Amato J. Giaccia,Janine T. Erler
SUMMARY OF KEY POINTS
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INTRODUCTION
Tumors are described as benign or malignant. Malignant tumors can spread by invasion and metastasis, whereas benign tumors cannot and remain localized. One of the hallmarks of cancer cells is their ability to grow and divide without undergoing senescence, provided they have sufficient oxygen, nutrients, and space. As tumors grow, oxygen and nutrients can quickly become limiting in large part as a result of an inadequate vascular supply. Cancer cells will adapt to these growth-limiting environments and also seek out fresh terrain to take up residence, where neither space nor nutrients are (initially) limiting. The spread of cancer from its primary site to secondary sites in the body is defined as metastasis, which comes from the Greek word meaning “change of state.” These secondary sites may be located in a new organ or in a different region of the same organ. In reductionist terms, cancer cells metastasize by dislodging from the primary tumor, penetrating through lymphatic and blood vessels, and establishing new growth at a new site in normal tissue. Cancer cells must acquire the capability for invasion and metastasis to escape the primary tumor mass and colonize new terrain in the body where nutrients and space are not limiting. Acquisition of this capability for invasion and metastasis is another of the hallmarks of cancer[1] and is significantly influenced by changes in gene expression and by microenvironmental factors. It is the ability to spread to other tissues and organs that makes cancer a potentially life-threatening disease, in that metastases are responsible for 90% of cancer patient deaths.[2] Very few treatment options exist for patients with metastatic cancer, and furthering our understanding of the process of metastasis will aid in the development of new approaches to treat metastatic disease.[3]
MULTISTEP PROCESS OF METASTASIS
Metastasis is a multistep process ( Fig. 3-1 ) consisting of a series of discrete biological processes. These steps allow primary tumor cells to invade the surrounding tissue, intravasate through blood vessels to enter the circulatory or lymph system, acquire mutations to survive fluctuating environmental changes, extravasate from the circulatory or lymph system into new tissue, proliferate at secondary sites, and develop a vascular system to support growth of the metastases. In some cases, metastases can also give rise to new metastases. There is a propensity for certain tumors to seed in particular organs in part as a result of blood flow. However, blood flow alone cannot explain the different patterns of metastases found for different primary tumors. The most popular theory to explain the patterns of metastases is the “seed and soil” theory put forth by Stephen Paget over a century ago in 1889.[4] Paget described tumor cells as “seeds” and the host environment as the “soil,” and proposed that their interaction determines metastatic outcome.
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Figure 3-1 Metastasis is a multistep process consisting of a series of discrete biological events. These allow primary tumor cells to invade the surrounding tissue, intravasate the circulatory system and survive this harsh environment, extravasate into new tissue, proliferate at secondary sites, and develop a vascular system to support growth of the metastases. See text for details. BM, basement membrane; ECM, extracellular matrix. (Adapted from Le QT, Denko NC, Giaccia AJ: Hypoxic gene expression and metastasis. Cancer Metastasis Rev 2004;23:293–310.) |
Although the Paget theory is appealing, we know that metastasis is a very inefficient process, because very few cells that escape the primary tumor take up residence in new tissues. For this reason, the tumor cells that survive the process have been termed by Fidler as the “decathlon champions,” because they excel at all the steps in the metastatic process.[5] Metastasis is strongly influenced by the interactions between tumor and host cells, and by both the immediate and extended tumor microenvironments. There is ever more evidence demonstrating that these interactions between tumor and host cells are key determinants for the success of metastatic growth and spread.
The Tumor Microenvironment Acts as a Selection Pressure for Metastatic Tumor Cells
The first step in tumorigenesis is transformation, where cells accumulate mutations in proto-oncogenes that result in dysregulated cell growth and increased life span, and loss of tumor suppressor genes that normally act to limit cell growth and viability. In addition, alterations in DNA damage sensing and repair pathways result in decreased genomic stability and can promote tumor progression. In contrast to cellular transformation, tumor cells must overcome a different set of barriers to metastasize. These are external barriers created by the tumor microenvironment that limit tumor progression ( Fig. 3-2 ). External forces include physical barriers such as extracellular matrix (ECM) components and basement membranes, as well as physiologic barriers such as limited oxygen (hypoxia) and nutrients, changes in pH, and immunologic barriers by the immune system.[2] Cells respond to external microenvironmental influences by altering gene expression such that they are able to adapt and survive. The tumor microenvironment thus exerts a selection pressure for cells capable of overcoming these barriers, driving tumor progression.
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Figure 3-2 Barriers for tumor progression. The first step in tumorigenesis is cellular transformation where cells must overcome several internal barriers. To metastasize, cells must overcome external barriers put in place by the tumor microenvironment. (Adapted from Gupta GP, Massague J: Cancer metastasis: building a framework. Cell 2006;127:679–695.) |
Tumor hypoxia is a potent microenvironmental influence and is associated with metastasis and poor survival in cancer patients. [6] [7] [8] [9] Hypoxia selects for cells with low apoptotic potential [10] [11] [12]and increases genomic instability, allowing rapid mutational adaptations. [13] [14] Hypoxia additionally increases the expression of genes involved in glucose transportation, angiogenesis, anaerobic metabolism, cell survival, invasion, and metastasis (a list is given in Table 3-1 ). [15] [16] All of these changes allow cells to adapt to oxygen-deprived conditions and permit cells to escape these conditions by establishing new blood supplies or by physically moving from an oxygen-poor environment to an oxygen-rich environment. A large number of gene expression changes are mediated by hypoxia-inducible factor (HIF)-1, a helix-loop-helix transcription factor that is activated by oxygen-deprived conditions. HIF-1 is often found overexpressed in cancer cells as a result of the hypoxia microenvironment of solid tumors as well as oncogene and tumor suppressor gene mutations, and is associated with metastasis and poor survival.[17] Several HIF-1 targets have been shown to be mediators of metastasis, such as CXCR4, which promotes organ-specific metastasis in renal cancer,[18] and c-met, which increases tumor cell invasion (see section on Invasion). [19] [20]
Table 3-1 -- Hypoxia-Regulated Genes
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Adapted from Le QT, Denko NC, Giaccia AJ: Hypoxic gene expression and metastasis. Cancer Metastasis Rev 2004;23:293–310.
