The tumor microenvironment and the extracellular matrix of developing organisms are chock-full of messages that are read by the cancer and developing cells in order for them to grow and avoid immune surveillance (for the former) and to reach the level of specialization so that various organs and tissues are generated (for the latter). Indeed, there is a long history of research focused on the signaling events evoked by the tumor stroma/extracellular milieu.1 Decorin, a small leucine-rich proteoglycan (SLRP) primarily synthesized by fibroblasts and myofibroblasts,2, 3 regulates collagen fibrillogenesis both in vitro and in vivo,4–6 and it is involved in a number of physiological and pathological processes including control of osteogenic stem cells and muscular development,7, 8 wound healing,9 myocardial infarction,10 Lyme disease,11 tubulointerstitial fibrosis and diabetic nephropathy,12–14 muscular dystrophy8 and cancer growth.15 Lumican is another SLRP substituted with a keratan sulfate side chain.16 It binds Type I collagen and regulates fibrillogenesis similar to decorin.17, 18 In recent years, a role for lumican in tumor progression has come to light. Decorin and lumican are typically located around growing neoplasms, the so called tumor microenvironment.19 The main biological functions of decorin are conveyed by interactions of its protein core, whereas the involvement of the glycosaminoglycan side chains is not well studied. Decorin protein core structure is composed of 10 central leucine-rich repeats, which take part in biological interactions with collagen,20–22 transforming growth factor β (TGFβ)23 and receptor tyrosine kinases.15, 24 Decorin represents a powerful tumor cell growth and migration inhibitor by both modulating tumor stroma deposition and cell signaling pathways. It directly binds to the epidermal growth factor receptor (EGFR) and downregulates its activity as well as the activity of other members of the ErbB family of receptor tyrosine kinases, such as ErbB2.25–27 These receptors are overexpressed and/or mutated in many cancers driving tumor progression. Decorin causes growth arrest of various tumor cell lines by inducing expression of p2127–30 and p27,31 2 inhibitors of cyclin-dependent kinases. Mice with a targeted disruption of the decorin gene do not develop spontaneous tumors, but when both the decorin gene and the tumor-suppressor gene p53 are disrupted, mice show an acceleration of malignant lymphomas, which leads to early mortality.32 This represents genetic evidence that lack of decorin is “permissive” for tumor development. A more recent publication brings forward the idea that decorin could indeed act as a “tumor suppressor gene” in the intestinal epithelium, insofar as 30% of decorin-deficient mice develop spontaneous intestinal tumors.33 The mechanism of action involves downregulation of p21 and p27 and upregulation of the β-catenin signaling. The involvement of other SLRPs in cancer has not nearly been investigated to the extent of decorin and lumican. Certainly, a role for other molecules of this family in tumor development cannot be excluded but the lack of data on their mechanism of action prevents us from including them in this short review. For a detailed overview of the SLRPs biological functions, please refer to a recent review.34
Decorin, the prototype member of the small leucine-rich proteoglycans, resides in the tumor microenvironment and affects the biology of various types of cancer by downregulating the activity of several receptors involved in cell growth and survival. Decorin binds to and modulates the signaling of the epidermal growth factor receptor and other members of the ErbB family of receptor tyrosine kinases. It exerts its antitumor activity by a dual mechanism: via inhibition of these key receptors through their physical downregulation coupled with attenuation of their signaling, and by binding to and sequestering TGFβ. Decorin also modulates the insulin-like growth factor receptor and the low-density lipoprotein receptor-related protein 1, which indirectly affects the TGFβ receptor pathway. When expressed in tumor xenograft-bearing mice or injected systemically, decorin inhibits both primary tumor growth and metastatic spreading. In this review, we summarize the latest reports on decorin and related molecules that are relevant to cancer and bring forward the idea of decorin as an anticancer therapeutic and possible prognostic marker for patients affected by various types of tumors. We also discuss the role of lumican and LRIG1, a novel cell growth inhibitor homologous to decorin. © 2008 Wiley-Liss, Inc.
