Therapeutic Potential of Matrix Metalloproteinase Inhibition in Breast Cancer

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that cleave nearly all components of the extracellular matrix as well as many other soluble and cell‐associated proteins. MMPs have been implicated in normal physiological processes, including development, and in the acquisition and progression of the malignant phenotype. Disappointing results from a series of clinical trials testing small molecule, broad spectrum MMP inhibitors as cancer therapeutics led to a re‐evaluation of how MMPs function in the tumor microenvironment, and ongoing research continues to reveal that these proteins play complex roles in cancer development and progression. It is now clear that effective targeting of MMPs for therapeutic benefit will require selective inhibition of specific MMPs. Here, we provide an overview of the MMP family and its biological regulators, the tissue inhibitors of metalloproteinases (TIMPs). We then summarize recent research from model systems that elucidate how specific MMPs drive the malignant phenotype of breast cancer cells, including acquisition of cancer stem cell features and induction of the epithelial–mesenchymal transition, and we also outline clinical studies that implicate specific MMPs in breast cancer outcomes. We conclude by discussing ongoing strategies for development of inhibitors with therapeutic potential that are capable of selectively targeting the MMPs most responsible for tumor promotion, with special consideration of the potential of biologics including antibodies and engineered proteins based on the TIMP scaffold. J. Cell. Biochem. 118: 3531–3548, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Matrix metalloproteinases 2 and 9 as the factors of head and neck tumor metastases

The levels of metalloproteinases (MMP-2,-9), their tissue inhibitors (TIMP-1,-2) and extracellular matrix metalloproteinase inducer (EMMPRIN) were studied in tumor tissue and blood serum from patients with head and neck squamous cell carcinoma. Immunohistochemical investigation showed much higher expression of MMP-9 and TIMP-1 in tumor tissue compared with MMP-2 and TIMP-2. There was different distribution of the investigated parameters (except TIMP-1) in cancer cells and stroma. Accumulation of MMP-2, MMP-9, and TIMP-2 was found mainly in cell elements (fibrocytes, leukocytes, etc.) and in stromal extracellular space. Expression of EMMPRIN was significantly higher in tumor cells than in stromal cells. It is possible that carcinoma cells express EMMPRIN, which may increase MMP production by surrounding cells. There was significant decrease of TIMP-1 expression in carcinoma cells with N1 grade of metastasis than in tumors without metastasis. The level of TIMP-1 in blood serum from patients with tumor metastases to regional lymph nodes was lower than in serum from patients without metastases. Thus, MMP-9 and TIMP-1 play an important role in the development of head and neck squamous cell carcinoma and the TIMP-1 level in blood serum and cancer tissues is linked to the first grade of regional lymph node metastasis.     

Herpes simplex virus type 1 preferentially targets human colon carcinoma: role of extracellular matrix

Viral therapy of cancer (viral oncolysis) is dependent on selective destruction of the tumor tissue compared with healthy tissues. Several factors, including receptor expression, extracellular components, and intracellular mechanisms, may influence viral oncolysis. In the present work, we studied the potential oncolytic activity of herpes simplex virus type 1 (HSV-1), using an organ culture system derived from colon carcinoma and healthy colon tissues of mouse and human origin. HSV-1 infected normal colons ex vivo at a very low efficiency, in contrast to high-efficiency infection of colon carcinoma tissue. In contrast, adenoviral and lentiviral vectors infected both tissues equally well. To investigate the mechanisms underlying the preferential affinity of HSV-1 for the carcinoma tissue, intracellular and extracellular factors were investigated. Two extracellular components, collagen and mucin molecules, were found to restrict HSV-1 infectivity in the healthy colon. The mucin layer of the healthy colon binds to HSV-1 and thereby blocks viral interaction with the epithelial cells of the tissue. In contrast, colon carcinomas express small amounts of collagen and mucin molecules and are thus permissive to HSV-1 infection. In agreement with the ex vivo system, HSV-1 injected into a mouse colon carcinoma in vivo significantly reduced the volume of the tumor. In conclusion, we describe a novel mechanism of viral selectivity for malignant tissues that is based on variance of the extracellular matrix between tumor and healthy tissues. These insights may facilitate new approaches to the application of HSV-1 as an oncolytic virus.     

