Invasion and Metastasis

Generation of a Metastatic Cell

Metastasis is defined as the dissemination of neoplastic cells to discontiguous nearby or distant secondary sites where they proliferate to form a mass. But how did tumor cells acquire the ability to metastasize? The answer to this question requires examination of the mechanisms underlying how tumors arose and progressed toward increasingly aggressive behavior.

By the time a neoplasm is diagnosed, it comprises at least 10 ⁹ cells. Yet, even cursory examination of a tumor histologically reveals that the cells are pleiomorphic. Furthermore, if one isolates single cell clones from a tumor, they vary dramatically in terms of biological behavior.

Tumor heterogeneity exists for virtually every phenotype measured. There are three types of heterogeneity within a tumor: positional, temporal, and genetic. Positional heterogeneity is determined by the accessibility of a cell to external stimuli (e.g., oxygen [O ₂ ] levels). For example, radiation sensitivity is proportional to oxygenation; therefore, two identical cells would exhibit differences in radioresponse depending on distance from a capillary. Temporal heterogeneity is relevant with regard to changes in cells due to cyclical signals. Cells in the G ₀ /G ₁ phase of the cell cycle would be less sensitive than cells in the S phase to drugs targeting DNA replication. Genetic heterogeneity is the result of inherent properties of tumor cells themselves. Isolation of single-cell clones confirms that there are inherent differences between subpopulations comprising a single tumor mass.

The heterogeneity of tumors raises an important question regarding tumor origin: Are tumors of unicellular or multicellular origin? Tumors express maternal or paternal isoenzymes, but rarely both, strongly suggesting that they arose from a single cell. Analysis of karyotypes reveals that virtually all cells within a tumor share a common abnormal chromosomal change (e.g., all CML cells have t[9;22]). Additional karyotypic abnormalities may be superimposed on the shared ones. If tumors are monoclonal, how, then, does heterogeneity arise?

The generation of heterogeneity requires divergence of single transformed cells into multiple phenotypically distinct progeny. The process appears to be fundamental to tumor progression, but it also occurs in normal physiology. For example, pluripotent hematopoietic stem cells can generate cells along multiple lineages, and a single fertilized egg yields a multicellular organism with organs and tissues. Although stem cell theory accommodates diversification, the molecular mechanisms underlying differentiation and diversification of both normal and cancer cells are still being elucidated (see Chapter 10 ). The journey of a metastatic cell is described in the next sections.

Tumor Invasion

Tumor invasion, the capacity for tumor cells to disrupt the basement membrane and penetrate underlying stroma, is the distinguishing feature of malignancy. Invasion requires major changes in cell morphology and phenotype, in particular for epithelial cells that represent the precursors to over 90% of human cancers. Normal epithelial cells form polarized sheets maintained by tight junctions, adherens junctions that organize the actin (microfilament) and tubulin (microtubule) cytoskeleton, and desmosomes attached to keratin-containing intermediate filaments. They are anchored to the basement membrane by hemidesmosomes and their associated intermediate filaments and integrin contacts that organize actin. Invasion requires alterations in cell-cell and cell-matrix adhesion, coordinated with matrix degradation and cellular motility ( Figure 18-2 ). The structural and regulatory proteins that control cell adhesion and migration are key downstream targets of oncogene and tumor suppressor-controlled signaling pathways, providing insights into how oncogenic transformation results in progression to an invasive phenotype. An interesting observation has been that many of the molecules implicated in tumor invasion also affect other processes involved in tumor progression, including cell survival, growth, apoptosis, and angiogenesis, highlighting the intricacy of the network of interrelated pathways that controls cellular behavior.

