Douglas Hanahan, Robert A. Weinberg. Hallmarks of cancer: the next generation. Cell, Volume 144, Issue 5, p646–674, 4 March 2011
In 2000, the mechanisms underlying invasion and metastasis were largely an enigma. It was clear that as carcinomas arising from epithelial tissues progressed to higher pathological grades of malignancy, reflected in local invasion and distant metastasis, the associated cancer cells typically developed alterations in their shape as well as in their attachment to other cells and to the extracellular matrix (ECM). The best characterized alteration involved the loss by carcinoma cells of E-cadherin, a key cell-to-cell adhesion molecule. By forming adherens junctions with adjacent epithelial cells, E-cadherin helps to assemble epithelial cell sheets and maintain the quiescence of the cells within these sheets. Increased expression of E-cadherin was well established as an antagonist of invasion and metastasis, whereas reduction of its expression was known to potentiate these phenotypes. The frequently observed downregulation and occasional mutational inactivation of E-cadherin in human carcinomas provided strong support for its role as a key suppressor of this hallmark capability.
Additionally, expression of genes encoding other cell-to-cell and cell-to-ECM adhesion molecules is demonstrably altered in some highly aggressive carcinomas, with those favoring cytostasis typically being downregulated. Conversely, adhesion molecules normally associated with the cell migrations that occur during embryogenesis and inflammation are often upregulated. For example, N-cadherin, which is normally expressed in migrating neurons and mesenchymal cells during organogenesis, is upregulated in many invasive carcinoma cells. Beyond the gain and loss of such cell-cell/matrix attachment proteins, the master regulators of invasion and metastasis were largely unknown or, when suspected, lacking in functional validation.
The multistep process of invasion and metastasis has been schematized as a sequence of discrete steps, often termed the invasion-metastasis cascade. This depiction envisions a succession of cell-biologic changes, beginning with local invasion, then intravasation by cancer cells into nearby blood and lymphatic vessels, transit of cancer cells through the lymphatic and hematogenous systems, followed by escape of cancer cells from the lumina of such vessels into the parenchyma of distant tissues (extravasation), the formation of small nodules of cancer cells (micrometastases), and finally the growth of micrometastatic lesions into macroscopic tumors, this last step being termed “colonization.”
Research into the capability for invasion and metastasis has accelerated dramatically over the past decade as powerful new research tools and refined experimental models have become available, and as critical regulatory genes were identified. While still an emerging field replete with major unanswered questions, significant progress has been made in delineating important features of this complex hallmark capability. An admittedly incomplete representation of these advances is highlighted below.
EMT программа широко регулирует инвазию и метастазирование
A developmental regulatory program, referred to as the “epithelial-mesenchymal transition” (EMT), has become prominently implicated as a means by which transformed epithelial cells can acquire the abilities to invade, to resist apoptosis, and to disseminate. By co-opting a process involved in various steps of embryonic morphogenesis and wound healing, carcinoma cells can concomitantly acquire multiple attributes that enable invasion and metastasis. This multifaceted EMT program can be activated transiently or stably, and to differing degrees, by carcinoma cells during the course of invasion and metastasis.
A set of pleiotropically acting transcriptional factors, including Snail, Slug, Twist, and Zeb1/2, orchestrate the EMT and related migratory processes during embryogenesis; most were initially identified by developmental genetics. These transcriptional regulators are expressed in various combinations in a number of malignant tumor types and have been shown in experimental models of carcinoma formation to be causally important for programming invasion; some have been found to elicit metastasis when ectopically overexpressed. Included among the cell-biological traits evoked by such transcription factors are loss of adherens junctions and associated conversion from a polygonal/epithelial to a spindly/fibroblastic morphology, expression of matrix-degrading enzymes, increased motility, and heightened resistance to apoptosis—all traits implicated in the processes of invasion and metastasis. Several of these transcription factors can directly repress E-cadherin gene expression, thereby depriving neoplastic epithelial cells of this key suppressor of motility and invasiveness.
The available evidence suggests that these transcription factors regulate one another as well as overlapping sets of target genes. No rules have yet been established to describe their interactions and the conditions that govern their expression. Evidence from developmental genetics indicates that contextual signals received from neighboring cells in the embryo are involved in triggering expression of these transcription factors in those cells destined to pass through an EMT; in an analogous fashion, increasing evidence suggests that heterotypic interactions of cancer cells with adjacent tumor-associated stromal cells can induce expression of the malignant cell phenotypes that are known to be choreographed by one or more of these transcriptional regulators. Moreover, cancer cells at the invasive margins of certain carcinomas can be seen to have undergone an EMT, suggesting that these cancer cells are subject to microenvironmental stimuli distinct from those received by cancer cells located in the cores of these lesions.
