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Abstract

  1. Top of page
  2. Abstract
  3. ALTERATIONS IN THE TUMOR MICROENVIRONMENT
  4. PROTEOLYSIS OF ECM
  5. ROLE OF ECM IN RESPONSE TO THERAPY
  6. CONCLUSION
  7. Acknowledgements
  8. LITERATURE CITED

Recently, a view of the tumor as a functional tissue interconnected with the microenvironment has recently been described. For many years, the stroma has been studied in the context of the malignant lesion, and only rarely has its role been considered before carcinogenic lesions appear. Recent studies have provided evidence that stromal cells and their products can cause the transformation of adjacent cells through transient signaling that leads to the disruption of homeostatic regulation, including control of tissue architecture, adhesion, cell death, and proliferation. It is now well established that tumor progression requires a continually evolving network of interactions between neoplastic cells and extracellular matrix. A relevant step of this process is the remodeling of microenvironment which surrounds tumors leading to the release of ECM-associated growth factors which can then stimulate tumor and/or endothelial cells. Finally, tumor cells reorganizing the extracellular matrix to facilitate communications and escape the homeostatic control exerted by the microenvironment modify response to cytotoxic treatments. © 2002 Wiley-Liss, Inc.

The notion of cancer as a progression of genetic mutations in an aberrant tissue mass has allowed the identification and characterization of tumor suppressor genes and oncogenes involved in cancer susceptibility (Cahill et al., 1999). However, a different view of the tumor as a functional tissue interconnected with the microenvironment has recently been described (Radisky et al., 2001). In fact, tissue architecture and function are thought to be maintained by a dynamic interplay between epithelial cells and a microenvironment consisting of an insoluble extracellular matrix (ECM), a stroma composed of fibroblasts, adipose, vasculature and resident immune cells, and a milieu of cytokines and growth factors. The ECM is defined as a complex mixture of proteins, proteoglycans, and adhesive glycoproteins that provides structural and mechanical support to cells and tissues. The structural and regulatory proteins of the ECM function cooperatively to regulate a wide variety of cellular processes, both positively and negatively (Yurchenco and Schittny, 1990; Damsky and Werb, 1992; Mosher et al., 1992). Each cell type displays surface receptors appropriate for its tissue environment, and interaction of these receptors with ECM components influences cell shape, behavior, and the response to soluble molecules including cytokines and growth factors (Lin and Bissell, 1993). Indeed, there is evidence that ECM, depending on its context, can actively regulate cellular processes such as growth, death, adhesion, migration, invasion, gene expression, and differentiation (Ingber and Folkman, 1989; Juliano and Haskill, 1993; Assoian and Marcantonio, 1996). These cellular events, in turn, regulate physiological processes such as embryonic development, tissue morphogenesis, and angiogenesis and probably also pathological processes such as transformation and metastasis (Ingber and Folkman, 1989; Yurchenco and Schittny, 1990; Damsky and Werb, 1992; Mosher et al., 1992; Juliano and Haskill, 1993; Assoian and Marcantonio, 1996). In general, signals eliciting these cellular responses are interpreted in a context dictated by cell association with the basement membrane, a highly organized and specialized ECM, whose composition differs from that of stromal ECM and to which epithelial cells attach. ECM-controlled signaling ensures that cells divide and differentiate only as needed by the organism. Thus, tumor cells must remodel the matrix to facilitate communication and escape control by the microenvironment. Remodeling can also include interactions with “alternative” ECM, leading to cellular proliferation, structural disruption, and circumvention of apoptosis. Current findings obtained through a variety of approaches increasingly point to the contribution of stromal components to oncogenic signals that mediate both phenotypic and genomic changes in epithelial cells (Tlsty and Hein, 2001).

