Proteomics discovery of metalloproteinase substrates in the cellular context by iTRAQ labeling reveals a diverse MMP-2 substrate degradome

Elucidation of protease substrate degradomes is essential for understanding the function of proteolytic pathways in the protease web and how proteases regulate cell function. We identified matrix metalloproteinase-2 (MMP-2) cleaved proteins, solubilized pericellular matrix, and shed cellular ectodomains in the cellular context using a new multiplex proteomics approach. Tryptic peptides of intact and cleaved proteins, collected from conditioned culture medium of Mmp2(-/-) fibroblasts expressing low levels of transfected active human MMP-2 at different time points, were amine-labeled with iTRAQ mass tags. Peptide identification and relative quantitation between active and inactive protease transfectants were achieved following tag fragmentation during tandem MS. Known substrates of MMP-2 were identified thereby validating this technique with many novel MMP-2 substrates including the CX(3)CL1 chemokine fractalkine, osteopontin, galectin-1, and HSP90alpha also being identified and biochemically confirmed. In comparison with ICAT-labeling and quantitation, 8-9-fold more proteins and substrates were identified by iTRAQ. "Peptide mapping," the location of multiple peptides identified within a particular protein by iTRAQ in combination with their relative abundance ratios, enabled the domain shed and general location of the cleavage site to be identified in the native cellular substrate. Hence this advance in degradomics cell-based screens for native protein substrates casts new light on the roles for proteases in cell function.     

Extracellular matrix composition and remodeling in human abdominal aortic aneurysms: a proteomics approach

Abdominal aortic aneurysms (AAA) are characterized by pathological remodeling of the aortic extracellular matrix (ECM). However, besides the well-characterized elastolysis and collagenolysis little is known about changes in other ECM proteins. Previous proteomics studies on AAA focused on cellular changes without emphasis on the ECM. In the present study, ECM proteins and their degradation products were selectively extracted from aneurysmal and control aortas using a solubility-based subfractionation methodology and analyzed by gel-liquid chromatography-tandem MS and label-free quantitation. The proteomics analysis revealed novel changes in the ECM of AAA, including increased expression as well as degradation of collagen XII, thrombospondin 2, aortic carboxypeptidase-like protein, periostin, fibronectin and tenascin. Proteomics also confirmed the accumulation of macrophage metalloelastase (MMP-12). Incubation of control aortic tissue with recombinant MMP-12 resulted in the extensive fragmentation of these glycoproteins, most of which are novel substrates of MMP-12. In conclusion, our proteomics methodology allowed the first detailed analysis of the ECM in AAA and identified markers of pathological ECM remodeling related to MMP-12 activity.     

Protease Nexin-1 affects the migration and invasion of C6 glioma cells through the regulation of urokinase Plasminogen Activator and Matrix Metalloproteinase-9/2

Protease Nexin-1 (PN-1) or Serpine2 is a physiological regulator of extracellular proteases as thrombin and urokinase (uPA) in the brain. Besides, PN-1 is also implicated in some human cancers and further identified as a substrate for Matrix Metalloproteinase (MMP)-9, a key enzyme in tumor invasiveness. Our aim was to study the role of PN-1 in the migration and invasive potential of glioma cells, using the rat C6 glioma cell line as stable clones transfected with pAVU6 + 27 vector expressing PN-1 short-hairpin RNA. We find that PN-1 knockdown enhanced the in vitro migration and invasiveness of C6 cells which also showed a strong gelatinolytic activity by in situ zymography. PN-1 silencing did not alter prothrombin whereas increased uPA, MMP-9 and MMP-2 expression levels and gelatinolytic activity in a conditioned medium from stable C6 cells. Selective inhibitors for MMP-9 (Inhibitor I), MMP-2 (Inhibitor III) or exogenous recombinant PN-1 added to the culture medium of C6 silenced cells restored either the migration and invasive ability or gelatinolytic activity thus validating the specificity of PN-1 silencing strategy. Phosphorylation levels of extracellular signal-related kinases (Erk1/2 and p38 MAPK) involved in MMP-9 and MMP-2 signaling were increased in PN-1 silenced cells. This study shows that PN-1 affects glioma cell migration and invasiveness through the regulation of uPA and MMP-9/2 expression levels which contribute to the degradation of extracellular matrix during tumor invasion.     

