cancer invasion: watch your neighbourhood!

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Tumori, 89: 343-348, 2003

CANCER INVASION: WATCH YOUR NEIGHBOURHOOD! Vito Quaranta1 and Gianluigi Giannelli2 1 Department of Cell Biology, The Scripps Research Institute, USA; 2Department of Internal Medicine, Immunology, and Infectious Diseases, Section of Internal Medicine, University of Bari Medical School, Bari, Italy

The critical event in neoplastic diseases is the invasion of surrounding tissue by cancer cells. This event greatly reduces treatment options, and makes cancer a lethal disease. Factors that initiate cancer invasion are not well understood, neither do we have mechanistic insights in the process itself. Recently, a new concept has emerged: the tissue surrounding tumor cells, ie, the tumor microenvironment, may play an important, if not decisive role in triggering invasion. This concept is based on data from many laboratories working on the cell biology of

cancer invasion. In this review, we survey several components of the tumor microenvironment, including extracellular matrix macromolecules, metalloproteinases and soluble factors, and discuss their potential involvement in stimulating cancer cell motility. These novel views may have far-reaching consequences, since “normal” tissue microenvironment components, rather than the traditional tumor cells themselves, may eventually become targets for devising new treatments that prevent, inhibit or block cancer invasion and metastasis.

Key words: cancer, extracellular matrix proteins, metastasis, microenvironment, tissue remodelling.

Introduction

Cancer metastasis is the most significant event influencing clinical outcome of patients with neoplastic disease. Its occurrence affects prognosis severely and dramatically reduces survival. No treatments to prevent or block cancer spread are available, possibly because the responsible mechanisms are still unclear. An important goal in this field is to understand these mechanisms at the molecular level. Histopathological features of tumors have been widely studied: while less differentiated tumors are commonly associated with worse prognosis, no strong correlations can be drawn with invasive behavior. Recently, there has been strong emphasis on identifying genes strictly related to, or possibly directly responsible for cancer invasion1. In spite of several promising candidates, results along these lines appear to have failed to fulfill expectations. The general consensus is now that cancer invasion is not necessarily based on the same mechanisms that lead to deregulation of growth or apoptosis, ie, accumulation of mutations in critical genes. Though this possibility has not been ruled out, invasion may indeed occur via epigenetic mechanisms, that is, inappropriate use of perfectly normal signaling pathways controlling cell motility2. Thus, the hope of identifying mechanisms of cancer invasion has not declined but rather a critical review of the literature has led to reconsideration of approaches to the problem. In the last few years, growing interest has been focused on the role of surrounding tissue, the tumor microenvironment, as a regulator of the epithelial mesenchymal transition (EMT), believed to be responsible for many invasive traits of tumor cells3,4. The term microenvironment refers to a varied mix of

different cell types (including fibroblasts, endothelial cells, tissue macrophages etc), extracellular matrix (ECM) proteins (including laminins, fibronectin, collagens, etc), growth factors and chemokines associated to ECM proteins. Tissue homeostasis, including processes of remodelling or neoplastic degeneration, represents the balance between the epithelial cells and the surrounding microenvironment. A modification of this balance can determine the acquisition of a malignant and aggressive phenotype by cancer cells. In fact, once cancer cells have lost their cell-cell contact and have crossed basement membrane (BM) structures, they are in a position to penetrate the surrounding microenvironment. During such movement, cancer cells become engaged with other cell types as well as with the ECM proteins present in the neighbouring tissues, and the result of this continuous cross-talk determines the ability of the tumor cells to penetrate through tissues and eventually metastasize in other organs. In this review we will consider how some of these microenviromental factors may trigger the invasive phenotype. Tissue remodelling and matrix metalloproteinases (MMPs)

MMPs are a family of zinc endopeptidases secreted as pro-enzymes, with proteolytic activity toward ECM components5. They have been implicated in the process of breaking down tissue boundaries leading to changes in histological borders. Such changes commonly occur in situation of tissue remodelling, such as in breast tissue during pregnancy, or uterine mucosa during the ovarian cycle6-11. MMPs have been implicated in cancer cell dissemination because of their ability to degrade

Correspondence to: Dr Gianluigi Giannelli, Dipartimento di Clinica Medica, Immunologia e Malattie Infettive, Clinica Medica “Cesare Frugoni”, Piazza G Cesare 11, 70124, Bari, Italy. Tel +39 (080) 5478-858; fax +39 (080) 5478-670; e-mail [email protected] Received April 2, 2003; accepted May 2, 2003.