ALTERATIONS IN THE TUMOR MICROENVIRONMENT

  1. Top of page
  2. Abstract
  3. ALTERATIONS IN THE TUMOR MICROENVIRONMENT
  4. PROTEOLYSIS OF ECM
  5. ROLE OF ECM IN RESPONSE TO THERAPY
  6. CONCLUSION
  7. Acknowledgements
  8. LITERATURE CITED

During the transition from normal tissue to in situ and invasive carcinoma, the microenvironment of the local host stroma is an active player. Interaction between the epithelial and mesenchimal compartments produces a particular area of interrupted epithelial basement membrane from which the malignant cell emerges and invades other tissue. Events that potentially create “reactive” stroma or stroma with an altered homeostatic balance might contribute to the risk of malignant disease. In fact, stroma and tumor cells can interchange growth factors, for example, transforming growth factor-β (TGF-β) and epidermal growth factor (EGF), cytokines and chemoattractants, for example, interleukin-6 (IL-6) and scatter factor/hepatocyte growth factor (SF/HGF), angiogenesis factors, for example, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and proteases, for example, matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), to activate the adjacent ECM and, in turn, induce the selection and expansion of neoplastic cells (Brown et al., 1999; Liotta and Kohn, 2001).

Oncogenic functions of stromal cells

For many years, the stroma has been studied in the context of the malignant lesion, and only rarely has its role been considered before carcinogenic lesions appear. Recent studies have provided evidence that stromal cells and their products can cause the transformation of adjacent cells through transient signaling that leads to the disruption of homeostatic regulation, including control of tissue architecture, adhesion, cell death and proliferation (Tlsty and Hein, 2001). In particular, biochemical, molecular, and immunohistochemical analyses have demonstrated a functional role for surrounding stroma in the development of solid neoplasia, such as breast, colon and prostate carcinomas (Ronnov-Jessen et al., 1996; Olumi et al., 1999). Stromal cells produce a vast number of specific molecules that directly or indirectly affect the epithelial cell phenotype. Recently, oncogenic signals from carcinoma-associated fibroblasts have been shown to stimulate the transformation of normal epithelial cells. In one study, carcinoma-associated prostatic fibroblasts engrafted in athymic nude mice injected with immortalized human prostatic epithelial cells actively promoted tumor growth (Olumi et al., 1999). The histology and growth characteristics of carcinoma-associated fibroblasts differ from those of fibroblasts associated with normal epithelial cells (Sappino et al., 1990; Tlsty, 2001) and several altered properties, including enhanced collagen production (Bauer et al., 1979) and stimulation of hyaluronate synthesis have been reported for fibroblasts in the vicinity of tumors (Knudson et al., 1984). Altered invasive properties have been detected not only in skin fibroblasts from cancer patients (Olumi et al., 1999), but also in fibroblasts from patients with hereditary cancers (Antecol et al., 1986). Indeed, it has been suggested that fibroblast abnormalities, including uncontrolled growth, may influence stromal-epithelial interactions and elicit tumor formation in the presence of congenital defects (Kopelovich, 1982).

The inflammatory microenvironment of tumors is characterized by the presence of host leukocytes in both the supporting stroma and the tumor areas. Cross-talk between tumor cells and altered microenvironment is represented in Figure 1. Although, tumor-infiltrating lymphoid cells are generally considered as cytotoxic for the tumor cells, in some cases such lymphoid cells can contribute to cancer growth and spread, and to the immunosuppression associated with malignant disease (Balkwill and Mantovani, 2001). For example, Coussens et al. (1999), using a mouse model of epithelial carcinogenesis, showed that inflammatory mast cells are involved in activating premalignant neovascularization in the skin and proposed a key functional role for these infiltrating cells during the premalignant stages of squamous carcinogenesis due to the release of specific pro-angiogenic proteases. In other studies, the production of growth factors (such as IL-6) by herpesvirus-infected dendritic cells and macrophages was shown to drive the initial proliferation of hematopoietic malignant cells and autoimmune deficiency syndrome-associated neoplasms (McGrath et al., 1995; Rettig et al., 1997). In both studies, herpesvirus sequences were detected only in the tumor-associated stromal cells (dendritic cells and macrophages) and not in the malignancies.

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Figure 1. Dynamic interplay between epithelial tumor cells and their microenvironment. 1: Production of altered ECM proteins by tumor cells; (2) altered ECM in stroma; (3) recruitment of immune cells directed against altered ECM proteins; (4) release of cytokines in altered microenvironment; (5) increased tumor growth by cytokines release.

