Role of tenascins in the ECM of gliomas

Abstract

Tenascins are a family of extracellular matrix molecules that are mainly expressed in embryonic development and down-regulated in adulthood. A re-expression in the adult occurs under pathological conditions such as inflammation, regeneration or neoplasia. As the most prominent member of the tenascin family, TN-C, is highly expressed in glioma tissue and rising evidence suggests that TN-C plays a crucial role in cell migration or invasion – the most fatal characteristics of glioma – also the other members of this protein family have been investigated with regard to their impact on glioma biology. For all tenascins correlations between the expression levels of the different family members and the degree of malignancy and invasiveness of glial tumors could be detected. Overall, the former and recent results in the research on glioma and tenascins point at distinct roles of each of the molecules in glioma biology and the devastating properties of these tumors.

Glioma

The most common type of primary brain tumors is represented by the class of glioma with an incidence of ∼5/100,000 patients.^1,2 Despite intensive research, advanced diagnostics and improved therapy strategies the prognosis of high-grade glioma remains devastating.³ The survival time still varies between 15 months (high-grade glioma) and 3 y (low-grade glioma), depending on the grade of malignancy which is categorized by the World Health Organization (WHO) as follows: Tumors of grade I and II were referred to as low grade tumors with a 5-years survival rate of 58–72% as shown by the NCCTG trial, EORTC 22844 and EORTC 22845.⁴ Glial malignancies of grade III and IV are classified as high-grade glioma, with the worst survival rate indicated above.² Particularly, patients diagnosed with the severest glial tumor glioblastoma multiforme (GBM, grade IV) who survive more than 3 y after diagnosis were referred to as long time survivors. Certainly, this group comprises only 3–5% of patients with a GBM⁵ and this underlines that the GBM is not only the most frequently recognized glioma (>51% of all glioma), but also the malignant endpoint of this cancer type.⁶ Comparing primary and secondary glioblastoma both GBM-types share similarities concerning histologic and morphologic features, but differ in their genetic profiles. Primary glioblastoma arise de novo and are characterized by EGFR amplification, mutations in rb1, cdkn2a, p14arf and PTEN and monosomia 10.^7-9 Secondary glioblastoma develop from former low-grade glioma and show unique alterations such as mutations of TP53 or IDH1, or the loss of chromosome 19q and 13q and the overexpression of PDGFRα.^8,10-12

Histologically primary and secondary glioblastoma display characteristic attributes, including high mitotic activity, cellular and nuclear atypia, strong microvascular proliferation and extended areas of necrosis.^6,10 Even though these atypias arise from different genomic alterations both GBM types respond similarly to therapeutic approaches¹³ with a slightly better prognosis for patients suffering from secondary glioblastomas.⁸ The high heterogeneity within the classification of glioma tumors gained renewed attention in conjunction with the highly promising research field of glioma-initiating cells^14,15 in order to develop new, personalized therapy strategies.

Additionally, the highly invasive behavior of glioma cells leads to quick spreading of the tumor throughout both hemispheres. This feature dramatically shortens the lifespan of glioma patients. Despite accurate surgery it is impossible to remove all malignant cells⁶ and the recurrent tumors exhibit explicit resistance to chemotherapy and radiation.^16,17 The invasion of glioma cells is characterized by their ability to migrate as single cells even to distant parts of the brain. With regard to migration pathways, the tumor cells display a preference for white matter tracts, subependymal layers and blood vessel basement membranes as leading structures.^10,18,19 To initiate this migration process glioma cells degrade the ECM into a migration favorable microenvironment.

The Extracellular Matrix

Cells of each tissue are surrounded by a dynamic molecular meshwork filling the extracellular space. This extracellular matrix (ECM) provides a scaffold for the organization of tissues and supports the cohesion of cells.²⁰ While the structural task represents an important function of the ECM, numerous additional features of the ECM have been uncovered in recent years. The maintenance of cell-cell-communications^21,22 and the construction of favorable substrates for cell migration²⁰ illustrate central tasks of the ECM which could be observed ubiquitously in the context of differentiation, proliferation, survival and polarity in the regulation of embryonic development as well as in the homeostasis and tissue remodeling in pathological incidents.^23,24

