Chapter 13. Alterations in Cell Proliferation, Cell Death, Or Nutrient Supply Jane C

part 2. mechanisms of carcinogenesis

chapter 13. Alterations in cell proliferation, cell death, or nutrient supply Jane C. Caldwell PART 2 CHAPTER 13

Introduction these agents – that can be used to 1986; Jayat and Ratinaud, 1993; identify and organize mechanistic in- Stacey and Hitomi, 2008; Irish and Mechanistic data have been information related to cancer induction Doxie, 2014). Although for many of cluded in Volume 100 of the IARC (see Chapter 10, by Smith; see also the human carcinogens Volume 100 Monographs, and they vary with the Smith et al., 2016). One of these 10 of the IARC Monographs contains agent studied. These data are espe- mechanistic categories of data is a more descriptive data under this cat- cially dependent on the type of study composite that includes information egory, primarily as changes in cell and the contemporary understand- on the ability of a carcinogen to alter proliferation, these changes are in- ing of the state of the science at the cell proliferation, cell death, or nutri- herently related to alterations in cell time of publication of the study. ent supply. signalling and/or cell-cycle control. As an outcome of the two-part Alteration of cell proliferation is Many challenges are associated IARC Workshop on Tumour Site identified through assays that detect with the use of data described for Concordance and Mechanisms replicative DNA synthesis, 5-bro- of Carcinogenesis, a mechanistic mo-2′-deoxyuridine (BrdU) labelling, alterations in cell proliferation, cell database was assembled for the proliferating cell nuclear antigen death, or nutrient supply to examine IARC Group 1 carcinogens (see (PCNA) labelling, and hyperplasia mechanistic and tumour site concor- Chapter 22, by Krewski et al.). These or the occurrence of multinucleated dance between humans and experi- agents were examined with regard to cells by light microscopy, and through mental animals. Several key mecha- 10 key characteristics – one or more analysis of some of these end-points nistic characteristics can result in or of which are commonly exhibited by by use of flow cytometry (Gray et al., arise from changes in cell signalling

Part 2 • Chapter 13. Alterations in cell proliferation, cell death, or nutrient supply 117 (e.g. inflammation, genotoxicity, and This chapter focuses on issues as- ellers, growth factors, growth factor epigenetic alterations) and can have sociated with the understanding and receptors, signal transducers, and both genetic and epigenetic origins. interpretation of available data for apoptosis regulators (Narayanan There are other levels of interde- this key mechanistic characteristic. et al., 2015). In response to mito- pendence between the key mecha- gens, cell proliferation is triggered by nistic characteristics. Inflammation, Genetic drivers of cell increased translocation into the nu- excessive oxidative stress, and ge- proliferation and apoptosis: cleus of ERK 1 and 2 (ERK1/2), the complex relationships and nomic instability are related (see last proteins in the MAPK/ERK cas- pleiotropic roles of cell Chapter 17, by Kane). Mutagenesis cade. Activating mutations upstream signalling molecules may also underlie some epigenetic or within the ERK1/2 cascade are events that change cell signalling. Cell signalling is a process whereby present in several human cancers, For example, mutations in genes proteins or other chemical messen- but ERK1/2 activation also occurs in involved in the methylation of DNA, gers activate receptors at the cell cancers without mutation of compo- modification of histones, and bind- surface and then transmit signals nents of the cascade (Plotnikov et al., ing of microRNAs to the genome or inside the cell via membrane-to-nu- 2011). to other RNAs may initiate epige- cleus pathways. In healthy adults, Ras, a small upstream guano- netic changes (see Chapter 12, by cell proliferation, cell differentiation, sine-5′-triphosphate (GTP)-binding DeMarini, and Chapter 20, by Rice and cell death determine the size protein in several signalling path- and Herceg). of the proliferating cell population ways, has two isoforms, H-Ras and Cell signalling pathways that reg- in soft tissues, for example surface K-Ras, with different potencies to ulate cell proliferation are not inde- epithelia, mucosal lining cells of ex- activate the MAPK/ERK pathway; pendent of those associated with cretory ducts, columnar epithelia the KRAS gene is more frequent- other key mechanistic characteris- lining the gastrointestinal tract and ly mutated in human cancer, which tics of IARC Group 1 carcinogens. uterus, transitional epithelium of the can result in constitutive activation Many of the genes associated with urinary tract, and bone marrow and (McCubrey et al., 2007). H-Ras cell proliferation are also linked with haematopoietic cells. These pro- has been implicated as contributing apoptosis, inflammation, and several liferating cells replace dead cells to the cancer phenotype, through pleiotropic responses. Dysregulation throughout life. Pathological effects evasion of anti-growth signalling, in the mitogen-activated protein (e.g. injury resulting from hepato- angiogenesis, genetic instability, kinase (MAPK) pathway affects cellular necrosis or partial hepatec- tissue invasion and metastasis, tu- most, if not all, processes involved tomy) and physiological conditions mour-promoting inflammation, and in cancer (Dhillon et al., 2007). The (e.g. estrogen-induced effects on the changes in the tumour microenviron- extracellular signal-regulated kinase endometrium during the menstrual ment (Engström et al., 2015). K-Ras (ERK) pathway of the MAPK family cycle) can involve stimulation of cell promotes metabolic reprogramming, is most commonly associated with proliferation (Engström et al., 2015). activation of proliferative signalling regulation of cell proliferation (Reuter The relationships between cell pathways, glycolysis, reduction of et al., 2010). signalling molecules and pathways oxidative metabolism in the tricar- The ability to use this character- that control cell proliferation and pro- boxylic acid cycle, and channelling of istic to evaluate both mechanistic grammed cell death (apoptosis) are glucose intermediates into anabolic and tumour site concordance is in- complex. Numerous enzymes and pathways, such as the hexosamine fluenced by recent developments in cell signalling pathways are modulat- biosynthesis pathway. cancer research, by the overarching ed during apoptosis, and dysfunction The tumour suppressor protein issue of how carcinogens may ex- of cell death pathways is associated p63, which is activated by DNA dam- press certain characteristics, and with initiation and progression of tum- age, cellular stress, and oncogenic by the question as to the biological origenesis; the products of proto-on- signal transduction, has pleiotropic basis of the differences between cogenes (genes that encode proteins anti-proliferative and metabolic ef- species, strains, target organs, and that stimulate cell proliferation, inhibit fects that include metabolic cell-cy- target cells in cell signalling and apoptosis, or do both) include tran- cle arrest (Robey et al., 2015). cell-cycle control. scription factors, chromatin remod- Numerous pathways have been

