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17-Isovalerate esters of ferrediol and sideritriol (compounds 1 and 5 ) and 17-(2-methylbutyrate) ferrediol (compound 2 ), which have not been earlier described, were isolated along with five known ent -15-kaurene diterpenes (siderol, sideridiol, sideroxol, eubotriol, and epi -candicandiol), one ent -beyerene (flavovirol), and one phytosterol (β-sitosterol) from the aerial parts of Sideritis clandestina subsp. peloponnesiaca (Boiss. & Heldr.) Baden that is a mountain tea species endemic to Northern Peloponnese in Greece. Compounds 1 - 5 exhibited weak antiproliferative activity against triple-negative MDA-MB-231 and Hs578T breast cancer cells. In MCF-7 breast cancer cells, compounds 1 , 2 , siderol, and sideridiol were equipotent to 5-fluorouracil (moderate antiproliferative activity)., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: Testicular germ cell tumors (TGCTs) exhibit diverse biological and pathological features and are divided in two main types, seminomas and nonseminomatous germ cell tumors (NSGCTs). CD44 is a cell surface receptor, which is highly expressed in malignancies and is implicated in tumorigenesis affecting cell-matrix interactions and cell signaling., Methods and Results: Here, we examined the expression of CD44 in tumor cell lines and in patients' material. We found that CD44 is over-expressed in TGCTs compared to normal tissues. Immunohistochemical staining in 71 tissue specimens demonstrated increased expression of CD44 in some patients, whereas CD44 was absent in normal tissue. In seminomas, a high percentage of tumor and stromal cells showed cytoplasmic and/or cell surface staining for CD44 as well as increased staining for CD44 in the tumor stroma was found in some cases. The increased expression of CD44 either in tumor cells or in stromal components was associated with tumor size, nodal metastasis, vascular/lymphatic invasion, and disease stage only in seminomas. The increased stromal expression of CD44 in TGCTs was positively associated with angiogenesis., Conclusions: CD44 may exhibit diverse biological functions in seminomas and NSGCTs. The expression of CD44 in tumor cells as well as in tumor stroma fosters an aggressive phenotype in seminomas and should be considered in disease treatment., (© 2024. The Author(s).)

Serglycin (SRGN) is a pro-tumorigenic proteoglycan expressed and secreted by various aggressive tumors including glioblastoma (GBM). In our study, we investigated the interplay and biological outcomes of SRGN with TGFβRI, CXCR-2 and inflammatory mediators in GBM cells and fibroblasts. SRGN overexpression is associated with poor survival in GBM patients. High SRGN levels also exhibit a positive correlation with increased levels of various inflammatory mediators including members of TGFβ signaling pathway, cytokines and receptors including CXCR-2 and proteolytic enzymes in GBM patients. SRGN-suppressed GBM cells show decreased expressions of TGFβRI associated with lower responsiveness to the manipulation of TGFβ/TGFβRI pathway and the regulation of pro-tumorigenic properties. Active TGFβRI signaling in control GBM cells promotes their proliferation, invasion, proteolytic and inflammatory potential. Fibroblasts cultured with culture media derived by control SRGN-expressing GBM cells exhibit increased proliferation, migration and overexpression of cytokines and proteolytic enzymes including CXCL-1, IL-8, IL-6, IL-1β, CCL-20, CCL-2, and MMP-9. Culture media derived by SRGN-suppressed GBM cells fail to induce the above properties to fibroblasts. Importantly, the activation of fibroblasts by GBM cells not only relies on the expression of SRGN in GBM cells but also on active CXCR-2 signaling both in GBM cells and fibroblasts.

Breast cancer is the leading cause of cancer deaths for women worldwide. Endocrine therapies represent the cornerstone for hormone-dependent breast cancer treatment. However, in many cases, endocrine resistance is induced with poor prognosis for patients. In the current study, we have developed MCF-7 cell lines resistant to fulvestrant (MCF-7Fulv) and tamoxifen (MCF-7Tam) aiming at investigating mechanisms underlying resistance. Both resistant cell lines exerted lower proliferation capacity in two-dimensional (2-D) cultures but retain estrogen receptor α (ERα) expression and proliferate independent of the presence of estrogens. The established cell lines tend to be more aggressive exhibiting advanced capacity to form colonies, increased expression of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and heterodimerization of ERBB family receptors and activation of EGFR downstream pathways like MEK/ERK1/2 and PI3K/AKT. Tyrosine kinase inhibitors tested against resistant MCF-7Fulv and MCF-7Tam cells showed moderate efficacy to inhibit cell proliferation, except for lapatinib, which concomitantly inhibits both EGFR and HER2 receptors and strongly reduced cell proliferation. Furthermore, increased autophagy was observed in resistant MCF-7Fulv and MCF-7Tam cells as shown by the presence of autophagosomes and increased Beclin-1 levels. The increased autophagy in resistant cells is not associated with increased apoptosis, suggesting a cytoprotective role for autophagy that may favor cells' survival and aggressiveness. Thus, by exploiting those underlying mechanisms, new targets could be established to overcome endocrine resistance. NEW & NOTEWORTHY The development of resistance to hormone therapy caused by both fulvestrant and tamoxifen promotes autophagy with concomitant apoptosis evasion, rendering cells capable of surviving and growing. The fact that resistance also triggers ERBB family signaling pathways, which are poorly inhibited by tyrosine kinase inhibitors might attribute to cells' aggressiveness. It is obvious that the development of endocrine therapy resistance involves a complex interplay between deregulated ERBB signaling and autophagy that may be considered in clinical practice.

Background: Osteoarthritis (OA) is a common degenerative chronic disease accounting for physical pain, tissue stiffness and mobility restriction. Current therapeutic approaches fail to prevent the progression of the disease considering the limited knowledge on OA pathobiology. During OA progression, the extracellular matrix (ECM) of the cartilage is aberrantly remodeled by chondrocytes. Chondrocytes, being the main cell population of the cartilage, participate in cartilage regeneration process. To this end, modern tissue engineering strategies involve the recruitment of mesenchymal stem cells (MSCs) due to their regenerative capacity as to promote chondrocyte self-regeneration., Methods and Results: In the present study, we evaluated the role of type II collagen, as the main matrix macromolecule in the cartilage matrix, to promote chondrogenic differentiation in two MSC in vitro culture systems. The chondrogenic differentiation of human Wharton's jelly- and dental pulp-derived MSCs was investigated over a 24-day culture period on type II collagen coating to improve the binding affinity of MSCs. Functional assays, demonstrated that type II collagen promoted chondrogenic differentiation in both MSCs tested, which was confirmed through gene and protein analysis of major chondrogenic markers., Conclusions: Our data support that type II collagen contributes as a natural bioscaffold enhancing chondrogenesis in both MSC models, thus enhancing the commitment of MSC-based therapeutic approaches in regenerative medicine to target OA and bring therapy closer to the clinical use., (© 2023. The Author(s).)

