Nikos K. Karamanos

Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting

Matrix metalloproteinases (MMPs) consist of a multigene family of zinc‐dependent extracellular matrix (ECM) remodeling endopeptidases implicated in pathological processes, such as carcinogenesis. In this regard, their activity plays a pivotal role in tumor growth and the multistep processes of invasion and metastasis, including proteolytic degradation of ECM, alteration of the cell–cell and cell–ECM interactions, migration and angiogenesis. The underlying premise of the current minireview is that MMPs are able to proteolytically process substrates in the extracellular milieu and, in so doing, promote tumor progression. However, certain members of the MMP family exert contradicting roles at different stages during cancer progression, depending among other factors on the tumor stage, tumor site, enzyme localization and substrate profile. MMPs are therefore amenable to therapeutic intervention by synthetic and natural inhibitors, providing perspectives for future studies. Multiple therapeutic agents, called matrix metalloproteinase inhibitors (MMPIs) have been developed to target MMPs, attempting to control their enzymatic activity. Even though clinical trials with these compounds do not show the expected results in most cases, the field of MMPIs is ongoing. This minireview critically evaluates the role of MMPs in relation to cancer progression, and highlights the challenges, as well as future prospects, for the design, development and efficacy of MMPIs.

Proteoglycans in health and disease: novel roles for proteoglycans in malignancy and their pharmacological targeting

The expression of proteoglycans (PGs), essential macromolecules of the tumor microenvironment, is markedly altered during malignant transformation and tumor progression. Synthesis of stromal PGs is affected by factors secreted by cancer cells and the unique tumor‐modified extracellular matrix may either facilitate or counteract the growth of solid tumors. The emerging theme is that this dual activity has intrinsic tissue specificity. Matrix‐accumulated PGs, such as versican, perlecan and small leucine‐rich PGs, affect cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Furthermore, expression of cell‐surface‐associated PGs, such as syndecans and glypicans, is also modulated in both tumor and stromal cells. Cell‐surface‐associated PGs bind various factors that are involved in cell signaling, thereby affecting cell proliferation, adhesion and motility. An important mechanism of action is offered by a proteolytic processing of cell‐surface PGs known as ectodomain shedding of syndecans; this facilitates cancer and endothelial cell motility, protects matrix proteases and provides a chemotactic gradient of mitogens. However, syndecans on stromal cells may be important for stromal cell/cancer cell interplay and may promote stromal cell proliferation, migration and angiogenesis. Finally, abnormal PG expression in cancer and stromal cells may serve as a biomarker for tumor progression and patient survival. Enhanced understanding of the regulation of PG metabolism and the involvement of PGs in cancer may offer a novel approach to cancer therapy by targeting the tumor microenvironment. In this minireview, the implication of PGs in cancer development and progression, as well as their pharmacological targeting in malignancy, are presented and discussed.

Glycosaminoglycans: key players in cancer cell biology and treatment

Glycosaminoglycans are natural heteropolysaccharides that are present in every mammalian tissue. They are composed of repeating disaccharide units that consist of either sulfated or non‐sulfated monosaccharides. Their molecular size and the sulfation type vary depending on the tissue, and their state either as part of proteoglycan or as free chains. In this regard, glycosami‐noglycans play important roles in physiological and pathological conditions. During recent years, cell biology studies have revealed that glycosaminoglycans are among the key macromolecules that affect cell properties and functions, acting directly on cell receptors or via interactions with growth factors. The accumulated knowledge regarding the altered structure of glycosaminoglycans in several diseases indicates their importance as biomarkers for disease diagnosis and progression, as well as pharmacological targets. This review summarizes how the fine structural characteristics of glycosaminoglycans, and enzymes involved in their biosynthesis and degradation, are involved in cell signaling, cell function and cancer progression. Prospects for glycosaminoglycan‐based therapeutic targeting in cancer are also discussed.