118 identified to be involved in disruption trinucleotides that identify amino protein-coding genes, which are like- of resistance to cell death, and p53 acids, and the regulatory code that ly to mediate species differences (Lin has been described as implicated determines how DNA sequences diet al., 2014). in cell-cycle arrest, apoptosis, reg- rect gene expression are highly con- Human-to-mouse transgenic ex- ulation of metabolism, DNA repair, served between species. However, periments demonstrate recapitu- and every pathway linked to these species differ in the composition and lation of human gene regulation in processes (Narayanan et al., 2015). the length of the DNA sequences mice, even in the case of human Disruption of the MAPK cascades, that use this language in the regu- genes that lack murine orthologues. which are central signalling pathways latory regions of their genes (Nitta However, for distinct biological path- that regulate a wide variety of cellular et al., 2015). ways, the expression profiles of processes, is associated with induc- The Mouse ENCODE (Encyclo- many mouse genes diverged from tion and progression of various dis- pedia of DNA Elements) Consortium those of human orthologues (Yue eases, including not only cancer but reported that the degree of conser- et al., 2014). A core set of candidate also diabetes, autoimmune disease, vation is high: the mouse genome is regulatory sequences were con- and developmental abnormalities similar to the human genome in size, served and display similar activity (Plotnikov et al., 2011). structure, and sequence composi- profiles in humans and mice: expres- The role of gene activation in tion, and more than 80% of mouse sion patterns for genes that encode carcinogenesis is also complex and

genes have human orthologues. proteins in the nuclear and intracel- PART 2 evolving. Activation of protein kinase

