Bioactive TNIIIA2 Sequence in Tenascin-C Is Responsible for Macrophage Foam Cell Transformation; Potential of FNIII14 Peptide Derived from Fibronectin in Suppression of Atherosclerotic Plaque Formation.

One of the extracellular matrix proteins, tenascin-C (TN-C), is known to be upregulated in age-related inflammatory diseases such as cancer and cardiovascular diseases. Expression of this molecule is frequently detected, especially in the macrophage-rich areas of atherosclerotic lesions; however, the role of TN-C in mechanisms underlying the progression of atherosclerosis remains obscure. Previously, we found a hidden bioactive sequence termed TNIIIA2 in the TN-C molecule and reported that the exposure of this sequence would be carried out through limited digestion of TN-C by inflammatory proteases. Thus, we hypothesized that some pro-atherosclerotic phenotypes might be elicited from macrophages when they were stimulated by TNIIIA2. In this study, TNIIIA2 showed the ability to accelerate intracellular lipid accumulation in macrophages. In this experimental condition, an elevation of phagocytic activity was observed, accompanied by a decrease in the expression of transporters responsible for lipid efflux. All these observations were mediated through the induction of excessive ß1-integrin activation, which is a characteristic property of the TNIIIA2 sequence. Finally, we demonstrated that the injection of a drug that targets TNIIIA2's bioactivity could rescue mice from atherosclerotic plaque expansion. From these observations, it was shown that TN-C works as a pro-atherosclerotic molecule through an internal TNIIIA2 sequence. The possible advantages of clinical strategies targeting TNIIIA2 are also indicated.

Heat shock-induced heme oxygenase-1 expression in a mouse hepatoma cell line is dependent on HSF1 and modified by NRF2 and BACH1.

The induction mechanism of heme oxygenase-1 (HO-1) by heat shock (HS) is still unknown. Here, we discovered that HS activates the HO-1 expression in a mouse hepatoma cell line (Hepa 1-6). Knockdown experiments showed that the HS-induced HO-1 expression was dependent on HS factor 1 (HSF1). A chromatin immunoprecipitation (ChIP) assay demonstrated that the HS-activated HSF1 bound to the HS elements (HSEs) in the upstream enhancer 1 region (E1). Unexpectedly, HS also facilitates the BTB and CNC homology 1 (BACH1) binding to the Maf recognition elements (MAREs) in E1. We examined the effects of a catalytically inactive CRISPR-associated 9 nucleases (dCas9) with short guide RNAs (sgRNAs), and demonstrated that the HSF1 binding to HSEs in E1 was indispensable for the HS-induced HO-1 expression. Heme treatment (HA) dissociates BACH1 from MAREs and facilitated the binding of nuclear factor-erythroid-2-related factor 2 (NRF2) to MAREs. Following treatment with both HS and HA, the HO-1 induction and the HSF1 binding to HSEs in E1 were most notably observed. These results indicate that the HS-induced HO-1 expression is dependent on the HSF1 binding to HSEs in E1, although modulated by the BACH1 and NRF2 binding to MAREs within the same E1.

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.

Wound Healing Promotion by Hyaluronic Acid: Effect of Molecular Weight on Gene Expression and In Vivo Wound Closure.

Hyaluronic acid (HA) has been known to play an important role in wound healing process. However, the effect of molecular weight (MW) of exogenously administered HA on the wound healing process has not been fully understood. In this study, we investigated HA with different MWs on wound healing process using human epidermal keratinocytes and dermal fibroblasts. Cell proliferation and migration ability were assessed by water soluble tetrazolium (WST) assay and wound scratch assay. We examined the effect of HA addition in a full-thickness wound model in mice and the gene expression related to wound healing. Proliferation and migration of HaCaT cells increased with the increase of MW and concentration of HA. Interleukin (IL-1ß), IL-8 and vascular endothelial growth factor (VEGF) as well as matrix metalloproteinase (MMP)-9 and MMP-13 were significantly upregulated by high molecular weight (HMW) HA in keratinocytes. Together with VEGF upregulation and the observed promotion of HaCaT migration, HA with the MW of 2290 kDa may hold potential to improve re-epithelialization, a critical obstacle to heal chronic wounds.

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.

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.

Biologically Active TNIIIA2 Region in Tenascin-C Molecule: A Major Contributor to Elicit Aggressive Malignant Phenotypes From Tumors/Tumor Stroma.