Protease nexin-2/amyloid beta-protein precursor in blood is a platelet-specific protein

The protease inhibitor, protease nexin-2 (PN-2), is the secreted form of the amyloid beta-protein precursor (APP) which contains the Kunitz protease inhibitor domain. PN-2/APP is an abundant platelet alpha-granule protein which is secreted upon platelet activation. PN-2/APP mRNA is present in cultured endothelial cells and the protein has been detected in plasma. In the present studies we quantitated PN-2/APP in platelets, plasma and several different cell types of the vasculature to identify the repository of the protein in the circulatory system. We report that PN-2/APP is predominantly a platelet protein in the vascular compartment. Lysates of unstimulated umbilical vein endothelial cells, granulocytes or monocytes contained little PN-2/APP based on sensitive functional protease binding and immunoblotting assays. Quantitative immunoblotting studies demonstrated that normal citrated-plasma contains less than or equal to 60 pM PN-2/APP. In contrast, platelets can contribute up to 30 nM PN-2/APP, indicating that they are the major source of the protein in blood.     

Synaptic cell adhesion molecule-2 and collapsin response mediator protein-2 are novel members of the matrix metalloproteinase-9 degradome

The elucidation of entire sets of protease substrates ("proteodegradomes") is important for understanding proteolytic pathways, their networks, and their role in the regulation of cell function. Matrix metalloproteinase-9 (MMP-9) is an extracellularly operating protease that is expressed and released in the brain in response to enhanced neuronal activity. Under physiological conditions, MMP-9 is involved in neuronal plasticity, including long-term potentiation, learning, and memory. This function may be related to its activity at the synapse. Under pathological conditions (e.g., during excitotoxicity, stroke, and traumatic brain injury), when the concentration of glutamate is persistently increased, MMP-9 is detrimental to brain tissue. To assess the MMP-9 degradome, we used synaptoneurosomal fractions isolated from the hippocampus of wildtype and MMP-9 knockout mice. To induce MMP-9 activity, the synaptoneurosomal fractions were treated with 50 μM glutamate for 30 min at 37°C. To investigate MMP-9 targets, two-dimensional fluorescence difference gel electrophoresis was performed. This approach enabled the accurate analysis of differences in protein abundance between samples. The differential spots that contained potential MMP-9 substrates were excised from the gel, and proteins of interest were identified using mass spectrometry. Two novel MMP-9 targets were identified: synaptic cell adhesion molecule-2 and collapsin response mediator protein-2. The MMP-9-driven processing of the newly identified substrates was confirmed by western blot in primary hippocampal neurons after stimulation with either N-methyl-D-aspartate or glutamate or incubation with recombinant autoactivating MMP-9 and use of a specific inhibitor.     

Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediate mitochondrial impairment after ionising radiation

Qualitative proteome profiling of formalin-fixed, paraffin-embedded (FFPE) tissue is advancing the field of clinical proteomics. However, quantitative proteome analysis of FFPE tissue is hampered by the lack of an efficient labelling method. The usage of conventional protein labelling on FFPE tissue has turned out to be inefficient. Classical labelling targets lysine residues that are blocked by the formalin treatment. The aim of this study was to establish a quantitative proteomics analysis of FFPE tissue by combining the label-free approach with optimised protein extraction and separation conditions. As a model system we used FFPE heart tissue of control and exposed C57BL/6 mice after total body irradiation using a gamma ray dose of 3 gray. We identified 32 deregulated proteins (p≤0.05) in irradiated hearts 24h after the exposure. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation. In good agreement with our previous results using fresh-frozen tissue, the analysis indicated radiation-induced alterations in three main biological pathways: respiratory chain, lipid metabolism and pyruvate metabolism. The label-free approach enables the quantitative measurement of radiation-induced alterations in FFPE tissue and facilitates retrospective biomarker identification using clinical archives.     

