Underexpression of MMP-2 and its regulators, TIMP2, MT1-MMP and IL-8, is associated with prostate cancer

OBJECTIVE: Extracellular matrix homeostasis is strictly maintained by a coordinated balance between the expression of metalloproteinases (MMPs) and their regulators. The purpose of this study was to investigate whether MMP-2 and its specific regulators, TIMP-2, MT1-MMP and IL-8, are expressed in a reproducible, specific pattern and if the profiles are related to prognosis and clinical outcome of prostate cancer (PCa). MATERIALS AND METHODS: MMP-2, TIMP-2, MT1-MMP and IL-8 expression levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in freshly frozen malignant and benign tissue specimens collected from 79 patients with clinically localized PCa who underwent radical prostatectomies. The control group consisted of 11 patients with benign prostate hyperplasia (BPH). The expression profile of the MMP-2 and its regulators were compared using Gleason scores, pathological stage, pre-operative PSA levels and the final outcome of the PCa. RESULTS: The analysis of 79 specimens of PCa revealed that MMP-2, TIMP-2, MT1-MMP and IL-8 were underexpressed at 60.0%, 72.2%, 62.0% and 65.8%, respectively, in malignant prostatic tissue in relation to BPH samples. Considering the prognostic parameters, we demonstrated that high Gleason score tumors (> 7) overexpressed MMP-2 (p = 0.048) and TIMP-2 (p = 0.021), compared to low Gleason score tumors (< 7). CONCLUSION: We have demonstrated that MMP-2 and its regulators are underexpressed in PCa. Alternatively, overexpression of MMP-2 and TIMP-2 was related to higher Gleason score tumors. We postulate that alterations in metalloproteinase expression may be important in the control of tissue homeostasis related to prostate carcinogenesis and tumor behavior.

MMP-2 and MMP-9 in lymph-node-positive bladder cancer

Matrix metalloproteinases (MMP), particularly MMP-2 and MMP-9, participate in tumour progression and metastasis in various cancers. Their significance in urothelial cancer of the bladder (UCB) is unclear. Expression analysis of MMP-2 and MMP-9 in tissue microarrays (TMA) constructed of corresponding samples from histopathological normal urothelium, tumour centre and invasion front of primary tumours and lymph-node (LN) metastases might help to elucidate their relevance in UCB.

Increased invasive potential and up-regulation of MMP-2 in MDA-MB-231 breast cancer cells expressing the beta3 integrin subunit

Integrins are a family of transmembrane adhesion receptors that might transduce signals from the extracellular matrix into the inside of cells after ligand binding. In order to investigate whether beta3 integrins expressed in tumor cells might mediate such outside-in signaling, human MDA-MB-231 breast cancer cells that were stably transfected with either beta3 integrin or mock-transfected were investigated in a matrigel degradation assay and a grafting experiment was performed on the developing chicken chorioallantoic membrane (CAM). After cultivation on matrigel for time periods between one and five days, more matrigel was digested in the wells in which beta3 integrin expressing cells were incubated than in wells of mock-transfected cells. Furthermore, extracts of beta3 integrin expressing cells contained higher levels of MMP-2 protein as determined by immunoblotting and more MMP-2 associated gelatinase activity as detected by zymography than extracts of mock-transfected cells. Matrigel degradation and gelatinase activity as well as MMP-2 expression were elevated when beta3 integrin expressing cells were incubated in the presence of the RGD peptide (mimicking an integrin ligand). After grafting on 10 day-old embryonic chicken CAM for three to five days, beta3 integrin expressing cells assembled in spheroids showed higher rates of spreading on the CAM surface and CAM invasion as well as a significant MMP-2 up-regulation compared to mock-transfected cells. The results from the in vivo and in vitro experiments allow the conclusion that the presence of beta3 integrin in MDA-MB-231 breast cancer cells induced an increased MMP-2 expression and activity that might contribute to the enhanced invasive potential observed.

Expression of the Recessive Glomerulosclerosis Gene Mpv17 Regulates MMP-2 Expression in Fibroblasts, the Kidney, and the Inner Ear of Mice

The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.