The chromatin landscape in a cell lular organelle compartments, and CHAPTER 13 C, which acts as a catalyst for sever- lineage is relatively stable in both genes involved in RNA processing, al cellular functions that are related humans and mice, transcription fac- nucleic acid metabolism, chromatin to cancer (e.g. cell survival, proliferation, apoptosis, and migration), tor networks are substantially more organization, and other intracellular has been thought to enable tumour conserved, and both the human and processes. However, less interspe- development. However, protein ki- mouse genomes are pervasively cies concordance was observed for nase C isozymes have recently been transcribed (Vierstra et al., 2014; Yue genes involved with the extracellular reported to be suppressed in human et al., 2014). The pattern of chromatin matrix, cellular adhesion, signalling cancers, possibly through loss of states (defined by histone modifica- receptors, and immune responses function that suppresses other on- tions) and the large-scale chromatin (Yue et al., 2014). cogenic signals (Antal et al., 2015). domains are highly similar between Within orthologous mouse and hu- The gene for an RNA-binding protein mice and humans, but there is a di- man cell types, there is conservation that is highly active in blood cancers vergence in the regulatory landscape across species of the global fraction (i.e. RNA-binding protein Musashi that confers plasticity both between of regulatory DNA sequences that homolog 2) is not directly mutated cell types and between species (Yue encode recognition sites for each in tumours, but its activation affects et al., 2014). transcription factor (Vierstra et al., the ability of RNA to be translated Organ-specific genes are more 2014). However, between humans into proteins (Wang et al., 2015), and highly expressed than housekeep- and mice there is variation in regu- consequently its role in cancer has ing genes (i.e. those present in all latory regions that govern individual not been identified through mutation tissues), and the highest organ-spe- gene systems and the occupancy or gene expression patterns. cific gene expression is observed in pattern of transcription factors, with the testes, brain, liver, muscle (car- extensive cis-regulatory “rewiring”, Genetic variability in cell diac and/or skeletal), and kidney (Lin mediated by elements that recognize signalling between species, strains, and target organs et al., 2014). Comparisons of gene transcription factors. Although they expression between human and mu- have a common language in regula- Genetic variability between species rine tissues showed similarities in tion, active elements in one species has been described in terms of their gene expression profiles at the tissue may be reassigned to a different genomic content and the regulation and organ level. However, there were tissue in another species (Vierstra and expression of their genes. Both greater similarities within each spe- et al., 2014). Thus, differences in the the genetic code, which specifies cies for non-coding and conserved regulation of gene expression and

Part 2 • Chapter 13. Alterations in cell proliferation, cell death, or nutrient supply 119 cell signalling between species and mutations, one chemically induced i.e. it was lower in areas of active tissues may affect mechanistic and and the other genetically engineered chromatin and transcription (Polak tumour site concordance. transgenic, produced tumours with et al., 2015). different gene expression patterns To create a comprehensive cat- Variability in mutation targets (Westcott et al., 2015) and showed alogue of genes responsible for the and cell signalling across differences not only in tumour sus- initiation and progression of cancer, tissues and in tumours ceptibility but also in model-de- 27 types of cancer were studied through sequencing of matched tu- Effects from activation of the MAPK/ pendent signalling pathways. (See mour and normal tissue samples as ERK pathway, such as cell growth, Chapter 19, by Caldwell et al., for part of the Cancer Genome Atlas and prevention of apoptosis, and cell-cy- a discussion of host susceptibility the International Cancer Genome cle arrest, depend on the cell line- factors that influence tumour site Consortium (Lawrence et al., 2013). age; for example, activation of this concordance.) However, as sample sizes increase, pathway is associated with prolifera- As noted above, human tumours the number of putative significant tion and drug resistance in haemato- carry mutations in genes that en- genes also increases and the risk of poietic cells, but the activation is sup- code components of cell signalling false positives resulting from tissue pressed in some prostate cancer cell pathways associated with cell prolif- lines (McCubrey et al., 2007). heterogeneity between cancer types eration and cell death. High mutation The difficulty in predicting in mutation type, distribution, and frequencies have been associated cell-specific effects on cell signal- frequency is highly variable. Across with tumours induced by particular ling is also illustrated by differences the 27 types of cancer, the median carcinogens or mixtures of carcin- in certain biological responses be- frequency of non-synonymous mu- ogens, for example melanoma in- tween histological subtypes of lung tations varied over more than three duced by exposure to ultraviolet light cancer, i.e. adenocarcinoma versus orders of magnitude; half of the var- squamous cell carcinoma, in human and lung cancer induced by exposure iation in mutation frequency was as- patients and in chemically induced to tobacco smoke (Lawrence et al., sociated with tissue type of origin. lung cancer in mouse models. In 2013). Within cancer types, patient-specific A/J mice treated with urethane to However, cancer is not a disease mutation frequencies also spanned induce adenocarcinoma, or with of uniform origin, progression, or cell three orders of magnitude. This mu- N-nitrosotris-(2-chloroethyl)urea to biology. Different types of cancer tational heterogeneity was strongly induce squamous cell carcinoma, show variation in overall mutation correlated with DNA replication tim- inhibition of vascular endothelial rate, predominant mutation type, ing and with transcriptional activity, growth factor has the opposite ef- and distribution of mutations along i.e. it was higher in late-replicating DNA regions and lower in highly fect in these two tumour subtypes the genome. Epigenetic patterns expressed genes. Higher mutation (increased apoptosis vs increased of chromatin accessibility, histone frequencies occurring in late-rep- proliferation) (Larrayoz et al., 2014). modification, gene expression, and licating genes may be responsible In another example, gene ex- DNA replication timing are also cell pression profiling was poor at dis- for potentially false-positive putative lineage-specific (Polak et al., 2015). tinguishing histological subtype and cancer genes, such as the olfacto- A study of 173 cancer genomes from cell type of origin for human breast ry receptor genes and some genes eight different types of cancer, rep- cancer, but a mouse model could cited in association with lung cancer resenting a wide range of tissues of demonstrate the correct genetic le- (Lawrence et al., 2013). Thus, the origin, carcinogenic mechanisms, sion and cell type to model human tissue of origin greatly affects mu- disease, by confirming that the ori- and mutational signatures, showed tation patterns and is linked to DNA gin of BRCA1 mutation-associated that chromatin features of the cell replication timing and tissue-specific breast cancer is a luminal estrogen type of origin, and not of matched transcriptional activity. receptor (ER)-negative mammary cancer cell lines, were the best pre- Genomic sequencing of estab- epithelial progenitor (Molyneux and dictors of local frequency of somatic lished tumours to study their caus- Smalley, 2011). However, different mutations. Mutation density was as- es has its limitations, because such mouse models with the same K-ras sociated with epigenomic features, an approach is unable to study the