Thrombin induces tumor invasion through the induction and association of matrix metalloproteinase-9 and beta1-integrin on the cell surface

The procoagulatory serine protease, thrombin, is known to induce invasion and metastasis in various cancers, but the mechanisms by which it promotes tumorigenesis are poorly understood. Because the 92-kDa gelatinase (MMP-9) is a known mediator of tumor cell invasion, we sought to determine whether and how thrombin regulates MMP-9. The thrombin receptor, PAR-1, and MMP-9 are expressed in osteosarcomas, as determined by immunohistochemistry. Stimulation of U2-OS osteosarcoma cells with thrombin and a thrombin receptor-activating peptide induced pro-MMP-9 secretion as well as cell surface-associated pro-MMP-9 expression and proteolytic activity. This was paralleled by an increase in MMP-9 mRNA and MMP-9 promoter activity. Thrombin-induced invasion of U2-OS cells through Matrigel was mediated by the phosphatidylinositol 3-kinase signaling pathway and could be inhibited with an MMP-9 antibody. The stimulation of MMP-9 by thrombin was paralleled by an increase in beta1-integrin mRNA and beta1-integrin expression on the cell surface, which was also mediated by phosphatidylinositol 3-kinase and was required for invasion. Thrombin activation induced and co-localized both beta1-integrin and pro-MMP-9 on the cell membrane, as evidenced by co-immunoprecipitation, confocal microscopy, and a protein binding assay. The thrombin-mediated association of these two proteins, as well as thrombin-mediated invasion of U2-OS cells, could be blocked with a cyclic peptide and with an antibody preventing binding of the MMP-9 hemopexin domain to beta1-integrin. These results suggest that thrombin induces expression and association of beta1-integrin with MMP-9 and that the cell surface localization of the protease by the integrin promotes tumor cell invasion.     

Probing the efficiency of proteolytic events by positional proteomics

Several mass spectrometry-driven techniques allow to map the substrate repertoires and specificities of proteases. These techniques typically yield long lists of protease substrates and processed sites with (potential) physiological relevance, but in order to understand the primary function of a protease, it is important to discern bystander substrates from critical substrates. Because the former are generally processed with lower efficiency, data on the actual substrate cleavage efficiency could assist in categorizing protease substrates. In this study, quantitative mass spectrometry following metabolic proteome labeling (SILAC), combined with the isolation of N-terminal peptides by Combined Fractional Diagonal Chromatography, was used to monitor fluxes in the concentration of protease-generated neo-N-termini. In our experimental setup, a Jurkat cell lysate was treated with the human serine protease granzyme B (hGrB) for three different incubation periods. The extensive list of human granzyme B substrates previously catalogued by N-terminal Combined Fractional Diagonal Chromatography (1) was then used to assign 101 unique hGrB-specific neo-N-termini in 86 proteins. In this way, we were able to define several sites as getting efficiently cleaved in vitro and consequently recognize potential physiologically more relevant substrates. Among them the well-known hGrB substrate Bid was confirmed as being an efficient hGrB substrate next to several other potential regulators of hGrB induced apoptosis such as Bnip2 and Akap-8. Several of our proteomics results were further confirmed by substrate immunoblotting and by using peptide substrates incubated with human granzyme B.     