Proteoglycans (PGs) are pivotal components of extracellular matrices, involved in a variety of processes such as migration, invasion, morphogenesis, differentiation, drug resistance, and epithelial-to-mesenchymal transition (EMT). Cellular plasticity is a crucial intermediate phenotypic state acquired by cancer cells, which can modulate EMT and the generation of cancer stem cells (CSCs). PGs affect cell plasticity, stemness, and EMT, altering the cellular shape and functions. PGs control these functions, either by direct activation of signaling cascades, acting as co-receptors, or through regulation of the availability of biological compounds such as growth factors and cytokines. Differential expression of microRNAs is also associated with the expression of PGs and their interplay is implicated in the fine tuning of cancer cell phenotype and potential. This review summarizes the involvement of PGs in the regulation of EMT and stemness of cancer cells and highlights the molecular mechanisms.

Dehydroabietic Acid (DHA, 1 ) derivatives are known for their antiproliferative properties, among others. In the context of this work, DHA was initially modified to two key intermediates bearing a C18 methyl ester, a phenol moiety at C12, and an acetyl or formyl group at C13 position. These derivatives allowed us to synthesize a series of DHA-chalcone hybrids, suitable for structure-activity relationship studies (SARS), following their condensation with a variety of aryl-aldehydes and methyl ketones. The antiproliferative evaluation of the synthesized DHA-chalcone hybrids against three breast cancer cell lines (the estrogen-dependent MCF-7 and the estrogen-independent MDA-MB-231 and Hs578T) showed that eight derivatives ( 33, 35, 37, 38, 39, 41, 43, 44 ) exhibit low micromolar activity levels (IC 50 2.21-11.5 μΜ/MCF-7). For instance, some of them showed better activity compared to the commercial anticancer drug 5-FU against MCF-7 cells ( 33, 41, 43, 44 ) and against MDA-MB231 ( 33 and 41 ). Hybrid 38 is a promising lead compound for the treatment of MCF-7 breast cancer, exhibiting comparable activity to 5-FU and being 12.9 times less toxic (SI = 22.7). Thus, our findings suggest that DHA-chalcone hybrids are drug candidates worth pursuing for further development in the search for novel breast cancer therapies.

Serglycin is a proteoglycan highly expressed by immune cells, in which its functions are linked to storage, secretion, transport, and protection of chemokines, proteases, histamine, growth factors, and other bioactive molecules. In recent years, it has been demonstrated that serglycin is also expressed by several other cell types, such as endothelial cells, muscle cells, and multiple types of cancer cells. Here, we show that serglycin expression is upregulated in transforming growth factor beta (TGF-β) induced epithelial-mesenchymal transition (EMT). Functional studies provide evidence that serglycin plays an important role in the regulation of the transition between the epithelial and mesenchymal phenotypes, and it is a significant EMT marker gene. We further find that serglycin is more expressed by breast cancer cell lines with a mesenchymal phenotype as well as the basal-like subtype of breast cancers. By examining immune staining and single cell sequencing data of breast cancer tissue, we show that serglycin is highly expressed by infiltrating immune cells in breast tumor tissue., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with the author ADT., (Copyright © 2022 Tellez-Gabriel, Tekpli, Reine, Hegge, Nielsen, Chen, Moi, Normann, Busund, Calin, Mælandsmo, Perander, Theocharis, Kolset and Knutsen.)

Extracellular matrix (ECM) is a dynamic 3-dimensional network of macromolecules that provides structural support for the cells and tissues. Accumulated knowledge clearly demonstrated over the last decade that ECM plays key regulatory roles since it orchestrates cell signaling, functions, properties and morphology. Extracellularly secreted as well as cell-bound factors are among the major members of the ECM family. Proteins/glycoproteins, such as collagens, elastin, laminins and tenascins, proteoglycans and glycosaminoglycans, hyaluronan, and their cell receptors such as CD44 and integrins, responsible for cell adhesion, comprise a well-organized functional network with significant roles in health and disease. On the other hand, enzymes such as matrix metalloproteinases and specific glycosidases including heparanase and hyaluronidases contribute to matrix remodeling and affect human health. Several cell processes and functions, among them cell proliferation and survival, migration, differentiation, autophagy, angiogenesis, and immunity regulation are affected by certain matrix components. Structural alterations have been also well associated with disease progression. This guide on the composition and functions of the ECM gives a broad overview of the matrisome, the major ECM macromolecules, and their interaction networks within the ECM and with the cell surface, summarizes their main structural features and their roles in tissue organization and cell functions, and emphasizes the importance of specific ECM constituents in disease development and progression as well as the advances in molecular targeting of ECM to design new therapeutic strategies., (© 2021 Federation of European Biochemical Societies.)

The extracellular matrix is engaged in an ever-evolving and elegant ballet of dynamic reciprocity that directly and bi-directionally regulates cell behavior. Homeostatic and pathophysiological changes in cell-matrix signaling cascades manifest as complex matrix phenotypes. Indeed, the extracellular matrix can be implicated in virtually every known human disease, thus, making it the most critical and dynamic "organ" in the human body. The overall goal of this Special Issue is to provide an accurate and inclusive functional definition that addresses the inherent complexity of matrix phenotypes. This goal is summarily achieved via a corpus of expertly written articles, reviews and original research, focused at answering this question empirically and fundamentally via state-of-the-art methods and research strategies., Competing Interests: The authors declare no conflicts of interest., (© 2020 The Author(s).)

Numerous studies point out serglycin as an important regulator of tumorigenesis in a variety of malignancies. Serglycin expression correlates with the aggressive phenotype of tumor cells and serves as a poor prognostic indicator for disease progression. Although serglycin is considered as an intracellular proteoglycan, it is also secreted in the extracellular matrix by tumor cells affecting cell properties, oncogenic signaling and exosomes cargo. Serglycin directly interacts with CD44 and possibly other cell surface receptors including integrins, evoking cell adhesion and signaling. Serglycin also creates a pro-inflammatory and pro-angiogenic tumor microenvironment by regulating the secretion of proteolytic enzymes, IL-8, TGFβ2, CCL2, VEGF and HGF. Hence, serglycin activates multiple signaling cascades that drive angiogenesis, tumor cell growth, epithelial to mesenchymal transition, cancer cell stemness and metastasis. The interference with the tumorigenic functions of serglycin emerges as an attractive prospect to target malignancies., (Copyright © 2019. Published by Elsevier Ltd.)