120 evolution of the clones, the accumu- 2013). Two distinct expression ar- It is difficult to identify associa- lation of mutations in normal somatic rays of breast cancer cells, with al- tions that directly support a primary cells, the variability among individ- most no genes – and thus no pro- metabolic link between environmen- uals in driver mutation profiles, and tein changes – in common, can be tal exposures and cancer, for several the variability among cancer genes equally useful for predicting clinical reasons: metabolic control does not in clonal dynamics. In a study of 74 behaviour, and analyses with gene occur in a single step in a metabol- cancer genes in sun-exposed skin, expression arrays may not provide a ic pathway; controlling factors differ i.e. a polyclonal quilt of mutations true understanding of cancer biology between intact cells and in vitro cell- in key cancer genes consistent (Weinberg, 2014). free systems; observed changes in with damage from ultraviolet light, Sequencing of entire tumour ge- individual pathway elements do not multiple cancer genes were found nomes has yet to demonstrate de- always lead to changes in metabol- to be under strong positive selec- ic flux; and cancer cell phenotypes finitively the number of somatic mu- tion even in physiologically normal are neither fixed nor cancer-specific. tations required to create a human skin (Martincorena et al., 2015). The review also noted the function- tumour. A few conceptual insights Positively selected mutations were al interdependence of dysregulated into cell and tissue behaviour have found in 18–32% of normal skin metabolism and other hallmarks of resulted from elaborate maps of cells. The size of clonal expansions cancer, considering that, for exam- varied across genes, and gene size interacting signalling components

ple, proliferating cancer cells have PART 2 was not necessarily correlated with and computer models of signalling

shared regulatory factors associ- CHAPTER 13 the potential of the somatic mutation (Weinberg, 2010). The paradigm ated with the fundamental anabol- to induce malignant transformation. of somatic evolution and multistep ic and catabolic demands of the Consistent with findings in tumours, tumorigenesis does not provide a hallmark “sustaining proliferative the mutation rate also varied along logical reason why oncogenesis signalling” (Robey et al., 2015). the genome, with higher rates found recapitulates ontogenesis (Huang Increased body mass index has in less frequently expressed genes et al., 2009). been associated with increased risk and in repressed chromatin. These of cancer: obesogens – chemicals findings were inconsistent with the Alterations in nutrient supply that disrupt normal development assumption that driver mutations oc- and the balance of lipid metabol- cur infrequently in long-lived cell line- Although dysregulated metabolism ism – are able to cause permanent ages and that those arising in cancer is one of the most common and changes in metabolism that may ren- are too small to be detected clinically recognizable features of cancer and der the subject more susceptible to (Martincorena et al., 2015). is associated with other phenotyp- cancer later in life (see Chapter 19, If a gene signature – a group of ic indicators, the results of a recent by Caldwell et al.). Inflammation is genes with a characteristic com- literature review attempting to link associated with metabolic changes bined expression pattern – is associ- cancer development and dysregulat- ated with a prognosis, it is assumed and has been linked with several ed metabolism suggested that there to be likely to encode a biological chronic diseases, including cancer. are major gaps in the understanding signature driving carcinogenesis. Extracellular pro-inflammatory met- of exposure-related carcinogenesis However, this assumption has been abolic signals are adenine nucleo- and metabolic reprogramming, for questioned in view of findings that tides, succinate, oxidized nicotin- example with respect to the specif- + random changes in gene sets are amide adenine dinucleotide (NAD ), ic causal and temporal relationships associated with prognosis and and urate (McGettrick and O’Neill, that prognostic signatures in ER- between exposures, dysregulated 2013; Tannahill et al., 2013). The negative breast cancer – associated metabolism, and the development gut microbiome has an important with hypoxia and angiogenesis – of cancer and the associated phe- role in carbohydrate absorption and are more similar to those in ovarian notypic hallmarks of cancer (Robey metabolism in humans and plays a cancer than to those in ER-positive et al., 2015). This review did not con- significant part in inflammatory re- breast cancer, which are driven by sider lifestyle-related exposures and sponses as well (see Chapter 19, by proliferation pathways (Beck et al., IARC Group 1 carcinogens. Caldwell et al.).