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the ECM components of BM12,13. In the last few years, however, the role of MMPs in cancer invasion has been revisited: MMPs are no longer considered simply as molecular shears that cut the ECM molecules, opening spaces where cancer cells can penetrate, but rather as molecular tools able to rearrange the biochemical structure of ECM molecules, thus stimulating motility and invasion via mechanisms whose details remain to be elucidated14-17. Cleavage of ECM macromolecules by MMPs is kept in check by the presence of tissue inhibitors of MMPs (TIMPs), which presumably contain overly excessive and uncontrolled proteolysis12,18. Remodelling of the ECM is restricted to the pericellular space, so that MMPs are often activated and focalized at cellular protrusions named invadopodia, where other classes of proteases such as serin protease uPA and bone morphogenetic protein-1 type metalloproteinases have also been detected19-21. Whatever the cell type responsible for the production of proteinases, they are frequently concentrated along the advancing edge of different types of cancer, as reported by a number of studies22,23. This leaves a number of questions still open, such as which receptors are in charge of concentrating and focalizing the floating proteases. Membrane type-1 MMP (MT1-MMP) is a transmembrane MMP expressed on the cellular surface that binds TIMP-2, and this complex binds and activates MMP-2. MT1-MMP is unique in this sense, since it is connected with the intracellular space because of its intra-cytoplasmic tail, it has a true proteolytic activity toward ECM proteins and, finally, it has a function in the activation mechanism for MMP224,25. The integrins, a class of transmembrane receptors known to bind ECM macromolecules have also been implicated in MMP mediated mechanisms of cancer spread. In particular, αVβ3 integrin has been shown to bind MMP-2 with a high affinity and to direct this MMP along the advancing edge of cancer cells23. Lately, α3β1 integrin has been shown to be implicated in the activation of MMP-9 and MMP-2 in breast and liver cancer, respectively26,27. It has therefore been proposed as a potential MMP focusing receptor, although no data have so far been reported to indicate that this integrin can directly bind MMP-2 and/or MMP-9. In conclusion, MMPs could have an essential role in changing the structure and consequently the biochemical functions of ECM macromolecules. This may alter interactions between cancer cells and the microenvironment, possibly pushing the balance in favor of cancer invasion. While MMPs are perhaps the best understood proteinases in this context, much still remains to be investigated, and it is virtually certain that other classes of proteinases also play a role in these mechanisms. ECM macromolecules and integrins

BMs are a dominant theme in tissue organization, particularly epithelia, muscle and vessels. Their supramolecular structure as well as specific functions

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are still ill-defined. Structurally, BM can be viewed as complex mixtures of ECM macromolecules interposed between two distinctly differentiated cell types (eg, epithelial cells and mesenchyme in epithelia, myotubes and tendon or mesenchyme in muscle, endothelial cells and smooth muscle in vessels). This role of BM as physical barriers between differentiated cell types has long been recognized but, more recently, it has been increasingly appreciated that the BM also provide instructive cues regulating cell adhesion, motility, proliferation and survival. In certain tissues, such as the epidermis, gut and mammary gland, BM may represent a unique communication interface between the epithelial compartment and the rest of the organism. As such, they may play an essential role in synchronizing epithelial cell functions within tissues, particularly for the purposes of self-renewal, remodeling, wound healing and regeneration. Thus, BMs are dynamic structures in tune with the tissue environment and conditions, including pathological states, and their molecular composition may change according to the specialization and requirements of a particular tissue. ECM macromolecules play a key role in the processes of invasion and metastasis for a number of reasons. First of all, cancer cells require interaction with ECM, so that loss of contact determines cell death due to anoikis. The interaction between cancer cells and the ECM is mediated, as for other epithelial cells, by the integrins, that not only ensure physical adhesion to the protein substrate but also deliver signals to the cells serving to modulate cellular functions and behavior28-30. Integrins are also localized at the advancing edge on the pseudopodia structures, where remodelling of the ECM occurs. This is the case for Ln-5, which promotes static adhesion and hemidesmosomes formation, so that epithelial cells such as keratinocytes are normally almost immobile cells. However, under certain circumstances, as during wound healing, hemidesmosomes are disassembled and epithelial cells acquire strong motility31. In this scenario, Ln-5 is cleaved by MMPs such as MMP-2 and/or MT1-MMP, on the γ2 chain and this induces migration of epithelial cells such as keratinocytes, breast and colon epithelial cells, as shown in cell culture systems14,15. Other proteinases, including serine proteinases such as plasmin, have been shown to cleave the Ln-5 α3 chain and this converts Ln-5 from a migratory to an adhesive substrate32. Therefore, it is likely that, after deposition, Ln-5 can undergo distinct rearrangements, some mediated by MMPs and others by serine proteinases, that change its biological function to either more migratory or more adhesive. Therefore, according to the particular situation, Ln-5 can promote distinct functions and induce different cellular behaviors depending on which type of proteinase is mainly active. Although it is well established that MMP-cleaved Ln5 induces cell migration and invasion, the mechanisms underlying this acquired function has not yet been completely elucidated. Recently, it has been reported that af-