Although the evidence is still incomplete, it would appear that EMT-inducing transcription factors are able to orchestrate most steps of the invasion-metastasis cascade save the final step of colonization. We still know rather little about the various manifestations and temporal stability of the mesenchymal state produced by an EMT. Although expression of EMT-inducing transcription factors has been observed in certain nonepithelial tumor types, such as sarcomas and neuroectodermal tumors, their roles in programming malignant traits in these tumors are presently poorly documented. Additionally, it remains to be determined whether invasive carcinoma cells necessarily acquire their capability through activation of parts of the EMT program, or whether alternative regulatory programs can also enable this capability.
Гетеротипическое содействие стромальных клеток инвазии и метастазированию
It is increasingly apparent that crosstalk between cancer cells and cells of the neoplastic stroma is involved in the acquired capability for invasive growth and metastasis. Such signaling may impinge on carcinoma cells and act to alter their hallmark capabilities as suggested above. For example, mesenchymal stem cells (MSCs) present in the tumor stroma have been found to secrete CCL5/RANTES in response to signals released by cancer cells; CCL5 then acts reciprocally on the cancer cells to stimulate invasive behavior.
Macrophages at the tumor periphery can foster local invasion by supplying matrix-degrading enzymes such as metalloproteinases and cysteine cathepsin proteases; in one model system, the invasion-promoting macrophages are activated by IL-4 produced by the cancer cells. And in an experimental model of metastatic breast cancer, tumor-associated macrophages (TAMs) supply epidermal growth factor (EGF) to breast cancer cells, while the cancer cells reciprocally stimulate the macrophages with CSF-1; their concerted interactions facilitate intravasation into the circulatory system and metastatic dissemination of the cancer cells.
Observations like these indicate that the phenotypes of high-grade malignancy do not arise in a strictly cell-autonomous manner, and that their manifestation cannot be understood solely through analyses of tumor cell genomes. One important implication, still untested, is that the ability to negotiate most of the steps of the invasion-metastasis cascade may be acquired in certain tumors without the requirement that the associated cancer cells undergo additional mutations beyond those that were needed for primary tumor formation.
Пластичность программы инвазивного роста
The role of contextual signals in inducing an invasive growth capability (often via an EMT) implies the possibility of reversibility, in that cancer cells that have disseminated from a primary tumor to a more distant tissue site may no longer benefit from the activated stroma and invasion/EMT-inducing signals that they experienced while residing in the primary tumor; in the absence of ongoing exposure to these signals, carcinoma cells may revert in their new homes to a noninvasive state. Thus, carcinoma cells that have undergone an EMT during initial invasion and metastatic dissemination may pass through the reverse process, termed the mesenchymal-epithelial transition (MET). This plasticity may result in the formation of new tumor colonies of carcinoma cells exhibiting a histopathology similar to those of carcinoma cells in the primary tumor that never underwent an EMT. Moreover, the notion that cancer cells routinely pass through a complete EMT program is likely to be simplistic; instead, in many cases, cancer cells may enter into an EMT program only partially, thereby acquiring new mesenchymal traits while continuing to express residual epithelial traits.
Отличительные формы инвазии могут лежать в основе различных типов рака
The EMT program regulates a particular type of invasiveness that has been termed «mesenchymal». In addition, two other distinct modes of invasion have been identified and implicated in cancer cell invasion. «Collective invasion» involves nodules of cancer cells advancing en masse into adjacent tissues and is characteristic of, for example, squamous cell carcinomas; interestingly, such cancers are rarely metasatic, suggesting that this form of invasion lacks certain functional attributes that facilitate metastasis. Less clear is the prevalence of an «amoeboid» form of invasion, in which individual cancer cells show morphological plasticity, enabling them to slither through existing interstices in the extracellular matrix rather than clearing a path for themselves, as occurs in both the mesenchymal and collective forms of invasion. It is presently unresolved whether cancer cells participating in the collective and amoeboid forms of invasion employ components of the EMT program, or whether entirely different cell-biological programs are responsible for choreographing these alternative invasion programs.
Another emerging concept, noted above, involves the facilitation of cancer cell invasion by inflammatory cells that assemble at the boundaries of tumors, producing the extracellular matrix-degrading enzymes and other factors that enable invasive growth; these functions may obviate the need of cancer cells to produce these proteins through activation of EMT programs. Thus, cancer cells may secrete the chemoattractants that recruit the proinvasive inflammatory cells rather than producing the matrix-degrading enzymes themselves.
Пугающая сложность метастатической колонизации
Metastasis can be broken down into two major phases: the physical dissemination of cancer cells from the primary tumor to distant tissues, and the adaptation of these cells to foreign tissue microenvironments that results in successful colonization, i.e., the growth of micrometastases into macroscopic tumors. The multiple steps of dissemination would seem to be in the purview of the EMT and similarly acting migratory programs. Colonization, however, is not strictly coupled with physical dissemination, as evidenced by the presence in many patients of myriad micrometastases that have successfully disseminated but never progress to macroscopic metastatic tumors.