Part 2 • Chapter 13. Alterations in cell proliferation, cell death, or nutrient supply 121 The transgenerational character modes of action, and the third de- out the need for either continued of metabolic disturbances and ef- scribes the ability of a transformed exposure or sustained proliferation fects on cell signalling is demonstrat- cell to escape normal cell-cycle con- during exposure (see Chapter 19, by ed by studies of multigenerational trol and to continue replication. The Caldwell et al.). It has been noted for undernutrition in rats (i.e. for 50 gen- interpretation of mechanistic data some time that enhanced cell divi- erations). The undernourished rats for cell proliferation and cell death sion does not always predict carcino- were predisposed to insulin resis- is dependent on the development genesis (Melnick et al., 1993). After tance, had altered levels of several of appropriate animal models (see exposure to a carcinogen, the de- metabolic regulators (e.g. circulating Chapter 19, by Caldwell et al.), and velopment of cancer in experimental insulin, homocysteine, endotoxin, although cell proliferation has been animal models is influenced by the leptin, adiponectin, vitamin B12, and used in descriptions of hypothesized circumstances under which expo- folate), and had a higher susceptibili- modes of action (Wood et al., 2015), sure occurs (e.g. sustained vs tran- ty to streptozotocin-induced diabetes it should be viewed in the context of sient) and by the presence or ab- compared with properly fed control the newer understandings of cancer sence of inflammatory mediators or rats. These changes were not re- mechanisms. DNA damage. versed by feeding rats a normal diet For DNA damage to lead to a Liver cancer in the two subsequent generations mutation, DNA replication and cell (Hardikar et al., 2015). division are typically required (see The complex interactions between Studies on altered cell signalling Chapter 12, by DeMarini). As noted proliferation, mutation, and inflam- have traditionally been performed in the United States Environmental matory cell signalling have been on putative target cells of cancer, Protection Agency guidance assess- studied extensively for liver cancer. but the contribution of the gut mi- ing the risk of cancer from early-life In humans, hepatocellular carcinoma crobiome (i.e. the microbiota living exposures (EPA, 2005), more fre- (HCC) is markedly heterogeneous, on and in humans) has only recently quent cell division during develop- both histomorphologically (Yeh et al., been investigated as a factor in can- ment can result in enhanced fixation 2007) and genetically, with a wide cer susceptibility (see Chapter 19, of mutations because of the reduced diversity in gene expression patterns by Caldwell et al.). The microbiome time available for repair of DNA le- (Chen et al., 2002). Histopathological plays a role in the control of nutri- sions, and clonal expansion of a variability is also associated with ge- ent supply (e.g. the gut microbiome mutated cell produces a larger pop- ographical region: slow-growing, dif- is highly enriched in carbohydrate ulation of mutant cells. For adult or- ferentiated HCC nodules surrounded metabolism genes, compared with ganisms, sustained cell proliferation by a fibrous capsule are common the human genome overall; Bultman, has also been postulated to increase in this type of cancer in Japanese 2014), in metabolic pathways, and in risk of cancer, based on the same ra- patients, whereas a febrile form of host susceptibility to metabolic dis- tionale, and it has been proposed as HCC, characterized by leukocytosis, ease (Suez et al., 2014). a factor in increased cancer suscep- fever, and necrosis within a poorly tibility. Sustained cell proliferation differentiated tumour, is common in Cell proliferation as a component or cause of is a feature of several hypothesized this cancer type among black peo- cancer modes of action for cancer develop- ple in South Africa (Feitelson et al., ment, for example the induction of 2002).

There are at least three scenarios kidney cancer via alpha2u-globulin HCC signature genes vary con- related to cancer and mechanisms of accumulation (EPA, 1991). siderably and depend on etiologi- cancer induction in which alterations Although alterations in cellu- cal and accompanying pathological in cellular replication and/or cell-cy- lar replication or cell-cycle control conditions, such as viral infection, cle control have been described. The are important features of carcino- cirrhosis, inflammation, and fibrosis. first invokes the predisposition of genesis, cell proliferation in and of The study of tumour formation in the replicating cells with unrepaired DNA itself is not able to induce cancer. liver is also affected by continuous damage to develop into cancer cells. Several carcinogenic substances changes in the transcriptome that The second identifies sustained can cause cancer in humans after accompany hepatectomy and age replication as a key event in various perinatal or prenatal exposure with- (Colak et al., 2010). A comparison of