Despite the functional role of serglycin as an intracellular proteoglycan, a variety of malignant cells depends on its expression and constitutive secretion to advance their aggressive behavior. Serglycin arose to be a biomarker for glioblastoma, which is the deadliest and most treatment-resistant form of brain tumor, but its role in this disease is not fully elucidated. In our study we suppressed the endogenous levels of serglycin in LN-18 glioblastoma cells to decipher its involvement in their malignant phenotype. Serglycin suppressed LN-18 (LN-18 shSRGN ) glioblastoma cells underwent astrocytic differentiation characterized by induced expression of GFAP, SPARCL-1 and SNAIL, with simultaneous loss of their stemness capacity. In particular, LN-18 shSRGN cells presented decreased expression of glioma stem cell-related genes and ALDH1 activity, accompanied by reduced colony formation ability. Moreover, the suppression of serglycin in LN-18 shSRGN cells retarded the proliferative and migratory rate, the invasive potential in vitro and the tumor burden in vivo. The lack of serglycin in LN-18 shSRGN cells was followed by G2 arrest, with subsequent reduction of the expression of cell-cycle regulators. LN-18 shSRGN cells also exhibited impaired expression and activity of proteolytic enzymes such as MMPs, TIMPs and uPA, both in vitro and in vivo. Moreover, suppression of serglycin in LN-18 shSRGN cells eliminated the activation of pro-tumorigenic signal transduction. Of note, LN-18 shSRGN cells displayed lower expression and secretion levels of IL-6, IL-8 and CXCR-2. Concomitant, serglycin suppressed LN-18 shSRGN cells demonstrated repressed phosphorylation of ERK1/2, p38, SRC and STAT-3, which together with PI3K/AKT and IL-8/CXCR-2 signaling control LN-18 glioblastoma cell aggressiveness. Collectively, the absence of serglycin favors an astrocytic fate switch and a less aggressive phenotype, characterized by loss of pluripotency, block of the cell cycle, reduced ability for ECM proteolysis and pro-tumorigenic signaling attenuation., Competing Interests: The authors declare that no conflict of interest exists., (© 2020 The Authors.)

Extracellular matrices (ECMs) are highly specialized and dynamic three-dimensional (3D) scaffolds into which cells reside in tissues. ECM is composed of a variety of fibrillar components, such as collagens, fibronectin, and elastin, and non-fibrillar molecules as proteoglycans, hyaluronan, and glycoproteins including matricellular proteins. These macromolecular components are interconnected forming complex networks that actively communicate with cells through binding to cell surface receptors and/or matrix effectors. ECMs exert diverse roles, either providing tissues with structural integrity and mechanical properties essential for tissue functions or regulating cell phenotype and functions to maintain tissue homeostasis. ECM molecular composition and structure vary among tissues, and is markedly modified during normal tissue repair as well as during the progression of various diseases. Actually, abnormal ECM remodeling occurring in pathologic circumstances drives disease progression by regulating cell-matrix interactions. The importance of matrix molecules to normal tissue functions is also highlighted by mutations in matrix genes that give rise to genetic disorders with diverse clinical phenotypes. In this review, we present critical and emerging issues related to matrix assembly in tissues and the multitasking roles for ECM in diseases such as osteoarthritis, fibrosis, cancer, and genetic diseases. The mechanisms underlying the various matrix-based diseases are also discussed. Research focused on the highly dynamic 3D ECM networks will help to discover matrix-related causative abnormalities of diseases as well as novel diagnostic tools and therapeutic targets., (© 2019 Federation of European Biochemical Societies.)

Extracellular matrix (ECM) maintains the structural integrity of tissues and regulates cell and tissue functions. ECM is comprised of fibrillar proteins, proteoglycans (PGs), glycosaminoglycans, and glycoproteins, creating a heterogeneous but well-orchestrated network. This network communicates with resident cells via cell-surface receptors. In particular, integrins, CD44, discoidin domain receptors, and cell-surface PGs and additionally voltage-gated ion channels can interact with ECM components, regulating signaling cascades as well as cytoskeleton configuration. The interplay of ECM with recipient cells is enriched by the extracellular vesicles, as they accommodate ECM, signaling, and cytoskeleton molecules in their cargo. Along with the numerous biological properties that ECM can modify, autophagy and angiogenesis, which are critical for tissue homeostasis, are included. Throughout development and disease onset and progression, ECM endures rearrangement to fulfill cellular requirements. The main responsible molecules for tissue remodeling are ECM-degrading enzymes including matrix metalloproteinases, plasminogen activators, cathepsins, and hyaluronidases, which can modify the ECM structure and function in a dynamic mode. A brief summary of the complex interplay between ECM macromolecules and cells in tissues and the contribution of ECM in tissue homeostasis and diseases is given.

Extracellular matrix is a highly dynamic macromolecular network. Proteoglycans are major components of extracellular matrix playing key roles in its structural organization and cell signaling contributing to the control of numerous normal and pathological processes. As multifunctional molecules, proteoglycans participate in various cell functions during morphogenesis, wound healing, inflammation and tumorigenesis. Their interactions with matrix effectors, cell surface receptors and enzymes enable them with unique properties. In malignancy, extensive remodeling of tumor stroma is associated with marked alterations in proteoglycans' expression and structural variability. Proteoglycans exert diverse functions in tumor stroma in a cell-specific and context-specific manner and they mainly contribute to the formation of a permissive provisional matrix for tumor growth affecting tissue organization, cell-cell and cell-matrix interactions and tumor cell signaling. Proteoglycans also modulate cancer cell phenotype and properties, the development of drug resistance and tumor stroma angiogenesis. This review summarizes the proteoglycans remodeling and their novel biological roles in malignancies with particular emphasis to the underlying molecular mechanisms., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The overall structure and architecture of the extracellular matrix undergo dramatic alterations in composition, form, and functionality over time. The stochasticity begins during development, essential for maintaining organismal homeostasis and is heavily implicated in many pathobiological states including fibrosis and cancer. Modeling and remodeling of the matrix is driven by the local cellular milieu and secreted and cell-associated components in a framework of dynamic reciprocity. This collection of expertly-written reviews aims to relay state-of-the-art information concerning the mechanisms of matrix modeling and remodeling in physiological development and disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Serglycin is an intracellular proteoglycan that is expressed and constitutively secreted by numerous malignant cells, especially prominent in the highly-invasive, triple-negative MDA-MB-231 breast carcinoma cells. Notably, de novo expression of serglycin in low aggressive estrogen receptor α (ERα)-positive MCF7 breast cancer cells promotes an aggressive phenotype. In this study, we discovered that serglycin promoted epithelial to mesenchymal transition (EMT) in MCF7 cells as shown by increased expression of mesenchymal markers vimentin, fibronectin and EMT-related transcription factor Snail2. These phenotypic traits were also associated with the development of drug resistance toward various chemotherapy agents and induction of their proteolytic potential as shown by the increased expression of matrix metalloproteinases, including MMP-1, MMP-2, MMP-9, MT1-MMP and up-regulation of urokinase-type plasminogen activator. Knockdown of serglycin markedly reduced the expression of these proteolytic enzymes in MDA-MB-231 cells. In addition, serglycin expression was closely linked to a pro-inflammatory gene signature including the chemokine IL-8 in ERα-negative breast cancer cells and tumors. Notably, serglycin regulated the secretion of IL-8 in breast cancer cells independently of their ERα status and promoted their proliferation, migration and invasion by triggering IL-8/CXCR2 downstream signaling cascades including PI3K, Src and Rac activation. Thus, serglycin promotes the establishment of a pro-inflammatory milieu in breast cancer cells that evokes an invasive mesenchymal phenotype via autocrine activation of IL-8/CXCR2 signaling axis., (Copyright © 2018 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

The extracellular matrix (ECM) constitutes a highly dynamic three-dimensional structural network comprised of macromolecules, such as proteoglycans/glycosaminoglycans (PGs/GAGs), collagens, laminins, fibronectin, elastin, other glycoproteins and proteinases. In recent years, the field of PGs has expanded rapidly. Due to their high structural complexity and heterogeneity, PGs mediate several homeostatic and pathological processes. PGs consist of a protein core and one or more covalently attached GAG chains, which provide the protein cores with the ability to interact with several proteins. The GAG building blocks of PGs significantly influence the chemical and functional properties of PGs. The primary goal of this comprehensive review is to summarize major achievements and paradigm-shifting discoveries made on the PG/GAG chemistry-biology axis, focusing on structural variability, structure-function relationships, metabolic, molecular, and epigenetic mechanisms underlying their synthesis. Recent insights related to exosome biogenesis, degradation, and cell signaling, their status as diagnostic tools and potential pharmacological targets in diseases as well as current applications in nanotechnology and biotechnology are addressed. Moreover, issues related to docking studies, molecular modeling, GAG/PG interaction networks, and their integration are discussed.

