General mechanisms of metastasis†
Presented at the Skeletal Complications of Malignancy Symposium, Bethesda, Maryland, April 19-20, 1997.
In the present article, the steps involved in the process of tumor metastasis are discussed. Several events are required for malignant cells to leave the primary tumor and proliferate at a distant site: vessel formation (angiogenesis), cell attachment, invasion (matrix degradation, cell motility), and cell proliferation. Molecular mechanisms underlying each of these steps are described. Based on blocking these processes, new anti-metastasis therapies are being developed. Cancer 1997; 80:1529-37. © 1997 American Cancer Society.
Tumor metastasis is the leading cause of death in cancer patients. Metastasis is the process by which a tumor cell leaves the primary tumor, travels to a distant site via the circulatory system, and establishes a secondary tumor. A deeper understanding of the genetic and molecular steps that are necessary for a tumor cell to invade and metastasize is the starting point for new diagnostic and therapeutic strategies. Inhibition of invasion constitutes a new class of targets for chemoprevention. Intervention could commence between the period of tumor proliferation and the onset of invasion, preventing the series of events leading to metastasis.
Although the genetic basis of tumorigenesis can vary greatly, the steps required for metastasis are similar for all tumor cells. Therefore, clinical therapies that block metastasis will be useful in treating cancers of various genetic origins. Several steps can be recognized in the process of metastasis (Table 1). The generation of blood vessels (angiogenesis) is an essential step for the primary tumor to grow. The rich vascularization increases the chance for tumor cells to reach the bloodstream and colonize secondary sites. During the metastatic process, tumor cells need to attach to other cells and/or matrix proteins. Adhesion molecules play a central role in cellular attachment. Translocation of neoplastic cells across extracellular matrix barriers (invasion) is also part of the metastatic process. Lysis of matrix proteins by specific proteinases is required for invasion. Many factors, including matrix components, stimulate tumor cell migration. Finally, colonization of the secondary site by tumor cells requires tumor cell proliferation. Growth factors that stimulate metastatic cells to proliferate at secondary sites have been identified. Several antimetastasis therapies are currently being tested clinically, including those that target cell surface and extracellular proteins that mediate sensing, extracellular matrix degradation, adhesion, and motility (i.e., adhesion receptors, degradative enzymes and their inhibitors, and proliferative/motility-stimulating cytokines).
Table 1. Steps in Metastasis and Examples of Proteins Involved at Each Stage of Progression
| Stimulation||Fibroblast growth factor family|
| ||Transforming growth factor-α|
| Basement membrane disruption||Basement membrane|
| || e.g., Collagens|
| || Fibronectin|
| || Elastin|
| || Proteoglycans|
| Proteolysis of matrix||Matrix metalloproteinases|
| || Interstitial collagenases|
| || Stromelysins|
| || Gelatinases|
| ||Type I collagen|
| ||Type IV collagen|
Bone is the third most common site of metastasis. Certain tumors--including breast, prostate, and lung--frequently metastasize to bone. In fact, 90% of patients that die of breast carcinoma have bone metastases.1 Metastatic tissue in the bone causes severe pain and a dramatic reduction in the quality of life of these patients. Furthermore, no effective treatments have been developed that specifically target metastases to bone.
The distribution pattern of metastases can be predicted in part by the pattern of regional venous drainage. Most metastases develop in the first capillary bed encountered after discharge from the primary tumor. However, tumors also metastasize to distant locations that are not predictable based on blood flow patterns. The high proportion of bone metastases in breast, prostate, and lung cancers are examples of selective homing of tumor cells to a specific organ. Three major types of homing mechanisms have been proposed. The first is selective growth. Under this mechanism, tumor cells extravasate ubiquitously but selectively grow only in the organs that have the appropriate growth factors or extracellular matrix environment. The second mechanism is selective adhesion to sites on the endothelial lumenal surface only at the site of organ homing. The third major mechanism is selective chemotaxis of circulating tumor cells to the organ producing soluble attraction factors. All of these mechanisms have been found to play a role in experimental metastasis.2, 3
Angiogenesis, or the generation of new blood vessels, is important for the proliferation of primary tumors. A correlation has been found between angiogenesis and tumor thickness.4 Angiogenesis also appears to be important for metastasis. A negative correlation was observed between patient survival and the degree of vascularization of several tumor types including gastric,5 breast,6-8 prostate,9 esophageal,5 vulvar,10 and melanoma.11 A high degree of tumor vascularization increases the chance for tumor cells to enter the circulatory system and metastasize. It is also possible that the newly formed blood vessels are more permeable to tumor cells and may contribute to new metastases.12
The process of angiogenesis can be divided into the following three steps that parallel those required for invasiveness of tumor cells: (1) proliferation of endothelial cells, (2) breakdown of the extracellular matrix, and (3) migration of endothelial cells.12 These steps can be promoted by growth factors secreted by tumor cells. These factors are termed angiogenic factors and include the heparin-binding growth factor or fibroblast growth factor family, transforming growth factor-α, angiogenin, vascular permeability growth factor (VPF), vascular endothelial growth factor (VEGF), and others (reviewed in Denijn and Ruiter12). The production of these growth factors leads to tumor growth and causes a concomitant increase in vascularization. Angiogenesis requires interactions among the tumor cells, endothelial cells, and the extracellular matrix. The enzymes that mediate these interactions are serine proteases and metalloproteinases. The expression of these enzymes can be affected by angiogenic factors.