The ECM is composed of a complex mixture of matrix molecules. Glycoproteins like fibronectin, laminins or tenascins contribute to it, as well as glycosaminoglycans.^24,25 With regard to the content of collagens the ECM of the central nervous system (CNS) differs from the classical matrix in other organs. Whereas the ECM of numerous tissues contains a high amount of collagen fibers this element is rigorously restricted to blood vessels and the glia limitans²⁶ in the brain. The proteins of the ECM interact with each other and their neighboring cells mainly via the specialized matrix receptors of the integrin superfamily. As a result of this interaction the ECM is able to influence different signaling pathways and to give impulses to the behavior of cells by varying its mechanical properties.^27-29 During this remodeling process the ability of sulfated proteoglycans to bind growth factors allows the ECM to function as a pool of growth factors which can be released if possible.³⁰

Especially the involvement of the ECM in the regulation of cell motility constitutes an area of interest in the glioma research field because of the known high motility of these cells that results in invasion and recurrence of the tumor. It has been discovered long ago that proteins of the ECM such as laminins, fibronectin or tenascins influence the behavior of glioma cells.^31-35 Several ECM components including collagens I, II and IV as well as laminin, fibronectin and tenascin-c (TN-C) could be detected within the basal lamina of tumor blood vessels.^23,36-38 With exception of tenascin-c the ECM molecules are not synthesized by the tumor cells themselves. Rather, the tumor cells induce cells of the surrounding brain tissue to produce these proteins.³⁹ In contrast, tenascin-c is autonomously expressed by the glioma cells.³⁵ Hereby the tumor is able to generate an individual overexpression of matrix components whose secretion alters the ECM in various manners. On the one hand it could lead to the stimulation of adhesion and migration of the glioma cells, but on the other hand the ECM could be condensed in a way that a highly concentrated ECM might diminish the diffusion of neuroactive molecules or therapeutical agents.^40,41

Tenascins

Tenascin-C

Tenascin-C (TN-C) is a member of the glycoprotein family of tenascins (Fig. 1) mainly expressed during embryonic development, downregulated in the adult and re-expressed under pathological conditions.^42,43 The discovery of TN-C occurred in parallel in different fields of research (e.g., embryonic development, tumor biology, neurobiology). From this follows that TN-C was originally known under various names. In 1983 it was introduced by Bourdon & Wikstrand as glial/mesenchymal extracellular matrix protein GMEM.⁴⁴ Shortly thereafter TN-C was named myotendinous antigen,^45,46 Hexabrachion,⁴⁷ Cytotactin,⁴⁸ J1220/200⁴⁹ and neuronectin.⁵⁰ Erickson & Inglesias introduced the name hexabrachion, refering to the structural composition of the TN-C molecule. TN-C consists of 6 polypeptide monomers which are combined into the hexamer at their N-termini.⁴⁷ Each monomer of human TN-C comprises a cysteine-rich domain on the N-terminus followed by 14.5 EGF-like repeats. The EGF-like repeats are connected to 8 constitutively expressed fibronectin type III (FNIII) domains (1–8). In between the fifth and sixth FNIII domain up to 9 alternatively spliced FNIII domains (A1-A4, B, AD2, AD1, C, D) may be integrated. At the C-terminus a fibrinogen-like globe terminates each monomer (Fig. 1A).^51-53 The alternative splicing of the FNIII domains leads to various isoforms which influence different cell types in varying manners, depending on the individual set up of FNIII domains.^54,55

Figure 1.

Monomer structure of tenascin family members. A Tenascin-C, B Tenascin-R, C Tenascin-W, D Tenascin-X EGF/R epidermal growth factor; N-Cad, N-Cadherin; α/β, α/β-Integrin; GPC-1, Glypican-1; FGF/R, fibroblast growth factor; Syn-4, Syndecan-4; ANXII, Annexin II; Cont, Contactin, CASP; Contactin associated Protein; Cat, Catenin; PI3K, Phospho-Inositol-3-Kinase; GPα, G-Protein α; FAK, Focal adhesion kinase; PAX, Paxillin; PIP2, Phosphatidyl-Inositol-Biphosphate, α-Act, α-Actinin; PKC, Proteinkinase C.

