Invasion and Metastases

Lance A Liotta; Elise C Kohn

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Bast RC Jr, Kufe DW, Pollock RE, et al., editors. Holland-Frei Cancer Medicine. 5th edition. Hamilton (ON): BC Decker; 2000.

See "Cancer Medicine, 6th Edition"

Holland-Frei Cancer Medicine. 5th edition.

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Chapter 8Invasion and Metastases

Lance A Liotta, MD and Elise C Kohn, MD.

Invasion and metastasis are the most insidious and life-threatening aspects of cancer.^1–³ The capacity for invasion may not be expressed initially or in all tumors, hence our ability to cure most in situ lesions with a local intervention. However, most cancers unmask their invasive potential, thus progressing to frank malignancy from pre-existing carcinoma in situ, adenomas, or disorders of epithelial proliferation. Once the neoplasm becomes invasive, it can disseminate readily through whatever means or conduits are available to it, via the lymphatics and/or vascular channels. Invasion and metastases kill hosts through two processes: local invasion and distant organ injury. Local invasion can compromise the function of involved tissues by local compression, local destruction, or prevention of normal organ functioning. The most significant turning point in the disease, however, is the establishment of metastasis. At this stage, the patient can no longer be cured by local therapy alone. The patient with metastatic disease most commonly succumbs to injury caused by cancer dissemination or less often to complications associated with cytotoxic therapies.

Our understanding of the processes of invasion and metastases has improved, but our ability to detect occult metastatic disease or metastatic potential prior to development of occult disease still lags. Some patients with cancer have benefited from improved screening techniques wherein they are being diagnosed earlier, such as in breast cancer. Despite this, approximately 30% or patients still will have clinically detectable metastases at the time of initial diagnosis, and a further 30 to 40% of patients will harbor occult metastases. Continuing with the breast cancer example, the relapse rate in stage I, with less than 1 cm disease, remains 20 to 25%, indicating that the unveiling of metastatic potential is an earlier event than had been thought (Fig. 8.1). Thus, less than one-third of newly diagnosed cancer patients potentially can be cured by local therapeutic modalities alone. The size and age variation in metastases, their dispersed anatomic locations, and their heterogeneous composition over time hinder complete surgical extirpation of disease and can limit the effectiveness of many systemic anticancer drugs.

Figure 8.1

Temporal progression of cancer. Diagnosis and treatment of cancer occurs generally late in the course of disease. At this time, a high proportion of patients have obvious or occult meatastases. Acquisition of the invasive and angiogenic phenotypes occur (more...)

Tumors of comparable size and histology can have widely divergent metastatic potential, depending on their genotype and local environmental influences. Metastatic potential is influenced by the local microenvironment, angiogenesis, stroma–tumor interactions, and elaboration of cytokines by the local tissue, and more significantly by its molecular phenotype. This underscores the importance of understanding the molecular metastatic process and using that understanding to develop marker panels, through which to predict presence and location of active metastatic disease, and, where possible, occult metastases and to identify and develop therapeutic targets. The malignant phenotype is the culmination of a series of genetic changes in the primary tumor and its metastases through which investigation of the activation, regulation, and manipulation of regulatory elements can be exploited as a new frontier for metastases research.

Recent evidence has indicated that angiogenesis, tumor-induced neovascularization, and invasion are obligate early events.¹ An angiogenic switch has been postulated as an early event, perhaps occurring earlier than actual malignant transformation,⁴ and will be discussed in detail elsewhere. Local microinvasion has been shown to occur early, even though distant dissemination may not be evident or may not yet have occurred. Invasion is a more efficient process than metastasis, with millions of cells shed into circulation daily, but only a small fraction successful at initiating colonies.⁵ There are also examples where invasion and dissemination are extensive early in the process when the primary remains small or even microscopic, such as in ocular melanoma. Thus, further understanding and molecular dissection of the processes of invasion and metastasis are needed to speed up our progress against cancer (Table 8.1).

Table 8.1

Metastasis Facts.

Tumor–Host and Tumor–Stromal Interactions

The process of metastasis is a cascade of linked sequential steps involving multiple host–tumor interactions (Table 8.2). A cell or group of cells must be able to leave the primary tumor, invade the local host tissue, and survive at the secondary sites and while in transit, in order to proliferate successfully at the metastatic site. This complex process requires the cells to enter into the vascular or lymphatic circulation, arrest at a distant vascular or lymphatic bed, actively extravasate into the organ interstitium and parenchyma, and proliferate as a secondary colony. A large foundation of experimental work suggests that during each stage of the process, only the fittest tumor cells survive.^1,⁶ A very small percentage (0.01%) of circulating tumor cells ultimately initiate successful metastatic colonies. Thus, metastasis is a hightly selective competition, favoring the survival of a minor subpopulation of metastatic tumor cells that pre-exist within the primary tumor.

Table 8.2

Tumor-Host Interactions During the Metastatic Cascade.

The distribution of metastases varies widely depending on the histologic type and anatomic location of the primary tumor. On the one hand, the most frequent organ location of distant metastases in many types of cancers still appears to be the first capillary bed or lymphatic tree encountered by the circulating cells. This explains why lung and liver metastases are the first parenchymal metastases seen from most systemic cancers. On the other hand, there are many metastatic sites that cannot be predicted on the basis of anatomic considerations alone and might be considered examples of organ tropism. Clear cell carcinoma of the kidney often metastasizes to the thyroid, breast cancer to the ovary, and ocular melanoma to the liver. The mechanisms for this homing are not yet understood. The predilection of breast and prostate cancers for bone may also reflect a degree of organ tropism and may, in part, be due to the production and sequestration of osteopontin by the tumor cells.^7,⁸ A pilot study of breast cancer patients suggested that the cells either synthesize osteopontin or bind and sequester it from the microenvironment. This behavior was correlated with tumor aggressiveness and poor prognosis. There are several theoretic mechanisms for organ tropism. First, tumor cells disseminate equally in all organs, but selectively grow only in specific organs. Data to support this can be found in the different patterns of growth and dissemination using subcutaneously implanted xenografts, compared with orthotopic implantation.⁶ Preferential growth and homing may be induced by the local microenvironment. Second, circulating tumor cells may adhere specifically to the endothelial luminal surface only in the targeted organ.

Differential expression of attachment and adhesion factors has been shown, and the endothelia themselves have different properties in different organs. It has, therefore, been postulated that there be recognized signals on the endothelial cells that determine the organ specificity. Last, circulating tumor cells may respond to soluble factors diffusing locally out of the target organs.¹ Such factors could act in a chemotactic fashion to attract the tumor cells to extravasate. Endothelial surface antigens have been identified that may mediate selective adhesion of circulating tumor cells to endothelium of particular organs.⁹ Attempts to characterize these keys are under way.

To induce successful metastasis to a site distant from the primary tumor, neoplastic cells migrate from the primary tumor mass and successfully traverse tissue barriers. This may involve simple cell locomotion from the primary into the interstitial stroma or may require penetration and proteolysis of tissue obstacles (Figs. 8.2 and 8.3). Further, tumor cells have to survive the stage of vascular transport and arrest in the capillary beds of distant organs to engage in a second round of invasion—extravasation—whereby neoplastic cells exit from the vessel lumen into the local angiogenesis in this new environment to grow from micrometastases into the progressively enlarging tumors that will threaten the survival of the host.

Figure 8.2

Active pseudopodial progression. A B16F10 murine melanoma cell is invading the basement membrane. Arrowheads point to the rent in the basement membrane at the site of the pseudopodial protrusion. Bar = 1 μm (9,600X)

Figure 8.3

Cell invasion of the extracellular matrix. Physiologic or malignant invasion is motility coupled to adhesion and proteolysis. The advancing pseudopod, extended through calcium-regulated actin polymerization, may focus the action of cell surface proteinases, (more...)

The mammalian organism is divided into a series of tissue compartments separated by the extracellular matrix. The basement membrane and its underlying interstitial stroma constitute the extracellular matrix and are the major connective tissue units separating organ compartments. Tumor cells penetrate the epithelial basement membrane and enter the underlying interstitial stroma during the transition from in situ to invasive carcinoma.^2,¹⁰ The basement membrane is a dense meshwork of type IV collagen, glycoproteins, such as laminin and fibronectin, proteoglycans, and embedded growth factors. Once the tumor cells invade the underlying stroma, they gain access to the lymphatics and blood vessels for distant dissemination. General and widespread changes occur in the organization, distribution, and quantity of the epithelial basement membrane during the transition from benign to invasive carcinoma. Loss of continuity of the basement membrane is the distinguishing feature for malignancy. By example, benign proliferative disorders of the breast, such as fibrocystic disease, sclerosing adenosis, and intraductal byperplasia, all have disorganized epithelial stromal architecture. Extreme forms can mimic the appearance of invasive carcinoma. These benign disorders always contain a continuous basement membrane separating the epithelium from the stroma. In contrast, invasive ductal and lobular carcinomas consistently possess a defective extracellular basement membrane. Thus, the sine qua non of cancer is invasion, not proliferation.

Three seminal events must occur for the invasive process to proceed. Molecular analysis has allowed more detailed dissection of these events as discussed below. The first event on activation of the sprouting endothelial cell or invasive tumor cell is to change its adhesive interaction with its basement membrane and also for tumor cells to interact with the exposed vascular or lymphatic basement membrane. Following that, the cells create a rent in the basement membrane through which locomotion can occur in response to the basement membrane protein fragments, released or local cytokines, or tissue-associated attractants. The invasive process is a dynamic one involving cyclic repetition of these steps (see Fig. 8.3).

Adhesion

Both cell–cell interactions and cell–stroma interactions play an important role during the invasive cascade. Connections through cell adhesion molecules, integrins, and cadherins stabilize tissue integrity, whereas loss or alteration of these cell surface proteins has been shown to be associated with increased metastatic potential. Cell polarity and organization during spreading and migration is regulated by cell interaction with extracellular matrix proteins through the integrin family and with other cells, through the transmembrane glycoprotein cadherins. Activation of these cell surface receptors passes signals from the outside into the cell and thus directs cell behavior.