Because angiogenesis is a complex, multistep process, a large number of potential inhibitors exist. More than 25 inhibitors have been identified; these may serve as cancer treatments because angiogenesis is necessary to support tumor growth and metastasis. One example of an antiangiogenic factor is a protein that was derived from cartilage called the cartilage-derived inhibitor (CDI).13 Cartilage-derived inhibitor functions by inhibition of collagenase activity and was demonstrated to inhibit angiogenesis as well as endothelial cell migration and proliferation. In a similar manner, tissue inhibitors of metalloproteinases are also antiangiogenic due to the inhibition of collagenases. Other inhibitors of angiogenesis are platelet factor 4 (PF4) and angiostatin. Platelet factor 4 is likely to function by inhibition of the heparin-binding basic fibroblast growth factor (bFGF).14 Angiostatin is a 38 kDa plasminogen fragment with a capacity to suppress growth of metastasis.15
The ability of tumor cells to form transient attachments is necessary for metastasis. Metastasizing tumor cells must be able to attach to extracellular matrix components and to other cells (of the same or different type). Integrins may play a critical role in the attachment of tumor cells to the extracellular matrix.16 Integrins are transmembrane receptors that bind to a variety of extracellular matrix molecules including laminin, fibronectin, vitronectin, and collagens. Integrins are involved in cell signaling pathways through association with cytoskeletal components inside the cell. The signals are transduced through phosphorylation of cytoskeletal components and Ca2+ influx.17 Integrins are heterodimeric molecules (composed of an α and β subunit) that bind the extracellular matrix through the Arg-Gly-Asp (RGD) peptide sequence present in extracellular matrix proteins. Addition of RGD peptides that compete for binding to integrins was found to inhibit metastasis of melanoma cells.18
Specific integrins have been recognized to play an important role in the metastasis of tumor cells to bone. The αVβ3 integrin (the vitronectin receptor) is an essential component of the bone attachment apparatus of osteoclasts, transmitting signals across the osteoclast plasma membrane.19, 20 Recently, this integrin was shown to be highly expressed in bone-residing breast carcinoma cells, suggesting a relationship of the αVβ3 integrin to the ability of this cancer to metastasize to bone.21 The αVβ1 integrin mediates binding through adhesion to vascular cell adhesion molecule (VCAM)-1 and fibronectin. Transfection of Chinese hamster ovary cells with human α4 cDNA increased bone metastasis in mice,22 suggesting an important role of this integrin in retention of metastatic cells to bone.
Three other adhesion molecules, CD44, VLA-4, and ICAM-1, have been implicated in the binding of tumor cells to bone marrow elements. CD44 and VLA-4 play a role in the adhesion to bone marrow fibroblasts and endothelial cells, whereas ICAM-1 is involved in the binding of tumor cells to bone marrow macrophages.23
Osteopontin (OPN) is an acidic phosphoprotein produced at high levels by many transformed cell lines in comparison to their untransformed counterparts.24, 25 Osteopontin has the GRGDS (Gly-Arg-Gly-Asp-Ser) motif and binds to the vitronectin receptor. Through this mechanism, OPN may play a role in the homing of metastatic cells to bone. OPN may also play a role in a signaling process, suppressing the oxidative burst, inhibiting nitric oxide production, and thus protecting the tumor cells from killing by the cytotoxic host cells.26
An adhesion molecule, E-cadherin, may play a role in the suppression of metastasis. E-cadherin appears to maintain homotypic cell-cell interactions. Decreased expression of E-cadherin has been found to be associated with cancer progression.27 Transfection of the E-cadherin cDNA into highly metastatic epithelial tumor lines results in decreased invasiveness of cells in vitro and more differentiated tumor cells in vivo28 and suppresses the development of osteolytic bone metastasis.29 The effect on metastatic epithelial cells could be blocked by treatment with anti-cadherin antibodies.28 These experiments demonstrate the ability of E-cadherin to suppress invasion and metastasis.
