Membrane Type 1-MMP Expression Research Articles

Discretionary Transduction of MMP-Sensitized Tousled in Head and Neck Cancer

Oral radiotoxicity is often a limiting factor in cancer treatment. Previously, we demonstrated that transfer of cell-permeable, TAT-fusion Tousled-like kinase 1B (TLK1B) protein in salivary glands effectively mitigates radiation-induced salivary dysfunction. However, similar to most radioprotectors, TLK1B can carry the risk of limiting cancer treatment efficacy. The central goal of the study was, therefore, to reengineer TLK1B as a selective radioprotector of normal cells. Degradation of the extracellular matrix by proteases such as matrix metalloproteinases (MMPs) is a hallmark of aggressive tumors. Increased expression of membrane type 1-MMP (MT1-MMP; also called MMP14) is observed in a variety of cancers including head and neck squamous cell carcinoma (HNSCC). To limit TLK1B transduction to normal cells, we rendered the protein susceptible to MT1-MMP cleavage on the premise that high expression of MT1-MMP on the cell surface of HNSCC will suppress TLK1B internalization. Two optimal MT1-MMP-sensitive sequences (MS) were identified that when incorporated in TAT-TLK1B excluded its cellular entry in HNSCC, SCC40, but not immortalized salivary acinar cells, NS-SV-AC. Importantly, administration of MS-harboring TAT-TLK1B did not affect the sensitivity of tumors to radiation in a nude mouse xenograft tumor model. We conclude that a MMP-sensitive TLK1B can be an attractive therapeutic to allay salivary radiotoxicity without compromising cancer treatment efficacy.

Effects of loss of occlusal contact on the expression of matrix metalloproteinase-2, membrane type 1-MMP, tissue inhibitor of the MMP-2, eruption rate, organization and resistance of collagen fibers of the rat incisor periodontal ligament.

This study investigated the effect of occlusal contact loss (induced by tooth shortening), on matrix metalloproteinase (MMP)-2, membrane type 1-MMP (MT1-MMP) and tissue inhibitor of the MMP-2 (TIMP-2) expressions in the periodontal ligament of the rat incisor, as well as eruption rate, resistance and collagen organization. Male Wistar rats were distributed into a control group, denominated normofunctional group, whose lower teeth underwent a normal eruption process; and a hypofunctional group, whose lower left incisor teeth were shortened every 2days during 14days. Parameters were evaluated on the first, seventh and 14th days and the eruption rate was determined according to the size of the incisor during the eruption process. MMP-2 activity was determined by zymography and the expressions of the MT1-MMP and TIMP-2 proteins were quantitated by western blot. Collagen protein organization, as indicated by the birefringence of the periodontal ligament, was analyzed under polarized light and the periodontal ligament's resistance was determined from the load necessary to inject the incisor into its alveolar space, before extraction. Loss of occlusal contact, in rats submitted to hypofunctional eruption, increased MMP-2 activity and eruption rate, but decreased MT1-MMP and TIMP-2 expression and disrupted collagen organization in the periodontal ligament, consequently reducing periodontal ligament resistance. We conclude that, after incisor eruption, occlusal contact may be an important factor for regulating the remodeling and the physiological resistance of the periodontal ligament against the continuous eruption process observed in rat incisors.

Differential spatio-temporal regulation of MMPs in the 5xFAD mouse model of Alzheimer's disease: evidence for a pro-amyloidogenic role of MT1-MMP.