In some types of cancer, the primary tumor may release systemic suppressor factors that render such micrometastases dormant, as revealed clinically by explosive metastatic growth soon after resection of the primary growth. In others, however, such as breast cancer and melanoma, macroscopic metastases may erupt decades after a primary tumor has been surgically removed or pharmacologically destroyed; these metastatic tumor growths evidently reflect dormant micrometastases that have solved, after much trial and error, the complex problem of tissue colonization.
One can infer from such natural histories that micrometastases may lack other hallmark capabilities necessary for vigorous growth, such as the ability to activate angiogenesis; indeed the inability of certain experimentally generated dormant micrometastases to form macroscopic tumors has been ascribed to their failure to activate tumor angiogenesis. Additionally, recent experiments have shown that nutrient starvation can induce intense autophagy that causes cancer cells to shrink and adopt a state of reversible dormancy; such cells may exit this state and resume active growth and proliferation when changes in tissue microenvironment, such as access to more nutrients, permit. Other mechanisms of micrometastatic dormancy may involve anti-growth signals embedded in normal tissue extracellular matrix and tumor-suppressing actions of the immune system.
Most disseminated cancer cells are likely to be poorly adapted, at least initially, to the microenvironment of the tissue in which they have landed. Accordingly, each type of disseminated cancer cell may need to develop its own set of ad hoc solutions to the problem of thriving in the microenvironment of one or another foreign tissue. These adaptations might require hundreds of distinct colonization programs, each dictated by the type of disseminating cancer cell and the nature of the tissue microenvironment in which colonization is proceeding. As further discussed below, however, certain tissue microenviroments may be preordained to be intrinsically hospitable to disseminated cancer cells.
Metastatic dissemination has long been depicted as the last step in multistep primary tumor progression, and indeed for many tumors that is likely the case, as illustrated by recent genome sequencing studies that present genetic evidence for clonal evolution of pancreatic ductal adenocarcinoma to metastasis. On the other hand, evidence has recently emerged indicating that cells can disseminate remarkably early, dispersing from ostensibly noninvasive premalignant lesions in both mice and humans. Additionally, micrometastases can be spawned from primary tumors that are not obviously invasive but possess a neovasculature lacking in lumenal integrity. Although cancer cells can clearly disseminate from such pre-neoplastic lesions and seed the bone marrow and other tissues, their capability to colonize these sites and develop into pathologically significant macrometastases remains unproven. At present, we view this early metastatic dissemination as a demonstrable phenomenon in mice and humans whose clinical significance is yet to be established.
Beyond the timing of their dissemination, it also remains unclear when and where cancer cells develop the ability to colonize foreign tissues as macroscopic tumors. This capability may arise during primary tumor formation as a result of a tumor’s particular developmental path prior to any dissemination, such that primary tumor cells entering the circulation are fortuitously endowed with the ability to colonize certain distant tissue sites. Alternatively, the ability to colonize specific tissues may only develop in response to the selective pressure on already disseminated cancer cells to adapt to growth in foreign tissue microenvironments.
Having developed such tissue-specific colonizing ability, the cells in metastatic colonies may proceed to disseminate further, not only to new sites in the body but also back to the primary tumors in which their ancestors arose. Accordingly, tissue-specific colonization programs that are evident among cells within a primary tumor may originate not from classical tumor progression occurring within the primary lesion but instead from emigrants that have returned home. Such reseeding is consistent with the aforementioned studies of human pancreatic cancer metastasis. Stated differently, the phenotypes and underlying gene expression programs of the populations of cancer cells (and of the cancer stem cells discussed below) within primary tumors may be significantly modified by reverse migration of their distant metastatic progeny.
Implicit in this self-seeding process is another notion: the supportive stroma that arises in a primary tumor and contributes to its acquisition of malignant traits may intrinsically provide a hospitable site for reseeding and colonization by circulating cancer cells emanating from metastatic lesions.
Clarifying the regulatory programs that enable metastatic colonization represents an important agenda for future research. Substantial progress is being made, for example, in defining sets of genes («metastatic signatures») that correlate with and appear to facilitate the establishment of macroscopic metastases in specific tissues. The challenge is considerable, given the apparent multitude of distinct colonization programs cited above. Moreover, colonization is unlikely to depend exclusively on cell-autonomous processes. Instead, it almost certainly requires the establishment of a permissive tumor microenvironment composed of critical stromal support cells. For these reasons, the process of colonization is likely to encompass a large number of cell-biological programs that are, in aggregate, considerably more complex and diverse than the preceding steps of metastatic dissemination.