the carcinogens and other exogenous Duuren, 1980; Pitot, 1990; Xue and hallmarks (Hanahan and Weinberg, agents that may contribute to can- Warshawsky, 2006; Cohen and 2011) enable the specification of cer development. However, in iden- Arnold, 2011) have not been based tens – if not hundreds – of genes tifying the hallmarks, Hanahan and on any generally agreed categori- whose expression contributes to the Weinberg did not pursue this matter. zation according to mechanism of malignant phenotype. At the single-gene level, mutation Identifying mechanisms of action. Rather, the common theme carcinogenesis has been the enumeration of bio- of TP53, specified with reference to logical parameters that may deter- particular transitions and transver- As mentioned above, chemical car- mine whether tumours develop in sions, is attributable to miscoding, cinogens have been categorized response to carcinogens in general. which in turn is a consequence of primarily with reference to whether DNA adduct formation from rele- Genotoxicity: progress and vant carcinogens, including those in they exhibit genotoxicity. This mech- problems anistic distinction began with many (Soussi, 2011). The then-known carcinogens being iden- Multiple indicators of genotoxicity data provide evidence of particular tified as in vitro by use of have been recognized and catego- exposures, but it remains unclear particular bacterial strains and after rized as involving data generated how tumorigenesis is enhanced metabolic activation (the ) either in vitro or in vivo (Montesano by such mutation, beyond the (McCann et al., 1975). The term et al., 1976). In vitro test systems consideration that a functional

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 97 Table 11.2. Chemicals cited by Ashby (1992) and Eastmond (2012) as examples of compounds with equivocal genotoxicity

Chemicals identified byAshby (1992) Chemicals identified byEastmond (2012)

3-Amino-4-ethoxyacetanilide Bromate

3-Amino-9-ethylcarbazole.HCl

Chlorinated paraffins

CI Acid Orange 3

CI Basic Red 9.HCl (III)

Cinnamyl anthranilate Chromium(VI)

1,2-Dibromo-3-chloropropane 1,3-Dichloro-2-propanol

di-Menthol 1,4-Dioxane

Methyldopa sesquihydrate Ethylene glycol monobutyl ether

5-Nitroacenaphthene

4-Nitro-o-phenylenediamine 2-Nitrotoluene

Piperonyl butoxide Trichloroacetic acid

Piperonyl sulfoxide 1,2,3-Trichloropropane

1,2-Propylene

Sulfallate

induces apoptosis, cell-cy- 10 times that in lung tumours from not evident from genomic analysis cle arrest, and , and never-smokers (Govindan et al., (Muzny et al., 2012). In short, the role that these processes are compro- 2012). of mutation as contributing to cancer mised after TP53 mutation (Bieging However, analysis of lung cancer development may be elucidated with- and Attardi, 2012). The hallmarks genomics does not require immedi- out reference to any genotoxic agent, offer a broadened perspective as ate reference to smokers and nev- even when the role of such an agent to signalling pathways that may be er-smokers to present relevant data has been otherwise established. affected by mutation of TP53 or any (Liu et al., 2012; Peifer et al., 2012). Distinguishing genotoxic tumour suppressor gene. Moreover, the recognition of tobac- from non-genotoxic In the first such determination co-induced genomic injury does not carcinogens made, genotoxic injury by tobac- necessarily extend to other sites; for Even though molecular process- co smoke in one individual case of example, on the basis of individual es associated with genotoxicity are lung cancer accounted for 22 910 genomic analysis, it is not possible being defined in steadily greater base substitutions, of which to differentiate between cases of detail, it is not always possible to 134 were in coding sequences in smokers and in immediately discriminate between (Pleasance et al., 2010). The role never-smokers (Wei et al., 2012). individual chemicals on the basis of tobacco smoke as a determinant More generally, although mutation of whether particular substances of the genomic landscape of lung of TP53 is highly relevant to colorec- should be categorized as genotox- cancer has been confirmed, with an tal cancer, the impact of exogenous ic. Difficulties are evident when rel- average mutation frequency in lung influences or causal factors on the evant chemicals are considered on tumours from smokers of more than development of this tumour type is a case-by-case basis. More than