Matrix metalloproteinases (MMPs) are pleiotropic endopeptidases involved in a variety of neurodegenerative/neuroinflammatory processes through their interactions with a large number of substrates. Among those, the amyloid precursor protein (APP) and the beta amyloid peptide (Aβ) are largely associated with the development of Alzheimer’s disease (AD). However, the regulation and potential contribution of MMPs to AD remains unclear. In this study, we investigated the evolution of the expression of MMP-2, MMP-9, and membrane-type 1-MMP (MT1-MMP) in the hippocampus at different stages of the pathology (asymptomatic, prodromal-like and symptomatic) in the 5xFAD transgenic mouse AD model. In parallel we also followed the expression of functionally associated factors. Overall, the expression of MMP-2, MMP-9, and MT1-MMP was upregulated concomitantly with the tissue inhibitor of MMPs-1 (TIMP-1) and several markers of inflammatory/glial response. The three MMPs exhibited age- and cell-dependent upregulation of their expression, with MMP-2 and MMP-9 being primarily located to astrocytes, and MT1-MMP to neurons. MMP-9 and MT1-MMP were also prominently present in amyloid plaques. The levels of active MT1-MMP were highly upregulated in membrane-enriched fractions of hippocampus at 6 months of age (symptomatic phase), when the levels of APP, its metabolites APP C-terminal fragments (CTFs), and Aβ trimers were the highest. Overexpression of MT1-MMP in HEK cells carrying the human APP Swedish mutation (HEKswe) strongly increased β-secretase derived C-terminal APP fragment (C99) and Aβ levels, whereas MMP-2 overexpression nearly abolished Aβ production without affecting C99. Our data consolidate the emerging idea of a regulatory interplay between MMPs and the APP/Aβ system, and demonstrate for the first time the pro-amyloidogenic features of MT1-MMP. Further investigation will be justified to evaluate this MMP as a novel potential therapeutic target in AD.

Effect on the Expression of MMP-2, MT-MMP in Laryngeal Carcinoma Hep-2 Cell Line by Antisense Glucose Transporter-1

Glucose transporter protein-1 (Glut-1) is correlated with biological behaviors of malignant tumors. However, there was no evidence that overexpression of Glut-1 mechanistically lead to invasion or metastasis of cancer cells. We hypothesized that Glut-1 regulates the expression of membrane type 1-MMP (MT1-MMP) and matrix metalloproteinase-2 (MMP-2). Analysis of the expression of Glut-1, MMP-2, β-actin, and MT1-MMP was performed using RT-PCR. Expression of Glut-1 protein, MMP-2, and MT1-MMP was detected by Western blotting. At mRNA and protein levels, Glut-1 and MMP-2 were co-expressed in the Hep-2 laryngeal carcinoma cell line. After transfection, Glut-1 antisense oligodeoxynucleotide (AS-ODN) decreased the expression of MMP-2 mRNA and protein as well as Glut-1 mRNA and protein. Glut-1 AS-ODN also decreased the expression of MT1-MMP mRNA. Co-expression of Glut-1 and MMP-2 in Hep-2 laryngeal carcinoma cells and Glut-1 may regulate MMP-2 and MT1-MMP expression.

Clinical relevance of cyclooxygenase-2 and matrix metalloproteinases (MMP-2 and MT1-MMP) in human breast cancer tissue

Breast cancer (BC) is the most common neoplasm among women in most developed countries, including Egypt. Elevated levels of certain proteins in human BC are associated with unfavorable prognosis and progressive stages of the disease. The aim of our study was to evaluate the protein expression profile and prognostic significance of cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP-2), MMP-9 and membrane type 1-MMP (MT1-MMP) and their interaction in operable BC patients. The protein expression of COX-2, MMP-2 and MT1-MMP were evaluated by western blot technique, whereas enzymatic activity of MMP-2 and MMP-9 was determined by zymography in 47 breast cancer patients as well as normal adjacent tissues. Also, the correlation between these proteins and age, tumor size, LN stage, TNM stage, estrogen receptor, progesterone receptor, disease-free survival, and overall survival (OS) has been investigated. As compared to adjacent normal tissues, COX-2, MMP-2 and MT1-MMP were over-expressed in 43, 64, and 60 % of tumor tissues, respectively. In the same pattern, the activity of MMP-2 (62 %) and MMP-9 (45 %) was elevated in BC tissues. Multivariate analysis showed a positive correlation between the protein expression of COX-2, MMP-2, and MT1-MMP and the activity of MMP-2 and MMP-9 in BC patients. However, the enzymatic activity showed no correlation with clinicopathological features. This study confirms the preclinical evidence that COX-2 increased the expression of MT1-MMP, which in turn activates MMP-2. The lack of correlation with clinicopathological features, OS or disease-free survival ascertains the complexity of tumor progression and metastasis with many pro- and counter regulatory factors.

