Involvement of Matricellular Proteins in Cellular Senescence: Potential Therapeutic Targets for Age-Related Diseases.

Senescence is a physiological and pathological cellular program triggered by various types of cellular stress. Senescent cells exhibit multiple characteristic changes. Among them, the characteristic flattened and enlarged morphology exhibited in senescent cells is observed regardless of the stimuli causing the senescence. Several studies have provided important insights into pro-adhesive properties of cellular senescence, suggesting that cell adhesion to the extracellular matrix (ECM), which is involved in characteristic morphological changes, may play pivotal roles in cellular senescence. Matricellular proteins, a group of structurally unrelated ECM molecules that are secreted into the extracellular environment, have the unique ability to control cell adhesion to the ECM by binding to cell adhesion receptors, including integrins. Recent reports have certified that matricellular proteins are closely involved in cellular senescence. Through this biological function, matricellular proteins are thought to play important roles in the pathogenesis of age-related diseases, including fibrosis, osteoarthritis, intervertebral disc degeneration, atherosclerosis, and cancer. This review outlines recent studies on the role of matricellular proteins in inducing cellular senescence. We highlight the role of integrin-mediated signaling in inducing cellular senescence and provide new therapeutic options for age-related diseases targeting matricellular proteins and integrins.

Anoikis resistance conferred by tenascin-C-derived peptide TNIIIA2 and its disruption by integrin inactivation.

Glioblastoma multiforme (GBM), the most common brain tumor in adults, has an extremely poor prognosis, which is attributed to the aggressive properties of GBM cells, such as dysregulated proliferation and disseminative migration. We recently found that peptide TNIIIA2, derived from tenascin-C (TNC), which is highly expressed in GBM, contributes to the acquisition of these aggressive properties through ß1-integrin activation. In general, cancer cells often acquire an additional malignant property that confers resistance to apoptosis due to loss of adhesion to the extracellular matrix, termed anoikis resistance. Our present results show that regulation of ß1-integrin activation also plays a key role in both the development and loss of anoikis resistance in GBM cells. Despite being derived from a GBM with an extremely poor prognosis, the human GBM cell line T98G was susceptible to anoikis but became anoikis resistant via treatment with peptide TNIIIA2, which is able to activate ß1-integrin. The TNIIIA2-conferred anoikis resistance of T98G cells was disrupted by further addition of peptide FNIII14, which has the ability to inactivate ß1-integrin. Moreover, anchorage-independent survival of GBM cells in suspension culture was abrogated by peptide FNIII14, but not by RGD and CS-1 peptides, which are antagonistic for integrins α5ß1, αvß3, and α4ß1. These results suggest that GBM cells develop anoikis resistance through activation of ß1-integrin by TNC-derived peptide TNIIIA2, which is abundantly released into the tumor microenvironment of GBM. Inactivation of ß1-integrin may provide a promising strategy to overcome the apoptosis resistance of cancer cells, including GBM.

A novel method to purify neutrophils enables functional analysis of zebrafish hematopoiesis.

Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5, a DNA-staining fluorescent dye. In adult kidney marrow, we purified neutrophil-like PHA-E4low DRAQ5low cells, which neutrophil-type granules. Specifically, at 96-hr post-fertilization, we sorted large-sized cells from larvae using forward scatter and found that they consisted of PHA-Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B-positive and expressed high levels of neutrophil-specific (csf3r and mpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (Zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA-E and DRAQ5-based flow cytometry serves as a tool to purify zebrafish neutrophils.

Involvement of Integrin-Activating Peptides Derived from Tenascin-C in Cancer Aggression and New Anticancer Strategy Using the Fibronectin-Derived Integrin-Inactivating Peptide.

Matricellular proteins, which exist in association with the extracellular matrix (ECM) and ECM protein molecules, harbor functional sites within their molecular structures. These functional sites are released through proteolytic cleavage by inflammatory proteinases, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and the peptides containing these functional sites have unique biological activities that are often not detected in the parent molecules. We previously showed that tenascin-C (TNC) and plasma fibronectin (pFN), examples of matricellular proteins, have cryptic bioactive sites that have opposite effects on cell adhesion to the ECM. A peptide containing the bioactive site of TNC, termed TNIIIA2, which is highly released at sites of inflammation and in the tumor microenvironment (TME), has the ability to potently and persistently activate ß1-integrins. In the opposite manner, the peptide FNIII14 containing the bioactive site of pFN has the ability to inactivate ß1-integrins. This review highlights that peptide TNIIIA2 can act as a procancer factor and peptide FNIII14 can act as an anticancer agent, based on the regulation on ß1-integrin activation. Notably, the detrimental effects of TNIIIA2 can be inhibited by FNIII14. These findings open the possibility for new therapeutic strategies based on the inactivation of ß1-integrin by FNIII14.

