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www.nature.com/npjbcancer All rights reserved 2374-4677/15 ARTICLE OPEN Breast cancer cell adhesome and degradome interact to drive metastasis 1 1 1,2 1 1 1,2 Asif Rizwan , Menglin Cheng , Zaver M Bhujwalla , Balaji Krishnamachary , Lu Jiang and Kristine Glunde BACKGROUND: Although primary breast tumors are detected early in most cases, it is inevitable that many patients remain at risk for future recurrence and death due to micrometastases. We investigated interactions between the degradome and the adhesome that drive metastasis, and have focused on matrix metalloproteases (MMPs) within the degradome and integrins and E-cadherin within the adhesome. AIMS: The aim of this study is to identify interaction networks between adhesion molecules and degradative enzymes in breast cancer metastasis. METHODS: We compared non-metastatic (BT-474, T47D, MCF7) and metastatic (MDA-MB-231, SUM149, SUM159) human breast cancer cell lines and xenografts, in which we measured growth rate, migration, invasion, colony formation, protein expression, and enzyme activity in vitro and in vivo. RESULTS: The metastatic breast cancer lines and xenografts displayed higher expression and activity levels of MMPs, which was also conﬁrmed by noninvasive imaging in vivo. These metastatic breast cancer models also displayed elevated heterophilic cell-extracellular matrix (ECM) and lower homophilic cell–cell adhesion compared with those of non-metastatic models. This was conferred by an increased expression of the heterophilic cell adhesion molecule integrin β1 (ITGB1) and a decreased expression of the homophilic cell adhesion molecule E-cadherin. Inhibition of MMPs in metastatic cells led to a reduced expression of ITGB1, and stimulation of ITGB1 resulted in higher MMP activities in metastatic cancer cells, demonstrating reciprocal dependencies between degradome and adhesome. Re-expression of E-cadherin (CDH1) led to an increased expression of the precursor form of ITGB1. CONCLUSIONS: Our results point toward a concerted interdependence of MMPs, ITGB1, and CDH1 that is critical for breast cancer metastasis. npj Breast Cancer (2015) 1, 15017; doi:10.1038/npjbcancer.2015.17; published online 28 October 2015 INTRODUCTION signaling complexes with oncogenic Her2, Met, and EGF receptors, and it can recruit MMPs to the cell surface for local degradation of The occurrence of metastasis is the most life-threatening aspect of ECM in human breast cancer. E-cadherin promotes homotypic breast cancer. In the ﬁrst 10 years after diagnosis, up to 12% of tumor cell–cell adhesion and provides intercellular contacts that Stage I/II breast cancer patients, whose cancers are locally conﬁne tumor cells to the primary tumor site. MMPs, integrins, contained within the breast, die. However, 60% of Stage III 8,9 and E-cadherin are all implicated in breast cancer metastasis. patients, whose cancers have invaded nearby lymph nodes, and Although previous studies have shown that integrin activation over 90% of Stage IV patients, whose cancers have spread to controls metastasis in human breast cancer and that the cancer distant organs, die within 10 years after diagnosis. Cancer degradome contributes to tumor progression, invasion, cell–ECM metastasis is driven by networks of proteolytic enzymes, 11,12 communication, and the metabolome, the interaction collectively referred to as the degradome, and networks of between these two groups of molecular players and its role in adhesion molecules, collectively referred to as the adhesome. metastasis is just emerging. Our hypothesis that cell adhesion Matrix metalloproteases (MMPs) are key players of the tumor molecules interact with the degradome is supported by degradome. MMPs act as molecular scissors for cancer cells to (i) co-localized expression of adhesion molecules such as integrins chop and dice components of the extracellular matrix (ECM), such and E-cadherin and degradative enzymes such as MMPs, as collagen1 (COL1) ﬁbers, leading eventually to a remodeled ECM (ii) regulation of MMP activities by integrins and E-cadherin, that provides an escape path for cancer cells to metastasize. More (iii) regulation of the integrin-mediated adhesome by MMPs, than 23 MMPs have been identiﬁed in humans, including 6 (iv) binding of key integrins as well as MMPs to the same membrane-type MMPs. extracellular matrix (ECM) components, and (v) interaction Cancer cell to ECM and cell-to-cell adhesion is, to a large extent, between integrins, E-cadherin, and membrane-bound MMPs in conferred by integrins and E-cadherin. Integrin β1 facilitates cell– 16,18 ECM adhesion and movement of tumor cells into the stroma and cell communication in several types of cancer. Recent ﬁndings thus assists in local invasion within the primary site and growth in also revealed that several types of cancer cells have associations metastatic sites. At least 24 different integrin complexes are between adhesion and degradome molecules. For example, in capable of binding to distinct ECM ligands. Integrin can also form melanoma cells, integrin α β protein binds directly with MMP-2 V 3 The Johns Hopkins University In Vivo Cellular and Molecular Imaging Center, Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD, USA and The Johns Hopkins University School of Medicine, The Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, USA. Correspondence: K Glunde (email@example.com) Received 26 June 2015; revised 2 September 