Role of matrix metalloproteinases in development of diabetic nephropathy

This review considers molecular mechanisms that underlie disorders in the structure and metabolism of renal extracellular matrix in diabetic nephropathy. The contribution of the increased synthesis of renal extracellular matrix proteins in the accumulation of renal mesangial matrix is considered, and the important role of the degradation system of the extracellular matrix proteins in the development of fibrosis is also shown. Data on changes in mRNA expression for the matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in various forms of diabetic nephropathy are presented. A correlation is established between changes in the balance of MMP proteolytic activity and TIMP activity and the accumulation of extracellular matrix.     

Elevated membrane-type matrix metalloproteinases in gliomas revealed by profiling proteases and inhibitors in human cancer cells

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate proteolysis of the extracellular matrix and other extracellular proteins, including growth factors and their receptors. The aberrant expression of these genes is common in most cancers. We profiled the RNA levels of every human MMP and TIMP in a variety of cell types (fibroblast, endothelial, hematopoietic, carcinoma, melanoma, and glioma) using quantitative PCR, with the aim of identifying novel expression patterns. Almost all members of the membrane-type (MT-) MMP and TIMP families were elevated in glioma lines compared to carcinomas. In clinical glioma specimens, there were positive correlations between glioma grade and RNA levels of MT-1, MT-2, and MT-6 MMP, TIMP-1 and TIMP-2, and for several growth factors and receptors. These findings suggest that advanced malignant gliomas have elevated levels of membrane-associated MMPs and TIMPs, which may potentially regulate vascularization and invasion. Concurrent elevation of signaling molecules suggests potential bidirectional relationships that enhance tumor aggressiveness.     

In vitro and in vivo matrix metalloproteinase expression after photodynamic therapy with a liposomal formulation of aminolevulinic acid and its methyl ester

Photodynamic therapy (PDT) is a well-known method for the treatment of malignant tumors, and its principles have been well established over the past 30 years. This therapy involves the application of a chemical called a photosensitizer and its subsequent excitation with light at the appropriate wavelength and energy. Topical photodynamic therapy with aminolevulinic acid (5-ALA) is an alternative therapy for many malignant processes, including nonmelanoma skin cancers such as basal-cell carcinoma (BCC). Our novel approach for this study was to use a liposomal formulation of 5-ALA and its methyl ester (commercially available as metvix) both in vitro and in vivo, and to check whether the liposome-entrapped precursors of photosensitizers can induce the expression of metalloproteinases (MMPs) in animal tumor cells and in other tissues from tumor-bearing rats and in selected cell lines in vitro. We also checked whether the application of tissue inhibitors of matrix metalloproteinases (TIMPs) has any effect on MMPs in the above-mentioned experimental models, and if they can cause complete inhibition of MMP expression. Immunohistochemical studies revealed that after the PDT, the intensity of expression of MMPs in healthy animals was very low and seen in single cells only. After the PDT in tumor-bearing rats, MMP-3 was expressed in the tumor cells with the highest intensity of staining in the tissues directly adjacent to the tumors, while MMP-2 and -9 were not found. In the control groups, there was no observed expression of MMPs. In vitro studies showed that MMP-3 was expressed in MCF-7 cells after PDT, but MMP-9 was not observed and MMP-2 was only seen in single cases. Our studies confirmed that the application of an MMP-3 inhibitor may block an induction of MMP-3 expression which had previously been initiated by PDT. The preliminary data obtained from cancer patients revealed that new precursors are effective in terms of PDT, and that using MMP inhibitors should be considered as a potential enhancing factor in clinical PDT.     

Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells.

