The multifaceted role of Matricellular Proteins in health and cancer, as biomarkers and therapeutic targets.

The extracellular matrix (ECM) is composed of a mesh of proteins, proteoglycans, growth factors, and other secretory components. It constitutes the tumor microenvironment along with the endothelial cells, cancer-associated fibroblasts, adipocytes, and immune cells. The proteins of ECM can be functionally classified as adhesive proteins and matricellular proteins (MCP). In the tumor milieu, the ECM plays a major role in tumorigenesis and therapeutic resistance. The current review encompasses thrombospondins, osteonectin, osteopontin, tenascin C, periostin, the CCN family, laminin, biglycan, decorin, mimecan, and galectins. The matrix metalloproteinases (MMPs) are also discussed as they are an integral part of the ECM with versatile functions in the tumor stroma. In this review, the role of these proteins in tumor initiation, growth, invasion and metastasis have been highlighted, with emphasis on their contribution to tumor therapeutic resistance. Further, their potential as biomarkers and therapeutic targets based on existing evidence are discussed. Owing to the recent advancements in protein targeting, the possibility of agents to modulate MCPs in cancer as therapeutic options are discussed.

Functions of Matricellular Proteins in Dental Tissues and Their Emerging Roles in Orofacial Tissue Development, Maintenance, and Disease.

Matricellular proteins (MCPs) are defined as extracellular matrix (ECM) associated proteins that are important regulators and integrators of microenvironmental signals, contributing to the dynamic nature of ECM signalling. There is a growing understanding of the role of matricellular proteins in cellular processes governing tissue development as well as in disease pathogenesis. In this review, the expression and functions of different MP family members (periostin, CCNs, TSPs, SIBLINGs and others) are presented, specifically in relation to craniofacial development and the maintenance of orofacial tissues, including bone, gingiva, oral mucosa, palate and the dental pulp. As will be discussed, each MP family member has been shown to have non-redundant roles in development, tissue homeostasis, wound healing, pathology and tumorigenesis of orofacial and dental tissues.

ECM deposition is driven by caveolin-1-dependent regulation of exosomal biogenesis and cargo sorting.

The composition and physical properties of the extracellular matrix (ECM) critically influence tumor progression, but the molecular mechanisms underlying ECM layering are poorly understood. Tumor-stroma interaction critically depends on cell communication mediated by exosomes, small vesicles generated within multivesicular bodies (MVBs). We show that caveolin-1 (Cav1) centrally regulates exosome biogenesis and exosomal protein cargo sorting through the control of cholesterol content at the endosomal compartment/MVBs. Quantitative proteomics profiling revealed that Cav1 is required for exosomal sorting of ECM protein cargo subsets, including Tenascin-C (TnC), and for fibroblast-derived exosomes to efficiently deposit ECM and promote tumor invasion. Cav1-driven exosomal ECM deposition not only promotes local stromal remodeling but also the generation of distant ECM-enriched stromal niches in vivo. Cav1 acts as a cholesterol rheostat in MVBs, determining sorting of ECM components into specific exosome pools and thus ECM deposition. This supports a model by which Cav1 is a central regulatory hub for tumor-stroma interactions through a novel exosome-dependent ECM deposition mechanism.

Investigating cell-type specific functions of tenascin-C.

The extracellular matrix molecule tenascin-C (TNC) has received a lot of attention since its discovery 30 years ago because of its multiple roles in tissue repair, and in pathologies such as chronic inflammation, fibrosis, and cancer. Mouse models with high or no TNC expression have enabled the validation of key roles for TNC in immunity and angiogenesis. In parallel, many approaches including primary cell or organ cultures have shed light on the cellular and molecular mechanisms by which TNC exerts its multiple actions in vivo. Here, we will describe assays that investigate its antiadhesive properties and that measure the effect of TNC on the actin cytoskeleton, cell survival, proliferation, and migration. We will also describe assays to assess the impact of TNC on endothelial and immune cells in cell and organ culture, and to compare the responses of fibroblasts from normal and diseased tissues.

Bone Marrow-Derived Tenascin-C Attenuates Cardiac Hypertrophy by Controlling Inflammation.

