The basement membrane matrix in malignancy

Authors

  • Jean A Engbring,

    1. Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland, USA
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  • Hynda K Kleinman

    Corresponding author
    1. Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland, USA
    • Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, 30 Convent Dr, MSC 4370, Bethesda, MD 20892-4370, USA.

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Abstract

Cancer is the second most common cause of death among Americans, although for several age groups it ranks first. Most of these deaths are not due to the primary tumour but rather to tumour cell metastases to distant organs. There are many steps that lead to metastasis, all of which are being studied with the goal of preventing these fatalities. Normally, cells attach to the extracellular matrix to maintain tissue integrity. During cancer progression, cells become more motile and acquire invasive qualities. Tumour cells move along blood and lymph vessels or invade into them to travel to distant sites. Then, the tumour cells must attach to the vessel wall, extravasate from the vessel, invade the new tissue, proliferate, and form a secondary tumour. Angiogenesis, the formation of new blood vessels, is critical to survival of these cells at the new site and is also important for primary tumour growth and spread. Tumour cell metastasis is a complex cascade of sequential steps, each of which is not yet fully understood. Progress has been made in identifying several key activators, one of which is the extracellular matrix. A major tumour promoter is the glycoprotein laminin, which is predominantly found in the extracellular matrix produced by endothelial and epithelial cells. This review will follow the metastatic process with particular attention to the effect of laminin on tumour cells. Copyright © 2003 John Wiley & Sons, Ltd.

Basement membrane and Matrigel

A major barrier to tumour cell extravasation and invasion is the basement membrane extracellular matrix, which underlies the endothelium of the vessel wall. This thin extracellular matrix is encountered twice during metastases 1, 2. Proteases specific for the basement membrane are important in invasion since inhibitors of these proteases block metastases in experimental models 3, 4. Invasion requires cell adhesion, migration, and protease activity. Very few cells accomplish invasion, but those that do are more aggressive in their growth and in their ability to generate additional metastases. During new blood vessel formation (angiogenesis), degradation of the basement membrane also occurs as the first step, followed by migration of endothelial cells. The degradation of the basement membrane during tumour invasion into tissues and during angiogenesis likely releases active molecules and active fragments of matrix components which promote tumour cell growth, spread, and angiogenesis.

The major components of the basement membrane include laminin, collagen IV, perlecan (a heparan sulphate-containing proteoglycan), entactin, and various growth factors and proteases, all of which promote malignancy and/or angiogenesis 5. The amount and type of these components vary depending on the stage of development and the tissue type. Additional components are found in certain basement membranes, and many of these are tissue specific. These components interact with each other to form the matrix. Many growth factors and cytokines are ‘stored’ in the basement membrane matrix and are only released upon its dissolution 6. Many are stored in inactive forms that are active when released.

Matrigel is an extract of basement membrane derived from a murine tumour 7. The components of this tumour basement membrane are identical, both chemically and immunologically, to authentic basement membrane components. Using Matrigel as a barrier for tumour cell invasion, a number of inhibitors and stimulators of invasion have been identified 8. Of greatest interest are those inhibitors of invasion that have the potential to be developed as clinical therapeutics. Furthermore, when tumour cells and basement membrane (Matrigel) are subcutaneously co-injected into mice, the incidence of tumour take and growth are greatly increased, due in part to angiogenesis 9. Thus, basement membranes are not just support structures or barriers to cell invasion, but also impart functional signals to tumour cells.

Laminin

Laminin was first described in 1979 as a large (Mr = 800 000) basement membrane-derived glycoprotein consisting of three chains held together by disulphide bonds. Since then, several isomeric forms, which now form the laminin family, have been identified 10. Important biological functions have been identified for several of the isoforms. At present, five α, three β, and three γ chains have been described, which are assembled into at least 12 distinct isoforms of laminin, with laminin-1 being composed of α1β1γ1 chains, laminin-2 composed of α2β1γ1 chains, etc. These laminin isoforms have different tissue- and development-specific localizations, suggesting important functions. For example, laminins-8 and -10 (α4β1γ1 and α5β1γ1) have been described in the endothelial cell basement membrane, while laminin-2 is localized in muscle basement membrane 10, 11.

Laminins are very biologically active and have been found to promote cell adhesion, migration, protease activity, proliferation, tumour growth, angiogenesis, and metastasis 2, 10. Laminin increases the malignant phenotype by inducing proteases in tumour cells. It also promotes tumour cell adhesion and migration. Laminin adhesion-selected tumour cells are more tumorigenic and more malignant than either the parental cells or fibronectin adhesion-selected cells 12. More than 20 cell surface receptors have been identified for laminin, including integrins, a 32/67 kDa protein, proteoglycans, sulphatides, gangliosides, amyloid precursor protein, lectins, and galactosyltransferases 13. The specificity of the cellular response is dependent on the cell type, the receptor, and the laminin isoform. Less is known about the signalling pathways, but these are also critical to the active cellular response.