2015; accepted 17 September 2015 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Cancer adhesome and degradome interact to drive metastasis A Rizwan et al and thereby localizes MMP-2 to cell surfaces, and hence assists in Descriptions of cell-speciﬁc culture media are given in Supplementary Materials and Methods under ‘Cell type speciﬁc media used for breast ECM, speciﬁcally collagen, degradation. In human melanoma cancer cell lines’. tdTomato protein expression was detected by cells, MMP-2 cleaves ﬁbronectin into small fragments to enhance ﬂuorescence microscopy using a × 20 objective attached to a Nikon cell adhesion mediated by α β integrin. The co-localization of v 3 inverted microscope, equipped with a ﬁlter set for 528 to 553 nm MMP-14 (MT1-MMP) and integrin β1 is necessary for local invasion excitation and 600 to 660 nm emission and a Nikon COOLPIX digital by human adenocarcinoma, epidermoid carcinoma, and ﬁbro- camera (Nikon Instruments, Inc, Melville, NY, USA). sarcoma cells. It was also shown that MMP-14 and integrin form protein complexes that regulate ﬁbronectin remodeling in MMP expression and relapse-free survival in breast cancer patient murine myoblast cells. In human osteosarcoma cell lines, The relationship of MMP gene expression and relapse-free survival was integrin α β positively regulates the expression of MMP-1 and 2 1 evaluated in an integrated multi-study breast cancer transcriptomic data collagen-1α1. set using Kmplotter (http://kmplot.com). Kaplan–Meier estimates of Over the past 40 years, several generations of MMP inhibitors 10-year relapse-free survivals (RFS) were calculated with data collected have been investigated to potentially treat cancer development from patients with systemic treatment. The median of gene expression and metastasis. The ﬁrst-generation of MMP inhibitors such as was used to dichotomize data into high- and low-expression groups. marimastat, ilomastat (GM6001) and batimastat were hydroxamate- Differences in survival curves were evaluated by log-rank test. Signiﬁcantly based inhibitors based on the structure of collagen. They inhibit different Kaplan–Meier estimators indicate longer RFS for patients with low expression of MMPs compared to the high-expressing group. several MMPs, such as MMP-1, MMP-2, MMP-7 and MMP-9, by 2+ 16 directly binding to Zn ions in the active site. Clinical studies with these hydroxamate-based broad-spectrum MMP inhibitors Gene analysis of adhesome and degradome in breast cancer cell were ineffective at doses associated with musculoskeletal toxicity lines in a phase III trial for metastatic breast cancers that were stable A breast cancer microarray data set (GSE-16975) was analyzed where the after chemotherapy. The second generation of hydroxamate- breast cancer cell lines were grown to optimal cell densities for RNA extraction and hybridization on Affymetrix microarrays. The heat map based MMP inhibitors was more selective for speciﬁc groups of was generated using the Gene-e matrix visualization and analysis platform MMPs, such as for example MMI-166, which is a selective inhibitor (http://www.broadinstitute.org). The heat map represents changes in of MMP-2, MMP-9, and MMP-14. However, they still suffered relative content of adhesome and degradome gene expression levels in from the general limitation of hydroxamate-based MMP inhibitors 17 metastatic breast cancer cell lines (MDA-MB-231, SUM149, SUM159, that drug metabolism leads to the loss of the hydroxamate Zn MDA-MB-468, MDA-MB-436, MDA-MB-157, MDA-MB-175VII, MDA-MB-361, + 22 -binding group and thus were ineffective in human trials. The MDA-MB-435s, BT20, BT549, DU4475, HCC1937, Hs578T, SK-BR-7, SUM102, second generation of non-hydroxamate MMP inhibitors such as SUM1315MO2) and 11 non-metastatic breast cancer cell lines (BT-474, rebimastat, tanomastat, and SB-3CT were developed to avoid T-47D, MCF-7, BT-483, MDA-MB-415, MPE-600, SUM52PE, SUM44PE, ZR-75-1, MDA-MB-134VI, CAMA-1). metabolic inactivation. However, their efﬁcacies were incon- sistent and opposing outcomes were obtained depending on the timing of administration. The third generation of MMP inhibitors, Protein–protein interaction network which are speciﬁc to only one target and possess no zinc-binding Network analysis of potential protein interactions among adhesome and group, is currently being evaluated preclinically. MMPs are also degradome proteins, whose expressions were different in metastatic involved in cytokine and chemokine activation and inactivation, versus non-metastatic cell lines in the Gene-e analysis, was carried out using the STRING-9.1 (http://string-db.org) computational tool and database cell-surface-receptor cleavage and release. The full potential of with a high conﬁdence interval of 0.7. The STRING network, composed of MMP inhibitors can only be explored once all MMP functions and the proteins of interest, is constructed based on genomic context, high- all MMP interactions with other important molecules in cancer throughput experiments, co-expression, and scientiﬁc reports. The network such as growth factors, apoptotic mediators, and adhesion nodes are proteins and edges represent the predicted functional associa- molecules are fully understood. tions. A red line indicates the presence of fusion evidence; a green line In this paper, we have performed comparative studies with a set neighborhood evidence; a blue line co-ocurrence evidence; a purple line of non-metastatic (BT-474, T-47D, MCF-7) versus