A catalogue of putative HIV-1 protease host cell substrates

Abstract Processing of human immunodeficiency virus (HIV) proteins by the HIV-1 protease is essential for HIV infectivity. In addition, several studies have revealed cleavage of human proteins by this viral protease during infection; however, no large-scale HIV-1 protease degradomics study has yet been performed. To identify putative host substrates in an unbiased manner and on a proteome-wide scale, we used positional proteomics to identify peptides reporting protein processing by the HIV-1 protease, and a catalogue of over 120 cellular HIV-1 protease substrates processed in vitro was generated. This catalogue includes previously reported substrates as well as recently described interaction partners of HIV-1 proteins. Cleavage site alignments revealed a specificity profile in good correlation with previous studies, even though the ELLE consensus motif was not cleaved efficiently when incorporated into peptide substrates due to subsite cooperativity. Our results are further discussed in the context of HIV-1 infection and the complex substrate recognition by the viral protease.     

MMP-9 Sheds the ��2 Integrin Subunit (CD18) from Macrophages

Activated macrophages are essential effectors of immunity and a rich source of matrix metalloproteinase-9 (MMP-9; gelatinase B). To search for cellular substrates of the enzyme, we subjected wild-type macrophages and macrophages expressing an autoactivating form of pro-MMP-9 (M9A macrophages) to proteomics analysis. Two-dimensional liquid chromatography together with tandem mass spectrometry identified 467 proteins in medium conditioned by M9A and/or wild-type macrophages. Subtractive proteomics identified 18 candidate MMP-9 substrates. Biochemical studies confirmed that two transmembrane proteins, β₂ integrin subunit (CD18) and amyloid protein precursor (APP), were enriched in the medium of M9A macrophages. To identify potential cleavage sites, we synthesized an overlapping library of peptides that spanned 60 residues of the ectodomain and transmembrane domain of β₂ integrin. Active MMP-9 cleaved a single peptide, ECVKGPNVAAIVGGT, at residues corresponding to Ala⁷⁰⁵ and Ile⁷⁰⁶ of the β₂ integrin. Peptides corresponding to this cleavage site were detected by tandem mass spectrometric analysis only in medium from M9A macrophages, strongly supporting the proposal that β₂ integrin is shed by autoactivating MMP-9. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a powerful approach for identifying proteolytic substrates and suggest that MMP-9 plays previously unsuspected roles in the regulation and shedding of β₂ integrin.Matrix metalloproteinases (MMPs),¹ a subfamily of metazincins, are a structurally related group of zinc-dependent proteases (1). They are synthesized in latent form as pro-MMPs, and their prodomain must be removed or modified before they are proteolytically active. Some MMPs are secreted, whereas others are anchored to the cell surface, but their proteolytic activity is thought to be confined locally within the secretory pathway at the cell surface and nearby extracellular space (1–3). Individual MMPs have distinct substrate specificities and act on diverse extracellular and membrane proteins, such as chemokines, cell surface adhesion proteins, and extracellular matrix components. Proteolysis by MMPs plays an important role in a wide variety of normal and pathological processes, such as host defense, inflammation, and tumor progression (1–9).High levels of MMP-9 (gelatinase B) are expressed by activated macrophages (10), which are key effector cells of both innate and acquired immunity. In addition to having homeostatic functions, MMP-9 secreted by macrophages has been implicated in aneurysm formation, tumor progression, and disruption of atherosclerotic plaques (8, 9, 11, 12). Although the pathogenesis of those processes is generally thought to involve inappropriate degradation of extracellular matrix proteins, it has become increasingly clear that MMPs cleave a number of diverse substrates to mediate their varied functions (3, 13). Because MMP-9 can accumulate on the cell surface (14), it is likely to act on membrane proteins.To understand the specific roles of individual MMPs in inflammatory and immune responses, it is critical to identify their physiological substrates (3, 15–17). Most studies have focused on identifying substrates by their ability to be cleaved in defined in vitro reactions (18, 19), but this approach is biased in two ways. First, the candidate substrate must be selected a priori. Second, in vitro reactions fail to account for the complexity of the pericellular environment. Another method is to identify sequences in synthetic peptides that MMPs can cleave (20, 21). However, individual MMPs cleave different proteins at a variety of sites rather than at a consensus site. Moreover MMPs often interact with substrates through domains remote from the active site (exosites) (22), and exosites of MMP-2 have been used in a yeast two-hybrid system to trap candidate substrates (23). However, some substrates may bind weakly or not at all to exosites, limiting the utility of this approach for global substrate screening.An emerging strategy for finding MMP substrates is to conduct an unbiased, global search by coupling gel electrophoresis or liquid chromatography with MS-based protein identification. For example, two-dimensional (2D) gel electrophoresis (24) and derivatization of cysteine-containing peptides with an isotope affinity tag (25) have identified candidate substrates for membrane type-1 MMP (MT1-MMP) in plasma and cultured cells. Quantitative approaches using 2D difference gel electrophoresis have identified potential substrates of MMP-2 and MMP-9 in bronchoalveolar lavage fluid (26) and of MMP-9 and the related metalloproteinases ADAM-10 and ADAM-17 in cancer cells (27, 28). Lectin affinity chromatography detected glycosylated proteins that were selectively enriched in medium from a monocyte cell line expressing ADAM-17 and in phorbol ester-stimulated monocytes (16). Recently iTRAQ (isobaric tags for relative and absolute quantitation) labeling was used to identify substrates of MMP-2 (29). It is important to note, however, that proteases can affect protein abundance by pathways not involving proteolysis. Thus, an important limitation of many of these studies is that they fail to provide evidence that proteins with altered abundance in cells expressing a protease are direct substrates for proteolytic cleavage.In the current studies, we used subtractive proteomics to identify proteins enriched in the medium of a macrophage cell line. Subtractive proteomics compares two or more proteomes to identify proteins that are specifically enriched or depleted under certain conditions (30, 31). Our biochemical studies confirmed that two integral membrane proteins, amyloid precursor protein (APP) and the β₂ integrin subunit (CD18), were shed by macrophages expressing autoactivating MMP-9. We next used a peptide substrate mapping strategy to identify potential MMP-9 cleavage sites in β₂ integrin subunit. Targeted MS/MS analysis demonstrated that β₂ integrin subunit peptides with the same cleavage site were detected only in the medium of macrophages expressing autoactivating MMP-9, providing strong evidence that β₂ integrin is a direct substrate for proteolysis. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a robust, high throughput technique for identifying cellular substrates that are proteolytically shed from macrophages.     