Cadherins

Cadherins are transmembrane glycoproteins that mediate extracellular calcium-dependent cell–cell interactions.^11,¹² E-(epithelial) cadherin, the most extensively studied, is involved in epithelial cell–cell communication. It is found at the cell membrane in adherens junctions in complex with a family of distinct by related cytoplasmic proteins, the catenins, α, β and γ catenin and armadillo; γ catenin is also known as plakoglobin.^13,¹⁴ Cadherin–catenin complexes are linked to the cytoskeleton through direct interactions between α-catenin and α-actinin.^15,¹⁶ E-cadherin has been demonstrated to function as a metastasis suppressor molecule in several cell lines.^17,¹⁸ In these studies, loss of gene expression has been correlated with increased invasiveness and metastatic potential, and replacement or augmentation of gene expression has resulted in suppression of the invasive phenotype. Loss of E-cadherin in ovarian surface epithelial cells is associated with loss of cell–cell contact and increase in motile and metastatic potential during progression of the transformation of ovarian surface epithelium to ovarian carcinoma.¹⁹ Noninvasive (MDCK) cells acquired an invasive phenotype after treatment with antibodies that blocked E-cadherin.²⁰ This was measured by enhanced migration through collagen I gels and invasion in a chick fragment model. Human breast adenocarcinoma cells (MCF-7), but not normal mammary epithelial cells, induced endothelial cell dissociation, which correlated with the loss of E-cadherin expression at the site of tumor cell–endothelial cell contact.²¹

There are situations where E-cadherin is normal but other membranes of the complex are not. For example, when E-cadherin was expressed in invasive breast cancer MCF-7-10A cells, they exhibited the ability to aggregate, but their morphology was unaltered and the cells remain invasive. These cells had reduced the expression of plakoglobin and less phosphorylation of β-catenin as compared with the less invasive MCF-7 human breast cancer cells.²² β-catenin, under normal conditions, binds to the tumor suppressor gene product, adenomatous polyposis coli, APC.²³ APC—β-catenin interactions promote APC hyperphosphorylation resulting in targeted degradation of β-catenin. Thus, intercellular adhesion mediated by cadherin–catenin complexes plays a role in both structural morphology and functional differentiation. Any loss of this control mechanism may facilitate the invasive process.

Integrins

The integrins are a family of transmembrane glycoproteins that are expressed by the cell as αβ heterodimers.^24,²⁵ Although originally identified as cell adhesion molecules, integrins are recognized as signaling molecules for regulation of apoptosis,^26–²⁹ gene expression,³⁰ cell proliferation,³¹ invasion and metastasis,^25,²⁷ and angiogenesis.^32,³³ Extracellular matrix (EMC) ligands for integrins include a variety of molecules, such as collagens, laminin, tenascin, fibronectin, vitronectin, von Willebrand’s factor, and thrombospondin.¹⁵ Cell–cell interactions are mediated also through heterophilic association, as seen in the interaction with mucosal endothelial addressin cell molecule-1 (MAdCAM-1) and with E-cadherin.

Peptides of the Arg-Gky-Asp (RDG) group inhibit the functions of many of the integrins. Collagen type I in its native form can be bound by two integrins, α1β1 and α2β1 through interactions with the tetrapeptide, RGDA. Denaturation of collagen I results in exposure of cryptic RGD sites that bind to alternative integrins expressed by melanoma cells, αvβ3. This duality may allow the cell to bind collagen under different circumstances, such as tissue remodeling or metastatic infiltration. Integrin–ligand interactions are accompanied by activation and clustering of integrins on the cell surface and transduction of this activation into intracellular signal transduction pathways that mediate a variety of intracellular events. Integrin signaling depends on the dynamic formation of cellular focal contacts or focal adhesions. Focal adhesions are specialized sites at which cells form signaling complexes with the extracellular matrix proteins and in which cytoskeletal and signaling proteins are concentrated. These signaling complexes regulate cell shape and migration and create a framework for the association of important signaling molecules.³⁴ Protein phosphorylation, mobilization of calcium, and (GTP) exchange are common signals involved in propagation of integrin-mediated information.³⁵

The integrin αvβ3 plays a fundamental role in angiogenesis and invasion.³⁶ It mediates cellular adhesion to vitronectin, von Willebrand’s factor, fibrinogen, fibronectin, and laminin. Activation intitiates a calcium-dependant signaling pathway leading to increase in cell motility and proteolysis.³⁷ αvβ3 is expressed minimally in normal or resting blood vessels but is upregulated significantly on the endothelium of neovessels.³⁸ Antagonists of αvβ3, such as the murine monoclonal antibody LM609, induce vascular cell apoptosis and potently inhibit angiogenesis. The humanized form of LM609 is now in clinical trial.

Proteolysis

The process of invasion is not a passive one due to pressure from excessive cellular proliferation alone but is an active, dynamic process that requires protein synthesis and degradation.³⁹ A critical proteolytic event early in the metastatic cascade appears to be the degradation of basement membrane collagen.² A chorioallantoic membrane invasion assay was used to characterize the importance of proteolysis in the invasive process.⁴⁰ Tumor cells in this model invaded the mesenchymal connective tissue through an active and progressive process. The highly malignant breast carcinoma cell lines Hep-3 and MDA-231 intravasated, while MCF-7 a less aggressive breast tumor cell line did not. PC-3, a highly malignant, hormone-resistant prostate tumor cell, entered the chorioallantoic vasculature, while LNCaP, a weakly malignant hormone requiring prostate tumor cell, did not. Tumor cells have to secrete enzymes to degrade the extracellular matrix barriers in order to intravasate successfully. Almost all cells of the tumor and host environment overexpress one or more of these enzymes.⁴¹ Degradation of the basement membrane is not dependent solely on the amount of proteolytic enzymes present but on the balance of activated proteases and their naturally occurring inhibitors. A positive correlation with tumor aggressiveness has been shown for a variety of degradative enzymes, including heparanases, seryl-, thiol- (cathepsins), and metal-dependent enzymes.

Matrix Metalloproteinases

Matrix metalloproteases (MMPs) are a family of neutral metalloenzymes secreted as latent proenzymes. They require activation through proteolytic cleavage of the amino-terminal domain, and their activity depends on the presence of Zn⁺⁺ and Ca⁺⁺.^42,⁴³ Five MMP subclasses have been defined, grouped according to substrate specificity: interstitial collagenases, gelatinases, stromelysins, membrane type-MMPs (MT-MMPs), and elastases.⁴⁴ There is structural similarity between members of the family.⁴⁴ Increased MMP activity has been detected and shown to correlate with invasion and metastatic potential in a wide range of cancers, including ovary, lung, prostate, breast, and pancreas cancers.^42,^45,⁴⁶ Type IV collagen is a critical component of the basement membrane architectural scaffolding, on which laminin, heparan sulfate, proteoglycan, and minor components of the basement membrane are assembled. Two MMPs, the gelatinases, degrade type IV collagen as a primary substrate and are distinguished by their capacity to degrade gelatin as well. It has been shown that co-localization of MMP-2 and αvβ3 on angiogenic blood vessels and melanoma cells was associated with presentation of MMP-2 in a proteolytically active form, facilitating collagen degradation.³⁷ Other metalloproteases, such as stromelysin, matrilysin, and interstitial collagenases, are also important in metastasis; they proteolyze other matrix proteins and may degrade type IV collagen in the pepsin-sensitive nonhelical domains in a less specific fashion.⁴⁴

There are at lease two gelatinases: the 72-kD MMP-2 and the 92-kD MMP-9.^2,^41,⁴⁴ MMP-2 and MMP-9 are distinguishable by immunologic, molecular, regulation, and biochemical criteria, but not by substrate specificity. Both are secreted as latenet proenzymes, can be activated by organomercurial compounds with the concomitant autoproteolytic removal of and amino-terminal fragment. In addition, they are inhibited by members of the endogenous tissue inhibitor of metalloproteinase (TIMP) family.^47–⁵¹ There are now five members of the TIMP family.⁵² The relationship between the levels of activated MT-MMPs, MMPs, and free TIMPs determines the balance between matrix degradation and matrix formation or stabilization. There are selective and specific interactions between TIMPs and MMPs, as well as more general interactions, such as those between TIMP-1 and TIMP-2 and the activated MMPs, where both may be inhibitory. Selectivity can be shown by the example of the action of TIMP-2 but not TIMP-1 on basic fibroblastic growth factor (bFGF) –induced stimulation of endothelial cell proliferation that is also independent of its ability to inhibit MMP.⁵³ Both MMPs and TIMPs can be found in the same tissue or serum samples; they can be made by the same tumor or stromal cell, or the induction of one or the other can occur as a function of the local microenvironment. A positive correlation among MMP-2 activity, TIMP, and tumor cell invasion has been demonstrated.^51,^54,⁵⁵

The function of TIMP-1 and TIMP-2 may not be limited to metalloptroeinase blockade. These proteins may also act as cytokines and recognize specific receptors. It was demonstrated that TIMP inhibited angiogenesis in vivo and both capillary endothelial cell proliferation and migration in vitro.^53,⁵⁶ These results suggest that the TIMPs may have profound biologic effects that extend well beyond their role as inhibitors of the collagenases.

Metastatic potential has been shown to correlate in a positive fashion with type IV collagenolytic activity in murine tumor models. Low levels of MMP-2 have been shown to be produced by normal, nontumorigenic, nonmetastatic cells, such as the myopeithelial cells of the human breast.⁵⁷ Benign proliferative lesions of the breast have some increase in MMP-2 restricted to the myoepithelial cells. An increase in MMP-2 was associated with the dysplastic or neoplastic cells in increasing amount with progressive severity of the breast lesions from atypical hyperplasia through carcinoma in situ to frankly invasive carcinoma. Similarly, benign polyps of the colon and normal colorectal and gastric mucosa all showed negligible immunoreactivity for MMP-2, whereas almost all invasive colonic and gastric adenocarcinomas were positive.⁵⁸ These results are also found when gene expression was studied at the RNA level and for other cancers as well. Endothelial cells in culture secrete a readily detectable quantity of MMP-2 activity.³⁹ Antisera against MMP-2 can inhibit bFGF-induced endothelial cell invasion of human amnion membrane in vitro.⁵⁹ These observations suggest that MMP-2 may also function in normal physiologic processes, such as basement membrane turnover by myoepithelial cells and angiogenesis by endothelial cells. MMP-specific enzyme-linked immunoabsorbent assays are now commercially available to measure MMP-2 and MMP-9 in serum.⁶⁰ These measurements have been shown to correlate with the degree of invasiveness and are being used as potential surrogate markers of the effectiveness of synthetic MMP inhibitors (MMPIs) now in clinical trial.