Combination of MEK and SRC inhibition suppresses melanoma cell growth and invasion

The RAS-RAF-MEK-ERK pathway is deregulated in over 90% of malignant melanomas and targeting MEK as central kinase of this pathway is currently tested in clinical trials. However, dose-limiting side effects are observed, and MEK inhibitors that sufficiently reduce ERK activation in patients show a low clinical response. Apart from dose-limitations, a reason for the low response to MEK targeting drugs is thought to be the up-regulation of counteracting signalling cascades as a direct response to MEK inhibition. Therefore, understanding the biology of melanoma cells and the effects of MEK inhibition on these cells will help to identify new combinatorial approaches that are more potent and allow for lower concentrations of drug being used. We have discovered that in melanoma cells MEK inhibition by selumetinib (AZD6244, ARRY-142886) or PD184352 while efficiently suppressing proliferation stimulates increased invasiveness. Inhibition of MEK suppresses actin-cortex contraction and increases integrin-mediated adhesion. Most importantly, and surprisingly MEK inhibition results in a significant increase in MMP-2 and MT1-MMP expression. All together MEK inhibition in melanoma cells induces a ‘mesenchymal’ phenotype that is characterised by protease driven invasion. This mode of invasion is dependent on integrin-mediated adhesion, and because SRC kinases are main regulators of this process, the SRC kinase inhibitor saracatinib (AZD0530) completely abolished the MEK inhibitor induced invasion. Moreover, the combination of saracatinib and selumetinib effectively suppressed the growth and invasion of melanoma cells in a 3D environment, suggesting that combined inhibition of MEK and SRC is a promising approach to improve the efficacy of targeting the ERK/MAP kinase pathway in melanoma.

Kuguacin J, a triterpeniod from Momordica charantia leaf, modulates the progression of androgen-independent human prostate cancer cell line, PC3

In this study, we focused on the in vitro effects of Kuguacin J (KuJ), a purified component of bitter melon (Momordica charantia) leaf extract (BMLE), on the androgen-independent human prostate cancer cell line PC3 and the in vivo effect of dietary BMLE on prostate carcinogenesis using a PC3-xenograph model. KuJ exerted a strong growth-inhibitory effect on PC3 cells. Growth inhibition was mainly through G1-arrest: KuJ markedly decreased the levels of cyclins (D1 and E), cyclin-dependent kinases (Cdk2 and Cdk4) and proliferating cell nuclear antigen. Interestingly, KuJ also dramatically decreased the levels of survivin expressed by PC3 cells. In addition, KuJ exerted anti-invasive effects on PC3 cells, significantly inhibiting migration and invasion: KuJ inhibited secretion of the active forms of MMP-2, MMP-9 and uPA by PC3 cells. In addition, KuJ treatment significantly decreased the expression of membrane type 1-MMP (MT1-MMP) by PC3 cells. In vivo, 1% and 5% BMLE in the diet resulted in 63% and 57% inhibition of PC3 xenograft growth without adverse effect on host body weight. Our results suggest that KuJ is a promising new candidate chemopreventive and chemotherapeutic agent for prostate cancer.

MT1-MMP Plays a Critical Role In the Modulation of Hematopoiesis.