98 Table 11.3. Examples of categories of non-genotoxic carcinogens as variously proposed over more than three decadesa

Weisburger and Weisburger (1989) Marquardt (1999) Hernández et al. Benigni et al. (2013) Williams (1981) (2009)

Solid-state Halogenated Cytotoxic Endocrine modifiers Peroxisome proliferators carcinogens compounds carcinogens

Hormones Immunosuppressants Tumour promoters Receptor-mediated Gap-junction inhibitors

Immunosuppressants Hormones Hormones Non-receptor- DNA-methylating agents mediated

Co-carcinogens Solid-state materials Immunosuppressants Promoters Agonists/antagonists of the aryl receptor

Promoters Certain Peroxisome Tissue-specific toxicity Oxidative inducers hypolipidaemic proliferators and inflammation carcinogens inducers

Phthalate ester Solid bodies or Cytotoxic agents and Hormonal imbalance particles immunosuppressants inducers PART 2 CH A PTER 11 Gap-junction inhibitors

a Typically, the listings have been provided by the respective authors for illustrative purposes, without necessarily specifying an intent to be comprehensive.

20 years ago, Ashby (1992) reported • the agent’s metabolism and Non-genotoxicity: multiple on “practical examples of instances toxicokinetics; mechanisms and pathways in which the term genotoxic is both • structural similarities to recognized Regardless of any difficulty with par- needed and capable of having differ- mutagenic carcinogens; ticular agents as discussed in the ent meanings”. Two decades later, • the origin of or mechanisms under- previous section, the conceptual ba- Eastmond (2012) provided insight by lying the observed effects; and sis of genotoxicity is unequivocally summarizing data for another set of • in vivo data, particularly in the focused on a particular pathway to chemicals, different from those dis- target organ. . No such cussed by Ashby (Table 11.2). Eastmond (2012) illustrated each Hence, there are some chemicals single focus is available for non- of these points with two or more that are not readily categorized in re- genotoxic carcinogens, as illustrat- examples. lation to genotoxicity because, for ex- ed by the designation “epigenetic”, Specifying genotoxicity is com- ample, they produce positive results which, although previously applied plex, as becomes evident when when assessed by use of in vitro to these agents (Weisburger and all available mechanistic data are genotoxicity tests but after their ad- Williams, 1981; Benigni et al., 2013), identified, as occurs, for example, ministration to intact animals, they do can no longer be unequivocally used in IARC Monographs evaluations. In not cause structural DNA damage or in this context. other manifestations of genotoxicity. some instances, the totality of avail- Epigenetic processes are relevant As described by Eastmond (2012), able mechanistic data may indicate to both genotoxic and non-genotoxic apparently contradictory findings that the categorization of a carcino- agents (Pogribny et al., 2008), and can be reconciled when, for differ- gen as genotoxic is equivocal. There epigenetic change may be deter- ent individual chemicals, account is does not appear to be a context in mined by mutation (You and Jones, taken of: which awareness of the hallmarks 2012). From a different perspective, • the chemical properties of the would provide an improved basis for when discussing non-genotoxic car- agent, its metabolites, and/or its identifying genotoxic carcinogens cinogens, Meza et al. (2010) iden- degradation products; specifically. tified tobacco smoke and in