metastatic (MDA- experimental evidence; a yellow line text-mining evidence; a light blue line database evidence; a black line co-expression evidence. Clustering MB-231, SUM149, SUM159) human breast cancer cell lines and algorithms (K = 2) were used to extract relevant modules. Inter- means xenografts. Experiments were performed to determine growth cluster edges are represented by dashed-lines. rate, migration and invasion, colony formation, adhesion, aggrega- tion, protein expression (western blot) and activity (zymography). In vitro assays Enzymatically activatable optical imaging probes were utilized to study in vivo tumor degradome activity and angiogenesis. The We performed a number of assays to characterize the tdTomato-expressing breast cancer cell lines used in our studies. Cell proliferation was assessed results provided novel insights into the molecular networks that with the WST-1 assay. A series of protease activity and adhesion assays were comprise the cancer adhesome and degradome in breast cancers, performed such as zymography to assess MMP activities, cell adhesion and tested how their combined expression and activation drives assays, hanging drop assays, cell aggregation assays, clonogenic assays, cancer growth, invasion, ECM remodeling, and metastasis. Our quantitative reverse transcription PCR (qRT-PCR), immunoblotting protein results also suggest that, collectively, the adhesome and degra- assays, cell migration, and invasion assays. E-cadherin transfection was done dome molecules expressed in a given tumor can predict the using E-cadherin-GFP, which was a kind gift from Jennifer Stow (Addgene metastatic risk of this primary tumor. plasmid # 28009). Experimental details are provided in Supplementary Materials and Methods under ‘In vitro assays’. MATERIALS AND METHODS In vivo and ex vivo ﬂuorescence imaging Cell culture Optical imaging was carried out using the Xenogen IVIS 200 Spectrum Human breast cancer cell lines, MDA-MB-231, SUM149, SUM159, BT-474, system. Enzymatically activatable optical imaging probes MMPSense-680 T-47D, and MCF-7, were obtained from the American Type Culture (NEV10126), and AngioSense-750 (NEV10011EX) from PerkinElmer Collection (ATCC, MD, USA). Cells were stably transfected with a construct (Waltham, MA, USA) were injected into the tail veins of mice growing containing cDNA of tdTomato as outlined in the Supplementary Materials orthotopic tumor xenografts according to the manufacturer’s protocol. and Methods under ‘Cell lines’. Human mammary ﬁbroblasts were a kind MMPSense is activated by MMP-2, -7, -9, -12, -13, and -14. AngioSense gift from Dr Gary Luker at the University of Michigan. All cells were injection enables imaging of tumor blood vessels and was used for incubated at 37 °C with 5% CO in a humidiﬁed incubator. normalization of uneven delivery of the probes to the tumor. Each mouse npj Breast Cancer (2015) 15017 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Cancer adhesome and degradome interact to drive metastasis A Rizwan et al was imaged at 24 h after the injection with the IVIS camera settings at 1 protein interaction data from multiple sources for a large number and 2 s exposure time, binning factor of 8, ﬁeld of view of 18.8 cm, and f of organisms. Using STRING, we identiﬁed several members of the number of 2. Then, the animals were killed and tumor xenografts and MMP family (MMP-1, -2, -3, -7, -9, -11, -13, -14, -16, -19), integrin lungs were excised for ex vivo ﬂuorescence imaging. Four to six 2-mm thick family (ITGA-1, -3, -4-, -5, -6, -V, ITGB-1, -3) and the E-Cadherin fresh tissue sections were cut from the primary tumor using an adjustable molecule that have direct and indirect associations with each tissue slicer (Braintree Scientiﬁc, Braintree, MA, USA). Tumor sections and other, as well as associations with major ECM components such as whole lungs were imaged with IVIS camera settings at 1- and 2-s exposure collagen, laminin, and ﬁbronectin (Figure 1c). E-cadherin directly time, binning factor of 8, ﬁeld of view of 6.4 cm, and f number of 2. All interacts with MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-14, experiments were carried out according to the approved guidelines of the and MMP-19. ITGB-1 directly and indirectly interacts with MMP-2, Institutional Animal Care and Use Committees (IACUCs) of the Johns -9, and -14. Overall, E-cadherin and the MMPs have a larger Hopkins University. number of interactions and thus form a cluster. The integrins form a cluster with the major ECM molecules such as collagen, Ex vivo preparation of tumor sections and histopathology ﬁbronectin, and laminin. Following ex vivo imaging, tissues were ﬁxed in 4% paraformaldehyde for 24 h andembeddedinparafﬁn blocks. Serial sections of 5-μm thickness were cut from the formalin ﬁxed, parafﬁn embedded tissue blocks and ﬂoated onto Clinical relationship between the degradome and metastasis in charged glass slides (Super-Frost Plus, Fisher Scientiﬁc, Pittsburgh, PA, breast cancer 31,32 USA). A hemotoxylin and eosin stained section was obtained from each Breast tumors with an overexpression of MMPs are associated with tissue block. Unstained sectioned were further processed for immunohisto- 9 invasion and metastases. In our analysis of clinical data sets taken chemical detection of MMP-1, MMP-9, and ITGB-1 as detailed in 36 from the KM-Plotter database, we show that signiﬁcantly Supplementary Materials and Methods under ‘Immunohistochemistry’. increased