Hypoxia-regulated genes represent potentially specific therapeutic targets that should be highly tumor or metastases specific. [9] [21] Recent studies have suggested that lysyl oxidase (LOX) is a hypoxia-induced gene that is a very promising target for metastatic disease. Research has demonstrated that inhibition of the secreted protein can prevent both invasion and metastatic growth.[22] In addition, LOX and other HIF-1α targets such as CA-IX have been shown to be independent markers of prognosis. [22] [23] [24] [25]
Animal imaging studies have revealed that tumor cells can move rapidly along collagen fibers in the ECM that act as “highways” for metastasis.[26] This process is facilitated by host macrophage cells.[27]Furthermore, increased fiber deposition enhances ECM stiffness, which has been shown to increase cancer cell malignancy.[28] These events occur through activation of ERK and Rho by integrin clustering (see section on Cell Motility).[29] Production of reactive oxygen and nitrogen species by host immune cells and rapidly proliferating tumor cells not only increases genomic instability but has also been proposed to upregulate the expression of metastasis-promoting genes.[30]
Invasion
Changes in Cell Adhesion
The first step of metastasis is invasion. Cells must undergo changes in their cell-cell and cell-matrix adhesion interactions to dissociate themselves from the tumor.[31] Acquisition of an invasive phenotype requires changes in expression of genes that control cell-cell adhesion as well as proteolytic degradation of the ECM.[32] Cell-cell adhesions are mediated primarily by E-cadherin proteins expressed at junctions between cells.[31] Cadherins bind cells through protein-protein interactions at their extracellular domains, whereas their intracellular domains signal to catenins and the actin cytoskeleton. Changes in E-cadherin expression allow cells to detach from their neighbors and begin their migratory route toward the circulatory or lymphatic system to seek out new terrain. Reduced expression of E-cadherin is often observed in aggressive cancers through epigenetic silencing, proteosomal degradation, proteolytic cleavage, or mutation. In fact, inactivating mutations of E-cadherin have been shown to predisposepatients to gastric cancer, implicating E-cadherin as a tumor suppressor gene.[31]
Loss of E-cadherin is highly associated with epithelial to mesenchymal transition (EMT), a program that is essential for numerous developmental processes.[33] The acquisition of the invasive phenotype has many similarities to EMT, including loss of cell-cell adhesion mediated by E-cadherin repression and an increase in cell mobility. During EMT, there is a switch from E-cadherin (an epithelial cell marker) to N-cadherin expression (a mesenchymal cell marker), which promotes cell-matrix adhesion instead of cell-cell adhesion.[33]
Several signal transduction pathways, such as the Ras-MAPK and Wnt pathways, have been shown to regulate EMT ( Fig. 3-3 ). In particular, the Ras-MAPK pathway activates two related transcription factors known as Snail and Slug. [34] [35] Both of these proteins act as transcriptional repressors of E-cadherin, and their expression induces EMT in cancer cells.[36] Studies have indicated that Slug is an independent prognostic parameter for poor survival in colorectal carcinoma patients.[37] Twist, another basic helix-loop-helix transcription factor that is necessary for proper embryonic development, has also been shown to induce EMT through the repression of E-cadherin.[38] Both Twist and Snail expression levels are elevated in breast cancer patients, and are associated with poor prognosis. [38] [39]Dysregulation of Wnt signaling is common in many types of human cancers and regulates EMT in part through Snail activation, an important early step in metastasis.[40]
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Figure 3-3 Signaling pathways involved in epithelial to mesenchymal transition (EMT). EMT is a program of development of biological cells essential for numerous developmental processes. Tumor cell invasion has many phenotypic similarities to EMT, including a loss of cell-cell adhesion mediated by E-cadherin repression and an increase in cell mobility. Several signal transduction pathways have been shown to be involved in regulation of EMT. These include Ras-MAPK and Wnt. These pathways are activated by the binding of ligands to transmembrane receptors. These include: TGFβ binding to TGFβRI and TGFβRII; HGF binding to c-Met; Wnt binding to Frizzled; and IGF binding to IGF-1R. Activation of these pathways results in transcriptional repression of E-cadherin, and transcriptional activation of Snail, Slug, and Twist. These transcription factors regulate expression of genes involved in EMT. An important repressor of E-cadherin is β-catenin, which is normally targeted for degradation by GSK-3β. Activation of the Wnt pathway inhibits GSK-3β activity, resulting in stabilization of the β-catenin and translocation to the nucleus. (Adapted from Lee JM, Dedhar S, Kalluri R, Thompson EW: The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 2006;172:973–981.) |
Cell Motility
Cancer cells are able to take advantage of many mechanisms to migrate and invade, including both individual and collective cell-migration strategies ( Table 3-2 ).[32] Most cancer cells of epithelial origin undergo EMT and acquire invasive migration capacity to enter the circulatory or lymphatic system. Invasive migration is a dynamic and complex process involving changes in cell-matrix adhesion and the cytoskeleton ( Fig. 3-4 ). Changes in cell-matrix adhesion are necessary for the leading edge of the cell to grab onto the matrix surrounding it and pull itself forward in a movement similar to an inchworm. This invasive migration can be viewed as cycles of adhesion and detachment, allowing the cell to bind, then detach after pulling forward. Cell-matrix adhesions are in large part regulated by integrin proteins. Integrins are heterodimers of one of 18 alpha and 8 beta transmembrane proteins that bind to specific components of the ECM.[41] They can transmit signals into or out of the cell and are important mediators of malignant transformation.
