Seminars in Cancer Biology 18 (2008) 322–329
Contents lists available at ScienceDirect
Seminars in Cancer Biology journal homepage: www.elsevier.com/locate/semcancer
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
323 324 324 324 325 326 326 326 327 327 328 328
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]
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?
E. Laconi et al. / Seminars in Cancer Biology 18 (2008) 322–329
progression. Broadly speaking, tumor promotion consists of the selective (clonal) expansion of altered cells to form focal lesions . 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 . 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 , 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  (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 . 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 . 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 . 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 , 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 . 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.
E. Laconi et al. / Seminars in Cancer Biology 18 (2008) 322–329
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 . 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  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 . 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 . 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  and orotic acid  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 . 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 , possibly as a consequence of the inﬂicted DNA-damage , 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 . 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 .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  (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 ; however, at lower concentrations it can stimulate proliferation of mammary epithelial cells through an estrogen receptor-dependent pathway . 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 . 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
E. Laconi et al. / Seminars in Cancer Biology 18 (2008) 322–329
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 , 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 . 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 , 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  (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 , 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 . Studies from our laboratories have further indicated that the aged rat liver stimulates the clonal expansion of transplanted normal hepatocytes . 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 . 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 . 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  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 . 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 . 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 , the stomach [80,81], the intestine , the prostate , 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 . 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 , suggesting their possible regenerative signiﬁcance . 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 . It is reasonable to assume that such randomly inﬂicted damage will generally impair genome function , rather than improve it, in analogy to what has been discussed with reference to genotoxic agents ; in fact, accelerated aging has also been associated with altered DNA repair capacity . 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  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 .
E. Laconi et al. / Seminars in Cancer Biology 18 (2008) 322–329
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. ). 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) . 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.  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.  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 , 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 . Survivors will all carry the mutation. In this transition MMR plays a central role, being inhibited during the stationary-phase . Similar modulation in stationary-phase has been shown in human cells . 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 . Interestingly, the selected stress-induced mutator alleles are positively correlated with the strength of selection and negatively with the frequency of such stresses . 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.  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 . 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,
E. Laconi et al. / Seminars in Cancer Biology 18 (2008) 322–329