Whereas in the adult the smallest isoform with only one alternatively spliced FNIII domain is found in static tissues (e.g., cartilage),⁵⁶ the embryonic development^57,58,59,60 as well as pathological situations like inflammation, regeneration or tumorigenesis^{61,62,42,63,60} is marked by the dominant expression of large isoforms.

By binding to integrins as their main receptor type TN-C affects the cell behavior in a direct way.⁴³ Indirectly, TN-C acts via binding to other ECM molecules like brevican or neurocan.^54,64 This leads to a variety of processes TN-C could be effective in: cell migration,^65,66 inhibition of focal adhesion assembly,⁶⁷ promotion of angiogenesis,⁴³ increase in proliferation^68,69 and changes in gene expression to modulate the composition of the ECM.⁷⁰

The discovery in 1983 of TN-C as GMEM in glioblastoma tissue and glioma cell lines by Bourdon⁴⁴ highlighted the interest of this protein as a characteristic component of tumors. Nearly all kinds of solid tumors express high levels of TN-C but the highest concentrations were found in glioma (Fig. 2).^{71 72} This is coupled to the correlation of a high TN-C expression with elevated malignancy and poor patients' survival.^18,73,74

Figure 2.

Tenascin-C strongly expressed in cells and tissues of glioblastoma multiforme (A–C) Staining of glioblastoma tissue specimen probed for Tenascin-C with monoclonal antibodies mAb 20A1_TN-C, mAb 608_TN-C and mAb606_TN-C (D–F) Staining of glioblastoma cell lines examined with monoclonal anti-tenascin-C antibodies mAb 578_TN-C, mAB 608_TN-C and mAb 19H12_TN-C.

The deleterious influence of TN-C could be associated with 3 main areas TN-C plays a crucial role in: angiogenesis, proliferation and cell migration. Each of these stands for prospects of high malignancy and in combination they represent the “evil face” of glioma.

TN-C in tumor angiogenesis

Glioma – like all other cancer types – are in need of receiving nutrients and disposing metabolic waste. Therefore they trigger the generation of new blood vessels from pre-existing vessels. In the last years it could be concordantly revealed that TN-C is highly associated with tumor blood vessels (Fig. 2A–C).^38,75,76 The amount of TN-C in tumor blood vessels is correlated with the malignancy of the tumor as it is found in higher concentration in high-grade glioma than in low-grade glioma.⁷⁵ Certainly, the blood vessels of glioma do not resemble the architecture of normal blood vessels. Within one tumor different phenotypes like incipient proliferation of endothelial cells or sarcomatous structures are found.⁷⁷

Especially the alternatively spliced domains TNfnC^78,79 and TNfnA2³⁸ could be found highly up-regulated in glioma blood vessels, but also the EGF-type repeats as well as the fibrinogen globe are supposed to play a role in the formation of tumor blood vessels.⁸⁰ The TN-C-induced generation of tumor blood vessels could be related to the binding to endothelial cells⁸¹ and its stimulating effect on this cell type.^82,83 Besides TGFbeta as an expected candidate involved in the underlying pathway,⁸⁴ it could be revealed that the expression of VEGF is strongly correlated with the expression of tenascin-C in perivascular zones. Tanaka et al. discovered that TN-C regulates the expression of VEGF⁸⁵ and Behrem et al. showed that TN-C has an influence on VEGF action and that the microvascular density shows a dependency of TN-C expression.⁸⁶ Additionally to the high expression of TN-C in glioma blood vessels the known receptor for TN-C, integrin α v, is found in elevated levels in glioma tissue, as well as the protein periostin.⁷⁷ Periostin was detected as a promoter of TN-C incorporation into the ECM and to organize the architecture of the ECM.⁸⁷ For murine pancreatic neuroendocrine tumorigenesis it could be recently shown that the composition of upregulated matrisomal genes in pericytes could be correlated to genes overexpressed also in glioma. Furthermore an abrogation of TN-C from this matrix diminishes the number and proportion of angiogenic islets formed during the progression of this tumor. ⁸⁸