Adhesion

Invasion of epithelial cell–derived carcinomas often involves dramatic changes in cell shape. Conversion from an epithelial morphology to a nonpolarized, motile, spindle-shaped cell resembling a fibroblast is referred to as the epithelial-mesenchymal transition (EMT). EMT is characterized by the loss of epithelial-specific E-cadherin from the adherens junctions and a switch from the expression of keratins as the major intermediate filament to the mesenchymal intermediate-filament vimentin. EMT is not cancer specific; it is a normal process that occurs during embryonic development and wound healing. EMT is influenced by the tumor microenvironment and is observed primarily at the edge of the tumor in contact with tumor stroma. Soluble factors, in particular transforming growth factor-β and hepatocyte growth factor/scatter factor, are regulators of EMT. Tumor cells may reverse the process and undergo a mesenchymal-epithelial transition (MET) in the absence of EMT-inducing signals. The transient nature of EMT helps explain why metastatic cells can morphologically resemble cells in the primary tumor despite the fact that they by necessity have accomplished all the steps of the metastatic cascade.

Epithelial cell-cell interactions are mediated primarily by cadherins, transmembrane glycoproteins that form calcium-dependent homotypic complexes. The epithelial-specific cadherin, E-cadherin, functions as a tumor suppressor and a metastasis suppressor. Loss of E-cadherin correlates with increased invasion and metastatic potential in most tumor types. Reexpression of E-cadherin in experimental models can block invasion, suggesting that E-cadherin loss is indeed causative. Loss of E-cadherin in cancer occurs through several mechanisms, including transcriptional repression and proteolytic degradation. The zinc finger transcriptional repressors Snail and Slug, in particular, have been implicated in regulating EMT by virtue of their ability to repress E-cadherin transcription. Cadherins are regulated by catenins (α-, β-, γ-, and p120 catenins), cytoplasmic proteins that functionally link the cadherin complex to the actin cytoskeleton. β-Catenin is a cell adhesion protein and a transcription factor. In addition to its role in adherens junctions, it participates in canonical Wnt signaling, a signaling pathway important in development and cancer. E-cadherin levels and function are also disrupted by loss of p120 catenin, which occurs in many tumor types and may also contribute to tumor metastasis.

Loss of function of cell-cell adhesion molecules other than E-cadherin is associated with the ability of tumor cells to invade and metastasize. Neural cell adhesion molecule (NCAM), a member of the immunoglobulin-like cell adhesion molecule Ig-CAM family, is downregulated in several tumor types, and NCAM loss results in an increased ability of tumor cells to disseminate. Other Ig-CAMs, such as DCC (deleted in colorectal carcinoma), CEACAM1 (carcinoembryonic antigen CAM1), and Mel-CAM (melanoma-CAM), also demonstrate reduced expression in specific cancer types. However, not all cell-cell adhesion molecules can be viewed as potential invasion suppressors. N-cadherin promotes motility in some cell types, and Ig-CAMs such as L1, CEA (carcinoembryonic antigen), and ALCAM (activated leukocyte CAM) are often overexpressed in advanced cancers and have functions associated with cancer progression. This complexity may be explained by signaling functions for these molecules, either direct or indirect, that are distinct from their role in cell-cell adhesion. The interrelatedness of tumor growth and tumor invasion, and limitations of experimental model systems, often does not allow a distinction between growth effects that influence the appearance of an invasive phenotype and an effect on cellular invasion per se.

The extracellular matrix (ECM) provides a scaffold for the organization of cells and spatial cues that dictate cell behavior. The ECM is composed of proteins, primarily triple-helical collagens, glycoproteins such as laminins and fibronectin, and proteoglycans. The basement membrane is an organized ECM that separates polarized epithelial, endothelial, and muscle cells from the underlying tissue. Interstitial matrix provides the structure characteristic of connective tissues. The molecular composition of the ECM varies between tissues and organs and provides contextual information to cellular constituents. In addition, the ECM serves as a repository for secreted regulatory proteins and growth factors. Finally, ECM proteins themselves can be active signaling molecules, activities that frequently are only revealed after proteolysis reveals cryptic sites. Thus, the interaction of cells with ECM molecules determines their capacity for survival, growth, differentiation, and migration.

