Glycogen synthase kinase 3ß: the nexus of chemoresistance, invasive capacity, and cancer stemness in pancreatic cancer.

The treatment of pancreatic cancer remains a significant clinical challenge due to the limited number of patients eligible for curative (R0) surgery, failures in the clinical development of targeted and immune therapies, and the pervasive acquisition of chemotherapeutic resistance. Refractory pancreatic cancer is typified by high invasiveness and resistance to therapy, with both attributes related to tumor cell stemness. These malignant characteristics mutually enhance each other, leading to rapid cancer progression. Over the past two decades, numerous studies have produced evidence of the pivotal role of glycogen synthase kinase (GSK)3ß in the progression of over 25 different cancer types, including pancreatic cancer. In this review, we synthesize the current knowledge on the pathological roles of aberrant GSK3ß in supporting tumor cell proliferation and invasion, as well as its contribution to gemcitabine resistance in pancreatic cancer. Importantly, we discuss the central role of GSK3ß as a molecular hub that mechanistically connects chemoresistance, tumor cell invasion, and stemness in pancreatic cancer. We also discuss the involvement of GSK3ß in the formation of desmoplastic tumor stroma and in promoting anti-cancer immune evasion, both of which constitute major obstacles to successful cancer treatment. Overall, GSK3ß has characteristics of a promising therapeutic target to overcome chemoresistance in pancreatic cancer.

Deactivation of glycogen synthase kinase-3ß by heat shock­inducible tumor small protein attenuates hyperthermia­induced pro­migratory activity in colorectal cancer cells.

Hyperthermia is a promising approach for improving cancer treatment in combination with chemotherapy, radiotherapy and/or immunotherapy; however, its molecular mechanisms remain unclear. Although heat shock proteins (HSPs) are involved in hyperthermia via antigen presentation and immune activation, major HSPs including HSP90 are associated with cancer progression via tumor cell migration and metastasis. The present study showed that heat shock­inducible tumor small protein (HITS) could counteract the pro­migratory effects of HSPs in colorectal cancer (CRC) cells, which represents a novel function. Western blotting analysis revealed that overexpression of HITS increased the protein level of glycogen synthase kinase­3ß (GSK3ß) phosphorylated (p) at the serine 9 (pGSK3ßS9; inactive form) in HCT 116, RKO and SW480 CRC cells. GSK3ßS9 phosphorylation was reported to suppress migration in some cancer types; therefore, by using the wound healing assay, the present study revealed that HITS overexpression decreased the migration activity of CRC cells. Induction of HITS transcription was observed at 12 and 18 h after heat shock (HS) by using semi­quantitative reverse transcription­PCR analysis, followed by increased levels of pGSK3ßS9 protein at 24 and 30 h in CRC cells in western blotting. Thus, HS induced not only HSPs to promote cell migration, but also HITS to counteract the migratory activity of these HSPs in CRC cells. HITS knockdown in CRC cells subject to HS showed increased cell migration in wound healing assay, which was decreased by the GSK3ß inhibitor AR­A014418, confirming the anti­migratory effect of HITS via the deactivation of GSK3ß. The present findings indicated that the deactivation of GSK3ß sufficiently offset the pro­migratory effect of hyperthermia via major HSPs in CRC.

Discovery of a Novel Aminocyclopropenone Compound That Inhibits BRD4-Driven Nucleoporin NUP210 Expression and Attenuates Colorectal Cancer Growth.

Epigenetic deregulation plays an essential role in colorectal cancer progression. Bromodomains are epigenetic "readers" of histone acetylation. Bromodomain-containing protein 4 (BRD4) plays a pivotal role in transcriptional regulation and is a feasible drug target in cancer cells. Disease-specific elevation of nucleoporin, a component of the nuclear pore complex (NPC), is a determinant of cancer malignancy, but BRD4-driven changes of NPC composition remain poorly understood. Here, we developed novel aminocyclopropenones and investigated their biological effects on cancer cell growth and BRD4 functions. Among 21 compounds developed here, we identified aminocyclopropenone 1n (ACP-1n) with the strongest inhibitory effects on the growth of the cancer cell line HCT116. ACP-1n blocked BRD4 functions by preventing its phase separation ability both in vitro and in vivo, attenuating the expression levels of BRD4-driven MYC. Notably, ACP-1n significantly reduced the nuclear size with concomitant suppression of the level of the NPC protein nucleoporin NUP210. Furthermore, NUP210 is in a BRD4-dependent manner and silencing of NUP210 was sufficient to decrease nucleus size and cellular growth. In conclusion, our findings highlighted an aminocyclopropenone compound as a novel therapeutic drug blocking BRD4 assembly, thereby preventing BRD4-driven oncogenic functions in cancer cells. This study facilitates the development of the next generation of effective and potent inhibitors of epigenetic bromodomains and extra-terminal (BET) protein family.