expression levels of MMP-2 (P = 1.5e − 6), MMP-9 (P = 0.027), and MMP-14 (P = 0.00051) were predictive of a Quantiﬁcation and statistical analysis decreased RFS in chemotherapy treated ER- human breast cancer Statistically signiﬁcant differences between quantitative measurements patients (n = 211). High expression levels of MMP-1 (P = 0.00012) were analyzed by unpaired Student’s t-test. Po0.05 was considered and MMP-9 (P = 0.00022) were predictive of a decreased RFS in statistically signiﬁcant. Box-and-Whisker plots for cell migration, invasion, endocrine treated ER+ human breast cancer patients (n = 690) as and adhesion were generated by BoxPlotR (Montréal, Québec, Canada), shown in Figure 1d. Overall, our data analysis supports the roles of where the center lines show the medians, box limits indicate the 25th and MMPs in breast cancer aggressiveness. High expression of MMP-2 75th percentiles as determined by R software (Vienna, Austria), whiskers (P = 1.5e−6), MMP-9 (P = 0.027), and MMP-14 (P = 0.00051) is extend 1.5 times the interquartile range from the 25th to 75th percentiles, predictive of lower RFS in chemotherapy treated for ER − human and outliers are represented by dots. A one-way analysis of variance was breast cancer patients. High expression of MMP-1 (P = 0.00012), calculated from the number of migrated or invaded cells in all experimental groups. Post hoc comparisons using the Fisher least and MMP-9 (P = 0.00022) is predictive of lower RFS in endocrine signiﬁcant difference were explored to compare the mean of one group treated ER+ human breast cancer patients. These Kaplan–Meier with the mean of another group. Po0.05 was considered statistically curves were generated using KM plotter from http://kmplot.com. signiﬁcant. In vivo MMP activity was measured for each pixel by calculating the ratio of MMPsense divided by Angiosense optical signal intensity using Characterization of constitutively tdTomato-expressing metastatic in-house software written in Matlab (Natick, MA, USA). Box plots for MMP and non-metastatic breast cancer models activity were also generated using Matlab. We have performed comparative in vitro and in vivo/ex vivo experiments with human breast cancer cell lines and xenografts as RESULTS outlined in Supplementary Figure 1A. All cell lines such as MDA- Adhesome and degradome molecules in metastatic versus non- MB-231, SUM149, SUM159, BT-474, T-47D, MCF-7 were engineered metastatic breast cancers to constitutively express tdTomato ﬂuorescent protein for asses- sing tumor growth and metastatic spread by optical imaging In an analysis of 1,144 genes from a panel of 28 human breast in vivo and ex vivo (Supplementary Figures 1B–D). Proliferation cancer cell lines given in the GEO data set GSE16795, we have assays of the tdTomato-expressing breast cancer cell lines demonstrated that metastatic compared with non-metastatic demonstrated that all three metastatic lines grew signiﬁcantly breast cancer cells contain signiﬁcantly (Po0.01) increased faster than the non-metastatic lines (Supplementary Figure 1E). messenger RNA (mRNA) gene expression levels of integrin α-1, Clonogenic assays of the tdTomato-expressing breast cancer cell α-4, α-5, α-6, α-V, and β-1, a decreased level of E-cadherin, and lines were performed to examine whether a single cell can grow increased levels of MMP-2, -3, -11, -14, -16, and -19 as shown in into a colony in uncoated, collagen1 coated, and matrigel-coated Figure 1a. Some genes display signiﬁcant changes in more than plates within 2 weeks (Supplementary Figures 2A and B ). one microarray probe. To compare metastatic nodules with the Metastatic tdTomato-expressing MDA-MB-231 and SUM159 cells corresponding primary breast cancers, we have analyzed the GEO grew a signiﬁcantly (Po0.01) higher number of colonies data set GSE2603, where the lung-metastatic nodules of tail-vein- compared with all three tdTomato-expressing non-metastatic cell injected MDA-MB-231 breast tumor xenografts were expanded in lines on uncoated, collagen1 coated, and matrigel-coated plates. cell culture. The lung-metastatic (LM) cell lines showed an Metastatic tdTomato-expressing SUM149 cells formed fa signiﬁ- increased gene expression of integrin α-1, α-4, α-5, α-6, α-V, and cantly (Po0.01) increased number of colonies on uncoated β-1, E-cadherin,and MMP-1, -2, -3, -11, -16, and -24 compared with surfaces, but not in collagen1, and matrigel-coated surfaces, wild-type MDA-MB-231 breast cancer cells (WT) as shown in Figure 34 compared with all three non-metastatic cell lines. 1b. It should be noted here that tail vein injected breast cancer We also tested the migration and invasion capabilities of all cells represent a subpopulation of cancer cells with increased tdTomato-expressing cell lines. In transwell migration and invasion tendency to colonize a particular organ. assays, signiﬁcantly fewer non-metastatic tdTomato-expressing cell lines (BT-474, T-47D, MCF-7) migrated or invaded through the Analysis of interacting proteins within the degradome and 8 μm pores of a transwell chamber compared with metastatic adhesome tdTomato-expressing cell lines (MDA-MB-231, SUM149, SUM159; To identify known and potential protein–protein interactions Figures 2a and b). In these migration and invasion assays, around relevant to the adhesome and degradome, we used the STRING 90% of tdTomato-expressing SUM149 and SUM159 cells were 9.1 software and database. STRING quantitatively integrates detected on the bottom