The microenvironments of multistage carcinogenesis

The microenvironments of multistage carcinogenesis

Seminars in Cancer Biology 18 (2008) 322–329 Contents lists available at ScienceDirect Seminars in Cancer Biology journal homepage:

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Seminars in Cancer Biology 18 (2008) 322–329

Contents lists available at ScienceDirect

Seminars in Cancer Biology journal homepage:


The microenvironments of multistage carcinogenesis Ezio Laconi a,∗ , Silvia Doratiotto a , Paolo Vineis b,∗ a b

Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Patologia Sperimentale, Universit` a di Cagliari, 09125 Cagliari, Italy Department of Epidemiology and Public Health, Imperial College London, St Mary’s Campus, Norfolk Place, W2 1PG London, United Kingdom

a r t i c l e Keyword: Carcinogenesis

i n f o

a b s t r a c t Overt neoplasia is often the result of a chronic disease process encompassing an extended segment of the lifespan of any species. A common pathway in the natural history of the disease is the appearance of focal proliferative lesions that are known to act as precursors for cancer development. It is becoming increasingly apparent that the emergence of such lesions is not a cell-autonomous phenomenon, but is heavily dependent on microenvironmental cues derived from the surrounding tissue. Specific alterations in the tissue microenvironment that can foster the selective growth of focal lesions are discussed herein. Furthermore, we argue that a fundamental property of focal lesions as it relates to their precancerous nature lies in their altered growth pattern as compared to the tissue where they reside. The resulting altered tissue architecture translates into the emergence of a unique tumor microenvironment inside these lesions, associated with altered blood vessels and/or blood supply which in turn can trigger biochemical and metabolic changes fueling tumor progression. A deeper understanding of the role(s) of tissue and tumor microenvironments in the pathogenesis of cancer is essential to design more effective strategies for the management of this disease. © 2008 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3. 4. 5. 6. 7. 8.

The origin of focal proliferative lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The tissue environment can drive tumor promotion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Selection of cells expressing a resistant phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The promoting potential of a growth-constrained tissue microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The molecular analysis of selectogenic microenvironments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiologic evidence: early origins of genomic instability? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stress and the mutator phenotype—bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stress and the mutator phenotype—human cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . From tissue microenvironment to tumor microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Overt neoplasia is often the result of a chronic disease process encompassing an extended segment of the lifespan of any species. During this process, a series of sequential changes take place in the affected tissue, collectively defining what is referred to as the natural history of the disease. One of the most common pathways for the development of cancer unfolds through the appearance of focal proliferative lesions. The latter (papillomas, polyps, adenomas, nodules) are not mere temporal precursors of neoplastic

disease; rather, the material continuity between focal lesions and cancer has been demonstrated beyond any reasonable doubt in several systems, both in experimental animals and in the clinical setting [1,2]. Thus, such lesions represent at least one of the possible sites from which cancer can arise. In light of these facts, two questions emerge as particularly relevant to the biology of cancer development in these systems:

∗ Corresponding authors. E-mail addresses: [email protected] (E. Laconi), [email protected] (P. Vineis).

According to classical multistage modelling of carcinogenesis, the first question refers to the process of tumor promotion or selection, while the second encompasses the long phase of tumor

1044-579X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2008.03.019

(i) How do focal proliferative lesions arise? (ii) How do they evolve towards cancer?