Multiple decorin receptors and signaling pathways
The main target of decorin on the cell surface of tumor cells is the EGFR. After binding, the receptor dimerizes and is subsequently internalized and degraded in the lysosomes35, 36 (Fig. 1). From a physiological point of view, it is relevant to note that decorin can compete with EGF, the EGFR natural ligand, for receptor binding. Decorin inhibits tumor cell proliferation by evoking a signaling cascade that is different than the one evoked by EGF, possibly by inducing a different EGFR conformation and selectively activating phosphotyrosines in the receptor autophosphorylation domain. Upon decorin treatment or de novo decorin expression, mitogen-activated protein kinases (MAPK) are activated in a protracted fashion leading to growth arrest (Fig. 1) and EGFR levels are reduced by 40–50%.27, 37 Decorin targets the EGFR to degradation via caveolin-mediated internalization (Fig. 2), whereas the canonical internalization pathway evoked by EGF is primarily via clathrin-coated endocytic vescicles.36 The only exception to this model reported so far has been in MG-63 human osteosarcoma cells, where the decorin-expressing tumor cells show overexpression paired to constitutive activation of the EGFR.38 In these cells, decorin is not capable of reducing tumor cell growth and shows to favor cell migration by blocking TGFβ2 effects. This model could provide insights of a possible mechanism by which tumor cells escape decorin-mediated cytostatic effects and needs further investigation.
Decorin induces apoptosis in a squamous cell carcinoma model via activation of caspase-3 and this effect is dependent on the ability to phosphorylate the EGFR39 (Fig. 1). Caspase-3 can cleave the intracellular domain of the EGFR, an additional mechanism by which decorin could downregulate the receptor activity. Decorin also suppresses the activity of ErbB226, 40 and ErbB4 receptors via degradation. This effect is most likely achieved indirectly by binding to the EGFR and affecting EGFR/ErbB2 and EGFR/ErbB4 heterodimerization equilibrium.
IGF-I receptor and LRP-1
The insulin-like growth factor receptor (IGF-IR) and the low-density lipoprotein receptor-related protein (LRP) are novel partners for decorin. Decorin affects IGF-IR signaling by specifically binding to the receptor with a nanomolar range affinity in endothelial cells41 and renal fibroblasts.42 In addition, decorin binds to and sequesters the IGF-I, the natural ligand of this receptor tyrosine kinase. The signaling cascade evoked by decorin has been dissected and involves activation of phosphoinositide-3 kinase (PI3K), Akt and mTOR (mammalian target of rapamycin). Parallel to this pathway, the involvement of pyruvate dehydrogenase kinase-1 (PDK-1) has been reported and both cascades result in the transcription of specific genes (Fig. 3). The relevance of decorin in the IGF-IR pathway is reinforced in 2 experimental animal models of inflammatory angiogenesis in the cornea and unilateral ureteral obstruction. In both cases, decorin-deficient mice show a significant increase in IGF-IR levels as compared to mock-treated controls.42 Considering that IGF-IR signaling plays an important role in angiogenesis41, 43, 44 and that decorin affects both angiogenesis and IGF-IR biology, further investigation in this field of research will be of interest.
Decorin binds to the low-density lipoprotein receptor-related protein (LRP-1) on the cell surface of C2C12 myoblast and CHO cells.45 LRP-1 is a gigantic receptor that mediates the binding and internalization of numerous ligands. In C2C12 myoblasts, decorin is endocytosed through LRP-1 and metabolized, and decorin-deficient C2C12 cells exhibit an attenuated response to exogenous TGFβ, which is promptly restored by re-expressing decorin.46 Indeed, binding of decorin to LRP-1 activates PI3K and indirectly modulates TGFβ receptor signaling through Smad proteins46 (Fig. 3). Notably, decorin gene transfer promotes muscle cell differentiation and muscle regeneration,47 and decorin expression is associated with quiescence of muscle satellite cells.48
Modulation of the IGF-IR pathway and the TGFβ receptor signaling, via LRP-1, by decorin has not yet been shown in cancer cells. However, it is well established that, when deregulated, these 2 signaling pathways are implicated in the pathobiology of cancer by enhancing tumor growth and survival. It is likely, therefore, that the abnormal expression of decorin and related SLRPs in several tumor stromas49, 50 might influence these 2 signaling pathways thereby regulating tumor cell growth.