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Oncogenic signals of ECM components

A strong desmoplastic reaction is a common aspect of many solid tumors, including those of the breast, prostate, colon, and lung. This reaction represents a peculiar phenotypic change due to a morphologically and functionally altered stroma frequently associated with inflammatory cells. TGF-β, present in a latent form in the ECM and often produced by tumor cells, and platelet-derived growth factor (PDGF), a potent mitogen and chemoattractant for mesenchymal cells, are two reasonable candidates for involvement in the induction of the desmoplastic response through different signaling routes (Elenbaas and Weinberg, 2001; Tlsty, 2001). Ultrastructural and immunohistochemical analyses support the notion that typical markers of the desmoplastic reaction are altered expression levels of α-smooth-muscle actin, smooth muscle myosin, vimentin and desmin in desmoplastic fibroblasts (Sappino et al., 1990), and altered production of several ECM proteins such as collagen types III, V, and tenascin (Sappino et al., 1990), laminin (Tagliabue et al., 1996), MMPs and their inhibitors (Chang and Werb, 2001), and growth factors (Nakamura et al., 1997). Additionally, the distribution and expression levels of laminin, a molecule critical for the architectural integrity of tissue, is altered and reduced in fibroblasts associated with malignant cells (Tlsty, 2001). Recent studies using a gene expression profiling technique, have shown that the gene for osteopontin a secreted integrin-binding ECM glycoprotein, is differentially expressed during colon cancer progression (Agrawal et al., 2002). In keeping, we found that glycoprotein fibulin-1 (FBLN-1), a cysteine-rich, calcium-binding ECM and plasma molecule (Argraves et al., 1990), is aberrantly overtranscribed in epithelial tumor cell lines and breast surgical specimens, and that the protein is accumulated in the breast cancer-associated stroma, as detected immunohistochemically (Forti et al., 2002). Augmented expression of FBLN-1 has been also reported in human ovarian cancer cells stimulated with estrogens, and the resulting overexpression facilitated ovarian tumor cell invasion and progression (Clinton et al., 1996; Moll et al., 2002). FBLN-1 self-associates and binds to other ECM proteins, including fibronectin, laminin and nidogen, and to fibrinogen (Roark et al., 1995). Since FBLN-1 under physiological conditions participates to laminin polymerization, its excess in ECM might induce disruption of laminin polymers, in turn changing the signal mediated by the interaction of tumor cells with ECM. FBLN-1 might also promote tumor cell extravasation, since this protein has been shown to increase platelet adhesion by interacting with fibrinogen (Tran et al., 1995). By analysis of recombinant cDNA expression library using breast cancer patients' sera, we detected humoral immunity to FBLN-1 (Forti et al., 2002), indicating that altered ECM components can also be immunogenic in tumor patients.

Hyaluronic acid (HA), an ECM polysaccharide involved in wound healing by promoting cell migration through its cell surface receptor, is also frequently overexpressed in malignant tumors. The intensity of HA in the stroma was found to be an independent prognostic factor for overall survival according to Cox's multivariate analysis (Auvinen et al., 2000). HA interacts with tumor cells through the hyaluran-binding protein RHAMM by which it regulates ras signaling. Accordingly, RHAMM expression has been associated with lymph node metastasis (Wang et al., 1998).

Recent studies suggest that oncogenic activity displayed by stroma can reflect many different processes, including: exposure to carcinogens, which not only induce genetic changes in the cell that directly damage the epithelium, but also alter ECM composition; altered growth factor activities, mainly mediated by the pleiotropic cytokine TGF-β, whose effects include phenotypic changes in adhesion, migration, differentiation and cell death, and altered expression of receptors that mediate cell–cell interactions (Barcellos-Hoff and Ravani, 2000); and direct cellular communications with the ECM, especially β1-integrins (Ruoslahti, 1999). Together, these findings point to the significant contribution of stromal alterations to tumorigenesis.

As a potentially oncogenic agent, the stroma can drive tumorigenicity in adjacent cells with the acquisition of genomic changes and in the absence of pre-existing tumor cells. As a physically supportive and responsive structure, the stroma can be induced by tumor cells to express critical signals that drive proliferation, angiogenesis, and motility and that suppress apoptosis.