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ter having lost their cell-cell contact, colon cancer cells show a down regulation of E-cadherin resulting in detectable β-catenin oncoprotein in the nucleus33,34. This seems to be responsible for up-regulation of the LAMC2 gene encoding the γ2 chain of Ln-5. On this basis, the Ln-5 γ2 chain seems to be a downstream effect of an invasive signaling cascade. An MMP fragment of the γ2 chain of Ln-5 has recently been shown to be a ligand of the epidermal growth factor receptor (EGF) that stimulates cell motility35. Furthermore, the γ2 monomeric chain has been detected at the advancing edge of different malignancies and it has recently been reported to be secreted by ovarian and melanoma cells36. All these data suggest a new role for the γ2 chain that is quite intriguing, since the functions of Ln-5 have so far been considered to be ensured through the entire molecule rather than a single chain and/or portion of the molecule. While these findings are new for Ln-5, similar data have been observed for other ECM proteins where small fragments such as the RGD sequence have been shown to be responsible for the biological functions of fibronectin (Fn), fibrinogen (Fg), and vitronectin (Vn). These RGD peptides are currently under investigation for their possible therapeutic applications in the treatment of thrombosis, osteoporosis, and cancer. Integrins are heterodimers formed by the non-covalent association of two transmembrane subunits, α and β. There are at least 12 α and 9 β subunit genes. Restrictions in the permitted combinations of α and β chains result in approximately 20 distinct integrin heterodimers in man, whose functions may be further diversified by alternative splicing, in both the extracellular and cytoplasmic domains. In several resting epithelia, the main integrin in contact with Ln-5 is α6β4, whereas α3β1 is mostly confined to the lateral plasmamembrane. However, during wound healing, α3β1 is redistributed to the basal cell surface and mediates rapid adhesion, spreading and migration of keratinocytes. Once a confluent cell layer is

Static cancer cell Laminin-5 High MMPs High serine proteinases

EMT

Low E-cadherins

Latent TGF-β1

High β-catenin TGF-β1 Integrins ECM

Invasive cancer cell Figure 1 - Schematic representation of microenvironment components involved in triggering a more invasive and aggressive phenotype of epithelial cancer cells.

reconstituted, the basal localization of α3β1 is lost and replaced by integrin α6β4, concomitantly with the formation of strong adhesion structures, the hemidesmosomes. This phenomenon has also been observed in cultured keratinocytes, where α3β1 is the main receptor driving cell adhesion and spreading on Ln-5, but is gradually displaced by α6β4 as the hemidesmosome structures appear37,38. However, although the two Ln-5 receptors have been demonstrated to have different functions, no studies have so far documented how such functions may be coordinated. According to several reports, cancer cells, including breast, colon and liver, use integrin α3β1 to migrate and invade in in vitro models39. On the other hand, some reports suggest that down-regulation of α3β1 correlates with poor prognosis in lung carcinoma, facilitating cancer growth and invasion40. Consistent with these latter reports, the N-myc oncogene modulates the post-transcriptional level of this integrin expression in neuroblastoma cells, suggesting a link to EMT, responsible for a more malignant and aggressive phenotype41. In all of these situations, integrin α3β1 has been studied in association with the Ln-5 substrate, However, α3β1 was shown to affect cell behavior regardless of any without direct engagement with ECM, by virtue of physical association with members of the tetraspanin family which can affect cell motility. Integrin α3β1 was also implicated in the expression of urokinase-type plasminogen activator receptors (uPAR) by keratinocytes, and in the regulation of MMP-2 activation, as observed for the αVβ3 integrin42,27,23. However, other studies have emphasized the role of α6β4 as the main receptor in migration of lung cancer cells. These studies are only apparently in conflict, since it is very possible that mechanical, adhesive and signaling actions can be modulated differently and in a synergistic manner to enable cancer cells to migrate and invade39. Soluble factors