The involvement of decorin in angiogenesis and particularly tumor angiogenesis is still open to investigation and far from being resolved. In some experimental settings, decorin shows to be proangiogenic.30, 43 Decorin is expressed during endothelial cell sprouting in vitro,51, 52 a phenomenon that occurs when endothelial cells become postconfluent mimicking angiogenesis, and is produced by capillary endothelial cells during inflammation-associated angiogenesis.53 Moreover, paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation in collagen lattices.54 Finally, decorin and lumican are 2 of the major transcripts that progressively increase during capillary-like formation induced by Matrigel.55 A very recent publication brings forward the idea that decorin can play a proangiogenic role by favoring endothelial cell adhesion and migration on collagen Type I.56 Specifically, decorin mediates adhesion by binding to the α2β1 integrin and promoting integrin-collagen interaction. Interestingly, it favors endothelial cell migration by modulating IGF-IR signaling, a result confirming the involvement of the decorin/IGF-IR interaction in angiogenesis showed in different models presented above.
In other experimental settings, however, decorin is antiangiogenic via 2 potential mechanisms: by interfering with thrombospondin-157 or by suppressing the endogenous tumor cell production of vascular endothelial growth factor (VEGF), which in turns attenuates migration and capillary-like formation of endothelial cells.58 The leucine-rich repeat 5 was shown to be the main mediator of decorin antiangiogenic activity.59 Notably, neutralizing antibodies against EGFR and ErbB2 downregulate VEGF production by tumor cells,60 in analogy to the decorin activity mentioned above. Interestingly, decorin's expression has been associated with human benign vascular tumors compared to malignant vascular tumors, which are void of decorin.61 This study, though small in the number of patient samples, suggests that decorin might have a role in inhibiting human tumor angiogenesis. In an in vivo squamous carcinoma model, systemically-delivered decorin does not significantly reduce tumor angiogenesis, as measured by CD31 staining of frozen tumor sections.39 Remarkably, decorin synthesized by osteoblasts has an important role in reducing myeloma cells proliferation and inducing apoptosis.62 This outcome has been linked to decorin's ability to antagonize myeloma-cell induced angiogenesis by human vascular endothelial cells. Thus, one of decorin's roles in the microenvironment is the modulation of angiogenesis albeit in a cell-specific context, also depending on the physiological or pathological scenario.
Decorin expression in cancer patients
Decorin expression is altered in various types of cancer. Therefore, decorin has been taken into consideration as a possible prognostic marker in cancer patients. Decorin gene expression is found highly upregulated in pancreatic cancer tissue63, 64vis-à-vis normal pancreas. The pancreas stromal component, fibroblasts and stellate cells, maintain this up-regulation. This is expected considering the strong desmoplastic reaction occurring in pancreatic cancer, where fibroblasts and myofibroblasts are activated to produce extracellular matrix components such as decorin. On the contrary, decorin expression is found downregulated by at least 50% in 2 types of nonsmall cell lung cancer, adenocarcinoma and squamous cell carcinoma.65 Although this study is limited by the small sample size, only 7 patients with each kind of carcinoma were analyzed, decorin was found significantly downregulated in 4 out 7 adenocarcinomas and 6 out of 7 squamous carcinomas. It is important to note that decorin expression levels are directly proportional to the amount of tumor stroma. A tissue in which tumor cells are predominant over the stromal components is expected to accumulate less decorin. This could explain the opposite results in terms of decorin expression in the 2 studies presented above.