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 99 this context. Despite such ambigu- of cell differentiation and cycling, equally recognized (Schetter et al., ity, 45 non-genotoxic carcinogens hormonal and nutritional homeosta- 2010). Cell proliferation in this con- were recognized in 2009 among sis, coordination of cellular stress text does not pertain to proliferation 371 agents classified by IARC in responses (including inflammation after toxic injury by genotoxic agents. Group 1, Group 2A (probably carci- and apo­), immune responses, Proliferative activity induced by ge- nogenic to humans), and Group 2B and ageing. Therefore, it is difficult nomic injury may be considered in (possibly carcinogenic to humans) to identify AhR-mediated processes relation to the pluripotent stem cells (Hernández et al., 2009). with a specific hallmark. (Cohen and Arnold, 2011), further in- Grouping agents on the basis of a The adoption of a mechanistic ap- dicating how a characteristic – such default criterion – i.e. that the agent proach to categorize non-genotoxic as the hallmark “sustaining prolifer- is not genotoxic – implies uncertain- carcinogens to incongruities ative signalling” – cannot readily be ty. The scope of uncertainty can be if definitive and exclusive specifica- assigned or restricted to a particular seen from differences between re- tions are sought. Thus, TCDD may category of carcinogens. ports indicating categories of agents be readily identified as a that are reasonably considered (Ray and Swanson, 2009) while also Public decision- to be non-genotoxic carcinogens; being recognized as a complete car- making: the definitive Table 11.3 shows selected examples cinogen on the basis of bioassay and consideration from 1981 to 2013. epidemiological data (Baan et al., Parameters used to identify 2009). Similarly, although PAHs can This IARC Scientific Publication is non-genotoxic carcinogens include be identified with the genotoxicity of, based on evaluations made in Volume either the nature of the agent or for example, tobacco smoke, Puga 100 of the IARC Monographs. Two some indicator of a putative mecha- et al. (2009) noted that exposure to broad issues are addressed: (i) the nism of action. The terminology is far toxic PAHs raises several toxic and extent to which the occurrence and from definitive. Thus, while the term carcinogenic responses in experi- anatomical site of agent-attributable “promoter” may be used to identify a mental animals and humans, medi- cancer in humans may be correlat- non-carcinogen that contributes to ated for the most part by AhR. Such ed with the occurrence and, where tumour development, tumour promo- apparent paradoxes indicate that relevant, organ site of tumours in tion may be identified with the action although mechanistic categorization animals treated with the same agent; of many non-genotoxic carcinogens of many genotoxic carcinogens is and (ii) whether known mechanisms (Schulte-Hermann et al., 1999). definitive and exclusive, the same of action of the carcinogenic agents in question, considered together The role of receptors has long process applied to non-genotoxic with current knowledge of cancer been recognized as key to the car- agents may to outcomes de- etiology, reveal options for catego- cinogenicity of many non-genotoxic termined by context. The relevant rizing carcinogens, so as to better agents (Lucier, 1992) and under- agents cannot be identified with a indicate the risk posed to humans by pins current commentaries (Klaunig, single path to malignancy. exposure. 2010). Relevant receptors include The role of cell proliferation in re- These two considerations are inti- the aryl hydrocarbon receptor (AhR), lation to non-genotoxic agents also mately related. Thus, the occurrence the peroxisome proliferator-activated depends on the context (Preston- or absence of tumours in rodents receptor (PPAR), and various hor- Martin et al., 1990; Marquardt, treated with particular agents may mone receptors. 1999). With respect to chemicals, the be wholly dependent on biological Arguably, AhR is recognized original focus was on , in- mechanisms operating, or not oper- mainly as mediating the carcino- cluding peroxisome-proliferating car- ating, in particular species. Until now, genicity of 2,3,7,8-tetrachlorodiben- cinogens (Butterworth et al., 1992). mechanistic assessment of carcino- zo-para-dioxin (TCDD). However, This approach now identifies inflam- gens has not established a compre- as specified by Matsumura et al. mation as contributing to cancer de- hensive basis for determining wheth- (2009), apart from mediating toxic velopment, and auto-inflammatory er particular agents are capable of effects of some pollutants, AhR is disease and the impact of various causing cancer in humans. This situ- involved in development, regulation cancer-causing infectious agents are ation confirms that evaluations of the