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progression. Broadly speaking, tumor promotion consists of the selective (clonal) expansion of altered cells to form focal lesions [3]. Within this definition, the process of promotion is mainly a quantitative phenomenon (many cells arising from a single cell), while no qualitative changes are necessarily implied; it is a fact that cell populations in early nodules, papillomas, or polyps are rather homogeneous in size and shape, or in the expression of specific biochemical markers [1]. However, these latter properties are lost during tumor progression, which is typically characterized by increasing levels of tumor cell heterogeneity. This implies that qualitative changes are now dominant [4], generating distinct cellular sub-clones with different phenotypes. Such a background represents the landscape for the full deployment of tumor progression. We would like to propose that these two fundamentally distinct biological processes, i.e. the mainly quantitative process of tumor promotion and the intrinsically qualitative process of tumor progression, are driven by two distinct microenvironments, i.e. the tissue and the tumor microenvironments, as they were recently defined [5] (Fig. 1). The tissue microenvironment specifically refers to the local environment surrounding altered cells during their selective clonal expansion to form focal proliferative lesions. Conversely, the tumor microenvironment describes the unique biological milieu that emerges inside focal proliferative lesions as a consequence of their altered growth pattern [5]. Such new biological niche is characterized by a tissue architecture which is not developmentally programmed and is bound to pose major challenges for cell survival, due to altered/inadequate supply of oxygen and nutrients [6]. This in turn can lead to biochemical and metabolic alterations that can profoundly impact on the fate of the cell populations inside focal lesions [7,8]. The latter topic will be the focus of other papers in this issue (Refs. [9,10]), while emphasis in this chapter will be given to the analysis of possible mechanisms mediating the effect of the surrounding tissue environment on the initial growth of altered cells until the emergence of focal proliferative lesions.

1. The origin of focal proliferative lesions The available evidence is consistent with the hypothesis that focal lesions result from the clonal expansion of altered cells [11,12].


If this is so, the first and simplest mechanistic question to be answered is how does a focal lesion develop from those rare altered cells [3]. Theoretically, at least two different (and not mutually exclusive) possibilities should be considered:

(i) The altered/initiated cell is already endowed with some degree of inherent growth autonomy and starts to replicate unchecked, forming a focal lesion and then, after a number of further steps, a full blown cancer. Conceptually, this possibility is still widely postulated and entertained [13–16], although, in essence, it lacks any solid support and is unlikely to be of any major relevance in the vast majority of human and experimental systems. For example, the analysis of several multistage models of cancer induction has led to the conclusion that initiation per se does not result in any significant growth of pre-neoplastic and/or neoplastic lesions, and the appearance of the latter is heavily dependent on the presence of a promoting/selective environment [17–19]. Furthermore, transplantation experiments have convincingly demonstrated that different types of altered cells do not display any evidence of growth autonomy when transferred in a normal tissue environment of young animals in vivo [20–23]. By analogy, altered, putative initiated cells can be found in the skin of several healthy human subjects [24], suggesting that their presence per se is not necessarily associated with selective clonal growth, and additional (promoting/selective) influences must be enforced when the latter does occur [25]. Also epidemiologic evidence on smoking and lung cancer suggests that clonal expansion of cells is much more relevant than early mutations [26,27]. (ii) The other possibility is that the single altered cell does not express any significant degree of growth autonomy and is still under the control of normal homeostatic mechanisms; if this is the case, its selective clonal growth must be linked to the dynamics of cell turnover typical of the tissue where it resides. Thus, specific alterations of these dynamics could translate into a promoting effect for any putative altered cells present in that tissue. It is self-apparent that, within this perspective, the tissue microenvironment surrounding rare initiated/altered cells is given a central role in their selective emergence as focal pro-

Fig. 1. Schematic representation of the microenvironmental influences during multistage carcinogenesis; see text for details.