Monoclonal antibodies to protease nexin 1 that differentially block its inhibition of target proteases

Protease nexin 1 (PN-1) is a protease inhibitor secreted by cultured fibroblasts that forms complexes with certain serine proteases; the complexes bind back to the cells and are internalized and degraded. In the present studies, a panel of PN-1 monoclonal antibodies (mAbs) was isolated; none showed detectable cross-reactivity with four related plasma protease inhibitors. Four purified mAbs (mAbp1, mAbp6, mAbp9, and mAbp18) were tested for their ability to block the formation of complexes between PN-1 and target proteases. mAbp1, as well as a rabbit polyclonal anti-PN-1 IgG preparation, did not block formation of 125I-thrombin-PN-1 complexes. mAbp6, mAbp9, and mAbp18 blocked the formation of 125I-thrombin-PN-1 and 125I-urokinase-PN-1 complexes at stoichiometric concentrations of mAb and PN-1. Studies on their ability to block formation of 125I-trypsin-PN-1 complexes showed that mAbp18 also blocked this reaction at stoichiometric concentrations with PN-1 whereas mAbp6 and mAbp9 blocked less effectively. Thus, mAbp18 appears to bind at or close to the reactive center of PN-1. The blocking mAbs should be useful in studies to probe physiological functions of PN-1.     