TAp63 represses transcription of MYCN/NCYM gene and its high levels of expression are associated with favorable outcome in neuroblastoma.

TAp63 is an isoform of p63 gene, a p53 family gene that suppresses tumorigenesis via transcriptional regulation. TAp63 represses transcription of MYC oncogene in glioblastomas; however, its role in another MYC family gene, MYCN, has remained elusive. In this study, we showed that TAp63 repressed transcription of the MYCN gene in human cancer cells. Overexpression of TAp63 in HeLa cells suppressed MYCN expression, whereas knockdown of TAp63 had the opposite effect. By binding to exon 1 of MYCN gene, TAp63 suppressed the promoter activities of MYCN and its cis-antisense gene, NCYM. Other p53 family members, p53 and TAp73, showed lesser ability to suppress MYCN/NCYM promoter activities compared with that of TAp63. All-trans-retinoic acid (ATRA) treatment of MYCN/NCYM-amplified neuroblastoma CHP134 cells induced TAp63 and reduced p53 expressions, accompanied by downregulation of MYCN/NCYM expressions. Meanwhile, TAp63 knockdown inhibited ATRA-induced repression of NCYM gene expression. Blocking the p53 family binding sites by CRISPR-dCas9 system in CHP134 cells induced MYCN/NCYM expression and promoted apoptotic cell death. Expression levels of TAp63 mRNA inversely correlated with those of MYCN/NCYM expression in primary neuroblastomas, which was associated with a favorable prognosis. Collectively, TAp63 repressed MYCN/NCYM bidirectional transcription, contributing to the suppression of neuroblastoma growth.

Autocrine Production of PDGF Stimulated by the Tenascin-C-Derived Peptide TNIIIA2 Induces Hyper-Proliferation in Glioblastoma Cells.

Expression level of tenascin-C is closely correlated to poor prognosis in glioblastoma patients, while the substantial role of tenascin-C responsible for aggressive progression in glioblastoma cells has not been clarified. We previously found that peptide TNIIIA2, which is derived from the tumor-associated tenascin-C variants, has the ability to promote cell adhesion by activating ß1-integrins. Our recent study demonstrated that potentiated activation of integrin α5ß1 by TNIIIA2 causes not only a dysregulated proliferation in a platelet-derived growth factor (PDGF)-dependent manner, but also disseminative migration in glioblastoma cells. Here, we show that TNIIIA2 enhances the proliferation in glioblastoma cells expressing PDGF-receptorß, even without exogenous PDGF. Mechanistically, TNIIIA2 induced upregulated expression of PDGF, which in turn stimulated the expression of tenascin-C, a parental molecule of TNIIIA2. Moreover, in glioblastoma cells and rat brain-derived fibroblasts, tenascin-C upregulated matrix metalloproteinase-2, which has the potential to release TNIIIA2 from tenascin-C. Thus, it was shown that autocrine production of PDGF triggered by TNIIIA2 functions to continuously generate a functional amount of PDGF through a positive spiral loop, which might contribute to hyper-proliferation in glioblastoma cells. TNIIIA2 also enhanced in vitro disseminative migration of glioblastoma cells via the PKCα signaling. Collectively, the tenascin-C/TNIIIA2 could be a potential therapeutic target for glioblastoma.

Peptide TNIIIA2 Derived from Tenascin-C Contributes to Malignant Progression in Colitis-Associated Colorectal Cancer via ß1-Integrin Activation in Fibroblasts.