100 IARC Monographs are appropriate available data are ordered according correlates with the enabling charac- for identification, as distinct to these end-points, it is evident that teristic “genomic instability and mu- from any simple categorization of for many agents, simple categoriza- tation”. The end-points “alterations in relevant agents. The fact that agents tion according to a single mechanism length” and “immortaliza- may be classified into Groups does is not possible or appropriate. tion” address the hallmark “enabling not alter the need to make evalua- An important consideration is the replicative immortality”. tions on a case-by-case basis. discrepancy between the extents It would appear that the hallmark The determination of whether a to which end-points have been as- “evading growth suppressors” cor- chemical induces cancer through sessed. DNA damage and gene responds to end-points identified by a genotoxic mechanism frequently mutations have been studied most cell-cycle effects taken together with plays an important role in evaluating extensively, and agents for which a subset within the end-point “gene the risks associated with low expo- there is unequivocal evidence of ge- mutations”: the subset of mutation of sures (Eastmond, 2012). For low notoxicity across in vitro and in vivo tumour suppressor genes as distin- levels of exposure to non-genotoxic guished from mutation of oncogenes carcinogens, there is expected to systems have rarely been studied in or other genes. The default position be a dose–response threshold for relation to, for example, epigenetic the carcinogenic effects; this does alterations. Epigenetic alterations would then be to identify “sustaining not apply to genotoxic carcinogens have been described for estrogenic proliferative signalling” – arguably the premier hallmark – with the re- (Klaunig, 2010). Low-dose models of hormones (Imamura, 2011), arse- PART 2 induction in by geno­ nic (Jensen et al., 2008), and maining end-points. However, ref- CH A PTER 11 toxic carcinogens indicate further (Costa et al., 2005), although each erence to those end-points leads to levels of complexity (Williams, 2012), of these agents had also been char- the recognition that end-points such and ongoing controversy about acterized as causing DNA damage. as “epigenetic alterations” are the non-monotonic responses means Evidence of means through which many, if not all, that such issues remain pertinent may have been considered as a sin- of the hallmarks may emerge. (Fagin, 2012). Mechanisms that un- gular mechanism of carcinogenesis, Finally, “deregulating cellular en- derpin, for example, dose–response but while can be char- ergetics” remains as the hallmark curves may become amenable to ge- acterized as immunosuppressive, not addressed through the char- nomic and related analyses. this agent also causes DNA damage. acteristics identified, because this Having been adopted as de- parameter has not been recognized Systematic appraisal in systematic efforts to character- of mechanisms of scribed, the 10 key characteristics ize mechanisms of carcinogenesis. carcinogenesis warrant review with reference to the hallmarks as cataloguing a broad Overall, no particular insight ap- Information about mechanisms of biological basis for malignancy pears to be gained by attempting to carcinogenesis for the Group 1 (Hanahan and Weinberg, 2011). One relate the 10 key characteristics with agents in the IARC Monographs hallmark, “activating invasion and specific hallmarks. is summarized in this Scientific metastasis”, is not recognized as a Tobacco smoke, cancer of the Publication with initial reference to mechanistic end-point because few, lung, and the hallmarks 24 mechanistic end-points, which if any, agents are identified primarily were then merged into 10 key with metastatic growth, given that no Generalizing across tumour types, characteristics (see Chapter 10, by such hazard needs to be established genomic and comparable analyses Smith). These end-points – which over and above carcinogenicity. are concerned little, if at all, with ex- include DNA damage, changes in Some hallmarks are singularly iden- ogenous agents that mediate malig- , receptor-mediated tified as mechanistic end-points or nant transformation. Paradoxically, effects, and inhibition of gap junc- enabling characteristics, i.e. those the first tumour document- tional intercellular communication – corresponding to chronic inflam- ed was described with a total focus have been adopted on the basis of mation, immune effects, , on mutations attributable to tobac- their wide use to investigate mech- and angiogenic effects. Arguably, co smoke (Pleasance et al., 2010). anisms of carcinogenesis. Once the the end-point “DNA repair alteration” Although genomic analysis revealed

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 101 Fig. 11.1. Hallmarks of lung . Left: The prevalence of mutation or somatic copy number alterations of genes mapping to cancer hallmarks defined by Hanahan and Weinberg (2011) based on tumour specimens from a cohort of 183 patients of whom more than 85% had a history of . Top right: Genes comprising the mutated genes in the hallmark “sustaining proliferative signalling” are shown. Bottom right: A proposed new hallmark of “epigenetic or RNA deregulation” is shown, depicted as above. Genes shown in grey are candidate lung adenocarcinoma genes identified in the study of Imielinski et al. (2012) that may additionally contribute to the hallmark. Reprinted from Imielinski et al. (2012), copyright 2012, with permission from Elsevier.