Cells adhere to ECM via integrins, a family of transmembrane glycoproteins assembled as specific combinations of 18 α and 8 β subunits. Integrins bind to distinct but overlapping subsets of ECM components. During tumor progression, cancer cells tend to undergo a switch in their integrin expression pattern, downregulating the integrins that mediate adhesion and maintain a quiescent, differentiated state, and expressing integrins that promotes survival, migration, and proliferation. Although there is a cell-type dependency on integrin function, in general integrins α ₂ β ₁ and α ₃ β ₁ are viewed as suppressors of tumor progression, whereas α _v β ₃ , αβ ₆ , and α ₆ β ₄ promote cellular proliferation and migration. Integrins mediate both “outside-in” and “inside-out” signaling, so that changes in cellular adhesion can alter cellular phenotype, and changes in intracellular signaling pathways can modulate cellular adhesion. A well-described and important mechanism whereby integrin-ECM interactions modulate cell function is by cooperative signaling with different growth factor receptors. Many of the cellular responses induced by activation of tyrosine kinase growth factor receptors are dependent on the cells being able to adhere to an ECM substrate in an integrin-dependent fashion. Signaling in response to ECM ligation usually activates focal adhesion kinase (FAK) and nonreceptor tyrosine kinases of the src family.

Matrix Degradation

Disruption of basement membrane is a hallmark of malignancy. Degradative enzymes produced by the tumor cells, and by resident and infiltrating cells as a response to the tumor, contribute to matrix degradation and facilitate tumor cell invasion. Proteolytic enzymes of many classes have been implicated in tumor cell invasion, including the serine proteinases plasmin, plasminogen activator, seprase, hepsin, several kallikreins, the cysteine proteinase cathepsin-B, the aspartyl proteinase cathepsin-D, and metal-dependent proteinases of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) families. Other matrix-degrading enzymes such as heparanase, which cleaves heparin sulfate proteoglycans, and hyaluronidase cleavage of its substrate hyaluronic acid have also been causally associated with tumor progression and invasion.

Liotta and colleagues observed that metastatic potential correlates with the degradation of type IV basement membrane collagen and focused attention on the metal-dependent gelatinases. These enzymes are now recognized as MMP2 and MMP9, and many of the 23 members of the MMP family of matrix-degrading metalloproteinases have been associated with tumor progression. Elevated MMP levels correlate with invasion, metastasis, and poor prognosis in many cancer types, and animal models provide evidence for a causal role for MMP activity in cancer progression. The plasminogen activator/plasmin system has also been causally implicated in cancer invasion, and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are validated prognostic and predictive markers for breast cancer.

The regulation of matrix proteolysis is complex and can involve the concerted action of multiple proteinases and proteinase classes from both tumor cells and adjacent resident and infiltrating cells ( Figure 18-3 ). The conversion of pro-MMP2 to active MMP2 requires membrane-type MT1-MMP (MMP14), a transmembrane MMP that is activated intracellularly by the proprotein convertase family member, furin. There is evidence for a cascade of cathepsin-D–cathepsin-B–uPA–plasmin–MMP activation that results in activated enzymes capable of degrading all components of the ECM. Proteolysis is also regulated by the production of specific endogenous protease inhibitors, including the tissue inhibitors of metalloproteinases (TIMPs), serine proteinase inhibitors (serpins), and cysteine protease inhibitors (cystatins). These inhibitory activities are produced and secreted by tumor or stromal cell types, and some proteinase inhibitors are stored in high concentrations in the ECM. Proteinase activity cascades can function via proteolytic degradation of some of these proteinase inhibitors in addition to activation of other proteinases.

The original view that proteolytic enzymes function predominantly to remove physical ECM barriers has been expanded with the realization that proteolysis is a key regulator of multiple steps of tumor progression. For example, MMP substrates in the matrix or on the cell surface that modulate cellular growth, differentiation, apoptosis, angiogenesis, chemotaxis, and migration have been identified. The abundant evidence for a role for MMPs in tumor progression led to the design and testing of synthetic MMP inhibitors for cancer therapy. These inhibitors proved to be ineffective in clinical trials, results that have been explained by problems with inhibitor or clinical trial design and a lack of understanding of the broad range of MMP activities resulting in both cancer-promoting and cancer-inhibitory effects.