Decorin expression in cancer can also be altered by transcriptional, post-transcriptional and post-translational modifications. Decorin appears to be upregulated and abnormally retained inside the cells obtained from dysplastic oral mucosa.66 This outcome is explained by the production of longer decorin transcripts. In ovarian tumors, decorin is expressed by the myofibroblasts, whereas the tumor cells only express decorin transcripts but no protein.67 Notably, decorin production is re-established following treatment with a proteasome inhibitor, suggesting that ovarian cancer cells developed a mechanism for rapid degradation of decorin.67
There are very few studies that analyze the prognostic significance of decorin expression levels in terms of patient survival. In one study, high expression of decorin in patients with advanced ovarian cancer was associated with poor response to treatment and a greater incidence of relapse for those patients that initially responded.68 Reduced amounts of decorin were, for the first time, associated with poor prognosis in node-negative invasive breast cancer69 and some types of soft tissue tumors.70 Specifically, lower decorin levels correlated with larger primary breast tumor burden, higher risk of early recurrence and overall poor prognosis. Breast cancer patients with tumors expressing high EGFR and low decorin levels resulted into an even poorer outcome. This reinforces the notion that decorin is a key player in EGFR signaling modulation in vivo. Low decorin levels in liposarcomas and malignant peripheral nerve sheath tumors are also associated with lower disease-free and survival rates. The fact that decorin levels were found in secondary lesions at levels even lower than primary lesions in soft tissue tumors, suggests that loss of decorin is permissive for metastatic spreading.
In normal tissues, the ratio of chondroitin and dermatan sulfate side chains associated with decorin is balanced. In tumor tissue, commonly, the chondroitin sulfate chains become predominant. This post-translational modification has been reported in colon,49, 71 ovarian,67 gastric72 and pancreatic carcinoma.73 Dermatan sulfate appeared more recently in the evolutionary tree than chondroitin sulfate as the former is chemically more complex and requires additional enzymes to be synthesized. Therefore, it is reasonable to think that the synthesis of the chemically simpler chondroitin sulfate is favored in tumor tissue. In line with this concept, chondroitin sulfate side chains have been proposed to be more permissive to cell migration favoring tumor aggressiveness.74 It is not surprising that tumor cells, with very few exceptions, do not express decorin, a cytostatic and apoptotic protein, but the mechanism by which decorin expression is switched off is unclear and needs further investigation. Potentially, epigenetic control including hypermethylation of the promoter region might play a role in silencing decorin gene. In the tumor stroma, on the contrary, hypomethylation of the decorin promoter has been previously documented.49, 75 In addition, tumor cells can synthesize soluble factors that repress decorin expression by stromal cells, as in the case of pancreatic tumor cells affecting the surrounding stellate cells.64
We foresee that decorin will attract more interest in the future as an anticancer therapeutic. Considering that chemotherapy is still the leading therapy for cancer patients and that decorin could be administered in concomitance with various compounds, it is relevant to understand their biological interaction. Decorin shows to have synergistic effect with carboplatin in inhibiting ovarian cancer cell growth,76 whereas it antagonizes carboplatin and gemcitabine effects against pancreatic cancer cells.77 Because of lack of data and somewhat contrasting results, this area definitely needs more investigation.