Plasminogen Activator Family

Plasminogen activators (PA) are serine-specific proteases that convert inactive plasminogen to active plasmin, a trypsin-like enzyme that degrades a variety of proteins, including fibrin, fibronectin, type IV collagen, vitronectin, and laminin. PA exists as tissue-type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). uPA is involved primarily in cell-mediated proteolysis during macrophage invasion, wound healing, embryogenesis, invasiveness, and metastasis, where it has the ability to activate latent collagenases and proplasminogen activators, and to degrade TIMPs.^61,⁶² uPA has been shown to directly activate latent growth factors, such as the precursor form of hepatocyte growth factor/scatter factor (pro-HGF/SF) and indirectly latent transforming growth factor (TGF) -β through activation of plasminogen.^63,⁶⁴ The ability of uPA to activate such growth factors suggests not only a role in modulating extracellular matrix degradation but also in tumor cell migration and proliferation. The interaction of uPA and its receptor plays an important role in direct and indirect extracellular matrix degradation, thus potentiating invasive events.⁶⁵ uPA has been shown to bind to specific cell surface receptors (uPAR).⁶⁶ Both uPA and its inactive zymogen (pro-uPA) bind with high affinity to uPAR. Expression of uPAR is upregulated by a variety of growth factors and tumor promoters including TGF-β, epidermal growth factor (EGF), HGF/SF and phorbol ester.^67–⁶⁹

Direct evidence for the role of these enzymes in invasion has been demonstrated using an in vitro invasion assay. It was demonstrated that expression of mutant uPA in PC3 human prostate cancer cells resulted in a dominant negative suppression of metastasis in nude mice xenografts. This inhibition occurred through the displacement of active uPA from its receptor by the inactive mutant enzyme.⁷⁰ A positive correlation between plasminogen activator activity and metastatic potential has been established for the B16 murine melanoma line. Highly metastatic cells of the F10 generation showed high levels in the primary tumors and even higher levels in the pulmonary metastases.

High levels of uPA have been observed in both human tumors and cell lines of bladder, breast, lung, prostate, and ovarian cancers.^71–⁷³ Elevated expression of uPA and plasminogen activator inhibitor-1 (PAI-1) in tumor extracts of ovarian and breast cancers has been correlated with increased invasion, increased incidence of relapse, and shorter overall survival.⁷⁴ Tumor progression and recurrence were associated with high uPA content in bladder cancer.⁷⁵ Breast cancer expressing high uPA levels had an increased risk for early recurrence and had a poor prognosis, while in primary breast cancer, metastasis-free survival could be predicted by uPA levels.^76,⁷⁷ Moser and colleagues showed that production of uPA in normal ovarian epithelial cells was 17- to 38-fold lower than that found in ovarian carcinoma cells.⁷⁸ It also has been demonstrated that stimulation of PC3 human prostatic carcinoma cells with EGF not only increases their uPA expression but also increases the invasive ability of the cells.⁷⁹

The action of uPA can be counteracted by naturally occurring inhibitors of the uPA/plasmin system, such as members of the SERPIN (serine protease inhibitor) family, PAI-1, PAI-2, and protease nexin-1 (PN-1).^80,⁸¹ Receptor-bound active uPA is inhibited by PAI-1, PAI-2, and PN-1.⁸² Unlike the uPAR–uPA complex which remains stable at the cell surface, the uPAR–uPA–inhibitor complex is internalized quickly by the cell and degraded. PAI-1 is produced primarily by endothelial cells but also by a number of other cell types.⁸³ Inhibition of endothelial cell migration indicates a role for PAI-1 in blocking neoangiogenesis.^84,⁸⁵ Cancer cells that overexpress PAI-1 have a decreased ability to degrade ECM and reduced invasive potential. PAI-2 is a less efficient but more specific inhibitor uPA that also inhibits invasion and metastasis.⁸⁶ PN-1 is an inhibitor of uPA, tPA, plamin, and thrombin that can bind to ECM proteins and heparin. As with PAI-1 and 2 receptor-bound active uPA in complex with PN-1 is rapidly internalized and degraded.⁶² The net balance between inhibitor and the protease is a determinant of the degradative ability of the tumor cells. Therefore, an increase in the inhibitor may not always result in a reduction of active uPA as uPA production may also be upregulated by the tumor cells.

Tumor Cell Migration

Tumor cell migration is necessary at the initiation of the metastatic cascade, at which time the tumor cells leave the primary and gain access to the circulation and also at the end of invasion, when they are entering the secondary site. To achieve forward locomotion, the invading cell couples local proteolysis with coordinated and temporally limited attachment and deattachment. Tumor cells have been found to respond to a variety of agents in a motile fashion, including host-derived motility and growth factors, EMC components, and tumor-secreted factors.^87–⁸⁹ These agents may stimulate both the initiation and maintenance of tumor cell motility and the “directedness” of that migration. Pseudopodia protruding in response to chemoattractants may serve multiple functions, including acting as sense organs for the migrating cell to locate directional clues, to secrete motility-stimulating factors, to provide propulsive traction for locomotion, and to induce matrix proteolysis to assist in the penetration of the matrix (see Fig. 8.2).⁹⁰ The complexity of tumor cell migration suggests that more than one agent is involved in the direction, location, and magnitude of the migratory response. The reliance on the host for migration stimulation would not favor the sustained migration seen in highly metastatic populations of tumor cells, thus emphasizing the importance of tumor-derived chemoattractants.

The demonstrated importance of autocrine growth factors lead to the hypothesis that tumor cells also secrete autocrine motility-stimulating factors.^88,⁹¹ The first described autocrine motility factor (AMF) was isolated and characterized from the conditioned media of the A2058 human melanoma cell line.⁸⁸ The initial AMF activity autotaxin (ATX) was purified and characterized as a cell surface–associated ectokinase^92,⁹³ and shown to stimulate motility directly through a pertussis toxin–dependent mechanism.⁹⁴ Recent studies demonstrated that its phosphodiesterase activity is necessary for motility induction, and that transfection of ATX produces and autocrine motile phenotype.⁹⁵ AMF-like activity has now been described in a number of systems.^96,⁹⁷ Gp78, an AMF receptor, has been described and shown to be associated with worse prognosis in multiple cancers.^97,⁹⁸ A multivariate analysis of expression in colorectal cancer patients showed it to be a predictor of disease recurrence along with lymph node status, another marker of invasive potential.⁹⁷

Examples of growth factors that stimulate tumor cell motility include the insulin-like growth factors (IGF), HGF, FGF, and TGF-β, among others. IGF-I has been shown to induce a chemotactic response in melanomas, prostate cancers, and human ovarian cancer.⁹⁹ HGF/SF is a paracrine motility factor that stimulates motility of epithelial and endothelial cells.^100,¹⁰¹ HGS/SF, the preferred ligand for the c-met proto-oncogene product, induces the scatter or chemokinetic locomotion of epithelial colonies, resulting in an invasive phenotype in vivo.^89,^102,¹⁰³ Transfection of a mutated met oncogene into the human osteosarcoma cell line, HOS, induced an invasive phenotype in vitro and tumorigenic and metastatic ability in vivo. In another experimental setting, transfection of murine met into NIH-3T3 cells producing endogenous HGF/SF caused the cells to become highly tumorigenic and metastatic by completing the autocrine loop. These results indicate that HGF/SF and its receptor c-met can play an important role during tumor progression by stimulating the growth and motility of cancer cells.

These factors primarily stimulate chemotactic, or directed, motility and may play a role in the tumor cell homing to secondary sites. Therefore, the response of the tumor cell to autocrine motility stimulation and endocrine or paracrine stimulation by matrix components and host-derived growth factors is important in the initiation of tumor cell locomotion, its directedness, and potentially the determination of the location of the metastatic focus.

Angiogenesis

Neovascularization, or angiogenesis, covered in more detail elsewhere in this compendium, is a prerequisite for the local expansion of tumor colonies beyond the size restricted by oxygen and nutrient diffusion. Tumor vascularization is, thus, one of the rate-limiting steps for tumor metastasis and growth.^4,^104,¹⁰⁵ New capillaries also provide cancer cells with conduits for entry into the circulation. Extravasated cancer cells will later require neovascularization in order to grow and form new metastatic foci. Therefore, angiogenesis is necessary at the beginning and end of the metastatic cascade. The process of blood vessel formation is fuctionally similar to tumor cell invasion and can be considered as a form of regulated invasion,¹ with the independent events of adhesion, proteolysis, and migration that characterize the spreading of cancer cells and that are also displayed by activated endothelial cells. Many autocrine growth factors for tumors may also act as angiogenic factors causing a pleiotropic response of enzyme production, migration, and/or proliferation in endothelial cells. It has been hypothesized that unvascularized primary tumors may be maintained as dormant small tumor nodules, their volume kept constant by a balance of cell proliferation and apoptosis.¹⁰⁶ A tumor mass larger than 0.125 mm² has outgrown its capacity to acquire nutrients by simple diffusion and must initiate angiogenesis through host vessel initiation of capillary sprouts in the direction of the tumor.¹⁰⁷ Neovascularization is a permissive event that allows metastatic dissemination of invasion-competent cells. Histologic and ultrastructural anaylses of tumor vessels have revealed pronounced differences in tumor vessels, compared with normal vessels found in mature tissues. The distinction includes differences in the cellular composition of tumor vessels, the basement membrane composition and integrity, and differences in permeability.¹⁰⁸ Due to a discontinuous basement membrane, tumor vessels are leaky and easily penetrated by cancer cells entering circulation at a high rate. The parallel between the cell and molecular biology of the physiologic invasion of angiogenesis and the malignant invasion of metastasis stresses how therapeutics targeted at invasion can also have potential benefits as antiangiogenic agents.

Genetic Regulation of Invasion and Metastasis

Invasion and metastasis are very complicated multi-step processes. Consequently, one gene product is not sufficient for metastasis. In order to exhibit the metastatic phenotype, individual tumor cells must have either a deficiency in the negative factors (loss of function) and/or an augmentation in the positive factors (gain of function). The metastatic phenotype may require additional genetic changes over and above those resulting in uncontrolled proliferation. Two classes of metastasis suppressor gene products can be identified: (1) those that act outside the cell to block key aspects of metastasis, such as proteolysis, and (2) those that have their action inside the cell in a regulatory pathway. Examples of both will be presented.

