Matrix metalloproteinase-1 (MMP-1) is critical for mediating breast cancer metastasis to bone. We investigated the role of MMP-1 in breast cancer invasion of soft tissues and bone using human MDA MB-231 breast cancer cells stably transfected with shRNAs against MMP-1 and a novel murine model of bone invasion. MMP-1 produced by breast cancer cells with control shRNA facilitated invasion of tumors into soft tissue in vivo, which correlated with enhanced blood vessel formation at the invasive edge, compared to tumors with silenced MMP-1 expression. Tumors expressing MMP-1 were also associated with osteolysis in vivo, whereas tumors with inhibited MMP-1 levels were not. Additionally, tumor-secreted MMP-1 activated bone-resorbing osteoclasts in vitro. Together, these data suggest a mechanism for MMP-1 in the activation of osteoclasts in vivo. We conclude that breast cancer-derived MMP-1 mediates invasion through soft tissues and bone via mechanisms involving matrix degradation, angiogenesis, and osteoclast activation.
Breast cancer; Bone; Invasion; Metastasis; MMP; Murine model
Matrix metalloproteinase-2 (MMP-2) is known to degrade the collagen IV, play a role in radiation-induced lung injury. We therefore investigated the anti-tumor effects of combining MMP-2 inhibition using an adenovirus expressing siRNA against MMP-2 (Ad-MMP-2-Si) with radiation therapy (IR) on A549 lung cancer cells in vitro and in vivo. IR increased MMP-2 mRNA, protein and activity in lung cancer cells. MMP-2 inhibition along with IR enhanced radiosensitivity as determined by clonogenic assay, flow cytometry, and TUNEL assay. We show that MMP-2 inhibition prior to irradiation reduced p53 phosphorylation, with a corresponding reduction in the expression of the p53 downstream target gene p21Cip1/Waf1. Irradiated tumor cells induced the FoxM1-mediated DNA repair gene, XRCC1 and Checkpoint-2/1, which were abrogated with combined treatment of Ad-MMP-2-Si and IR. Further, the combination of Ad-MMP-2-Si with radiotherapy significantly increased anti-tumor efficacy in vivo compared to either agent alone. Indeed, histological analysis of tumor sections collected from the combination group revealed more apoptotic cells. These studies suggest that MMP-2 inhibition in combination with radiotherapy abrogates G2 cell cycle arrest leading to apoptosis and provide evidence of the anti-tumor efficacy of combining MMP-2 inhibition with irradiation as a new therapeutic strategy for the effective treatment of NSCLC patients.
MMP-2; FoxM1; G2/M arrest; radiosensitization; A549
Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult.
We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state.
In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma.
We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.
Invasion and metastasis are the main causes of treatment failure and death in breast cancer. Thus, novel invasion-based therapies such as those involving natural agents are urgently required. In this study, we examined the effects of magnolol (Mag), a compound extracted from medicinal herbs, on breast cancer cells in vitro and in vivo. Highly invasive cancer cells were found to be highly sensitive to treatment. Mag markedly inhibited the activity of highly invasive MDA-MB-231 cells. Furthermore, Mag significantly downregulated matrix metalloproteinase-9 (MMP-9) expression, an enzyme critical to tumor invasion. Mag also inhibited nuclear factor-κB (NF-κB) transcriptional activity and the DNA binding of NF-κB to MMP-9 promoter. These results indicate that Mag suppresses tumor invasion by inhibiting MMP-9 through the NF-κB pathway. Moreover, Mag overcame the promoting effects of phorbol 12-myristate 13-acetate (PMA) on the invasion of MDA-MB-231 cells. Our findings reveal the therapeutic potential and mechanism of Mag against cancer.
The serine protease urokinase-type plasminogen activator (uPA) plays a significant role in tumor cell invasion and metastasis when bound to its specific receptor, uPAR (also known as CD87). In addition to the uPA-uPAR system, matrix metalloproteinases (MMPs) are involved in tumor cell invasion and metastasis. In this study, we achieved specific inhibition of uPAR and MMP-9 using RNAi technology. We introduced small interfering RNA (siRNA) to downregulate the expression of uPAR and MMP-9 (pUM) in breast cancer cell lines (MDA MB 231 and ZR 75 1). In vitro angiogenesis studies indicated a decrease in the angiogenic potential of the treated cells; in particular, a remarkable decrease was observed in the cells treated with bicistronic construct (pUM) in comparision to the controls. Additionally, bicistronic construct inhibited the formation of capillary-like structures in in vivo models of angiogenesis. Similarly, the invasive potential and migration decreased dramatically when treated with the bicistronic construct as shown by matrigel invasion and migration assays. These results suggest a synergistic effect from the simultaneous downregulation of uPAR and MMP-9. We also assessed the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules and found reduction in the levels of these molecules in cells treated with the bicistronic construct as compared to the control cells. Furthermore, targeting both uPAR and MMP-9 totally regressed orthotopic breast tumors in nude mice. In conclusion, our results provide evidence that the simultaneous downregulation of uPAR and MMP-9 using RNAi technology may provide an effective tool for breast cancer therapy.