BACKGROUND: Tenascin-C (TNC) is a highly conserved matricellular protein with a distinct expression pattern during development and disease. Remodeling of the left ventricle (LV) in response to pressure overload leads to the re-expression of the fetal gene program. OBJECTIVES: The aim of this study was to investigate the function of TNC in cardiac hypertrophy in response to pressure overload. METHODS: Pressure overload was induced in TNC knockout and wild-type mice by constricting their abdominal aorta or by infusion of angiotensin II. Echocardiography, immunostaining, flow cytometry, quantitative real-time polymerase chain reaction, and reciprocal bone marrow transplantation were used to evaluate the effect of TNC deficiency. RESULTS: Echocardiographic analysis of pressure overloaded hearts revealed that all LV parameters (LV end-diastolic and -systolic dimensions, ejection fraction, and fractional shortening) deteriorated in TNC-deficient mice compared with their wild-type counterparts. Cardiomyocyte size and collagen accumulation were significantly greater in the absence of TNC. Mechanistically, TNC deficiency promoted rapid accumulation of the CCR2+/Ly6Chi monocyte/macrophage subset into the myocardium in response to pressure overload. Further, echocardiographic and immunohistochemical analyses of recipient hearts showed that expression of TNC in the bone marrow, but not the myocardium, protected the myocardium against excessive remodeling of the pressure-overloaded heart. CONCLUSIONS: TNC deficiency further impaired cardiac function in response to pressure overload and exacerbated fibrosis by enhancing inflammation. In addition, expression of TNC in the bone marrow, but not the myocardium, protected the myocardium against excessive remodeling in response to mild pressure overload.

Quantitative proteomics identify Tenascin-C as a promoter of lung cancer progression and contributor to a signature prognostic of patient survival.

The extracellular microenvironment is an integral component of normal and diseased tissues that is poorly understood owing to its complexity. To investigate the contribution of the microenvironment to lung fibrosis and adenocarcinoma progression, two pathologies characterized by excessive stromal expansion, we used mouse models to characterize the extracellular matrix (ECM) composition of normal lung, fibrotic lung, lung tumors, and metastases. Using quantitative proteomics, we identified and assayed the abundance of 113 ECM proteins, which revealed robust ECM protein signatures unique to fibrosis, primary tumors, or metastases. These analyses indicated significantly increased abundance of several S100 proteins, including Fibronectin and Tenascin-C (Tnc), in primary lung tumors and associated lymph node metastases compared with normal tissue. We further showed that Tnc expression is repressed by the transcription factor Nkx2-1, a well-established suppressor of metastatic progression. We found that increasing the levels of Tnc, via CRISPR-mediated transcriptional activation of the endogenous gene, enhanced the metastatic dissemination of lung adenocarcinoma cells. Interrogation of human cancer gene expression data revealed that high TNC expression correlates with worse prognosis for lung adenocarcinoma, and that a three-gene expression signature comprising TNC, S100A10, and S100A11 is a robust predictor of patient survival independent of age, sex, smoking history, and mutational load. Our findings suggest that the poorly understood ECM composition of the fibrotic and tumor microenvironment is an underexplored source of diagnostic markers and potential therapeutic targets for cancer patients.

Composition and adaptation of human myotendinous junction and neighboring muscle fibers to heavy resistance training.

The myotendinous junction (MTJ) is a common site of strain injury and yet understanding of its composition and ability to adapt to loading is poor. The main aims of this study were to determine the profile of selected collagens and macrophage density in human MTJ and adjoining muscle fibers, and to investigate whether heavy exercise loading would alter this profile. Fifteen individuals scheduled for anterior cruciate ligament repair surgery were randomized into three groups: control, acute or 4 weeks heavy resistance training. MTJ samples were collected from the semitendinosus and gracilis muscles and were sectioned and stained immunohistochemically for collagen types I, III, VI, XII, XIV, XXII, Tenascin-C and CD68. Macrophage density and distribution was evaluated and the amount of each collagen type in muscle and MTJ was graded. Collagen XXII was observed solely at the MTJ, while all other collagens were abundant at the MTJ and in muscle perimysium or endomysium. The endomysial content of collagen XIV, macrophages and Tenascin-C increased following 4 weeks of training. These findings illustrate the heterogeneity of collagen type composition of human MTJ. The increase in collagen XIV following 4 weeks of training may reflect a training-induced protection against strain injuries in this region.

High expression of intratumoral stromal proteins is associated with chemotherapy resistance in breast cancer.