Glycogen synthase kinase-3ß participates in acquired resistance to gemcitabine in pancreatic cancer.

Acquisition of resistance to gemcitabine is a challenging clinical and biological hallmark property of refractory pancreatic cancer. Here, we investigated whether glycogen synthase kinase (GSK)-3ß, an emerging therapeutic target in various cancer types, is mechanistically involved in acquired resistance to gemcitabine in human pancreatic cancer. This study included 3 gemcitabine-sensitive BxPC-3 cell-derived clones (BxG30, BxG140, BxG400) that acquired stepwise resistance to gemcitabine and overexpressed ribonucleotide reductase (RR)M1. Treatment with GSK3ß-specific inhibitor alone attenuated the viability and proliferation of the gemcitabine-resistant clones, while synergistically enhancing the efficacy of gemcitabine against these clones and their xenograft tumors in rodents. The gemcitabine-resensitizing effect of GSK3ß inhibition was associated with decreased expression of RRM1, reduced phosphorylation of Rb protein, and restored binding of Rb to the E2 transcription factor (E2F)1. This was followed by decreased E2F1 transcriptional activity, which ultimately suppressed the expression of E2F1 transcriptional targets including RRM1, CCND1 encoding cyclin D1, thymidylate synthase, and thymidine kinase 1. These results suggested that GSK3ß participates in the acquisition of gemcitabine resistance by pancreatic cancer cells via impairment of the functional interaction between Rb tumor suppressor protein and E2F1 pro-oncogenic transcription factor, thereby highlighting GSK3ß as a promising target in refractory pancreatic cancer. By providing insight into the molecular mechanism of gemcitabine resistance, this study identified a potentially novel strategy for pancreatic cancer chemotherapy.

Potential therapeutic effect of targeting glycogen synthase kinase 3ß in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a common gastrointestinal cancer and is often refractory to current therapies. Development of efficient therapeutic strategies against ESCC presents a major challenge. Glycogen synthase kinase (GSK)3ß has emerged as a multipotent therapeutic target in various diseases including cancer. Here we investigated the biology and pathological role of GSK3ß in ESCC and explored the therapeutic effects of its inhibition. The expression of GSK3ß and tyrosine (Y)216 phosphorylation-dependent activity was higher in human ESCC cell lines and primary tumors than untransformed esophageal squamous TYNEK-3 cells from an ESCC patient and tumor-adjacent normal esophageal mucosa. GSK3ß-specific inhibitors and small interfering (si)RNA-mediated knockdown of GSK3ß attenuated tumor cell survival and proliferation, while inducing apoptosis in ESCC cells and their xenograft tumors in mice. GSK3ß inhibition spared TYNEK-3 cells and the vital organs of mice. The therapeutic effect of GSK3ß inhibition in tumor cells was associated with G0/G1- and G2/M-phase cell cycle arrest, decreased expression of cyclin D1 and cyclin-dependent kinase (CDK)4 and increased expression of cyclin B1. These results suggest the tumor-promoting role of GSK3ß is via cyclin D1/CDK4-mediated cell cycle progression. Consequently, our study provides a biological rationale for GSK3ß as a potential therapeutic target in ESCC.

Glycogen Synthase Kinase 3ß in Cancer Biology and Treatment.