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liferative lesions. The evidence supporting this view will be discussed and considered next in the context of the overall sequence of cancer development. 2. The tissue environment can drive tumor promotion 2.1. Selection of cells expressing a resistant phenotype The hypothesis that cell populations arising during carcinogenesis could represent clones of rare cells that were able to expand in an otherwise growth-limiting environment was first proposed by Haddow [28]. Noting that most carcinogenic agents are toxic and cause growth suppression in the target organ, Haddow postulated that the end product of the exposure to such agents, i.e. cancer, could only originate from cells that had found ways to overcome those toxic effects, i.e. they expressed a resistant phenotype. This simple intuition was also one the first interpretations of neoplastic development as a true biological process, i.e. a process that is not directly caused by the inciting agent acting on a passive target, but results from the interaction of living structures (cells, tissues and organs) with those agents. Stated otherwise, cancer began to be viewed as the outcome of a pattern of tissue reaction to specific types of injury, a concept that has since received increasing support in the literature [7,8,17,18,29–32]. However, it was not until the seminal work of Solt and Farber [33] that a direct link was established between growth inhibitory effects and the emergence of phenotypically resistant cell populations during carcinogenesis. It was found that, following a single exposure to an initiating carcinogen, rare cells were induced in the target organ that could withstand certain types of growth suppression imposed on surrounding normal cells, thereby acquiring a proliferative advantage under appropriate “selective” conditions. Furthermore, the growth of such resistant cells to form focal proliferative lesions could be induced by physiological homeostatic stimuli, as it occurs during tissue regeneration and/or turnover, and these rare clones could emerge because the bulk of surrounding normal cells were unable to respond to those stimuli [33]. Incidentally, the work of Farber was the first to provide evidence that tumor promotion could be interpreted (and in fact defined) as a process of cell selection, thereby introducing a Darwinian perspective in the analysis of carcinogenesis [19,34]. Thus, early phases of cancer development, far from being exclusively cell-autonomous, appeared to be heavily dependent on environmental influences and in fact could be interpreted as adaptive reactions to altered conditions in the surrounding tissue [29]. Specifically, if the growth potential of normal cells in a given tissue was severely impaired, this could translate into a driving force for any altered/initiated cells to expand and compensate for the (relative) impairment of their surrounding counterparts. As a consequence, cancer development was a process to be considered and analyzed at tissue/organ level, not just at single cell level, and a role for the tissue microenvironment in this process was clearly defined [29,33]. This “new principle” has received support from the analysis of other experimental models of carcinogenesis and is likely to be at play during cancer development in humans as well (Table 1). Classical tumor promoters, including phorbol esters in the skin Table 1 Tissue microenvironments associated with tumor promotion Type of alteration


Growth-constraint Chronic, widespread cytotoxicity Aging Chronic inflammation Atrophy

[23,29,33,35–39,71–74,76] [26,41–43,45–53] [22,60,67–69] [71–73,77–83] [80,81,83–85]

[35,36] and phenobarbital [37] and orotic acid [38] in the liver, were reported to exert cytotoxic and/or mito-inhibitory effects on their target tissue, thereby selecting for the emergence of putative resistant cell clones. Similarly, an interesting model of leukemogenesis in dogs was postulated to involve the selection of radio-resistant progenitors following chronic exposure to myelo-toxic doses of radiation [39]. As a general consideration, it is important to point out that any model of cancer induction based on the chronic exposure to a genotoxic carcinogen (including human exposure) is likely to involve a similar sequence of events. Given that the large majority of these agents can both (i) exert growth-suppression in the target tissue [28], possibly as a consequence of the inflicted DNA-damage [40], and (ii) induce rare altered/initiated cells with a resistant phenotype [24,33], it is clear that the outcome of such combined effects includes the possibility of a selective expansion of the resistant cell population [33,34,40]. As it has been pointed out, random mutations are more likely to damage the function of the genome rather than to improve it [34]. This implies that the same genotoxic agent can both initiate the carcinogenic process in a given tissue and exert a promoting/selective effect on rare initiated/altered cells by limiting the proliferative potential and/or impose other cytotoxic effects in the bulk of the surrounding cells in that tissue [30,41]. Several lines of evidence suggest, albeit indirectly, that a similar scenario might be relevant to the pathogenesis of human cancer also. Thus, exposure to UV, which is associated with an increased risk for the development of skin cancer, can exert widespread toxicity on epidermal cells, thereby selecting for the expansion of rare clones resistant to this effect [42,43]. It has also been shown that both human HPV-immortalized keratinocytes and oral cancer cells are relatively refractory to UV-induced cell cycle arrest and cell death, compared to their normal counterparts [44].Furthermore, chronic toxicity of cigarette smoke on the airway epithelium is well documented, and includes cell death, inflammation, DNA damage and growth inhibition [45–50]; these effects run in parallel with the induction of rare altered cells potentially resistant to cytotoxicity (e.g. p53-mutated) [26,51,52]. Studies in vitro have shown that both cigarette smoke extracts and purified carcinogens present in these extracts are powerful inhibitors of normal cell growth; however, lung cancer cells were less sensitive to these effects [46,53]. Thus, such findings are suggestive for a role the “resistance phenotype pathway” in the origin of the deadliest type of cancer in humans [30] (Table 1). Yet another possible case in point is the aromatic amine 2amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is one of the major mutagenic products formed in cooked meat. Exposure to PhIP has been implicated with an increased risk of cancer in humans at various sites, including colon, pancreas, prostate and breast, although the evidence is still scanty and contradictory. Upon metabolism, PhIP becomes genotoxic and induces a cell cycle block on cultured cells [54]; however, at lower concentrations it can stimulate proliferation of mammary epithelial cells through an estrogen receptor-dependent pathway [55]. The combined effects of genotoxicity, together with suppression or enhancement of proliferation, depending on the dose of exposure to PhIP, could provide the appropriate environment for the selection of any altered cell clones resistant to PhIP-induced growth inhibition [56]. It will be discussed later how exposure to PhPI can result in the emergence of PhIP-resistant cells. 2.2. The promoting potential of a growth-constrained tissue microenvironment A direct testing of the hypothesis that a growth-constrained microenvironment can represent a powerful driving force during