PROTEOLYSIS OF ECM

  1. Top of page
  2. Abstract
  3. ALTERATIONS IN THE TUMOR MICROENVIRONMENT
  4. PROTEOLYSIS OF ECM
  5. ROLE OF ECM IN RESPONSE TO THERAPY
  6. CONCLUSION
  7. Acknowledgements
  8. LITERATURE CITED

Tumor progression requires a continually evolving network of interactions between neoplastic cells and ECM. A relevant step of this process is the remodeling of microenvironment, which surrounds tumors. This phenomenon occurring also in physiological events as embryonic development and wound healing, leads to structural disruption of ECM components which, in turn, modifies cell survival, proliferation, and migration.

Enzymes

Tumor remodeling of the microenvironment appears to require proteolysis of ECM proteins. The principal enzymes that degrade the ECM include MMPs (Stetler-Stevenson et al., 1996; Giannelli et al., 1997), cysteine proteinases (cathepsin B) (Yan et al., 1998; Lah et al., 2000), aspartic proteinases (cathepsin D) (Yan et al., 1998; Tetu et al., 1999), and serine proteinases (elastase, uPA) (Heck et al., 1990; Andreasen et al., 1997). Most of these enzymes are lysosomal proteases that are involved in physiological process such as turnover of cellular proteins. Immunohistochemical and enzymatic analyses of protease expression in different tumors have shown that increased expression or changes in cell localization are important prognostic factors and correlated with tumor progression (Heck et al., 1990; Kolkhorst et al., 1998; Yan et al., 1998; Tetu et al., 1999). However, in the remodeling process, most enzymes are produced by stromal cells of the host (Crawford and Matrisian, 1996; Saren et al., 1996). In human breast cancer, stromal fibroblasts surrounding tumor cells per se produce MMP-11 (Basset et al., 1990) and uPA (Pyke et al., 1991, 1993; Nielsen et al., 1996). Similarly, in situ hybridization analysis of human breast carcinoma has demonstrated the localization of MMP-1, -2, -3 mRNA primarily in stromal fibroblasts in proximity to invading cancer cells, but not in the carcinoma cells (Nelson et al., 2000). When stimulated by proinflammatory cytokines present in an inflammatory cancer, macrophages also show up-regulated expression of some MMPs, which can actively participate in ECM destruction (Saren et al., 1996). In turn, the expression of matrix proteinases becomes regulated by tumor cells. For example, the extracellular matrix metalloproteinase inducer (EMMPRIN) (Biswas et al., 1995) is an intrinsic plasma membrane glycoprotein produced at high levels by cancer cells (Polette et al., 1997), which stimulates adjacent stromal cells to synthesize matrix MMPs (Guo et al., 2000).

Immunolocalization studies using specific antibodies to identify proteinases, have generally revealed MMP-2 and MMP-9 protein on the cancer cell surface bound to collagen type IV components (Olson et al., 1998), CD44 (Yu and Stamenkovic, 2000) and αvβ3 integrin (Brooks et al., 1996). Cathepsin B has also been detected on the plasma membrane of tumor cells and is associated with consequent focal dissolution of ECM proteins and tumor cell invasion (Sloane et al., 1986). Insertion of cathepsin B in the tumor cell membrane depends on the cell surface expression of heparan sulfate proteoglycans (Almeida et al., 2001), which protect the enzyme from alkaline pH-induced inactivation. An example of a breast cancer matrix protease specifically expressed in fibroblasts is uPA, which interacts with its receptor (uPAR) on carcinoma cells and macrophages close to fibroblasts (Pyke et al., 1993) and (Pyke et al., 1991; Tan et al., 1995; Nielsen et al., 1996) localizes its degradative activity to particular regions of the cell membrane. Recent studies have demonstrated that EMMPRIN stimulation of MMP-1 synthesis in fibroblasts is followed by proteinase binding to its inducer on the tumor cell surface, arming tumor cells for degradation of ECM (Guo et al., 2000). Besides, ECM remodeling, proteolytic enzymes also influence tumor initiation and risk for neoplastic development, as demonstrated by the formation of mesenchymal-like tumors in mice transgenically expressing MMP3 in normal mammary epithelial cells (Sternlicht et al., 1999). MMP3/stromelysin-1 is known to degrade numerous ECM substrates, including collagens III, IV, V, IX, X, and XI, laminins, elastin, entactin, fibronectin, fibrin, fibrillins, fibulin, osteonectin, tenascin, vitronectin, and ECM proteoglycans, and can also release cell surface molecules, including E-cadherin, L-selectin, and TNF-α. Moreover, MMP3 can activate other MMPs and inactivate several serine proteinase inhibitors.