surface of the insert and the remaining © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15017 Cancer adhesome and degradome interact to drive metastasis A Rizwan et al 10% were attached to the plate well. For tdTomato-expressing attached to the well. All three metastatic cell lines showed higher MDA-MB-231 cells, around 20% of the cells migrated or invaded to migration and invasion compared with the migration and invasion the bottom surface of the insert, while the remaining 80% were by the non-metastatic cell lines (Supplementary Table 1). It should Metastatic Non-metastatic WT LM ER- patients, Chemotherapy treated - - 100 - 100 100 - 100 - - - 80 - 80 80 80 - - - 60 - 60 60 60 - - - 40 - 40 40 40 - - - 20 - 20 20 20 MMP-1 Low MMP-2 Low MMP-9 Low MMP-14 Low - - MMP-9 High - 0 - MMP-1 High 0 MMP-2 High 0 0 MMP-14 High P = 0.073 P = 1.5e-6 P = 0.027 P = 0.00051 0 20 40 60 80 100 120 020 40 60 80 100 120 0 20 40 60 80 100 120 020 40 60 80 100 120 Months ER+ patients, Endocrine treated - - - - 100 100 100 100 - - - - 80 80 80 80 - - - - 60 60 60 60 - - - - 40 40 40 40 - - - - 20 20 20 20 MMP-1 Low MMP-2 Low MMP-9 Low MMP-14 Low MMP-2 High MMP-9 High MMP-14 High - MMP-1 High - - - 0 0 0 0 P = 0.14 P = 0.00022 P = 0.00012 P = 0.032 Months 020 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Figure 1. Analysis of publicly available microarray messenger RNA (mRNA) expression data from clinical breast tumors and breast cancer cell lines. (a) Expression levels of key adhesion molecules and key degradative enzymes discriminate between metastatic (n= 17) and non-metastastic (n= 11) human breast cancer cell lines (GSE16795). (b) MMPs, integrins and E-cadherin that are increased in subpopulations of lung-metastatic MDA-MB-231 (LM) compared with parental MDA-MB-231 (WT) cells (GSE2603). (c) Protein interaction network of major cell adhesion and degradome molecules differentially expressed in metastatic versus non-metastatic cell lines. (d) Kaplan–Meier curves show that high expression levels of MMP-2, MMP-9, and MMP-14 are predictive of decreased relapse-free survival in chemotherapy treated ER − human breast cancer patients, while high expression levels of MMP-1 and MMP-9 are predictive of decreased relapse-free survival in endocrine treated ER+ human breast cancer patients. npj Breast Cancer (2015) 15017 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Percent of relapse Percent of relapse free survival free survival Cancer adhesome and degradome interact to drive metastasis A Rizwan et al Migration Invasion 100 100 50 50 150 150 100 100 50 50 0 0 Pro-MMP-9 MMP-9 Active MMP-9 β-Actin Pro-MMP-2 Active MMP-2 Pro-MMP-9 Active MMP-9 Pro-MMP-2 Active MMP-2 Figure 2. Invasion and MMP expression and activity in human breast cancer cell lines. (a) Breast cancer cell lines were allowed to migrate or invade matrigel for 48 h, and were stained with crystal violet. Representative photographs of the membrane insert and well from the transwell migration and invasion assays are shown. Average numbers of migrating and invading cells per ﬁeld of view were plotted as Box-and-Whisker plots. Statistical testing for signiﬁcant differences is shown in Supplementary Table 1. (b) In vitro MMP-2 and MMP-9 activity were measured using gelatin zymography. Conditioned media were collected after 24 h of incubation. Photographs show pro-MMP-2 (72 kDa), active MMP-2 (62 kDa), pro-MMP-9 (90–100 kDa), and active MMP-9 (82 kDa). (c) MMP-2 and MMP-9 activity measured by gelatin zymography of breast cancer cells that were co-cultured with HMF for 24 h. (d) Western blots showing MMP-9 expression in breast cancer cell lines. High MMP-9 expression was observed in metastatic lines, whereas non-metastatic lines and ﬁbroblasts expressed small amounts or no MMP-9. ß-actin was used as loading control. (e) Immunohistochemistry analysis showing MMP-9 expression in sections from metastatic breast tumor xenografts. Scale bars, 100 μm. be noted here that the migration and invasion properties of associated with increased activities of MMP-2 and MMP-9. To tdTomato-expressing cells and wild-type cells were comparable investigate the possible involvement of these proteases in our cell lines, gelatin zymography analyses from serum-free conditioned for all of the six cell lines that we have used in our experiments media were performed. As shown in Figure 2b, active MMP-9 was (data not shown). detected in medium conditioned by MDA-MB-231 cell and MMP-2 was detected in medium conditioned by SUM149 and SUM159 MMP expression and activity proﬁles in metastatic versus cells. Cancer-associated ﬁbroblasts are also known to contribute to non-metastatic breast cancer cell lines and xenografts MMP activity in vivo. We have examined the conditioned media Breakdown of basement membrane is a critical step for tumor of cancer cells co-cultured at a 1:1 ratio with human mammary invasion. Loss of basement membrane type IV collagen is ﬁbroblasts (HMF) with gelatin zymography. HMF alone are able to © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15017 Migration-2 Migration-1 (Falls to bottom (Below the of the well) Transwell) #Cells per field #Cells per field HMF+ MDA-MB-231 MDA-MB-231 MDA-MB-231 HMF+ SUM149 SUM149 SUM149 HMF+ SUM159 SUM159 SUM159 HMF+ BT-474 BT-474 BT-474 T-47D T-47D HMF+ T-47D HMF+ MCF-7 MCF-7 MCF-7 HMF Invasion-2 Invasion-1 SUM149 MDA-MB-231 (Falls to bottom (Bellow the of the well) Transwell) #Cells per field #Cells per field MDA-MB-231 MDA-MB-231 SUM149 