Hyaluronan–CD44 interactions as potential targets for cancer therapy

It is becoming increasingly clear that signals generated in tumor microenvironments are crucial to tumor cell behavior, such as survival, progression and metastasis. The establishment of these malignant behaviors requires that tumor cells acquire novel adhesion and migration properties to detach from their original sites and to localize to distant organs. CD44, an adhesion/homing molecule, is a major receptor for the glycosaminoglycan hyaluronan, which is one of the major components of the tumor extracellular matrix. CD44, a multistructural and multifunctional molecule, detects changes in extracellular matrix components, and thus is well positioned to provide appropriate responses to changes in the microenvironment, i.e. engagement in cell–cell and cell–extracellular matrix interactions, cell trafficking, lymph node homing and the presentation of growth factors/cytokines/chemokines to co‐ordinate signaling events that enable the cell responses that change in the tissue environment. The potential involvement of CD44 variants (CD44v), especially CD44v4–v7 and CD44v6–v9, in tumor progression has been confirmed for many tumor types in numerous clinical studies. The downregulation of the standard CD44 isoform (CD44s) in colon cancer is postulated to result in increased tumorigenicity. CD44v‐specific functions could be caused by their higher binding affinity than CD44s for hyaluronan. Alternatively, CD44v‐specific functions could be caused by differences in associating molecules, which may bind selectively to the CD44v exon. This minireview summarizes how the interaction between hyaluronan and CD44v can serve as a potential target for cancer therapy, in particular how silencing CD44v can target multiple metastatic tumors.

Extracellular matrix structure

Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented.

The impact of zoledronic acid therapy in survival of lung cancer patients with bone metastasis.

Bone metastases occur in 20–40% of patients with lung cancer. Recent studies demonstrate a direct antiproliferative effect of 3rd generation bisphosphonates (BPs) on lung tumors, which may influence the survival. Therefore, we examined the clinical impact of zoledronic acid (ZOL; Zometa®), a 3rd generation BP, with a focus on the survival, time to progression and pain effect in lung cancer patients with bone metastases. Lung cancer patients (n = 144, Stage IV) with evidence of metastasis bone scan were included. Eighty‐seven of 144 experienced bone pain and received ZOL, 4 mg i.v. every 21 days (Group A), whereas the other 57 patients received no ZOL (Group B). All patients were treated with a combination chemotherapy consisted of docetaxel 100 mg/m2 and carboplatin AUC = 6. It was found that Group A had a statistically significant longer survival (p < 0.01) when compared to Group B. A statistically significant positive correlation was found between the number of cycles of therapy with ZOL and total patient survival (p < 0.01, Pearson correlation) and time to progression (p < 0.01). Pain effect of ZOL had no significant difference between the 2 groups of patients (p > 0.05). Urine N‐telopeptide of type I collagen (NTx) levels decreased in patients with NTx ≤ 29 nM BCE/mM creatinine at baseline after treatment with ZOL. The results of our study suggest that the addition of ZOL increases overall survival in lung cancer patients with bone metastases. The longer period of receiving ZOL, the better effect on survival and time to progression.

Ion-pair high-performance liquid chromatography for determining disaccharide composition in heparin and heparan sulphate.

In this report we describe a convenient and sensitive HPLC method for separating and determining the non- and variously sulphated delta-disaccharides derived from heparan sulphate, heparin and Fragmin, using heparin- and heparan sulphate lyases. This method is superior to others since it can separate and determine twelve different non-, mono-, di- and trisulphated delta-disaccharides containing either N-sulphated, N-acetylated or unsubstituted glucosamine in a single HPLC run. The various types of delta-disaccharides are separated by an ion-pair reversed-phase chromatographic procedure on a Supelcosil LC-18 column, using a binary acetonitrile gradient system with tetrabutylammonium as the ion-pairing reagent. The eluted peaks were recorded by dual wavelength at 232 and 226 nm and a linear detector response was obtained over the entire interval tested, i.e., to 50 micrograms of delta-disaccharides. As little as 0.8-5 ng of delta-disaccharides can be reliably detected and accurately determined. Following separate digestion with the heparin- and heparan sulphate lyases (heparin lyases I, II and III), the characteristic heparin delta-disaccharides in the heparan sulphate chain, as well as the heparan sulphate delta-disaccharides in the heparin polymer, can be identified. Using combined digestions with these three lyases, the glycosaminoglycan chains are degraded almost completely (> 90%) to delta-disaccharides, which are then determined by direct injections into the HPLC system and thus an almost complete spectrum of disaccharide composition can be obtained. By this method, it is possible to analyse and confirm that the heparan sulphate chain is defined as a glycosaminoglycan dominated by GlcNAc(+/- 6S)-GlcA disaccharides and by some copolymeric disaccharides, such as GlcNS-IdoA2S and GlcNS6S-IdoA2S, otherwise most common in heparin. Fragmin, which is a controlled cepolymerized heparin fragment of M(r) 5000, is made up mainly of trisulphated disaccharides of the GlcNS6S-IdoA2S type (88.8%). Using separate digestions with the specific heparin lyases, one can also distinguish between heparin and heparan sulphate.