Table 3-2 -- Mechanisms of Cancer Cell Migration
Adapted from Friedl P, Wolf K: Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 2003;3:362–374.
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Figure 3-4 Invasive migration. Invasive migration is a dynamic and complex process involving changes in cell-matrix adhesion and the cytoskeleton. It begins with pseudopod protrusion at the leading edge. This increases cell-matrix interaction, stimulating integrin receptors. Integrin stimulation promotes formation of focal adhesion contacts, focal adhesion kinase (FAK) activation through phosphorylation, and formation of FAK-Src complexes. Intracellular signaling mediated by FAK activates Rac, RhoC, cdc42, and other GTPases that mediate cellular changes required for invasion. These include actomycin contraction that propels the cell forward, and recruitment of proteases to focal adhesion sites where they degrade the ECM, allowing the cell to glide forward when focal adhesion complexes are disassembled after contraction. The remodeled matrix tracks left behind the cell have been shown to facilitate movement of subsequent cells. (Adapted from Friedl P, Wolf K: Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 2003;3:362–374.) |
Integrins are stimulated when they come into contact with specific ECM substrates, or through growth factor-stimulated signaling where they interact with receptor tyrosine kinases. [42] [43] [44] For example, hepatocyte growth factor (HGF, also known as scatter factor) influences invasion by signaling through its receptor c-met.[45] Integrin stimulation also promotes formation of focal adhesion contacts, focal adhesion kinase (FAK) activation through phosphorylation, and formation of FAK-Src complexes.[44] It is noteworthy that Src mutations that have been implicated in tumor cell motility are often observed in human cancers such as adenocarcinoma of the colon.[46] Intracellular signaling mediated by FAK activates Rac, RhoC, cdc42, and other guanosine triphosphatases (GTPases) that mediate cellular changes required for invasion.[42] These include actin-myosin contraction that propels the cell forward, and recruitment of matrix metalloproteases (MMPs) to focal adhesion sites where they degrade the ECM, allowing the cell to glide forward when focal adhesion complexes are disassembled after contraction.[32] In addition, the remodeled matrix tracks left behind the cell have been shown to facilitate movement of subsequent cells, similar to the generation of ski tracks by the first cross-country skier that allow following skiers to move more easily.[26]
Disruption of the Basement Membrane
The basement membrane provides a physical barrier between epithelial cells and the stroma. It is composed of numerous glycoproteins and proteoglycans that provide ligands for integrins permitting control of cell orientation and outside to inside signaling. Epithelial and stromal cells produce a mixture of these components that form a dense meshwork underlying the epithelial cells.
The basement membrane is not normally permeable to cells. Tumor cells overcome this barrier by altering the expression of their cell surface receptors such that they can now adhere to basement membrane components. [32] [47] [48] For example, tumor cells will increase expression of integrins that can bind laminin and collagen IV.[49] Increased CD44 expression permits cell binding to hyaluronan, a basement membrane proteoglycan, and is observed in several types of human cancer including metastatic colon carcinomas.[50] In addition, cancer cells modify the components of the basement membrane to ease penetration. For example, reduced laminin expression is observed in poorly differentiated human colon carcinomas.[51]
ECM protease activity is tightly regulated by proteins that inhibit their functions. [45] [52] Tumor cells proteolytically disrupt the basement membrane by altering the balance between ECM proteases and their inhibitory proteins. For example, elevated MMP expression and activity increases degradation of the basement membrane. MMP-1 (also known as collagenase or gelatinase) degrades collagen IV and is increased in highly metastatic cancer cells.[53] MMP degradation of the ECM not only facilitates cell movement but additionally generates a large number of bioactive cleaved peptides, and releases growth factors and chemokines trapped within the ECM mesh.[52] For example, MMP activity releases active forms of proteoglycans including heparin, hyaluronate, and chondroitin sulfate. [54] [55]
Intravasation
The entry of tumor cells into the circulation (intravasation) and the exit of tumor cells from the circulation (extravasation) to host tissue represent critical steps in the metastatic process. One clear difference between intravasation and extravasation has to do with the composition of the blood vessels. Tumor blood vessels are malformed and irregular, often possessing breaks in their thin lining that permit the easy access of tumor cells into the circulation. In contrast, the vasculature of normal tissue where tumor cells extravasate do not have these same features. This very observation suggests that the processes of intravasation and extravasation are distinct and probably require different gene functions. The abnormal vasculature found in tumors is the result of the dysregulated expression of proangiogenic growth factors, inhibition of antiangiogenic genes and pathways, recruitment of vascular progenitor cells from the bone marrow, and, in some cases, vascular memory by tumor cells.
Tumors typically do not possess abundant lymphatics and are under high interstitial pressure. Although tumors secrete lymphangiogenic factors such as vascular endothelial growth factor-C (VEGF-C), the development of lymphatics in tumors is also abnormal. In fact, the intravasation of tumor cells into lymphatics is probably easier than through vasculature in that lymphatic vessels function as a collection point for interstitial fluids.
Our knowledge of the genetic determinants involved in intravasation is limited. Gradients of chemo-attractant proteins such as chemokines have been proposed to guide cells toward the circulatory system.[26] In addition, tumor cells move along collagen fibers produced by invading cells, a process facilitated by host macrophages.[26] Whether or not genes such as LOX that are required for cross-linking collagen are involved in this process is still unknown. Interestingly, the transcription factor Twist that is implicated in EMT (as described previously) has also been shown to increase the ability of tumor cells to intravasate.[38] However, the underlying molecular mechanisms involved in promoting intravasation by Twist are as yet unknown.