Glycogen synthase kinase (GSK)3ß is a multifunctional serine/threonine protein kinase with more than 100 substrates and interacting molecules. GSK3ß is normally active in cells and negative regulation of GSK3ß activity via phosphorylation of its serine 9 residue is required for most normal cells to maintain homeostasis. Aberrant expression and activity of GSK3ß contributes to the pathogenesis and progression of common recalcitrant diseases such as glucose intolerance, neurodegenerative disorders and cancer. Despite recognized roles against several proto-oncoproteins and mediators of the epithelial-mesenchymal transition, deregulated GSK3ß also participates in tumor cell survival, evasion of apoptosis, proliferation and invasion, as well as sustaining cancer stemness and inducing therapy resistance. A therapeutic effect from GSK3ß inhibition has been demonstrated in 25 different cancer types. Moreover, there is increasing evidence that GSK3ß inhibition protects normal cells and tissues from the harmful effects associated with conventional cancer therapies. Here, we review the evidence supporting aberrant GSK3ß as a hallmark property of cancer and highlight the beneficial effects of GSK3ß inhibition on normal cells and tissues during cancer therapy. The biological rationale for targeting GSK3ß in the treatment of cancer is also discussed at length.

Glycogen synthase kinase 3ß as a potential therapeutic target in synovial sarcoma and fibrosarcoma.

Soft tissue sarcomas (STSs) are a rare cancer type. Almost half are unresponsive to multi-pronged treatment and might therefore benefit from biologically targeted therapy. An emerging target is glycogen synthase kinase (GSK)3ß, which is implicated in various diseases including cancer. Here, we investigated the expression, activity and putative pathological role of GSK3ß in synovial sarcoma and fibrosarcoma, comprising the majority of STS that are encountered in orthopedics. Expression of the active form of GSK3ß (tyrosine 216-phosphorylated) was higher in synovial sarcoma (SYO-1, HS-SY-II, SW982) and in fibrosarcoma (HT1080) tumor cell lines than in untransformed fibroblast (NHDF) cells that are assumed to be the normal mesenchymal counterpart cells. Inhibition of GSK3ß activity by pharmacological agents (AR-A014418, SB-216763) or of its expression by RNA interference suppressed the proliferation of sarcoma cells and their invasion of collagen gel, as well as inducing their apoptosis. These effects were associated with G0/G1-phase cell cycle arrest and decreased expression of cyclin D1, cyclin-dependent kinase (CDK)4 and matrix metalloproteinase 2. Intraperitoneal injection of the GSK3ß inhibitors attenuated the growth of SYO-1 and HT1080 xenografts in athymic mice without obvious detrimental effects. It also mitigated cell proliferation and induced apoptosis in the tumors of mice. This study indicates that increased activity of GSK3ß in synovial sarcoma and fibrosarcoma sustains tumor proliferation and invasion through the cyclin D1/CDK4-mediated pathway and enhanced extracellular matrix degradation. Our results provide a biological basis for GSK3ß as a new and promising therapeutic target for these STS types.

Identification of GSK3ß inhibitor kenpaullone as a temozolomide enhancer against glioblastoma.

Cancer stem cells are associated with chemoresistance and rapid recurrence of malignant tumors, including glioblastoma (GBM). Although temozolomide (TMZ) is the most effective drug treatment for GBM, GBM cells acquire resistance and become refractory to TMZ during treatment. Therefore, glioma stem cell (GSC)-targeted therapy and TMZ-enhancing therapy may be effective approaches to improve GBM prognosis. Many drugs that suppress the signaling pathways that maintain GSC or enhance the effects of TMZ have been reported. However, there are no established therapies beyond TMZ treatment currently in use. In this study, we screened drug libraries composed of 1,301 existing drugs using cell viability assays to evaluate effects on GSCs, which led to selection of kenpaullone, a kinase inhibitor, as a TMZ enhancer targeting GSCs. Kenpaullone efficiently suppressed activity of glycogen synthase kinase (GSK) 3ß. Combination therapy with kenpaullone and TMZ suppressed stem cell phenotype and viability of both GSCs and glioma cell lines. Combination therapy in mouse models significantly prolonged survival time compared with TMZ monotherapy. Taken together, kenpaullone is a promising drug for treatment of GBM by targeting GSCs and overcoming chemoresistance to TMZ.