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tumor promotion is provided by experiments involving cell transplantation. Hepatocytes isolated from chemically induced liver nodules were infused into the liver of syngenic recipients pretreated with retrorsine, an agent that causes a long lasting block on resident hepatocytes cell cycle [23,57]. Under such endogenous conditions of growth-constraint, transplanted altered cells could selectively expand in the recipient liver, forming hepatocyte nodules and eventually progressing to hepatocellular carcinoma. Importantly, no nodular growth was observed when a similar preparation of hepatocytes was injected into normal, untreated recipients [23], suggesting that nodular cells had no inherent growth autonomy. Furthermore, the microenvironment of the retrorsine-treated rat liver is also able to sustain the selective growth of transplanted normal hepatocytes [58]. The latter finding is particularly relevant, in that it is a clear indication that the growth of normal and nodular cells is under similar types of regulatory stimuli [59], i.e. the selective growth of altered cells in this system occurs under the influence of homeostatic mechanisms which are similar to, and possibly coincide with those controlling normal cell turnover. The concept illustrated by the retrorsine model for cell transplantation might well apply to other growth-constrained environments that are associated with increased risk for cancer development. One relevant situation to be considered in this context is aging. In fact, aging represents the major risk factor for neoplastic disease [60] (albeit it is not an avoidable risk); moreover, it is characterised, if not defined, by a generalized decrease in the functional proficiency of several organs and tissues, including a decline in their proliferative potential [61–63]. For example, lymphocyte production is reduced during aging [64], as it is the regenerative capacity of the liver [65,66]. Interestingly, it has been shown that the microenvironment of the aged liver is able to support the growth of a transplanted epithelial cell line, while that of the young recipient is not [22]. Studies from our laboratories have further indicated that the aged rat liver stimulates the clonal expansion of transplanted normal hepatocytes [67]. Although this latter phenomenon is not quantitatively comparable to that observed following exposure to retrorsine [57,58], it does suggest that the liver microenvironment associated with aging is also clonogenic, and could therefore foster the emergence of altered cell populations. It appears reasonable to propose that such clonogenic potential might be linked, at least in part, to the growth-constrained environment associated with aging, in analogy to the experimental conditions induced by retrorsine in rat liver [67]. It is likely that a role in such age-associated clonogenic effect is also played by stromal fibroblasts. Senescent fibroblasts can in fact stimulate early growth of both grafted normal and tumor epithelial cells, suggesting that they can mediate, at least in part, the effect of aging on the parenchymal component of various tissues [68,69]. However, this effect can translate into selective growth of rare altered cells if the majority of surrounding counterparts are relatively impaired in their proliferative capacity, as it occurs during aging [70]. Yet another notable case in point for the present discussion is liver cirrhosis, which is the strongest risk factor for the development of human hepatocellular carcinoma. Recent reports have indicated that hepatocytes are mito-inhibited during the evolution of cirrhosis [71] and in the fibrotic liver of hepatitis C virus patients [72,73]. These findings could in turn explain the impaired regenerative capacity of the cirrhotic liver [74]. Furthermore, preliminary data from our laboratories have suggested that the microenvironment of the fibrotic liver is able to sustain the growth of transplanted nodular hepatocytes (Laconi et al., unpublished observation). Thus, the message that derives from such combined evidence is that the