AbstractAbstract 3851Specific niches, in which hematopoietic stem cells (HSCs) reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. In the recent study, activation of the fibrinolytic pathway via matrix metalloproteinases (MMPs) including MMP-9 resulted in the release of kit ligand (KitL) in the BM niche. Membrane type 1-MMP (MT1-MMP) can activate MMP-9 in the process of mutual activation of MMP. It has already known that BM myeloablation with irradiation or anti-cancer drug induces MT1-MMP expression, but the role of MT1-MMP in hematopoiesis is not well-understood. We examined MT1-MMP deficient mice (MT1-MMP-/-) 12 days after birth. MT1-MMP-/- were suffering from pancytopenia, and are reduced numbers of bone marrow mononuclear cells (BMMCs), splenocytes and number of hematopoietic progenitor cells in the BM although the number of HSCs in BM showed no significant difference. BM cytospins from MT1-MMP-/- mice showed mild erythropoietic disturbance and severer impairment of myelopoiesis. Interestingly, a powerful hematopoietic factor, Kit-ligand (KitL) levels were significantly lower in MT1-MMP-/- mice than wild type. MT1-MMP knockdown by shRNA or/and siRNA impaired KitL expression and secretion in transfected stroma cells compared to Mock controls, demonstrating that reduced KitL plasma levels were due to impaired release/production and not due to reduced numbers of stromal cells in MT1-MMP-/-. Similarly, impaired proliferation and differentiation of MT1-MMP-/- BMMCs in vitro could be restored by exogenous sKitL. Others and we reported that BM ablation induces the production of stromal cell-derived factor-1 (SDF-1; CXCL12), which plays a key role in stem cell homing and B-cell lymphopoiesis. Reduced SDF-1 expression was observed in BMMCs of MT1-MMP-/- mice and genetic knockdown of MT1-MMP resulted in lower SDF-1 expression both on a transcriptional and protein level. BMMCs of MT1-MMP-/- showed a decrease in the percentage of mature B cells compare to controls. Knocking down of MT1-MMP in stromal cell reduced the number of adherent hematopoietic cells, but addition of rec. SDF-1 could reverse the phenotype. These results suggested stromal-derived MT1-MMP was functionally important to maintain HSC function in long-term cultures of WT HSCs. Thus, MT1-MMP is a critical modulator of hematopoiesis, as it alters the growth factor output of niche cells. Disclosures:No relevant conflicts of interest to declare.

Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg(98) and Arg(101) residues, whereas plasmin only cleaved the propeptide downstream of Arg(101). Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn(109)-Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation.

MT1-MMP and RECK are involved in human CD34+ progenitor cell retention, egress, and mobilization

The mechanisms governing hematopoietic progenitor cell mobilization are not fully understood. We report higher membrane type 1-MMP (MT1-MMP) and lower expression of the MT1-MMP inhibitor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK), on isolated circulating human CD34+ progenitor cells compared with immature BM cells. The expression of MT1-MMP correlated with clinical mobilization of CD34+ cells in healthy donors and patients with lymphoid malignancies. Treatment with G-CSF further increased MT1-MMP and decreased RECK expression in human and murine hematopoietic cells in a PI3K/Akt-dependent manner, resulting in elevated MT1-MMP activity. Blocking MT1-MMP function by Abs or siRNAs impaired chemotaxis and homing of G-CSF-mobilized human CD34+ progenitors. The mobilization of immature and maturing human progenitors in chimeric NOD/SCID mice by G-CSF was inhibited by anti-MT1-MMP treatment, while RECK neutralization promoted motility and egress of BM CD34+ cells. BM c-kit+ cells from MT1-MMP-deficient mice also exhibited inferior chemotaxis, reduced homing and engraftment capacities, and impaired G-CSF-induced mobilization in murine chimeras. Membranal CD44 cleavage by MT1-MMP was enhanced following G-CSF treatment, reducing CD34+ cell adhesion. Accordingly, CD44-deficient mice had a higher frequency of circulating progenitors. Our results reveal that the motility, adhesion, homing, and mobilization of human hematopoietic progenitor cells are regulated in a cell-autonomous manner by dynamic and opposite changes in MT1-MMP and RECK expression.