TN-C in tumor cell proliferation

It has been convincingly proven that TN-C stimulates the proliferation of different cell types.^86,89 Among them are not only endothelial cells with their importance for the tumor angiogenesis^82,83 mentioned above, but also the glioma cells themselves.^90-92 Until now only little is known about the detailed signaling pathways TN-C contributes to proliferation. Much less information has been obtained about the involvement of distinct domains of TN-C and their impact on individual signaling.^86,93 Nonetheless some signaling pathways could be revealed that are involved in TN-C-stimulated proliferation. One known way to induce proliferation in glioma cells concerns the interaction between TN-C and fibronectin. By blocking the adhesion of cells to fibronectin the activation of syndecan-4 is impaired, which leads to proliferation of the cells caused by prevention of cell adhesion and spreading.⁹⁴

In the alternatively spliced region of the FNIII-domains the highly overexpressed domains C, AD1 and AD2 in glioma^79,95,96 correlate with the proliferation rate of cancer cells.⁹⁷ More detailed information about underlying mechanisms of glioma proliferation has been obtained for the N-terminal EGF type domains^69,98 and the C-terminal fibrinogen knob.⁹⁷ These pathways including phospholipase Cγ1, Ras/MAPK, phosphatidyl inositol 3-kinase/Akt could be activated by binding of EGF domains to the EGF receptor^69,99-101 and increase cell proliferation. In line with this observation the inhibition of the phosphatidyl-inositol-3-kinase/Akt pathway via modulated suppression or inhibition of the EGFR leads to a decrease in the proliferation of glioma cells.^102,103 Additionally, the fibrinogen domain of TN-C plays a pivotal role in stimulating the proliferation of chondrocytes via the ERK/MAPK-pathway.¹⁰⁴ In contrast, the combination of all FNIII-domains decreases the proliferative effect of intact TN-C on glioma cells.³⁸

TN-C and tumor cell migration

The most fatal attribute of glioma is the capability to invade into healthy brain parenchyma as single cells to build new tumors.¹⁰ This leads to severe problems for therapeutic approaches because the secondary tumors could arise even in distant parts of the opposite hemisphere.^1,10 Cell migration in general can be separated into 4 distinct steps. After an initial polarization step the cells generate cell protrusions and in the third step create new contacts with the surrounding matrix. Finally, the previous cell matrix contacts are disrupted.¹⁰⁵ The ECM modulates this sequential mechanism.^106,107 As known for other cells types also in glioma cells TN-C leads to a highly motile and invasive phenotype.^108,109

The alternatively spliced domains of TN-C are thought to play a crucial role in the migratory behavior of glioma cells. Thus, the domain TNfnA2 has been reported to induce the generation of stress fibers and focal adhesion sites in dependency of β1 integrin¹¹⁰ – 2 highly important processes in the context of motility. Whereas the decrease of stress fibers and focal adhesion sites leads to weakening of the contractile forces, inducing a decrease in cell migration.^111,112 For example, the initiation of contractile forces due to strengthened stress fibers and an augmentation of focal adhesion sites by TNfnA2 could support an increase in cell motility. The underlying pathways of these morphological changes influencing migration are related to the activation of the focal adhesion kinase (FAK) and the GTPases of the RhoA-subfamily.¹¹³

Another mechanism by which TNfnA2 may boost cell migration could be mediated by the transmembrane heparan sulfate proteoglycan syndecan-4. This proteoglycan favors the activation of a cryptic binding site in TNfnA2 resulting from the action of MMP-2¹¹⁰ and leads to changes in stress fibers and focal adhesion sites which can produce alterations in cell migration in mouse embryonic stem cells.^114,115 Considering the high expression of TN-C^71,72 and MMP-2¹¹⁶ in glioma cells this pathway is a possible candidate to induce glioma migration.

Not only the alternatively spliced domains contribute to cell migration but also the constitutively expressed domains such as TNfn3 are involved. This domain contains an RGD-sequence and could mediate cell adhesion via different integrins to modulate cell motility (reviewed in⁴³). Yokosaki et al. have reported that the integrin α9β1 increases cell migration independently of the RGD-sequence.¹¹⁷

Another prominent pathway involved in cell migration includes signaling via the α2β1-integrin and can be neutralized by blocking this receptor.¹⁰⁹ Possibly, this integrin represents a potential candidate for the still unknown signaling cascade that is activated by the EGF-type domains of TN-C and induces glioma cells migration.⁶⁹ It is noteworthy that a HER2-specific phosphorylation of the EGFR could lead to an activation of this receptor and an N-Cadherin mediated stimulation of glioma cell migration.¹¹⁸ As the fibrinogen domain plays a role in the migration of vascular smooth muscle and bladder cancer cells through an ICAM-1-mediated pathway^119,120 including Akt- and MAPK-dependent signaling, it is conceivable that the fibrinogen domain of TN-C exhibits analogous properties. This hypothesis, however, remains to be examined in future studies.