102 an average mutation frequency in Possible inferences from oncogene activation was achieved lung tumours from smokers of more hallmark-based studies by genetic manipulation or after ex- than 10 times that in lung tumours posure to an alkylating N-nitroso from never-smokers (Govindan et al., Any malignancy is expected to ex- compound (Westcott et al., 2015). 2012), the genomic pattern of squa- hibit the hallmarks, whether it arises Hence, genomic analysis may reveal mous cell lung cancer, established spontaneously or upon exposure to a distinct patterns of tumour-associ- from 178 patients of whom 96% had carcinogen. Insight into mechanisms ated changes that are dependent of carcinogenesis is gained by the a history of smoking, was presented on etiology and relevant to the full demonstration of biological change, with no overt reference to tobacco scope of tumour-associated signal which may be aligned with a hallmark use (Hammerman et al., 2012). transduction as identified by the (He et al., 2014). The The genomic profile of lung ade- hallmarks. implications of such a discovery may nocarcinoma, involving a cohort of Apart from any mechanistic cate- apply to agents not recognized as patients of whom more than 85% gorization of carcinogens in relation carcinogenic but shown to be pro- had a history of smoking, was pre- to particular hallmarks, the hallmarks moters and/or inducers of inflamma- sented with reference to the hall- do provide a basis for innovation. tion or angiogenesis. Nicotine is an Genes identified from the perspec- marks, documenting the prevalence example of such an agent (Cardinale tive of each hallmark provide a ba- of the enabling characteristic “geno- et al., 2012; Schaal and Chellappan, sis on which to analyse both known mic instability and mutation” in 25 ad- 2014). In addition to its contribution carcinogens and agents of unknown PART 2 enoma genes adopted as indicators to a better understanding of tobacco status in that regard. An indication CH A PTER 11 (Imielinski et al., 2012). The findings smoke carcinogenesis, this informa- of agents worthy of attention may were not presented with reference tion about the properties of nicotine well be achieved by adding hall- to smoking status but indicated is relevant to appropriate regulation mark-related targets in the context of markedly different fractions of mu- of electronic (also known high-throughput assays, tation (Fig. 11.1), including 42% with as electronic nicotine delivery sys- as described by Kavlock and col- respect to “genomic instability and tems) (Dutra and Glantz, 2014). leagues (Kleinstreuer et al., 2013). mutation”. This result indicates the Nicotine may contribute to cancer The outcome may be the recognition requirement to distinguish between development, for example by stimu- of new classes of that contrib- gene mutation being relevant to eti- lating angiogenesis, in a manner not ute to increased risk of cancer. ology, whether or not it is caused by likely to result in the compound being designated a carcinogen. an exogenous agent, and frequency Summary of mutation being an indicator of ge- During the past 50 years, the nomic instability and thus a charac- understanding and use of the term Cancer was once described with ref- “carcinogenesis” has changed from teristic of malignancy. Also of note, erence to causative agents, and mul- that involving a necessary reference only 6% of tumours had alterations tistage development of tumours was to one or more exogenous carcin- assigned to all six original hallmarks. characterized through the impact of ogens to that involving intracellu- Mutation of genes that mediate particular chemicals. Subsequently, lar processes leading to malignant multistage development of cancer particular hallmarks and are at- transformation, with no necessary was identified with morphological tributable to, among other agents, or implied reference to exogenous change being correlated with altered N-nitroso derivatives of nicotine and agents. This understanding has genetic makeup. The more recent related compounds, and PAHs, is to recently included the description of description of eight hallmarks of ma- be expected. However, beyond lung random mutations arising from DNA lignancy is based not on morpholo- cancer, there are only few references replication in normal non-cancerous gy or on the impact of carcinogens to genomic analyses that enable indi- stem cells as accounting for sporadic but on changes in gene expression, vidual tumours attributable to smok- disease (Tomasetti and Vogelstein, sometimes mediated by mutation, ing to be distinguished from others. 2015). However, another recent and on selection for growth. Thus, genomic analysis did not re- development is the identification In parallel to this of our veal likely tobacco causation for par- of different mutational landscapes understanding of cancer, no gener- ticular pancreatic (Biankin between classes of K-ras-driven ally recognized mechanism-based et al., 2012). tumours, depending on whether scheme for classifying carcinogens

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 103 has evolved beyond categorization variously resulting in emergence signal transduction pathways as- of chemical carcinogens accord- of the hallmarks, with the relevant sociated with particular hallmarks ing to genotoxicity. When appropri- processes being facilitated by ge- may provide new understanding of ately studied, both genotoxic and nomic instability and inflammation. agent-related carcinogenesis. non-genotoxic agents may medi- Enhancing –omics-based screening ate genetic and epigenetic change, procedures to specifically include

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