Unmasking of cryptic sites

Cleavage of matrix components by proteinases produced by stromal cells upon cancer cell induction not only removes the physical barriers to cell migration, but also perturbes signaling cascades generated by interaction between matrix and cell surface receptors with consequences on cell survival, proliferation, and migration (Giancotti and Ruoslahti, 1999). Proteolysis of ECM proteins modifies integrin-mediated anchorage, focal adhesion, and cytoskeletal architecture, and triggers signaling molecules such as focal adhesion kinase (FAK) (Braga, 2000; Fashena and Thomas, 2000). Enzymatic cleavage of matrix molecules also reveals binding sites previously blocked from interaction with cell surface receptors. For example, MMP-2 degradation of collagen unveils integrin-binding sites that rescue melanoma cells from apoptosis (Montgomery et al., 1994). However, tumor cells can unmask adhesion molecule sites that are usually embedded within the matrix through overexpressed molecules in addition to ECM degradation. We demonstrated that the 67-kDa monomeric laminin receptor (67LR), which is overexpressed (Martignone et al., 1993; Ménard et al., 1998) and released by various tumor cell types (Karpatová et al., 1996; Starkey et al., 1999), changes the conformation of the laminin adhesion molecule upon binding to it (Magnifico et al., 1996). Because the integrin recognition domains of laminin appear to be conformation-dependent (Mercurio, 1995), 67LR-modified laminin interacts more readily with integrins and with other molecules that normally do not participate significantly in laminin binding to the cell surface (Ramos et al., 1990; Feldman et al., 1991; Kleinman et al., 1991; Magnifico et al., 1996). This mechanism provides the cells with a greater number of binding sites and modulates the interaction between tumor cells and laminin, with consequences for metastatic potential (Pellegrini et al., 1995).

Production of functional fragments

In addition to unmasking sites, proteolysis of the ECM also releases fragments that mediate new functional effects to modulate the biologic response. For example, cleavage of laminin-5, one of the ECM components that promotes stable cell attachment, by MMP-2 or MT1-MMP generates a proteolytic γ2-chain fragment which promotes migration of human breast epithelial cells (Giannelli et al., 1997; Koshikawa et al., 2000). In addition, an α1-chain fragment generated by elastase proteolysis of laminin-1 has been shown to play a role in regulation of the macrophage degradative phenotype and in tissue remodeling (Khan et al., 2002). Note that the ECM fragments derived by MMP cleavage can also act as pro-angiogenic molecules, for example, the trimeric NC1 domain of collagen XVIII induces endothelial cell migration involved in angiogenesis (Kuo et al., 2001). On the other hand, a C-terminal fragment of collagen XVIII derived through elastase-dependent cleavage is a potent inhibitor of angiogenesis and tumor growth (Wen et al., 1999). Our recent finding that cathepsin B cleavage of 67LR-treated laminin leads to an altered pattern of degradation products including the release of a new proteolytic fragment, provides evidence that the 67LR-laminin interaction induces exposure of a previously hidden cleavage site(s) (Ardini et al., 2002). Laminin digested by cathepsin B after modification by 67LR promotes cell motility (Fig. 2), whereas the cathepsin B cleavage of unmodified laminin removes physical barriers to cell migration but leads to complete loss of cell migration. Thus tumor cells can utilize an overexpressed adhesion receptor as a mechanism to invade the host microenvironment. Functional fragments are also released by proteolysis of cell surface proteins. MMPs cleave E-cadherin at the cell surface and release a soluble fragment that inhibits E-cadherin function, resulting in induction of tumor cell invasion (Davies et al., 2001; Noe et al., 2001). MT1-MMP has been found to act as a processing enzyme for CD44, a major receptor for hyaluronan, releasing the receptor as a soluble fragment that promotes cell migration (Kajita et al., 2001). A naturally occurring fragment of MMP-2, which comprises the C-terminal hemopexin-like domain (PEX), can be detected in vivo in conjunction with integrin αvβ3. The PEX-mediated block in MMP-2 binding to integrin on the surface of new blood vessels inhibits angiogenesis (Brooks et al., 1998).