Colorectal cancer cells require glycogen synthase kinase-3ß for sustaining mitosis via translocated promoter region (TPR)-dynein interaction.

Glycogen synthase kinase (GSK) 3ß, which mediates fundamental cellular signaling pathways, has emerged as a potential therapeutic target for many types of cancer including colorectal cancer (CRC). During mitosis, GSK3ß localizes in mitotic spindles and centrosomes, however its function is largely unknown. We previously demonstrated that translocated promoter region (TPR, a nuclear pore component) and dynein (a molecular motor) cooperatively contribute to mitotic spindle formation. Such knowledge encouraged us to investigate putative functional interactions among GSK3ß, TPR, and dynein in the mitotic machinery of CRC cells. Here, we show that inhibition of GSK3ß attenuated proliferation, induced cell cycle arrest at G2/M phase, and increased apoptosis of CRC cells. Morphologically, GSK3ß inhibition disrupted chromosome segregation, mitotic spindle assembly, and centrosome maturation during mitosis, ultimately resulting in mitotic cell death. These changes in CRC cells were associated with decreased expression of TPR and dynein, as well as disruption of their functional colocalization with GSK3ß in mitotic spindles and centrosomes. Clinically, we showed that TPR expression was increased in CRC databases and primary tumors of CRC patients. Furthermore, TPR expression in SW480 cells xenografted into mice was reduced following treatment with GSK3ß inhibitors. Together, these results indicate that GSK3ß sustains steady mitotic processes for proliferation of CRC cells via interaction with TPR and dynein, thereby suggesting that the therapeutic effect of GSK3ß inhibition depends on induction of mitotic catastrophe in CRC cells.

Efficacy of glycogen synthase kinase-3ß targeting against osteosarcoma via activation of ß-catenin.

Development of innovative more effective therapy is required for refractory osteosarcoma patients. We previously established that glycogen synthase kinase-3ß (GSK- 3ß) is a therapeutic target in various cancer types. In the present study, we explored the therapeutic efficacy of GSK-3ß inhibition against osteosarcoma and the underlying molecular mechanisms in an orthotopic mouse model. Expression and phosphorylation of GSK-3ß in osteosarcoma and normal osteoblast cell lines was examined, together with efficacy of GSK-3ß inhibition on cell survival, proliferation and apoptosis and on the growth of orthotopically-transplanted human osteosarcoma in nude mice. We also investigated changes in expression, phosphorylation and co-transcriptional activity of ß-catenin in osteosarcoma cells following GSK-3ß inhibition. Expression of the active form of GSK- 3ß (tyrosine 216-phosphorylated) was higher in osteosarcoma than osteoblast cells. Inhibition of GSK-3ß activity by pharmacological inhibitors or of its expression by RNA interference suppressed proliferation of osteosarcoma cells and induced apoptosis. Treatment with GSK-3ß-specific inhibitors attenuated the growth of orthotopic osteosaroma in mice. Inhibition of GSK-3ß reduced phosphorylation at GSK- 3ß-phospho-acceptor sites in ß-catenin and increased ß-catenin expression, nuclear localization and co-transcriptional activity. These results suggest the efficacy of GSK-3ß inhibitors is associated with activation of ß-catenin, a putative tumor suppressor in bone and soft tissue sarcoma and an important component of osteogenesis. Our study thereby demonstrates a critical role for GSK-3ß in sustaining survival and proliferation of osteosarcoma cells, and identifies this kinase as a potential therapeutic target against osteosarcoma.

Glycogen synthase kinase-3ß is a pivotal mediator of cancer invasion and resistance to therapy.