Estrogen Deficiency-Induced Alterations of Vascular MMP-2, MT1-MMP, and TIMP-2 in Ovariectomized Rats

Matrix metalloproteinases (MMPs) activity may modulate hypertension-related accumulation of extracellular matrix (ECM) in arteries. We tested whether estrogen deficiency induces alterations of vascular collagen, MMP-2, membrane-type 1-MMP (MT1-MMP), or tissue inhibitor of metalloproteinases-2 (TIMP-2) expression in ovariectomized rats, which may be associated with postmenopausal hypertension. Estrogen deficiency was induced by ovariectomy (Ovx) in female rats. Time-course changes of aortic MMPs protein expression were evaluated. Treatment with tempol or aminoguanidine was used to examine the role of oxidative stress and nitric oxide (NO) on these changes. The level of the active-form MMP-2 was markedly reduced during 1-4 weeks after Ovx, with a significant increase in collagen accumulation and increased MT1-MMP expression. Although active-form MMP-2 and collagen progressively returned to normal levels, the markedly increased collagen deposition appeared again at 8 weeks and persisted until 12 weeks, followed by induction of MMP-2 and MT1-MMP at 12 weeks. The TIMP-2 level reduced for 2 weeks after Ovx, but soon returned to normal. Treatment with 17beta-estradiol (E(2)), tempol, or aminoguanidine for 6 weeks prevented Ovx-induced blood pressure elevation and apparently reversed the MMPs changes. In an initial period, E(2) deficiency induces a reduction of active-form MMP-2 leading to collagen accumulation, and induction of MT1-MMP, which may be a compensatory response to degrade collagen. At a latter stage, the concurrent elevation of active-form MMP-2 and MT1-MMP expression may be adaptive responses to regulate ECM composition in the vascular wall. Oxidative stress and NO contribute to activity modulation of vascular MMPs in Ovx rats.

Regulation of vascular smooth muscle cell expression and function of matrix metalloproteinases is mediated by estrogen and progesterone exposure

Postmenopausal women receiving hormone replacement therapy (HRT) have been reported to have more adverse outcomes after vascular reconstructions, including increased intimal hyperplasia development and bypass graft failure. HRT may be affecting the pathway contributing to intimal hyperplasia. An important component of this pathway involves matrix metalloproteinases (MMPs), implicated in vascular remodeling due to their ability to degrade components of the extracellular matrix. We hypothesize that estrogen (Est) and progesterone (Prog) upregulate the MMP pathway in vascular smooth muscle cells (VSMCs) thereby increasing MMP activity and function. VSMCs were incubated with Est (5 ng/mL), Prog (50 ng/mL), Est + Prog combination (Est/Prog), and/or doxycycline (40 microg/mL; Doxy). Using reverse transcriptase polymerase chain reaction (RT-PCR) analysis we have previously shown membrane type 1-MMP (MT1-MMP) messenger ribonucleic acid (mRNA) levels are significantly increased by Est. Here, Western blot analyses indicated MT1-MMP and MMP-2 protein levels, not tissue inhibitor of MMP-2 (TIMP-2), were increased in response to Est and Est/Prog (P < .05 vs control). In-gel zymography revealed that Est and Est/Prog resulted in increased MMP-2 activity (hormone groups, P < .05 vs control) with no significant difference among the hormone groups. VSMC migration was increased by 45 +/- 14% in response to Est (P < .05 vs control), as measured using a modified Boyden chamber assay. Doxycycline significantly inhibited basal and Est/Prog-stimulated increases in MMP-2 activity (P < .05 vs control; P < .05 vs hormone groups), and partially blocked basal and hormonally stimulated migration (P < .05 vs control and Est). Estrogen and progesterone affects the MMP pathway by increasing MMP-2 enzymatic activity, possibly via the upregulation of MT1-MMP expression without a corresponding increase in TIMP expression. This increased collagenase activity increases VSMC motility and their ability to migrate through a collagen type IV lattice. Est/Prog upregulation of MT1-MMP may contribute to the adverse effect of HRT on vascular interventions.