蝎毒多肽提取物抑制DU- 145 细胞 COX- 2 和MMP- 9 表达的研究

目的：研究蝎毒多肽提取物（peptide extract from scorpion venom,PESV）对雄激素非依赖性人前列腺癌细胞株DU-145COX-2和MMP-9表达的影响,进一步探讨其抗血管生成的分子机制,为抗前列腺癌骨转移提供有效的治疗手段。方法：采用免疫组只化学方法检测PESV对COX-2、MMP-9蛋白表达的影响,应用RT-PCR检测PESV对MMP-9在mRNA水平表达的影响。结果：蝎毒多肽提取物（40μg／mL）作用于前列腺癌细胞后,COX-2、MMP-9蛋白表达水平明显下调（P〈0．05）,进一步检测发现MMP-9在mRNA水平亦明显下降（P〈0．05）。结论：蝎毒多肽提取物（PESV）通过抑制前列腺癌细胞血管生成因子COX-2的表达而发挥其抗血管生成作用,具有临床应用价值。     

Sodium hydrogen exchanger and phospholipase D are required for alpha1-adrenergic receptor stimulation of metalloproteinase-9 and cellular invasion in CCL39 fibroblasts

Matrix metalloproteinase 9 (MMP-9) plays a critical role in digesting the extracellular matrix and has a vital function in tumor metastasis and invasion; this protease activity is significantly increased in non-small cell lung cancers. The sodium hydrogen exchanger isoform 1 (NHE1) functions as a focal point for signal coordination and cytoskeletal reorganization. NHE1 is thought to play a central role in establishing signaling components at the leading edge of a migrating cell. Therefore, we studied the relationship between NHE1 and MMP-9 activity in Chinese hamster lung fibroblasts (CCL39) stimulated with phenylephrine (PE). We show that PE increases MMP-9 gelatinolytic activity in CCL39 cells. The inhibition of phospholipase D (PLD) signaling abrogated PE-induced MMP-9 activity. The role of PLD as an essential signaling intermediate was confirmed when the addition of permeable phosphatidic acid increased MMP-9 activity in the same cells. PE-induced invasion was increased 1.9-fold over controls and the PE response was lost when 1-butanol was used to block PLD signaling. Cells pre-treated with the NHE1 inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) prior to PE addition resulted in a notable decrease in MMP-9 activation and cell invasion as compared to untreated PE-stimulated cells. CCL39 NHE1 null cells demonstrated no increase in MMP-9 protease activity or cell invasion in response to PE treatment. Reconstitution of NHE1 expression recovered the PE-induced activation of protease activity and cell invasion. MMP-9 processing was altered in cells expressing a proton transport defective NHE1 but retained the ability to respond to PE. Conversely, cells expressing an ezrin, radixin, moesin (ERM)-binding deficient NHE1 had a lower MMP-9 activity and the protease did not respond to PE addition. Parallel studies on NCI-H358 non-small cell lung cancer (NSCL) cells showed that PE stimulated both MMP-9 activity and cell invasion in an NHE1 dependent manner. This work describes for the first time a PE-induced relationship between NHE1 and MMP-9 and a new potential mechanism by which NHE1 could promote tumor formation and metastasis.     

Protease nexin-1 complexes and inhibits T cell serine proteinase-1

The T cell serine proteinase-1 (TSP-1) which most probably is involved in cell killing by cytotoxic T cells is inhibited by protease nexin-1 (PN-1), an extravascular serine protease inhibitor. The inhibition is irreversible and correlates with formation of SDS-stable complexes between the two proteins. Two distinct species of complexes (91 and 122 kDa) are observed upon SDS-PAGE analysis of the reacted proteins, indicating that PN-1 is capable of complexing and inhibiting both subunits of the homodimeric TSP-1 molecule. Heparin (2 micrograms/ml) increases the association rate constant from 4.2 x 10(4) M-1 sec-1 to 4.8 x 10(5) M-1 sec-1. These observations suggest that PN-1 may function as a major extravascular inhibitor of TSP-1 released from cytotoxic T lymphocytes.     