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Figure 2. Migratory activity of laminin-1 proteolytic fragment. 1: Binding of tumor cells to laminin; (2) 67LR-induced change of laminin-1 conformation; (3) unmasking of a cryptic site for cathepsin B proteolytic cleavage; (4) release of a “new” proteolytic fragment that promotes cell motility.

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Release of signaling factors

Many cytokines such as members of the EGF family, TGFβ, PDGF, b-FGF and IFN-γ bind to various components of the normal tissue ECM (Streuli, 1999), where they are stored in an inactive form until released and activated by matrix proteases when needed. An increase in matrix protease activity during tumor matrix remodeling can lead to the release of ECM-associated growth factors which can then stimulate tumor and/or endothelial cells. For example, the matrix proteases MMP-11, MMP-1, plasmin and heparinase each degrade the angiogenic factor b-FGF (Whitelock et al., 1996). MMP-3 has been shown to cleave the matrix molecule decorin, thereby releasing TGFβ (Imai et al., 1997). Angiogenesis in tumors depends in part on the release of angiogenic factors stored in the ECM. Indeed, some matrix proteases might regulate the availability of VEGFs to endothelial cells, since these factors, once released by epithelial cells, can be stored in the ECM through interaction with heparan sulfate proteoglycans (Park et al., 1993). In addition to the release of growth-stimulating signals from the matrix, tumor cells can induce the release of extracellular domains of proteins from the cell surface by MMP-directed proteolysis. MMP-2 was shown to influence cell surface receptor-mediated signaling by releasing the active ectodomain of the FGF receptor (Levi et al., 1996). Thus far, more than 40-cell surface proteins have been shown to undergo ectodomain shedding by proteolytic cleavage (Chang and Werb, 2001). All these processes, initially guided by the tumor cells and gradually involving the microenvironment of the host tissue, lead to dysregulated intracellular signaling between the diverse cell types and the ECM within the tumor mass.

ROLE OF ECM IN RESPONSE TO THERAPY

  1. Top of page
  2. Abstract
  3. ALTERATIONS IN THE TUMOR MICROENVIRONMENT
  4. PROTEOLYSIS OF ECM
  5. ROLE OF ECM IN RESPONSE TO THERAPY
  6. CONCLUSION
  7. Acknowledgements
  8. LITERATURE CITED

Recent view of a tumor as a functional tissue interconnected with the microenvironment leads to consider that response to therapy is not only related to the tumor phenotype but is also associated to the composition of the tumoral stroma. Indeed, tumor cells reorganizing the extracellular matrix to facilitate communications and to escape the homeostatic control exerted by the microenvironment modify response to cytotoxic treatments.

Role of adhesion in response to therapy

The role of the interaction between tumor cells and the ECM in drug sensitivity is still poorly understood, in large part because most in vitro drug sensitivity studies are performed on cell monolayers instead of three-dimensional clones in which the interactions between tumor cells and the ECM are maintained. Nevertheless, some studies have shown that adhesion of tumor cells impairs drug-induced apoptosis. Indeed, normally doxorubicin (DXR)-sensitive myeloma cells become resistant when allowed to adhere to fibronectin; the resistance phenotype is not due to reduced drug accumulation or to upmodulated expression of anti-apoptotic Bcl-2 family members (Damiano et al., 1999). Fibronectin adhesion mediated by beta1 integrins appears to protect tumor cells from drug-induced DNA damage by reducing topoisomerase II activity secondary to alterations in the nuclear distribution of this enzyme (Hazlehurst et al., 2001). In addition, adhesion of small cell lung carcinoma cells to the ECM has been shown to confer drug resistance to chemotherapeutic agents as a result of beta1 integrin-stimulated tyrosine kinase activation which suppresses chemotherapy-induced apoptosis (Sethi et al., 1999).

