RECK‐mediated inhibition of glioma migration and invasion

RECK is an anti‐tumoral gene whose activity has been associated with its inhibitory effects regulating MMP‐2, MMP‐9, and MT1‐MMP. RECK level decreases as gliobastoma progresses, varying from less invasive grade II gliomas to very invasive human glioblastoma multiforme (GBM). Since RECK expression and glioma invasiveness show an inverse correlation, the aim of the present study is to investigate whether RECK expression would inhibit glioma invasive behavior. We conducted this study to explore forced RECK expression in the highly invasive T98G human GBM cell line. Expression levels as well as protein levels of RECK, MMP‐2, MMP‐9, and MT1‐MMP were assessed by qPCR and immunoblotting in T98G/RECK+ cells. The invasion and migration capacity of RECK+ cells was inhibited in transwell and wound assays. Dramatic cytoskeleton modifications were observed in the T98G/RECK+ cells, when compared to control cells, such as the abundance of stress fibers (contractile actin–myosin II bundles) and alteration of lamellipodia. T98G/RECK+ cells also displayed phosphorylated focal adhesion kinase (P‐FAK) in mature focal adhesions associated with stress fibers; whereas P‐FAK in control cells was mostly associated with immature focal complexes. Interestingly, the RECK protein was predominantly localized at the leading edge of migrating cells, associated with membrane ruffles. Unexpectedly, introduced expression of RECK effectively inhibited the invasive process through rearrangement of actin filaments, promoting a decrease in migratory ability. This work has associated RECK tumor‐suppressing activity with the inhibition of motility and invasion in this GBM model, which are two glioma characteristics responsible for the inefficiency of current available treatments. J. Cell. Biochem. 110: 52–61, 2010. © 2010 Wiley‐Liss, Inc.     

LGI1, a putative tumor metastasis suppressor gene, controls in vitro invasiveness and expression of matrix metalloproteinases in glioma cells through the ERK1/2 pathway

Gliomas take a number of different genetic routes in the progression to glioblastoma multiforme, a highly invasive variant that is mostly unresponsive to current therapies. Gliomas express elevated levels of matrix metalloproteinases (MMPs), which have been implicated in the control of proliferation and invasion as well as neovascularization. Progressive loss of LGI1 expression has been associated with the development of high grade gliomas. We have shown previously that the forced re-expression of LGI1 in different glioma cells inhibits proliferation, invasiveness, and anchorage-independent growth in cells null for its expression. Here, using Affymetrix gene chip analysis, we show that reexpression of LGI1 in T98G cells results in the down-regulation of several MMP genes, in particular MMP1 and MMP3. LGI1 expression also results in the inhibition of ERK1/2 phosphorylation but not p38 phosphorylation. Inhibition of the MAPK pathway using the pharmacological inhibitors PD98059, U0126, and SB203580 in T98G LGI1-null cells inhibits MMP1 and MMP3 production in an ERK1/2-dependent manner. Treatment of LGI1-expressing cells with phorbol myristate acetate prevents the inhibition of MMP1/3 and restores invasiveness and ERK1/2 phosphorylation, suggesting that LGI1 acts through the ERK/MAPK pathway. Furthermore, LGI1 expression promotes phosphorylation of AKT, which leads to phosphorylation of Raf1(Ser-259), an event shown previously to negatively regulate ERK1/2 signaling. These data suggest that LGI1 plays a major role in suppressing the production of MMP1/3 through the phosphatidylinositol 3-kinase/ERK pathway. Loss of LGI1 expression, therefore, may be an important event in the progression of gliomas that leads to a more invasive phenotype in these cells.     

MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators

Substantial data indicate that microRNA 21 (miR-21) is significantly elevated in glioblastoma (GBM) and in many other tumors of various origins. This microRNA has been implicated in various aspects of carcinogenesis, including cellular proliferation, apoptosis, and migration. We demonstrate that miR-21 regulates multiple genes associated with glioma cell apoptosis, migration, and invasiveness, including the RECK and TIMP3 genes, which are suppressors of malignancy and inhibitors of matrix metalloproteinases (MMPs). Specific inhibition of miR-21 with antisense oligonucleotides leads to elevated levels of RECK and TIMP3 and therefore reduces MMP activities in vitro and in a human model of gliomas in nude mice. Moreover, downregulation of miR-21 in glioma cells leads to decreases of their migratory and invasion abilities. Our data suggest that miR-21 contributes to glioma malignancy by downregulation of MMP inhibitors, which leads to activation of MMPs, thus promoting invasiveness of cancer cells. Our results also indicate that inhibition of a single oncomir, like miR-21, with specific antisense molecules can provide a novel therapeutic approach for “physiological” modulation of multiple proteins whose expression is deregulated in cancer.     