RNAi; uPAR; MMP-9; Invasion; Angiogenesis; Tumor growth
Lung cancer is currently the leading cause of cancer deaths in this country. Conventional therapeutic treatments including surgery, chemotherapy and radiation therapy, have achieved only limited success. The overexpression of proteases such as urokinase-type plasminogen activator (uPA), its receptor (uPAR) and matrix metalloproteinases (MMPs), is correlated with the progression of lung cancer. In the present study, we used a replication-deficient adenovirus capable of expressing antisense uPAR and antisense MMP-9 transcripts to simultaneously downregulate uPAR and MMP-9 in H1299 cells. Ad-uPAR-MMP-9 infection of H1299 cells resulted in a dose-and time-dependent decrease of uPAR protein levels and MMP-9 activity as determined by western blotting and gelatin zymography respectively. Corresponding immunohistochemical analysis also demonstrated that Ad-uPAR-MMP-9 infection inhibited uPAR and MMP-9 expression. As shown by Boyden chamber assay, Ad-uPAR-MMP-9 infection significantly decreased the invasive capacity of H1299 cells compared to mock and Ad-CMV (empty vector)-infected cells in vitro. Furthermore, Ad-uPAR-MMP-9 infection inhibited capillary-like structure formation in H1299 cells co-cultured with endothelial cells in a dose-dependent manner compared with mock- and Ad-CMV-infected cells. Ad-uPAR-MMP-9 injection caused the regression of subcutaneously induced tumors after subcutaneous injection with H1299 lung cancer cells and inhibited lung metastasis in the metastatic model with A549 cells. These data suggest that Ad-uPAR-MMP-9 demonstrates its antitumor activity against both established and early phases of lung cancer metastases by causing the destruction of the tumor vasculature. In summary, adenovirus-mediated inhibition of uPA-uPAR interaction and MMP-9 on the cell surface may be a promising anti-invasion and anti-metastasis strategy for cancer gene therapy.
lung; invasion; MMP-9; uPAR; antisense; angiogenesis; uPA (urokinase-type plasminogen activator); uPAR (uPA receptor); CMV (cytomegalovirus); SV40 (simian virus type 40); PBS (phosphate-buffered saline); MMP-9 (matrix metalloprotease-9); metastasis
Matrix metalloproteinase-2 (MMP-2) expression is often upregulated in advanced cancers and known to play important role in tumor angiogenesis. We previously showed that adenoviral-mediated delivery of siRNA for MMP-2 (Ad-MMP-2-Si) inhibited lung cancer growth, angiogenesis and metastasis. In this study, we investigated the signaling mechanisms involved in Ad-MMP-2-Si-mediated inhibition of angiogenesis. Ad-MMP-2-Si treatment inhibited neo-vascularization in vivo as determined by mouse dorsal air sac model, and conditioned medium from Ad-MMP-2-Si-infected A549 lung cancer cells (Ad-MMP-2-Si-CM) inhibited endothelial tube formation in vitro. Ad-MMP-2-Si-CM decreased proliferation as determined by Ki-67 immunofluorescence and induced apoptosis in endothelial cells as determined by TUNEL assay. Furthermore, Ad-MMP-2-Si-CM inhibited AKT phosphorylation and induced phosphorylation of ERK-MAPKs in endothelial cells. Overexpression of constitutively active-AKT reversed the Ad-MMP-2-Si-CM-mediated inhibition of tube formation and induction of ERK phosphorylation. Conversely, Ad-MMP-2-Si-CM induced TIMP-3 expression, and the interaction of VEGFR2 and TIMP-3 was determined by co-immunoprecipitation experiments. TIMP-3 induction was mediated by ERK activation. In addition, electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrate that Sp1 transcription factor mediated Ad-MMP-2-Si-CM-stimulated increase of TIMP-3. Vasculature destruction was confirmed with co-localization studies with TUNEL and an endothelial marker, CD31, in tumor sections of Ad-MMP-2-Si-treated mice. Our data collectively suggest MMP-2 inhibition induces endothelial apoptosis in vivo and inhibits endothelial tube formation. These experiments provide the first evidence that inhibition of p-AKT and induction of p-ERK1/2 are crucial events in the induction of TIMP-3-mediated endothelial apoptosis in MMP-2 inhibited lung tumors.