Survival in the Circulatory System
Tumor cells that have successfully entered the bloodstream through intravasation theoretically have access to most organs in the body. However, before these tumor cells can extravasate into a target tissue they must first survive the environment of the circulatory system. Tumor cells in the circulatory system are subjected to immune attack, circulatory forces, and anoikis (apoptosis induced by loss of adhesion).[2] Circulating tumor cells bind to platelets to protect themselves from these dangers, thus increasing their chance of survival. [56] [57] [58] Tumor cells also bind to coagulation factors including thrombin, fibrinogen, tissue factor, and fibrin, creating emboli.[59] These tumor cell emboli are more resilient to both circulatory forces and immune attack, and have been shown to have greater metastatic potential than single tumor cells.[57] In the circulation, aggregates of tumor cells are termed homotypic clumps, because they are homogeneous in their cellular composition, whereas those associated with platelets are termed heterotypic clumps and may possess greater metastatic potential for the reasons described previously. [56] [58]
The ability to resist apoptotic cell death is important at a variety of steps in the metastatic process. First, tumor cells must survive the lack of oxygen and nutrients in the primary tumor to be able to migrate and invade. This is particularly noteworthy, because hypoxia increases the metastatic potential of tumor cells. Apoptotic resistance in response to decreased oxygen and nutrients is achieved by loss of thep53 tumor suppressor gene, increased expression of antiapoptotic members of the Bcl-2 family and decreased expression of proapoptotic members, and increased activity of the HIF transcription factor. Hypoxic tumor cells that are resistant to apoptosis have a greater probability of surviving for sufficient periods of time to intravasate into the circulation.
Apoptosis can also play a role in anoikis, death induced by loss of cell adhesion. Obviously, resistance to anoikis is important both in the early phases of invasion as well as during intravasation and circulation. Although a variety of receptor tyrosine kinases can impart resistance to anoikis,[59] most probably the formation of homotypic and heterotypic cell aggregates promotes resistance to anoikis as well.
Arrest and Extravasation
Much of our knowledge of tumor cell extravasation is patterned after leukocyte transmigration through endothelium. It is well known that leukocytes arrest before transmigration. Similarly, tumor cell arrest can occur passively through mechanical lodging or can be allowed by cell-surface molecules. [60] [61] [62] Endothelial cells are constantly shed from the blood vessel walls, creating temporary gaps to which tumor cells can more easily attach because basement membrane components are exposed. [63] [64] [65] Vessel wall damage also attracts platelets and tumor cells associated with platelets, which is enhanced by fibrinogen expression on the endothelial cell surface. [66] [67] Fibrin blood clots at the sites of tumor cell arrest can further damage vessels, attracting more platelets and circulating tumor cells.[68]Increased blood coagulation is often observed in cancer patients as a result of elevated levels of thromboplastin, procoagulant A, and phosphatidylserine produced by tumor cells. [69] [70] The most severe manifestations of this hypercoagulation state were described by Trousseau many years ago. The induction of the enzymes involved in this state can also be enhanced by changes in the tumor microenvironment.[71]
Tumor cell arrest is allowed by endothelial cell P- and E-selectins that bind to the tumor cells [72] [73] and by tumor glycosylation patterns and cell-cell adhesion molecules such as integrins and CD44. [74] [75] [76] [77] [78] Increased cell-surface expression of mucin carbohydrate is associated with increased metastatic potential in human colon carcinoma.[79] Tumor clump formation additionally facilitates tumor cell arrest by increasing the number of adhesive interactions. ECM components such as fibronectin and laminin enhance tumor cell arrest, and administration of targeting peptides to fibronectin and laminin can reduce metastatic formation.[80] Tumor cells may reside and grow within the intravascular space until the metastatic lesion physically breaks through the vessel.[81] Tumor cells may also extravasate by inducing endothelial cell retraction permitting cell attachment to the ECM.[81] It is highly noteworthy that VEGF increases vascular permeability and may permit extravasation through Src activation. [82] [83] Thus, anti-VEGF therapy could potentially act to inhibit metastases by decreasing vascular permeability. In some cases, tumor cells direct their movement and invasion into new organ terrain by following migrating white blood cells and tissue motility factors.[84]
Proliferation
The final steps of metastasis involve the resumption of cell proliferation at the secondary site and induction of angiogenesis to supply oxygen and nutrients. Studies have shown that the host tissue can influence tumor growth through autocrine, paracrine, and endocrine signals. However, it is the net balance of positive and negative signals that determines metastatic proliferation. This can partially explain organ-specific metastasis, because only certain cells will be able to respond to tissue-specific proliferation-stimulating signals and leave their dormant state.[85] For example, insulin-like growth factor-1 (IGF-1), HGF, and transforming growth factor a (TGFa) are highly expressed in the liver, [86] [87] [88] and cancer cells from colon, breast, and bladder overexpress receptors for these ligands such as epidermal growth factor receptor (EGF-R) [89] [90] [91] [92] [93] and c-met receptor,[94] resulting in proliferation of metastatic cells in these tissues.
Angiogenesis
The formation of a new blood supply from pre-existing vasculature is stimulated by an angiogenic “switch” that occurs when the ratio of inducers to inhibitors is increased. Inhibitors of angiogenesis include ECM proteins thrombospondin and endostatin. [95] [96] [97] Angiogenic inducers include VEGF, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), TGFβ, and ephrin, and their family members.[3] Of these, VEGF is the best characterized and has successfully been targeted through the use of monoclonal antibodies and soluble receptors.[98] VEGF increases angiogenesis by stimulating endothelial cells, mobilizing endothelial progenitor cells, stimulating outgrowth of pericytes that line the walls of mature blood vessels, and increasing vascular permeability allowing macromolecules to traverse endothelium. [99] [100] Furthermore, VEGF is thought to be a key molecule for the homing of VEGFR-positive bone marrow-derived progenitor cells involved in premetastatic niche formation (see later discussion),[101] and for homing of VEGFR-positive tumor cells to metastatic sites.[102] Recruitment of bone marrow-derived circulating endothelial cells additionally increases angiogenesis.[103] Thus, angiogenesis is important both for primary tumor and metastatic tumor growth, making it an attractive target.