Decorin in vivo
Following the accumulating evidence that decorin is a potent inhibitor of tumor cell proliferation in vitro, considerable effort has been recently applied to prove that decorin can be an antitumor therapeutic in vivo. Adenoviral-mediated delivery of decorin slows the growth of lung, squamous and colon carcinoma tumor xenografts in immunocompromised mice.78, 79 The same decorin delivery system retards MTLn3 mammary adenocarcinoma growth and prevents metastatic spreading to the lungs.40 When MTLn3 cells are engineered to express decorin under an inducible promoter, they show a considerable reduction in ErbB2 receptor levels, which is overexpressed in this cell line as in ∼30% of human breast cancers. Moreover, these cells exhibit growth retardation in vivo and impaired ability to form pulmonary metastases. In line with these results, a wide-screen proteomic study of changes induced by decorin in a breast carcinoma cell line indicated that several key proteins involved in invasion are affected.80 Major decorin-evoked changes include suppression of several breast cancer-associated biomarkers, a reduction in membrane ruffling, usually linked to inhibition of cell motility/migration, and a concurrent increase in cell–cell adhesion.80 Moreover, decorin-expressing murine osteosarcoma cells show impaired ability to form lung metastases.81 Interestingly, in this model, the primary tumor volumes are not affected. This is explained by decorin's ability to impair only tumor cell migration and invasion and not proliferation. In a rat glioma model, ectopic expression of decorin prolongs the survival of the animals and the size of the tumor is directly proportional to how early and how much decorin is expressed.82
The first evidence that decorin can be administered as therapeutic agent against cancer was achieved in the A431 squamous carcinoma model. Systemically injected decorin protein core specifically localizes within the tumor, antagonizes EGFR activity and induces apoptosis of the tumor cells.39 The outcome is primary tumor growth inhibition due to slower growth rate combined with apoptosis and impaired tumor metabolism. This reduction in metabolism is likely to be due to less tumor angiogenesis and/or tumor glycolysis. A recent report from our group proved that decorin injected systemically can reduce breast tumor growth and metabolism and halt metastatic spread to the lungs.83 The finding that ectopic expression of decorin can revert the malignant phenotype in several cell lines of various histogenetic backgrounds28, 84 and can antagonize primary tumor growth and metastases in vivo further raises a hope for the postulated clinical application of decorin and related molecules.
LRIG1 and lumican
LRIG1 is a leucine-rich protein containing immunoglobulin-like repeats and is likely the human ortholog of the Drosophila Kekkon-1 gene.85Kekkon-1 is a transmembrane protein that was discovered as part of the EGFR negative feedback signaling loop.86, 87 EGF stimulation induces Kekkon-1 expression, which localizes at the cell surface and binds the EGFR leading to its downregulation. LRIG1 has also been implicated in the negative regulation of the EGFR and other members of the ErbB family of receptor tyrosine kinases88 by inducing their degradation.89 Recent evidence indicates that LRIG1 is a key regulator of the quiescent state of epidermal stem cells.90 In addition to downregulating ErbB receptors, LRIG1 has been shown to destabilize the Met receptor,91 which is an important player in tumor cells growth and invasion. Our group has shown that LRIG1 is homologous to decorin and has potential as a cancer growth inhibitor when used in a soluble form.92 LRIG1 has been proposed as a prognostic factor in astrocytic tumors and cutaneus squamous cell carcinomas, where its high expression is associated with well-differentiated lesions and overall improved survival rate.93, 94 Notably, LRIG1 expression levels are inversely related to the metastatic rate. The role of LRIG1 in colon cancer is dubious due to high heterogeneity of expression in patients and no evident correlation to EGFR expression.95 LRIG1 has been proposed to be a tumor suppressor whose expression is a predictor of survival in early stages of cervical cancer.96 Knock-out mice for the Lrig1 gene develop psoriasiform epidermal hyperplasia,97 characterized by deregulated epidermal proliferation and differentiation. In psoriatic lesions from patients, LRIG1 mRNA levels appear to be similar to control epidermis but the protein is not correctly transported to the cell surface,98 where it can function as negative regulator of the EGFR. Indeed, human psoriasis has been associated with EGFR overexpression as well as upregulated growth factor signaling. Remarkably, transgenic mice overexpressing TGF-α99 or amphiregulin,100 both EGFR ligands, show a phenotype resembling the Lrig1-/-mice. This model of disease represents further proof for the role of LRIG1 in balancing EGFR signaling and promoting epidermal homeostasis. The recent discovery that LRIG1 interacts and modulates another tyrosine kinase receptor, the Ret receptor,101 strongly suggests that these leucine-rich proteins, in analogy to decorin and other SLRPs, might represent an interactive layer of regulators for various tyrosine kinase receptors.