The relative expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is an important determinant in trophoblast invasion of the uterus and tumor invasion and metastasis. Our previous studies have shown that low oxygen levels increase the in vitro invasiveness of trophoblast and tumor cells. The present study examined whether changes in oxygen levels affect TIMP and MMP expression by cultured trophoblast and breast cancer cells. Reverse zymographic analysis demonstrated reduced TIMP-1 protein secretion by HTR-8/SVneo trophoblast cells as well as MDA-MB-231 and MCF-7 breast carcinoma cells cultured in 1% vs 20% oxygen for 24 h. While gelatin zymography revealed no changes in the levels of MMP-9 secreted by HTR-8/SVneo trophoblasts cultured under various oxygen concentrations for 24 h, human MDA-MB-231 breast carcinoma cells displayed increased MMP-9 secretion and human MCF-7 breast cancer cells exhibited reduced secretion of this enzyme when cultured under similar conditions. In contrast, MMP-2 levels remained unchanged in all cultures incubated under similar conditions. Western blot analysis of MMP-9 protein in cell extracts confirmed the results of zymography. To assess the contribution of enhanced MMP activity to hypoxia-induced invasion, the effect of an MMP inhibitor (llomastat) on the ability of MDA-MB-231 cells to penetrate reconstituted extracellular matrix (Matrigel) was examined. Results showed that MMP inhibition significantly decreased the hypoxic upregulation of invasion by these cells. These findings indicate that the increased cellular invasiveness observed under reduced oxygen conditions may be due in part to a shift in the balance between MMPs and their inhibitors favoring increased MMP activity.     

Extracellular Alkaline pH Leads to Increased Metastatic Potential of Estrogen Receptor Silenced Endocrine Resistant Breast Cancer Cells

Introduction

Endocrine resistance in breast cancer is associated with enhanced metastatic potential and poor clinical outcome, presenting a significant therapeutic challenge. We have established several endocrine insensitive breast cancer lines by shRNA induced depletion of estrogen receptor (ER) by transfection of MCF-7 cells which all exhibit enhanced expression profile of mesenchymal markers with reduction of epithelial markers, indicating an epithelial to mesenchymal transition. In this study we describe their behaviour in response to change in extracellular pH, an important factor controlling cell motility and metastasis.

Methods

Morphological changes associated with cell exposure to extracellular alkaline pH were assessed by live cell microscopy and the effect of various ion pumps on this behavior was investigated by pretreatment with chemical inhibitors. The activity and expression profile of key signaling molecules was assessed by western blotting. Cell motility and invasion were examined by scratch and under-agarose assays respectively. Total matrix metalloproteinase (MMP) activity and specifically of MMP2/9 was assessed in conditioned medium in response to brief alkaline pH exposure.

Results

Exposure of ER –ve but not ER +ve breast cancer cells to extracellular alkaline pH resulted in cell shrinkage and spherical appearance (termed contractolation); this was reversed by returning the pH back to 7.4. Contractolation was blocked by targeting the Na⁺/K⁺ and Na⁺/H⁺ pumps with specific chemical inhibitors. The activity and expression profile of key signaling molecules critical for cell adhesion were modulated by the exposure to alkaline pH. Brief exposure to alkaline pH enhanced MMP2/9 activity and the invasive potential of ER –ve cells in response to serum components and epithelial growth factor stimulation without affecting unhindered motility.

Conclusions

Endocrine resistant breast cancer cells behave very differently to estrogen responsive cells in alkaline pH, with enhanced invasive potential; these studies emphasise the crucial influence of extracellular pH and caution against indiscriminate application of alkalinising drug therapy.     

Redox regulation of matrix metalloproteinase gene family in small cell lung cancer cells