Extracellular matrix (ECM) is a dynamic network of macromolecules, playing a regulatory role in cell functions, tissue regeneration and remodeling. Wound healing is a tissue repair process necessary for the maintenance of the functionality of tissues and organs. This highly orchestrated process is divided into four temporally overlapping phases, including hemostasis, inflammation, proliferation and tissue remodeling. The dynamic interplay between ECM and resident cells exerts its critical role in many aspects of wound healing, including cell proliferation, migration, differentiation, survival, matrix degradation and biosynthesis. Several epigenetic regulatory factors, such as the endogenous non-coding microRNAs (miRNAs), are the drivers of the wound healing response. microRNAs have pivotal roles in regulating ECM composition during wound healing and dermal regeneration. Their expression is associated with the distinct phases of wound healing and they serve as target biomarkers and targets for systematic regulation of wound repair. In this article we critically present the importance of epigenetics with particular emphasis on miRNAs regulating ECM components (i.e. glycoproteins, proteoglycans and matrix proteases) that are key players in wound healing. The clinical relevance of miRNA targeting as well as the delivery strategies designed for clinical applications are also presented and discussed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are capable of degrading numerous extracellular matrix (ECM) components thus participating in physiological and pathological processes. Apart from the remodeling of ECM, they affect cell-cell and cell-matrix interactions and are implicated in the development and progression of various diseases such as cancer. Numerous studies have demonstrated that MMPs evoke epithelial to mesenchymal transition (EMT) of cancer cells and affect their signaling, adhesion, migration and invasion to promote cancer cell aggressiveness. Various studies have suggested MMPs as suitable targets for treatment of malignancies, and several MMP inhibitors (MMPIs) have been developed. Although initial trials have failed to establish MMPIs as anticancer agents due to lack of specificity and side effects, new MMPIs have been developed with improved action that are currently being investigated. Furthermore, novel strategies that target MMPs for improving drug delivery and regulating their activity in tumors are presented. This review summarizes the implication of MMPs in cancer progression and discusses the advancements in their targeting.

Glycosaminoglycans are integral part of the dynamic extracellular matrix (ECM) network that control crucial biochemical and biomechanical signals required for tissue morphogenesis, differentiation, homeostasis and cancer development. Breast cancer cells communicate with stromal ones to modulate ECM mainly through release of soluble effectors during cancer progression. The intracellular cross-talk between cell surface receptors and estrogen receptors is important for the regulation of breast cancer cell properties and production of ECM molecules. In turn, reorganized ECM-cell surface interface modulates signaling cascades, which regulate almost all aspects of breast cell behavior. Heparan sulfate chains present on cell surface and matrix proteoglycans are involved in regulation of breast cancer functions since they are capable of binding numerous matrix molecules, growth factors and inflammatory mediators thus modulating their signaling. In addition to its anticoagulant activity, there is accumulating evidence highlighting various anticancer activities of heparin and nano-heparin derivatives in numerous types of cancer. Importantly, heparin derivatives significantly reduce breast cancer cell proliferation and metastasis in vitro and in vivo models as well as regulates the expression profile of major ECM macromolecules, providing strong evidence for therapeutic targeting. Nano-formulations of the glycosaminoglycan heparin are possibly novel tools for targeting tumor microenvironment. In this review, the role of heparan sulfate/heparin and its nano-formulations in breast cancer biology are presented and discussed in terms of future pharmacological targeting.

IGF-IR is highly associated with the behaviour of breast cancer cells. In ERα-positive breast cancer, IGF-IR is present at high levels. In clinical practice, prolonged treatment with anti-estrogen agents results in resistance to the therapy with activation of alternative signaling pathways. Receptor Tyrosine Kinases, and especially IGF-IR, have crucial roles in these processes. Here, we report a nodal role of IGF-IR in the regulation of ERα-positive breast cancer cell aggressiveness and the regulation of expression levels of several extracellular matrix molecules. In particular, activation of IGF-IR, but not EGFR, in MCF-7 breast cancer cells results in the reduction of specific matrix metalloproteinases and their inhibitors. In contrast, IGF-IR inhibition leads to the depletion by endocytosis of syndecan-4. Global important changes in cell adhesion receptors, which include integrins and syndecan-4 triggered by IGF-IR inhibition, regulate adhesion and invasion. Cell function assays that were performed in MCF-7 cells as well as their ERα-suppressed counterparts indicate that ER status is a major determinant of IGF-IR regulatory role on cell adhesion and invasion. The strong inhibitory role of IGF-IR on breast cancer cells aggressiveness for which E2-ERα signaling pathway seems to be essential, highlights IGF-IR as a major molecular target for novel therapeutic strategies.

Syndecans are a small family of four transmembrane proteoglycans in mammals. They have similar structural organization, consisting of an N-terminal ectodomain, single transmembrane domain and C-terminal cytoplasmic domain. Over the years, the association between syndecans and the actin cytoskeleton has been established, which has consequences for the regulation of cell adhesion and migration. Specifically, ecto- and cytoplasmic domains are responsible for the interaction with extracellular matrix molecules and intracellular kinases, respectively. These interactions indicate syndecans as key molecules during cancer initiation and progression. Particularly syndecans interact with other cell surface receptors, such as growth factor receptors and integrins, which lead to activation of downstream signaling pathways, which are critical for the cellular behavior. Moreover, this review describes the key role of syndecans in intracellular calcium regulation and homeostasis. The syndecan-mediated regulation of calcium metabolism is highly correlated with cells' adhesion phenotype through the actin cytoskeleton and formation of junctions, with implications during differentiation and disease progression., (© 2016 Federation of European Biochemical Societies.)