ERK; TIMP-3; MMP-2; siRNA; angiogenesis; apoptosis; lung cancer
Despite continued improvements in diagnosis, surgical techniques, and chemotherapy, breast cancer patients are still overcome by cancer metastasis. Tumor cell proliferation, invasion and metastasis are mediated, at least in part, through degradation of basement membrane by neutral matrix metalloproteinases (MMP) produced by tumor and stromal cells. Evidence suggests that MMP-9 plays a significant role in breast tumor cell invasion and metastasis. DNAzymes or catalytic oligonucleotides are new classes of gene targeting molecules that bind and cleave a specific mRNA, resulting in decreased protein expression.
The application of anti-MMP-9 DNAzyme (AM9D) for the treatment of primary and metastatic breast cancer was evaluated in vitro and in vivo using MDA-MB-231 cells and the MMTV-PyMT transgenic breast cancer mouse model. Spontaneously developed mammary tumors in MMTV-PyMT transgenic mice were treated intratumorally with naked AM9D, once a week for 4 weeks. The stability of DNAzyme was determined in vitro and in vivo using fluorescently labeled DNAzyme.
AM9D specifically inhibited expression of MMP-9 in MDA-MB-231 cells resulting in reduced invasive property of these cells by 43%. Weekly intratumoral treatment of spontaneously developed mammary tumors in MMTV-PyMT transgenic mice was sufficient to significantly reduce the rate of tumor growth and final tumor load in a dose dependent and statistically significant manner (P < 0.05). This decrease in tumor growth was correlated with decreased MMP-9 protein production within the treated tumor tissues. Tumors treated with AM9D were also less vascularized and contained more apoptotic cells compared to control and untreated tumors.
These results show that targeting and down regulation of MMP-9 by AM9D could prove useful as a therapy against breast carcinoma tumor growth and invasion.
The matrix metalloproteinases (MMPs) play a key role in normal and pathological angiogenesis by mediating extracellular matrix degradation and/or controlling the biological activity of growth factors, chemokines, and/or cytokines. Specific functions of individual MMPs as anti- or proangiogenic mediators remain to be elucidated. In the present study, we assessed the impact of single or combined MMP deficiencies in in vivo and in vitro models of angiogenesis (malignant keratinocyte transplantation and the aortic ring assay, respectively). MMP-9 was predominantly expressed by neutrophils in tumor transplants, whereas MMP-2 and MMP-3 were stromal. Neither the single deficiency of MMP-2, MMP-3, or MMP-9, nor the combined absence of MMP-9 and MMP-3 did impair tumor invasion and vascularization in vivo. However, there was a striking cooperative effect in double MMP-2:MMP-9-deficient mice as demonstrated by the absence of tumor vascularization and invasion. In contrast, the combined lack of MMP-2 and MMP-9 did not impair the in vitro capillary outgrowth from aortic rings. These results point to the importance of a cross talk between several host cells for the in vivo tumor promoting and angiogenic effects of MMP-2 and MMP-9. Our data demonstrate for the first time in an experimental model that MMP-2 and MMP-9 cooperate in promoting the in vivo invasive and angiogenic phenotype of malignant keratinocytes.
angiogenesis; tumor invasion; proteolysis; gelatinases; stromal MMP
Proteolysis mediated by matrix metalloproteinases (MMPs) and serine proteinases is associated with cancer invasion and metastasis. Activation of latent proMMPs, and especially the proforms of the type IV collagen degrading gelatinases A and B (proMMP-2 and proMMP-9), is thought to be a critical step in this process. We have recently found that human tumour-associated trypsin-2 is a potent activator of proMMP-9 and it also activates proMMP-2 in vitro. Trypsinogen, MMP-2, and MMP-9 are expressed in ovarian cancer. To elucidate the function of trypsin in vivo, we studied whether high concentrations of trypsinogen-1, trypsinogen-2, their α1-proteinase inhibitor (API) complexes, and tumour-associated trypsin inhibitor (TATI) are associated with proMMP-2 and proMMP-9 activation in ovarian tumour cyst fluids. Zymography and immunofluorometric analysis of 61 cyst fluids showed a significant association between high trypsin concentrations and the activation of MMP-9 (P= 0.003–0.05). In contrast, the trypsin concentrations were inversely associated with the activation of MMP-2 (P= 0.01–0.02). Immunohistochemical analysis of ovarian tumour tissue demonstrated expression of trypsinogen-2 and TATI in the secretory epithelium. MMP-2 was detected both in stromal and epithelial cells whereas MMP-9 was detected in neutrophils and macrophage-like cells in stromal and epithelial areas. These results suggest that trypsin may play a role in the regulation of the MMP-dependent proteolysis associated with invasion and metastasis of ovarian cancer. © 2001 Cancer Research Campaign www.bjcancer.com
trypsin; TATI; MMP-2; MMP-9; ovarian cancer; cyst fluid