Several active sites on laminin-1 with various biological activities have been identified using proteolytic fragments, recombinant proteins, and synthetic peptides. Multiple sites have been found that promote tumour growth, angiogenesis, and metastasis. YIGSR (amino acids 828–933 on the β1 chain) inhibits melanoma lung cell colonization, solid tumour growth, and angiogenesis via the 32/67 kDa receptor (Figure 1) 14–16. Melanoma cells selected for adhesion to the YIGSR peptide form larger and more numerous tumours when injected into the tail vein, indicating the importance of the 32/67 kDa cellular receptor in malignancy 17. SIKVAV (amino acids 2099–2104 on the α1 chain) promotes malignancy via increased proteases and increased angiogenesis. This sequence can enhance plasminogen activator 22-fold 18–21. VAYI (amino acids 127–130 on the α1 chain) and the homologous peptide YVRL (amino acids144–147 on the γ1 chain) promote angiogenesis and tumour growth via integrins αvβ3 and α5β1 22–24. LQVQLSIR (amino acids 2623–2630 on the α1 chain) promotes metastasis (Figure 1) 25. This sequence recognizes cell surface proteoglycans, including syndecan-1 and a chondroitin–heparan sulphate proteoglycan on melanoma cells 26. Thus, multiple active sequences on laminin have been found that promote the malignant phenotype and regulate angiogenesis.

Figure 1.

Schematic of laminin-1 showing active sites

Genes

Certain genes have been implicated in the transition of normal cells to the metastatic phenotype. Several of these genes encode extracellular matrix proteins. An increase in fibronectin expression is correlated with metastasis and tumorigenesis. Also, expression of the Gla protein, collagen subunits α2(I) and α1(III), is elevated in highly metastatic cells 27. Several genes are involved in attachment to the basement membrane, the loss of which leads to tumour cell invasion and metastasis. For example, a decrease in expression of Dab2, a candidate tumour suppressor of ovarian cancer, is associated with a reduction of laminin in the basement membrane 28, 29. Loss of function of BRCA1, another tumour suppressor, leads to ovarian and breast cancer, and laminin 3A expression is associated with breast cancer and BRCA1 30. In addition, downregulation of E-cadherin has been linked to tumour invasion and metastasis 31, 32. Alternatively, high levels of hepatocyte growth factor (HGF) and the receptor Met are associated with tumour cell invasion 33. Overexpression of the laminin γ2 chain in tissues is considered a marker of invasion and metastasis 34. The laminin γ2 chain is cleaved by MMP-2, exposing a cryptic site important for cell migration 35, 36. Genes involved in invasion are also altered during metastasis. Matrix metalloproteinases (MMP) -1, -2, -9, and MT1 MMP are increased in aggressive carcinomas 37. Finally, angiogenesis is influenced by differential expression of genes by tumour cells. HGF, which is elevated in metastatic cells, promotes expression of angiopoietin, vascular endothelial growth factor (VEGF), and VEGF-C, each of which increases angiogenesis 38. Furthermore, HGF can also induce angiogenesis independently of VEGF 39, 40. These changes in gene expression allow tumour cells to become more malignant and to survive as metastatic lesions.

Invasion

The basement membrane is a barrier to tumour cell metastasis, separating the epithelium from connective tissue and the vascular endothelium. Remodelling, or loss of the basement membrane, is required for tumour cells to reach vessels, and thus allow for access to distant organs. This involves upregulation of proteinases that act on various components of the ECM. MMPs are a family of zinc-dependent endoproteinases whose activity is directed against the ECM 41. Most MMPs are produced by fibroblasts, inflammatory cells, and endothelial cells surrounding tumours. They are capable of degrading ECM. MMP-1, -2, and -9 have been shown by cDNA microarray to be increased in aggressive compared to non-aggressive melanomas 37. Also, type IV collagenase activity in melanoma and fibrosarcoma cells is upregulated by intact laminin 42. Expression of plasminogen activators, which are involved in activating matrix-degrading proteinases, is regulated by laminin-1 43. Laminin-5 normally promotes static adhesion and hemidesmosome formation between cells; however, MMP-1, -2, and MT1-MMP cleave laminin-5, and a resulting fragment stimulates cell migration and invasion 36, 44. Pancreatic tumour cells secreting laminin-5 have increased levels of surface β4 integrins that participate in hemidesmosome reorganization to form invading edges of malignant epithelial carcinomas 45. Laminin-6 may also play a role in tumour cell invasion. The G4-5 domain suppresses cell adhesion, but when released from the intact structure this region promotes adhesion 46. In addition, the expression and function of cell adhesion molecules termed cadherins may be altered, leading to metastasis 32. Invasion through the ECM via these proteinases and alteration of cell–cell and cell–matrix adhesion proteins allow for cells to contact blood and lymph vessels.