Antitumor activity of a membrane lytic peptide cyclized with a linker sensitive to membrane type 1-matrix metalloproteinase

Membrane lytic peptides are a novel class of anticancer agents that have the potential to overcome drug resistance. The limited selectivity against cancer cells, however, presents a major hurdle for the application. We aim to exploit the proteolytic activity of tumor-associated matrix metalloproteinases (MMP) to mediate the cytotoxicity of these peptides. We designed a membrane lytic peptide cyclized with a linker cleavable by membrane type 1-MMP (MT1-MMP). We showed that the cyclic peptide could be restored to the linear state on MT1-MMP digestion, and it preferentially killed MMP-overexpressing cells above a threshold concentration. Circular dichroism indicated that cyclization resulted in a more rigid structure, making it more difficult for the lytic peptide to transit from random coil to alpha-helix in a membrane-mimicking environment. Selective membrane activity of the cyclic peptide was shown by comparing cytotoxicity results on RBC and two human breast cancer cell lines of different malignancy and MT1-MMP expression: highly invasive MDA-MB-435 and noninvasive MCF-7. Above a concentration of 5 micromol/L, suppressed activity to MCF-7 and RBC was observed, whereas the toxicity against MDA-MB-435 was maintained. MMP inhibition experiments further showed that the membrane-lysing activity was enzyme dependent.

Expression of E1AF, an ets-oncogene transcription factor, highly correlates with malignant phenotype of malignant melanoma through up-regulation of the membrane-type-1 matrix metalloproteinase gene

Matrix metalloproteinase (MMP) is closely involved in the degradation of extracellular matrix and confers invasive and metastatic potential to malignant tumors. MMP-2 is a type-IV collagenase secreted as a proenzyme that is activated on the surface of the tumor cell by membrane-type 1-MMP (MT1-MMP). MT1-MMP plays a critical role during tumor progression and metastasis. We investigated the expression levels of E1AF and MT1-MMP in malignant melanoma cell lines and specimens from patients in order to clarify the mechanisms responsible for the invasion and metastasis of malignant melanoma. High levels of E1AF and MT1-MMP mRNA expression were observed in melanoma cells by Northern blotting and real-time PCR. The expression level was highly correlated with an invasive potential determined by an in vitro invasion assay. The down-regulation of MT1-MMP was identified when E1AF was knocked down by RNA interference. These results suggest that E1AF plays a crucial role in the invasion and metastasis of malignant melanoma through up-regulating the MT1-MMP expression.

Expression and distribution of MMPs and TIMPs in human uveal melanoma

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are involved in tumour invasion, metastasis and angiogenesis, and have been implicated as progression markers in uveal melanoma, although their topographical expression has not been fully described. In this study we compared the distribution and specificity of several classes of MMPs (MMP-1, -2, -9, -19, and MT1-MMP) and physiological MMP inhibitors (TIMP-2 and -3) in different regions of the tumour microenvironment and adjacent choroid in a series of primary uveal melanomas. Paraffin sections of untreated uveal melanomas ( n = 18, 3/18 spindle; 11/18 mixed, and 4/18 epithelioid) were examined for MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MT1-MMP (membrane-type 1-MMP), MMP-19, TIMP-2 and TIMP-3 (tissue inhibitors of MMPs), using indirect peroxidase immunohistochemistry. The distribution and intensity of immunolabelling was graded semi-quantitatively (0–3) by 2 independent observers. Non-parametric analyses were used to test for associations between tumour cell type, and the average grade of MMP or TIMP expression. Immunostaining for MMP-1, -9, -19 and MT1-MMP was ≥Grade 2 in more than 70% of specimens, and a heterogeneous pattern of MMP-1, -9, MT1-MMP and TIMP-3 expression was observed. At the tumour-scleral interface (TSI), melanoma cells had a flattened morphology and a much reduced MMP and TIMP expression, with a high expression in tumour areas adjacent to the TSI. Tumour vasculature and stromal cells strongly expressed MMP-2. We also observed heterogeneous immunostaining of the vasculature by MMP-1, -9, MT1-MMP and TIMP-2 antibodies, and of the extravascular matrix by MMP-9 antibody. The distinct immunostaining patterns observed for MMPs and TIMPs within uveal melanoma are consistent with their involvement in tumour growth and angiogenesis. In particular, the heterogeneous expression within regions of the tumours, and the localized expression in vasculature and stromal cells emphasises the importance of the tumour microenvironment in the pathogenesis of uveal melanoma (and other tumours).