Metastasis of Metastases
Tumor cells that have successfully colonized secondary organs are capable of further metastasis and colonization of other organs. Cells within the metastatic tumors are subjected to similar microenvironmental stresses experienced by the primary tumor, and adapt to overcome these external barriers and seed new terrain. These cells from metastases may have an intrinsic colonization capability allowing them to constantly reseed both primary and secondary tumors.[104]
LYMPHATIC METASTASIS
The vascular and lymphatic systems have numerous connections,[105] and metastasizing tumor cells can easily pass from one system to another. [106] [107] Invading tumor cells can additionally enter small lymphatic vessels directly and be passively transported to the lymph. Cancer cells may spread to lymph nodes near the primary tumor, known as the regional lymph nodes (RLNs). This is often referred to as nodal involvement, positive nodes, or regional disease. Tumor cells may become trapped in the first lymph node, or form distal nodal metastases referred to as “skip metastases” because they have bypassed the first draining lymph nodes in the area.[4] RLNs may become enlarged and are often removed to prevent cancer spread. Lymph node involvement and presence of micrometastases in the sentinel lymph node (the lymph node draining the tumor site) correlate with decreased survival.[108] Localized spread to RLNs near the primary tumor is not normally considered as metastasis per se, although it is also a sign of worse prognosis. [109] [110] Some malignancies, such as sarcomas in contrast to breast carcinomas, do not spread to the RLNs before metastasizing to distant sites. Thus, node status does not always correlate with metastasis.[2]
COLONIZATION BY METASTATIC TUMOR CELLS
The patterns of colonization cannot solely be explained by circulatory routes above. The propensity for certain tumors to seed in particular organs was first discussed as the “seed and soil” theory by Stephen Paget over a century ago in 1889.[4] For example, prostate cancer often metastasizes to the bones, and colon cancer has a tendency to metastasize to the liver. Colonization is an extremely inefficient process that is heavily dependent on the interactions between “seeding” tumor cells and the “soil” microenvironment of the secondary site. Many factors including formation of a premetastatic niche and organ specificity determine these patterns of colonization.
Premetastatic Niche
Recent in vivo data have suggested that the formation of a premetastatic niche is essential for the growth of extravasating metastatic tumor cells.[101] Factors secreted by primary tumor cells stimulate mobilization of bone marrow-derived cells that enter circulation and reside in sites of future metastasis. These bone marrow-derived cells express VEGFR-1, c-kit, CD133, and CD134, and increase angiogenesis at the premetastatic sites. Targeted inhibition of VEGFR-1 prevented niche formation and subsequent metastatic progression. This tissue preconditioning may thus represent a key step that could be targeted therapeutically, although studies with anti-VEGF therapy fail to show significant benefit in preventing metastatic growth for long periods of time. The role of hematopoietic progenitor cells and other bone marrow-derived cells in tumor progression is reviewed by Kaplan and colleagues[111] and shown in Figure 3-5 .
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Figure 3-5 The role of bone marrow-derived cells in tumor progression. Cells derived from the bone marrow niche are involved at many stages during tumor progression. Hematopoietic progenitor cells (HPCs), hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and stromal cells including macrophages and fibroblasts, permit tumor growth and development at primary and metastatic niches. (Adapted from Wong SY, Hynes RO: Lymphatic or hematogenous dissemination: how does a metastatic tumor cell decide? Cell Cycle 2006;5:812–817.) |
An additional function of the premetastatic niche is to guide metastases to specific organs. Kaplan and coworkers demonstrated that injection of secreted factors collected from cancer cells that metastasize to multiple organs could permit cancer cells that only metastasize to the lung when grown as subcutaneous tumors in mice, to display widespread metastasis through governing bone marrow-derived cell accumulation.[101] Elevated fibronectin expression by fibroblasts and fibroblast-like cells resident at premetastatic sites seems to be a critical factor in the development of the premetastatic niche. The key tumor-secreted factors that determine metastatic sites and mediate premetastatic niche formation have yet to be identified, although a role for tumor necrosis factor α (TNFα), TGFβ, and VEGF-α pathways has been demonstrated.[112]
MMPs may also play an important role in this process. For example, VEGF-R1 signaling has been shown to be required for premetastatic induction of MMP-9 expression in endothelial cells and macrophages of the lungs by distant primary tumors.[113] This is thought to make the lung microenvironment more compliant for invasion of metastasizing cells. This concept is supported by the finding that pericyte recruitment and angiogenesis are not observed in tumor-bearing mice with MMP-9 knockout bone marrow cells.[114] Furthermore, stromal-derived MMP-2 and MMP-9 have also been shown to contribute to establishment and growth of metastases.[115] Thus, whereas there is evidence that MMPs play multiple roles in metastases, clinical trials with MMP inhibitors have failed to show significant efficacy. In large part, this has been due to unexpected normal tissue toxicities and conflicting roles in metastases.
Organ Specificity
The organ distribution of metastases from a primary is not random. Minn and colleagues used bioluminescence imaging to reveal patterns of metastasis formation by human breast cancer cells in mice.[116]They also showed that individual cells from the pleural effusion of a breast cancer patient showed distinct patterns of organ-specific metastasis.[117] Single-cell progenies derived from this population demonstrated different abilities to metastasize to the bone, lung, or adrenal medulla. These studies indicate that there are particular requirements for metastasis to colonize specific organs. Some of the key molecules determining organ-specific metastasis have been identified and are briefly discussed in the following section.