Metastasis Suppressor Genes

The existence of tumor suppressor genes led to the demonstration of metastasis suppressor genes.^55,^109,¹¹⁰ The first nonimmunologically related metastasis suppressor gene was described by Pozzatti and colleagues.¹¹¹ Rat embryo fibroblasts transfected with c-Ha-ras were highly metastatic on intravenous injection, but co-transfection of rat embryo fibroblasts with c-Ha-ras and adenovirus 2 EIA resulted in transformed but virtually nonmetastatic cells. The EIA gene, therefore, suppressed the metastasis-inducing activity of the activated ras gene. Further, studies of TIMPs 1 and 2 show that they have metastasis suppressor and some tumor suppressor activity.^44,¹¹²

A major method of identification of candidate suppressor genes has been differential gene expression studies, where the toggle switch is invasive potential, not tumorigenesis. Two examples of differential expression yielding a metastasis suppressor gene are nm23 and KiSS-1.^109,^110,¹¹³ The nm23 gene was identified on the basis of its reduced expression at the mRNA level in a series of seven sublines derived from a single K-1735 murine melanoma. nm23 RNA and protein levels were shown to be reduced in metastatic infiltrating ductal breast carcinomas. Transfection experiments using the K-1735 melanoma and the MDA-231 breast cancer models confirmed the metastasis suppressor phenotype. nm23, an nucleotide disphosphate kinase, is the human homologue of the highly conserved Drosophila awd gene product involved during imaginal disk development.¹¹⁴ A second example is the KiSS gene, identified through somatic chromosome fusion followed by differential gene expression. Addition of chromosome 6 caused a prometastatic behavior. When those cells were analyzed against the nonmetastatic cells, a chromosome 1 gene, KiSS-1, was identified.¹¹³ Proof of principle came from transfection of full-length KiSS-1 cDNA into melanoma cells, yielding suppressed metastatic potential in an expression-dependent manner.¹¹⁰ Similar results were found in a breast cancer model as well.

Oncogene Induction of Metastasis

A long list of oncogenes has been described that, either singly or in combinations, confer anchorage-independent colony growth in soft agar and, in many cases, tumorigenicity in animal hosts.^13,⁶³ A growing body of evidence, however, indicates that some oncogenes may also independently induce the phenotype of invasion and metastasis. The best studied oncogene capable of inducing the metastatic phenotype is H-ras. Activated H-ras transfected into NIH-3T3 cells produced experimental metastases.¹¹⁵ These data were confirmed by direct transfection of the cloned activated ras oncogene in several systems, including primary rat embryo fibroblast cultures. When the same cells were tested for metastatic propensity, the cells transformed by the activated H-ras were more efficient in the production of metastases than the proto-oncogene transfected cells.¹¹¹ More recent data show that many oncogenic signaling proteins, receptors, and ligands, when transfected, can induce a metastatic property, although not all in isolation of a proliferative behavior. These findings suggest that metastatic potential may be the final step in the continuum of cancer progression or it may be an independently regulated event.

Novel Technologies for Identification of Metastasis and Invasion-Promoting Genes and Gene Products

As the list of expressed human genes expands, the major scientific challenge is to understand the molecular events that drive normal tissue morphogenesis and the evolution of pathologic lesions in actual tissue. Laser capture microdissection (LCM) has been developed to provide a reliable method to procure pure populations of cells from specific microscopic regions of tissue sections, in one step, under direct visualization.^116,¹¹⁷ The elegance of this technique is that no tissue is destroyed in the process; LCM operates by positive rather than negative selection. Cells of interest are transferred to a polymer film, which is activated by laser pulses and retained on the transfer film without damage to the morphology, DNA, RNA, or protein of the procured cells (Fig.8.4). LCM, first conceived at the National Cancer Institute, is now available as a commercial instrument (Arcturus Engineering, Inc). All standardized protocols for fixation, sectioning, LCM procurement, and molecular analysis of DNA and RNA are posted on the NIH LCM web page (http://dir.nichd.nih.gov/lcm/lcm.htm). LCM is being used in the Cancer Genome Anatomy Program (CGAP) to catalogue the genes which are expressed during human solid tumor progression from normal epithelium, through premalignant progression, transitioning into invasive and metastatic disease. With the advent of LCM, microhybridization arrays of thousands of genes may be used to examine gene expression in microdissected human tissue biopsies. This will yield a fingerprint of gene expresssion that may provide crucial clues for etiology, diagnostic markers, and novel therapies tailored individual patients.

Figure 8.4

Application of microdissection to the study of invasion and metastases. Isolation of involved and stromal cells independently can allow for dissection of the genotypic, gene expression, and proteomic events underlying acquisition of invasive behavior (more...)

Molecular analysis of pure cell populations in their native tissue environment will be an important component of the next generation of the study of invasion and metastasis. Like the importance of orthotopic implantation in tumor and metastasis assays, isolation of proteins and RNA in situ is necessary to maintain the influence of the local microenvironment. LCM is key to allowing isolation only of the cells of interest, epithelial or stromal, especially since the cell subpopulation of interest may constitute a tiny fraction of the total tissue volume. For example, a biopsy of breast tissue harboring malignant tumor can contain (1) fat cells in local adipose tissue, (2) normal epithelium and myoepithelium in uninvolved branching ducts, (3) stromal fibroblasts and endothelial cells, (4) premalignant cells in in situ lesions, and (5) clusters of invasive carcinoma. The output of studies such as sophisticated microarray hybridization technology will be compromised by the use of whole tissue digests containing some or all of these cell types and will thus provide misleading data. Culturing cells from fresh tissue is one approach to reduce contamination but is fraught with the changes in gene expression and selection biases that occur with the loss of the microenvironmental influences. Thus, the cellular heterogeneity within the test samples is a significant barrier to an accurate molecular representation of normal and diseased tissue.

Microdissection samples can then be applied for analysis of genomic DNA, such as was done for the cloning of the MEN-1 gene¹¹⁸ or loss of heterozygosity studies,^119,¹²⁰ gene expression through cDNA library analysis¹²¹ and cDNA arrays,¹²² and now in protein applications (Liotta and Petricoin, personal communication). The purity of the DNA, RNA, and protein extracted from LCM-targeted cells allow for qualitative and semiquantitative analyses of critical changes. The mRNA from microdissected cancer lesions has been used as the starting material to produce cDNA libraries,¹²¹ microchip microarrays, differential display,¹²³ and other techniques to find new genes or mutations. Efficient coupling of LCM of serial tissue sections with multiplex molecular analysis techniques should lead to sensitive and quantitative methods to visualize three-dimensional interactions between morphologic elements of the tissue.¹²⁴ It is hoped that it will be possible to trace gene expression patterns along the length of a prostate gland or breast duct, for example, in order to examine the progression of neoplastic development. The end result will be a new era in the integration of molecular biology with tissue morphogenesis and pathology for the identification and characterization of novel metastasis and invasion–associated genes.

Metastasis as a Therapeutic Target

Therapeutic Targets for Cancer Progession

The recognition that invasion and even metastasis are early events leads to the logical application of these disciplines to clinical translation. That angiogenesis uses the same cassette of proteins and genes during neovascularization as is used by the malignant and invasive tumor cell leads to the overlap potential for agents targeted at invasion to also be antiangiogenic.^1,¹²⁵ Thus, regulation of adhesion, proteolysis, migration, and targeted signaling may be directions for translational application. Brief examples of several of the targets for metastasis inhibitors will be discussed (Table 8.3 and Figure 8.5).

Table 8.3

Metastasis Therapeutic Targets and Agents.

Figure 8.5

Therapeutic uses of anti-invations therapies. A. Time course of solid tumor presentation, response, and progression. B. Prevention. Invasion and angiogenesis, which use the same molecular and biochemical processes as malignant invasion, occur early. As (more...)

Growth Factor Metastasis Targets

Many growth factors can stimulate both tumor and endothelial cell behaviors ranging from proliferation to attachment, motility, and proteolysis. For that reason, they are a logical target for therapeutic intervention. Vascular endothelial growth factor (VEGF) has been targeted through the genesis drug development approaches and the successes in that venue are discussed elsewhere in this compendium. Other growth factors, such as EGF and platelet-derived growth factor (PDGF), for example, have been addressed. Two classes of molecules have been developed against growth factor receptors: small molecule receptor antagonists and monoclonal antibodies. Tyrphostins have been developed that bind to a series of molecules, tyrosine kinase–containing growth factor receptors, such as those of EGF, VEGF, and PDGF.^126,¹²⁷ This class of molecules focused on the ligand and ATP–binding sites of the tyrosine kinase receptors. Molecules that have receptor specificity have now been developed and are in clinical trial. For example, SU101 is selective to the PDGF receptor.¹²⁸ Another approach is the use of directed monoclonal antibodies. An antibody directed against VEGF is in clinical trial and discussed elsewhere. A chimerized antiactivated EGFR antibody also has entered trials. It has documented antitumor and antiangiogenic activity.^129–¹³¹ These promising directions have also been shown to have improved efficacy when administered in combination with classic chemotherapeutics.

Antiadhesive Agents

Limited agents targeted at tumor or endothelial cell adhesion have entered clinical trials, and several are under development. Interventions include peptidomimetics and monoclonal antibodies targeted currently at integrins. Both therapeutic approaches have been taken against αvβ3, the vitronectin receptor integrin.¹³² αvβ3 is also important because it is selectively present in immature blood vessels.^33,¹³³ Antagonists of αvβ3, such as the murine monoclonal antibody LM609 and its newer humanized counterpart Vitaxin, induce vascular cell apoptosis and potently inhibit angiogenesis. This is related to the ability of these antagonists to selectively promote programmed cell death of newly sprouting blood vessels.¹³⁴ This is a new area that has not been fully exploited.