Inflammatory bowel diseases increase the risk of colorectal cancer and colitis-associated colorectal cancer (CAC). Tenascin-C, a matricellular protein, is highly expressed in inflammatory bowel diseases, especially colorectal cancer. However, the role of tenascin-C in the development of CAC is not yet fully understood. We previously showed that a peptide derived from tenascin-C, peptide TNIIIA2, induces potent and sustained activation of ß1-integrin. Moreover, we recently reported that peptide TNIIIA2 promotes invasion and metastasis in colon cancer cells. Here, we show the pathological relevance of TNIIIA2-related functional site for the development of CAC. First, expression of the TNIIIA2-containing TNC peptides/fragments was detected in dysplastic lesions of an azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model. In vitro experiments demonstrated that conditioned medium from peptide TNIIIA2-stimulated human WI-38 fibroblasts induced malignant transformation in preneoplastic epithelial HaCaT cells. Indeed, these pro-proliferative effects stimulated by peptide TNIIIA2 were abrogated by peptide FNIII14, which has the ability to inactivate ß1-integrin. Importantly, peptide FNIII14 was capable of suppressing polyp formation in the AOM/DSS model. Therefore, tenascin-C-derived peptide TNIIIA2 may contribute to the formation of CAC via activation of stromal fibroblasts based on ß1-integrin activation. Peptide FNIII14 could represent a potential prophylactic treatment for CAC.

The Promoting Effect of the Extracellular Matrix Peptide TNIIIA2 Derived from Tenascin-C in Colon Cancer Cell Infiltration.

The extracellular matrix (ECM) molecule tenascin C (TNC) is known to be highly expressed under various pathological conditions such as inflammation and cancer. It has been reported that the expression of TNC is correlated with the malignant potential of cancer. In our laboratory, it was found that the peptide derived from the alternative splicing domain A2 in TNC, termed TNIIIA2, has been shown to influence a variety of cellular processes, such as survival, proliferation, migration, and differentiation. In this study, we investigated the effect of TNC/TNIIIA2 on the invasion and metastasis of colon cancer cells, Colon26-M3.1, or PMF-Ko14, using an in vitro and in vivo experimental system. The degree of cell invasion was increased by the addition of TNC and TNIIIA2 in a dose-dependent manner. The invasion by TNC and TNIIIA2 were suppressed by an MMP inhibitor or TNIIIA2-blocking antibody. In an in vivo experiment, pulmonary metastasis was promoted conspicuously by the addition of TNIIIA2. In this study, we found that colon cancer cell invasion and metastasis was accelerated by TNC/TNIIIA2 via MMP induction. This result suggests the possibility of a new strategy targeting TNC/TNIIIA2 for colon cancer.

Induction of cellular senescence in fibroblasts through ß1-integrin activation by tenascin-C-derived peptide and its protumor effect.

Tenascin-C is upregulated during inflammation and tumorigenesis, and its expression level is correlated with a poor prognosis in several malignancies. Nevertheless, the substantial role of tenascin-C in cancer progression is poorly understood. Previously, we found that a peptide derived from tenascin-C, termed TNIIIA2, acts directly on tumor cells to activate ß1-integrin and induce malignant progression. Here, we show that ß1-integrin activation by TNIIIA2 in human fibroblasts indirectly contributes to cancer progression through the induction of cellular senescence. Prolonged treatment of fibroblasts with TNIIIA2 induced cellular senescence, as characterized by the suppression of cell growth and the induction of senescence-associated-ß-galactosidase and p16INK4a expression. The production of reactive oxygen species and subsequent DNA damage were responsible for the TNIIIA2-induced senescence of fibroblasts. Interestingly, peptide FNIII14, which inactivates ß1-integrin, inhibited fibroblast senescence induced not only by TNIIIA2 but also by H2O2, suggesting that ß1-integrin activation plays a critical role in the induction of senescence in fibroblasts. Moreover, TNIIIA2-induced senescent fibroblasts secreted heparin-binding epidermal growth factor-like growth factor (HB-EGF), which caused preneoplastic epithelial HaCaT cells to acquire malignant properties, including colony-forming and focus-forming abilities. Thus, our study demonstrates that tenascin-C-derived peptide TNIIIA2 induces cellular senescence in fibroblasts through ß1-integrin activation, causing cancer progression via the secretion of humoral factors such as HB-EGF.

Exposure of the cryptic de-adhesive site FNIII14 in fibronectin molecule and its binding to membrane-type eEF1A induce migration and invasion of cancer cells via ß1-integrin inactivation.