Tenascin-R

TN-R as a member of the tenascin family^121,122 was first discovered in 1985 as “low molecular weight J1 glycoprotein (J1–160/ 80)”.⁴⁹ Similar to TN-C also TN-R has originally been independently discovered in different species and designated with different names, that is janusin^123,124 and restrictin.¹²⁵ Structurally TN-R appears in 2 variants of the glycoprotein: TN-R 160 (160 kDa) and TN-R 180 (180 kDa) that differ by one alternatively spliced FNIII-domain.¹²⁶ Each molecule starts at the N-terminus with a cysteine-rich region followed by 4.5 EGF-like repeats and 8 constitutively expressed FNIII domains, possibly supplemented with one alternatively spliced FNIII domain. At the C-terminus the molecule is completed with a fibrinogen like domain (Fig. 1B).¹²⁷ The expression of TN-R is restricted to the nervous system. It could be found in motor neurons, on motor axons,¹²⁵ in the hippocampus, cerebellum, olfactory bulb, myelinating oligodendrocytes as well as type-2 astrocytes.¹²⁷

The function of TN-R depends on distinct ligands and receptors, for example chondroitinsulfate proteoglycans (CSPGs) of the lectican-family, the membrane-based part-time proteoglycan CALEB, or the Ig-superfamily-based receptor F3/contactin.¹²⁸ The resulting effects comprise influences on cell adhesion, neural cell migration, the size of the extracellular space, regulation of cell-matrix interaction and axon outgrowth (extensively reviewed in¹²⁸). Additionally the CS-GAG chains of TN-R are involved in an interaction with TN-C in a Ca²⁺-dependent manner and lead to a regulation of cell-matrix interactions in cases of tissue repair and neoplasms.^128-130 Although studies on TN-R-deficient mice reveal that they have a normal life span and display only few histological aberrations with mild behavioral changes^124,131,132 the outcome of EEG-examinations support the hypothesis that TN-R could play a crucial role in the development of epilepsy.¹²⁸ A possible role for TN-R in other CNS diseases is suggested by the observation that TN-R is reduced in tissue samples from patients suffering from multiple sclerosis.¹³³ Already in 1996 Carnemolla et al. have reported that TN-R is expressed in the healthy brain. Furthermore, TN-R was found in samples of human astrocytoma and meningioma, where the small isoform with one FNIII-repeat less amounted for 10% of the whole TN-R content.¹²⁷

Studies on TN-R in glioma have in common that tumors with non-invasive behavior and/or grading into WHO I or II (e.g. pilocytic astrocytoma,¹³⁴ medulloblastoma in children¹³⁵) show a high expression of TN-R. In contrast, a decrease of TN-R expression has been observed in correlation with increasing malignancy. At the endpoint of glioma progression, that is in the glioblastoma of WHO grade IV only a weak TN-R expression could be detected. Whether there is an inverse correlation between the grade of malignancy and the expression level of TN-R and whether TN-R plays a role in the non-invasive behavior of tumors is still unclear and remains to be clarified further.¹³⁴

Tenascin-W

Cloned from zebrafish and mice in the late 1990s¹³⁶ and early 2000s,^137,138 human TN-W was cloned and characterized in 2007.¹³⁹ Like TN-C it appears in hexamers composed of 2 × 3 monomers. Each monomer starts with the typical cysteine-rich region at the N-terminus. 3.5 EGF-like domains are connected to this region, followed by varying FNIII-repeats (in mice: 12, in human: 9 repeats). The sequence ends with a fibrinogen-like globe at the C-terminus (Fig. 1C).¹³⁸ Like the other tenascins TN-W is expressed during embryonic development and partially co-expressed with TN-C.^136,138 In adulthood it is nearly absent from most types of tissues but could be found re-expressed under pathological conditions, especially in tumor development. In human cancer TN-W was found highly enriched in colon carcinoma and breast tumors, whereas the healthy tissue is devoid of any TN-W.¹³⁹ In the special case of highly destructive brain tumors TN-W has been reported in elevated levels in astrocytoma, glioblastoma and oligodendroglioma while the healthy adult brain is devoid of TN-W expression.¹⁴⁰