Inhibition of hepatocellular carcinoma invasion by suppression of claudin-10 in HLE cells

Previously, we showed that down-regulation of claudin-10 (CLDN-10) in hepatocellular carcinoma is associated with prolonged disease-free survival after curative surgery. Claudins are important tight junction components. Increasing evidence shows that claudins are involved in cancer progression but each member of claudins is specifically expressed in a variety of malignancies. The biological role of CLDN-10 in hepatocellular carcinoma is unexplored. In the current study, we investigated the CLDN-10 function in two different hepatocellular carcinoma cell lines by in vitro assays with the CLDN-10 overexpression and small interfering RNA-mediated knockdown transfectants. We observed that overexpression of CLDN-10 conferred malignant phenotypes to hepatocellular carcinoma cells, Hep3B, which lack CLDN-10 expression, by promoting cancer cell survival, motility, and invasiveness. More importantly, matrix metalloproteinase 2 (MMP2) was up-regulated. Increase in mRNA transcription and protein expression of membrane type 1-MMP (MT1-MMP) was also observed in the CLDN-10 transfectants, where MT1-MMP was a protease shown to promote intrahepatic metastasis in hepatocellular carcinoma in our earlier study. In addition, CLDN-1, CLDN-2, and CLDN-4 was up-regulated in CLDN-10 overexpression transfectants, indicating that the expression of CLDN-10 in cancer cells might affect the expression levels of its family members. On the contrary, small interfering RNA-based knockdown of CLDN-10 in HLE, an invasive cell line with high level of CLDN-10 expression, abolished invasion and strongly decreased activation of MMPs and claudin members expression. These findings showed that CLDN-10 is functionally involved in hepatocellular carcinoma invasion and is a potential target for hepatocellular carcinoma therapy.

Immunohistochemical analysis of the progression of flat and papillary preneoplastic lesions in intrahepatic cholangiocarcinogenesis in hepatolithiasis

Two types of precursor lesions, flat-type 'biliary intraepithelial neoplasia (BilIN)' and papillary-type 'intraductal papillary neoplasm of the bile duct (IPNB)', are proposed in the tumorigenesis of intrahepatic cholangiocarcinoma (ICC) in hepatolithiasis. In this study, the participation of cancer-related molecules in the progression of these two precursor lesions was examined, using 64 hepatolithiatic livers with BilIN lesions (45 livers) and IPNB lesions (19 livers) and 10 hepatolithiatic livers without neoplastic lesions as a control. The expression of E-cadherin, beta-catenin, matrix metalloproteinase-7 (MMP-7), membrane type 1-MMP (MT1-MMP), cyclin D1 and c-myc was immunohistochemically examined. The membranous expression of beta-catenin decreased along with the progression in both BilIN and IPNB lineages. Membranous expression of E-cadherin was significantly decreased in invasive ICC with BilIN and IPNB in comparison with non-invasive counterparts. MMP-7 and MT1-MMP were commonly expressed in invasive ICC with BilIN (100%), while non-invasive lesions (BilIN-1, -2, -3) and the IPNB lineage were only occasionally and weakly positive for these molecules. Cyclin D1 and c-myc, target molecules of Wnt signalling, were frequently positive in the IPNB lineage (65 and 54% respectively), and interestingly nuclear beta-catenin staining, reflecting activation of Wnt signalling, was observed only in the IPNB lineage (22%) (P<0.05). Decreased membranous expression of beta-catenin and E-cadherin is an early event in the tumorigenesis of both BilIN and IPNB lineages. The expression of MMP-7 and MT1-MMP was closely associated with invasive growth of the BilIN lineage. The Wnt signalling pathway may play an important role in the tumorigenesis of the IPNB lineage.