It has been implicated that reactive oxygen species (ROS) play important roles in modulating tumor progression. However, the mechanisms by which redox-regulated tumor progression are largely unknown. We previously demonstrated that reduced intracellular redox conditions could be achieved in stably transfected small cell lung cancer cells with gamma-glutamylcysteine synthetase (gamma-GCSh) cDNA which encodes a rate-limiting enzyme in the biosynthesis of glutathione (GSH), a major physiological redox regulator. In the present study, using DNA microarray analyses, we compared the expression profiles between the gamma-GCSh-transfected cells and their nontransfected counterpart. We observed downregulation of several matrix metalloproteinases (MMPs), i.e., MMPI and MMP3, and MMP10 in the transfected cells. Dot blot and Northern blot hybridizations confirmed that, among the 18 MMP gene family members and four tissue inhibitors of matrix metalloprotein family (TIMP) analyzed, the expression levels of these three MMPs were consistently reduced. Transiently increased gamma-GCSh expression using tetracycline-inducible gamma-GCSh adenoviral expression system also showed down-regulation of MMP3 and MMP10, but not MMP1. Our results demonstrated that redox regulation of MMP1, MMP3 and MMP10 expression depend upon different modes of redox manipulation. These results bear implication that antioxidant modulation of antitumor progression may be contributed at least in part by the downregulation of a subset of metrix metalloproteins.     

Crystal structures of MMPs in complex with physiological and pharmacological inhibitors

Matrix Metalloproteinases (MMPs) are a family of multidomain zinc endopeptidases that function in the extracellular space or attached to the cell membrane. Their proteolytic activity is controlled by the presence of endogenous inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), alpha-macroglobulin and others. Disruption of the proteinase-inhibitor balance is observed in serious diseases such as arthritis, tumor growth and metastasis, rendering the MMPs attractive targets for drug intervention by pharmacological inhibitors. The determination of MMP structures is of critical importance in order to understand their substrate preferences, dimerization events, and their association with matrix components and inhibitors. Thus, MMP structures may contribute significantly to the development of specific MMP inhibitors, which should allow precise control of individual members of the MMP family without affecting all members or the closely related metalloproteinases such as ADAMs and ADAMTSs.     

Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-alpha-converting enzyme

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a dual inhibitor of the matrix metalloproteinases (MMPs) and some adamalysins, two families of extracellular and cell surface metalloproteinases that function in extracellular matrix turnover and the shedding of cell surface proteins. The mechanism of inhibition of MMPs by TIMPs has been well characterized, and since the catalytic domains of MMPs and adamalysins are homologous, it was assumed that the interaction of TIMP-3 with adamalysins is closely similar. Here we report that the inhibition of the extracellular region of ADAM-17 (tumor necrosis factor alpha-converting enzyme (TACE)) by the inhibitory domain of TIMP-3 (N-TIMP-3) shows positive cooperativity. Also, mutations in the core of the MMP interaction surface of N-TIMP-3 dramatically reduce the binding affinity for MMPs but have little effect on the inhibitory activity for TACE. These results suggest that the mechanism of inhibition of ADAM-17 by TIMP-3 may be distinct from that for MMPs. The mutant proteins are also effective inhibitors of tumor necrosis factor alpha (TNF-alpha) release from phorbol ester-stimulated cells, indicating that they provide a lead for engineering TACE-specific inhibitors that may reduce side effects arising from MMP inhibition and are possibly useful for treatment of diseases associated with excessive TNF-alpha levels such as rheumatoid arthritis.     

Interstitial collagenase, gelatinases A and B and their endogenous inhibitors in squamous cell cervical carcinomas

Interstitial collagenase and gelatinases are matrix metalloproteinases (MMP), which play the key role in tumor invasion and metastasis determining tumor malignancy. The aim of this study was to elucidate peculiarities of expression of interstitial collagenase (MMP-1), gelatinases A and B (MMP-2 and MMP-9) and their endogenous tissue inhibitors TIMP-1 and TIMP-2 as invasive factors of squamous cell carcinomas (SCC) of human cervical cancer. The study was carried out using 24 specimens of SCC and 11 specimens of morphologically normal tissue adjacent to tumor. All carcinoma specimens expressed the E7 HPV-16 gene. Results obtained indicate that the increased expression of MMP-1 and MMP-9 and low expression of TIMP-1 and TIMP-2 make the main contribution to the destructive (invasive) potential of SCC. Changes in MMP-2 expression are less important. In the specimens of morphologically normal tissue adjacent to the tumor, substantial expression of MMP-1, MMP-2 and MMP-9 was registered. This expression appears to make additional contribution to the tumor destructive potential.     