122 conserved genes between rats and simple paradigm to explain the differ- cell signalling and is consistent with humans (human orthologues) with ences in strain sensitivity, for exam- inflammation contributing to cancer respect to early expression profiles ple between C3H/HeJ and C57BL/6J development. After exposure of ro- of HCC signature genes showed mice, which show a difference of dents to trichloroethylene, hepato- some conservation between species up to 40-fold in multiplicity of liv- cyte proliferation is confined to only for components of the MAPK/ERK, er tumours (Hanigan et al., 1988; a small population of cells without phosphoinositide 3-kinase (P13K)/ Maronpot, 2009). regenerative hyperplasia, sustained Akt, and transforming growth factor The activation of oncogenic hepatocellular proliferation, and beta (TGF-β) pathways (Colak et al., pathways appears to be more het- hepatocellular necrosis. Any tran- 2010). erogeneous in human HCC than in sient DNA synthesis, peroxisome Development of liver cancer af- other types of cancer (El-Serag and proliferation, or cytotoxicity is not ter exposure to carcinogens is more Rudolph, 2007). The high degree of correlated with trichloroethylene-in- common in rodents, especially in heterogeneity in the ways in which duced liver carcinogenicity (EPA, the mouse, than in humans. There cell signalling is disturbed before 2011). Thus, induction of liver cancer are obvious differences between hepatocellular neoplasia may make by trichloroethylene is not a result of rodents, non-human primates, and induction of liver cancer a useful sustained cell proliferation. humans in background susceptibil- marker for changes that can lead to Exposure to one of the most ity to hepatocarcinogenesis and, cancer elsewhere, depending on cel- studied carcinogens, 2,3,7,8-tetra- as noted above, in the regulation of lular context and target (Vogelstein chlorodibenzo-para-dioxin (TCDD), PART 2 CHAPTER 13 gene expression and cell signalling. and Kinzler, 2004). induces liver cancer in rodents, but With respect to the ability to respond Thus, pathway concordance be- short-term effects do not include to a mitogenic stimulus such as par- tween species may not always result induction of hepatocellular prolifer- tial hepatectomy, the liver responds in site concordance for expression ation. Rather than simply inducing differently and much more slowly in of cancer. The analysis of liver tu- cell proliferation, TCDD is thought non-human primates and in humans mour site concordance is complicat- to cause cancer by altering the cel- compared with rodents (Gaglio et al., ed by the heterogeneity of disease lular ability to proliferate, migrate, 2002). in humans, as well as by rodent undergo apoptosis, senesce, and Global gene expression patterns susceptibility. terminally differentiate in a multistep in HCCs from seven different mouse For induction of cancer in the liv- process focused on the accumula- models were compared with those er in rodents, the nature of cell pro- tion of mutations and heritable epige- in human HCCs from groups with liferation also determines the risk of netic changes (Safe, 2001; Marlowe poorer survival and better survival. cancer (Caldwell et al., 2008). When and Puga, 2005; Ray and Swanson, Expression patterns in HCCs from both necrosis and inflammation are 2009). In addition, the upregulation Myc-Tgfa transgenic mice and in di- present, the resultant hepatocellular of drug-metabolizing enzymes by ethylnitrosamine-induced HCCs in proliferation is fundamentally differ- TCDD may enhance the formation of mice were most similar to those in ent from the transient proliferation highly reactive intermediates during human HCCs from the group with caused by peroxisome proliferators metabolic activation and/or trans- poorer survival, whereas the pat- or other primary mitogens. After formation of several key hormones terns in HCCs from Myc, E2f1, and treatment with a mutagenic agent, (e.g. enzyme induction as a source Myc-E2f1 transgenic mice were most transient proliferation induced by pri- of reactive oxygen species forma- similar to those in human HCCs from mary mitogens has not been shown tion, which is linked to decoupling of the group with better survival (Lee to lead to cancer induction, whereas the cytochrome P450 catalytic cy- et al., 2004). partial hepatectomy or necrogenic cle) and result in DNA damage and Many factors, such as diet, hor- treatments with carbon tetrachlo- mutations (IARC, 2012c). mones, oncogene activation, methyl- ride have (Ledda-Columbano et al., Although liver data provide an ation, imprinting, and cell prolifera- 1993; Gelderblom et al., 2001). example of the role of inflammatory tion or apoptosis, are modulators of The mechanism by which necro- signals under some circumstances, spontaneous and induced murine sis may enable cancer development inflammation in itself may not induce hepatocarcinogenesis. There is no involves concurrent inflammatory cancer without other concurrent