Recent Advances in the Structural Study of Functional Chondroitin Sulfate and Dermatan Sulfate in Health and Disease

Chondroitin sulfate (CS) dermatan sulfate (DS), and CS/DS hybrid chains are biologically active like heparan sulfate, and structurally the most complex species of the glycosaminoglycan family along with heparan sulfate. They exist at the cell surface and extracellular matrix in the form of proteoglycans. They function as regulators of functional proteins such as growth factors, cytokines, chemokines, adhesion molecules, and lipoproteins through interactions with the ligands of these proteins via specific saccharide domains. Structural alterations have been often implicated in pathological conditions, such as cancer and atherosclerosis. Recent microsequencing of CS/DS oligosaccharides that bind growth factors, such as pleiotrophin, and various monoclonal antibodies against CS/DS, have revealed a considerable number of unique oligosaccharide sequences. This review focuses on recent advances in the study of the structure-function relation of CS, DS and their hybrid chains in physiological and pathological conditions.

Determination of hyaluronan and galactosaminoglycan disaccharides by high-performance capillary electrophoresis at the attomole level. Applications to analyses of tissue and cell culture proteoglycans

A rapid, highly sensitive and reproducible HPCE method is described for the determination of all non- and variously sulphated disaccharides present in hyaluronan and vertebrate chondroitin sulphates and dermatan sulphates. Following chondroitinase digestion of glycosaminoglycans or proteoglycans, the non-, di- and tri-sulphated delta-disaccharides are completely separated and readily determined within 14 min on a fused-silica capillary in 15 mM sodium dihydrogen orthophosphate, pH 3.00, using reversed polarity at 20 kV and detection at 232 nm. The determination of the various delta-disaccharides derived from either glucuronic or iduronic acid and the presence of glucuronic and iduronic clustered structures in dermatan sulphate can also easily be made, using digests with chondroitinase AC or B. A linear detector response was obtained for the entire interval tested (up to 10 mg/l of delta-disaccharides). Concentrations as small as 32, 65, 100 and 250 pmol/l (22, 38, 50 and 98 ng/l) of tri-, di- and nonsulphated delta-disaccharides, respectively, can be reliably detected.

Determination of twelve heparin- and heparan sulfate-derived disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced fluorescence detection

In quest for high sensitivities, we developed an ultrahigh capillary electrophoresis (CE) method for the structural analysis of heparin and heparan sulfate (HS) in biologic samples. Heparin and HS were digested with an equi‐unit mixture of heparin lyases I, II and III and the obtained Δ‐disaccharides were derivatized with the fluorophore 2‐aminoacridone. All known twelve non‐, mono‐, di‐ and trisulfated Δ‐disaccharides were completely resolved in a single run, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV. Relative standard deviation in migration times and peak areas as well as day‐to‐day variance ranged from 0.9 to 2.4%, suggesting a reproducible and precise method. Detection of 2‐aminoacridone (AMAC)‐derivatives of Δ‐disaccharides by UV at 255 nm showed 2.8 and 10 times higher sensitivity than that of derivatized and nonderivatized ones at 232 nm. Laser‐induced fluorescence detection with an Ar‐ion laser source showed an approximately 100 times higher sensitivity than that obtained at 232 nm of the nonderivatized species. Application of this method to quantitative analysis of Δ‐disaccharides derived from porcine intestinal mucosa heparin and bovine kidney HS showed excellent agreement with previously published methods, suggesting an accurate method. The developed method can be easily applied for the disaccharide analysis of heparin/HS at the attomole level with high accuracy, for distinguishing between heparin and HS and may be of value for studying their interactions with matrix effective molecules.