Tenascin (TN)-C is highly expressed specifically in the lesions of inflammation-related diseases, including tumors. The expression level of TN-C in tumors and the tumor stroma is positively correlated with poor prognosis. However, no drugs targeting TN-C are currently clinically available, partly because the role of TN-C in tumor progression remains controversial. TN-C harbors an alternative splicing site in its fibronectin type III repeat domain, and its splicing variants including the type III-A2 domain are frequently detected in malignant tumors. We previously identified a biologically active region termed TNIIIA2 in the fibronectin type III-A2 domain of TN-C molecule and showed that this region is involved in promoting firm and persistent cell adhesion to fibronectin. In the past decade, through the exposure of various cell lines to peptides containing the TNIIIA2 region, we have published reports demonstrating the ability of the TNIIIA2 region to modulate distinct cellular activities, including survival/growth, migration, and invasion. Recently, we reported that the signals derived from TNIIIA2-mediated ß1 integrin activation might play a crucial role for inducing malignant behavior of glioblastoma (GBM). GBM cells exposed to the TNIIIA2 region showed not only exacerbation of PDGF-dependent proliferation, but also acceleration of disseminative migration. On the other hand, we also found that the pro-inflammatory phenotypic changes were promoted when macrophages are stimulated with TNIIIA2 region in relatively low concentration and resulting MMP-9 upregulation is needed to release of the TNIIIA2 region from TN-C molecule. With the contribution of TNIIIA2-stimulated macrophages, the positive feedback spiral loop, which consists of the expression of TN-C, PDGF, and ß1 integrin, and TNIIIA2 release, seemed to be activated in GBM with aggressive malignancy. Actually, the growth of transplanted GBM grafts in mice was significantly suppressed via the attenuation of ß1 integrin activation. In this review, we thus introduce that the TNIIIA2 region has a significant impact on malignant progression of tumors by regulating cell adhesion. Importantly, it has been demonstrated that the TNIIIA2 region exerts unique biological functions through the extremely strong activation of ß1-integrins and their long-lasting duration. These findings prompt us to develop new therapeutic agents targeting the TNIIIA2 region.

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.

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.

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.

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.

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.

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.

Control of cell adhesion and proliferation utilizing polysaccharide composite film scaffolds.

We have developed polysaccharide composite films made of anionic polysaccharides and chitosan (CHI) by utilizing hot press techniques. In order to demonstrate the versatility of these films as cell scaffolds, the present study investigated the adhesion and proliferation of fibroblasts on composite films prepared by using various kinds of anionic polysaccharides and that were modified with proteins. Cells were spread on heparin/CHI and alginic acid/CHI films and grew well, whereas those on chondroitin sulfate C (CS)/CHI and hyaluronic acid/CHI films were round in shape. The differences in adhesion and proliferation behaviors of cells could be explained by the differences in the biochemical function of the anionic polysaccharides and the physical properties of the films such as morphology, storage modulus, ζ-potentials, and swelling ratios. Among them, the number of cells on CS/CHI films remained almost unchanged. The mechanisms underlying growth suppression on CS/CHI films were investigated by using an integrin stimulator, the TNIIIA2 peptide, and platelet-derived growth factor-B. It was indicated that the growth suppression was due to the lack of fibronectin-integrin growth signaling. The surface modification of CS/CHI films with fibronectin promoted the adhesion and proliferation of cells. These results show that the chemical and physical properties of the polysaccharide composite films, which resulted from the chemical species of anionic polysaccharides or surface modifications of the films, can modulate cell adhesion and proliferation properties thereon.

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.

Coadministration of the FNIII14 Peptide Synergistically Augments the Anti-Cancer Activity of Chemotherapeutic Drugs by Activating Pro-Apoptotic Bim.

The acquisition of drug resistance mediated by the interaction of tumor cells with the extracellular matrix (ECM), commonly referred to as cell adhesion-mediated drug resistance (CAM-DR), has been observed not only in hematopoietic tumor cells but also in solid tumor cells. We have previously demonstrated that a 22-mer peptide derived from fibronectin, FNIII14, can inhibit cell adhesion through the inactivation of ß1 integrin; when coadministered with cytarabine, FNIII14 completely eradicates acute myelogenous leukemia by suppressing CAM-DR. In this study, we show that our FNIII14 peptide also enhances chemotherapy efficacy in solid tumors. Coadministration of FNIII14 synergistically enhances the cytotoxicity of doxorubicin and aclarubicin in mammary tumor and melanoma cells, respectively. The solid tumor cell chemosensitization induced by FNIII14 is dependent upon the upregulation and activation of the pro-apoptotic protein, Bim. Furthermore, the metastasis of tumor cells derived from ventrally transplanted mammary tumor grafts is suppressed by the coadministration of FNIII14 and doxorubicin. These results suggest that the coadministration of our FNIII14 peptide with chemotherapy could achieve efficient solid tumor eradication by increasing chemosensitivity and decreasing metastasis. The major causes of tumor recurrence are the existence of chemotherapy-resistant primary tumor cells and the establishment of secondary metastatic lesions. As such, coadministering FNIII14 with anti-cancer drugs could provide a promising new approach to improve the prognosis of patients with solid tumors.