Migration

Tumour cells must travel to distant sites to establish secondary tumours. These cells are generally thought to enter the circulation and extravasate, often in an organ-specific manner 47. Tumour cells migrate both on extracellular matrices (haptotaxis) and to components of the extracellular matrix (chemotaxis). Tumour cells generally interact with the extracellular matrix via integrins, which provide both the connection to the adhesive substrate and signalling to the cytoskeleton necessary for movement. Tumour cells encounter different types of extracellular matrices during metastasis and can adapt with different receptors and proteases to accomplish migration. Growth factors and proteases are generally associated with tumour cell motility 48. Growth factors are thought of as the chemotactic signals, although matrix molecules and their fragments also promote migration. Proteases are required to allow invasion of tissues. It is of interest to note that proteases and integrins have been found to co-localize on the cell surface 49, 50. Such migration/invasion can in some cases result in the release and/or generation of active migration stimuli 51. Recently, it has been shown that proteases may not be required for migration in tissues. If the tumour cell proteases are inhibited, tumour cells can still migrate effectively 50. Here, the tumour cells adopt a more amoeboid-type shape and squeeze through the gaps in the tissue matrices. Such plasticity of tumour cell movement is key to the metastatic process.

In addition to intravasation and extravasation from vessels as a process during tumour spread, it has recently been observed that tumour cells can also migrate along the outside of vessels to form distant metastases. This migration is observed both in vivo and in vitro and parallels glioma migration along nerve tracks in the brain. The β2 chain of laminin has been found at the interface of the tumour cells and vessels and appears to play a role in extravascular cell migration 52. An increase in laminin expression may facilitate migration along the endothelial cells of these vessels, since tumour cells associate with endothelial cells predominantly through laminin 53.

Attachment

Once the tumour cells arrive at the new organ they must attach to the basement membrane in order to establish a secondary tumor. Attachment to many of the same cell adhesion molecules that were involved in detachment prior to tumour cell invasion and migration now is critical for tumour cell survival at the new site. Many different cell surface receptors that interact with laminin, including integrins, have been identified and/or characterized.

Integrins are a family of transmembrane glycoproteins composed of α and β subunits that, when activated, trigger signalling pathways to regulate cell adhesion and movement. These proteins connect the cytoskeleton to the extracellular matrix. They are widely distributed and interact with many ligands, including laminin. The α3, α6, β1, and β4 subunits have been connected to metastasis, in particular, α3β1, α6β1, and α6β4 alone and in combination. The α3β1 and α6β4 integrins are probable receptors for laminin-5 and are involved in tumour cell proliferation and invasion 54. The α6β1 and α6β4 integrins are implicated in breast cancer progression, including invasion and cell survival 55. Also, lamellipodia and filopodia, which regulate motility, are associated with α6β4 56.

Tumour cells also attach to the extracellular matrix via the 32/67 kDa laminin receptor, which is implicated in many cancers including breast, lung, ovary, prostate, and lymphomas. This protein acts as an integrin accessory molecule to stabilize tumour cell binding to laminin through integrins 57. Increased expression of this receptor correlates with increased malignancy, and the binding site on laminin has been identified as the peptide YIGSR 2.

As expected, other proteins are involved in adherence to the extracellular matrix, some of which are heparan sulphate proteoglycans. For instance, downregulation of syndecans correlates with transformation to a tumorigenic phenotype 58. CD44, a hyaluronan receptor, is associated with homing of lymphocytes to lymph nodes 59. Galactosyltransferases, α-dystroglycan, and a heparan sulphate/chondroitin sulphate-containing proteoglycan have also been shown to be important in tumour metastasis 13, 26. Tumour cell attachment to the extracellular matrix through a variety of ligands aids in the metastatic process.