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. . 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 . 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 . 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  and by Fang et al.  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 . 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” . 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 . 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 . 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 , the inﬂuence of any inﬁltrating cell population, such as macrophages  or other white blood cells . Learning more insights into the spe-
E. Laconi et al. / Seminars in Cancer Biology 18 (2008) 322–329
ciﬁc determinants of tumor progression derived from the tumor microenvironment will enable to devise novel approaches for the management of pre-neoplastic and neoplastic disease. Acknowledgments This work was supported MIUR (Italian Ministry of University and Research, to EL), by AIRC (Italian Association for Cancer Research, to EL), by Compagnia di San Paolo (Torino, to PV) and by ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: “Food Quality and Safety” (Contract No. 513943, to PV). References  Foulds L. Neoplastic development, vol. 2. Academic Press; 1975.  Leslie A, Carey FA, Pratt NR, Steele RJ. The colorectal adenoma–carcinoma sequence. Br J Surg 2002;89:845–60.  Sarma DS, Rao PM, Rajalakshmi S. Liver tumour promotion by chemicals: models and mechanisms. Cancer Surv 1986;5:781–98.  Foulds L. Multiple etiological factors in cancer development. Cancer Res 1965;25:1339–47.  Laconi E. The evolving concept of tumor microenvironments. BioEssays 2007;29:738–44.  Baluk P, Hashizume H, McDonald DM. Cellular abnormalities of blood vessels as targets in cancer. Curr Opin Genet Dev 2005;15:102–11.  Huang LE, Bindra RS, Glazer PM, Harris AL. Hypoxia-induced genetic instability—a calculated mechanism underlying tumor progression. J Mol Med 2007;85:139–48.  Gillies RJ, Gatenby RA. Hypoxia and adaptive landscapes in the evolution of carcinogenesis. Cancer Metast Rev 2007;26:311–7.  Quaranta V, Rejniak KA, Gerlee P, Anderson ARA. Sem Cancer Biol 2008;18:338–48.  Fang JS, Gillies RD, Gatenby RA. Sem Cancer Biol 2008;18:330–7.  Iannaccone PM, Weinberg WC, Deamant FD. On the clonal origin of tumors: a review of experimental models. Int J Cancer 1987;39:778–84.  Preston SL, Wong WM, Chan AO, Poulsom R, Jeffery R, Goodlad RA, et al. Bottom-up histogenesis of colorectal adenomas: origin in the monocryptal adenoma and initial expansion by crypt ﬁssion. Cancer Res 2003;63:3819–25.  Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA. Creation of human tumour cells with deﬁned genetic elements. Nature 1999;400:464–8.  Sun B, Chen M, Hawks C, Hornsby PJ, Wang X. Tumorigenic study on hepatocytes coexpressing SV40 with Ras. Mol Carcinog 2006;45:213–9.  Yang G, Rosen DG, Mercado-Uribe I, Colacino JA, Mills GB, Bast Jr RC, et al. Knockdown of p53 combined with expression of the catalytic subunit of telomerase is sufﬁcient to immortalize primary human ovarian surface epithelial cells. Carcinogenesis 2007;28:174–82.  Brash DE. Photocopying cancer cells. J Invest Dermatol 2003;121:XII–V.  Farber E, Cameron RG. The sequential analysis of cancer development. Adv Cancer Res 1980;31:125–226.  Rubin H. Microenvironmental regulation of the initiated cell. Adv Cancer Res 2003;90:1–62.  Vineis P. Cancer as an evolutionary process at the cell level: an epidemiological perspective. Carcinogenesis 2003;24:1–6.  Hanigan MH, Pitot HC. Growth of carcinogen-altered rat hepatocytes in the liver of syngeneic recipients promoted with phenobarbital. Cancer Res 1985;45:6063–70.  Tatematsu M, Lee G, Hayes MA, Farber E. Progression in hepatocarcinogenesis: differences in growth and behavior of transplants of early and later hepatocyte nodules in the rat spleen. Cancer Res 1987;47:4699–705.  McCullough KD, Coleman WB, Smith GJ, Grisham JW. Age-dependent regulation of the tumorigenic potential of neoplastically transformed rat liver epithelial cells by the liver microenvironment. Cancer Res 1994;54:3668–71.  Laconi S, Pani P, Pillai S, Pasciu D, Sarma DSR, Laconi E. A growth constrained environment drives tumor progression in vivo. Proc Natl Acad Sci USA 2001;198:7806–11.  Jonason AS, Kunala S, Price GJ, Restifo RJ, Spinelli HM, Persing JA, et al. Frequent clones of p53-mutated keratinocytes in normal human skin. Proc Natl Acad Sci USA 1996;93:14025–9.  Mudgil AV, Segal N, Andriani F, Wang Y, Fusenig NE, Garlick JA. Ultraviolet B irradiation induces expansion of intraepithelial tumor cells in a tissue model of early cancer progression. J Invest Dermatol 2003;121:191–7.  Rodin SN, Rodin AS. Origins and selection of p53 mutations in lung carcinogenesis. Semin Cancer Biol 2005;15:103–12. ¨  Schollnberger H, Manuguerra M, Bijwaard H, Boshuizen H, Altenburg HP, Rispens SM, et al. Analysis of epidemiological cohort data on smoking effects and lung cancer with a multi-stage cancer model. Carcinogenesis 2006;27:1432–44.