Matrix metalloproteinase localisation by in situ-RT-PCR in archival human breast biopsy material

Utilising archival human breast cancer biopsy material we examined the stromal/epithelial interactions of several matrix metalloproteinases (MMPs) using in situ-RT-PCR ( IS-RT-PCR). In breast cancer, the stromal/epithelial interactions that occur, and the site of production of these proteases, are central to understanding their role in invasive and metastatic processes. We examined MT1-MMP (MMP-14, membrane type-1-MMP), MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) for their localisation profile in progressive breast cancer biopsy material (poorly differentiated invasive breast carcinoma (PDIBC), invasive breast carcinomas (IBC) and lymph node metastases (LNM)). Expression of MT1-MMP, MMP-1 and MMP-3 was observed in both the tumour epithelial and surrounding stromal cells in most tissue sections examined. MT1-MMP expression was predominantly localised to the tumour component in the pre-invasive lesions. MMP-1 gene expression was relatively well distributed between both tissue compartments, while MMP-3 demonstrated highest expression levels in the stromal tissue surrounding the epithelial tumour cells. The results demonstrate the ability to distinguish compartmental gene expression profiles using IS-RT-PCR. Further, we suggest a role for MT1-MMP in early tumour progression, expression of MMP-1 during metastasis and focal expression pattern of MMP-3 in areas of expansion. These expression profiles may provide markers for early breast cancer diagnoses and present potential therapeutic targets.

Stroma-Derived Matrix Metalloproteinase (MMP)-2 Promotes Membrane Type 1-MMP–Dependent Tumor Growth in Mice

Matrix metalloproteinase-2 (MMP-2) is a stroma-derived MMP belonging to the type IV collagenase family. It is believed to mediate tumor cell behavior by degrading deposits of type IV collagen, a major component of the basement membrane. The membrane type 1-MMP (MT1-MMP) is a highly potent activator of MMP-2 and is expressed in many tumor and stromal cells. However, the roles played by stromal MMP-2 in tumor progression in vivo remain poorly understood. We established a colon epithelial cell line from an Mt1-mmp(-/-) mouse strain and transfected these cells with an inducible expression system for MT1-MMP (MT1rev cells). Following s.c. implantation into Mmp-2(+/+) mice and induction of MT1-MMP expression, MT1rev cells grew rapidly, whereas they grew very slowly in Mmp-2(-/-) mice, even in the presence of MT1-MMP. This MT1-MMP-dependent tumor growth of MT1rev cells was enhanced in Mmp-2(-/-) mice as long as MMP-2 was supplied via transfection or coimplantation of MMP-2-positive fibroblasts. MT1rev cells cultured in vitro in a three-dimensional collagen gel matrix also required the MT1-MMP/MMP-2 axis for rapid proliferation. MT1rev cells deposit type IV collagen primarily at the cell-collagen interface, and these deposits seem scarce at sites of invasion and proliferation. These data suggest that cooperation between stroma-derived MMP-2 and tumor-derived MT1-MMP may play a role in tumor invasion and proliferation via remodeling of the tumor-associated basement membrane. To our knowledge, this is the first study demonstrating that MT1-MMP-dependent tumor growth in vivo requires stromal-derived MMP-2. It also suggests that MMP-2 represents a potential target for tumor therapeutics.

Lysophospholipids Enhance Membrane Type‐1 Matrix Metalloproteinase Expression Activity in Human Endothelial Cells

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low molecular weight lysophospholipids (LPLs), which promote cell proliferation, migration, and invasion via interaction with a family of specific G protein-coupled receptors. Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes, which are involved in degradation of the extracellular matrix. Among members of the MMP family, membrane type 1-MMP (MT1-MMP) has been recognized as an essential enzyme involved in matrix remodeling and in the migration of endothelial cells, which facilitates angiogenesis process. In our present study, we examined the effects of LPA and S1P on MT1-MMP expression and activity in human umbilical cord vein endothelial cells. By Western Blot analysis and Biotrak activity assay, we showed that both LPA and S1P enhanced MT1-MMP protein expression and enzymatic activity in a dose- and time-dependent manner. Maximal expression appeared at 1 μM post-ligand treatment at 4 h. These observations suggest that lysophospholipids might play important roles in endothelial cell invasion by regulating the expression and activity of MT1-MMP.