Equine laminitis: increased transcription of matrix metalloproteinase-2 (MMP-2) occurs during the developmental phase

Reasons for performing study: The dysadhesion and destruction of lamellar basement membrane of laminitis may be due to increased lamellar metalloproteinase activity. Characterising lamellar metalloproteinase-2 (MMP-2) and locating it in lamellar tissues may help determine if laminitis pathology is associated with increased MMP-2 transcription. Objectives: To clone and sequence the cDNA encoding lamellar MMP-2, develop antibody and in situ hybridisation probes to locate lamellar MMP-2 and quantitate MMP-2 transcription in normal and laminitis tissue. Methods: Total RNA was isolated, fragmented by RT-PCR, cloned into vector and sequenced. Rabbit anti-equine MMP-2 and labelled MMP-2 riboprobe were developed to analyse and quantitate MMP-2 expression. Results: Western immunoblotting with anti-MMP-2 detected 72 kDa MMP-2 in hoof tissue homogenates and cross-reacted with human MMP-2. Immunohistochemistry and in situ hybridisation detected MMP-2 in the cytoplasm of basal and parabasal cells in close proximity to the lamellar basement membrane. Northern analysis and quantitative real-time PCR showed MMP-2 expression significantly (P

Aggravation of ADAMTS and MMP production and ERK1/2 pathway in the interaction of osteoarthritic subchondral bone osteoblasts and articular cartilage chondrocytes : a possible pathogenic role in osteoarthritis

Introduction: Degradative enzymes, such as A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinases (MMPs), play key roles in osteoarthritis (OA) development. The aim of the present study was to investigate if cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of ADAMTS5, ADAMTS4, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13, and also to test the possible involvement of mitogen activated protein kinase (MAPK) signaling pathway during this process. Methods: ACCs and SBOs were isolated from normal and OA patients. An in vitro co-culture model was developed to study the regulation of ADAMTS and MMPs under normal and OA joint cross-talk conditions. MAPK-ERK inhibitor, PD98059 was applied to delineate the involvement of specific pathway during this interaction process. Results: Indirect co-culture of OA SBOs with normal ACCs resulted in significantly increased expression of ADAMTS5, ADAMTS4, MMP-2, MMP-3 and MMP-9 in ACCs, whereas co-culture of OA ACCs led to increased MMP-1 and MMP-2 expression in normal SBOs. The upregulation of ADAMTS and MMPs under these conditions was correlated with activation of the MAPK-ERK1/2 signaling pathway and the addition of the MAPK-ERK inhibitor, PD98059, reversed the overexpression of ADAMTS and MMPs in co-cultures. Conclusion: In summary, we believe, these results add to the evidence that in human OA, altered bi-directional signals transmitted between SBOs and ACCs significantly impacts the critical features of both cartilage and bone by producing abnormal levels of ADAMTS and MMPs. Furthermore, we have demonstrated for the first time that this altered cross-talk was mediated by the phosphorylation of MAPK-ERK1/2 signaling pathway.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

Effects of cyclosporin, phenytoin, and nifedipine on the synthesis and degradation of gingival collagen in tufted capuchin monkeys (Cebus apella): Histochemical and MMP-1 and -2 and collagen I gene expression analyses

Background: The purpose of this experimental study was to evaluate the collagen fiber distribution histologically after phenytoin, cyclosporin, or nifedipine therapy and to correlate it with collagen I and matrix metalloproteinase (MMP)-1 and -2 gene expression levels.Methods: Gingival samples from the canine area were obtained from 12 male monkeys (Cebus apella). The mesial part of each sample was assessed by reverse transcription-polymerase chain reaction, whereas the distal part was processed histologically for picrosirius red and hematoxylin and eosin stainings, as well as for collagen IV immunostaining. One week after the first biopsy, the animals were assigned to three groups that received daily oral dosages of cyclosporin, phenytoin, or nifedipine for 120 days. Additional gingival samples were obtained on days 52 and 120 of treatment from two animals from each group on the opposite sides from the first biopsies.Results: Picrosirius red staining showed a predominance of mature collagen fibers in the control group. Conversely, there was an enlargement of areas occupied by immature collagen fibers in all groups at days 52 and 120, which was not uniform over each section. There was a general trend to lower levels of MMP-1 gene expression on day 52 and increased levels on day 120. Phenytoin led to increased levels of MMP-2 and collagen I gene expression on day 120, whereas the opposite was observed in the nifedipine group.Conclusion: Cyclosporin, phenytoin, and nifedipine led to phased and drug-related gene expression patterns, resulting in impaired collagen metabolism, despite the lack of prominent clinical signs.