Estrogen receptors have pivotal roles in breast cancer growth and progression. ERα has been clearly shown to play key role in hormone-dependent breast cancer properties, but little is known for the isoform ERβ. To evaluate the role of ERβ, we established stably transfected ERβ-suppressed MDA-MB-231 breast cancer cells by knocking down the human ERβ gene, using specific shRNA lentiviral particles. As observed by scanning electron microscopy, the ERβ suppression induces significant phenotypic changes in these cells, as compared to the control cells. Notably, the down-regulation of ERβ decreases the expression of the mesenchymal markers fibronectin and vimentin, whereas it increases the expression levels of the epithelial marker E-cadherin and cell junctions. These alterations are followed by reduced levels of the functional cell properties that promote the aggressiveness of these cells, such as proliferation, migration, spreading capacity, invasion and adhesion on collagen I. Notably, the down-regulation of ERβ reduces the migration of breast cancer cells through the tyrosine kinase receptors EGFR/IGF-IR and the JAK/STAT signaling pathways. Moreover, ERβ has a crucial role on the gene expression of several matrix mediators, including the proteoglycans syndecans-2/-4 and serglycin, several matrix metalloproteinases, plasminogen activation system components and receptor tyrosine kinases. These data clearly show that ERβ plays a crucial role in the cell behavior and ECM composition of the highly aggressive MDA-MB-231 cells and opens a new area of research to further understand its role and to improve pharmaceutical targeting of the non-hormone dependent breast cancer., (Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Notch may behave as an oncogene or a tumor suppressor gene in lung cancer cells. Notch receptor undergoes cleavage by enzymes, including γ-secretase, generating the active Notch intracellular domain (NICD). The aim of the present study was to investigate the effect of DAPT, a γ-secretase inhibitor, in non-small cell lung cancer (NSCLC) cells, as well as the impact of epidermal growth factor (EGF) that is over-expressed by NSCLC cells, on Notch signaling. H23, A549, H661 and HCC827 human NSCLC cell lines were used, expressing various NICD and EGF receptor (EGFR) protein levels., Results: DAPT decreased the number of H661 cells in a concentration-dependent manner, while it had a small effect on H23 and A549 cells and no effect on HCC827 cells that carry mutated EGFR. Notch inhibition did not affect the stimulatory effect of EGF on cell proliferation, while EGF prevented DAPT-induced NICD decrease in H23 and H661 cells. The type of cell death induced by DAPT seems to depend on the cell type., Conclusions: Our data indicate that inhibition of Notch cleavage may not affect cell number in the presence of EGFR mutations and that EGFR may affect Notch signalling suggesting that a dual inhibition of these pathways might be promising in NSCLC.

Proteoglycans control numerous normal and pathological processes, among which are morphogenesis, tissue repair, inflammation, vascularization and cancer metastasis. During tumor development and growth, proteoglycan expression is markedly modified in the tumor microenvironment. Altered expression of proteoglycans on tumor and stromal cell membranes affects cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Despite the high complexity and heterogeneity of breast cancer, the rapid evolution in our knowledge that proteoglycans are among the key players in the breast tumor microenvironment suggests their potential as pharmacological targets in this type of cancer. It has been recently suggested that pharmacological treatment may target proteoglycan metabolism, their utilization as targets for immunotherapy or their direct use as therapeutic agents. The diversity inherent in the proteoglycans that will be presented herein provides the potential for multiple layers of regulation of breast tumor behavior. This review summarizes recent developments concerning the biology of selected proteoglycans in breast cancer, and presents potential targeted therapeutic approaches based on their novel key roles in breast cancer., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The 17β-estradiol (E2)/estrogen receptor alpha (ERα) signaling pathway is one of the most important pathways in hormone-dependent breast cancer. E2 plays pivotal roles in cancer cell growth, survival, and architecture as well as in gene expression regulatory mechanisms. In this study, we established stably transfected MCF-7 cells by knocking down the ERα gene (designated as MCF-7/SP10+ cells), using specific shRNA lentiviral particles, and compared them with the control cells (MCF-7/c). Interestingly, ERα silencing in MCF-7 cells strongly induced cellular phenotypic changes accompanied by significant changes in gene and protein expression of several markers typical of epithelial to mesenchymal transition (EMT). Notably, these cells exhibited enhanced cell proliferation, migration and invasion. Moreover, ERα suppression strongly affected the gene and protein expression of EGFR and HER2 receptor tyrosine kinases, and various extracellular matrix (ECM) effectors, including matrix metalloproteinases and their endogenous inhibitors (MMPs/TIMPs) and components of the plasminogen activation system. The action caused by E2 in MCF-7/c cells in the expression of HER2, MT1-MMP, MMP1, MMP9, uPA, tPA, and PAI-1 was abolished in MCF-7/SP10+ cells lacking ERα. These data suggested a regulatory role for the E2/ERα pathway in respect to the composition and activity of the extracellular proteolytic molecular network. Notably, loss of ERα promoted breast cancer cell migration and invasion by inducing changes in the expression levels of certain matrix macromolecules (especially uPA, tPA, PAI-1) through the EGFR-ERK signaling pathway. In conclusion, loss of ERα in breast cancer cells results in a potent EMT characterized by striking changes in the expression profile of specific matrix macromolecules highlighting the potential nodal role of matrix effectors in breast cancer endocrine resistance., (Copyright © 2015. Published by Elsevier B.V.)

ADAMTSs are a family of secreted proteinases that share the metalloproteinase domain with matrix metalloproteinases (MMPs). By acting on a large panel of extracellular substrates, they control several cell functions such as fusion, adhesion, proliferation and migration. Through their thrombospondin motifs they also possess anti-angiogenic properties. We investigated whether ADAMTSs participate in colorectal cancer progression and invasion. Their expression was investigated at both mRNA and protein levels. Using RT-PCR, the expression of ADAMTS-1, -4, -5 and ADAMTS-20 was estimated in colorectal tumors of different cancer stage and anatomic site and 3 cell lines of different aggressiveness. An overexpression of ADAMTS-4 and -5 was observed, especially in tissue samples, whereas ADAMTS-1 and -20 were found to be down-regulated. Western blot analysis further supported the RT-PCR findings, revealing in addition the degradation of ADAMTS-1 and -20 in cancer. In situ expression and localization of ADAMTS-1, -4, -5 and -20 was also investigated by immunohistochemical analysis. Our data suggest a positive correlation between ADAMTS-4 and -5 expression and cancer progression, in contrast with the anti-angiogenic members of the family, ADAMTS-1 and -20, which were found to be down-regulated. Our findings support the notion that overexpression of ADAMTS-4 and ADAMTS-5 in colorectal cancer might be a possible invasive mechanism of cancer cells in order to degrade proteoglycans of ECM.

Capillary electrophoresis is a separation technique with high resolving power and sensitivity with applications in glycosaminoglycan analysis. In this chapter, we present validated protocols for determining the variously sulfated chondroitin or dermatan sulfate-derived disaccharides. These approaches involve degradation of the polysaccharides with specific chondro/dermato-lyases and electrophoretic analysis with capillary zone electrophoresis in a low pH operating buffer and reversed polarity. This methodology has been applied to drug/nutraceutical formulations or to biologic samples (blood serum, lens capsule) and has been validated. Analysis of biologic tissue samples is often more demanding in terms of detection sensitivity, and thus concentration pretreatment steps and/or a derivatization step with 2-aminoacridone are often advisable.