Drug-induced alterations in ECM interactions

The central role of beta1 integrin in determining drug sensitivity is also demonstrated by its upmodulation in drug-resistant variants, which has been observed in a variety of tumors including glioma (Hikawa et al., 2000), myeloma (Damiano et al., 1999), and breast carcinoma (Nista et al., 1997; Narita et al., 1998). Production of ECM components such as fibronectin (Soose et al., 1993) is also upmodulated by drug treatment of tumor cells. In vitro data indicate that DXR perturbs the turnover of fibronectin secreted by human mesangial cells such that it accumulates extracellularly (Soose et al., 1993). We recently detected upmodulation of mRNA for fibulin-1, a molecule involved in laminin polymerization, after treatment of breast carcinoma cells with DXR (Fig. 3). The drug-induced upmodulation of ECM components is not restricted to tumor cells, since normal kidney cells from drug-treated rats also produced large amounts of fibronectin, laminin and collagen I and IV (Manabe et al., 2001). In parallel with the increased secretion of adhesion molecules, decreased activity of the collagen-degrading proteolytic enzyme MMP-1 resulted from DXR treatment (Benbow et al., 1999). Because only low non-toxic doses of DXR were used in that study, the decrease in MMP-1 cannot be attributed to selection of resistant cells expressing low MMP-1 levels, but instead to altered regulation of gene expression induced by the drug. Interestingly, the upmodulation of adhesion molecule synthesis likely rests in the stress induced by the drug since fibulin-1 upmodulation in tumor cells occurs with a similar kinetics after treatment with DXR or after thermal shock at 42°C (Fig. 3b).

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Figure 3. RT-PCR analyses of fibulin-1 expression (a) before and after DXR treatment of three human breast cancer cell lines (SKBR-3, MCF-7, and MDA-MB-157) and (c) kinetics of fibulin-1 expression in MDA-MB-157 cells after thermal shock at 42°C. (b and d) cDNAs tested for integrity by amplification of GAPDH transcripts as an internal control.

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In vitro, cells that adhere to the ECM are more resistant to drug-induced apoptosis. Consistent with a role for strong tumor cell–ECM interaction in decreased drug responsiveness is the finding that treatment with hyaluronidase, which degrades hyaluronic acid, a major components of tumor ECM, greatly increases the sensitivity of the tumor to drug-induced apoptosis (St Croix et al., 1998). On the other hand, treatment of tumor cells with vitronectin, another ECM component, decreases drug sensitivity (Uhm et al., 1999).

CONCLUSION

  1. Top of page
  2. Abstract
  3. ALTERATIONS IN THE TUMOR MICROENVIRONMENT
  4. PROTEOLYSIS OF ECM
  5. ROLE OF ECM IN RESPONSE TO THERAPY
  6. CONCLUSION
  7. Acknowledgements
  8. LITERATURE CITED

Malignancy is a state that emerges from a tumor-host microenvironment. Tumor cells modify stroma and vasculature through production and secretion of extracellular elements, growth factors, and cytokines. The locally changed host microenvironment, in turn, modifies the proliferative and invasive behaviors of tumor cells. Thus, manipulation of the interaction between cancer cells and the microenvironment might lead to novel stromal therapeutic targets. Indeed, whether the molecular signals relevant in tumor-host cross-talk are targeted by specific inhibitors such as tumor necrosis factor (TNF), chemokines, and IL-6 antagonists, or by regulatory reagents such as non-steroidal anti-inflammatory drugs (NSAIDs) (Balkwill and Mantovani, 2001), stromal therapy might represent a valid approach to cancer prevention and intervention while minimizing collateral toxicity of uninvolved tissues. Furthermore, consistent with a role for tumor-ECM interaction in drug sensitivity, the combination of traditional cytotoxic treatments with therapies targeting the tumor-host communication might hold future promise in anticancer therapeutic efforts.

Acknowledgements

  1. Top of page
  2. Abstract
  3. ALTERATIONS IN THE TUMOR MICROENVIRONMENT
  4. PROTEOLYSIS OF ECM
  5. ROLE OF ECM IN RESPONSE TO THERAPY
  6. CONCLUSION
  7. Acknowledgements
  8. LITERATURE CITED

We thank Dr. Fabio Castiglioni for his help with figures and Mrs. Laura Mameli for manuscript preparation.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. ALTERATIONS IN THE TUMOR MICROENVIRONMENT
  4. PROTEOLYSIS OF ECM
  5. ROLE OF ECM IN RESPONSE TO THERAPY
  6. CONCLUSION
  7. Acknowledgements
  8. LITERATURE CITED
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