Cell migration is a highly coordinated process that involves not only integrin-mediated adhesion but also de-adhesion. We previously found that a cryptic de-adhesive site within fibronectin molecule, termed FNIII14, weakens cell adhesion to the extracellular matrix by inactivating ß1-integrins. Surprisingly, eukaryotic translation elongation factor-1A (eEF1A), an essential factor during protein biosynthesis, was identified as a membrane receptor that mediates the de-adhesive effect of FNIII14. Here, we demonstrate that FNIII14-mediated de-adhesion causes enhanced migration and invasion in two types of highly invasive/metastatic cancer cells, resulting in the initiation of metastasis. Both in vitro migration and invasion of highly invasive human melanoma cell line, Mum2B, were inhibited by a matrix metalloproteinase (MMP)-2/9 inhibitor or a function-blocking antibody against FNIII14 (anti-FNIII14 Ab), suggesting that MMP-mediated exposure of the cryptic de-adhesive site FNIII14 was responsible for Mum2B cell migration and invasion. The MMP-induced FNIII14 exposure was also shown to be functional in the migration and invasion of highly metastatic mouse breast cancer cell line 4T1. Overexpression and knockdown experiments of eEF1A in Mum2B cells revealed that the migration and invasion were dependent on the membrane levels of eEF1A. In vivo experiments using tumor xenograft mouse models derived from Mum2B and 4T1 cell lines showed that the anti-FNIII14 Ab has a significant anti-metastatic effect. Thus, these results provide novel insights into the regulation of cancer cell migration and invasion and suggest promising targets for anti-metastasis strategies.

Erratum: Combining peptide TNIIIA2 with all-trans retinoic acid accelerates N-Myc protein degradation and neuronal differentiation in MYCN-amplified neuroblastoma cells.

[This corrects the article on p. 434 in vol. 9, PMID: 30906641.].

Inactivation of beta1 integrin induces proteasomal degradation of Myc oncoproteins.

The MYC family oncogenes (MYC, MYCN, and MYCL) contribute to the genesis of many human cancers. Among them, amplification of the MYCN gene and over-expression of N-Myc protein are the most reliable risk factors in neuroblastoma patients. On the other hand, we previously found that a peptide derived from fibronectin, termed FNIII14, is capable of inducing functional inactivation in ß1-integrins. Here, we demonstrate that inactivation of ß1-integrin by FNIII14 induced proteasomal degradation in N-Myc of neuroblastoma cells with MYCN amplification. This N-Myc degradation by FNIII14 reduced the malignant properties, including the anchorage-independent proliferation and invasive migration, of neuroblastoma cells. An in vivo experiment using a mouse xenograft model showed that the administration of FNIII14 can inhibit tumor growth, and concomitantly a remarkable decrease in N-Myc levels in tumor tissues. Of note, the activation of proteasomal degradation based on ß1-integrin inactivation is applicable to another Myc family oncoprotein, c-myc, which also reverses cancer-associated properties in pancreatic cancer cells. Collectively, ß1-integrin inactivation could be a new chemotherapeutic strategy for cancers with highly expressed Myc. FNIII14, which is a unique pharmacological agent able to induce ß1-integrin inactivation, may be a promising drug targeting Myc oncoproteins for cancer chemotherapy.

Acyclic Retinoid Combined With Tenascin-C-derived Peptide Reduces the Malignant Phenotype of Neuroblastoma Cells Through N-Myc Degradation.

BACKGROUND/AIM: Despite intensive chemotherapy, the survival rates for high-risk neuroblastoma, most of which have MYCN amplification, remain low. Overexpression of N-myc oncoprotein promotes expression of cancer-associated properties. We recently found that combination of all-trans retinoic acid (ATRA) with the ß1-integrin-activating peptide TNIIIA2 attenuated cancer-associated properties of neuroblastoma cells through N-Myc degradation. However, ATRA has serious side-effects and there are concerns about late adverse effects. The aim of this study was to examine the effects of the combination of acyclic retinoid (ACR) with TNIIIA2 on neuroblastoma. MATERIALS AND METHODS: The effects of ACR and TNIIIA2 were examined by neuroblastoma cell proliferation and survival assays as well as by using a neuroblastoma xenograft model. The levels of N-Myc and cancer-associated malignant properties were assayed by western blot and colony formation assay, respectively. RESULTS: Combining ACR, which is clinically safe, with TNIIIA2 induced proteasomal degradation of N-Myc and reduction of neuroblastoma cell malignant properties. An in vivo experiment showed therapeutic potential. CONCLUSION: ACR-TNIIIA2 combination treatment may be efficacious and clinical safe chemotherapy for high-risk neuroblastoma.