Broad-spectrum matrix metalloproteinase (MMP) inhibitors (MMPI) were unsuccessful in cancer clinical trials, partly due to side effects resulting from limited knowledge of the full repertoire of MMP substrates, termed the substrate degradome, and hence the in vivo functions of MMPs. To gain further insight into the degradome of MMP-14 (membrane type 1 MMP) an MMPI, prinomastat (drug code AG3340), was used to reduce proteolytic processing and ectodomain shedding in human MDA-MB-231 breast cancer cells transfected with MMP-14. We report a quantitative proteomic evaluation of the targets and effects of the inhibitor in this cell-based system. Proteins in cell-conditioned medium (the secretome) and membrane fractions with levels that were modulated by the MMPI were identified by isotope-coded affinity tag (ICAT) labeling and tandem mass spectrometry. Comparisons of the expression of MMP-14 with that of a vector control resulted in increased MMP-14/vector ICAT ratios for many proteins in conditioned medium, indicating MMP-14-mediated ectodomain shedding. Following MMPI treatment, the MMPI/vehicle ICAT ratio was reversed, suggesting that MMP-14-mediated shedding of these proteins was blocked by the inhibitor. The reduction in shedding or the release of substrates from pericellular sites in the presence of the MMPI was frequently accompanied by the accumulation of the protein in the plasma membrane, as indicated by high MMPI/vehicle ICAT ratios. Considered together, this is a strong predictor of biologically relevant substrates cleaved in the cellular context that led to the identification of many undescribed MMP-14 substrates, 20 of which we validated biochemically, including DJ-1, galectin-1, Hsp90α, pentraxin 3, progranulin, Cyr61, peptidyl-prolyl cis-trans isomerase A, and dickkopf-1. Other proteins with altered levels, such as Kunitz-type protease inhibitor 1 and beta-2-microglobulin, were not substrates in biochemical assays, suggesting an indirect affect of the MMPI, which might be important in drug development as biomarkers or, in preclinical phases, to predict systemic drug actions and adverse side effects. Hence, this approach describes the dynamic pattern of cell membrane ectodomain shedding and its perturbation upon metalloproteinase drug treatment.
Hypoxia, which is a loss of oxygen in tissues, is a common condition in solid tumors due to the tumor outgrowing existing vasculature. Under hypoxic conditions, hypoxia-inducible factor (HIF)-1α rapidly accumulates and transactivates hundreds of genes, such as matrix metalloproteinases (MMPs). MMPs contribute to invasion and metastasis of tumor cells by degrading the surrounding basement membrane and extracellular matrix barriers, which enables the easy migration and spread of cancer cells. We examined whether hypoxia increases tumor cell invasion, and whether increased invasiveness was due to HIF-1α and MMP-9 expression.
Transwell invasion assays were performed to demonstrate whether hypoxia enhance tumor invasion by use of MDA-MB-231 breast cancer cells. An immunofluorescence assay was used to demonstrate expression of HIF-1α and MMP-9 under hypoxic conditions. Luciferase and ChiP assays were performed to demonstrate that MMP-9 promoter activity was regulated by HIF-1α.
HIF-1α was stabilized under hypoxic conditions and stimulated MMP-9 expression, which affected the tumor invasiveness of breast cancer cells. HIF-1α transactivated the MMP-9 promoter by forming a transcriptional unit with p300, thus increasing expression of MMP-9 transcripts. Zymography indicated that MMP-9 had more gelatinase activity under hypoxic conditions than normoxic conditions. Furthermore, the small GTPase Ras was also activated in response to hypoxia, which then aids stabilization of HIF-1α, and in turn upregulates MMP-9 expression. We also demonstrate that MMP-9 is upregulated concurrently with HIF-1α in tumor tissues from patients with breast cancer.
These results suggest that HIF-1α promotes cell invasion through a MMP-9-dependent mechanism and that future antitumor agents could be used to target HIF-1α and MMP-9.
Angiogenesis; Breast neoplasms; Hypoxia-inducible factor 1 alpha subunit; Matrix metalloproteinases
Abl interactor 1 (Abi1) is a key regulator of actin polymerization/depolymerization. The involvement of Abi1 in the development of abnormal cytoskeletal functions of cancer cells has recently been reported. It remains unclear, however, how Abi1 exerts its effects in tumor cells and whether it contributes to tumor progression in vivo. We report here a novel function for Abi1 in the regulation of invadopodia formation and Src-inhibitor of differentiation protein 1 (Id1)-matrix metalloproteinase (MMP)-9 pathway in MDA-MB-231 human breast cancer cells. Abi1 is found in the invadopodia of MDA-MB-231 cells. Epigenetic silencing of the Abi1 gene by short hairpin RNA in MDA-MB-231 cells impaired the formation of invadopodia and resulted in downregulation of the Src activation and Id1/MMP-9 expression. The decreased invadopodia formation and MMP-9 expression correlate with a reduction in the ability of these cells to degrade extracellular matrix. Remarkably, the knockdown of Abi1 expression inhibited tumor cell proliferation and migration in vitro and slowed tumor growth in vivo. Taken together, these results indicate that the Abi1 signaling plays a critical role in breast cancer progression and suggest that this pathway may serve as a therapeutic target for the treatment of human breast cancer.