Tumor cell invasion is the active process of translocation of neoplastic cells across extracellular matrix barriers. Invasion requires local proteolysis of the extracellular matrix, pseudopodial extension, and cell migration.30 Expression of the malignant phenotype is not caused by one single gene or protein. The invasive process involves genetic deregulation of the tumor cells leading to an imbalance of stimulatory and inhibitory physiologic events. This deregulation occurs in a small subset of tumor cells; less than 0.05% of circulating tumor cells establish metastases.31, 32
The components of the invasion process are biological molecules that play important roles in physiologic events. At the biochemical level, the mechanism of invasion used by tumor cells appears to be similar to the ones used by nonmalignant cells to cross tissue boundaries under physiologic conditions. Angiogenesis,33, 34 embryogenesis, and morphogenesis35; nerve growth cone extension and homing36; and trophoblast implantation37 are examples of physiologic invasion. In contrast to malignant invasion, physiologic invasion is tightly regulated and ceases when the stimulus is removed.33, 36-39 A fundamental question in the study of metastasis is how metastatic cells activate the biochemical processes involved in invasion.
Invasion within a three-dimensional matrix requires the protrusion of a cylindrical pseudopod before the translocation of the whole cell body.40, 41 Several pieces of evidence point to pseudopodia as the organs of motility and invasion. First, pseudopodial cell fragments lacking nuclei were shown to retain sensing and directional locomotive capacity,40, 41 proving that they have all the sensory and motor equipment necessary for crawling. Also, cell surface degradative enzymes and adhesion receptors appear to aggregate at pseudopodia.42 Furthermore, pseudopodia protrude at the point of surface stimulation by ligands that cause migration.40-42 The cytoskeleton is critical for pseudopodial extension. The internal actin structures provide the mechanical basis for pseudopodia extension into free space at the area of binding of the ligands that induce migration.41, 43
The extension of pseudopodia is coupled with the other cellular events involved in invasion. A series of proteins on the surface of the pseudopod coordinate sensing, protrusion, burrowing, and traction. The invading cell must mesh local proteolysis38 with limited attachment and detachment.44 The leading edge of the cell must express activated proteinases. Then, as the cell moves into the zone of lysis, adhesion of the leading edge is required to grip the extracellular matrix and pull the cell forward, and proteolysis must stop. The rear of the cell must detach from previous attachment sites to allow the cells to move forward.
The basement membrane and interstitial stroma play an integral role in the regulation of tumor cell invasion. Basement membranes constitute barriers that must be overcome for tumor cell invasion. The extracellular matrix may also contain stored latent proteinases and cytokines that can be activated by the invading cell pseudopodia. Matrix metalloproteinases are a family of zinc-binding enzymes that cleave extracellular matrix components. The role of matrix metalloproteinases in tumor cell invasion has been the subject of extensive investigation because of the importance of the extracellular matrix as a tumor cell boundary. Also, the expression levels of matrix metalloproteinases are correlated with invasiveness. Metalloproteinases hold promise as therapeutic targets in the treatment of cancer.