In contrast to the best-known tenascin family member TN-C, TN-W is not expressed by glioma cells, but exclusively by endothelial cells in tumor blood vessels, where it colocalizes with von-Willebrand-factor.¹⁴⁰ TN-W could be associated with different cellular activities. Distinct from TN-C, TN-W is not able to mediate any adhesion to cells^138-140 or to induce proliferation.¹⁴¹ TN-W-dependent signaling could partially be attributed to the activation of the β1-integrin subunit^138,139 that is highly expressed in glioma tumors.^142,143 Therefore, it is not surprising that TN-W is able to induce the migration of tumor¹³⁹ as well as of endothelial cells.¹⁴⁰ Concerning tumors of the glioma type investigations of the influence of TN-W on cell motility or angiogenesis are still outstanding but the previous results point to a potential role of TN-W also in this cancer type.

Tenascin-X

The latest discovered member of the tenascin family TN-X possesses the same molecular organization than the other family members TN-C, TN-R and TN-W. Initiated by a N-terminal cysteine-rich assembly domain, 18.5 EGF-like domains are attached and followed by 32 FNIII domains (human). The C-terminus is formed by a fibrinogen like globe (Fig. 1D). In contrast to the other tenascins, the TN-X molecule integrates a proline-rich stretch of about 100 amino acids.¹⁴⁴ Via disulphide bonds TN-X is assembled into trimers¹⁴⁵ and achieved prominence as it is related to the human heritable disorder named Ehlers-Danlos syndrome.¹⁴⁶ The research on this syndrome has led to insights that TN-X is involved in network formation of the ECM as patients suffering from this disease show joint laxity and skin hyperextensibility.^146,147

Found in many adult tissues TN-X plays a crucial role in the modulation of cell-matrix communication.¹⁴⁵ By interacting with numerous ECM molecules like collagens^148,149 or decorin¹⁵⁰ it is involved in the adhesion and spreading of cells. The mechanisms behind these effects are still mostly unclear but 2 structures are thought to be part of the used signaling pathways: the C-terminal fibrinogen-like globe could interact with a still unknown integrin containing the β1 subunit whereas the signaling via heparansulfate proteoglycans might use the heparin binding site contained in the FNIII domains 10 and 11.¹⁵¹

Which of these signaling pathways – if any – is responsible for the inhibition of tumor cell invasion and metastasis¹⁵² is presently unknown. But considering the results by Hasegawa et al. showing that there is an inverse correlation between the expression level of TN-X and malignancy of glioma tumors¹⁵³ there is rising evidence that TN-X could play a role in the invasion of glioma cells. Comparing the expression patterns of TN-C which is found in the intercellular spaces and in tumor blood vessels of high grade glioma^38,153 and TN-X which is restricted to the tumor stroma and surrounds blood vessels¹⁵³ the question remains whether TN-X is a potential key for the restricted invasiveness of low-grade glioma.

Conclusion

Considering the data discussed above there is no doubt that the family of tenascin proteins is involved in regulating the behavior of glioma cells and/or the development of glial tumors. Although TN-C is so far the most intensely examined member of the tenascin family, a substantial portion of its mechanisms of action and signaling pathways still remains unclear. In particular, the effects of the FNIII-domains on tumor cell migration and invasion as well as on angiogenesis remain to be clarified. Ongoing research in this direction is very encouraging and seems on the right track toward elucidation. Concerning the other members of the family only few studies have been published and further investigations are required to understand the contribution to glioma development and inhibition of increasing malignancy due to expression of TN-R, TN-W and TN-X.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed

Funding

We acknowledge grant support by the Stem Cell Network Northrhine Westphalia, the German Research Foundation (DFG: SPP 1109, Fa 159/16-1, SFB 642, SPP 1757, GRK 736, GSC 98/1), The German Ministry of Education, Research and Technology (BMBF 01GN0503) and the Ruhr-University (President's special program call 2008).