Tumor cell invasion and resistance to therapy are the most intractable biological characteristics of cancer and, therefore, the most challenging for current cancer research and treatment paradigms. Refractory cancers, including pancreatic cancer and glioblastoma, show an inextricable association between the highly invasive behavior of tumor cells and their resistance to chemotherapy, radiotherapy and targeted therapies. These aggressive properties of cancer share distinct cellular pathways that are connected to each other by several molecular hubs. There is increasing evidence to show that glycogen synthase kinase (GSK)-3ß is aberrantly activated in various cancer types and this has emerged as a potential therapeutic target. In many but not all cancer types, aberrant GSK3ß sustains the survival, immortalization, proliferation and invasion of tumor cells, while also rendering them insensitive or resistant to chemotherapeutic agents and radiation. Here we review studies that describe associations between therapeutic stimuli/resistance and the induction of pro-invasive phenotypes in various cancer types. Such cancers are largely responsive to treatment that targets GSK3ß. This review focuses on the role of GSK3ß as a molecular hub that connects pathways responsible for tumor invasion and resistance to therapy, thus highlighting its potential as a major cancer therapeutic target. We also discuss the putative involvement of GSK3ß in determining tumor cell stemness that underpins both tumor invasion and therapy resistance, leading to intractable and refractory cancer with dismal patient outcomes.

Tip60 regulates MT1-MMP transcription and invasion of glioblastoma cells through NF-κB pathway.

A histone acetyltransferase Tat-interacting protein 60 kDa (Tip60) regulates the DNA damage response by acetylating histone and remodeling chromatin. In addition to histone acetyltransferase activity, Tip60 is known to regulate a variety of cellular functions, including gene expression, DNA damage response, cell migration and apoptosis. Lower expression of Tip60 is observed in lymphomas, melanomas, breast, colon, and lung cancer. It is widely accepted that Tip60 functions as a tumor suppressor. However, a role of Tip60 in gliomas still remains unclear. In this study, we investigated the role of Tip60 in the malignant behavior of human gliomas. By quantitative RT-PCR analysis using fresh human brain tumor tissues from 55 patients, we found that lower Tip60 expression and higher membrane-type 1 matrix metalloproteinase (MT1-MMP) expression are associated with advanced tumor grade in glioma tissues. Knockdown of Tip60 in glioblastoma cells promoted cell adhesion, spreading and MT1-MMP transcription and thereby invasion, which was suppressed by inhibition of MT1-MMP and nuclear factor-kappa B (NF-κB) activity. We demonstrate for the first time that tumor suppressor Tip60 down-regulates cell adhesion and MT1-MMP expression and thereby invasion of glioblastoma cells by suppressing NF-κB pathway.

Glycogen synthase kinase 3ß sustains invasion of glioblastoma via the focal adhesion kinase, Rac1, and c-Jun N-terminal kinase-mediated pathway.

The failure of current treatment options for glioblastoma stems from their inability to control tumor cell proliferation and invasion. Biologically targeted therapies offer great hope and one promising target is glycogen synthase kinase-3ß (GSK3ß), implicated in various diseases, including cancer. We previously reported that inhibition of GSK3ß compromises the survival and proliferation of glioblastoma cells, induces their apoptosis, and sensitizes them to temozolomide and radiation. Here, we explore whether GSK3ß also contributes to the highly invasive nature of glioblastoma. The effects of GSK3ß inhibition on migration and invasion of glioblastoma cells were examined by wound-healing and Transwell assays, as well as in a mouse model of glioblastoma. We also investigated changes in cellular microarchitectures, cytoskeletal components, and proteins responsible for cell motility and invasion. Inhibition of GSK3ß attenuated the migration and invasion of glioblastoma cells in vitro and that of tumor cells in a mouse model of glioblastoma. These effects were associated with suppression of the molecular axis involving focal adhesion kinase, guanine nucleotide exchange factors/Rac1 and c-Jun N-terminal kinase. Changes in cellular phenotypes responsible for cell motility and invasion were also observed, including decreased formation of lamellipodia and invadopodium-like microstructures and alterations in the subcellular localization, and activity of Rac1 and F-actin. These changes coincided with decreased expression of matrix metalloproteinases. Our results confirm the potential of GSK3ß as an attractive therapeutic target against glioblastoma invasion, thus highlighting a second role in this tumor type in addition to its involvement in chemo- and radioresistance.

Vinculin negatively regulates transcription of MT1-MMP through MEK/ERK pathway.