Matrix metalloproteinases are secreted as proenzymes that require extracellular activation. They are divided into the following three general classes: (1) interstitial collagenases, (2) stromelysins, and (3) gelatinases (type IV collagenases).45-47 Interstitial collagenases degrade triple helical regions of the fibrillar collagens types I, II, III, VII, VIII, and X into a 1/4 amino terminus and a 3/4 carboxyl terminus. The stromelysins include stromelysins-1, -2, and -3, and matrilysin and these enzymes cleave fibronectin, proteoglycans, and nonhelical regions of type IV collagen. The gelatinases cleave degraded collagen (gelatin) and native collagen types IV, V, VII, IX, and X in addition to fibronectin and elastin. A positive correlation between tumor aggressiveness and metalloproteinase expression levels has been established for each of the three classes (reviewed in Ray and Stetler-Stevenson48). Tumors metastasized to bone express high levels of the 92 kDa type IV collagenase (gelatinase B) as well as cathepsin B and L.49
Members of all three classes of the matrix metalloproteinase family are inhibited by endogenous inhibitors called tissue inhibitors of metalloproteinases (TIMPs). Three TIMP proteins have been identified: TIMP-1, TIMP-2, and TIMP-3. TIMP-1 specifically binds and inhibits activated interstitial collagenase, stromelysin-1, and both the activated and latent forms of gelatinase B.46, 50, 51 TIMP-2 is an inhibitor of the 72 kDa type IV collagenase (gelatinase A),52, 53 which can bind to either the latent or activated forms of the enzyme.54 The third member of the TIMP family is the TIMP-3 protein. Unlike TIMP-1 and TIMP-2 (which are soluble proteins), TIMP-3 is associated with the extracellular matrix.55 These inhibitors, in addition to their physiologic roles, appear to be important as regulators of metastasis. For example, overexpression of TIMP-2 in breast carcinoma cells is correlated with a decrease in the number of osteolytic bone lesions.56
Several approaches for inhibition of basement membrane degradation by matrix metalloproteinases are available. Because matrix metalloproteinases require activation of latent proenzymes for function, the prevention of activation may provide a future clinical therapy for the prevention of metastasis. Activation of the proenzyme is achieved by cleavage of an 80 residue amino terminal domain containing the consensus sequence PRCGXPDV.57-59 The latency of the proenzyme is maintained by the interaction between the metal atom and a sulfhydryl group on the cystinyl residue. Peptides containing the consensus sequence in the prodomain block enzyme activation and have been successful at halting invasion.57-59 The cystinyl-containing peptide prevented more than 80% of the invasion of reconstituted extracellular matrix by human fibrosarcoma, breast carcinoma, or melanoma cells.
Another approach for inhibition of metalloproteinases is to use collagen peptide analogs that have zinc-binding moieties that target the peptides to the metalloproteinase active sites.60 An example of such an inhibitor is the compound BB94 (Batimastat, British Biotechnology, Ltd., Cowley, Oxford, UK).60-62 Several studies have found that administration of BB94 reduces growth of the primary tumor, incidence of tumor local invasion, and incidence of spontaneous metastases.61, 62
A third approach is to use the endogenous metalloproteinase inhibitors TIMP-1 and TIMP-2, which inhibit metalloproteinases by forming inactive complexes with the enzymes.50, 52 Administration of native or recombinant TIMP has been found to inhibit the invasion of human amniotic membranes in vitro and in vivo metastases in animal models.63 Transfection of TIMP-1 antisense RNA into mouse 3T3 cells enhanced their ability to form metastatic tumors in athymic mice.64 Exogenous TIMP-2 was also found to inhibit tumor cell invasion in vitro.65, 66 Overexpression of TIMP-2 in ras-transformed rat fibroblasts suppressed the formation of lung metastases in nude mice.66
The motility of tumor cells is also an important component of invasion. Certain factors contribute to metastasis by stimulation of motility. These factors can be divided into three groups. The first group consists of factors that are secreted by the tumor cells themselves, or autocrine motility factors. At least 11 autocrine motility factors have been identified, although only 3 have been cloned and purified to homogeneity: hepatocyte growth factor/scatter factor (HGF/SF),67 insulin-like growth factor II (IGF-II),68 and autotaxin (ATX).69 HGF/SF normally acts as a paracrine growth factor, but in tumor cells it can act as an autocrine motility factor. The receptor for HGF/SF has been identified as the protein encoded by the c-Met oncogene.70, 71 When HGF/SF binds to the c-Met receptor, a tyrosine kinase, it causes autophosphorylation of c-Met followed by phosphorylation of PI-3 kinase, GAP, PLCγ, src, ras, and MAP kinase.72 The ATX autocrine motility factor is a potent stimulator of motility.69 ATX has been demonstrated to exhibit phosphodiesterase activity, although it is not known how this enzymatic activity translates into cell motility.73 The receptor for autotaxin has not been identified; however, the motility response is pertussis toxin sensitive, indicating that G proteins are involved in the signal transduction pathway.69
The second group of motility factors corresponds to extracellular matrix proteins. Matrix proteins that can induce motility are vitronectin,74 fibronectin,75 laminin75 type I collagen,76 type IV collagen,77 and thrombospondin.78 These proteins stimulate chemotaxis (motility toward a chemical gradient) and haptotaxis (motility stimulation toward a bound substrate). Several of these matrix proteins stimulate motility through integrin receptors.79 The motility stimulation by matrix proteins may be an important component of tumor metastasis, coupling matrix protein degradation to motility. Matrix proteins surround the tumor cells. As they are cleaved by degradative enzymes secreted by the tumors, the soluble matrix proteins can stimulate the tumor cells to migrate. The ability of matrix proteins to stimulate haptotaxis in tumor cells also indicates that the intact extracellular matrix proteins may provide a path for the migration of tumor cells. Type I collagen, which comprises 90% of the bone matrix, has been shown to stimulate the motility of tumor cells.80, 81 Other products of bone resorption, including α2HS glycoprotein, osteocalcin,80, 82 and transforming growth factor-β (TGF-β),83 can stimulate chemotaxis of cancer cells.