HPV16 oncoproteins induce MMPs/RECK-TIMP-2 imbalance in primary keratinocytes: possible implications in cervical carcinogenesis

Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome.     

The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis.

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.     

Inhibition of matrix metalloproteinase-9 gene expression by an isoflavone metabolite, irisolidone in U87MG human astroglioma cells

Matrix metalloproteinase-9 (MMP-9) plays an important role in mediating the invasion and angiogenic process of malignant gliomas. This study was undertaken to determine if an isoflavone metabolite, irisolidone, inhibits MMP-9 expression in human astroglioma cells. Irisolidone was found to inhibit the secretion and protein expression of MMP-9 induced by PMA in U87 MG glioma cells, accompanied by the inhibition of MMP-9 mRNA expression and promoter activity. Further mechanistic studies revealed that irisolidone inhibits the binding of NF-κB and AP-1 to the MMP-9 promoter and suppresses the PMA-induced phosphorylation of ERK and JNK, which are upstream signaling molecules in MMP-9 expression. The Matrigel-invasion assay showed that irisolidone suppresses the in vitro invasiveness of glioma cells. Therefore, the strong inhibition of MMP-9 expression by irisolidone might be a potential therapeutic modality for controlling the growth and invasiveness of gliomas.     

Analysis of the MMP-dependent and independent functions of tissue inhibitor of metalloproteinase-2 on the invasiveness of breast cancer cells

Matrix metalloproteinases (MMPs) are secreted endopeptidases that play an essential role in remodeling the extracellular matrix (ECM). MMPs are primarily active during development, when the majority of ECM remodeling events occurs. In adults, elevated MMP activity has been observed in many pathological conditions such as cancer and osteoarthritis. The proteolytic activity of MMPs is controlled by their natural inhibitors - the tissue inhibitor of metalloproteinases (TIMPs). In addition to blocking MMP-mediated proteolysis, TIMPs have a number of MMP-independent functions including binding to cell surface proteins thereby stimulating signaling cascades. TIMP-2, the most studied member of the family, can both inhibit and activate MMPs directly, as well as inhibit MMP activity indirectly by upregulating expression of RECK, a membrane anchored MMP regulator. While TIMP-2 has been shown to play important roles in breast cancer, we describe how the MMP-independent effects of TIMP-2 can modulate the invasiveness of MCF-7, T47D and MDA-MB-231 breast cancer cells. Using an ALA + TIMP-2 mutant which is devoid of MMP inhibition, but still capable of initiating specific cell signaling cascades, we show that TIMP-2 can differentially affect MMP activity and cellular invasiveness in both an MMP dependent and independent manner. More specifically, MMP activity and invasiveness is increased with the addition of exogenous TIMP-2 in poorly invasive cell lines whereas it is decreased in highly invasive cells lines (MDA-MB-231). Conversely, the addition of ALA + TIMP-2 resulted in decreased invasiveness regardless of cell line.     

Higher expression and activity of metalloproteinases in human cervical carcinoma cell lines is associated with HPV presence.

Matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in 3 human invasive cervical carcinoma cell lines. Two HPV16-positive cell lines (SiHa and CaSki) and an HPV-negative cell line (C33A) were cultured either onto a type-I collagen gel, Matrigel, or plastic, to recreate their three-dimensional growth environment and evaluate the expression of these genes using quantitative real-time PCR. We also analyzed the gelatinolytic activity of MMP-2 and MMP-9 by zymography. We found that HPV (human papillomavirus)-positive cell lines express higher levels of MMP-2, MT1-MMP, and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with higher pro-MMP-2 activity. Furthermore, Matrigel substrate influences MMP-2 expression in both SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP-2, MT1-MMP, and TIMP-2, and that pro-MMP-2 activity is higher in HPV-positive than in HPV-negative cells.     

The mechanical and pharmacological regulation of glioblastoma cell migration in 3D matrices

The invasion of glioblastoma is a complex process based on the interactions of tumor cells and the extracellular matrix. Tumors that are engineered using biomaterials are more physiologically relevant than a two-dimensional (2D) cell culture system. Matrix metalloproteinases and the plasminogen activator generated by tumor cells regulate a tumor’s invasive behavior. In this study, microtumors were fabricated by encapsulating U87 glioma cells in Type I collagen and then glioma cell migration in the collagen hydrogels was investigated. Crosslinking of collagen with 8S-StarPEG increased the hydrogel viscosity and reduced the tumor cell migration speed in the hydrogels. The higher migration speed corresponded to the increased gene expression of MMP-2, MMP-9, urokinase plasminogen activator (uPA), and tissue plasminogen activator (tPA) in glioma cells grown in non-crosslinked collagen hydrogels. Inhibitors of these molecules hindered U87 and A172 cell migration in collagen hydrogels. Aprotinin and tranexamic acid did not inhibit U87 and A172 migration on the culture dish. This study demonstrated the differential effect of pharmacologic molecules on tumor cell motility in either a 2D or three-dimensional culture environment.     