SUM149 SUM159 T-47D BT-474 SUM159 BT-474 BT-474 T-47D T-47D MCF-7 MCF-7 HMF Cancer adhesome and degradome interact to drive metastasis A Rizwan et al Tumor Bright Field MMPSense/ Tumor Bright Field MMPSense/ AngioSense AngioSense BT-474 MDA- MB-231 SUM149 T-47D Figure 3. Analysis of MMP activities in breast cancer xenograft models. (a) Ex vivo ﬂuorescence images of MMP activity in fresh breast tumor xenograft slices measured following MMPSense and AngioSense injection. Scale bar, 5 mm. (b) Box plots show the intensity of MMP ﬂuorescent signals normalized to AngioSense to normalize for uneven delivery of the probe. *Po0.05. (c) Lungs of mice growing metastatic breast cancer xenografts displayed increased MMP activity. Scale bar, 5 mm. Corresponding H&E stains showed micrometastases in these lungs as pointed out by arrows. Scale bar, 2.5 mm. produce pro-forms and active forms of MMP-2 and MMP-9. lungs of mice growing primary tumor xenografts were also The activity of active MMP-2 was enhanced when HMF were imaged for MMP activity. MMP activity in the lungs of mice with co-cultured with MDA-MB-231, and active MMP-9 activity was metastatic primary tumors was evident prior to metastatic seeding enhanced when HMF were co-cultured with SUM149 or SUM159 in the lungs, indicating that the secretion of degradative enzymes cancer cells (Figure 2c). Western blot analysis of cell lysates by the metastatic tumor or stroma occurs well in advance of revealed higher expression levels of MMP-9 in the metastatic cell metastatic seeding in distant organs, which is in good agreement lines, but no expression in the non-metastatic cell lines (Figure 2d). with previous studies. Immunohistochemical staining of tumor xenograft sections showed higher MMP-9 expression levels in the metastatic versus Strong heterophilic and weak hemophilic adhesion signature in non-metastatic xenograft models (Figure 2e). Collagenase-type primary metastatic breast cancer cell lines and tumor xenografts MMP-13 and membrane-type MMP-14 expression did not show Metastatic cells displayed increased adhesion compared to non- any signiﬁcant change across the tested panel of cell lines. metastatic cells on ECM surfaces as measured by cell adhesion However, collagenase MMP-1 and MMP-8 were signiﬁcantly assay shown in Figure 4a. The immunoblots of cells, as well as increased in metastatic cell lines as seen by immunohistochem- immunohistochemistry (IHC) of tumor xenografts, consistently istry (IHC) and western blot, respectively (Supplementary Figures demonstrated an increased expression of the heterophilic 3A and B). adhesion molecule integrin β1 (ITGB1) in metastatic tumors Using MMP-activatable ﬂuorescent imaging agents that are compared to non-metastatic tumors (Figures 4b and c). Metastatic activated by key MMPs, we observed that metastatic breast cell lines showed decreased cell aggregation in the hanging drop tumors displayed increased MMP activities compared with non- assay and in collagen1 gel (Figure 5a). When co-cultured with metastatic breast tumors, which was shown in fresh 2-mm thick HMF, metastatic cells displayed increased adhesion to ﬁbroblasts tumor sections ex vivo in Figure 3a. The resulting quantitative in the hanging drop assay (Figure 5b). Compared with non- MMP activities, normalized to perfusion to account for agent delivery to the tumor, are shown in a box plot in Figure 3b. The metastatic cells, metastatic cells had a signiﬁcantly decreased npj Breast Cancer (2015) 15017 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited MDA-MB-231 SUM149 SUM159 BT-474 T-47D MCF-7 Cancer adhesome and degradome interact to drive metastasis A Rizwan et al ITGB1-M- β-Actin Uncoated plate Collagen1 coated plate Matrigel coated plate MDA-MB-231 BT-474 Figure 4. Analysis of cell adhesion and integrin β1 expression in breast cancer cell lines and xenograft models. (a) Representative photographs of cell adhesion assays where plates were coated with collagen1 (50 μg/ml). The column graphs represent quantitative cell adhesion assays on uncoated, collagen 1 coated, and matrigel-coated plates. Light absorbance from the WST-1 assay, which is proportional to the number of cells adhering to the plate, is displayed on the y-axis. Values are mean± s.d. *Po0.05. (b) Immunoblots showed signiﬁcant differences in mature integrin β1 expression in highly expressing metastatic cells compared with non-expressing non-metastatic cells. (c) Immunohistochemistry analysis demonstrated strong integrin β1 expression in metastatic MDA-MB-231 tumor sections, but not in non-metastatic BT-474 tumor sections. Scale bar, 200 μm. 44,45 expression level of the homophilic adhesion molecule E-cadherin breast cancer cells. Nevertheless, we observed that E-cadherin (CDH1) both in vitro and in vivo (Figures 5c–e). re-expression at the mRNA level reduced protein expression of the mature form of integrin β1 in metastatic MDA-MB-231 and SUM159 cells (Figure 6d, Supplementary Figure 4). A signiﬁcantly Interaction of degradome and adhesome lower number of cells with E-cadherin re-expression were able to The major integrin β1 binding site is an Arg–Gly–Asp (RGD) migrate or invade in transwell assays (Figure 6e). peptide, which is present in a variety of ligands, which are part of the ECM such as collagen, laminin, and ﬁbronectin and thereby represents a major recognition system for cell adhesion. We DISCUSSION analyzed the effect of an RGD-containing peptide, namely Our study presents several important