Objective: Aberrant expression of individual matrix metalloproteinases has been associated with poor prognosis in various human carcinomas. The current study aimed at defining an RNA expression profile of various MMPs in breast cancer and correlating their expression with clinicopathological parameters.
Methods: The RNA expression patterns of 6 MMPs (MMP2, MMP8, MMP9, MMP10, MMP11, MMP13) were determined in 25 breast carcinomas using quantitative RT-PCR and correlated with clinicopathological parameters, including menopausal status, tumor size and grade, and lymph node involvement.
Results: We observed high MMP2 levels more frequently in premenopausal than in postmenopausal women (p = 0.02). Analysis of luminal A type invasive ductal carcinomas (19/25), revealed an even stronger association of MMP2 with menopausal status (p = 0.005). Within this subgroup, we also found a correlation between MMP11 and menopausal status (p = 0.02). No correlation was found between MMP expressions and other clinicopathological parameters. In co-expression analyses MMP2-MMP10 and MMP8-MMP9 showed a weak correlation of their expression.
Conclusions: Although this is a pilot study, our findings indicate that luminal A invasive ductal carcinomas commonly express high MMP2 and MMP11 levels in premenopausal breast cancer patients and suggest a co-regulation of MMP2-MMP10 and MMP8-MMP9.
Breast cancer; matrix metalloproteinases; menopausal status; luminal A; clinicopathological parameters
The c-Myb transcription factor is essential for the maintenance of stem-progenitor cells in bone marrow, colon epithelia, and neurogenic niches. c-Myb malfunction contributes to several types of malignancies including breast cancer. However, the function of c-Myb in the metastatic spread of breast tumors remains unexplored. In this study, we report a novel role of c-Myb in the control of specific proteases that regulate the matrix-dependent invasion of breast cancer cells.
Ectopically expressed c-Myb enhanced migration and ability of human MDA-MB-231 and mouse 4T1 mammary cancer cells to invade Matrigel but not the collagen I matrix in vitro. c-Myb strongly increased the expression/activity of cathepsin D and matrix metalloproteinase (MMP) 9 and significantly downregulated MMP1. The gene coding for cathepsin D was suggested as the c-Myb-responsive gene and downstream effector of the migration-promoting function of c-Myb. Finally, we demonstrated that c-Myb delayed the growth of mammary tumors in BALB/c mice and affected the metastatic potential of breast cancer cells in an organ-specific manner.
This study identified c-Myb as a matrix-dependent regulator of invasive behavior of breast cancer cells.
c-Myb; Metastasis; Breast cancer; Matrix metalloproteinase; Cathepsin D; Extracellular matrix
Bone metastasis of breast cancer induces severe osteolysis with increased bone resorption. Osteoclast differentiation regulated by the receptor activator of NF-κB ligand (RANKL) in osteoblasts and matrix degradation induced by matrix metalloproteinases (MMPs) are thought to be involved in the process of bone resorption. When nude mice were inoculated with human breast cancer cells, MDA-MB-231(MDA-231), numerous osteoclasts resorbed bone and the degradation of the bone matrix markedly progressed in the femur and tibia with metastasis of the MDA-231 tumour. The expression of RANKL, MMP-13 and membrane-type 1-MMP mRNA was markedly elevated in bone with metastasis. When MDA-231 cells were cocultured with mouse calvaria, MDA-231 markedly induced bone resorption measured by calcium release from the calvaria, and the expression of RANKL, MMP-2 and MMP-13 was elevated in the calvaria after the coculture. The separation of MDA-231 from the calvaria using filter insert showed decreased bone resorption, suggesting that cell-to-cell interaction is essential for cancer-induced bone resorption. Adding MDA-231 cells to bone marrow cultures markedly induced osteoclast formation, and the expression of RANKL in osteoblasts was enhanced by contact with the cell surface of MDA-231 cells. These results indicate that RANKL-induced osteoclast formation and MMP-dependent matrix degradation are associated with osteolysis because of bone metastasis of breast cancer.
bone metastasis; bone resorption; osteoclast; matrix metalloproteinases; breast cancer
Matrix metalloproteinase-2 (MMP-2) and MMP-7 have been implicated in tumor growth and metastasis. This study aimed to investigate the expressions of MMP-2 and -7 in colorectal cancer and to evaluate their values as prognostic markers.