Oct-3/4 promotes migration and invasion of glioblastoma cells

As a result of increased glioblastoma migration and invasion into normal brain parenchyma, treatment of local tumor recurrence following initial treatment in glioblastoma patients remains challenging. Recent studies have demonstrated increased Oct-3/4 expression, a self-renewal regulator in stem cells, in glioblastomas. However, little is known regarding the influence of Oct-3/4 in glioblastoma cell invasiveness. The present study established Oct-3/4-overexpressing glioblastoma cells, which were prepared from human glioblastoma patients, to assess migration, invasion, and mRNA expression profiles of integrins and matrix metalloproteinases (MMPs). Compared with control cells, Oct-3/4 expressing-glioblastoma cells exhibited increased migration and invasion in wound healing and Matrigel invasion assays. Oct-3/4 overexpression resulted in upregulated FAK and c-Src expression, which mediate integrin signals. Vinculin accumulated along the leading edges of Oct-3/4 expressing-glioblastoma cells and associated with membrane ruffles during cell migration. Oct-3/4 expressing-cells exhibited increased MMP-13 mRNA expression and MMP-13 knockdown by shRNA suppressed cell invasion into Matrigel and organotypic brain slices. These results suggested that Oct-3/4 enhanced degradation of surrounding extracellular matrix by increasing MMP-13 expression and altering integrin signaling. Therefore, Oct-3/4 might contribute to tumor promoting activity in glioblastomas.     

Tissue inhibitor of matrix metalloproteinase-2 regulates matrix metalloproteinase-2 activation by modulation of membrane-type 1 matrix metalloproteinase activity in high and low invasive melanoma cell lines.

Activation of pro-matrix metalloproteinase (MMP)-2 on the surface of malignant cells by membrane-bound MT1-MMP is believed to play a critical role during tumor progression and metastasis. In this study we present evidence that MT1-MMP plays a key role for the in vitro invasiveness of malignant melanoma. Melanoma cell lines secreted latent MMP-2 when cultured on plastic. However, when cells were grown in floating type I collagen lattices, only high invasive melanoma cells activated proMMP-2. Activation could be inhibited by antibodies against MT1-MMP, by addition of recombinant tissue inhibitor of metalloproteinases (TIMP)-2 and by inhibition of MT1-MMP cleavage. MT1-MMP protein was detected as an inactive protein in all cell lines cultured as monolayers, whereas in collagen gels, active MT1-MMP protein was detected in the membranes of both high and low invasive melanoma cells. Production of TIMP-2 was about 10-fold higher in low invasive cells as compared with high invasive melanoma cells and was further increased in the low invasive cells upon contact to collagen. Thus, in melanoma cells TIMP-2 expression levels might regulate MT1-MMP-mediated activation of proMMP-2. High invasive melanoma cells displayed increased in vitro invasiveness, which was inhibited by TIMP-2. These data indicate the importance of these enzymes for the invasion processes and support a role for MT1-MMP as an activator of proMMP-2 in malignant melanoma.     

Expression of matrix metalloproteinases-2 and -9 and RECK during alveolar bone regeneration in rat

MMPs are endopeptidases that play a pivotal role in ECM turnover. RECK is a single membrane-anchored MMP-regulator. Here, we evaluated the temporal and spatial expression of MMP-2, MMP-9, and RECK during alveolar bone regeneration. The maxillary central incisor of Wistar rats was extracted and the animals were killed at 1, 3, 7, 10, 14, 21, 28, and 42 days post-operatively (n = 3/period). The hemimaxillae were collected, demineralized and embedded in paraffin. Immunohistochemical analysis was performed by the immunoperoxidase technique with polyclonal antibodies. On day 1, polymorphonuclear cells in the blood clot presented mild immunolabeling for MMPs. During bone remodeling, osteoblasts facing new bone showed positive staining for gelatinases and RECK in all experimental periods. MMPs were also found in the connective tissue and endothelial cells. Our results show for the first time that inactive and/or active forms of MMP-2, MMP-9 and RECK are differentially expressed by osteogenic and connective cells during several events of alveolar bone regeneration. This may be important for the replacement of the blood clot by connective tissue, and in the formation, maturation and remodeling of new bone.     