Metastases to the Bone
There are two types of bone metastases: osteoblastic and osteolytic. [118] [119] Osteoblastic metastases are observed in patients with advanced prostate cancer. Both the differentiation of osteoblastic precursors as well as the activity of osteoblast cells are stimulated by tumor and microenvironmental signals such as bone morphogenetic protein (BMP), FGFRs, and IGF-1R.[120] Runx-2 is a key transcription factor that regulates the differentiation of osteoblasts and osteoblastic precursor cells,[121] and represents a potential new target for inhibiting osteoblastic metastases by preventing osteoblastic precursor differentiation. In contrast, osteolytic metastases are observed in patients with breast cancer or multiple myeloma, [122] [123] and in these patients interactions between tumor cells and the bone microenvironment result in bone resorption and metastatic growth due to the unique interplay between osteoblasts and osteoclasts ( Fig. 3-6 ). [118] [122] Parathyroid hormone-related protein (PTHrP) secreted by the tumor cells, stimulates osteoblasts to produce receptor activator of nuclear factor–κB (RANK) ligand (RANKL). Consequently, bone-resorbing osteoclast cells are activated by RANKL when it binds to the RANK receptor. Activated osteoclasts upregulate MMPs, which degrade the bone matrix-releasing growth factors such as TGFβ, IGFs, PDGF, FGFs, and BMP. [118] [124] [125] These factors stimulate tumor cells to release PTHrP, thus restarting this pathway of bone resorption. Gene profiling has identified other important mediators of osteoclastic bone metastases including CXCR4, MMP-1, CTGF, and osteopontin.[126] Tumor cells additionally induce osteoclast formation by overexpressing interleukins such as IL-8 and IL-11, and by downregulating macrophage colony-stimulating factor. [127] [128] All of these latter factors represent new targets for metastases, although the importance of each factor in osteoclastic bone metastases requires further clarification.
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Figure 3-6 The vicious cycle of osteoclastic bone metastasis. Interactions between the tumor cells and the bone microenvironment create a “vicious cycle” of osteolytic metastatic lesion development. Parathyroid hormone-related protein (PTHrP), secreted by the tumor cells, stimulates osteoblasts to produce RANK ligand (RANKL). Bone-resorbing osteoclast cells are activated by RANKL when it binds to the RANK receptor. The activated osteoclasts upregulate MMPs that degrade the bone matrix-releasing growth factors such as TGFβ, IGFs, PDGF, FGFs, and BMP. These factors stimulate tumor cells to release PTHrP, thus completing the vicious cycle. (Adapted from Steeg PS: Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 2006;12:895–904.) |
Metastases to the Brain
Brain metastases are most commonly observed in patients with breast cancer, lung cancer, and melanoma. Vascular access to the brain is strictly regulated by the blood-brain barrier, an endothelial layer surrounding the brain connected by tight junctions and further lined by a basement membrane, pericytes, and astrocytes.[129] Macromolecules are not usually able to traverse the blood-brain barrier, and it remains unclear how tumor cells are able to penetrate the blood-brain barrier. However, once the tumor cells are within the brain parenchyma, glial cells permit establishment and growth of metastases by secreting chemokines, cytokines, and growth factors.[130] Other neurotransmitter hormones in the brain such as norepinephrine have also been reported to increase tumor cell motility and metastatic spread.[131]
Little is known about the key factors that determine colonization of the brain, mostly because there is a lack of good in vivo models of brain metastasis. Overexpression of Stat3 increases melanoma metastasis to the brain and increases invasion of the melanoma cells and angiogenesis, although the pathways modulated by Stat3 signaling require elucidation.[132] The dependence of brain metastases on VEGF has been demonstrated experimentally in animals through inhibition studies where VEGF neutralization reduces brain metastases. [133] [134]
In general, patients with brain metastases have an extremely poor prognosis. It is of concern that there has been an increase in the incidence of brain metastases in patients whose systemic disease is well controlled. [135] [136] [137] For example, patients with breast tumors that overexpress Her-2 and who are treated with Her-2 targeting trastuzumab (see later discussion) have an incidence of brain metastases twice that of breast cancer patients who are treated with other agents.[135] This is thought to be because the brain offers a sanctuary when systemic disease is being controlled.[3] The development of drugs that can cross the blood-brain barrier and target brain metastases are of paramount importance in the development of new targeted therapies to tackle this problem. Currently, the best treatment for oligometastases to the brain is radiosurgery.
Metastases to the Lung
Pulmonary metastases are frequently observed in patients with sarcoma, breast, melanoma, gastrointestinal, and kidney cancers. Because cardiac output from the pulmonary artery circulates through the lungs, a high incidence of pulmonary metastases in cancer patients can be expected on the basis of blood flow alone. Metastases therefore often initiate in pulmonary arterioles and later traverse the basement membrane into the lung parenchyma. TGFβ and NF-κB facilitate this process in breast cancer, [138] [139] [140] as does osteopontin in hepatocellular cancer,[141] and ezrin in osteosarcoma and breast cancer. [142] [143] In vivo studies have identified a gene expression signature for lung metastasis including several membrane-localized and secreted proteins that has been validated in breast cancer patients.[116] Interestingly, this group of genes was able to induce lung metastasis when expressed together but not individually, suggesting essential cooperation between proteins. Increased expression of antiapoptotic proteins such as Bcl-2 and Bcl-xl is also observed in lung metastases, facilitating survival and providing resistance to therapy. [144] [145] [146] [147] [148] These studies suggest that multiple targets must be inhibited with combination therapy to effectively inhibit lung metastases.