Combining peptide TNIIIA2 with all-trans retinoic acid accelerates N-Myc protein degradation and neuronal differentiation in MYCN-amplified neuroblastoma cells.

Neuroblastoma is one of the common solid tumors of childhood. Nearly half of neuroblastoma patients are classified into the high-risk group, and their 5-year event-free survival (EFS) rates remain unsatisfactory in the range of 30-40%. High-risk neuroblastoma is characterized by amplification of the MYCN gene and excessive expression of its protein product, N-Myc. Because N-Myc is a transcription factor for various pro-proliferative proteins, the excessive expression causes aberrant or blocked neuronal differentiation during development of sympathetic nervous system, which is a central aspect of neuroblastoma genesis. The current main treatment for high-risk neuroblastoma is intensive chemotherapy using anti-cancer drugs that induce apoptosis in tumor cells, but intensive chemotherapy has another serious risk of long-lasting side effects, so-called "late effects", that occur many years after chemotherapy has ended. As a solution for such situation, differentiation therapy has been expected as a mild chemotherapy with a low risk of late effects, and an application of retinoic acid (RA) and its derivatives as treatment for high-risk neuroblastoma has long been attempted. However, the clinical outcome has not been sufficient with the use of retinoids, including all-trans retinoic acid (ATRA), mainly because of the inhibition of differentiation caused by N-Myc. In the present study, we succeeded in synergistically accelerating the ATRA-induced neuronal differentiation of MYCN-amplified neuroblastoma cells by combining a peptide derived from tenascin-C, termed TNIIIA2, which has a potent ability to activate ß1-integrins. Accelerated differentiation was caused by a decrease in N-Myc protein level in neuroblastoma cells after the combined treatment of TNIIIA2 with ATRA. That is, combination treatment using ATRA with TNIIIA2 induced proteasomal degradation in the N-Myc oncoprotein of neuroblastoma cells with MYCN gene amplification, and this caused acceleration of neuronal differentiation and attenuation of malignant properties. Furthermore, an in vivo experiment using a xenograft mouse model showed a therapeutic potential of the combination administration of ATRA and TNIIIA2 for high-risk neuroblastoma. These results provide a new insight into differentiation therapy for high-risk neuroblastoma based on N-Myc protein degradation.

Aggressive Progression in Glioblastoma Cells through Potentiated Activation of Integrin α5ß1 by the Tenascin-C-Derived Peptide TNIIIA2.

Tenascin-C is a member of the matricellular protein family, and its expression level is correlated to poor prognosis in cancer, including glioblastoma, whereas its substantial role in tumor formation and malignant progression remains controversial. We reported previously that peptide TNIIIA2 derived from the cancer-associated alternative splicing domain of tenascin-C molecule has an ability to activate ß1-integrin strongly and to maintain it for a long time. Here, we demonstrate that ß1-integrin activation by TNIIIA2 causes acquisition of aggressive behavior, dysregulated proliferation, and migration, characteristic of glioblastoma cells. TNIIIA2 hyperstimulated the platelet-derived growth factor-dependent cell survival and proliferation in an anchorage-independent as well as -dependent manner in glioblastoma cells. TNIIIA2 also strongly promoted glioblastoma multiforme cell migration, which was accompanied by an epithelial-mesenchymal transition-like morphologic change on the fibronectin substrate. Notably, acquisition of these aggressive properties by TNIIIA2 in glioblastoma cells was abrogated by peptide FNIII14 that is capable of inducing inactivation in ß1-integrin activation. Moreover, FNIII14 significantly inhibited tumor growth in a mouse xenograft glioblastoma model. More importantly, FNIII14 sensitized glioblastoma cells to temozolomide via downregulation of O6-methylguanine-DNA methyltransferase expression. Consequently, FNIII14 augmented the antitumor activity of temozolomide in a mouse xenograft glioblastoma model. Taken altogether, the present study provides not only an interpretation for the critical role of tenascin-C/TNIIIA2 in aggressive behavior of glioblastoma cells, but also an important strategy for glioblastoma chemotherapy. Inhibition of the tenascin-C/ß1-integrin axis may be a therapeutic target for glioblastoma, and peptide FNIII14 may represent a new approach for glioblastoma chemotherapy. SIGNIFICANCE: These findings provide a proposal of new strategy for glioblastoma chemotherapy based on integrin inactivation.