Arsenic trioxide (As2O3) inhibits invasion of HT1080 human fibrosarcoma cells: role of nuclear factor-kappaB and reactive oxygen species

In order to define the role of As2O3 in regulating the tumor cell invasiveness, the effects of As2O3 on secretion of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), and in vitro invasion of HT1080 human fibrosarcoma cells were examined. As2O3 inhibited cell adhesion to the collagen matrix in a concentration dependent manner, whereas the same treatment enhanced cell to cell interaction. In addition, As2O3 inhibited migration and invasion of HT1080 cells stimulated with phorbol 12-myristate 13-aceate (PMA), and suppressed the expression of MMP-2, -9, membrane type-1 MMP, uPA, and uPA receptor (uPAR). In contrast, As2O3 increased the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and PA inhibitor (PAI)-1, and reduced the MMP-2, -9, and uPA promoter activity in the presence and absence of PMA. Furthermore, the promoter stimulating and DNA binding activity of nuclear factor-kappaB (NF-kappaB) was blocked by As2O3, whereas the activator protein-1 activity was unchanged. Pretreatment of the cells with N-acetyl-L-cysteine (NAC) significantly prevented suppression of MMPs and uPA secretion, DNA binding activity of NF-kappaB, and in vitro invasion of HT1080 cells by As2O3, suggesting a role of reactive oxygen species (ROS) in this process. These results suggest that As2O3 inhibits tumor cell invasion by modulating the MMPs/TIMPs and uPA/uPAR/PAI systems of extracellular matrix (ECM) degradation. In addition, the generation of ROS and subsequent suppression of NF-kappaB activity by As2O3 might partly be responsible for the phenomena. Overall, As2O3 shows potent activity controlling tumor cell invasiveness in vitro.     

Genetic polymorphisms of matrix metalloproteinases and their inhibitors in potentially malignant and malignant lesions of the head and neck

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that are capable of cleaving all extra cellular matrix (ECM) substrates. Degradation of matrix is a key event in progression, invasion and metastasis of potentially malignant and malignant lesions of the head and neck. It might have an important polymorphic association at the promoter regions of several MMPs such as MMP-1 (-1607 1G/2G), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T) and TIMP-2 (-418 G/C or C/C). Tissue inhibitors of metalloproteinases (TIMPs) are naturally occurring inhibitors of MMPs, which inhibit the activity of MMPs and control the breakdown of ECM. Currently, many MMP inhibitors (MMPIs) are under development for treating different malignancies. Useful markers associated with molecular aggressiveness might have a role in prognostication of malignancies and to better recognize patient groups that need more antagonistic treatment options. Furthermore, the introduction of novel prognostic markers may also promote exclusively new treatment possibilities, and there is an obvious need to identify markers that could be used as selection criteria for novel therapies. The objective of this review is to discuss the molecular functions and polymorphic association of MMPs and TIMPs and the possible therapeutic aspects of these proteinases in potentially malignant and malignant head and neck lesions. So far, no promising drug target therapy has been developed for MMPs in the lesions of this region. In conclusion, further research is required for the development of their potential diagnostic and therapeutic possibilities.     

Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells.

The relative expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is an important determinant in trophoblast invasion of the uterus and tumor invasion and metastasis. Our previous studies have shown that low oxygen levels increase the in vitro invasiveness of trophoblast and tumor cells. The present study examined whether changes in oxygen levels affect TIMP and MMP expression by cultured trophoblast and breast cancer cells. Reverse zymographic analysis demonstrated reduced TIMP-1 protein secretion by HTR-8/SVneo trophoblast cells as well as MDA-MB-231 and MCF-7 breast carcinoma cells cultured in 1% vs 20% oxygen for 24 h. While gelatin zymography revealed no changes in the levels of MMP-9 secreted by HTR-8/SVneo trophoblasts cultured under various oxygen concentrations for 24 h, human MDA-MB-231 breast carcinoma cells displayed increased MMP-9 secretion and human MCF-7 breast cancer cells exhibited reduced secretion of this enzyme when cultured under similar conditions. In contrast, MMP-2 levels remained unchanged in all cultures incubated under similar conditions. Western blot analysis of MMP-9 protein in cell extracts confirmed the results of zymography. To assess the contribution of enhanced MMP activity to hypoxia-induced invasion, the effect of an MMP inhibitor (llomastat) on the ability of MDA-MB-231 cells to penetrate reconstituted extracellular matrix (Matrigel) was examined. Results showed that MMP inhibition significantly decreased the hypoxic upregulation of invasion by these cells. These findings indicate that the increased cellular invasiveness observed under reduced oxygen conditions may be due in part to a shift in the balance between MMPs and their inhibitors favoring increased MMP activity.