Matrix Metalloproteinase Inhibitors

Regulation of the TIMP/MMP balance is critical to the localization inhibition of matrix breakdown for both physiologic invasion of angiogenesis and the malignant invasion of metastasis. A key action of TIMP-2 is to regulate and, therefore, focus local proteolysis. The site of action of TIMP-2 and the site of metal binding in the MMPs were identified as the key targets for therapeutic intervention. The first class of MMPIs are the hydroxamate molecules, examples of which are batimastat (BB-94) and marimastat, targeted to interact with the MMPs at the activation site by blocking chelation of the metal ion, thus mimicking the physiologic action of TIMPs.^135,¹³⁶ The effective concentrations for BB-94 range from 3nM for interstitial collagenase (MMP-1) to 20 nM for stromelysin (MMP-3). Both anti-invasive and antiangiogenic activity has also been observed.^136–¹³⁸ Marimastat is now in phase III clinical trials for ovarian cancer.¹³⁹ BAY 12-9566, a specific inhibitor of MMP-2 and MMP-9, does not contain a hydroxamate moiety and has a different pattern of toxicity, lacking drug-induced arthritis; anticancer trials have recently been terminated. It shows anti-invasive properties in vitro and antiangiogenic properties in vivo. Many other MMPIs are under development and in phase I and II clinical trials.

Antiangiogenesis Therapies

Because of the overlap of the mechanisms underlying neovascularization, invasion, and metastasis, many of the discussed agents can and do have potential as antiangiogenesis agents. Below are some agents that do no fall into the anti-invasion overlap group. TNP-470 (AGM-1470), is the first of the now exploding group of antiangiogenesis agents. It has been found to have an inhibitory effect on endothelial cell proliferation and migration,^140,¹⁴¹ and newer data suggest that it is a cell-cycle regulator. Thalidomide was shown to have antiangiogenic activity¹⁴² and has clinical activity in Kaposi’s sarcoma,¹⁴³ with more studies ongoing. Hepatic metabolism is required to yield an active but not yet defined metabolite.¹⁴⁴ Additional endothelial cell–specific reagents include those targeted at VEGF, including the anti-VEGF antibody and the tyrphostin directed against the VEGF receptor (SU5416). Both agents, alone and in combination, are in clinical trials presently. Disease stabilization and some anecdotal responses have been reported.

Antimetastasis Signal Transduction Therapy

The loss of balance in the cellular communication process may allow for dysregulation leading to tumorigenicity, invasion, and metastasis.^1,² Therapeutic efforts in cancer prevention and treatment are being focused at the level of signaling pathways or selective modulatory proteins. Investigations into the signaling pathways underlying metastasis have suggested that protein kinase activity, calcium homeostasis, and ras activation are important signals and therefore may be key regulatory sites for therapeutic intervention.

Several natural products have been found that inhibit protein tyrosine kinase activity and may possess antiproliferative or anti-invasive properties. These include genistein, herbimycin, and lavendustin A.¹⁴⁵ Genistein is an isoflavinoid that inhibits endothelial cell proliferation and in vitro angiogenesis, most likely through its inhibition of tyrosine phosphorylation and ATP-induced calcium influx.¹⁴⁶ A role for calcium influx has been shown in the process of angiogenesis,³⁹ and genistein may mediate its antiangiogenic effects via this action. The tyrphostins, a group of synthetic compounds designed to block phosphorylation of tyrosine residues, have been shown to be potent inhibitors of cell proliferation in vitro.^126,¹²⁷ Substitutions in the structure of the tyrphostins confer specificity to different receptor tyrosine kinases. Agents have been developed against the receptors of EGF, PDGF, and VEGF; many are in clinical trial presently.

Protein kinases C (PKCs) form a family of serine/threonine kinases that mediate phosphorylation events involved in the pathway of many growth factors, matrix components, and neurotransmitters. Phorbol esters, initially described as tumor promoters, were found to stimulate a subset of PKCs in place of diacylglycerol, its endogenous activator.¹⁴⁷ PKC activity has been linked to metastasis in many ways.^148–¹⁵¹ Staurosporine, with general protein kinase inhibitory activity, was shown to inhibit PKC and secondarily inhibit metastasis.¹⁵² An analogue of staurosporine, UCN-01,¹⁵³ is now in clinical trials, and Salfingol, an optical isomer of dihydrosphingosine, a specific inhibitor of PKC, has been tested.^154,¹⁵⁵

Intracellular calcium homeostasis is a common regulator of transmembrane signal transduction and the process of invasion, metastasis, and angiogenesis.^1,^39,^156–¹⁵⁸ A novel inhibitor of calcium mobilization, CAI (carboxyamido-triazole), was identified through a screen for antimotility agents.¹⁵⁹ CAI inhibits calcium influx through nonvoltage-gated calcium channels, and it has also been shown to inhibit other calcium influx–dependent downstream signaling pathways.^158,^160,¹⁶¹ CAI inhibits proliferation, production of MMP-2, motility, and signaling of endothelial cells and a variety of human cancer cell lines in vitro and in vivo.^39,^125,^157–^159,^162,¹⁶³ Phase I clinical trials of CAI have been completed, and phase II and III and combination studies have opened.^164–¹⁶⁶ CAI was cytostatic with disease stabilization in almost half the patients treated and minor or partial remission in two patients. Consistent with the anti-invasive and antiangiogenic targets of CAI, over 80% of patients progressed at sites of existing disease at the time of progression. Predominant toxicity was formulation-dependent mild to moderate nausea and vomiting and sensory peripheral neuropathy.

Another recently developed therapeutic target in metastases is the ras oncoprotein signaling cascade.^167,¹⁶⁸ Several agents have been taken to clinical trial. Investigators have further demonstrated its utility as a therapeutic target through studies that tie ras to the actin cytoskeleton and its function.¹⁶⁹ ras requires post-translational isoprenylation to allow it to translocate and interact at the cell membrane. This isoprenylation can be inhibited by intervention at two points. First, the interruption is in the prenyl group synthetic pathway with agents such as lovostatin, an inhibitor or HMG-CoA reductase. Second, new agents have come to the clinic that directly target the enzymes responsible for geranylgeranylation and farnesylation of key signaling proteins, such as ras and rhoA. These trials are underway.

Summary

The study of molecular mechanisms underlying the metastatic cascade has led to dramatic advances in our understanding of the processes of invasion and metastasis. It has led to the recognition that neovascularization uses the same cassettes of genes and proteins for the invasive behavior underlying vascular sprout formation and has shown how anti-invasive drugs can also be antiangiogenic. The dissection of the invasive and metastatic processes has created new directions for cancer marker development and application. Importantly, molecular advances have allowed confirmation that invasion and acquisition of the metastatic phenotype are early events in cancer progression. Further application of this critical knowledge will advance our ability to identify those patients at highest risk for disseminated disease, to better develop therapeutics for those key patients, and, perhaps, to prevent many patients from receiving treatment that they might not have needed.