Part 2 • Chapter 13. Alterations in cell proliferation, cell death, or nutrient supply 123 cofactors. For many of the agents ducts in portal spaces, Mdr2 knock- Several key mechanistic charac- discussed in Volume 100C of the out mice rarely develop tumours of teristics of IARC Group 1 carcino- IARC Monographs (IARC, 2012a), the bile duct (Nickoloff et al., 2005). gens induce traits of cancer cells de- inflammation is a key characteristic The relationship between chron- scribed as the hallmarks of cancer, of their effects (see Chapter 17, by ic inflammation and cancer is com- including effects on cell proliferation, Kane). One of the most recognized plex: inflammation may have roles cell death, and nutritional status (see examples of how inflammation con- in initial genetic mutations or epi- Chapter 11, by Stewart). The descrip- tributes to neoplastic development is genetic changes that not only drive tion of the hallmarks attempts to bring the induction of mucosa-associated cell transformation but also provide together a fundamental understand- lymphoid tissue (MALT) lymphoma a microenvironment that enables ing of how cancer cells manifest a and gastric adenocarcinoma asso- progression and metastasis and distinct phenotype. More recently, a ciated with exposure to Helicobacter prevents immune responses against series of reviews in Carcinogenesis pylori. The MALT proliferations of the tumour. Chronic inflammation reported the findings from an inter- B-cell lymphoid follicles are the pre- favours accumulation of DNA dam- national team of cancer biologists cursor of a low-grade lymphoma of age and chromosomal damage and toxicologists who participated B cells. A large proportion (98%) of (see Chapter 12, by DeMarini, and in the Halifax Project (Harris, 2015). patients with gastric MALT lympho- Chapter 17, by Kane). They reviewed the literature on each ma are also infected with H. pylori; of the hallmarks of cancer to exam- The hallmarks of cancer however, only a small percentage of ine the carcinogenic potential of exposure to low doses and mixtures H. pylori-positive individuals devel- In their updated paper, Hanahan of chemicals. Relevant reviews for op MALT lymphoma (Bassig et al., and Weinberg (2011) noted that the alterations in cell proliferation, cell 2012). Little is known about the pos- most fundamental trait of cancer death, or nutrient supply included sible role of environmental cofactors cells involves their ability to sustain the potential of chemical mixtures to in the predisposition to H. pylori-in- chronic proliferation. As part of the enable sustained proliferative signal- duced gastric lymphomagenesis. “hallmarks” of cancer, alterations in ling (Engström et al., 2015), to confer Other factors are certainly involved, cellular replication and/or cell-cy- resistance to cell death (Narayanan including susceptibility (IARC, cle control figure prominently in the et al., 2015), and to induce metabol- 2012b). discussions of cell proliferation, in- ic reprogramming and dysregulated flammation, and changes in cell sig- Inflammation metabolism (Robey et al., 2015). A nalling that are part of cancer cell related review that encompasses Certain types of inflammatory pro- physiology. The authors noted that many aspects of cell signalling re- cesses in skin, and possibly in other the precise identities and sources ported on environmental immune tissues, may serve a tumour suppres- of the proliferative signals in general disruptors, inflammation, and risk sor function. Some clinical conditions remain poorly understood, but that of cancer (Thompson et al., 2015). show that inflammation is a critical mitogenic signalling in cancer cells The overlap in the descriptions of component of tumour progression, is characterized and known in some- pathway disruption and functions of for example reflux esophagitis be- what more detail. The mechanism by cell signalling molecules between fore oesophageal cancer, or inflam- which necrosis enables cancer in- these papers is striking and is con- matory bowel disease that precedes duction was also described in terms sistent with the discussion presented colorectal cancer. However, a condi- of the release of pro-inflammatory above with regard to the complex re- tion such as psoriasis is known as a signals by necrotic cells and the in- lationship and pleiotropic roles of cell chronic cutaneous inflammatory dis- fluence of cytokines on proliferation signalling molecules involved in cell ease that is seldom, if ever, accom- and invasiveness of cancer cells. proliferation and apoptosis. panied by cancer. Similarly, despite Thus, tumour-promoting inflamma- The paradigms of cytotoxicity, extensive inflammation, activation of tion was considered by Hanahan and cell proliferation, and initiation–pro- nuclear factor kappa-light-chain-en- Weinberg (2011) to be an enabling motion as mechanisms of carcino- hancer of activated B cells (NF-κB), characteristic for acquisition of core genesis have been superseded by a and abundant proliferation of bile hallmark capabilities. more nuanced understanding of the