Immunohistochemical staining for MMP-2 and -7 was done in 144 resected colorectal cancer specimens. Clinicopathological data and survival results were compared with regard to the expression results.
The expression rates of MMP-2 in tumor cells in the tumor center and the tumor border were 16.7% and 38.9%, respectively. That of MMP-2 in stromal cells was 27.8%. MMP-7 immunoreactivities of tumor cells in the tumor center and the tumor border were 6.9% and 23.6%. The expressions of MMP-2 and MMP-7 were correlated. MMP-2 expression in stromal cells was more increased in the distal part of the colorectum: 8.8% in right colon cancer, 29.5% in left colon cancer and 36.4% in rectal cancer. MMP-2 expression of tumor cells in the tumor border was correlated with T-stage. MMP-7 expression of tumor cells in the tumor border was increased in case of infiltrative cancer compared with fungating tumor. The expression patterns of MMP-2 and -7 were not correlated with other clinicopathological factors, including tumor markers, node metastasis, distant metastasis, lymphatic invasion, tumor differentiation, and recurrence. No significant associations between the overall and disease-free survival rates and the MMP-2 and -7 expression patterns were noted.
The high expression rates of MMP-2 and -7 in tumor borders suggest that MMP-2 and -7 have some role in tumor invasion, but in this study, MMP-2 and -7 did not appear to be significant predictors of prognosis in colorectal cancer.
Matrix metalloproteinases; Colorectal neoplasms; Prognosis; Immunohistochemistry
Brain metastasis is an increasingly common complication for breast cancer patients; approximately 15– 30% of breast cancer patients develop brain metastasis. However, relatively little is known about how these metastases form, and what phenotypes are characteristic of cells with brain metastasizing potential. In this study, we show that the targeted knockdown of MMP-1 in breast cancer cells with enhanced brain metastatic ability not only reduced primary tumor growth, but also significantly inhibited brain metastasis.
Two variants of the MDA-MB-231 human breast cancer cell line selected for enhanced ability to form brain metastases in nude mice (231-BR and 231-BR3 cells) were found to express high levels of matrix metalloproteinase-1 (MMP-1). Short hairpin RNA-mediated stable knockdown of MMP-1 in 231-BR and 231-BR3 cells were established to analyze tumorigenic ability and metastatic ability.
Short hairpin RNA-mediated stable knockdown of MMP-1 inhibited the invasive ability of MDA-MB 231 variant cells in vitro, and inhibited breast cancer growth when the cells were injected into the mammary fat pad of nude mice. Reduction of MMP-1 expression significantly attenuated brain metastasis and lung metastasis formation following injection of cells into the left ventricle of the heart and tail vein, respectively. There were significantly fewer proliferating cells in brain metastases of cells with reduced MMP-1 expression. Furthermore, reduced MMP-1 expression was associated with decreased TGFα release and phospho-EGFR expression in 231-BR and BR3 cells.
Our results show that elevated expression of MMP-1 can promote the local growth and the formation of brain metastases by breast cancer cells.
Breast cancer; Brain metastasis; MMP-1; TGFα; EGFR
Several lines of evidence support an important role for Snail, a transcriptional factor, in breast cancer. Overexpression of Snail has been associated with breast cancer metastasis, although the specific role of Snail in the process remains unclear. To address this issue, the expression levels of Snail, RhoA and fibronectin, as well as MMP-2, were reduced in the breast tumor cell lines MDA-MB-231 and MDA-MB-435S, and their biological responses were studied in vitro and in vivo. For the first time, it was observed that downregulated Snail expression is correlated with a significant inhibition of the expression and activity of RhoA GTPase, as well as MMP-2. The present data provide evidence that Snail promotes tumor cell motility and angiogenesis which is mainly mediated through the regulation of RhoA activity. In conclusion, the present findings demonstrate a key regulatory role for Snail in breast tumor growth and progression.