We studied the changes of intratumoral stromal proteins including THBS1, TNC, FN, SPARC and α-SMA, following neoadjuvant chemotherapy. The underlying mechanisms by which THBS1 and TNC regulated resistance to docetaxel were further studied using functional studies. 100 patients with newly diagnosed breast cancer were treated with alternating sequential doxorubicin and docetaxel. Immunohistochemistry (IHC) staining for stromal proteins was performed on pre- and post-treatment core biopsies respectively. THBS1 and TNC were further validated with IHC in an independent cohort of 31 patients. A high baseline combined expression score of the 5 stromal proteins predicted independently for poor progression-free (HRadjusted 2.22, 95% CI 1.06-4.64) and overall survival (HRadjusted 5.94, 95% CI 2.25-15.71). After 1-2 cycles of chemotherapy, increased expression of THBS1, TNC, FN, SPARC and α-SMA was seen in patients with subsequent pathological lymph node involvement at surgery. Increased expression of THBS1 and TNC compared to baseline was also seen in intrinsically resistant tumors, but not in sensitive ones. Both THBS1 and TNC-associated chemoresistance were confirmed in an independent validation cohort. Exogenous THBS1 and TNC protected MCF-7 cells against proliferation inhibition induced by docetaxel through activating integrin ß1/mTOR pathway. Thus, up-regulation of THBS1, TNC, FN, SPARC and α-SMA following neoadjuvant chemotherapy was associated with chemotherapy resistance in breast cancer patients. Functional studies showed THBS1 and TNC to mediate chemoresistance through the integrin ß1/mTOR pathway, suggesting that therapies targeting integrin ß1/mTOR pathway may be a promising strategy to overcome chemotherapy resistance.

[Advances in molecular mechanisms of tenascin-C in promoting tumor metastasis].

Tenascin-C (TNC) is an extracellular matrix glycoprotein, which is usually highly expressed in embryonic tissues and tumor tissues, but is not expressed or just lowly expressed in mature tissues. TNC is involved in various complex signaling pathways during tumor metastasis, especially through modulating FAK, RhoA, Wnt and Notch pathways by interacting with syndecan-4, integrin α5ß1, matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). As a result, TNC affects epithelial mesenchymal transition, tumor cell adhesion, proliferation and angiogenesis, which eventually enhances the invasion and metastasis ability of many tumors. Further studies have demonstrated that TNC could be used as prognosis or metastasis marker of patients with malignant tumor.

[Vascular Calcification - Pathological Mechanism and Clinical Application - . Extracellular matrix tenascin-X in calcific aortic valves].

We previously disclosed a novel extracellular matrix tenascin-X (TNX) , the largest member of the tenascin family. So far, we have made efforts to elucidate the roles of TNX. TNX is involved in collagen deposition, collagen fibrillogenesis, and modulation of collagen stiffness. Homozygous mutations in TNXB, the gene encoding TNX, cause a classic-type Ehlers-Danlos syndrome (EDS) , a heritable connective tissue disorder, whereas haploinsufficiency of TNXB and heterozygous mutations in TNXB are associated with hypermobility-type EDS. Recently, we performed proteomic analyses of calcific aortic valves (CAVs) compared with relatively adjacent normal tissues to understand the underlying molecular mechanisms of dystrophic valvular calcification. Interestingly, we found that TNX was the protein with the greatest decrease in expression among the differentially expressed proteins and that expression levels of proteins modulating collagen structure and function, such as type I collagen and decorin, were also decreased in CAVs. In this review, I will discuss about the decreased level of collagen due to the reduction of expression levels of proteins that play regulatory roles in collagen functions such as fibril organization and fibrillogenesis in CAVs.

Tenascin-C Protects Cancer Stem-like Cells from Immune Surveillance by Arresting T-cell Activation.