Angiogenesis

The extracellular matrix is vital for the maintenance (scaffold) and differentiation of many cell types including the endothelium, and it plays a role during the formation of new vessels from pre-existing ones in a process known as angiogenesis 60. The endothelial cells of blood vessels generally remain in a quiescent state. While neovascularization in physiological processes is finely tuned by a balance of stimulatory and inhibitory factors, persistent and unregulated growth of new capillaries plays a prominent role in the development of pathological processes such as tumour growth. The angiogenic signal activates the endothelium and elicits a cascade of events that leads to the formation of new vessels. One of the first steps of this cascade is the degradation of the extracellular matrix, followed by cell migration and proliferation. These events are common to tumour growth and angiogenesis and many, but not all, of the factors which regulate angiogenesis also regulate tumour growth 61. The basement membrane is biologically active with endothelial cells and Matrigel has been shown to promote capillary-like formation of endothelial cells in vitro 62. Since the basement membranes serve as a storage depot for a large number of angiogenic growth factors, such as bFGF and EGF, the degradation of the matrix leads to the release of angiogenic factors that can in turn promote angiogenesis 63. Several extracellular matrix protein fragments have been identified to modulate angiogenesis 64. For example, a 25 kDa thrombospondin fragment has been shown to promote angiogenesis, whereas several other ECM fragments inhibit it, including endostatin (derived from collagen XVIII), angiostatin (from plasminogen), the NCI domains of collagen IV, several thrombospondin peptides and endorepellin (derived from the C-terminus of perlecan) 65–70. Fragments and synthetic peptides derived from laminin have also been found to regulate angiogenesis 43. Laminin is present in the endothelial basement membrane, and more than 20 peptides from laminin-1 that can promote angiogenesis in vivo have been identified 22, 23. Interestingly, several of these have been found to also increase tumour growth, either by promoting angiogenesis or by having a direct effect on the tumour cells, affecting both endothelial and tumour cells. For example, initial studies identified the IKVAV (ile-lys-val-ala-val) sequence of the α1 chain as being active for angiogenesis and metastasis in mice and showed that the YIGSR (tyr-ile-gly-ser-arg) peptide from the β1 chain inhibited blood vessel formation and metastasis 14, 16, 18–21. Both of these peptides regulate tumour growth by direct effects on the tumour cells as well. Two of the most potent angiogenic sites, A13 (RQVFVAYIIIKA) and C16 (KAFDITYVRLKF), are redundant angiogenic sites present in homologous regions of the α1 and γ1 chains, respectively 22, 23. These sequences bind to the endothelial cell integrins αvβ3 and α5β1 24. Although A13 and C16 can bind to both of these integrins, neither one of the peptides contains the RGD sequence.

Degradation of the extracellular matrix is one of the first steps that occurs during tumour invasion and angiogenesis 71. During this process, laminin becomes cleaved, possibly allowing its angiogenic sequences, which might include many sites to become exposed, enabling them to induce an angiogenic response 35, 43, 72, 73. The γ1 chain, and therefore the active sequence within C16, is found in 10 of the 12 laminins known to date 74. This strongly suggests that the γ1 chain plays an important physiological role, since it is located in most tissues. The existence of cryptic sites with biological activity within larger molecules is not unusual 35. Altogether, these data suggest that angiogenesis is tightly regulated by several extracellular fine-tuning molecules, including growth factors, circulatory proteins, and extracellular matrix proteins present within the adjacent microenvironment.

Proliferation and growth

Finally, once the tumour cells arrive at the secondary site they must proliferate to form metastases. The extracellular matrix is a reservoir for many molecules, including growth factors, cytokines, and angiogenic factors. The use of Matrigel as an in vitro model of metastasis is widely accepted and demonstrates the tumorigenic effects on growth of the extracellular matrix (Table 1). Heparanase, an enzyme involved in release of proteins from the extracellular matrix, is preferentially expressed in tumours and may therefore aid in tumour growth 63. Also, the expression of laminin-5 is associated with proliferation of tumour cells. The α3β1 and α6β4 integrins on epithelial cells are putative receptors for laminin-5 and are involved in proliferation 54. However, the proteoglycan glypican 1 has been found to be a negative regulator of cell proliferation 58.

Table 1. Basement membrane Matrigel promotes B16F10 melanoma growth
Amount of Matrigel (mg/ml)Tumour volume (mm 3)
Day 12Day 20
  1. B16F10 melanoma cells (100 000 per mouse) were injected subcutaneously with the indicated amount of Matrigel diluted in serum-free DMEM in a final volume of 0.5 ml. Tumour volume (l × w) was monitored by caliper measurements.

  2. N = 6 per group.

None 4001050
0.1 9001600
1.215006050
2.517506800
5.020009050

Summary

Tumour cell malignancy leading to metastases involves many processes and molecules, some of which are coordinately regulated. Many genes are up- or downregulated that enable tumour cells to be more motile and invasive, allowing for translocation to, and growth in, distant organs. Since the extracellular matrix, in particular laminin, has such a profound influence on tumour cell metastasis, it continues to be an area of active research for understanding mechanisms of metastasis and for developing therapeutics.

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