ﬁndings: (i) Increased Arg–Gly–Asp–Ser (RGDS), on the regulation of MMP secretion in expression and activity of MMP-2 and MMP-9 among others in cultured cells. When RGDS peptide was added to cell culture metastatic breast cancer cell lines, xenografts, and lungs as medium, the secreted gelatinases MMP-2 and MMP-9 increased in compared with non-metastatic lines. (ii) Heterophilic adhesion, the metastatic MDA-MB-231 and SUM159 cell lines, as shown in likely mediated by integrin β1, is increased in metastatic Figure 6a. However, cellular integrin β1 protein, as analyzed by compared to non-metastatic lines. (iii) Homophilic adhesion, likely western blot, did not show any differences in expression level mediated by E-cadherin, is increased in non-metastatic compared when the cells were treated with various concentrations of RGDS to metastatic lines. (iv) Re-expression of E-cadherin reduced the (Figure 6b). On the other hand, the mature form of integrin β1 expression of the mature form of integrin β1 in metastatic breast protein signiﬁcantly decreased (Po0.01) when metastatic cancer cells. (v) A reciprocal interaction exists between integrin β1 MDA-MB-231 and SUM159 cells were treated with the broad- and MMP-2/MMP-9 in metastatic breast cancer cells. spectrum MMP inhibitor marimastat or with the MMP-2 and We observed that MMP-2 and MMP-9 activities, among other MMP-9 inhibitor SB-3CT for 48 h (Figure 6c). This suggests a MMP activities, are increased in metastatic breast cancer cell lines, reciprocal regulatory relationship between MMP-2/MMP-9 and xenografts, and lungs as compared with non-metastatic lines. Our integrin β1. It should be noted that the fourth amino-acid S in analysis of publicly available clinical and cellular expression data RGDS contributes toward the stability of the RGDS conﬁrmation to revealed that low levels of MMP-1, -2, -9, and -14 are important for ﬁt the integrin receptors. Cells that were cultured in medium breast cancer survival. However, a slew of other MMPs also confer containing RGD sequence alone did not show any changes in the breast cancer metastasis, depending on the particular cell lines amount of MMP secretion. studied. Since elevated MMPs are an important component of Next, we transfected MDA-MB-231 and SUM159 cells to many aggressive tumors, it is a potential drug target for cancer re-express the E-cadherin gene. Although we were able to therapy. Despite promising preclinical data, clinical trials using detect E-cadherin gene expression by qRT-PCR in the transfected MMP inhibitors resulted in inconsistent outcomes. As evident from cells (Supplementary Figure 3), E-cadherin protein was not our data and the data of others, a key issue is that the types and detected by western blot, most likely due to post-translational levels of MMP expression and activity are quite variable across E-cadherin degrading mechanisms that are present in metastatic different breast tumors and the derived breast cancer cell lines, © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15017 Absorbance Absorbance Absorbance MDA-MB-231 SUM149 SUM159 BT-474 T-47D MCF-7 ITGB1 MDA-MB-231 SUM149 SUM159 BT-474 T-47D MCF-7 Cancer adhesome and degradome interact to drive metastasis A Rizwan et al Hanging Drop Hanging Drop (Cancer Cells/ Fibroblasts) Collagen Gel Phase Contrast E-Cadherin β-actin E-Cadherin MDA-MB-231 BT-474 Figure 5. Homophilic and heterophilic cell adhesion characteristics and E-cadherin expression in breast cancers. (a) Metastatic cell lines showed lower cell aggregation in hanging drop assays and collagen 1 gel as compared to non-metastatic cell lines. Scale Bar, 200 μm. (b) Equal numbers of red-labeled breast cancer cells and green-labeled ﬁbroblasts were mixed and evaluated in the hanging drop assay. Metastatic cells adhered to ﬁbroblasts and formed a mixed population. Non-metastatic cells remained aggregated in a sphere and separated from ﬁbroblast cells. Scale Bar, 200 μm. (c) Western blots showed signiﬁcant differences in E-cadherin expression in lowly expressing metastatic cells compared with highly expressing non-metastatic cells. (d) Immunoﬂuorescence staining demonstrated lower expression of E-cadherin in metastatic cells as compared to non-metastatic cells. (e) Immunohistochemistry analysis showing weak E-cadherin expression in metastatic MDA-MB-231 tumor sections, and high E-cadherin expression in non-metastatic BT-474 tumor sections. Scale bar, 200 μm. which is one of the main reason for the inconsistent outcomes in which degrade mammary basement membrane, the dense ECM clinical trials using MMP inhibitors. However, systemic treatment surrounding the tumor, and the ECM of the walls of blood vessels. with MMP inhibitors would be a good way of treating breast In our study, E-cadherin was increased in non-metastatic breast cancer metastasis, as MMP activity in metastatic sites is cancer cell lines that adhere well to each other but not to upregulated during or even prior to metastatic seeding, as also ﬁbroblasts, as compared with metastatic breast cancer cell lines. observed in the lungs of our metastatic tumor xenograft model. This ﬁnding is in good agreement with the suggestion that We showed that mature active integrin β1 expression was dynamic E-cadherin-mediated cell–cell adhesions and integrin- elevated in metastatic breast cancer cell lines that adhered to the mediated cell–ECM adhesions govern the invasive and metastatic ECM components collagen 1 and matrigel, while