References

1.: Kohn E C, Liotta L A. Molecular insights into cancer invasion: strategies for prevention and intervention. Cancer Res. 1995;55:1865–1862. [PubMed: 7728753]
2.: Liotta L A, Stetler-Stevenson W G. Tumor invasion and metastasis: an imbalance of positive and negative regulation. Cancer Res. 1991;51:5054s–5059s. [PubMed: 1884381]
3.: Duffy M J. Inhibiting tissue invasion and metastasis as targets for cancer therapy. Biotherapy. 1992;4:45–52. [PubMed: 1543650]
4.: Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86:353–364. [PubMed: 8756718]
5.: Woodhouse E C, Chuaqui R F, Liotta L A. General mechanisms of metastasis. Cancer. 1997;80:1529–1537. [PubMed: 9362419]
6.: Fidler I J, Hart I R. Biologic diversity in metastatic neoplasms: origins and implications. Science. 1982;217:998. [PubMed: 7112116]
7.: Tuck A B, O’Malley F P, Singhal H. et al. Osteopontin expression in a group of lymph node negative breast cancer patients. Int J Cancer. 1998;79:502–508. [PubMed: 9761120]
8.: Tuck A B, Arsenault D M, O’Malley F P. et al. Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells. Oncogene. 1999;18:4237–4246. [PubMed: 10435636]
9.: Nicolson G L. Gene expression, cellular diversification and tumor progression to the metastatic phenotype. Bioessays. 1991;13:337. [PubMed: 1759975]
10.: Liotta L A. Tumor invasion: role of the extracellular matrix. Cancer Res. 1986;46:1–7. [PubMed: 2998604]
11.: Knudsen K A, Frankowski C, Johnson K R, Wheelock M J. A role for cadherins in cellular signaling and differentiation. J Cell Biochem Suppl. 1998;30-31:168–176. [PubMed: 9893268]
12.: Vleminckx K, Kemler R. Cadherins and tissue formation: integrating adhesion and signaling. Bioessays. 1999;21:211–220. [PubMed: 10333730]
13.: Jou T S, Stewart D B, Stappert J. et al. Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex. Proc Natl Acad Sci USA. 1995;92:5067–5071. [PMC free article: PMC41849] [PubMed: 7761449]
14.: Chen Y T, Stewart D B, Nelson W J. Coupling assembly of the E-cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells. J Cell Biol. 1999;144:687–699. [PMC free article: PMC2132940] [PubMed: 10037790]
15.: Pignatelli M. Integrins, cadherins, and catenins: molecular cross-talk in cancer cells. J Pathol. 1998;186:1–2. [PubMed: 9875132]
16.: Sandig M, Negrou E, Rogers K A. Changes in the distribution of LFA-1, catenins, and F-actin during transendothelial migration of monocytes in culture. J Cell Sci. 1997;110:2807–2818. [PubMed: 9427289]
17.: Semb H, Christofori G. The tumor-suppressor function of E-cadherin. Am J Hum Genet. 1998;63:1588–1593. [PMC free article: PMC1377629] [PubMed: 9837810]
18.: Vleminckx K, Vakaet L, Mareel M. et al. Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell. 1991;66:107–119. [PubMed: 2070412]
19.: Hoffman A G, Burghardt R C, Tilley R, Auersperg N. An in vitro model of ovarian epthelial carcinogenesis: changes in cell-cell communication and adhesion occurring during neoplastic progression. Int J Cancer. 1993;54:828–838. [PubMed: 8325708]
20.: Burridge K, Fath K, Kelly T. et al. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Ann Rev Cell Biol. 1988;4:487–525. [PubMed: 3058164]
21.: Lewalle J M, Bajou K, Desreux J. et al. Alterations of interendothelial adherens junctions following tumor cell-endothelial cell interaction in vitro. Exp Cell Res. 1997;237:347–356. [PubMed: 9434630]
22.: Sommors C L, Gelmann E P, Kemler R. et al. Alterations in beta-catenin phosphorylation and plakoglobin expression in human breast cancer cells. Cancer Res. 1994;54:3544–3552. [PubMed: 8012979]
23.: Rubinfeld B, Souza B, Albert I. et al. Association of the APC gene product with beta-catenin. Science. 1993;262:1731–1734. [PubMed: 8259518]
24.: Hynes R O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69:11–25. [PubMed: 1555235]
25.: Gille J, Swerlick R A. Integrins: role in cell adhesion and communication. Ann NY Acad Sci. 1996;797:93–107. [PubMed: 8993354]
26.: Schwartz M A, Schaller M D, Ginsberg M H. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol. 1995;11:549–599. [PubMed: 8689569]
27.: Varner A J, Cheresh D A. Integrins and cancer. Curr Opin Cell Biol. 1996;8:724–730. [PubMed: 8939661]
28.: Frisch S M, Ruoslahti E. Integrins and snoikis. Curr Opin Cell Biol. 1997;9:701–706. [PubMed: 9330874]
29.: Meredith J E, Schwartz M. Integrins, adhesion, and apoptosis. Trends Cell Biol. 1997;7:146–150. [PubMed: 17708932]
30.: Chicurel M E, Singer R H, Meyer C J, Ingber D E. Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions. Nature. 1998;392:730–733. [PubMed: 9565036]
31.: Giancotti F G. Integrin signaling: specificity and control of cell survival and cell cycle progression. Curr Opin Cell Biol. 1997;9:691–700. [PubMed: 9330873]
32.: Friedlander M, Brooks P C, Shaffer R W. et al. Definition of two angiogenic pathways by distinct alpha v integrins. Science. 1995;270:1500–1502. [PubMed: 7491498]
33.: Brooks P C, Clark R A F, Cheresh D A. Requirement of vascular integrin avb3 for angiogensis. Science. 1994;264:569–571. [PubMed: 7512751]
34.: Schwartz M A. Transmembrane signalling by integrins. Trends Cell Biol. 1992;2:304–308. [PubMed: 14731926]
35.: Keely P, Parise L, Juliano R. Integrins and GTPases in tumor cell growth, motility and invasion. Trends Cell Biol. 1998;8:101–106. [PubMed: 9695818]
36.: Seftor R E B. Role of the β3 integrin subunit in human primary melanoma progression. Am J Pathol. 1998;153:1347–1351. [PMC free article: PMC1853420] [PubMed: 9811323]
37.: Brooks P C, Stromblad S, Sanders L C. et al. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interactions with integrin αvβ3. Cell. 1996;85:683–693. [PubMed: 8646777]
38.: Eliceiri B P, Cheresh D A. The role of alpha integrins during angiogensis. Mol Med. 1998;4:741–750. [PMC free article: PMC2230391] [PubMed: 9990860]
39.: Kohn E C, Allessandro R, Spoonster J. et al. Angiogenesis: role of calcium-mediated signal transduction. Proc Natl Acad Sci USA. 1995;92:1307–1311. [PMC free article: PMC42508] [PubMed: 7533291]
40.: Kim J, Yu W, Kovalski K, Ossowski L. Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell. 1998;94:353–362. [PubMed: 9708737]
41.: Nagase H, Woessner J F. Matrix metalloproteinases. J Biol Chem. 1999;274:21491– 21494. [PubMed: 10419448]
42.: Coussens L M, Werb Z. Matrix metalloproteinases and the development of cancer. Chem Biol. 1996;3:895–904. [PubMed: 8939708]
43.: Kelly T, Yan Y, Osborne R L. et al. Proteolysis of extracellular matrix by invadopodia facilitates human breast cancer cell invasion and is mediated by matrix metalloproteinases. Clin Exp Metastasis. 1998;16:501–512. [PubMed: 9872598]
44.: Chambers A F, Matrisian L M. Changing views of the role of matrix mettaloproteinases in metastasis. J Natl Cancer Inst. 1997;89:1260–1270. [PubMed: 9293916]
45.: Liotta L A, Saidel M G, Kleinerman J. The significance of hematogenous tumor cell clumps in the metastatic process. Cancer Res. 1976;36:889–894. [PubMed: 1253177]
46.: Liotta L A, Tryggvason K, Garbisa S. et al. Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature. 1980;284:67–68. [PubMed: 6243750]
47.: Stetler-Stevenson W G, Krutzsch H C, Liotta L A. Tissue inhibitor of metalloproteinase (TIMP-2): a new member of the metalloproteinase inhibitor family. J Biol Chem. 1989;264:17374–17378. [PubMed: 2793861]
48.: Goldberg G I, Marmer B L, Grant G A. et al. Human 72-kilodalton type IV collagenase forms a complex with a tissue inhibitor of metalloproteinases designated TIMP-2. Proc Nat Acad Sci USA. 1989;86:8207. [PMC free article: PMC298249] [PubMed: 2554304]
49.: Apte S S, Olsen B R, Murphy G. The gene structure of tissue inhibitor of metalloproteinases (TIMP)-3 and its inhibitory activities define the distinct TIMP gene family. J Biol Chem. 1995;270:14313–14318. [PubMed: 7782289]
50.: Leco K J, Apte S S, Taniguchi G T. et al. Murine tissue inhibitor of metalloproteinases-4 (TIMP-4): cDNA isolation and expression in adult mouse tissues. FEBS Lett. 1997;401:213–217. [PubMed: 9013889]
51.: Reynolds J J. Coolagenases and tissue inhibitors of metalloproteinsases: a functional balance in tissue degradation. Oral Dis. 1996;2:70–76. [PubMed: 8957940]
52.: Gomez D E, Alonso D F, Yoshiji H, Thorgeirsson U P. Tissue inhibitor of metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol. 1997;74:111–122. [PubMed: 9352216]
53.: Murphy A N, Unsworth E J, Stetler-Stevenson W G. Tissue inhibitor of metalloproteinases-2 inhibits bFGF-induced human microvascular endothelial cell proliferation. J Cell Physiol. 1993;157:351–358. [PubMed: 7693724]
54.: Kawamata H, Kawai K, Kameyama S. et al. Over-expression of tissue inhibitor of matrix metalloproteinases (TIMP1 and TIMP2) suppresses extravasation of pulmonary metastasis of a rat bladder carcinoma. Int J Cancer. 1995;63:680–687. [PubMed: 7591285]
55.: Ray J M, Stetler-Stevenson W G. TIMP-2 expression modulates human melanoma cell adhesion and motility. Ann NY Acad Sci. 1994;732:233–247. [PubMed: 7978796]
56.: Schnaper H W, Grant D S, Stetler-Stevenson W G. et al. Type IV collagenase(s) and TIMPs modulate endothelial cell morphogenesis in vitro. J Cell Physiol. 1993;156:235–246. [PubMed: 8344982]
57.: Monteagudo C, Merino M, San-Juan J. et al. Immunohistologic distribution of type IV collagenase in normal, benign and malignant breast tissue. Am J Pathol. 1990;136:585. [PMC free article: PMC1877499] [PubMed: 2156430]
58.: Levy A, Cioce V, Sobel M. et al. Increased expression of he Mr 72,000 type IV collagenase in human colonic adenocarcinoma. Cancer Res. 1991;51:439–444. [PubMed: 1846313]
59.: Mignatti P, Tsuboi R, Robbins E, Rifkin D. In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases. J Cell Biol. 1989;108:671. [PMC free article: PMC2115414] [PubMed: 2465298]
60.: Zucker S, Hymowitz M, Conner C. et al. Measurement of matrix metalloproteinases and tissue inhibitors of metalloproteinases in blood and tissues. Ann NY Acad Sci. 1999;878:212–227. [PubMed: 10415733]
61.: Gottesman M. The role of proteases in cancer. Semin Cancer Biol. 1990;1:97–100.
62.: Conese M, Blasi F. The urokinase/urokinase-receptor system and cancer invasion. Baillieres Clin Haematol. 1995;8:365–389. [PubMed: 7549068]
