Galectin-4 Reduces Migration and Metastasis Formation of Pancreatic Cancer Cells

Galectin-4 (Gal-4) is a member of the galectin family of glycan binding proteins that shows a significantly higher expression in cystic tumors of the human pancreas and in pancreatic adenocarcinomas compared to normal pancreas. However, the putative function of Gal-4 in tumor progression of pancreatic cancer is still incompletely understood. In this study the role of Gal-4 in cancer progression was investigated, using a set of defined pancreatic cancer cell lines, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), as a model. These two cell lines are derived from the same liver metastasis of a human primary pancreatic adenocarcinoma, but differ in their growth characteristics and metastatic capacity. We demonstrated that Gal-4 expression is high in PaTu-S, which shows poor migratory properties, whereas much lower Gal-4 levels are observed in the highly metastatic cell line PaTu-T. In PaTu-S, Gal-4 is found in the cytoplasm, but it is also secreted and accumulates at the membrane at sites of contact with neighboring cells. Moreover, we show that Gal-4 inhibits metastasis formation by delaying migration of pancreatic cancer cells in vitro using a scratch assay, and in vivo using zebrafish (Danio rerio) as an experimental model. Our data suggest that Gal-4 may act at the cell-surface of PaTu-S as an adhesion molecule to prevent release of the tumor cells, but has in addition a cytosolic function by inhibiting migration via a yet unknown mechanism.     

Metalloproteinase MT1-MMP islets act as memory devices for podosome reemergence

Podosomes are dynamic cell adhesions that are also sites of extracellular matrix degradation, through recruitment of matrix-lytic enzymes, particularly of matrix metalloproteinases. Using total internal reflection fluorescence microscopy, we show that the membrane-bound metalloproteinase MT1-MMP is enriched not only at podosomes but also at distinct “islets” embedded in the plasma membrane of primary human macrophages. MT1-MMP islets become apparent upon podosome dissolution and persist beyond podosome lifetime. Importantly, the majority of MT1-MMP islets are reused as sites of podosome reemergence. siRNA-mediated knockdown and recomplementation analyses show that islet formation is based on the cytoplasmic tail of MT1-MMP and its ability to bind the subcortical actin cytoskeleton. Collectively, our data reveal a previously unrecognized phase in the podosome life cycle and identify a structural function of MT1-MMP that is independent of its proteolytic activity. MT1-MMP islets thus act as cellular memory devices that enable efficient and localized reformation of podosomes, ensuring coordinated matrix degradation and invasion.     

MT-LOOP-dependent Localization of Membrane Type I Matrix Metalloproteinase (MT1-MMP) to the Cell Adhesion Complexes Promotes Cancer Cell Invasion

Localization of membrane type I matrix metalloproteinase (MT1-MMP) to the leading edge is thought to be a crucial step during cancer cell invasion. However, its mechanisms and functional impact on cellular invasion have not been clearly defined. In this report, we have identified the MT-LOOP, a loop region in the catalytic domain of MT1-MMP (¹⁶³PYAYIREG¹⁷⁰), as an essential region for MT1-MMP to promote cellular invasion. Deletion of the MT-LOOP effectively inhibited functions of MT1-MMP on the cell surface, including proMMP-2 activation, degradation of gelatin and collagen films, and cellular invasion into a collagen matrix. This is not due to loss of the catalytic function of MT1-MMP but due to inefficient localization of the enzyme to β1-integrin-rich cell adhesion complexes at the plasma membrane. We also found that an antibody that specifically recognizes the MT-LOOP region of MT1-MMP (LOOP_Ab) inhibited MT1-MMP functions, fully mimicking the phenotype of the MT-LOOP deletion mutant. We therefore propose that the MT-LOOP region is an interface for molecular interactions that mediate enzyme localization to cell adhesion complexes and regulate MT1-MMP functions. Our findings have revealed a novel mechanism regulating MT1-MMP during cellular invasion and have identified the MT-LOOP as a potential exosite target region to develop selective MT1-MMP inhibitors.     