In the present pilot study, we examined the presence of serglycin in lung, breast, prostate, and colon cancer and evaluated its expression in cell lines and tissues. We found that serglycin was expressed and constitutively secreted in culture medium in high levels in more aggressive cancer cells. It is worth noticing that aggressive cancer cells that harbor KRAS or EGFR mutations secreted serglycin constitutively in elevated levels. Furthermore, we detected the transcription of an alternative splice variant of serglycin lacking exon 2 in specific cell lines. In a limited number of tissue samples analyzed, serglycin was detected in normal epithelium but was also expressed in higher levels in advanced grade tumors as shown by immunohistochemistry. Serglycin staining was diffuse, granular, and mainly cytoplasmic. In some cancer cells serglycin also exhibited membrane and/or nuclear immunolocalization. Interestingly, the stromal cells of the reactive tumor stroma were positive for serglycin, suggesting an enhanced biosynthesis for this proteoglycan in activated tumor microenvironment. Our study investigated for first time the distribution of serglycin in normal epithelial and cancerous lesions in most common cancer types. The elevated levels of serglycin in aggressive cancer and stromal cells may suggest a key role for serglycin in disease progression.

Proteoglycans are major constituents of extracellular matrices, as well as cell surfaces and basement membranes. They play key roles in supporting the dynamic extracellular matrix by generating complex structural networks with other macromolecules and by regulating cellular phenotypes and signaling. It is becoming evident, however, that proteolytic enzymes are required partners for matrix remodeling and for modulating cell signaling via matrix constituents. Proteinases contribute to all stages of diseases, particularly cancer development and progression, and contextually participate in either the removal of damaged products or in the processing of matrix molecules and signaling receptors. The dynamic interplay between proteoglycans and proteolytic enzymes is a crucial biological step that contributes to the pathophysiology of cancer and inflammation. Moreover, proteoglycans are implicated in the expression and secretion of proteolytic enzymes and often modulate their activities. In this review, we describe the emerging biological roles of proteoglycans and proteinases, with a special emphasis on their complex interplay. We critically evaluate this important proteoglycan-proteinase interactome and discuss future challenges with respect to targeting this axis in the treatment of cancer., (© 2014 FEBS.)

Background: ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression., Methods: Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules., Results: EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF-EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF-EGFR network. The EGF-EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2., Conclusions and General Significance: The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF-EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines., Methods: EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR., Results: The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells., Conclusion and General Significance: The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In hormone-dependent breast cancer, estrogen receptors are the principal signaling molecules that regulate several cell functions either by the genomic pathway acting directly as transcription factors in the nucleus or by the non-genomic pathway interacting with other receptors and their adjacent pathways like EGFR/IGFR. It is well established in literature that EGFR and IGFR signaling pathways promote cell proliferation and differentiation. Moreover, recent data indicate the cross-talk between ERs and EGFR/IGFR signaling pathways causing a transformation of cell functions as well as deregulation on normal expression pattern of matrix molecules. Specifically, proteoglycans, a major category of extracellular matrix (ECM) and cell surface macromolecules, are modified during malignancy and cause alterations in cancer cell signaling, affecting eventually functional cell properties such as proliferation, adhesion and migration. The on-going strategies to block only one of the above signaling effectors result cancer cells to overcome such inactivation using alternative signaling pathways. In this article, we therefore review the underlying mechanisms in respect to the role of ERs and the involvement of cross-talk between ERs, IGFR and EGFR in breast cancer cell properties and expression of extracellular secreted and cell bound proteoglycans involved in cancer progression. Understanding such signaling pathways may help to establish new potential pharmacological targets in terms of using ECM molecules to design novel anticancer therapies., (© 2013. Published by Elsevier B.V. All rights reserved.)

Extracellular matrix (ECM) components form a dynamic network of key importance for cell function and properties. Key macromolecules in this interplay are syndecans (SDCs), a family of transmembrane heparan sulfate proteoglycans (HSPGs). Specifically, heparan sulfate (HS) chains with their different sulfation pattern have the ability to interact with growth factors and their receptors in tumor microenvironment, promoting the activation of different signaling cascades that regulate tumor cell behavior. The affinity of HS chains with ligands is altered during malignant conditions because of the modification of chain sequence/sulfation pattern. Furthermore, matrix degradation enzymes derived from the tumor itself or the tumor microenvironment, like heparanase and matrix metalloproteinases, ADAM as well as ADAMTS are involved in the cleavage of SDCs ectodomain at the HS and protein core level, respectively. Such released soluble SDCs "shed SDCs" in the ECM interact in an autocrine or paracrine manner with the tumor or/and stromal cells. Shed SDCs, upon binding to several matrix effectors, such as growth factors, chemokines, and cytokines, have the ability to act as competitive inhibitors for membrane proteoglycans, and modulate the inflammatory microenvironment of cancer cells. It is notable that SDCs and their soluble counterparts may affect either the behavior of cancer cells and/or their microenvironment during cancer progression. The importance of these molecules has been highlighted since HSPGs have been proposed as prognostic markers of solid tumors and hematopoietic malignancies. Going a step further down the line, the multi-actions of SDCs in many levels make them appealing as potential pharmacological targets, either by targeting directly the tumor or indirectly the adjacent stroma.

Serglycin has been initially characterized as an intracellular proteoglycan expressed by hematopoietic cells. All inflammatory cells highly synthesize serglycin and store it in granules, where it interacts with numerous inflammatory mediators, such as proteases, chemokines, cytokines, and growth factors. Serglycin is implicated in their storage into the granules and their protection since they are secreted as complexes and delivered to their targets after secretion. During the last decade, numerous studies have demonstrated that serglycin is also synthesized by various non-hematopoietic cell types. It has been shown that serglycin is highly expressed by tumor cells and promotes their aggressive phenotype and confers resistance against drugs and complement system attack. Apart from its direct beneficial role to tumor cells, serglycin may promote the inflammatory process in the tumor cell microenvironment thus enhancing tumor development. In the present review, we discuss the role of serglycin in inflammation and tumor progression.

Extensive experimental evidence in cell and animal tumor models show that hyaluronan-CD44 interactions are crucial in both malignancy and resistance to cancer therapy. Because of the intimate relationship between the hyaluronan-CD44 system and tumor cell survival and growth, it is an increasingly investigated area for applications to anticancer chemotherapeutics. Interference with the hyaluronan-CD44 interaction by targeting drugs to CD44, targeting drugs to the hyaluronan matrix, or interfering with hyaluronan matrix/tumor cell-associated CD44 interactions is a viable strategy for cancer treatment. Many of these methods can decrease tumor burden in animal models but have yet to show significant clinical utility. Recent advances in nanomedicine have offered new valuable tools for cancer detection, prevention, and treatment. The enhanced permeability and retention effect has served as key rationale for using nanoparticles to treat solid tumors. However, the targeted and uniform delivery of these particles to all regions of tumors in sufficient quantities requires optimization. An ideal nanocarrier should be equipped with selective ligands that are highly or exclusively expressed on target cells and thus endow the carriers with specific targeting capabilities. In this review, we describe how the hyaluronan-CD44 system may provide such an alternative in tumors expressing specific CD44 variants., (© 2014 Elsevier Inc. All rights reserved.)