The third class of tumor motility factors are host-secreted growth factors such as insulin-like growth factor-I (IGF-I),84 interleukin-8 (IL-8),85 and histamine.86 These factors are paracrine motility factors and have been referred to as homing factors because they cause tumor cells to move toward the organs that produce them. Many of these factors also act as mitogens for the tumor cells in which they cause motility. Through a variety of mechanisms, motility factors may cause one or more of the following which contribute to motility: changes in cell shape, cytoskeletal rearrangements, and changes in cell adhesion and/or membrane fluidity.
GROWTH OF METASTASES/GROWTH FACTORS
Tumor proliferation at the secondary site is required to establish metastasis. Various growth factors function as regulators of tumor proliferation. As some tumors progress to malignancy, they are decreasingly dependent on exogenous growth factors. For example, melanoma cells (unlike melanocytes) produce many positive growth regulators.87 One positive growth regulator of melanoma cells is bFGF, which is produced by melanoma cells and functions as an autocrine growth factor.88-90 Another autocrine growth factor that stimulates the growth of tumors is IL-8.91 Examples of paracrine positive growth regulators of metastases are IGF-I92, 93 and epidermal growth factor.94
Cells that metastasize to bone are capable of affecting bone tissue. These effects can be on the osteoblasts that are stimulated to form new bone (osteosclerotic metastasis) or on osteoclasts that cause bone resorption (osteolytic metastasis). The growth of bone metastases can, in turn, be influenced by growth factors derived from bone marrow, osteoblasts, or from the bone matrix products released by osteoclastic bone resorption. Bone-marrow-derived growth factors and cytokines include TGF-β,95 IGF-I, and IGF-II.96 All are expressed at high levels in the bone. Insulin-like growth factor-I and IGF-II have been shown to affect the growth of colorectal,97 breast,98 and prostate99 cancer cell lines. Osteoblasts also produce growth factors that can positively influence growth of metastases. Osteoblasts produce bFGF100 and IL-6.101 The growth of prostate cancer cells is stimulated by bFGF.102 Prostate cancer cells express the IL-6 receptor103 and are therefore likely to respond to IL-6 produced by osteoblasts.
Several tumor-derived factors that stimulate bone resorption by osteoclasts have been recognized. Examples of such factors are parathyroid hormone related protein (PTHrP),104 prostaglandin E,105 IL-1,106 and tumor necrosis factor.107 Parathyroid hormone related protein is a potent osteoclast activator that is produced at higher levels in bone metastatic tissues than in primary tumors.108-110 It has been shown that cancer cell lines expressing PTHrP frequently metastasize to bone in nude mice. Furthermore, antibodies against PTHrP significantly reduced the size of the osteolytic lesions.111 Although the exact role of PTHrP in bone metastasis is not known, it has been proposed that PTHrP produced locally by metastatic cells stimulates osteolysis and the release of growth factors important for the metastatic tumor to grow.111
Advances in the molecular techniques used to analyze tumor tissues will most likely lead to the identification of many new genes involved in the progression to metastasis. Comparison of primary tumors and metastatic lesions may provide useful information about tumor progression to metastasis. Differential display,112 serial analysis of gene expression (SAGE),113 subtractive hybridization,114 and representational difference analysis of cDNA115 are all techniques that detect genes that are expressed in particular tissues or cells. Recently, it has been possible to construct cDNA libraries from small amounts of microdissected tissue. This method allows the genetic fingerprinting of different cancer progression stages such as normal epithelium, preinvasive lesions, invasive tumors, and metastasis.116 Contamination with normal, inflammatory, or stromal cells represents a significant problem when analyzing tissue samples, especially in genetic deletion or gene expression studies. Recently, it has been possible to procure pure cell populations from microscopic lesions by laser capture microdissection.117 This approach is suitable for DNA, RNA, and protein analysis. Automation of genetic analysis techniques will allow patient samples to be screened for the expression of a large number of genes in a routine manner. The development of DNA panels containing arrays of thousands of sequences for automated hybridization has been proposed.118 These genetic panels would allow tests that would provide a nearly complete genetic characterization of any tumor sample. Such advances will contribute to a more complete understanding of the genetic deregulation that leads to metastasis and, as a result, to more effective treatment of cancer patients.