Vinculin regulates a variety of cellular functions partly through stabilization of tumor suppressor PTEN. In order to study the role of vinculin in tumor progression other than PTEN stabilization, vinculin was knocked down in PTEN-deficient squamous cell carcinoma HSC-4 cells. Knockdown of vinculin induced phenotypical change by reducing cell-cell and cell-extracellular matrix adhesions, and enhanced MT1-MMP expression at transcription level and subsequent cell migration. Up-regulation of MT1-MMP transcription by vinculin knockdown was abrogated by ERK inhibition. These results suggest that vinculin negatively regulates malignant phenotype of tumor cells including MT1-MMP transcription through MEK/ERK pathway.

Membrane-type 1 matrix metalloproteinase regulates fibronectin assembly and N-cadherin adhesion.

Fibronectin matrix formation requires the increased cytoskeletal tension generated by cadherin adhesions, and is suppressed by membrane-type 1 matrix metalloproteinase (MT1-MMP). In a co-culture of Rat1 fibroblasts and MT1-MMP-silenced HT1080 cells, fibronectin fibrils extended from Rat1 to cell-matrix adhesions in HT1080 cells, and N-cadherin adhesions were formed between Rat1 and HT1080 cells. In control HT1080 cells contacting with Rat1 fibroblasts, cell-matrix adhesions were formed in the side away from Rat1 fibroblasts, and fibronectin assembly and N-cadherin adhesions were not formed. The role of N-cadherin adhesions in fibronectin matrix formation was studied using MT1-MMP-silenced HT1080 cells. MT1-MMP knockdown promoted fibronectin matrix assembly and N-cadherin adhesions in HT1080 cells, which was abrogated by double knockdown with either integrin ß1 or fibronectin. Conversely, inhibition of N-cadherin adhesions by its knockdown or treatment with its neutralizing antibody suppressed fibronectin matrix formation in MT1-MMP-silenced cells. These results demonstrate that fibronectin assembly initiated by MT1-MMP knockdown results in increase of N-cadherin adhesions, which are prerequisite for further fibronectin matrix formation.

MT1-MMP prevents growth inhibition by three dimensional fibronectin matrix.

The extracellular microenvironment plays a key role in regulation of cellular functions and growth control. We show here that membrane-type 1 matrix metalloproteinase (MT1-MMP) acts as a growth promoter in confluent culture. When MT1-MMP was silenced in HT1080 fibrosarcoma cells, cells created three dimensional (3D) fibronectin matrix in a confluent culture, and growth of cells embedded within it was retarded. Formation of 3D fibronectin matrix initiated by MT1-MMP silencing was impeded by knockdown of either FN or integrin ß1, which resulted in restoration of cell growth. When cells in 3D fibronectin matrix were treated with integrin ß1 inhibitory antibody, cells underwent S phase entry. These results suggest that MT1-MMP prevents growth suppression by 3D fibronectin matrix, which is mediated through integrin ß1.

Shedding of kidney injury molecule-1 by membrane-type 1 matrix metalloproteinase.

Co-expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) with kidney injury molecule-1 (KIM-1) in HEK293T cells resulted in cleavage and shedding of KIM-1 ectodomain. Analysis of cleavage products using KIM-1 mutants localized cleavage site at the juxtamembrane region. HT1080 cells were stably transfected with expression plasmid for KIM-1 or its mutant with deletion of the juxtamembrane region (Asp(261)-Gly(295)) to establish HT/KIM-1 or HT/ΔKIM-1 cells, respectively. KIM-1 protein appeared on cell surface at low level in HT/KIM-1 cells, and accumulated by the treatment with MMP inhibitor BB-94 or small interfering RNA (siRNA) to MT1-MMP, indicating that MT1-MMP is involved in cleavage and shedding of KIM-1. In contrast, HT/ΔKIM-1 cells expressed KIM-1 protein at high level regardless of BB-94 or siRNA treatment. Cells expressing high level KIM-1 protein exhibited phagocytosis of Escherichia coli and reduced cell adhesion and spreading on collagen-coated plate compared with KIM-1 negative cells. Control HT1080 and HT/KIM-1 cells showed significantly higher invasive growth in collagen gel, cell migration on collagen-coated plate and liver metastasis in chick embryo than HT/ΔKIM-1 cells. These results suggest that KIM-1 negatively regulates cellular function mediated through interaction with collagen, and MT1-MMP abrogates it through the cleavage and shedding of KIM-1.