63.: Odekon L E, Blasi F, Rifkin D B. Requirement for receptor-bound urokinase in plasmin-dependent cellular conversion of latent TGF-beta to TGF-beta. J Cell Physiol. 1994;158:398–407. [PubMed: 8126064]
64.: Naldini L, Tamagnone L, Vigna E. et al. Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor. EMBO J. 1992;11:4825–4833. [PMC free article: PMC556958] [PubMed: 1334458]
65.: Miyake H, Hara I, Yamanaka K. et al. Elevation of urokinase-type plasminogen activator and its receptor densities as new predictors of disease progression and prognosis in men with prostate cancer. Int J Oncol. 1999;14:535–541. [PubMed: 10024688]
66.: Nielsen L S, Kellerman G M, Behrendt N. et al. A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification. J Biol Chem. 1988;263:2358–2363. [PubMed: 2828365]
67.: Lund L R, Ellis V, Ronne E. et al. Transcriptional and post-transcriptional regulation of the receptor for urokinase-type plasminogen activator by cytokines and tumour promoters in the human lung carcinoma cell line A549. Biochem J. 1995;310:345–352. [PMC free article: PMC1135894] [PubMed: 7646466]
68.: Picone R, Kajtaniak E L, Nielsen L S. et al. Regulation of urokinase receptors in monocyte-like U937 cells by phorbol ester phorbol myristate acetate. J Cell Biol. 1989;108:693–702. [PMC free article: PMC2115427] [PubMed: 2537321]
69.: Pepper M S, Matsumoto K, Nakamura T. et al. Hepatocyte growth factor increases urokinase-type plasminogen activator (u-PA) and (u-PA) receptor expression in Madin-Darby canine kidney epithelial cells. J Biol Chem. 1992;267:20493–20496. [PubMed: 1328201]
70.: Crowley C W, Cohen R L, Lucas B K. et al. Prevention of metastasis by inhibition of the urokinase receptor. Proc Natl Acad Sci. 1993;90:5021–5025. [PMC free article: PMC46645] [PubMed: 8389464]
71.: Billstrom A, Lecander I, Dagnaes-Hansen F. et al. Differential expression of uPA in an aggressive (DU 145) and a nonaggressive (1013L) human prostate cancer xenograft. Prostate. 1995;26:94–104. [PubMed: 7531848]
72.: Heiss M M, Allgayer H, Gruetzner K U. et al. Individual development and uPA-receptor expression of disseminated tumour cells in bone marrow: a reference to early systemic disease in solid cancer. Nat Med. 1995;1:1035–1039. [PubMed: 7489359]
73.: Kuhn W, Pache L, Schmalfeldt B. et al. Urokinase (uPA) and PAI-1 predict survival in advanced ovarian cancer patients (FIGO III) after radical surgery and platinum-based chemotherapy. Gynecol Oncol. 1994;55:401–409. [PubMed: 7835780]
74.: Schmitt M, Wilhelm O, Janicke F. et al. Urokinase-type plasminogen activator (uPA) and its receptor (CD87): a new target in tumor invasion and metastasis. J Obstet Gynaecol. 1995;21:151–165. [PubMed: 8556577]
75.: Hasui Y, Marutsuka K, Asada Y, Osada Y. Prognostic value of urokinase-type plasminogen activator in patients with superficial bladder cancer. Urology. 1996;47:34–37. [PubMed: 8560659]
76.: Duffy M J, Reilly D, O’Sullivan C. et al. Urokinase-plasminogen activator, a new and independent prognostic marker in breast cancer. Cancer Res. 1990;50:6827–6829. [PubMed: 2119883]
77.: Spyratos F, Martin P M, Hacene K. et al. Multiparametric prognostic evaluation of biological factors in primary breast cancer. J Natl Cancer Inst. 1992;84:1266–1272. [PubMed: 1640487]
78.: Moser T L, Young T N, Rodriguez G C. Secretion of extracellular matrix-degrading proteinases is increased in epithelial ovarian carcinoma. Int J Cancer. 1994;56:552–559. [PubMed: 8112891]
79.: Jarrard D F, Blitz B F, Smith R C. et al. Effect of epidermal growth factor on prostate cancer cell line PC3 growth and invasion. Prostate. 1994;24:46–53. [PubMed: 8290389]
80.: Sharp A M, Stein P E, Pannu N S. et al. The active conformation of plasminogen activator inhibitor 1, a target for drugs to control fibrinolysis and cell adhesion. Structure Fold Des. 1999;7:111–118. [PubMed: 10368279]
81.: Potempa J, Korzus E, Travis J. The serpin superfamily of proteinase inhibitors: structure, function, and regulation. J Biol Chem. 1994;269:15957–15960. [PubMed: 8206889]
82.: Ellis V, Dano K, Ellis V. et al. Plasminogen activation by receptor-bound urokinase: inhibition of receptor-bound urokinase by plasminogen-activator inhibitors. Semin Thromb Hemost. 1991;17:194–200. [PubMed: 1665584]
83.: Dano K, Behrendt N, Brunner N. et al. The urokinase receptor: protein structure and role in plasminogen activation and cancer invasion. Fibrinolysis. 1994;8:189–203.
84.: Fazioli F, Blasi F. Urokinase-type plasminogen activator and its receptor: new targets for anti-metastatic therapy? Trends Pharmacol Sci. 1994;15:25–29. [PubMed: 8140655]
85.: Montesano R, Pepper M S, Mohle-Steinlein U. et al. Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene. Cell. 1990;62:435–445. [PubMed: 2379237]
86.: Reiter L S, Kruithof E K, Cajob J F, Sordat B. The role of the urokinase receptor in extracellular matrix degradation by HT29 human colon carcinoma cells. Int J Cancer. 1993;53:444–450. [PubMed: 8381394]
87.: Aznavoorian S, Stracke M L, Krutzsch H C. et al. Signal transduction for chemotaxis and haptotaxis by matrix macromolecules in tumor cells. J Cell Biol. 1990;110:1427–1438. [PMC free article: PMC2116083] [PubMed: 2324200]
88.: Liotta L A, Mandler R, Murano G. Tumor cell autocrine motility factor. Proc Natl Acad Sci USA. 1986;83:3302. [PMC free article: PMC323501] [PubMed: 3085086]
89.: Rong S, Segal S, Anver M. et al. Invasiveness and metastasis of NIH 3T3 cells induced by Met-hepatocyte growth factor/scatter factor autocrine stimulation. Proc Natl Acad Sci. 1994;91:4731–4735. [PMC free article: PMC43862] [PubMed: 8197126]
90.: Lapis K, Paku S, Liotta L A. Endothelialization of embolized tumor cells during metastasis formation. Clin Exp Metastasis. 1988;6:73–89. [PubMed: 3335082]
91.: Aaronson S A. Growth factors and cancer. Science. 1991;254:1146–1153. [PubMed: 1659742]
92.: Liotta L A, Murata J. cDNA cloning of the autocrine motility factor, autotaxin (ATX). Clin Exp Metastasis. 1994;12:3–4.
93.: Stracke M L, Krutzsch H C, Unsworth E J. et al. Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein. J Biol Chem. 1992;267:2524–2529. [PubMed: 1733949]
94.: Clair T, Lee H Y, Liotta L A, Stracke M L. Autotaxin is an exoenzyme possessing5’-necleotide phophodiesterase/ATP pyrophosphatase and ATPase activities. J Biol Chem. 1997;272:996–1001. [PubMed: 8995394]
95.: Stracke M L, Clair T, Liotta L A. Autotaxin, tumor motility-stimulating exophophodiesterase. Adv Enzyme Regul. 1997;37:135–144. [PubMed: 9381968]
96.: Timar J. Autocrine motility factor signals integrin-mediated metastatic melanoma cell adhesion and invasion. Cancer Res. 1996;56:1902–1908. [PubMed: 8620512]
97.: Nakamori S, Watanabe H, Kameyama M. et al. Expression of autocrine motility factor receptor in colorectal cancer as a predictor for disease recurrence. Cancer. 1994;74:1855–1862. [PubMed: 8082090]
98.: Shimizu K, Tani M, Watanabe H. et al. The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein. FEBS Lett. 1999;456:295–300. [PubMed: 10456327]
99.: Kohn E C, Francis A, Liotta L A, Schiffmann E. Heterogeneity of the motility responses in malignant tumor cells: a biological basis for the diversity and homing of metastatic cells. Int J Cancer. 1990;46:287. [PubMed: 2116998]
100.: Stoker M. Effect of scatter factor on motility of epithelial cells and fibroblasts. J Cell Physiol. 1989;139:565–569. [PubMed: 2525565]
101.: Stoker M, Gherardi E. Regulation of cell movement: the mitogenic cytokines. Biochim Biophys Acta. 1991;1072:81–102. [PubMed: 2018780]
102.: Naldini L, Weidner K M, Vigna E. et al. Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor. EMBO J. 1991;10:2867–2878. [PMC free article: PMC452997] [PubMed: 1655405]
103.: Hartmann G, Naldini L, Weidner K M. et al. A functional domain in the heavy chain of scatter factor/hepatocyte growth factor binds the c-Met receptor and induces cell dissociation but not mitogenesis. Proc Natl Acad Sci USA. 1992;89:11574– 11578. [PMC free article: PMC50595] [PubMed: 1280830]
104.: Folkman J, Hochberg M, Knighton D. Self-regulation of growth in three dimensions: the role of surface area limitations. In: Clarkson B, Baserga R, editors. Control of proliferation in animal cells. Cold Spring Harbor, CA: 1974. p. 833–842.
105.: Liotta L A, Kleinerman J, Saidel G. Quantitative relationships of intravascular tumor cells: tumor vessels and pulmonary metastases following tumor implantation. Cancer Res. 1974;34:997–1003. [PubMed: 4841969]
106.: Holmgren L, O’Reilly M S, Folkman J. Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression [see comments] Nat Med. 1995;1:149–153. [PubMed: 7585012]
107.: Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285:1182. [PubMed: 4938153]
108.: Jain R K. Vascular and interstitial barriers to delivery of therapeutic agents in tumors. Cancer Metastasis Rev. 1990;9:253–266. [PubMed: 2292138]
109.: Steeg P S, Bevilacqua G, Kopper L. Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst. 1988;80:200–204. [PubMed: 3346912]
110.: Lee J H, Welch D R. Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis. Cancer Res. 1997;57:2384–2387. [PubMed: 9192814]
111.: Pozzatti R, Muschel R, Williams J. et al. Primary rat embryo cells transformed by one or two oncogenes show different metastatic potentials. Science. 1986;232:223–227. [PubMed: 3456644]
112.: Alvarez O A, Carmichael D F, DeClerck Y A. Inhibition of collagenolytic activity and metastasis of tumor cells by a recombinant human tissue inhibitor or metalloproteinases. J Natl Cancer Inst. 1990;82:589–595. [PubMed: 2156082]
113.: Lee J H, Miele M E, Hicks D J. et al. KiSS-1, a novel human malignant melanoma meatstasis-suppressor gene. J Natl Cancer Inst. 1996;88:1731–1737. [PubMed: 8944003]
114.: Rosengard A M, Krutzsch H C, Shearn A. et al. Reduced nm23/Awd protein in tumour metastasis and aberrant Drosophila development. Nature. 1989;342:177–180. [PubMed: 2509941]
115.: Thorgeirsson U P, Turpeenniemi-Hujanen T, Williams J E. et al. NIH 3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice. Mol Cell Biol. 1985;5:259. [PMC free article: PMC366702] [PubMed: 3982418]
116.: Emmert-Buck M R, Bonner R F, Smith P D. et al. Laser capture microdissection. Science. 1996;274:998–1001. [PubMed: 8875945]
117.: Bonner R F, Emmert-Buck M, Cole K. et al. Laser capture microdissection: molecular analysis of tissue. Science. 1997;278:1481–1483. [PubMed: 9411767]
118.: Chandrasekharappa S C, Guru S C, Manickam P. et al. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science. 1997;276:404–407. [PubMed: 9103196]
119.: Vocke C D, Pozzatti R O, Bostwick D G. et al. Analysis of 99 microdissected prostate carcinomas reveals a high frequency of allelic loss on chromosome 8p12-21. Cancer Res. 1996;56:2411–2416. [PubMed: 8625320]
120.: Brown M R, Chuaqui R, Vocke C D. et al. Allelic loss on chromosome arm 8p: analysis of low malignant potential and invasive epithelial ovarian tumors. Gynecol Oncol. 1999;74:98–102. [PubMed: 10385558]
121.: Krizman D B, Chuaqui R F, Meltzer P S. Construction of a representative cDNA library from prostatic intraepithelial neoplasia. Cancer Res. 1996;56:5380–5383. [PubMed: 8968089]
122.: Schena M, Shalon D, Davis R W, Brown P O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. 1995;270:467–470. [PubMed: 7569999]
123.: Chuaqui R F, Englert C R, Strup S E. et al. Identification of a novel transcript up-regulated in a clinically aggressive prostate carcinoma. Urology. 1997;50:302–307. [PubMed: 9255310]
124.: Cole K A, Krizman D B, Emmert-Buck M R. The genetics of cancer—a 3D model. Nat Genet. 1999;21:38–41. [PubMed: 9915499]
125.: Masiero L, Lapidos K A, Ambudkar I, Kohn E C. Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx. J Cell Sci. 1999;112:3205–3213. [PubMed: 10504326]
126.: Levitzki A, Gilon C. Tryphostins as molecular tools and potential antiproliferative drugs. Trends Pharmacol Sci. 1991;12:171–174. [PubMed: 1862529]
127.: Levitzki A. Tryphostins: tyrosine kinase blockers as novel antiproliferative agents and dissectors of signal transduction. FASEB. 1992;6:3275–3282. [PubMed: 1426765]
128.: Shawver L K, Schwartz D P, Mann E. et al. Inhibition of platelet-derived growth factor-mediated signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl]5-methylisoxazole-4-carboxamide. Clin Cancer Res. 1997;3:1167–1177. [PubMed: 9815796]
129.: Perrotte P, Matsumoto T, Inoue K. et al. Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice. Clin Cancer Res. 1999;5:257–265. [PubMed: 10037173]
130.: Huang S M, Bock J M, Harari P M. Epidermal growth factor receptor blockade with C225 modulates proliferation, apoptosis, and radiosensitivity in squamous cell carcinomas of the head and neck. Cancer Res. 1999;59:1935–1940. [PubMed: 10213503]
131.: Goldstein N I, Prewett M, Zuklys K. et al. Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clin Cancer Res. 1995;1:1311–1318. [PubMed: 9815926]
132.: van den Brule F A, Berchuck A, Bast R C. et al. Differential expression of the 67-kD laminin receptor and 31-kD human laminin-binding protein in human ovarian carcinomas. Eur J Cancer. 1994;30A:1096–1099. [PubMed: 7654437]
133.: Gasparini G, Brooks P C, Biganzoli E. et al. Vascular integrin alpha(V)beta3: a new prognostic indicator in breast cancer. Clin Cancer Res. 1998;4:2625–2634. [PubMed: 9829725]
134.: Pluda J M. Tumor-associated angiogenesis: mechanisms, clinical implications, and therapeutic strategies. Semin Oncol. 1997;24:203–218. [PubMed: 9129690]
135.: Wojtowicz-Praga S M, Dickson R B, Hawkins M J. Matrix metalloproteinase inhibitors. Invest New Drugs. 1997;15:61–75. [PubMed: 9195290]
136.: Davies B, Brown P D, East N. et al. A synthetic matrix metalloproteinase inhibitor decreases tumor burden and prolongs survival of mice bearing human ovarian carcinoma xenografts. Cancer Res. 1993;53:2087–2091. [PubMed: 8347186]
137.: Taraboletti G, Garofalo A, Belotti D. et al. Inhibition of angiogenesis and murine hemangioma growth by batimastat, a synthetic inhibitor of matrix metalloproteinases. J Natl Cancer Inst. 1995;87:293–298. [PubMed: 7535861]
138.: Wang X, Fu X, Brown P D. et al. Matrix metalloproteinase inhibitor BB-94 (batimastat) inhibits human colon tumor growth and spread in a patient-like orthotopic model in nude mice. Cancer Res. 1994;54:4726–4728. [PubMed: 8062271]
139.: Gore M, A’Hern R, Stankiewicz M, Slevin M. Tumour marker levels during marimastat therapy. Lancet. 1996;348:263–264. [PubMed: 8684209]
140.: Yanase T, Tamura M, Fujita K. et al. Inhibitory effect of angiogenesis inhibitor TNP-470 on tumor growth and metastasis of human cell lines in vitro and in vivo. Cancer Res. 1993;53:2566. [PubMed: 7684319]
141.: Ingber D, Fujita T, Kishimoto S, Sudo K. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth. Nature. 1990;348:555. [PubMed: 1701033]
142.: D’Amato R J, Loughnan M S, Flynn E, Folkman J. Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci USA. 1994;91:4082–4085. [PMC free article: PMC43727] [PubMed: 7513432]
143.: Soler R A, Howard M, Brink N S. et al. Regression of AIDS-related Kaposi’s sarcoma during therapy with thalidomide Clin Infect Dis 199623501–503.; discussion 504–505. [PubMed: 8879772]
144.: Bauer K S, Dixon S C, Figg W D. Inhibition of angiogenesis by thalidomide requires metabolic activation, which is species-dependent. Biochem Pharmacol. 1998;55:1827–1834. [PubMed: 9714301]
145.: Nussbaumer P, Winiski A P, Cammisuli S. et al. Novel antiproliferative agents derived from lavendustin A. J Med Chem. 1994;37:4079–4084. [PubMed: 7990108]
146.: Kozawa O, Shinoda J, Suzuki A. Genistein inhibits Ca2+ influx by extracellular ATP in PC12 pheochromocytoma cells. Horm Metab Res. 1995;27:272–274. [PubMed: 7557837]
147.: Hata A, Akita Y, Suzuki K, Ohno S. Functional divergence of protein kinase C (PKC) family members. J Biol Chem. 1993;268:9122–9129. [PubMed: 8473351]
148.: Dumont J, Jones W, Bitonti A. Inhibition of experimental metastasis and cell adhesion of V16F1 melanoma cells by inhibitors of protein kinase C. Cancer Res. 1992;52:1195–1200. [PubMed: 1737379]
149.: Liu B, Renaud C, Nelson K. et al. Protein kinase C inhibitor calphostin C reduces B16 amelanotic melanoma cell adhesion to endothelium and lung colonization. Int J Cancer. 1992;52:147–152. [PubMed: 1379995]
150.: Stanwell C, Gescher A, Bradshaw T, Pettit G. The role of protein kinase C isoenzymes in the growth inhibition caused by bryostatin 1 in human 549 hung and MCF-7 breast carcinoma. Int J Cancer. 1994;56:585–592. [PubMed: 8112895]
151.: Kiley S C, Clark K J, Goodnough M. et al. Protein kinase C delta involvement in mammary tumor cell metastasis. Cancer Res. 1999;59:3230–3238. [PubMed: 10397270]
152.: Schwartz G K, Redwood S M, Ohnuma T. et al. Inhibition of invasion of invasive human bladder carcinoma cells by protein kinase C inhibitor staurosporine. J Natl Cancer Inst. 1990;82:1753–1756. [PubMed: 2231770]
153.: Gescher A. Analogs of staurosporine: potential anticancer drugs? Gen Pharmacol. 1998;31:721–728. [PubMed: 9809468]
154.: Schwartz G K, Jiang J, Kelsen D, Albino A P. Protein kinase C: a novel target for inhibiting gastric cancer cell invasion. J Natl Cancer Inst. 1993;85:402–407. [PubMed: 8433394]
155.: Schwartz GK, Ward D, Saltz L, et al. A Phase 1 study of the protein kinase c (pkc) specific inhibitor safingol (Saf) alone and in combination with doxorubicin (Dox) [meeting abstract]. Proc Annu Meet Am Soc Clin Oncol 1995;14:.
156.: Cole K A, Kohn E C. Calcium-mediated signal transduction: biology, biochemistry, and therapy. Cancer Metastasis Rev. 1994;13:33–41. [PubMed: 8143344]
157.: Kohn E C, Felder C C, Jacobs W. et al. Structure-function analysis of signal and growth inhibition by carboxyamido-triazole, CAI. Cancer Res. 1994;54:935–942. [PubMed: 8313384]
158.: Kohn E C, Jacobs W, Kim Y. et al. Calcium influx modulates expression of matrix metalloproteinases-2 (72-kDa type IV collagenase, gelatinase A). J Biol Chem. 1994;269:21505–21511. [PubMed: 8063786]
159.: Kohn E C, Sandeen M A, Liotta L A. In vivo efficacy of a novel inhibitor of selected signal transduction pathways including calcium, arachidonate, and inositol phosphates. Cancer Res. 1992;52:3208–3212. [PubMed: 1591730]
160.: Felder C C, Ma A L, Liotta L A, Kohn E C. The antiproliferative and antimetastatic compound L651582 inhibits muscarinic acetylcholine receptor-stimulated calcium influx and arachidonic acid release. J Pharmacol Exp Ther. 1991;257:967–971. [PubMed: 1646332]
161.: Gusovsky F, Lueders J E, Kohn E C, Felder C C. Muscarinic receptor-mediated tyrosine phosphorylation of phospholipase C-gamma. An alternative mechanism fo cholinergic-induced phophoinositide breakdown. J Biol Chem. 1993;268:7768–7772. [PubMed: 7681827]
162.: Alessandro R, Spoonster J, Wersto R P, Kohn E C. Signal transduction as a therapeutic target. Curr Top Microbiol Immunol. 1996;213:167–188. [PubMed: 8815004]
163.: Luzzi KJ, Varghese HJ, MacDonald IC, et al. Inhibition of angiogenesis in liver metastases by carboxyamidotriazole (CAI): computer assisted analysis. Angiogenesis 1999. [PubMed: 14517457]
164.: Kohn E C, Reed E, Sarosy G. et al. Clinical investigation of a cytostatic calcium influx inhibitor in patients with refractory cancers. Cancer Res. 1996;56:569–573. [PubMed: 8564973]
165.: Kohn E C, Figg W D, Sarosy G A. et al. Phase I trial of micronized formulation carboxyamidotriazole in patients with refractory solid tumors: pharmacokinetics, clinical outcome, and comparison of formulations. J Clin Oncol. 1997;15:1985–1993. [PubMed: 9164210]
166.: Berlin J, Tutsch K, Hutson P. et al. Phase I clinical and pharmacokinetic study of oral carboxyamidotriazole, a signal transduction inhibitor. J Clin Oncol. 1997;15:781–789. [PubMed: 9053505]
167.: Gibbs J B, Oliff A, Kohl N E. Farnesyltransferase inhibitors: ras research yields a potential cancer therapeutic. Cell. 1994;77:175–178. [PubMed: 8168127]
168.: Gibbs J B. Ras C-terminal processing enzymes—new drug targets? Cell. 1991;65:1–4. [PubMed: 2013090]
169.: Predergast G C, and Gibbs J B. Pathways of Ras function: connections to the actin cytoskeleton. Adv Cancer Res. 1993;62:19–64. [PubMed: 8109319]

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