Detection of the Heterogeneous O-Glycosylation Profile of MT1-MMP Expressed in Cancer Cells by a Simple MALDI-MS Method

Background

Glycosylation is an important and universal post-translational modification for many proteins, and regulates protein functions. However, simple and rapid methods to analyze glycans on individual proteins have not been available until recently.

Methods/Principal Findings

A new technique to analyze glycopeptides in a highly sensitive manner by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using the liquid matrix 3AQ/CHCA was developed recently and we optimized this technique to analyze a small amount of transmembrane protein separated by SDS-PAGE. We used the MALDI-MS method to evaluate glycosylation status of membrane-type 1 matrix metalloproteinase (MT1-MMP). O-glycosylation of MT1-MMP is reported to modulate its protease activity and thereby to affect cancer cell invasion. MT1-MMP expressed in human fibrosarcoma HT1080 cells was immunoprecipitated and resolved by SDS-PAGE. After in-gel tryptic digestion of the protein, a single droplet of the digest was applied directly to the liquid matrix on a MALDI target plate. Concentration of hydrophilic glycopeptides within the central area occurred due to gradual evaporation of the sample solution, whereas nonglycosylated hydrophobic peptides remained at the periphery. This specific separation and concentration of the glycopeptides enabled comprehensive analysis of the MT1-MMP O-glycosylation.

Conclusions/Significance

We demonstrate, for the first time, heterogeneous O-glycosylation profile of a protein by a whole protein analysis using MALDI-MS. Since cancer cells are reported to have altered glycosylation of proteins, this easy-to-use method for glycopeptide analysis opens up the possibility to identify specific glycosylation patterns of proteins that can be used as new biomarkers for malignant tumors.     

Neurokinin-1 Receptor Directly Mediates Glioma Cell Migration by Up-regulation of Matrix Metalloproteinase-2 (MMP-2) and Membrane Type 1-Matrix Metalloproteinase (MT1-MMP)

Neurokinin-1 receptor (NK1R) occurs naturally on human glioblastomas. Its activation mediates glioma cell proliferation. However, it is unknown whether NK1R is directly involved in tumor cell migration. In this study, we found human hemokinin-1 (hHK-1), via NK1R, dose-dependently promoted the migration of U-251 and U-87 cells. In addition, we showed that hHK-1 enhanced the activity of MMP-2 and the expression of MMP-2 and MT1-matrix metalloproteinase (MMP), which were responsible for cell migration, because neutralizing the MMPs with antibodies decreased cell migration. The involved mechanisms were then investigated. In U-251, hHK-1 induced significant calcium efflux; phospholipase C inhibitor U-73122 reduced the calcium mobilization, the up-regulation of MMP-2 and MT1-MMP, and the cell migration induced by hHK-1, which meant the migration effect of NK1R was mainly mediated through the G_q-PLC pathway. We further demonstrated that hHK-1 boosted rapid phosphorylation of ERK, JNK, and Akt; inhibition of ERK and Akt effectively reduced MMP-2 induction by hHK-1. Meanwhile, inhibition of ERK, JNK, and Akt reduced the MT1-MMP induction. hHK-1 stimulated significant phosphorylation of p65 and c-JUN in U-251. Reporter gene assays indicated hHK-1 enhanced both AP-1 and NF-κB activity; inhibition of ERK, JNK, and Akt dose-dependently suppressed the NF-κB activity; only the inhibition of ERK significantly suppressed the AP-1 activity. Treatment with specific inhibitors for AP-1 or NF-κB strongly blocked the MMP up-regulation by hHK-1. Taken together, our data suggested NK1R was a potential regulator of human glioma cell migration by the up-regulation of MMP-2 and MT1-MMP.     