evolution of fibrosis and cirrhosis is associated with a progressive decline in the ability of hepatocytes to respond to physiologic growth stimuli; such relative impairment could translate into a selective driving force for the emergence of rare cells with a normal or an altered phenotype [59,75,76]. In this perspective, the presence of both bona fide regenerative nodules and dysplastic nodules in the cirrhotic livers can be interpreted as the two sides of the same coin, i.e. the clonal growth of either phenotypically normal or altered hepatocytes in a background of widespread reduced proliferative capacity [59]. From a more general standing point, liver cirrhosis is nothing but one of several settings in which chronic tissue injury and inflammation lead to both impaired function and an increased risk of neoplastic disease in the target organ [77,78]. Examples include the pancreas [79], the stomach [80,81], the intestine [82], the prostate [83], to mention but a few of the most common sites. It is reasonable to consider that a common factor in all these instances could be the progressive exhaustion of the functional and/or proliferative capacity of parenchymal cells, paving the way to the selection of rare variant cells with an altered phenotype [75]. Consistent with this interpretation is the finding of diffuse or focal parenchymal atrophy concomitant with proliferative lesions of putative clonal origin in the tissues referred to above [80–84]. As an example, the “proliferative inflammatory atrophy” of the prostate, which has been recognised as a risk factor for prostate carcinogenesis, is characterized by clusters of proliferating prostatic cells arising in areas of atrophic epithelium [85], suggesting their possible regenerative significance [83]. In the context of the preceding paragraphs, it is also of interest to consider the issue of genetically inherited defects in DNA repair pathways, which are known to be associated with increased risk of cancer. It is generally assumed that such higher risk is related to the increased probability that a series of critical genetic alterations might occur in rare cells, due to defective repair, and this will lead to the emergence of the neoplastic phenotype. However, such interpretation largely overlooks the consequences that the defective DNA repair might have on the bulk of the tissue, and their possible contribution to the increased carcinogenic risk that is seen in these conditions. Defects in DNA repair pathways can be associated with accumulation of widespread DNA damage [86]. It is reasonable to assume that such randomly inflicted damage will generally impair genome function [86], rather than improve it, in analogy to what has been discussed with reference to genotoxic agents [34]; in fact, accelerated aging has also been associated with altered DNA repair capacity [87]. Thus, the above considerations suggest that carcinogenesis related to defective DNA repair is also interpretable as the end result of at least two main biological components: (i) induction of rare altered cells and (ii) selection of such cells within a functionally impaired tissue environment.

3. The molecular analysis of selectogenic microenvironments Given that altered cells can be selected in a tissue microenvironment which is otherwise growth-inhibitory to surrounding counterparts, a relevant question pertains to the biochemical and molecular basis of such phenotypic resistance. Blagosklonny [88] has proposed the existence of two broad types of resistance: nononcogenic and oncogenic resistance. The former type relates to changes in drug metabolism and/or uptake, such that the rare altered cell is able to withstand toxicity compared to the rest of the population in that tissue. Such phenotypic resistance would still translate in the clonal growth of that rare cell, but no increased risk of neoplastic disease would be implied [88].