Cleavage of hepatocyte growth factor activator inhibitor-1 by membrane-type MMP-1 activates matriptase.

Co-expression of membrane-type 1 (MT1)-MMP with hepatocyte growth factor activator inhibitor-1 (HAI-1) in HEK293T cells resulted in cleavage of HAI-1 to produce three fragments. Recombinant MT1-MMP was shown to cleave HAI-1 protein in vitro. Hepatocyte growth factor activator inhibitor-1 was initially identified as the cognate inhibitor of matriptase, a transmembrane serine protease that processes urokinase-type plasminogen activator (uPA). Co-expression of HAI-1 with matriptase suppressed matriptase protease activity, and co-expression of MT1-MMP with them resulted in recovery of matriptase activity by stimulating shedding of HAI-1 fragments. Matriptase protein was detected in squamous carcinoma-derived HSC-4 cells, however, matriptase protease activity was undetectable. Transfection of siRNA for HAI-1 enhanced serine protease activity, which was suppressed by cotransfection of matriptase siRNA. Collagen-gel culture or treatment with concanavalin A (ConA) of HSC-4 cells enhanced MT1-MMP activity, which induced shedding of HAI-1 fragments and conversely stimulated uPA activation by these cells. Serine protease activity, including uPA activation of cells treated with ConA, was abrogated by downregulation of either matriptase or MT1-MMP through the transfection of each siRNA. These results suggest that MT1-MMP induced by collagen-gel culture or ConA treatment causes cleavage and shedding of HAI-1 protein, which allows activation of matriptase in HSC-4 cells. HSC-4 cells showed a characteristic invasive growth by forming vacuole-like structures in collagen gel, which was suppressed by transfection of siRNA for either MT1-MMP or matriptase, suggesting that activation of matriptase through the cleavage of HAI-1 is one of the MT1-MMP multifunctions essential for invasive growth of HSC-4 cells.

Membrane-type 1 matrix metalloproteinase regulates fibronectin assembly to promote cell motility.

Fibronectin (FN) matrix assembly is an essential process in normal vertebrate development, which is frequently lost in tumor cells. Here we show that membrane-type 1 matrix metalloproteinase (MT1-MMP) regulates FN matrix assembly. MT1-MMP knockdown induced FN assembly in breast carcinoma cells. Ectopic expression of MT1-MMP reduced specifically the assembled FN matrix level without affecting whole FN production in fibroblasts. Treatment of fibrosarcoma HT1080 cells with dexamethasone (DEX) enhanced FN synthesis, resulting in short fibrils but not dense matrix formation. Combined treatment of DEX and MT1-MMP inhibitor accelerated FN matrix assembly, which mediated cellular adhesion and reduced cell migration and invasion. These results indicate that MT1-MMP stimulates cell migration and invasion by negatively regulating FN assembly.

GI24 enhances tumor invasiveness by regulating cell surface membrane-type 1 matrix metalloproteinase.

GI24, an immunoglobulin superfamily member, has been cloned from a placenta cDNA library as a gene product that promoted activation of matrix metalloproteinase (MMP)-2 mediated by membrane type (MT) 1-MMP. Co-expression of GI24 with MT1-MMP in HEK293T cells increased the cell-surface level of MT1-MMP concomitant with the cleavage of the GI24 at the juxtamembrane site to shed the extracellular domain. HT1080 fibrosarcoma cells stably transfected with the GI24 gene expressed a higher level of MT1-MMP and showed more invasive ability in collagen gel than the control cells. GI24 was cleaved in HT1080 cells, which was blocked by the administration of MMP inhibitor BB94 or transfection of small interfering RNA (siRNA) targeting MT1-MMP. GI24 expression is relatively high in some squamous carcinoma and hepatocarcinoma cell lines. Transfection of siRNA for GI24 into oral squamous carcinoma-derived HSC-4 cells, which express GI24 and MT1-MMP genes reduced the expression of not only GI24 but also MT1-MMP, and attenuated invasive growth in the collagen matrix. These results suggest that GI24 contributes to tumor-invasive growth in the collagen matrix by augmenting cell surface MT1-MMP.