MT1-MMP: an enzyme with multidimensional regulation

The activity of membrane-type 1 matrix metalloproteinase (MT1-MMP) is a double-edged sword--it is crucial for both physiological processes and disease progression. MT1-MMP modifies various cellular functions and it is, sthus, regulated precisely as a proteinase and as a membrane protein. Recent studies have further revealed that the function of MT1-MMP is modified and regulated by O-glycosylation, interaction with CD44, internalization and recycling. Such multidimensional mechanisms enable MT1-MMP to be regulated spatially and temporally, and are essential for its proper functioning on the cell surface.     

Noncanonical Activation of Notch1 Protein by Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Controls Melanoma Cell Proliferation

Notch1 is an evolutionarily conserved signaling molecule required for stem cell maintenance that is inappropriately reactivated in several cancers. We have previously shown that melanomas reactivate Notch1 and require its function for growth and survival. However, no Notch1-activating mutations have been observed in melanoma, suggesting the involvement of other activating mechanisms. Notch1 activation requires two cleavage steps: first by a protease and then by γ-secretase, which releases the active intracellular domain (Notch1^NIC). Interestingly, although ADAM10 and -17 are generally accepted as the proteases responsible of Notch1 cleavage, here we show that MT1-MMP, a membrane-tethered matrix metalloproteinase involved in the pathogenesis of a number of tumors, is a novel protease required for the cleavage of Notch1 in melanoma cells. We find that active Notch1 and MT1-MMP expression correlate significantly in over 70% of melanoma tumors and 80% of melanoma cell lines, whereas such correlation does not exist between Notch1^NIC and ADAM10 or -17. Modulation of MT1-MMP expression in melanoma cells affects Notch1 cleavage, whereas MT1-MMP expression in ADAM10/17 double knock-out fibroblasts restores the processing of Notch1, indicating that MT1-MMP is sufficient to promote Notch1 activation independently of the canonical proteases. Importantly, we find that MT1-MMP interacts with Notch1 at the cell membrane, supporting a potential direct cleavage mechanism of MT1-MMP on Notch1, and that MT1-MMP-dependent activation of Notch1 sustains melanoma cell growth. Together, the data highlight a novel mechanism of activation of Notch1 in melanoma cells and identify Notch1 as a new MT1-MMP substrate that plays important biological roles in melanoma.     

Mutation of Membrane Type-1 Metalloproteinase, MT1-MMP, Causes the Multicentric Osteolysis and Arthritis Disease Winchester Syndrome

The "vanishing bone" syndromes represent a group of rare skeletal disorders characterized by osteolysis and joint destruction, which can mimic severe rheumatoid arthritis. Winchester syndrome was one of the first recognized autosomal-recessive, multicentric forms of the disorder. It was originally described nearly 50 years ago in two sisters with a severe crippling osteolysis. Using cultured fibroblasts from the proband, we have now identified homozygous mutations in membrane type-1 metalloproteinase (MT1-MMP or MMP14). We demonstrate that the resulting hydrophobic-region signal-peptide substitution (p.Thr17Arg) decreases MT1-MMP membrane localization with consequent impairment of pro-MMP2 activation, and we propose a structure-based mechanism for this effect.     

MT1-MMP modulates the mechanosensitivity of osteocytes

Membrane-type matrix metalloproteinase-1 (MT1-MMP) is expressed by mechanosensitive osteocytes and affects bone mass. The extracellular domain of MT1-MMP is connected to extracellular matrix, while its intracellular domain is a strong modulator of cell signaling. In theory MT1-MMP could thus transduce mechanical stimuli into a chemical response. We hypothesized that MT1-MMP plays a role in the osteocyte response to mechanical stimuli. MT1-MMP-positive and knockdown (siRNA) MLO-Y4 osteocytes were mechanically stimulated with a pulsating fluid flow (PFF). Focal adhesions were visualized by paxillin immunostaining. Osteocyte number, number of empty lacunae, and osteocyte morphology were measured in long bones of MT1-MMP(+/+) and MT1-MMP(-/-) mice. PFF decreased MT1-MMP mRNA and protein expression in MLO-Y4 osteocytes, suggesting that mechanical loading may affect pericellular matrix remodeling by osteocytes. MT1-MMP knockdown enhanced NO production and c-jun and c-fos mRNA expression in response to PFF, concomitantly with an increased number and size of focal adhesions, indicating that MT1-MMP knockdown osteocytes have an increased sensitivity to mechanical loading. Osteocytes in MT1-MMP(-/-) bone were more elongated and followed the principle loading direction, suggesting that they might sense mechanical loading. This was supported by a lower number of empty lacunae in MT1-MMP(-/-) bone, as osteocytes lacking mechanical stimuli tend to undergo apoptosis. In conclusion, mechanical stimulation decreased MT1-MMP expression by MLO-Y4 osteocytes, and MT1-MMP knockdown increased the osteocyte response to mechanical stimulation, demonstrating a novel and unexpected role for MT1-MMP in mechanosensing.     