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However, a second type of resistance is linked to the inability of the cell to sense or repair DNA damage and/or to activate effector mechanisms leading to cell cycle arrest and/or cell death. If the latter kind of resistance occurs, then the affected cell is susceptible to acquire a “mutator phenotype”, i.e. the tendency to undergo a cascade of further mutations [34,89]. A minority of cancers are related to an inherited form of mutator phenotype: for example colon cancer in carriers of the condition known as HNPCC, in which a defect in mismatch repair (MMR) genes is inherited, so that a cascade of mutations occurs in cancer-related genes. To justify the onset of a mutator phenotype in “sporadic cancers” (which are in fact the vast majority) we have to revisit some theories of carcinogenesis and their evidence base. In sporadic cancers the origin of the mutator phenotype has been attributed to chance, or to mutagens that selectively affect specific genes similar to MMR genes, or to a combination of the two. However, MMR is clearly mutated only in a minority of cases: for example, colon cancers characterized by the presence of microsatellites (MIN) are a small minority compared to cancers characterized by chromosome instability (CIN), whose onset has not yet been attributed to the failure of any specific gene repair such as MMR (although several candidates have been proposed, Ref. [89]). To explain the most common type of lesions that are found in non-hereditary cancers, chromosome aberrations and CIN, we have to explain how the mutator phenotype originates. In addition, a key concept that has also emerged recently is that mutations –or instability – themselves are irrelevant if there is not a microenvironmental change that selects the cells carrying such mutations. Therefore, we will discuss first some examples of such a “selectogenic” microenvironment. 4. Epidemiologic evidence: early origins of genomic instability? A striking recent observation was the finding of a very high proportion, in healthy newborns, of the fusion genes TEL-AML1 and AML-ETO associated with lymphocytic leukemia (the mutation rate was about 100 times higher than the cumulative incidence of leukemia) [90]. While the origin of such mutations is not known – but could express exposure to in utero stressors – it is clear that mutations per se are insufficient to explain the onset of leukemia, which is probably due to further “hits” that select cells having a selective advantage. In another investigation in humans, Finette et al. [91] found a high prevalence of hprt mutations at birth in healthy children, coming to similar conclusions as Mori et al. In a series of well-designed experiments, Somers et al. [92] reported increased mutation rates in herring gulls and mice exposed to air pollution at levels that characterize normal urban environments. In mice, in fact, mutations were transmitted trans-generationally, i.e. they were attributed to DNA damage in sperm cells. Somatic mutations in newborns have been related to air pollutants [93], and mutations in germ cells have been attributed to air pollution or cigarette smoking [94,95]. Therefore, one can speculate that some forms of genomic instability can be already present at birth and can confer some kind of selective advantage to cells under specific environmental conditions. 5. Stress and the mutator phenotype—bacteria Bacteria under environmental stress undergo a condition which is called SLAM, or stressful lifestyle associated mutation (or stationary-phase mutability). This condition requires functional proteins of the double-strand break repair recombination system, i.e. recombination is part of a mechanism by which stationary-

phase mutations form. In conditions of stress, mutations occur at a rate that is 10–1000 times higher than in control bacteria populations [96,97]. However, mutations do not occur in all cells, but only in subpopulations, which leave the hypermutable state when the adaptive mutation(s) is generated [98]. Survivors will all carry the mutation. In this transition MMR plays a central role, being inhibited during the stationary-phase [99]. Similar modulation in stationary-phase has been shown in human cells [99]. Among the conditions that trigger the stationary-phase there are starvation and physical and chemical stress. The small minority of heritable mutator mutants resembles that seen in several examples of adaptive evolution [96]. Interestingly, the selected stress-induced mutator alleles are positively correlated with the strength of selection and negatively with the frequency of such stresses [97]. At least for one environmental contaminant, cadmium, at the dose levels typical of human exposure, mutagenesis has been shown to be indirect and related to inhibition of MMR. Jin et al. [100] found that chronic exposure of yeast to environmentally relevant concentrations of cadmium resulted in extreme hypermutability. In extracts of human cells cadmium inhibited mismatch removal.