Traditionally, the studies on cancer growth and progression have been focused on the transformed, malignant cells. However, it is now well recognized that the tumour stroma represents a crucial parameter in tumour development, growth and progression. Indeed, several cancers are characterized by a prominent stromal compartment and it is the interactions between cancer cells, stromal cells and extracellular matrix (ECM) components that control the overall tumour growth. Among stromal cells, fibroblasts represent the most important type. They are responsible for deposition and remodeling of ECM components, as well as for the release of cytokines and growth factors, including platelet-derived growth factor (PDGF), acting in a paracrine manner on cancer cells. In this review we elucidate the role of tumor stroma interactions, the roles of PDGF receptor signaling in cancer-associated fibroblasts via alteration of stromal matrix composition and the mitogenic effects of cancer-derived PDGFs. Focus on the targeting of tumor microenvironment at the level of PDGF/PDGF receptor (PDGFR) is also presented as to stimulate further studies for designing and development of novel pharmaceutical agents and combined pharmaceutical interventions. Conclusively, PDGF/PDGFR axis is of paramount importance in the tumour microenvironment context and the inhibition of PDGF receptors' activation represents a major target for future anticancer therapies.

Serglycin is a proteoglycan expressed by some malignant cells. It promotes metastasis and protects some tumor cells from complement system attack. In the present study, we show for the first time the in situ expression of serglycin by breast cancer cells by immunohistochemistry in patients' material. Moreover, we demonstrate high expression and constitutive secretion of serglycin in the aggressive MDA-MB-231 breast cancer cell line. Serglycin exhibited a strong cytoplasmic staining in these cells, observable at the cell periphery in a thread of filaments near the cell membrane, but also in filopodia-like structures. Serglycin was purified from conditioned medium of MDA-MB-231 cells, and represented the major proteoglycan secreted by these cells, having a molecular size of ~ 250 kDa and carrying chondroitin sulfate side chains, mainly composed of 4-sulfated (~ 87%), 6-sulfated (~ 10%) and non-sulfated (~ 3%) disaccharides. Purified serglycin inhibited early steps of both the classical and the lectin pathways of complement by binding to C1q and mannose-binding lectin. Stable expression of serglycin in less aggressive MCF-7 breast cancer cells induced their proliferation, anchorage-independent growth, migration and invasion. Interestingly, over-expression of serglycin lacking the glycosaminoglycan attachment sites failed to promote these cellular functions, suggesting that glycanation of serglycin is a pre-requisite for its oncogenic properties. Our findings suggest that serglycin promotes a more aggressive cancer cell phenotype and may protect breast cancer cells from complement attack supporting their survival and expansion.

Epithelial-mesenchymal transition (EMT) is a developmental program, which can be adopted by cancer cells to increase their migration and ability to form metastases. Transforming growth factor β (TGFβ) is a well-studied inducer of EMT. We demonstrate that TGFβ potently stimulates hyaluronan synthesis via upregulation of hyaluronan synthase 2 (HAS2) in NMuMG mammary epithelial cells. This stimulatory effect requires the kinase active type I TGFβ receptor and is dependent on Smad signaling and activation of the p38 mitogen-activated protein kinase. Knockdown of HAS2 inhibited the TGFβ-induced EMT by about 50%, as determined by the phase contrast microscopy and immunostaining using the EMT marker ZO-1. Furthermore, real-time PCR analysis of the EMT markers fibronectin, Snail1 and Zeb1 revealed decreased expressions upon HAS2 suppression, using specific small interfering RNA (siRNA) for HAS2. Removal of the extracellular hyaluronan by Streptomyces hyaluronidase or inhibiting the binding to its cell surface receptor CD44 by blocking antibodies, did not inhibit TGFβ-induced EMT. Interestingly, HAS2 suppression completely abolished the TGFβ-induced cell migration, whereas CD44 knockdown did not. These observations suggest that TGFβ-dependent HAS2 expression, but not extracellular hyaluronan, has an important regulatory role in TGFβ-induced EMT.

Previously, we have shown that a proportion of the matrix metalloproteinase-9 (MMP-9) synthesized by the macrophage cell line THP-1 binds to a chondroitin sulfate proteoglycan (CSPG) core protein to form a reduction-sensitive heteromer. It was also shown that the hemopexin-like (PEX) domain and the fibronectin-like (FnII) module in the enzyme are involved in heteromer formation. In this paper, we show that reduction-sensitive and SDS-stable heteromers may be reconstituted in vitro by mixing proMMP-9 with either serglycin, versican or CSPGs isolated from various monocytic cell lines. In addition, a strong but SDS-soluble proMMP-9·CSPG heteromer was formed. The two macromolecules in the SDS-stable reduction-sensitive heteromers were not linked together by disulfide bonds. As for the heteromer isolated from THP-1 cells, in vitro reconstituted SDS-stable and SDS-soluble heteromers showed weaker binding to gelatin than the proMMP-9 monomer. Furthermore, gelatin inhibited in vitro reconstitution of the heteromers, showing that the FnII module is involved in the complex formation. Tissue inhibitor of metalloproteinase (TIMP)-1 was not be detected in the proMMP-9·CSPG complexes. However, the presence of TIMP-1 inhibited formation of the SDS-soluble heteromer, but not the SDS-stable reduction-sensitive heteromer. This indicates that different regions in the PEX domain are involved formation of these heteromers., (© 2013 FEBS.)

The epidermal growth factor receptor (EGFR) is a member of the HER family receptors and its activation induced by its natural ligand EGF results in colon cancer growth and progression. Panitumumab (pmAb) is a fully human IgG2 anti-EGFR antibody that blocks the EGFR actions. In the present study, we evaluated the effects of pmAb on the EGF-mediated cellular responses in a panel of colon cancer cells (HCT-8, HT-29, DLD-1 and HCT-116). HCT-1116 and DLD-1 cells showed no significant EGF-dependent cell proliferation; HT-29 and HCT-8 exhibited an EGF-dependent proliferation, with HCT-8 cells to be the most responsive with significant EGFR phosphorylation upon treatment with EGF. The effects of pmAb were then evaluated in the most EGF-responsive cells, HCT-8. In that respect, pmAb impedes the signaling cascade mediated by EGFR intracellular phosphorylation and activity of focal adhesion kinase (FAK) as well as the EGF-induced invasive and migratory potential of colon cancer cells. At the level of matrix effectors implicated in colon cancer progression we report that pmAb is a potent inhibitor of constitute and EGF-mediated gene expression of certain matrix effectors, such as membrane-type 1 metalloproteinase (MT1-MMP), extracellular metalloproteinases inducer (EMMPRIN), urokinase plasminogen activator (uPA) and syndecan-4. The obtained data demonstrated that pmAb is a specific blocker of EGF-mediated EGFR activation, resulting in a significant inhibition of colon cancer cell proliferation in early stages of growth, migration and invasiveness as well as of matrix effector implicated in cancer progression.