Development of a Radiolabeled Peptide-Based Probe Targeting MT1-MMP for Breast Cancer Detection

Breast cancer is one of the most frequent and aggressive primary tumors among women of all races. Matrix metalloproteinase (MMPs), a family of zinc- and calcium-dependent secreted or membrane anchored endopeptidases, is overexpressed in varieties of diseases including breast cancer. Therefore, noninvasive visualization and quantification of MMP in vivo are of great interest in basic research and clinical application for breast cancer early diagnosis. Herein, we developed a ^99mTc labeled membrane type I matrix metalloproteinase (MT1-MMP) specific binding peptide, [^99mTc]-(HYNIC-AF7p)(tricine)(TPPTS), for in vivo detection of MDA-MB-231 breast tumor by single photon emission computed tomography (SPECT). [^99mTc]-(HYNIC-AF7p)(tricine)(TPPTS) demonstrated nice biostability and high MT1-MMP binding affinity in vitro and in vivo. Tumor-to-muscle ratio was found to reach to the highest (4.17±0.49) at 2 hour after intravenously administration of [^99mTc]-(HYNIC-AF7P)(tricine)(TPPTS) into MDA-MB-231 tumor bearing mice. Overall, [^99mTc]-(HYNIC-AF7P)(tricine)(TPPTS) demonstrated great potential for MT1-MMP targeted detection in vivo and it would be a promising molecular imaging probe that are probably beneficial to breast cancer early diagnoses.     

TIMP-2 Interaction with MT1-MMP Activates the AKT Pathway and Protects Tumor Cells from Apoptosis

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components. In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms. We have previously shown that binding of tissue inhibitor of metalloproteinases-2 (TIMP-2) to MT1-MMP controls cell proliferation and migration, as well as tumor growth in vivo by activating the Ras—extracellular signal regulated kinase-1 and -2 (ERK1/2) pathway through a mechanism that requires the cytoplasmic but not the proteolytic domain of MT1-MMP. Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP. Fibroblast growth factor receptor-1 mediates TIMP-2 induction of ERK1/2 but not of AKT activation; however, Ras activation is necessary to transduce the TIMP-2-activated signal to both the ERK1/2 and AKT pathways. ERK1/2 and AKT activation by TIMP-2 binding to MT1-MMP protects tumor cells from apoptosis induced by serum starvation. Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells. Thus, TIMP-2 interaction with MT1-MMP provides tumor cells with either pro- or anti-apoptotic signaling depending on the extracellular environment and apoptotic stimulus.     

miR-96通过RECK促进结直肠癌细胞的迁移和侵袭

目的:研究miRNA-96在结直肠癌转移中的作用及其机制。方法:采用Transwell试验分析结直肠癌Lo Vo细胞的迁移和侵袭能力,采用荧光素报告基因及蛋白免疫印迹试验研究结直肠癌中miR-96的作用靶点。结果:miR-96抑制剂处理后下调miR-96的表达并抑制Lo Vo细胞的迁移和侵袭。荧光素报告基因试验显示RECK是miR-96的作用靶点,且RECK沉默能够部分阻碍miR-96抑制剂所导致的Lo Vo细胞迁移和侵袭减少。结论:miRNA-96可通过作用于RECK促进结直肠癌细胞转移,这可能成为治疗结直肠癌转移的新靶点。     