6. Stress and the mutator phenotype—human cells Chromosome instability (CIN) and microsatellite instability (MIN) have been described as two alternative pathways to cancer. Chromosomal instability is generally defined as the ability of a cell to gain and lose chromosomes and is a feature of many (both sporadic and hereditary) types of cancer. Conversely, microsatellite instability is mainly associated with hereditary non-polyposis colorectal cancer and sporadic gastrointestinal cancers carrying a defect in the DNA mismatch repair machinery (MIN cancers). We have conducted a study in vitro aimed at investigating the ability of different carcinogens to cause CIN or MIN, respectively, in cell lines [101]. We took advantage of the cells treated with the different carcinogens to assess whether the “genetic instability phenotype” was carcinogen-specific. The arylamine 4-aminobiphenyl (4-ABP) is a tobacco smoke constituent, an environmental contaminant, and a well-established carcinogen in humans. Bladder, lung, colon and breast cancers have been associated to 4-ABP. We hypothesized that in addition or independently from its activity as direct DNA mutagen 4-ABP could affect tumor occurrence by inducing genetic instability. Our hypothesis was based on the evidence that bulky-adduct-forming agents, such as 4-ABP, can induce chromosome breaks through nucleotide excision–repair processes, leading to the suggestion that cells with defects in DNA repair or mitotic checkpoints might be selected after exposure to such agents. We have investigated the effects of 4-ABP and N-methyl-N nitro-N-nitrosoguanidine (MNNG) on genetically stable colorectal (HCT116) and bladder (RT112) cancer cells. Cells were treated with carcinogens to generate resistant clones that were then subjected to genetic analysis to assess whether they displayed either chromosomal (CIN) or microsatellite (MIN) instability. We found that 50–60% of cells treated with 4-ABP developed CIN but none developed MIN as confirmed by their ability to gain and lose chromosomes. In contrast, all MNNG-treated clones (12/12) developed MIN but none developed CIN as showed by the microsatellite assay. Because MIN has previously been linked to mismatch repair defects, we have used Western blotting to analyze the level and pattern of expression of MLH1 and MSH2 in clones resistant to the carcinogens. The results suggested that the acquired mechanism of MIN resistance in the MNNG-treated colorectal cells is associated with the reduction or the complete loss of MLH1 expression. The net result of CIN is the deregulation of chromosome number (aneuploidy) and an enhanced rate of loss of heterozygosity,

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which is an important mechanism of inactivation of tumor suppressor genes. Cytogenetic studies of bladder, lung and colon tumors have shown that karyotype complexity, cell ploidy, and the number of structural changes found are closely associated with tumor grade and stage. We have proposed that chromosomal instability in tumors such as those affecting the bladder and the colon might be the result of exposure to the tobacco smoke constituent and environmental pollutant 4-ABP or chemicals with a similar mechanism of action. Furthermore, we suggested that different environmental carcinogens can induce specific forms of genetic instability. This suggestion came from the investigation of MNNG and 4-ABP in the paper we have summarized, but also of the bulky-adduct-forming agent 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), already mentioned above, in a previous experiment by Bardelli et al. [102]. They found that cells resistant to PhIP exhibited chromosomal instability, whereas cells resistant to MNNG exhibited microsatellite instability like in Saletta’s experiment. Therefore, they also found that cells purposely made into CIN cells were resistant to PhIP, whereas MIN cells were resistant to MNNG. These data demonstrate that exposure to specific carcinogens can indeed select for tumor cells with distinct forms of genetic instability and vice versa. These data offer potential clues to one of the remaining unsolved problems in cancer research, the relationship between environmental factors and the genetic abnormalities that effect tumorigenesis.