Background: The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties., Methods: Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts' adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL., Results: ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin β3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption., Conclusions: ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis., General Significance: The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Serglycin (SG) is mainly expressed by hematopoetic cells as an intracellular proteoglycan. Multiple myeloma cells constitutively secrete SG, which is also localized on the cell surface in some cell lines. In this study, SG isolated from myeloma cells was found to interact with collagen type I (Col I), which is a major bone matrix component. Notably, myeloma cells positive for cell-surface SG (csSG) adhered significantly to Col I, compared to cells lacking csSG. Removal of csSG by treatment of the cells with chondroitinase ABC or blocking of csSG by an SG-specific polyclonal antibody significantly reduced the adhesion of myeloma cells to Col I. Significant up-regulation of expression of the matrix metalloproteinases MMP-2 and MMP-9 at both the mRNA and protein levels was observed when culturing csSG-positive myeloma cells on Col I-coated dishes or in the presence of soluble Col I. MMP-9 and MMP-2 were also expressed in increased amounts by myeloma cells in the bone marrow of patients with multiple myeloma. Our data indicate that csSG of myeloma cells affects key functional properties, such as adhesion to Col I and the expression of MMPs, and imply that csSG may serve as a potential prognostic factor and/or target for pharmacological interventions in multiple myeloma., (© 2013 The Authors Journal compilation © 2013 FEBS.)

Estradiol (E2)-estrogen receptor (ER) actions are implicated in initiation, growth and progression of hormone-dependent breast cancer. Crosstalk between ERs, epidermal growth factor receptor (EGFR) and/or insulin-like growth factor receptor (IGFR) is critical for the observed resistance to endocrine therapies. Cell surface heparan sulfate proteoglycans (HSPGs) are principal mediators of cancer cell properties and the E2-ER pathway as well as those activated by EGFR and IGFR have significant roles in regulating the expression of certain cell surface HSPGs, such as syndecan-2 (SDC-2), syndecan-4 (SDC-4) and glypican-1. In this study, we therefore evaluated the role of EGFR-IGFR signaling on the constitutive expression and E2-mediated expression of ERs and HSPGs as well as the effect of E2-ERs and IGFR/EGFR-mediated cell migration in ERα+ (MCF-7) and ERβ+ (MDA-MB-231) breast cancer cells using specific intracellular inhibitors of EGFR and IGFR. We report that the expression of ERα is mainly enhanced by IGFR, whereas ERβ expression is mainly coordinated by EGFR. Moreover, constitutive SDC-2 expression in ERα+ and ERβ+ cells is mainly mediated through the IGFR, whereas in ERα+ E2-treated cells EGFR is the active one. In contrast, SDC-4 expression is regulated by IGFR in the presence and absence of E2. E2 also seems to diminish the inhibitory effect of EGFR and IGFR inhibitors in breast cancer cell migration. These data suggest that the coordinated action of ERs with EGFR and/or IGFR is of crucial importance, providing potential targets for designing and developing novel multi-potent agents for endocrine therapies., (© 2013 The Authors Journal compilation © 2013 FEBS.)

Cell surface heparan sulfate proteoglycans (HSPGs), syndecans and glypicans, play crucial roles in the functional properties of cancer cells, such as proliferation, adhesion, migration and invasion. Platelet-derived growth factor (PDGF)/PDGF receptor (PDGF-R) mediated signaling, on the other hand, is highly associated with cancer progression. Specifically, PDGF-Rα and PDGF-Rβ expressions documented in breast cancer tissue specimens as well as breast cancer cell lines are correlated with tumor aggressiveness and metastasis. Imatinib (Glivec(®)) is a tyrosine kinase inhibitor specific for PDGF-Rs, c-ΚΙΤ and BCR-ABL. In this study we evaluated the effects of imatinib on the properties of breast cancer cells as well as on the expression of HSPGs in the presence and absence of PDGF-BB. These studies have been conducted in a panel of three breast cancer cell lines of low and high metastatic potential. Our results indicate that imatinib exerts a significant inhibitory effect on breast cancer cell proliferation, invasion and migration as well as on the cell surface expression of HSPGs even after exposure of PDGF. These effects depend on the aggressiveness of breast cancer cells and the type of HSPG. It is suggested that imatinib may be of potential therapeutic usefulness in breast cancer regimes., (© 2013 The Authors Journal compilation © 2013 FEBS.)

Although syndecan-4 is implicated in cancer progression, there is no information for its role in testicular germ cell tumours (TGCTs). Thus, we examined the expression of syndecan-4 in patients with TGCTs and its correlation with the clinicopathological findings. Immunohistochemical staining in 71 tissue specimens and mRNA analysis revealed significant overexpression of syndecan-4 in TGCTs. In seminomas, high percentage of tumour cells exhibited membranous and/or cytoplasmic staining for syndecan-4 in all cases. Stromal staining for syndecan-4 was found in seminomas and it was associated with nodal metastasis (P = 0.04), vascular/lymphatic invasion (P = 0.01), and disease stage (P = 0.04). Reduced tumour cell associated staining for syndecan-4 was observed in nonseminomatous germ cell tumours (NSGCTs) compared to seminomas. This loss of syndecan-4 was associated with nodal metastasis (P = 0.01), vascular/lymphatic invasion (P = 0.01), and disease stage (P = 0.01). Stromal staining for syndecan-4 in NSGCTs did not correlate with any of the clinicopathological variables. The stromal expression of syndecan-4 in TGCTs was correlated with microvessel density (P = 0.03). Our results indicate that syndecan-4 is differentially expressed in seminomas and NSGCTs and might be a useful marker. Stromal staining in seminomas and reduced levels of syndecan-4 in tumour cells in NSGCTs are related to metastatic potential, whereas stromal staining in TGCTs is associated with neovascularization.

Background: The extracellular matrix (ECM) components play key roles in the multistep process of cancer growth and progression. Preclinical and clinical data show that bisphosphonates (BPs) may exert direct or indirect antitumoral effects. Despite proven efficiency in cancer treatment, the mechanism by which BPs can interfere with cancer progression remains elusive., Methods: We investigated the effects of the third generation BP, zoledronate (zoledronic acid, Zometa®), in the expression of ECM macromolecules as well as the functional properties (proliferation, adhesion, migration and invasion) in two breast cancer cell lines (MDA-MB-231 and MCF-7) with different metastatic potentials., Results: The data highlight that zoledronate effectively inhibits growth of breast cancer cells, functional invasion migration and adhesion to various matrices. At the level of ECM interacting molecules, the expression of specific heparan sulfate proteoglycans implicated in cancer progression, such as syndecan-1, -2 and glypican-1 is downregulated, whereas syndecan-4 expression is upregulated upon treatment with zoledronate. The levels of integrins ανβ3, ανβ5 and α5β1 were significantly reduced following treatment with zoledronate which is in accordance with the reduced cell adhesion on various ECM matrices. The expression of hyaluronan and its receptor CD44 was also significantly suppressed. Moreover, ZOL suppressed the expression of metalloproteinases MMP-2, -9, the membrane type MT1- and MT2-MMP, whereas it increased the expression of their endogenous tissue inhibitors., Conclusions and General Significance: The obtained results demonstrate that zoledronate is a critical modulator of ECM gene expression and powerful anticancer agent inhibiting growth, migration and the matrix-associated invasion of breast cancer cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)