Contrasting expression of membrane metalloproteinases, MT1-MMP and MT3-MMP, suggests distinct functions in skeletal development

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is the most ubiquitous and widely studied of the membrane-type metalloproteinases (MT-MMPs). It was thus surprising to find no published data on chicken MT1-MMP. We report here the characterization of the chicken gene. Its low sequence identity with the MT1-MMP genes of other species, high GC content, and divergent catalytic domain explains the absence of data and our difficulties in characterizing the gene. The absence of structural features in the chicken gene that have been suggested to be critical for the activation of MMP-2 by MT1-MMP; for the effect of MT1-MMP on cell migration and for the recycling of MT1-MMP suggest these features are either not essential or that MT1-MMP does not perform these functions in chickens. Comparison of the expression of chicken MT1-MMP with MT3-MMP and with MMP-2 and MMP-13 has confirmed the previously recognized co-expression of MT1-MMP with MMP-2 and MMP-13 in fibrous and vascular tissues, particularly those surrounding the developing long bones in other species. By contrast, MT3-MMP expression differs markedly from that of MT1-MMP and of both MMP-2 and MMP-13. MT3-MMP is expressed by chondrocytes of the developing articular surface. Similar expression patterns of this group of MT-MMPs and MMPs have been observed in mouse embryos and suggest distinct and specific functions for MT1-MMP and MT3-MMP in skeletal development.     

The Metastatic Potential and Chemoresistance of Human Pancreatic Cancer Stem Cells

Cancer stem cells (CSCs) typically have the capacity to evade chemotherapy and may be the principal source of metastases. CSCs for human pancreatic ductal carcinoma (PDAC) have been identified, but neither the metastatic potential nor the chemoresistance of these cells has been adequately evaluated. We have addressed these issues by examining side-population (SP) cells isolated from the Panc-1 and BxPC3 lines of human PDAC cells, the oncogenotypes of which differ. SP cells could be isolated from monolayers of Panc-1, but only from spheroids of BxPC3. Using orthotopic xenografts into the severely immunocompromised NSG mouse, we found that SP cells isolated from both cell lines produced tumors that were highly metastatic, in contrast to previous experience with PDAC cell lines. SP cells derived from both cell lines expressed the ABCG2 transporter, which was demonstrably responsible for the SP phenotype. SP cells gave rise to non-SP (NSP) cells in vitro and in vivo, a transition that was apparently due to posttranslational inhibition of the ABCG2 transporter. Twenty-two other lines of PDAC cells also expressed ABCG2. The sensitivity of PDAC SP cells to the vinca alkaloid vincristine could be greatly increased by verapamil, a general inhibitor of transporters. In contrast, verapamil had no effect on the killing of PDAC cells by gemcitabine, the current first-line therapeutic for PDAC. We conclude that the isolation of SP cells can be a convenient and effective tool for the study of PDAC CSCs; that CSCs may be the principal progenitors of metastasis by human PDAC; that the ABCG2 transporter is responsible for the SP phenotype in human PDAC cells, and may be a ubiquitous source of drug-resistance in PDAC, but does not confer resistance to gemcitabine; and that inhibition of ABCG2 might offer a useful adjunct in a therapeutic attack on the CSCs of PDAC.     

Effect of NeuroD Gene Silencing on the Migration and Invasion of Human Pancreatic Cancer Cells PANC-1