7. From tissue microenvironment to tumor microenvironment We shall now get back to the proposed distinction between tissue and tumor microenvironments, and to their complementary role in the pathogenesis of neoplastic disease [5]. To this end, it is important to re-consider the intimate nature of tumor promotion, i.e. to re-propose the following question: why is it biologically relevant to the process of cancer development that single initiated cells undergo selective clonal expansion? A widely held view posits, almost axiomatically, that clonal amplification of altered cells fuels carcinogenic process by increasing the likelihood that further genetic changes will occur in those dividing cells, towards the acquisition of a fully malignant phenotype [103,104]. Thus, according to this view, tumor promotion consists essentially, if not exclusively, in the clonal amplification of altered cells, which is per se sufficient to increase the risk for additional genetic hits, thereby fostering cancer development. While such interpretation might be theoretically appealing, it is pertinent to point out that early focal lesions resulting from tumor promotion (i.e. polyps, nodules, papillomas) are generally not associated with the emergence of cellular sub-clones, as the hypothesis above would predict. It is also worth reiterating that promotion entails a mainly quantitative phenomenon of selective amplification [3,4,17] and appears to be driven in many cases by physiological mechanisms involved in normal tissue turnover and/or reaction to injury. Instead, we would like to propose that the essence of tumor promotion, as it relates to carcinogenic process, is not clonal growth per se, but rather the formation of proliferative focal lesions resulting from the selective expansion of initiated/altered cells (Fig. 1). In this context, the term focal lesion refers to a discrete collection of cells displaying a growth pattern and/or histological appearance which are sufficiently distinct from that of the surrounding tissue [105]. According to this view, the focal nature of early lesions represents the critical end point of tumor promotion in that it forms the basis for the establishment of a new biological niche, with a fundamentally altered tissue architecture, which is generally referred to as the tumor microenvironment [5,6,106]. In other words, the emer-


gence of the tumor microenvironment from the clonal growth of altered cells represents indeed the quantum change brought about by the tumor promotion phase in the natural history of cancer development. As discussed by Quaranta-Anderson [9] and by Fang et al. [10] in this issue of the journal, a series of biochemical and metabolic changes are typically associated with the tumor microenvironment, being attributable, at least in part, to the altered blood and nutrient supply [7]. These changes can both induce and select for cell variants within the original focal cell population, setting the stage for the emergence and evolution of cell clones which represent the biological hallmarks of tumor progression [5,7,8,32,106,107]. Consistent with this interpretation, the analysis of multistage models of carcinogenesis has clearly documented that the long phase of tumor progression, leading from discrete focal lesions to the overt neoplastic phenotype, is a self-perpetuating process and does not depend on external manipulation, as it is the case for tumor promotion [3,17]. Thus, the unique microenvironment inside focal lesions appears to be sufficient to drive tumor progression. Foulds, in his comprehensive treatise on neoplastic development, referred to different types of “precancerous” or “premalignant” lesions, arguing that the latter terms are often used inappropriately; in fact, many of those lesions “are not preanything” [1]. He described four types of lesions, based on their fate: (i) those that progress to more advanced stages, including cancer; (ii) those that continue to grow without qualitative change; (iii) those persisting with no or minimal growth and no qualitative change; (iv) those that may regress [1]. Obviously, it is a main challenge for cancer researchers to gain insights into the biological and molecular bases for such different phenotypic behaviors. In the context of the present discussion, we would like to propose that the successful establishment of a tumor microenvironment, with its associated biochemical and metabolic alterations [7,8,32,105,106], represents an important determinant to consider in this type of analysis.

8. Summary and future perspectives Cancer development commonly proceeds through a series of steps with underlying distinct pathogenetic mechanism. In the preceding discussion, we have presented evidence to suggest how specific changes in the surrounding tissue microenvironment can foster the selective expansion of putative initiated/altered cells. Such selective growth (classically defined as tumor promotion) is often sustained by mechanisms similar to those responsible for normal tissue turnover and/or repair [3,30,43,59]. However, the salient feature of tumor promotion is not the clonal amplification of initiated cells per se, but rather the specific growth pattern resulting from such amplification, i.e. the formation of focal proliferative lesions, or discrete collections of cells with a tissue architecture distinct from that of surrounding counterparts. This represents a critical step in cancer development, in that it forms the basis for the emergence of the tumor microenvironment, a new and unique biological niche inside focal lesions that can act as the landscape for tumor progression to occur [7,8,32,106,107]. As pointed out by Foulds, not all focal lesions are equal and truly preneoplastic [1]. Accordingly, an important area of cancer research is to explore how such differences in phenotypic behavior can be accounted for by different microenvironmental cues arising inside the specific milieu of focal lesions. The latter include biochemical and metabolic changes related to altered blood supply, the fundamental interaction with stromal components [108], the influence of any infiltrating cell population, such as macrophages [109] or other white blood cells [110]. Learning more insights into the spe-


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