Tenascin-C-derived peptide TNIIIA2 highly enhances cell survival and platelet-derived growth factor (PDGF)-dependent cell proliferation through potentiated and sustained activation of integrin α5ß1.

Tenascin-C is an adhesion modulatory matrix protein that is highly expressed in tumors; however, its biochemical activity involved in tumorigenesis is not fully understood. On the other hand, increasing evidence indicates the importance of integrin α5ß1 in cancer development. We previously demonstrated that tenascin-C harbors a functional site that can be released as a proadhesive peptide such as TNIIIA2. Peptide TNIIIA2 is capable of inducing activation of ß1-integrins including α5ß1 via syndecan-4. In this study the proadhesive effect of TNIIIA2 was characterized by potentiated and sustained activation of integrin α5ß1. Based on this effect, TNIIIA2 rendered nontransformed fibroblasts (NIH3T3) resistant to serum deprivation-elicited anoikis through activation of the Akt/Bcl-2 pathway. Moreover, TNIIIA2 hyperstimulated PDGF-dependent proliferation of NIH3T3 by activating integrin α5ß1. Tenascin-C, a parental protein of TNIIIA2, also stimulated PDGF-dependent proliferation, which was blocked by a matrix metalloproteinase-2/9 inhibitor and an anti-TNIIIA2 function-blocking antibody, suggesting proteolytic exposure of the proadhesive effect of TNIIIA2. Mechanistic analyses revealed that TNIIIA2 induced a lateral association of PDGF receptor ß with the molecular complex of activated integrin α5ß1 and syndecan-4 in the membrane microdomains enriched with cholesterol/caveolin-1, resulting in prolonged activation of PDGF receptor ß and the subsequent Ras/mitogen-activated protein kinase pathway in a PDGF-dependent manner. Of note, TNIIIA2 induced continuous proliferation in NIH3T3 in an integrin α5ß1-dependent manner even after they formed a confluent monolayer. Thus, it was proposed that tenascin-C might be involved in deregulated cell growth through potentiated and sustained activation of integrin α5ß1 after exposure of the proadhesive effect of TNIIIA2.

A novel mechanism underlying the basic defensive response of macrophages against Mycobacterium infection.

Following inhalation of Mycobacterium tuberculosis, including bacillus Calmette-Guérin (BCG), pathogens enter and grow inside macrophages by taking advantage of their phagocytic mechanisms. Macrophages often fail to eliminate intracellular M. tuberculosis, leading to the induction of host macrophage death. Despite accumulating evidence, the molecular mechanisms underlying M. tuberculosis infection-induced cell death remain controversial. In this study, we show the involvement of two distinct pathways triggered by TLR2 and ß2 integrin in BCG infection-induced macrophage apoptosis. First, BCG infection induced activation of ERK1/2, which in turn caused phosphorylation/activation of the proapoptotic protein Bim in mouse macrophage-like Raw 264.7 cells. BCG-infected Raw cells treated with U0126, an MEK/ERK inhibitor, led to the suppression of Bim phosphorylation alongside a remarkable increase in the number of viable macrophages. Small interfering RNA-mediated knockdown of Bim rescued the macrophages from the apoptotic cell death induced by BCG infection. Stimulation with Pam3CSK, a TLR2 agonist, induced macrophage apoptosis with a concomitant increase in the phosphorylation/activation of MEK/ERK and Bim. These observations indicate the important role of the TLR2/MEK/ERK/Bim pathway in BCG infection-induced macrophage apoptosis. Second, we used the ß2 integrin agonists C3bi and fibronectin to show that the ß2 integrin-derived signal was involved in BCG infection-induced apoptosis, independent of MEK/ERK activation. Interestingly, latex beads coated with Pam3CSK and C3bi were able to induce apoptosis in macrophages to the same extent and specificity as that induced by BCG. Taken together, two distinct pattern-recognition membrane receptors, TLR2 and ß2 integrin, acted as triggers in BCG infection-induced macrophage apoptosis, in which MEK/ERK activation played a crucial role following the engagement of TLR2.