 Haddow A. Cellular inhibition and the origin of cancer. Acta Unio Int Contra Cancrum 1938;3:342–53.  Farber E, Rubin H. Cellular adaptation in the origin and development of cancer. Cancer Res 1991;51:2751–61.  Laconi E, Pani P, Farber E. The resistance phenotype in the development and treatment of cancer. Lancet Oncol 2000;1:235–41.  Crespi B, Summers K. Evolutionary biology of cancer. Trends Ecol Evol 2005;20:545–52.  Cristini V, Frieboes HB, Gatenby R, Caserta S, Ferrari M, Sinek J. Morphologic instability and cancer invasion. Clin Cancer Res 2005;11:6772–9.  Solt D, Farber E. New principle for the analysis of chemical carcinogenesis. Nature 1976;263:701–3.  Breivik J. The evolutionary origin of genetic instability in cancer development. Semin Cancer Biol 2005;15:51–60.  Wille Jr JJ, Pittelkow MR, Scott RE. Normal and transformed human prokeratinocytes express divergent effects of a tumor promoter on cell cycle-mediated control of proliferation and differentiation. Carcinogenesis 1985;6:1181–7.  Willey JC, Moser Jr CE, Lechner JF, Harris CC. Differential effects of 12-Otetradecanoylphorbol-13-acetate on cultured normal and neoplastic human bronchial epithelial cells. Cancer Res 1984;44:5124–6.  Barbason H, Rassenfosse C, Betz EH. Promotion mechanism of phenobarbital and partial hepatectomy in DENA hepatocarcinogenesis cell kinetics effect. Br J Cancer 1983;47:517–25.  Sheikh A, Yusuf A, Laconi E, Rao PM, Rajalakshmi S, Sarma DS. Effect of orotic acid on in vivo DNA synthesis in hepatocytes of normal rat liver and in hepatic foci/nodules. Carcinogenesis 1993;14:907–12.  Seed TM, Kaspar LV. Acquired radioresistance of hematopoietic progenitors (granulocyte/monocyte colony-forming units) during chronic radiation leukemogenesis. Cancer Res 1992;52:1469–76. ` A, Medema RH. Restarting the cell cycle when the check van Vugt MA, Bras point comes to a halt. Cancer Res 2005;65:7037–40.  Kraemer KH. Sunlight and skin cancer: another link revealed. Proc Natl Acad Sci USA 1997;94:11–4.  Ziegler A, Jonason AS, Leffell DJ, Simon JA, Sharma HW, Kimmelman J, et al. Sunburn and p53 in the onset of skin cancer. Nature 1994;372:773– 6.  Brash DE, Zhang W, Grossman D, Takeuchi S. Colonization of adjacent stem cell compartments by mutant keratinocytes. Semin Cancer Biol 2005;15: 97–102.  Gujuluva CN, Baek JH, Shin KH, Cherrick HM, Park NH. Effect of UV-irradiation on cell cycle, viability and the expression of p53, gadd153 and gadd45 genes in normal and HPV-immortalized human oral keratinocytes. Oncogene 1994;9:1819–27.  Willey JC, Grafstrom RC, Moser Jr CE, Ozanne C, Sundquvist K, Harris CC. Biochemical and morphological effects of cigarette smoke condensate and its fractions on normal human bronchial epithelial cells in vitro. Cancer Res 1987;47:2045–9.  Pfeifer AM, Lechner JF, Masui T, Reddel RR, Mark GE, Harris CC. Control of growth and squamous differentiation in normal human bronchial epithelial cells by chemical and biological modiﬁers and transferred genes. Environ Health Perspect 1989;80:209–20.  Hussain SP, Amstad P, Raja K, Sawyer M, Hofseth L, Shields PG, et al. Mutability of p53 hotspot codons to benzo(a)pyrene diol epoxide (BPDE) and the frequency of p53 mutations in nontumorous human lung. Cancer Res 2001;61:6350–5. ¨ CM, Zhu Y, Kohyama T, et al. Cigarette smoke  Wang H, Liu X, Umino T, Skold inhibits human bronchial epithelial cell repair processes. Am J Respir Cell Mol Biol 2001;25:772–9.  Baginski TK, Dabbagh K, Satjawatcharaphong C, Swinney DC. Cigarette smoke synergistically enhances respiratory mucin induction by proinﬂammatory stimuli. Am J Respir Cell Mol Biol 2006;35:165–74.  van der Toorn M, Slebos DJ, de Bruin HG, Leuvenink HG, Bakker SJ, Gans RO, et al. Cigarette smoke-induced blockade of the mitochondrial respiratory chain switches lung epithelial cell apoptosis into necrosis. Am J Physiol, Lung Cell Mol Physiol 2007;292:L1211–8.  Hainaut P, Pfeifer GP. Patterns of p53 G → T transversions in lung cancers reﬂect the primary mutagenic signature of DNA-damage by tobacco smoke. Carcinogenesis 2001;22:367–74.  Pfeifer GP, Denissenko MF, Olivier M, Tretyakova N, Hecht SS, Hainaut P. Tobacco smoke carcinogens DNA damage and p53 mutations in smokingassociated cancers. Oncogene 2002;21:7435–51.  Miyashita M, Willey JC, Sasajima K, Lechner JF, LaVoie EJ, Hoffmann D, et al. Differential effects of cigarette smoke condensate and its fractions on cultured normal and malignant human bronchial epithelial cells. Exp Pathol 1990;38:19–29.  Gooderham NJ, Zhu H, Lauber S, Boyce A, Creton S. Molecular and genetic toxicology of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Mutat Res 2002;506–507:91–9.  Lauber SN, Ali S, Gooderham NJ. The cooked food derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine is a potent oestrogen: a mechanistic basis for its tissue-speciﬁc carcinogenicity. Carcinogenesis 2004;25:2509–17.  Gooderham NJ, Creton S, Lauber SN, Zhu H. Mechanisms of action of the carcinogenic heterocyclic amine PhIP. Cancer Lett 2007;168:269–77.
E. Laconi et al. / Seminars in Cancer Biology 18 (2008) 322–329  Laconi S, Curreli F, Diana S, Pasciu D, De Filippo G, Sarma DS, et al. Liver regeneration in response to partial hepatectomy in rats treated with retrorsine: a kinetic study. J Hepatol 1999;31:1069–74.  Laconi S, Pillai S, Porcu PP, Shafritz DA, Pani P, Laconi E. Massive liver replacement by transplanted hepatocytes in the absence of exogenous growth stimuli in rats treated with retrorsine. Am J Pathol 2001;158:771–7.  Marongiu F, Doratiotto S, Montisci S, Pani P, Laconi E. Carcinogenesis and liver repopulation: two sides of the same coin? Am J Pathol 2008;172:857–64.  Campisi J. Aging and cancer cell biology. Aging Cell 2007;6:261–3.  Ackermann M, Chao L, Bergstrom CT, Doebeli M. On the evolutionary origin of aging. Aging Cell 2007;6:235–44.  Schumacher B, Garinis GA, Hoeijmakers JH. Age to survive: DNA damage and aging. Trends Genet 2008;24:77–85.  Sharpless NE, DePinho RA. How stem cells age and why this makes us grow old. Nat Rev Mol Cell Biol 2007;8:703–13.  Min H, Montecino-Rodriguez E, Dorshkind K. Effects of aging on early B- and T-cell development. Immunol Rev 2005;205:7–17.  Ikegami T, Nishizaki T, Yanaga K, Shimada M, Kishikawa K, Nomoto K, et al. The impact of donor age on living donor liver transplantation. Transplantation 2000;70:1703–7.  Gupta S. Hepatic polyploidy and liver growth control. Semin Cancer Biol 2000;10:161–71.  Pasciu D, Montisci S, Greco M, Doratiotto S, Pitzalis S, Pani P, et al. Aging is associated with increased clonogenic potential in rat liver in vivo. Aging Cell 2006;5:373–7.  Krtolica A, Parrinello S, Lockett S, Desprez PY, Campisi J. Senescent ﬁbroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci USA 2001;98:12072–7.  Liu D, Hornsby PJ. Senescent human ﬁbroblasts increase the early growth of xenograft tumors via matrix metalloproteinase secretion. Cancer Res 2007;67:3117–26.  Trost TM, Lausch EU, Fees SA, Schmitt S, Enklaar T, Reutzel D, et al. Premature senescence is a primary fail-safe mechanism of ERBB2-driven tumorigenesis in breast carcinoma cells. Cancer Res 2005;65:840–9.  Lunz 3rd JG, Tsuji H, Nozaki I, Murase N, Demetris AJ. An inhibitor of cyclin-dependent kinase, stress-induced p21Waf-1/Cip-1, mediates hepatocyte mito-inhibition during the evolution of cirrhosis. Hepatology 2005;41:1262–71.  Marshall A, Rushbrook S, Davies SE, Morris LS, Scott IS, Vowler SL, et al. Relation between hepatocyte G1 arrest, impaired hepatic regeneration, and ﬁbrosis in chronic hepatitis C virus infection. Gastroenterology 2005;128:33–42.  Clouston AD, Powell EE, Walsh MJ, Richardson MM, Demetris AJ, Jonsson JR. Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis. Hepatology 2005;41:809–18.  Richardson MM, Jonsson JR, Powell EE, Brunt EM, Neuschwander-Tetri BA, Bhathal PS, et al. Progressive ﬁbrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction. Gastroenterology 2007;133:80–90.  Prehn RT. Regeneration versus neoplastic growth. Carcinogenesis 1997;18:1439–44.  El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132:2557–76.  Philip M, Rowley DA, Schreiber H. Inﬂammation as a tumor promoter in cancer induction. Semin Cancer Biol 2004;14:433–9.  Perwez Hussain S, Harris CC. Inﬂammation and cancer: an ancient link with novel potentials. Int J Cancer 2007;121:2373–80. ¨ H, Treiber M, Lesina M, Schmid RM. Mechanisms of disease: chronic  Algul inﬂammation and cancer in the pancreas—a potential role for pancreatic stellate cells? Nat Clin Pract Gastroenterol Hepatol 2007;4:454–62.  Fox JG, Wang TC. Inﬂammation atrophy, and gastric cancer. J Clin Invest 2007;117:60–9.  Correa P, Houghton J. Carcinogenesis of Helicobacter pylori. Gastroenterology 2007;133:659–72.  Harpaz N. Neoplastic precursor lesions related to the development of cancer in inﬂammatory bowel disease. Gastroenterol Clin North Am 2007;36:901–26, vii–viii.  De Marzo AM, Nakai Y, Nelson WG. Inﬂammation, atrophy, and prostate carcinogenesis. Urol Oncol 2007;25:398–400.
 Brune K, Abe T, Canto M, O’Malley L, Klein AP, Maitra A, et al. Multifocal neoplastic precursor lesions associated with lobular atrophy of the pancreas in patients having a strong family history of pancreatic cancer. Am J Surg Pathol 2007;31:645–6.  De Marzo AM, Marchi VL, Epstein JI, Nelson WG. Proliferative inﬂammatory atrophy of the prostate: implications for prostatic carcinogenesis. Am J Pathol 1999;155:1985–92.  Rass U, Ahel I, West SC. Defective DNA repair and neurodegenerative disease. Cell 2007;130:991–1004.  de Boer J, Andressoo JO, de Wit J, Huijmans J, Beems RB, van Steeg H, et al. Premature aging in mice deﬁcient in DNA repair and transcription. Science 2002;296:1276–9.  Blagosklonny MV. Oncogenic resistance to growth-limiting conditions. Nat Rev Cancer 2002;2:221–5.  Bielas JH, Loeb LA. Mutator phenotype in cancer: timing and perspectives. Environ Mol Mutagen 2005;45:206–13.  Mori H, Colman SM, Xiao Z, Ford AM, Healy LE, Donaldson C, et al. Chromosome translocations and covert leukemic clones are generated during normal fetal development. Proc Natl Acad Sci USA 2002;99:8242–7.  Finette BA, Homans AC, Rivers J, Messier T, Albertini RJ. Accumulation of somatic mutations in proliferating T cell clones from children treated for leukemia. Leukemia 2001;15:1898–905.  Somers CM, Yauk CL, White PA, Parfett CL, Quinn JS. Air pollution induces heritable DNA mutations. Proc Natl Acad Sci USA 2002;99:15904–7.  Perera F, Hemminki K, Jedrychowski W, Whyatt R, Campbell U, Hsu Y, et al. In utero DNA damage from environmental pollution is associated with somatic gene mutation in newborns. Cancer Epidemiol Biomarkers Prev 2002;11:1134–7.  Rubes J, Selevan SG, Sram RJ, Evenson DP, Perreault SD. GSTM1 genotype inﬂuences the susceptibility of men to sperm DNA damage associated with exposure to air pollution. Mutat Res 2007;625:20–8.  Samet JM, DeMarini DM, Malling HV, Biomedicine. Do airborne particles induce heritable mutations? Science 2004;304:1008–10.  Rosenberg SM, Thulin C, Harris RS. Transient and heritable mutators in adaptive evolution in the lab and in nature. Genetics 1998;148:1559–66.  Bjedov I, Tenaillon O, Gerard B, Souza V, Denamur E, Radman M, et al. Stressinduced mutagenesis in bacteria. Science 2003;300:1404–9.  Hall BG. Spontaneous point mutations that occur more often when advantageous than when neutral. Genetics 1990;126:5–16.  Harris RS, Feng G, Ross KJ, Sidhu R, Thukin C, Longerich S, et al. Mismatch repair is diminished during stationary-phase mutation. Mutat Res 1999;437: 51–60.  Jin YH, Clark AB, Slebos RJC, Al-Refai H, Taylor JA, Kunzel TA, et al. Cadmium is a mutagen that acts by inhibiting mismatch repair. Nat Genet 2003;14:326–9.  Saletta F, Matullo G, Manuguerra M, Arena S, Bardelli A, Vineis P. Exposure to the tobacco smoke constituent 4-aminobiphenyl induces chromosomal instability in human cancer cells. Cancer Res 2007;67:7088–94.  Bardelli A, Cahill DP, Lederer G, Speicher MR, Kinzler KW, Vogelstein B, et al. Carcinogen speciﬁc induction of genetic instability. Proc Natl Acad Sci USA 2001;98:5770–5.  Simpson AJ. The natural somatic mutation frequency and human carcinogenesis. Adv Cancer Res 1997;71:209–40.  Arends JW. Molecular interactions in the Vogelstein model of colorectal carcinoma. J Pathol 2000;190:412–6.  Libbrecht L, Desmet V, Roskams T. Preneoplastic lesions in human hepatocarcinogenesis. Liver Int 2005;25:16–27.  Bindra RS, Glazer PM. Genetic instability and the tumor microenvironment: towards the concept of microenvironment-induced mutagenesis. Mutat Res 2005;569:75–85.  Anderson AR, Weaver AM, Cummings PT, Quaranta V. Tumor morphology and phenotypic evolution driven by selective pressure from the microenvironment. Cell 2006;127:905–15.  Ingber DE. Sem Cancer Biol 2008;18:356–64.  Sica A, Larghi P, Mancino A, Rubino L, Porta C, Totaro MG, et al. Sem Cancer Biol 2008;18:349–55.  Noonan DM, De Lerma Barbaro A, Vannini N, Mortara L, Albini A. Inﬂammation, inﬂammatory cells and angiogenesis: decisions and indecisions. Cancer Metast Rev 2008;27:31–40.