Snail; RhoA GTPase; metastasis; breast cancer
Following removal of the primary breast tumour by conservative surgery, patients may still have additional malignant foci scattered throughout the breast. Radiation treatments are not designed to eliminate all these residual cancer cells. Rather, the radiation dose is calculated to optimise long-term results with minimal complications. In a tumour, cancer cells are surrounded by a basement membrane, which plays an important role in the regulation of gene expression. Using an invasion chamber, we have shown that irradiation before cell plating of a reconstituted basement membrane (Matrigel; Becton Dickinson, Bedford, MA, USA) increased the invasiveness of the breast cancer cells MDA-MB-231. This radiation enhancement of invasion was associated with the upregulation of the pro-invasive gene matrix metalloproteinase (MMP)-2. The expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP), which are required to activate the MMP-2, were also increased. Confirming the role of MMP-2 and MT1-MMP, radiation enhancement of cancer cell invasion was prevented by an MMP-2 inhibitor and an anti-MT1-MMP antibody. This study also demonstrated that radiation can potentially enhance the invasion ability by inducing the release of pro-invasive factors stored in the Matrigel. Conversely, no enhancement of invasiveness was observed with the low metastatic cell line MCF-7. This lack of invasiveness correlated with the absence of the MMP-2 activator MT1-MMP in the MCF-7 cells. Radiotherapy is an efficient modality to treat breast cancer which could be further improved by inhibiting the pro-invasive gene upregulated by radiation.
breast cancer; basement membrane; invasion; matrix metalloproteinase; matrikine; radiation
Altered expression of Twist, matrix metalloproteinase (MMP)-2 and MMP-9 proteins has been identified in various types of human cancers. However, the correlation between Twist and these gelatinases in breast cancer remains unclear. In this study, immunohistochemical analysis of Twist, MMP-2 and MMP-9 expression was performed on tissue microarrays from 200 breast cancer cases. The association of Twist and gelatinase expression with clinicopathological factors and patient survival was analyzed. Altered expression of Twist, MMP-2 and MMP-9 proteins was observed in breast cancer tissue. The positive rates of Twist, MMP-2 and MMP-9 protein expression were 75.5, 97.0 and 96.0%, respectively. Increased expression of Twist was positively correlated with the status of axillary lymph node metastasis and higher tumor-node-metastasis (TNM) stage (P<0.01). Moreover, increased expression of Twist was correlated with poor overall survival (OS) and post-operative relapse-free survival (RFS), compared with those for the patients with reduced expression levels of Twist (P<0.05, P<0.01). The expression of MMP-2 and MMP-9 was positively correlated with Twist expression (P<0.001). Our results indicate that Twist may play an important role in the invasion, metastasis and prognosis of breast cancer. Additionally, our results suggest that Twist may be a regulator of gelatinases (MMP-2 and MMP-9).
breast neoplasms; pathology; gelatinases; matrix metalloproteinase 2; matrix metalloproteinase 9; immunohistochemistry
Accumulating evidence suggests that Raf kinase inhibitor protein (RKIP), which negatively regulates multiple signaling cascades including the Raf and nuclear factor κB (NF-κB) pathways, functions as a metastasis suppressor. However, the basis for this activity is not clear. We investigated this question in a panel of breast cancer, colon cancer and melanoma cell lines. We found that RKIP negatively regulated the invasion of the different cancer cells through three-dimensional extracellular matrix barriers by controlling the expression of matrix metalloproteinases (MMPs), particularly, MMP-1 and MMP-2. Silencing of RKIP expression resulted in a highly invasive phenotype and dramatically increased levels of MMP-1 and MMP-2 expression, while overexpression of RKIP decreased cancer cell invasion in vitro and metastasis in vivo of murine tumor allografts. Knockdown of MMP-1 or MMP-2 in RKIP-knockdown cells reverted their invasiveness to normal. In contrast, when examining migration of the different cancer cells in a two-dimensional, barrier-less environment, we found that RKIP had either a positive regulatory activity or no activity, but in no case a negative one (as would be expected if RKIP suppressed metastasis at the level of cell migration itself). Therefore, RKIP’s function as a metastasis suppressor appears to arise from its ability to negatively regulate expression of specific MMPs, and thus invasion through barriers, and not from a direct effect on the raw capacity of cells to move. The NF-κB pathway, but not the Raf pathway, appeared to positively control the invasion of breast cancer cells. A regulatory loop involving an opposing relationship between RKIP and the NF-κB pathway may control the level of MMP expression and cell invasion.
Raf kinase inhibitor protein; Raf/MEK/ERK, NF-κB, matrix metalloproteinases; cancer cell migration, invasion and metastasis
Stromal fibroblasts can contribute to tumor invasion through the release of matrix metalloproteinases (MMPs). Population studies have suggested that single nucleotide polymorphisms (SNPs) in MMP genes influence levels of expression and may be associated with breast cancer risk and with disease progression. This study directly examined the impact of MMP SNP genotype on the ability of host fibroblasts to promote tumor cell invasion.
Primary breast fibroblasts were isolated from patients with (n = 13) or without (n = 19) breast cancer, and their ability to promote breast cancer cell invasion was measured in in vitro invasion assays. Fibroblast invasion-promoting capacity (IPC) was analyzed in relation to donor type (tumor or non-tumor patient), MMP-1, MMP-3, and MMP-9 SNP genotype and MMP activity using independent samples t test and analysis of variance. All statistical tests were two-sided.