Motility

Cellular locomotion occurs as the result of coordinated polymerization and depolymerization of the actin cytoskeleton to extend a pseudopod at the leading edge of the cell, followed by contraction associated with disassembly of cell-matrix adhesive contacts at the trailing edge. Lamellipodial protrusions at the leading edge are nucleated by a branched actin network involving the Arp2/3 complex and its regulators, the WASp (Wiskott-Aldrich syndrome protein) family, cortactin, and the GTPase Rac. Actin contractility is regulated by myosin light-chain kinase and upstream small GTPases, in particular Rho and its effector Rho-kinase (ROCK). Single cells migrate with a spindle-shaped morphology, referred to as mesenchymal migration , or with the less adhesive ellipsoid shape used by leukocytes and Dictyostelium termed amoeboid migration ( Figure 18-4 ). Collective migration can occur when the cells retain cell-cell junctions and clusters of cells move in single file through a tissue.

Tumor cells can secrete factors that stimulate motility in an autocrine fashion. Tumor cell–produced lysophospholipase D (autotaxin) stimulates motility, as does lysophosphatidic acid (LPA), which can be produced by lysophospholipase D activity on lysophosphatidylcholine. Hepatocyte growth factor/scatter factor (HGF/SF) interacts with its receptor, c-met, to induce chemokinetic activity of epithelial cells, resulting in an invasive phenotype. Directional motility is a chemotactic or haptotactic effect in response to a gradient of soluble or localized factors, respectively. Chemotaxis is often the result of growth factors such as IGF, and chemokines of the CCR and CXC family. Haptotaxis is characterized as a response to gradients of ECM components such as laminin-5 and fibronectin and can be modulated positively or negatively by proteolysis.

Coordination of Cancer Invasion

The coordination of cell-cell and cell-matrix adhesion, matrix degradation, and cytoskeletal activity is required for cellular invasion. The type of cell migration (i.e., collective, mesenchymal, or amoeboid) is influenced by the relative levels of adhesion mediated by cadherins and integrins, proteolytic activity, and actin contractility. Modulation of any of these factors can convert one type of motility into another.

Invadopodia is the name that has been given to structures identified in invading cells that represent the physical convergence of the adhesive, proteolytic, and motility components of invasion ( Figure 18-5 ). Invadopodia are actin-rich organelles that protrude from the plasma membrane and contact and locally degrade the ECM. Invadopodia contain adhesion molecules, including several β1 integrins and CD44, the serine proteinases seprase and dipeptidyl dipeptidase IV, and several MMP and ADAM metalloproteinases. Inside the plasma membrane, invadopodia contain actin and actin assembly molecules and multiple signaling molecules including focal adhesion kinase (FAK), src- associated proteins such as p130Cas and Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains), and the small GTPases cdc42, Arf1, and Arf6. Thus, invadopodia are implicated as key cellular structures that are used to coordinate and regulate the various components of the process of cancer invasion.

The Metastatic Cascade

Although invasion is required for metastasis, the ability to invade is not sufficient for metastasis (see Figure 18-1 ). Some tumors are highly aggressive, forming secondary lesions with high frequency (e.g., small-cell carcinoma of the lung, melanoma, pancreatic carcinoma), whereas others are rarely metastatic despite being locally invasive (e.g., basal cell carcinomas of the skin, glioblastoma multiforme). Fidler and colleagues have proposed an analogy regarding metastasis that is highly illustrative. Metastatic cells are likened to athletes participating in the decathlon. Each cell must be capable of completing every step of the metastatic cascade. If a cell cannot complete any step, it cannot go on to subsequent steps and cannot form a metastasis.

Metastasis is primarily thought of as developing via dissemination in the bloodstream, although other routes of spread occur. Carcinoma cells tend to escape and spread initially to draining lymph nodes, becoming trapped and proliferating. The thoracic duct links the lymphatic system to the bloodstream, connecting lymphatic to hematogenous spread. Metastases can also develop by spreading across body cavities. For example, ovarian carcinoma cells most frequently establish secondary tumors by dissemination in the peritoneum while rarely forming metastases via hematogenous spread. Other routes of spread also exist but are far less common (e.g., dissemination of melanoma cells along the space between endothelium and basement membrane or perineural spread in pancreatic and prostatic carcinomas). Thus, the route of dissemination is not inherent to a definition of metastasis.