Human thyroid carcinoma cell invasion is controlled by the low density lipoprotein receptor-related protein-mediated clearance of urokinase plasminogen activator

The low density lipoprotein receptor-related protein (LRP), a large scavenger receptor reported to mediate the uptake and degradation of various ligands, emerges as a promising receptor for targeting the invasive behaviour of human cancer cells. However, the accurate function of LRP during tumor invasion seems to be highly dependent on cellular context and remains controversial. The expression patterns of both this receptor and the main proteolytic systems involved in cell invasion were examined in two follicular thyroid carcinoma cell lines exhibiting different invasive phenotypes. We established that a low expression of LRP at the cell surface was associated to elevated extracellular MMP2 and urokinase plasminogen activator (uPA) activities as well as to high invasiveness properties. Surprisingly, neither exogenously added receptor-associated protein, an antagonist of LRP, nor LRP blocking antibodies significantly modified the amount of extracellular MMP2. Furthermore, the invasive phenotype of thyroid carcinoma cells was not related to their matrix metalloproteinases amount since different specific inhibitors of these proteases failed to affect the invasive properties of both cell lines. Additionally, blocking LRP-mediated clearance led to a further increase of the uPA amount and activities and to increased invasiveness in both cell lines. Finally thyroid carcinoma cells aggressiveness was widely increased by exogenous uPA; and anti-uPA antibodies treatments abolished both basal and receptor-associated protein-induced thyroid cell invasion. Overall our results identified the LRP-mediated clearance of uPA as one of the mechanisms involved during the control of human thyroid carcinoma cell invasion.     

Development and characterization of a new polyclonal antibody specifically against tissue inhibitor of metalloproteinases 4 in human breast cancer

Tissue inhibitors of metalloproteinases (TIMPs) may regulate extracellular matrix turnover and cellular functions by modulating matrix metalloproteinase (MMP) activity and cell proliferation and apoptosis. To investigate the locations and functions of TIMP-4 in human breast cancer, a highly specific polyclonal anti-TIMP-4 peptide antibody (pAb-T4-S61) was developed. The potency and specificity of the purified IgG were characterized by an enzyme-linked immunosorbent assay, immunoblot, and immunohistochemistry. The optimal IgG concentration range was 0.1-10 microg/ml. pAb-T4-S61 did not cross-react with TIMP-1 and TIMP-2 and should not react with TIMP-3 according to the sequence analysis. Parental MDA-MB-435 breast cancer cells were TIMP-4 negative and a TIMP-4 transfected clone, TIMP-4-435-12, produced TIMP-4. Membrane type-1 MMP was detected although TIMP-2 was not found in these cells. Interestingly, the TIMP-4 protein was detected by immunohistochemical staining in infiltrating breast carcinoma cells in tumor tissues. Thus, pAb-T4-S61 is a useful tool to investigate expression patterns and functions of TIMP-4 in cancers.     

Increased TIMP-1 activity results in increased expression of gelatinases and altered cell motility.

Matrix metalloproteinases are proteolytic enzymes which play a major role in resorption of collagen and other components of the extracellular matrix. They are controlled by specific inhibitors, so-called tissue inhibitors of metalloproteinases (TIMPs). The balance between matrix metalloproteinases and TIMPs seems to play a major role in controlling extracellular matrix homeostasis and cell migration. The influence of TIMP-1 on migration behaviour was explored in human hepatoma cells transiently and stably transfected with mouse TIMP-1, and incubated with biologically active TIMP-1. Transfection and biosynthesis were verified by Northern blotting, Western blotting, metabolic labeling, and reverse zymography. Overexpression of and incubation with TIMP-1 resulted in suppressed migration and seemed to enhance cell-cell contact. Using gelatin zymography and Western blotting we measured a significant increase of matrix metalloproteinases-2 and matrix metalloproteinases-9 in cells transfected with TIMP-1. This new phenomenon may be of important physiological significance in modulating TIMP and MMP expression. Our results indicate a functional involvement of TIMP-1 in matrix homeostasis and some automatic control in matrix turnover.