Metastases to the Liver
Liver metastases are observed in patients with breast, lung, and pancreatic cancers. However, liver metastases are most commonly found in patients with metastatic colorectal cancer, because the liver is the first capillary bed encountered by the metastasizing cells. The circulatory system of the liver, in particular the liver sinusoids, does not have a barrier limiting macromolecule flux, and it is well perfused and highly permeable, permitting metastasizing cancer cells to establish themselves and grow. Thus, tumor cell invasion and survival are probably the key determinants in metastatic colonization of the liver.[149] There are two types of liver metastases: a nonangiogenic “replacement” of liver cells with tumor cells that preserves the stroma,[150] and a “pushing” type of metastasis[3] whereby the liver stroma is not preserved and has higher levels of endothelial cell proliferation. [151] [152] In light of the “pushing” type of metastases that stimulate angiogenesis, targeting the VEGF pathway experimentally in vivo has been shown to prevent liver metastases, [153] [154] [155] and is effective when combined with cytotoxic agents in patients with metastatic colon cancer. [156] [157] Other molecules thought to be important in colonization to the liver and that could be targeted therapeutically are COX-2, [158] [159] integrins,[160] and Src.[161]
HOST-TUMOR CELL INTERACTIONS
Tumor progression requires collaboration between tumor and host cells. Research has revealed much cross-talk between cancer cells and bone marrow-derived host immune and stromal cells.[162] For example, disruption of TGFβ signaling in fibroblasts can induce stomach and prostate cancer in mice.[163] Mutations commonly found in cancer cells, such as p53 and PTEN mutations, can also be found in cancer-associated stroma and have been hypothesized to be important during tumor progression. [164] [165] In fact, gene expression profiling of activated fibroblasts in vitro generated a signature that could predict primary tumor metastasis.[166] For example, the chemokine CXCL12 is produced by breast cancer-associated fibroblasts[167] and increases tumor cell migration and recruitment of endothelial progenitor cells expressing CXCR4,[168] a CXCL12 receptor.
Cells that respond to tissue injury (such as leukocytes and lymphocytes) are often associated with tumor cells and enhance their progression, for example by assisting travel in the bloodstream. The tumor-suppressing activities of cells in charge of immune attack (such as natural killer cells and antigen-presenting cells) can be dampened by overexpression of tumor-derived immunosuppressive cytokines such as TGFβ and interleukins, [169] [170] [171] and by lack of necessary costimulatory signals from tumor cells.[172] Thus, host cells can directly promote tumor progression by secreting growth factors and cytokines that stimulate tumor metastases and suppress tumor immune surveillance.
Inflammation and Metastases
Ironically, cells involved in chronic inflammation facilitate tumor formation and progression, mostly mediated by NF-κB and COX-2. [173] [174] In particular, infiltration of activated macrophages into tumors correlates with poor prognosis. [27] [175] The tumor-suppressing and tumor-promoting roles of these tumor-associated macrophages (TAMs) are shown in Table 3-3 . TAMs are especially attracted to regions of hypoxia, where they secrete abundant angiogenic inducers and proteases, including VEGF and MMPs, [175] [176] that have been reported to enhance angiogenesis.[27] TAMs express high levels of the HIF-2 transcription factor that has been found to be an independent prognostic factor of outcome.[177] Interestingly, work by Cramer and associates[178] has shown that in fact HIF-2 is needed for myeloid cell infiltration and activation. Furthermore, TAMs release growth factors such as PDGF, EGF, and HGF, which enhance proliferation, survival, and invasion.[175] Mutation of the macrophage colony-stimulating factor-1 gene that affects macrophage differentiation has been shown to prevent metastasis in mice bearing aggressive breast cancer tumors.[179] Targeting TAMs may be a viable mechanism for antimetastatic therapies.[180]
Table 3-3 -- Conflicting Roles of Tumor-Associated Macrophages
|
Pro-Tumor |
Anti-Tumor |
|
Proangiogenic cytokines |
Tumor cell lysis |
|
Immunosuppressive cytokines |
Immunostimulatory cytokines |
|
Protumorigenic chemokines |
Immunostimulatory chemokines |
|
Reactive oxygen species (ROS) |
ROS |
|
Elevated MMPs, TF, and uPA |
Adapted from Condeelis J, Pollard JW: Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 2006;124:263–266.
|
MMPs, matrix metalloproteinases; TF, tissue factor; uPA, urokinase plasminogen activator. |
DORMANT CELLS
The prolonged survival of single cells or micrometastases with no apparent progression is referred to as dormancy.[181] Dormant cells are frequently observed in patients with prostate, melanoma, and breast cancer, [182] [183] [184] and are found to reside in the lungs, liver, and bone marrow. These micrometastases represent minimal residual disease that results from the inefficiency of metastasizing tumor cells to colonize organs properly following extravasation.[185] Incompatibilities between tumor cells and their tissue soil, or inability of tumor cells to generate sufficient angiogenesis result in cell-cycle arrest and dormancy.[181] What genes and pathways are important in controlling metastatic dormancy are unknown and are important to identify, because they represent a “metastatic tumor suppressor mechanism.”
The presence of dormant tumor cells is associated with poor patient prognosis.[186] Isolation and reimplantation of dormant cells can generate primary tumors, demonstrating that these cells are viable. [185] [187] [188] In vivo experiments have indicated that growth of dormant metastatic cells can be activated by angiogenesis or removal of the primary tumor,[189] suggesting that limited levels of growth factors or cytokines may induce this dormant state. The angiogenic switch required for dormant cells to grow into tumors may be detectable by markers in the blood, such as VEGF, and could thus be used to monitor undetectable and asymptomatic disease in patients.[190] Interestingly, circulating tumor cells can be detected in breast cancer patients as long as 22 years after mastectomy.[191]
CANCER STEM CELLS AND METASTASIS
Stem cells are primal cells that retain the ability to renew themselves through cell division and can differentiate into a wide range of specialized cell types. They give rise to all tissues during embryogenesis and control tissue homeostasis in the adult. Cells with stem cell properties have been identified in some cancer types. [192] [193] [194] These cancer stem cells (CSCs) are able to self-renew and differentiate into multiple cell types and are believed to arise either by mutation of an adult stem cell or fusion of an adult stem cell with a CSC.[195] It is hypothesized that tumors arise from CSCs and that these cells persist in tumors as a distinct population that is responsible for disease relapse and metastasis. Because CSCs are the only cells capable of giving rise to new tumors by themselves, targeting this subpopulation may eradicate cancer.