Precociously disseminated cancer cells may seed quiescent sites of future metastasis if they can protect themselves from immune surveillance. However, there is little knowledge about how such sites might be achieved. Here, we present evidence that prostate cancer stem-like cells (CSC) can be found in histopathologically negative prostate draining lymph nodes (PDLN) in mice harboring oncogene-driven prostate intraepithelial neoplasia (mPIN). PDLN-derived CSCs were phenotypically and functionally identical to CSC obtained from mPIN lesions, but distinct from CSCs obtained from frank prostate tumors. CSC derived from either PDLN or mPIN used the extracellular matrix protein Tenascin-C (TNC) to inhibit T-cell receptor-dependent T-cell activation, proliferation, and cytokine production. Mechanistically, TNC interacted with α5ß1 integrin on the cell surface of T cells, inhibiting reorganization of the actin-based cytoskeleton therein required for proper T-cell activation. CSC from both PDLN and mPIN lesions also expressed CXCR4 and migrated in response to its ligand CXCL12, which was overexpressed in PDLN upon mPIN development. CXCR4 was critical for the development of PDLN-derived CSC, as in vivo administration of CXCR4 inhibitors prevented establishment in PDLN of an immunosuppressive microenvironment. Taken together, our work establishes a pivotal role for TNC in tuning the local immune response to establish equilibrium between disseminated nodal CSC and the immune system.

ADAM-9 is a novel mediator of tenascin-C-stimulated invasiveness of brain tumor-initiating cells.

BACKGROUND: Tenascin-C (TNC), an extracellular matrix protein overexpressed in malignant gliomas, stimulates invasion of conventional glioma cell lines (U251, U87). However, there is a dearth of such information on glioma stemlike cells. Here, we have addressed whether and how TNC may regulate the invasiveness of brain tumor-initiating cells (BTICs) that give rise to glioma progenies. METHODS: Transwell inserts coated with extracellular matrix proteins were used to determine the role of TNC in BTIC invasion. Microarray analysis, lentiviral constructs, RNA interference-mediated knockdown, and activity assay ascertained the role of proteases in TNC-stimulated BTIC invasion in culture. Involvement of proteases was validated using orthotopic brain xenografts in mice. RESULTS: TNC stimulated BTIC invasiveness in a metalloproteinase-dependent manner. A global gene expression screen identified the metalloproteinase ADAM-9 as a potential regulator of TNC-stimulated BTIC invasiveness, and this was corroborated by an increase of ADAM-9 protein in 4 glioma patient-derived BTIC lines. Notably, RNA interference to ADAM-9, as well as inhibition of mitogen-activated protein kinase 8 (c-Jun NH2-terminal kinase), attenuated TNC-stimulated ADAM-9 expression, proteolytic activity, and BTIC invasiveness. The relevance of ADAM-9 to tumor invasiveness was validated using resected human glioblastoma specimens and orthotopic xenografts where elevation of ADAM-9 and TNC expression was prominent at the invasive front of the tumor. CONCLUSIONS: This study has identified TNC as a promoter of the invasiveness of BTICs through a mechanism involving ADAM-9 proteolysis via the c-Jun NH2-terminal kinase pathway.

Tenascins in stem cell niches.

Tenascins are extracellular matrix proteins with distinct spatial and temporal expression during development, tissue homeostasis and disease. Based on their expression patterns and knockout phenotypes an important role of tenascins in tissue formation, cell adhesion modulation, regulation of proliferation and differentiation has been demonstrated. All of these features are of importance in stem cell niches where a precise regulation of growth versus differentiation has to be guaranteed. In this review we summarize the expression and possible functions of tenascins in neural, epithelial and osteogenic stem cell niches during normal development and organ turnover, in the hematopoietic and pro-inflammatory niche as well as in the metastatic niche during cancer progression.

[Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance]. / Proteinas inhibidoras de la regeneracion neuritica en la matriz extracelular: estructura, interacciones moleculares y sus funciones. Mecanismos del balance extracelular.

After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

Teneurin protein family: an emerging role in human tumorigenesis and drug resistance.

Using a chemoproteomic strategy, we recently demonstrated the expression of teneurin-2, a transmembrane glycoprotein, in the majority of malignant mesothelioma cell lines. This finding was unexpected since no formally organized evidence existed to implicate teneurins in human malignancy. For this reason, here we provide a comprehensive review on the expression of teneurins in human tumors and cell lines. Current evidence supports the aberrant expression of teneurins in various tumor types, their involvement in cancer-related regulatory networks, and their potential participation in drug resistance. Structural attributes of teneurins could enable the detection of shedded forms in body fluids for clinical applications.

Tenascin-C produced by oxidized LDL-stimulated macrophages increases foam cell formation through Toll-like receptor-4.