Tumor-derived fibroblasts promoted higher levels of invasion than normal fibroblasts (p = 0.041). When IPC was related to genotype, higher levels of IPC were generated by tumor fibroblasts with the high-expressing MMP-3 5A/5A genotype compared with the 5A/6A and 6A/6A genotypes (p = 0.05 and 0.07, respectively), and this was associated with enhanced MMP-3 release. The functional importance of MMP-3 was demonstrated by enhanced invasion in the presence of recombinant MMP-3, whereas reduction occurred in the presence of a specific MMP-3 inhibitor. An inverse relationship was demonstrated between fibroblast IPC and the high-expressing MMP-1 genotype (p = 0.031), but no relationship was seen with MMP-9 SNP status. In contrast, normal fibroblasts showed no variation in IPC in relation to MMP genotype, with MMP-3 5A/5A fibroblasts exhibiting significantly lower levels of IPC than their tumor-derived counterparts (p = 0.04).
This study has shown that tumor-derived fibroblasts exhibit higher levels of IPC than normal fibroblasts and that the MMP-3 5A/5A genotype contributes to this through enhanced MMP-3 release. Despite a high-expressing genotype, normal fibroblasts do not exhibit higher IPC or enhanced MMP release. This suggests that more complex changes occur in tumor-derived fibroblasts, enabling full expression of the MMP SNP genotype and these possibly are epigenetic in nature. The results do suggest that, in women with breast cancer, a high-expressing MMP-3 genotype may promote tumor progression more effectively.
The increased bone degradation in osteolytic metastases depends on stimulation of mature osteoclasts and on continuous differentiation of new pre-osteoclasts. Metalloproteinases (MMP)-13 is expressed in a broad range of primary malignant tumours and it is emerging as a novel biomarker. Recent data suggest a direct role of MMP-13 in dissolving bone matrix complementing the activity of MMP-9 and other enzymes. Tumour-microenvironment interactions alter gene expression in malignant breast tumour cells promoting osteolytic bone metastasis. Gene expression profiles revealed that MMP-13 was among the up-regulated genes in tumour-bone interface and its abrogation reduced bone erosion. The precise mechanism remained not fully understood. Our purpose was to further investigate the mechanistic role of MMP-13 in bone osteolytic lesions.
MDA-MB-231 breast cancer cells that express MMP-13 were used as a model for in vitro and in vivo experiments. Conditioned media from MDA-MB-231 cells were added to peripheral blood mononuclear cultures to monitor pre-osteoclast differentiation and activation. Bone erosion was evaluated after injection of MMP-13-silenced MDA-MB-231 cells into nude mice femurs.
MMP-13 was co-expressed by human breast tumour bone metastases with its activator MT1-MMP. MMP-13 was up-regulated in breast cancer cells after in vitro stimulation with IL-8 and was responsible for increased bone resorption and osteoclastogenesis, both of which were reduced by MMP inhibitors. We hypothesized that MMP-13 might be directly involved in the loop promoting pre-osteoclast differentiation and activity. We obtained further evidence for a direct role of MMP-13 in bone metastasis by a silencing approach: conditioned media from MDA-MB-231 after MMP-13 abrogation or co-cultivation of silenced cells with pre-osteoclast were unable to increase pre-osteoclast differentiation and resorption activity. MMP-13 activated pre-MMP-9 and promoted the cleavage of galectin-3, a suppressor of osteoclastogenesis, thus contributing to pre-osteoclast differentiation. Accordingly, MMP-13 abrogation in tumour cells injected into the femurs of nude mice reduced the differentiation of TRAP positive cells in bone marrow and within the tumour mass as well as bone erosion.
These results indicate that within the inflammatory bone microenvironment MMP-13 production was up-regulated in breast tumour cells leading to increased pre-osteoclast differentiation and their subsequent activation.
Matrix metalloproteinases (Mmps) stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2) in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mmp10 is highly expressed in oncosphere cultures enriched in CSCs and RNAi-mediated knockdown of Mmp10 leads to a loss of stem cell marker gene expression and inhibition of oncosphere growth, clonal expansion, and transformed growth in vitro. Interestingly, clonal expansion of Mmp10 deficient oncospheres can be restored by addition of exogenous Mmp10 protein to the culture medium, demonstrating a direct role for Mmp10 in the proliferation of these cells. Oncospheres exhibit enhanced tumor-initiating and metastatic activity when injected orthotopically into syngeneic mice, whereas Mmp10-deficient cultures show a severe defect in tumor initiation. Conversely, oncospheres implanted into syngeneic non-transgenic or Mmp10−/− mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human cancers reveals a strong positive correlation between tumor Mmp10 expression and metastatic behavior in many human tumor types. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells.