Adaptation versus Selection: The Origins of Metastatic Behavior (2007)

During the formation of a primary tumor, premalignant cells acquire a series of genetic and epigenetic changes that enable them to proliferate in the absence of growth factors, to resist proapoptotic stimuli, and to stimulate angiogenesis (1). Each of these acquired traits confers a distinct selective advantage on those cells that develop it. In contrast, it is less clear how the initial steps of the invasion-metastasis cascade can be acquired purely as a result of selection in the primary tumor. These steps include the loss of cell-to-cell adhesion, invasion into the local microenvironment, intravasation into the blood and lymphatic vasculature, and extravasation into the parenchyma of distant tissues. Accordingly, in the absence of such local growth advantage, cells acquiring these malignant traits will not undergo clonal expansion within the primary tumor, and the alleles favoring invasion and metastasis will not be represented in large numbers of neoplastic cells within this tumor. Hence, as argued below, in most human tumors, acquisition of these malignant traits is unlikely to arise as a consequence of selection occurring in the context of the primary tumor.Most, and possibly all, carcinoma cells that disseminate from primary tumors are poorly suited to survive and proliferate in the foreign tissue environments in which they happen to have landed. This suggests that Darwinian selection is indeed important during the last step of the invasion-metastasis cascade, termed colonization, which depends on the ability of the disseminated cancer cells forming a micrometastasis to adapt to the foreign tissue microenvironments at the site of final dissemination. It is clear that the vast majority of micrometastases fail to devise the means to survive and thrive in such novel microenvironments and eventually disappear. Here, enormous selective pressure operates to favor the outgrowth of the cells in a rare micrometastasis that can indeed solve the problem of adaptation. Still, this colonization step does not illuminate the processes occurring during the preceding steps of the invasion-metastasis cascade—the topics of this article.As we argue here, many of the cellular phenotypes associated with the earlier steps of this cascade, such as motility and invasiveness, do not arise as purely cell-autonomous processes. Instead, recruited stromal cells seem to play key roles in the acquisition of these traits by carcinoma cells, and adaptation of cancer cells to these signals, rather than selection, seems to be key to initiating these phenotypes (2).Our mechanistic insights into how adaptation may operate have been greatly increased by the discovery that within normal and neoplastic cells, a normally latent transcriptional circuitry resides that can confer on cancer cells many of the traits needed to complete the initial steps of the invasion-metastasis cascade. Developmental genetics has revealed that this circuitry is controlled by a set of master regulators that induce the transdifferentiation program termed the epithelial-mesenchymal transition (EMT), through which epithelial cells lose their epithelial traits and acquire instead many of the attributes of mesenchymal cells, including a loss of association with epithelial cell sheets and acquisition of cell motility, invasiveness, and resistance to apoptosis (3, 4).Normally, EMT programs are used to effect a number of critical morphogenetic steps during development, such as the formation of mesoderm and the migration of neural crest cells. Reflecting this complexity, this transdifferentiation program can be choreographed by a number of distinct, pleiotropically acting transcription factors, such as Snail, Twist, Slug, and Goosecoid (5–8). When active, each of these factors can initiate the complex transcriptional program that imparts to cancer cells many of the traits associated with high-grade malignancy. The involvement of these transcription factors in cancer pathogenesis exemplifies a situation in which cancer cells opportunistically co-opt cell biological programs that are normally operative during early embryogenesis (and perhaps during certain types of wound healing). Because expression of these transcription factors by embryonic cells is provoked by contextual signals that these cells receive from their surroundings, it is likely that such signals act similarly during cancer pathogenesis to trigger expression of these EMT-inducing transcription factors in cancer cells.Although hardly proven, it is plausible at present that most human carcinoma cells access and exploit components of the EMT program to acquire malignant cell traits. Indeed, it is even possible that nonepithelial cancer cells also use this program when they progress to highly malignant growth states.At the biochemical level, the EMT program involves the down-regulation of epithelial protein expression, notably E-cadherin and cytokeratins, and the induction of mesenchymal protein expression, including vimentin, N-cadherin, fibronectin, platelet-derived growth factor receptor, and matrix metalloproteinases. These changes are accompanied by the aforementioned cell biological changes, such as acquisition of motility and invasiveness (reviewed in ref. 5). When active in cancer cells, this program should enable them to complete the initial steps of the invasion-metastasis cascade, specifically local invasion, intravasation, survival while in the circulation, and extravasation. Although a transition from a completely epithelial to a completely mesenchymal state may occur in some human tumors, it seems likely that, more often than not, cancer cells advance partway into the EMT program, shedding some but not all epithelial markers and acquiring concomitant expression of some but not all mesenchymal markers. Importantly, the EMT is reversible, and carcinoma cells that have acquired a mesenchymal phenotype can revert to an epithelial state via a mesenchymal-epithelial transition (9).The question of whether invasive and metastatic behavior is acquired as a consequence of selection or adaptation can best be addressed by studying the state of the EMT program in a variety of tumors, posing the question of whether the EMT program is turned on reversibly in tumor cells or is constitutively activated in such cells. As argued here, if the EMT program is reversibly activated in carcinoma cells, then the case for adaptation is strongly supported; conversely, if the EMT program is activated irreversibly (constitutively), then the case for selection is favored.We actually know rather little about the signals and circumstances that are responsible for activating the EMT program in cancer cells. The seminal work of the Beug group showed that this program can be activated in ras-transformed mouse mammary carcinoma cells simply by the application of transforming growth factor-β to cells in vitro (10). This discovery represented the first demonstration that the EMT program in cancer cells can be triggered through signals that they receive from their microenvironment. Hence, cancer cells bearing multiple mutated genomes need not sustain additional mutations to activate this program, doing so instead in response to contextual signals that they receive from nearby cells, ostensibly those forming the tumor-associated stroma.This model has been sustained by many other subsequent reports, indicating that a variety of extracellular signals can contribute to triggering the EMT in the context of neoplasia; included here are the signals conveyed by hepatocyte growth factor (11), tumor necrosis factor-α (12), Notch (13), Hedgehog (14), Wnts (15), and platelet-derived growth factor (16). These reports and others suggest that an EMT is usually triggered by a convergence of several of these signals. (Because the EMT is used to effect morphogenetic steps in a variety of tissue sites during embryogenesis, it seems unlikely that a common, invariant set of heterotypic signals is responsible for inducing this program in all types of tumor cells.).Implicit in these models is an important assumption—specifically that such EMT-inducing signals do not originate in the cancer cells themselves, and therefore do not form a self-initiating autocrine signaling loop. Instead, they seem to originate in other cell types, for example those forming the stroma. As such, the signals responsible for activation of the EMT would seem to exemplify paracrine signaling, more specifically, heterotypic signals flowing from stroma to epithelium within a carcinoma.The notion that microenvironmental signals are responsible for activating the EMT holds a number of important further implications. Because the putative inductive signals are likely to act only over short distances, as is the case with most paracrine factors, it seems likely that only a subset of cells within an epithelial cell island will experience these signals and undergo an EMT. In fact, in the context of colon cancer pathogenesis, the EMT has been associated specifically with the cells at the invasive front at the epithelial-stromal interface and with individual carcinoma cells that have left the invasive front and invaded singly into the nearby stroma (17). Moreover, in the authors' laboratory, when studying the behavior of human tumor xenografts, the EMT can be clearly observed to affect a cell layer one to two cells deep that is directly apposed to the stroma; cells lying more internally in epithelial cells islands maintain their characteristic epithelial morphology and continue to express epithelial markers.11K.A. Hartwell and T.A. Ince, unpublished observations.The notion that contextual signals trigger the EMT suggests its transience and reversibility: When cancer cells within a primary tumor disseminate to distant sites within the body, they will no longer encounter the mix of contextual signals that they experienced in the primary tumor, specifically the signals released by the activated stroma within the primary tumor. In the absence of an ongoing flux of these inductive signals, the disseminated cancer cells may then revert, via a mesenchymal-epithelial transition, to the epithelial phenotype of their ancestors within the core of the primary tumor (5).Yet a third idea is implied in this model: Although hardly addressed experimentally, it seems likely that certain tumor cells are responsive to the paracrine, EMT-inducing signals whereas others are not. Such responsiveness may be dictated by the differentiation program expressed by the cancer cells, and may thus be reflective of their normal cell of origin. Even more important, however, may be the spectrum of genetic and epigenetic lesions that the cancer cells have accumulated during the course of multistep tumor pathogenesis. Included here are both mutations as well as DNA methylation events and resulting silencing of gene promoters. One set of these somatically acquired changes may render a cancer cell responsive to these EMT-inducing signals, whereas another set may not confer such responsiveness, leaving the cancer cells refractory to induction of an EMT. Moreover, it seems likely that fully normal epithelial cells will not be responsive to the EMT-inducing heterotypic signals that succeed in eliciting an EMT from derived carcinoma cells.To summarize, successful induction of an EMT would seem to be dictated by a confluence of at least three factors: (a) the differentiation program inherited by tumor cells from their normal cell of origin; (b) the set of heritable, somatically acquired changes accumulated during the course of tumor development; and (c) the mix of heterotypic signals that islands of carcinoma cells receive from the nearby activated stroma.It remains to be seen how often the EMT program is activated reversibly in commonly occurring human tumors. Progress in addressing this issue has been held back by several obstacles. First, antibodies that can be used to detect EMT-inducing transcription factors have been lacking. Second, in the event that epithelial cancer cells undergo a full EMT, it is difficult to resolve them from the mesenchymal cell types that exist naturally within the tumor-associated stroma. Third, and perhaps most importantly, as depicted above, the EMT may only involve a small subset of carcinoma cells—the minority that reside at the interface between epithelium and stroma within a carcinoma. Consequently, analytic procedures that are used to assess the importance of EMTs may often fail to do so, simply because they analyze entire carcinoma cell populations within a tumor, rather than the small minority of carcinoma cells at the invasive edge.Validation of the frequent and critical role of the EMT in carcinoma pathogenesis holds important implications for the adaptation versus selection debate. We imagine that during the early stages of multistep tumor progression, preneoplastic cells successively acquire a range of alleles that confer on them increasing proliferative and survival advantage. As mentioned, at this stage, the role of selection is clear and unambiguous. Importantly, these cells are undergoing such Darwinian evolution in a tissue microenvironment that still contains relatively normal stroma. Hence, these cells are not evolving in a context defined by EMT-inducing heterotypic signals.All this changes as the primary tumor reaches its final preinvasive state. The tumor cells may have acquired an array of alleles that render them responsive to EMT-inducing heterotypic signals. Equally important, at a certain point during progression, the surrounding stroma becomes “activated,” as indicated by the accumulation of myofibroblasts and a variety of inflammatory cells; for the first time, the stroma can release EMT-inducing signals, and only now does the induction of an EMT in the carcinoma cells become possible.This scenario represents a clear example of the acquisition of highly malignant cell biological traits through an adaptive mechanism. Thus, as preneoplastic cells proceed through the multistep evolution that leads them to the final preinvasive stage, these cells cannot anticipate future encounters by their descendants with EMT-inducing signals that will eventually be released by the nearby stroma. Moreover, because these preinvasive cells do not yet display the phenotypes of an EMT, such phenotypes cannot affect their reproductive fitness during these earlier, preinvasive steps of tumor progression. Finally, because the EMT is often exhibited by hundreds of cells at the outer edges of an epithelial cell island, it is highly unlikely that these cells are the results of a stably heritable trait (which is required for selection to operate), rather than a localized adaptation to a stromal signaling environment.Use of gene expression arrays to provide prognostic information about primary human breast cancers sheds further light on the adaptation versus selection debate. It is clear that in a subset of human breast cancers, the spectrum of expressed genes provides a clear indication of a greatly increased risk of eventual metastatic relapse (18). Importantly, because the pattern of expressed genes is readily detectable, it seems highly likely that this set of genes is expressed by the dominant cell clone in each primary tumor. (Conversely, it is experimentally unlikely that this pattern reflects the gene expression pattern of a small minority subclone of neoplastic cells residing in the primary tumor.) Hence, for whatever reason, the clonal expansion of cells expressing this set of genes was favored during multistep tumorigenesis.In these primary tumors, histopathologic analyses will quite often fail to reveal any of the cellular and tissue phenotypes that are associated with aggressive invasion and metastasis. (Indeed, success in detecting invasiveness and metastatic ability at the histologic level would obviate the use of gene expression arrays as a prognostic tool.) Accordingly, some tumors have a low probability of metastasizing, whereas other histopathologically identical tumors have a high tendency to do so.Here, one is confronted with cell clones that have undergone clonal expansion in the absence of any demonstrable phenotypic advantage conferred by high-grade invasiveness and metastasis. Hence, to the extent that they eventually become invasive and metastatic, these are unselected phenotypes that have been acquired adventitiously—unintended side products of tumor progression. Indeed, it seems to us increasingly likely that the metastatic powers of such breast cancer cells are strongly predetermined by the differentiation programs of their respective normal cells of origin as well as by mutations that are sustained in the early steps of tumor progression, long before invasive and metastatic traits could possibly be operative. These early influences will, by necessity, become widely represented in the descendant cell clones arising years later, when invasion and metastasis do indeed become phenotypically apparent.The scenario depicted above argues strongly for adaptation as the prime driving force that underlies the acquisition of highly malignant traits by neoplastic cells. (Yet other terms, such as “induction” or “education,” may ultimately prove to be more useful than “adaptation.”) However, adaptation cannot hold true for all types of human cancer, because of a highly interesting subset of these tumors that enter into a state in which the EMT is expressed constitutively (i.e., essentially irreversibly). We refer here to situations in which genetic or epigenetic lesions within the cancer cell genome create a cell autonomous, stably expressed EMT state.The most apparent of these states derives from numerous types of human carcinoma cells in which the expression of E-cadherin is compromised, either by reading-frame mutations or by methylation of the CDH1 gene promoter (19). Although we have depicted E-cadherin as one of the downstream effectors of epithelial phenotype and a target of repression by EMT-inducing transcription factors (see above), in fact, E-cadherin, on its own, may function as a pleiotropically acting regulator to elicit most, if not all, of an EMT. More specifically, work from our own laboratory and earlier work of others indicates that when E-cadherin is lost, many of the changes associated with an EMT are triggered (20). In the event that E-cadherin loss is essentially transdifferentiation to a mesenchymal state by carcinoma cells may also of inducing an EMT clearly depends on a cell-autonomous that a acquired by the carcinoma cells In this it with the of EMTs that are by heterotypic signals in the tumor-associated stroma. Because this is cell autonomous, it as a possible of Darwinian if E-cadherin loss is in many if not all neoplastic cells within a this loss have conferred a cell biological the clonal expansion of the the downstream of acquiring an EMT may the acquisition of local invasiveness within a primary tumor. It seems highly at this that cells that are invasive are well to multiple additional steps of the invasion-metastasis cascade, including intravasation, through vasculature, and we still cannot how metastatic can be a trait during the course of primary tumor This us to the that localized invasiveness than metastatic is indeed to certain primary tumor cells. it be so is hardly dictated by However, we can on at least into the stroma carcinoma cells greatly access to many of the and factors that are usually released by stromal cells and through to the invasiveness may also be by cancer cells to from the of primary tumors. suggests that may be an of the EMT It is possible to that the EMT program is turned it not only cells to but confers them the ability to resist additional stimuli, them a clear selective advantage. In fact, it has become increasingly that in the EMT program by master regulators such as and is the ability to resist apoptosis the of the adaptation versus selection is from We this that both may be is likely to be favored in the majority of in the EMT is reversibly (i.e., selection may be favored in the minority of tumors in which the EMT is by in the E-cadherin by the present is the that the EMT may be and in a small minority of tumors by a of alleles including that have been acquired during tumor progression, been for their and survival remains to be by provides an of epithelial-mesenchymal transition as a in the metastatic the by these two are several of first is the that metastasis is a complex and the that of this is critical to the of as in the by is between tumor cells and and cells. Although these are by the as epithelial cancer cells that have undergone EMT, it is also in their that such an origin is to from mesenchymal cells within the tumor-associated stroma. it is argued that to EMT, epithelial cells lose their epithelial traits and acquire mesenchymal attributes including an increased malignant a and by morphology and cellular of cells the of mesenchymal cells is associated with by inflammatory and has been a in the in those of and and In the present are on and cell In our we have the clonal selection to the over which this We from this that many into the clonal selection that the of tumor cell circulation, and growth at a site results in the survival of only a minority of tumor cells with the formation of a metastatic One important point of between the two is the that clonal selection during metastasis for the survival of tumor cells of the metastatic and not for tumor cells that have tumor Although this may seem as a it is a critical to the clonal selection of role of EMT in tumor and metastasis provides an from which to the of metastasis. However, as in the by it is only by from in (1). Indeed, the cellular within tumors, including by and cells provides an origin for the and processes as EMT within metastasis. as by some tumor cell to be important to the metastatic may also primary tumor a point of with the clonal selection unlikely for all metastatic invasive cancer cells are histopathologically apparent as cellular yet to the primary tumor. some metastatic cells can be as and growth as Thus, the histopathologic of primary tumors, and metastatic can provide prognostic as well as mechanistic into the of metastasis. Indeed, most notably of the have been to have prognostic to in a of the role clonal selection has in the of metastasis is It is our that these will stimulate and most as this However, we would to the need for any of metastasis to from models with from human tissue and the the of the the and of