Atherosclerosis is a chronic inflammatory disease in which both innate and adaptive immunity are involved. Although there have been major advances in the involvement of toll-like receptor 4 (TLR4) and CD36 in the initiation and development of this disease, detailed mechanisms remain unknown. Here, we show that tenascin-C (TN-C) can stimulate foam cell formation and this can be inhibited by a TLR4-blocking antibody or CD36 gene silencing. Our results identify TN-C-TLR4 activation as a common molecular mechanism in oxLDL-stimulated foam cell formation and atherosclerosis. In addition, CD36 is the major scavenger receptor responsible for the TN-C-mediated foam cell formation. Taken together, we have identified that TNC produced by oxLDL-stimulated macrophages increases foam cell formation through TLR4 and scavenger receptor CD36.

Matricellular proteins in cardiac adaptation and disease.

The term matricellular proteins describes a family of structurally unrelated extracellular macromolecules that, unlike structural matrix proteins, do not play a primary role in tissue architecture, but are induced following injury and modulate cell-cell and cell-matrix interactions. When released to the matrix, matricellular proteins associate with growth factors, cytokines, and other bioactive effectors and bind to cell surface receptors transducing signaling cascades. Matricellular proteins are upregulated in the injured and remodeling heart and play an important role in regulation of inflammatory, reparative, fibrotic and angiogenic pathways. Thrombospondin (TSP)-1, -2, and -4 as well as tenascin-C and -X secreted protein acidic and rich in cysteine (SPARC), osteopontin, periostin, and members of the CCN family (including CCN1 and CCN2/connective tissue growth factor) are involved in a variety of cardiac pathophysiological conditions, including myocardial infarction, cardiac hypertrophy and fibrosis, aging-associated myocardial remodeling, myocarditis, diabetic cardiomyopathy, and valvular disease. This review discusses the properties and characteristics of the matricellular proteins and presents our current knowledge on their role in cardiac adaptation and disease. Understanding the role of matricellular proteins in myocardial pathophysiology and identification of the functional domains responsible for their actions may lead to design of peptides with therapeutic potential for patients with heart disease.

Tenascin-C and its functions in neuronal plasticity.

The extracellular matrix glycoprotein tenascin-C (TN-C), a molecule highly conserved in vertebrates, is widely expressed in neural and non-neural tissue during development, repair processes in the adult organism, and tumorigenesis. In the developing central nervous system (CNS), in different brain regions TN-C is expressed in specific spatial and temporal patterns. In the adult CNS, its expression remains in areas of active neurogenesis and areas that exhibit neuronal plasticity. Understanding of the contribution of this extracellular matrix constituent to the major developmental processes such as cell proliferation and migration, axonal guidance, as well as synaptic plasticity, is derived from studies on TN-C deficient mice. Studies on these mice demonstrated that TN-C plays an important role in neuronal plasticity in the cerebral cortex, hippocampus and cerebellum, possibly by modulating the activity of L-type voltage-dependent Ca(2+) channels.

Titin-based stiffening of muscle fibers in Ehlers-Danlos Syndrome.

OBJECTIVE: tenascin-X (TNX) is an extracellular matrix glycoprotein whose absence leads to Ehlers-Danlos Syndrome (EDS). TNX-deficient EDS patients present with joint hypermobility and muscle weakness attributable to increased compliance of the extracellular matrix. We hypothesized that in response to the increased compliance of the extracellular matrix in TNX-deficient EDS patients, intracellular adaptations take place in the elastic properties of the giant muscle protein titin. METHODS: we performed extensive single muscle fiber mechanical studies to determine active and passive properties in TNX-deficient EDS patients. Gel-electrophoresis, Western blotting, and microarray studies were used to evaluate titin expression and phosphorylation. X-ray diffraction was used to measure myofilament lattice spacing. RESULTS: passive tension of muscle fibers from TNX-deficient EDS patients was markedly increased. Myofilament extraction experiments indicated that the increased passive tension is attributable to changes in the properties of the sarcomeric protein titin. Transcript and protein data indicated no changes in titin isoform expression. Instead, differences in posttranslational modifications within titin's elastic region were found. In patients, active tension was not different at maximal activation level, but at submaximal activation level it was augmented attributable to increased calcium sensitivity. This increased calcium sensitivity might be attributable to stiffer titin molecules. CONCLUSION: in response to the increased compliance of the extracellular matrix in muscle of TNX-deficient EDS patients, a marked intracellular stiffening occurs of the giant protein titin. The stiffening of titin partly compensates for the muscle weakness in these patients by augmenting submaximal active tension generation.