The aim of this study is to investigate the influence of Lenti-EGFP-NeuroD-miR, RNAi lentiviral expression vector, on the expression level of NeuroD and migration, and invasion of PANC-1 cell line. PANC-1 cells were cultured and cotransfected with Lenti-EGFP-NeuroD-miR and Lenti-GFP. The infection rate of lentivirus was determined by fluorescence. The interfering effection by the expression of NeuroD mRNA in PANC-1 cells was analyzed by real-time PCR after transfected. Biological behavior of PANC-1 cells transinfected was observed, and the migration and invasion were studied by transwell assay. Intrapancreatic allografts model in nude mice was established to observe the effects of NeuroD on tumorigenesis, tumor growth, and invasion in vivo. The expression of NeuroD mRNA decreased significantly after RNAi lentivirus transinfecting PANC-1 cell. The cell’s migration and invasion ability decreased obviously as soon as down regulate of NeuroD in PANC-1 cells. Comparing with control group, the tumors were smaller in size and the invasiveness was inhibited after 8 weeks intrapancreatic allografts in nude mice. Lenti-EGFP-NeuroD-miR transfected into PANC-1 cells shows a stable, effective, and especial blocking expression of NeuroD in mRNA level. The RNAi of lentiviral vector target NeuroD can reduce the migration and invasion abilities of PANC-1 cells.     

MT1-MMP hemopexin domain exchange with MT4-MMP blocks enzyme maturation and trafficking to the plasma membrane in MCF7 cells

The hemopexin-like domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) enables MT1-MMP to form oligomers that facilitate the activation of pro-matrix metalloproteinase-2 (pro-MMP-2) at the cell surface. To investigate the role of the MT1-MMP hemopexin domain in the trafficking of MT1-MMP to the cell surface we have examined the activity of two MT1-MT4-MMP chimaeras in which the hemopexin domain of MT1-MMP has been replaced with that of human or mouse MT4-MMP. We show that MT1-MMP bearing the hemopexin domain of MT4-MMP was incapable of activating pro-MMP-2 or degrading gelatin in cell based assays. Furthermore, cell surface biotinylation and indirect immunofluorescence show that transiently expressed MT1-MT4-MMP chimaeras failed to reach the plasma membrane and were retained in the endoplasmic reticulum. Functional activity could be restored by replacing the MT4-MMP hemopexin domain with the wild-type MT1-MMP hemopexin domain. Subsequent analysis with an antibody specifically recognising the propeptide of MT1-MMP revealed that the propeptides of the MT1-MT4-MMP chimaeras failed to undergo proper processing. It has previously been suggested that the hemopexin domain of MT4-MMP could exert a regulatory mechanism that prevents MT4-MMP from activating pro-MMP-2. In this report, we demonstrate unambiguously that MT1-MT4-MMP chimaeras do not undergo normal trafficking and are not correctly processed to their fully active forms and, as a consequence, they are unable to activate pro-MMP-2 at the cell surface.     

PTK6 Promotes Cancer Migration and Invasion in Pancreatic Cancer Cells Dependent on ERK Signaling

Protein Tyrosine Kinase 6 (PTK6) is a non-receptor type tyrosine kinase that may be involved in some cancers. However, the biological role and expression status of PTK6 in pancreatic cancer is unknown. Therefore in this study, we evaluated the functional role of PTK6 on pancreatic cancer invasion. Five pancreatic cancer cell lines expressed PTK6 at varying levels. PTK6 expression was also observed in human pancreatic adenocarcinomas. PTK6 suppression by siRNA significantly reduced both cellular migration and invasion (0.59/0.49 fold for BxPC3, 0.61/0.62 for Panc1, 0.42/0.39 for MIAPaCa2, respectively, p<0.05 for each). In contrast, forced overexpression of PTK6 by transfection of a PTK6 expression vector in Panc1 and MIAPaCa2 cells increased cellular migration and invasion (1.57/1.67 fold for Panc1, 1.44/1.57 for MIAPaCa2, respectively, p<0.05). Silencing PTK6 reduced ERK1/2 activation, but not AKT or STAT3 activation, while PTK6 overexpression increased ERK1/2 activation. U0126, a specific inhibitor of ERK1/2, completely abolished the effect of PTK6 overexpression on cellular migration and invasion. These results suggest that PTK6 regulates cellular migration and invasion in pancreatic cancer via ERK signaling. PTK6 may be a novel therapeutic target for pancreatic cancer.