Soluble factors can also participate in the development of metastasis by directing the migration of cancer cells through the surrounding microenvironment once they have crossed ECM tissue boundaries such as BM43. It is well known that target sites for metastases are not random. Rather, each tumor type has a preference for sites and/or organs. The mechanisms regulating “homing” of cancer cells are currently being intensely investigated. For example, it has been shown that different chemokines drive directional migration of breast adenocarcinoma cells, as well as of large-cell lymphoma and kidney cancer cells. In agreement, receptors for certain chemokines, such CXCR4, can participate in the preferential localization of some cancer cells in certain target tissues44. Growth factors such as transforming growth factor (TGF)-β1 represent another type of soluble factors involved in metastasis. TGF-β1 is a multifunctional growth factor secreted in a latent form that becomes ac-

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tive after proteolytic cleavage. Several studies reported increased serum levels of TGF-β1 in a number of malignancies including hepatocellular carcinoma (HCC)45,46. Furthermore, TGF-β1 is stored, in latent form, in the context of the ECM. During proteolytic remodelling of the ECM it is very possible that TGF-β1 can also be processed and become active. This hypothesis is supported by the fact that serine proteases, such as plasmin and MMPs including MMP-2 and MMP-9, have been shown to activate latent TGF-β147. This proteolytic activation could be responsible for local increase in the TGF-β1 concentration, sustained or transient, possibly without concomitant elevation of serum levels. All these data further emphasize the importance of the microenvironment in the metastatic process, since during proteolytic remodelling of the ECM proteins, stored TGF-β1 can be activated and participate in the invasive process. It has been reported that TGF-β1 can trigger EMT, inducing a more invasive and aggressive phenotype. In the case of HCC, TGF-β1 stimulates the expression of integrin α3β1 on HCC cancer cells and this regulates the acquired migration and invasion ability 48. Consistently, blocking antibodies against TGF-β1 reduce α3β1 expression as well as the migratory and invasive properties of HCC cells. In other experimental systems, TGF-β1 has been shown to induce αVβ3 integrin, up-regulating EMT , rearranging the cytoskeleton, and finally stimulating the invasive ability of cervical squamous carcinoma cells49. Similar results have been reported in a number of other cancers including breast, trachea, and colon50-53. Furthermore, the role of EMT induced by TGF-β1 has also been documented in keratinocytes during re-epithelialization of wound healing, a normal invasive process that resembles cancer invasion54. Molecular approaches to study the tumor microenvironment

As discussed above, the role of the microenvironment in the process of cancer invasion is expected to be important, but the main difficulty in this field is identifying the specific roles of a number of different factors that, as constituents of the microenvironment, can be si-

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multaneously involved in the metastatic process55. The other main problem is the difficulty in isolating each component from the complexity of the microenvironment. Recently, the use of microdissection using laser capture technology has greatly improved the possibility of obtaining more homogenous material56. In combination with laser capture microdissection, DNA microarrays offer the possibility of screening a great number of genes at the same time, in order to select those more likely to be involved. While these approaches open new avenues of research, many concerns remain regarding the correct use of such powerful tools, in particular the use of microarrays57. False positive and negative results are currently an important issue, so that standardization of these techniques has recently become a true field of research in its own right, whose future applications not only to research but also clinical practice are quite promising57. Is the microenvironment a target for new drugs?

The question as to whether or not new drugs can be targeted against components of the microenvironment is of vital importance to novel treatments for cancer invasion. This may not only represent a novel therapeutic strategy, but could also circumvent limitations imposed by drugs exclusively targeted to the cancer cell itself. That is, cancer cells may be constantly stimulated to assume an invasive and aggressive phenotype by different components of the surrounding microenvironment, while drugs against cancer cells could at best have a short-term role in limiting proliferation. In contrast, new biological weapons might be targeted at silencing the cross-talk between cancer cells and the microenvironment and thus stop or reverse the EMT responsible for invasiveness. In this regard, MMP inhibitors were successful in preclinical in vivo models, though the current generation of these inhibitors has unacceptable side-effects in humans. Other examples include blocking antibodies against integrin αVβ3, anti-angiogenic factors and, finally, the pirfenidone molecule used to block stroma remodelling and TGF-β stimulation. Additional targets and corresponding drugs will hopefully emerge by further studies on the tumor microenvironment.

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