124 process of carcinogenesis (Hanahan The biological basis for how cell delayed cancer induction after expo- and Weinberg, 2011). As stated signalling and cell-cycle control dif- sure to toxicants and the bystander above, cell proliferation, inflamma- fer between species, organs, and effect of radiation on tumour devel- tion, or cytotoxicity alone do not lead tumour cells, as well as the variabil- opment. With regard to cell signal- to cancer, and they are interrelated ity in mutation targets, are also dis- ling, spontaneous or environmentally through changes in cell signalling. cussed here. Determining the caus- induced epigenetic alterations are The hallmarks of cancer describe es of cancer through examination of increasingly recognized as early mo- gene expression profiles in tumours lecular events in cancer formation, well the characteristics that are is difficult, especially in terms of in- and these alterations may potential- manifested after the development of creased and sustained cell prolif- ly be more adverse than nucleotide cancer. However, by the time of di- eration, which is a characteristic of mutations, because their effects on agnosis, tumour cells already carry cancer itself. Alteration in nutrient regional chromatin structure can large numbers of mutations and are supply is a common and recogniza- spread out, thereby affecting multiple also very heterogeneous in gene ble feature of cancer, and is also not genetic loci (Weidman et al., 2007). product profiles; the multiple cell di- independent of activities associated The key characteristics of the visions and the consequent damage with increased cell proliferation or IARC Group 1 carcinogens have processing obscure the initial lesion, the hallmarks of cancer. some overlap with the hallmarks rendering it difficult, if not impossi- Some of the information col- of cancer and perhaps can provide ble, to make a distinction between lected in Volume 100 of the IARC insight into the “environment” that PART 2 CHAPTER 13 causal and consequential events in Monographs was presented in the creates the neoplastic cell. The elu- carcinogenesis. context of the older hypotheses for cidation and understanding of sus- mechanisms of cancer induction. ceptibility factors may help determine Conclusions The understanding of cancer mech- what parts of that environment are anisms and the descriptive data already present in individuals, spe- Among the 10 mechanistic charac- associated with them continues to cies, or target tissues where cancer teristics more commonly observed evolve (see Chapter 11, by Stewart). develops as a result of exposure to for IARC Group 1 carcinogens is a The discussion of mechanistic data environmental carcinogenic agents. composite that includes information for ionizing radiation in Volume 100D This may also help in evaluating on alteration of cell proliferation, of the IARC Monographs (IARC, mechanistic and tumour site concor- cell death, and nutrient supply. This 2012d) and Chapter 12, by DeMarini, dance between species. However, chapter examines many of the chal- provide more current discussions the mechanistic data provided on lenges associated with the use of about the understanding of cancer agents identified as carcinogenic this type of information to determine and the key mechanistic character- to humans need to be examined in mechanistic and tumour site con- istics of IARC Group 1 carcinogens. the context of more recent informa- cordance between humans and ex- With the present state of knowledge, tion on carcinogenesis. Cancer is a carcinogenesis cannot be confident- heterogeneous disease, even with- perimental animals, and discusses ly attributed to an underlying purely in the same target site. Because, how this mechanistic characteristic genetic or purely epigenetic pro- among other considerations, some shows interdependence with others, cess. Mechanistically, it is probably a epigenetic events may have a mu- such as genotoxicity and inflamma- mixture of the two. tational basis, the dichotomy once tion. Many of the indicators of chang- Epigenetic alterations may identified between genotoxic and es in cell proliferation or cell death precede DNA sequence mutations, non-genotoxic carcinogens should are non-specific for the induction with subsequent mutations occurring be reconsidered. of cancer, and although they result not in a random fashion but in re- Enhanced cell proliferation and re- primarily from effects on the MAPK/ sponse to specific types of epigenet- duced cell death are key mechanistic ERK cell signalling pathway, they are ic changes induced by the environ- characteristics of IARC Group 1 car- influenced by a large array of cell ment (Karpinets and Foy, 2005). This cinogens, are a hallmark of cancer, signalling molecules with pleiotropic selection for enhanced growth has and are necessary for DNA damage effects. been suggested to explain both the to be processed into a mutation.

Part 2 • Chapter 13. Alterations in cell proliferation, cell death, or nutrient supply 125 However, such changes alone are Acknowledgements Approval does not signify that the not sufficient to induce cancer. A key contents reflect the views of the The author thanks D.M. DeMarini, mechanistic question emerges as to agency, nor does mention of trade M.V. Evans, and S. Vulimiri for their what events or cellular environment names or commercial products con- helpful comments on this manuscript. may precede or cause such chang- stitute endorsement or recommen- es and may stimulate the formation Disclaimer dation for use. and selection of DNA sequence mutations and epigenetic changes that This article was reviewed and induce a cell and its descendants to approved for publication by the acquire the hallmarks of cancer, in- National Center for Environmental cluding increased cell proliferation Assessment, United States En- and evasion of apoptosis. vironmental Protection Agency.

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