Protease nexin-1 expression is altered in human breast cancer

Background  

Urokinase-type Plasminogen Activator (uPA), a serine protease, plays a pivotal role in human breast cancer metastasis by mediating the degradation of extracellular matrix proteins and promoting cell motility. In more advanced breast cancers, uPA activity is significantly up regulated and serves as a prognostic indicator of poor patient outcome. Classically, regulation of uPA activity, especially in breast cancers, is thought to be mediated by Type 1 Plasminogen Activator Inhibitor (PAI-1). However, we have recently found that a lesser known natural inhibitor of uPA, Protease Nexin 1 (PN-1), is expressed in normal human mammary tissue. Based on this observation, we investigated if PN-1 is also expressed in human breast cancers where it may contribute to the regulation of uPA and participate in the development of a metastatic phenotype.     

Inactivation of protease nexin-1 by xanthine oxidase-derived free radicals

Neuronal viability is affected by reactive oxygen species. Lipid peroxidation is often defined as a major reason for cellular breakdown. Additionally, certain indispensable proteins are possible targets for excessively formed reactive oxygen species. Evidence is given here that protease nexin-1(PN-1), an endogenous thrombin inhibitor and neurite outgrowth promoter, is inactivated by xanthine oxidase-derived free radicals. Varying protection by superoxide dismutase and catalase was observed, depending on the reaction conditions. The water-soluble a-tocopherol analogues MDL 74,406 (R(+)-3,4-dihydro-6-hydroxy-N,N,N- 2,5,7,8-heptamethyl-2H-1-benzopyran-2-ethanaminium 4-methylbenzenesulfonate), MDL 74,180DA (2,3- dihydro-2,2,4,6,7-pentamethyl-3-(4-methyl-piperazino)-1-benzofuran-5-ol dihydro-chloride) and trolox also protected PN-1. Neurodegeneration may be triggered by oxidative inactivation of protease inhibitors such as PN-1. Protection of PN-1 in Alzheimer's or Parkinson's diseases, could be a possible target for a therapeutic function of antioxidants in these diseases.     

Ectopic expression of CC chemokine receptor 7 promotes prostate cancer cells metastasis via Notch1 signaling

There currently exists no satisfactory treatment for patients with prostate cancer with local evolution and distant metastasis. Previous studies have confirmed the importance of CC chemokine receptor 7 (CCR7) in the invasion and metastasis of prostate cancer. And increasing evidence prove that Notch1 can play diametrically opposite roles in the development and progression of different tumors. To demonstrate the correlation between CCR7 and Notch1, PC-3 cells were transfected with pcDNA3.1-CCR7 or CCR7 si-RNA, respectively. Then Western blot analysis was used to detect the expressions of Notch1, ERK, P38, JNK, NF-κB, MMP-9, and epithelial-mesenchymal transition (EMT)-related proteins. Moreover, matrigel invasion assays were performed to assess the migratory and invasive activities of PC-3 cells. PcDNA3.1-CCR7 increased the expression of Notch1, phospho-MAPK, phospho-P65, MMP-9, N-cadherin, and Snail in PC-3 cells, but decreased the expression of E-cadherin. PcDNA3.1-CCR7 also promoted the migration and invasion of PC-3 cells. However, CCR7 si-RNA reversed the effect of pcDNA3.1-CCR7 in PC-3 cells. And MAPK and NF-κB pathway inhibitors were used to testify that activation of Notch1 induces EMT through MAPK and NF-κB pathway. All these results indicate that upregulation of Notch1 by CCR7 can accelerate the evolution of EMT and develop the invasion and metastasis in prostate cancer cells by activating MAPK and NF-κB signaling pathways in prostate cancer cells, which provides a new molecular evidence for targeted therapy in metastatic prostate cancer.