The recent discovery of CSCs has revolutionized our way of thinking about cancer. Cancer is classically thought of as a disease of progression, facilitated through the accumulation of mutations and driven by microenvironmental signals. Whereas the stem cell microenvironment or niche is thought to be the key determinant for stem cell regulation, the role of CSCs in multistage tumor progression, particularly with respect to metastasis, is poorly understood. There may exist a subset of CSCs with the inherent property to metastasize ( Fig. 3-7 ). Research into these metastatic CSdCs is greatly anticipated.
|
Figure 3-7 The role of cancer stem cells (CSCs) in metastasis. CSCs are able to self-renew and differentiate into multiple cell types and are believed to arise either by mutation of an adult stem cell or fusion of an adult stem cell with a cancer stem cell. It is hypothesized that a pool of CSCs develops and gives rise to the primary tumor. A subpopulation of CSCs is believed to exist with the inherent property to metastasize. Microenvironmental signals stimulate primary tumor cells to secrete factors involved in premetastatic niche formation, and additionally stimulate invasion and dissemination of the metastatic CSCs (mCSCs). These mCSCs are attracted to homing and anchorage signals produced by bone marrow-derived cells at the premetastatic niche. (Adapted from Al-Hajj M, Clarke MF: Self-renewal and solid tumor stem cells. Oncogene 2004;23:7274–7282.) |
ANTIMETASTATIC THERAPY
The literature is replete with genes that have been implicated in the metastatic process. [196] [197] Because of the immense heterogeneity of metastatic cells, metastatic selective therapeutic targets have been difficult to identify and develop for targeted therapy. Clinically, there have been two success stories thus far: bevacizumab and trastuzumab. However, both agents affect both primary and metastatic tumor growth. Bevacizumab targets VEGF and has displayed activity in several metastatic cancer types, especially when administered in combination with cytotoxic compounds.[157] Other small-molecule inhibitors to VEGF-R have also shown some antimetastatic effectiveness. Trastuzumab is a recombinant monoclonal antibody to Her-2 that is very effective against metastatic breast cancer, again, particularly in combination with cytotoxic agents.[198] However, only 30% to 40% of breast cancer patients overexpress Her-2 and are suitable for treatment.[198] In addition, whereas MMP inhibitors demonstrated good antimetastatic effects in vivo, these compounds failed in clinical trials, and additional research has revealed their conflicting roles in metastasis.[199]
A recent study by Gupta and associates[200] analyzed gene expression profiles of metastases from a breast cancer cell line, and identified four genes: epiregulin, which encodes a ligand that binds to the EGFR; cyclooxygenase, which encodes an enzyme that is involved in inflammatory responses and wound healing; and two MMPs that encode proteins involved in tissue remodeling and angiogenesis that affect both primary and metastatic tumor growth. The investigators found that inhibition of each gene individually through genetic knockdown resulted in a modest effect on lung metastases. In contrast, if combinations of these genes were inhibited, there was a significantly greater effect on the metastatic process, suggesting that combination therapy is more effective in controlling metastases. However, many of these reported genes, such as Her-2/Neu and EGFR, as described previously, affect both primary as well as metastatic tumor growth, somewhat obfuscating their roles as direct modulators of tumor metastases. What the field is desperately in need of is new candidate genes and proteins that specifically affect the metastatic process and have little effect on primary tumor growth.
One very promising target is LOX, a hypoxia-induced secreted protein involved in multiple stages of metastasis ( Fig. 3-8 ).[201] LOX contributes to tumor cell invasion by cross-linking collagens in the ECM, which stimulates integrin-mediated cell-matrix adhesion and activation of FAK, and additionally provides a route (“highway”) by which tumor cells may travel. Furthermore, LOX is involved in the formation and maintenance of the metastatic niche, allowing metastatic dissemination and growth. Targeting secreted LOX through antibody or small-molecule inhibition significantly reduced formation and growth of metastases to the lung, liver, bone, and brain, in several models of cancer. These data provide hope for the future, because there is an urgent need for new metastasis-targeting therapies.
|
Figure 3-8 The role of lysyl oxidase in metastasis. Lysyl oxidase (LOX) is a hypoxia-induced secreted protein involved in many stages of metastasis. LOX contributes to early-stage metastasis by increasing tumor cell invasion through the cross-linking of collagens in the ECM, which stimulates integrin-mediated cell-matrix adhesion and activation of focal adhesion kinase. LOX is expressed at the leading edge on invasive cells and extends along hairlike fibers in the ECM. Collagen cross-linking additionally provides a route (“highway”) by which tumor cells may travel. LOX secreted by hypoxic cells in the primary tumor is involved in premetastatic niche formation at distant sites. Furthermore, LOX is involved in later stages of metastasis where cell-matrix adhesion interactions are again required for arrest and extravasation, and invasive migration. LOX is further required for the formation of a mature ECM, which is essential for metastatic tumor cell growth. |
CONCLUSION
Metastatic disease, not the primary tumor, kills the majority of cancer patients. For such an important determinant of long-term survival, progress has been slow in understanding the crucial genes and pathways that drive metastatic progression. The reasons for this slow progress have been numerous, including inadequate animal models that reflect the metastatic process in humans, failure to identify genes that specifically affect metastatic tumor growth, the complexity of host and metastatic tumor interactions, and premature clinical trials focusing on “attractive” gene targets. The future for metastasis research looks very promising in large part because we understand the mistakes of the past and are using multiple genomic and proteomic approaches to target what has for so long seemed to be an untractable problem. It is only when we are able to attack the problem of metastases that we will make significant inroads in our war against cancer.
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