Lumican inhibits cell proliferation by inducing p21 expression and antagonizes anchorage-independent cell growth as well as cell migration.102 Lack of lumican has been shown to favor mouse embryonic fibroblasts and stromal keratocytes proliferation, and at the same time, inhibit apoptosis via downregulation of p53 and Fas.103 Furthermore, cleavage of lumican by the membrane-type matrix metalloprotease-1 abrogates lumican-evoked suppression of colony formation.102 Interestingly, and on the whole contrary to decorin, lumican is expressed by some tumor cell lines. The expression of lumican is 7-fold higher in well-differentiated less metastatic rather than moderately differentiated highly metastatic human osteosarcoma cells, Saos2 and MG-63, respectively.104 When lumican expression is inhibited via RNA interference, Saos2 cell growth is promoted, whereas MG-63 cells do not show any significant changes in their behavior. In the same study, lumican effects on cell growth and migration were shown to be linked to the TGFβ receptor pathway.104 B16F1 mouse melanoma cells do not express lumican. Melanoma cells transfected to express lumican or treated with recombinant protein show impaired anchorage-independent growth and capacity to invade the extracellular matrix.105 In this experimental setting, lumican did not affect cell proliferation in vitro; however, it induced apoptosis in vivo. Moreover, lumican may regulate vertical progression of human malignant melanoma.50 From the in vitro data using melanoma cells, it appears as lumican's mechanism of action is different from decorin's regarding the effects on cell proliferation. To understand the differences between decorin and lumican effects, more detailed studies on lumican-evoked signaling are needed, which most likely will reveal the involvement of various cell surface receptors.
Lumican-expressing melanoma cells are less tumorigenic in vivo and form significantly smaller tumors. Lumican has been reported to be more abundant than decorin in human breast carcinomas,106 even though the clinical relevance of this observation has not yet been explained. More importantly, lumican expression has been proposed as a prognostic factor in lymph node-negative breast69 and colorectal107 cancers. Both studies were performed on fairly large cohorts and report rather contrasting results. Low lumican levels were associated with larger breast tumor size and overall poorer survival, whereas, in colon cancer, higher lumican expression correlated with tumor invasion and metastatic spreading. These data could be explained by differences in lumican role in both cellular and matrix components, differences between the 2 types of cancer and by different growth factors and hormones available, which may affect lumican expression. It will be interesting in the future to test the effects of recombinant lumican on tumor growth and metastases. Additional studies carried out with different cancer types are required to dissect the mechanism of action of lumican.
Small proteoglycans and related proteins harboring leucine-rich tandem motifs are emerging as key modulators of the tumor microenvironment, especially of “solid” tumors where the development of the neoplastic mass requires months to years, thereby allowing a complex stromal network to emerge and concurrently develop. The take-home message from the studies summarized above is that all these molecules interact with a number of tyrosine kinase receptors which are often deregulated during cancer growth, due to overexpression, genome amplification or activating mutations. Thus, decorin and related molecules provide an additional layer of growth control toward the malignant phenotype.
Decorin biological activity is rather complex because of the fact that it regulates multiple processes in the extracellular matrix as well as in the tumor cells. Decorin mainly signals through binding the EGFR, and this interaction leads to prolonged inhibitory effects and apoptosis in tumor cells. Decorin is not only a pan-ErbB receptors inhibitor, but can also signal through the IGF-IR and TGFβ pathways. This multitude of activities can in part explain the dramatic in vivo effects in halting tumor growth and metastatic spreading. Decorin expression is generally antagonized and modified in cancer patients. It is no surprise that low decorin levels are associated with poor prognosis. The data collected from the studies conducted in mice and the few clinical statistics on decorin expression in cancer patients suggest that decorin might be utilized in the near future as an adjunct “protein therapeutic” for solid tumors in which receptor tyrosine kinases play a key role. Additional in vivo studies with various tumor models are desirable as well as studies to assess the functional interaction between decorin and existing anticancer chemotherapeutics to evaluate potential limitations. Lumican displays similar activity and appears to be a promising antitumor molecule, although more specific information about its mode of action needs to be gained. Another emerging member of the leucine-rich family of proteins is LRIG1, which binds and downregulates the EGFR, Met and Ret receptors. Thus, we support the hypothesis that leucine-rich proteins or derived peptides could be utilized in the fight against cancer, and we hope to attract interest in this field of natural inhibitors of cancer growth.
We thank Ms. Angela McQuillan for providing help with the illustrations.