TGF-beta 1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

Background: Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-beta 1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. Methods: The mRNA expression levels of TGF-beta isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-beta 1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. Results: In general, TGF-beta 2, T beta RI and T beta RII are over-expressed in more aggressive cells, except for T beta RI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-beta 1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-beta 1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-beta 1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-beta 1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-beta 1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-beta 1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. Conclusion: Altogether, our results support that TGF-beta 1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-beta 1 still remains a promising target for breast cancer treatment.

TGF-β1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

Abstract Background Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. Methods The mRNA expression levels of TGF-β isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-β1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. Results In general, TGF-β2, TβRI and TβRII are over-expressed in more aggressive cells, except for TβRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-β1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-β1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-β1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-β1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-β1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. Conclusion Altogether, our results support that TGF-β1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-β1 still remains a promising target for breast cancer treatment.

An assessment of MMP and TIMP gene expression in cell lines and stroma : tumour differences in microdissected breast cancer biopsies

To examine matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) mRNA levels in archival breast cancer biopsies, we employed microdissection to separate tumour tissue from the surrounding breast tissue, or stroma and RT-PCR to determine gross qualitative and small quantitative differences in the patterns of expression. In this study, a significant correlation (p < 0.05, by Mann-Whitney U analysis) between TIMP-2 expression and lymph node involvement was identified, while MMP-11 and TIMP-1 expression patterning also significantly (p < 0.05) differed between those tumours showing calcification and those that did not. When compared by Spearmans’ ρ correlation analysis, a significant association (p < 0.05, ρ = 0.404) was identified in the pattern of MMP-2 and MMP-9 gene expression. In this study, the use of microdissection and a systematic strategy of RT-PCR analysis have allowed us to investigate localized MMP and MMP inhibitor expression within breast tumours. We have identified patterns of gene expression that may further reveal aspects of breast carcinogenesis, and a robust method for examining changes in clinically important genes using archival biopsies and across stroma-tumour boundaries.

Matrix metalloproteinases 2 and 9 (gelatinases A and B) expression in malignant mesothelioma and benign pleura

Matrix metalloproteinases (MMPs), in particular the gelatinases (MMP-2 and -9), play a significant role in tumour invasion and angiogenesis. The expression and activities of MMPs have not been characterised in malignant mesothelioma (MM) tumour samples. In a prospective study, gelatinase activity was evaluated in homogenised supernatants of snap frozen MM (n = 35), inflamed pleura (IP, n = 12) and uninflammed pleura (UP, n = 14) tissue specimens by semiquantitative gelatin zymography. Matrix metalloproteinases were correlated with clinicopathological factors and with survival using Kaplan-Meier and Cox proportional hazard models. In MM, pro- and active MMP-2 levels were significantly greater than for MMP-9 (P = 0.006, P<0.001). Active MMP-2 was significantly greater in MM than in UP (P=0.04). MMP-2 activity was equivalent between IP and MM, but both pro- and active MMP-9 activities were greater in IP (P=0.02, P=0.009). While there were trends towards poor survival with increasing total and pro-MMP-2 activity (P=0.08) in univariate analysis, they were both independent poor prognostic factors in multivariate analysis in conjunction with weight loss (pro-MMP-2 P = 0.03, total MMP-2 P = 0.04). Total and pro-MMP-2 also contributed to the Cancer and Leukemia Group B prognostic groups. MMP-9 activities were not prognostic. Matrix metalloproteinases, and in particular MMP-2, the most abundant gelatinase, may play an important role in MM tumour growth and metastasis. Agents that reduce MMP synthesis and/or activity may have a role to play in the management of MM. © 2003 Cancer Research UK.

SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines

Activation of the matrix metalloproteinase 2 (MMP-2) has been shown to play a major role in the proteolysis of extracellular matrix (ECM) associated with tumor invasion. Although the precise mechanism of this activation remains elusive, levels of the membrane type 1-MMP (MT1-MMP) at the cell surface and of the tissue inhibitor of MMP-2 (TIMP-2) appear to be two important determinants. Induction of MMP-2 activation in cells cultivated on collagen type I gels indicated that the ECM is important in the regulation of this process. In this study, we show that SPARC/osteonectin, a small ECM- associated matricellular glycoprotein, can induce MMP-2 activation in two invasive breast cancer cell lines (MDA-MB-231 and BT549) but not in a noninvasive counterpart (MCF7), which lacks MT1-MMP. Using a set of peptides from different regions of SPARC, we found that peptide 1.1 (corresponding to the NH2-terminal region of the protein) contained the activity that induced NIMP-2 activation. Despite the requirement for MT1-MMP, seen in MCF-7 cells transfected with MT1-MMP, the activation of MMP-2 by SPARC peptide 1.1 was not associated with increased steady-state levels of MT1-MMP mRNA or protein in either MT1-MMP-transfected MCF-7 cells or constitutively expressing MDA- MB-231 and BT549 cells. We did, however, detect decreased levels of TIMP-2 protein in the media of cells incubated with peptide 1.1 or recombinant SPARC; thus, the induction of MMP-2 activation by SPARC might be due in part to a diminution of TIMP-2 protein. We conclude that SPARC, and specifically its NH2-terminal domain, regulates the activation of MMP-2 at the cell surface and is therefore likely to contribute to the proteolytic pathways associated with tumor invasion.

Pro-matrix metalloproteinase-2 transfection increases orthotopic primary growth and experimental metastasis of MDA-MB-231 human breast cancer cells in nude mice

The ability to activate pro-matrix metalloproteinase (pro-MMP)-2 via membrane type-MMP is a hallmark of human breast cancer cell lines that show increased invasiveness, suggesting that MMP-2 contributes to human breast cancer progression. To investigate this, we have stably transfected pro-MMP-2 into the human breast cancer cell line MDA-MB-231, which lacks MMP-2 expression but does express its cell surface activator, membrane type 1-MMP. Multiple clones were derived and shown to produce pro-MMP-2 and to activate it in response to concanavalin A. In vitro analysis showed that the pro-MMP-2-transfected clones exhibited an increased invasive potential in Boyden chamber and Matrigel outgrowth assays, compared with the parental cells or those transfected with vector only. When inoculated into the mammary fat pad of nude mice, each of the MMP-2-tranfected clones grew faster than each of the vector controls tested. After intracardiac inoculation into nude mice, pro-MMP-2-transfected clones showed a significant increase in the incidence of metastasis to brain, liver, bone, and kidney compared with the vector control clones but not lung. Increased tumor burden was seen in the primary site and in lung metastases, and a trend toward increased burden was seen in bone, however, no change was seen in brain, liver, or kidney. This data supports a role for MMP-2 in breast cancer progression, both in the growth of primary tumors and in their spread to distant organs. MMP-2 may be a useful target for breast cancer therapy when refinement of MMP inhibitors provides for MMP-specific agents.

Tyrosine phosphorylation mediates ConA-induced membrane type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in MDA-MB-231 human breast carcinoma cells

ConA-induced cell surface activation of pro-matrix metalloproteinase-2 (pro-MMP-2) by MDA-MB-231 human breast cancer cells is apparently mediated by up-regulation of membrane type 1 MMP (MT1-MMP) through transcriptional and posttranscriptional mechanisms. Here, we have explored the respective roles of cell surface clustering and protein tyrosine phosphorylation in the ConA- induction effects. Treatment with succinyl-ConA, a variant lacking significant clusterability, partially stimulated MT1-MMP mRNA and protein levels but did not induce MMP-2 activation, suggesting that clustering contributes to the transcriptional regulation by ConA but appears to be critical for the nontranscriptional component. We further found that genistein, an inhibitor of tyrosine phosphorylation, blocked ConA-induced pro-MMP-2 activation and ConA-induced MT1-MMP mRNA level in a dose-dependent manner, implicating tyrosine phosphorylation in the transcriptional aspect. This was confirmed by the dose-dependent promotion of pro-MMP-2 activation by sodium orthovanadate in the presence of suboptimal concentrations of ConA (7.5 μg/ml), with optimal effects seen at 25 μg/g orthovanadate. Genistein did not inhibit the ConA potentiation of MMP-2 activation in MCF-7 cells, in which transfected MT1-MMP is driven by a heterologous promoter, supporting the major implication of phosphotyrosine in the transcriptional component of ConA regulation. These data describe a major signaling event upstream of MT1- MMP induction by ConA and set the stage for further analysis of the nontranscriptional component.