non-metastatic potential of tumors. Loss of E-cadherin results in the weakening cell lines contained no integrin β1 and did not adhere well. We of cell–cell adhesion. On the other hand, increase in integrin-β1 also observed that metastatic breast cancer cell lines displayed subunits mediates cell–ECM interactions by linking signals from increased heterophilic adhesion. It is known that integrin the environment to the actin cytoskeleton. Simultaneously, these heterodimers containing β1 subunits are receptors for various two processes enhance the agility of metastatic breast cancer types of ECM molecules such as collagens, laminins, ﬁbronectin, cells, which let them respond to external signals and execute and tenascin, and thus have an essential role in cell–ECM successful migration and invasion. adhesion. In addition, β1 integrins initiate signaling cascades We observed that E-cadherin gene re-expression in metastatic in the cell in response to extracellular chemokines (outside-in), breast cancer cells reduced the amount of mature integrin β1 and also transmit intracellular signals that change the way the protein in metastatic breast cancer cells. In the clinical setting, it cells interact with the ECM (inside-out). These signaling pathways was shown that the secondary metastatic site can induce the regulate cell adhesiveness by changing the conformation of β1 re-expression of E-cadherin in cancer cells, which is a critical step 5 47 integrin binding to the ECM. These dynamic adhesion processes in the survival of cancer cells in the new microenvironment. The are crucial for conferring the migration abilities of cancer cells, effect of re-expressing E-cadherin on integrin β1-subunits has, to npj Breast Cancer (2015) 15017 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited MDA-MB-231 E-Cadherin IHC SUM149 MDA-MB-231 SUM159 SUM149 BT-474 SUM159 T-47D BT-474 MCF-7 T-47D MCF-7 HMF+ MDA-MB-231 MDA-MB-231 HMF+ SUM149 SUM149 HMF+ SUM159 SUM159 HMF+ BT-474 BT-474 HMF+ T-47D T-47D HMF+ MCF-7 MCF-7 Cancer adhesome and degradome interact to drive metastasis A Rizwan et al the best of our knowledge, not been studied so far. Our data and switch back to binding to cells instead of ECM when they suggest that reducing the amount of active integrin β1 increases arrest and colonize distant sites. the expression of E-cadherin. Taken together, our data indicate We found that RGD-stimulation of metastatic breast cancer cells upregulated the expression of MMP-2 and MMP-9. Vice versa, that a reciprocal interaction exists between E-cadherin and integrin β1, which couples homophilic cell–cell and heterophilic when we inhibited MMP activities in cell culture by either Marimastat or SB-3CT, the expression of integrin β1 was cell–ECM adhesion in metastatic breast cancer cells. This will enable metastatic breast cancer cells to lose contact to other cells signiﬁcantly reduced. Our experiments with the MMP inhibitors Marimastat and SB-3CT showed for the ﬁrst time that at a time when they start binding to the ECM while they migrate, MDA-MB-231 SUM159 MDA-MB-231 SUM159 ITGB1 β-Actin MMP9 MMP2 RGDS (μM) 0 10 100 010 100 RGDS (μM) 0 10 100 0 10 100 MDA-MB-231 SUM159 ITGB1-m ITGB1-pre β-Actin ITGB1-m β-actin 12 3 1 2 3 1. No Treatment 2. Marimastat 3. SB-3CT Migration Invasion P=0.02 P=0.0008 P=0.02 P=0.002 200 100 P=2E-5 NS P=0.03 NS 200 100 0 0 Figure 6. Interaction of MMPs, integrin β1, and E-cadherin in metastatic breast cancer cells. (a) MMP-9 and MMP-2 activities in conditioned media from metastatic breast cancer cell lines were analyzed at 48 h after treatment with the integrin β1 binding tetrapeptide RGDS. Secreted MMPs increased with increasing concentrations of RGDS. Values are mean± s.d. *Po0.05. (b) Cellular integrin β1 protein, as analyzed by western blot, did not show any differences when the cells were treated with RGDS. (c) Cellular integrin β1 protein levels signiﬁcantly decreased when metastatic breast cancer cells were treated with the broad-spectrum MMP inhibitor Marimastat or with the MMP-2 and MMP-9 speciﬁc inhibitor SC-3BT for 48 h. Values are mean± s.d. *Po0.05. (d) E-cadherin re-expression in metastatic MDA-MB-231 and SUM159 breast cancer cells resulted in reduced expression of mature integrin β1 and elevated expression of the precursor form of integrin β1as compared with empty vector (ev) transfected control cells. (e) E-cadherin re-expressing metastatic cell lines displayed reduced migration and invasion as compared with the corresponding empty vector (ev) control cells. © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15017 Relative signal (A.U.) Relative signal mature ITGB1 (%) Migration-2 Migration-1 (Falls to bottom (Below the of the well) Transwell) #Cells per field #Cells per field MDA-MB-231-ev MDA-MB-231-ECad SUM159-ev SUM159-ECad Invasion-2 Invasion-1 (Falls to bottom (Bellow the of the well) Transwell) #Cells per field #Cells per field ITGB1 mature MDA-MB-231-ev (A.U.) MDA-MB-231-ECad 231-ev SUM159-ev 231-Ecad 159-ev SUM159-ECad 159-Ecad0 Cancer adhesome and degradome interact to drive metastasis A Rizwan et al inhibition of MMP activities can reduce the expression of active 2 Okegawa T, Pong RC, Li Y, Hsieh JT. The role of cell adhesion molecule in cancer progression and its application in cancer therapy. Acta Biochim Pol 2004; 51: integrin β1, suggesting an association between active 445–457. MMP-2/MMP-9 and integrin β1 protein expression in metastatic 3 Birkedal-Hansen H, Moore WG, Bodden MK, Windsor LJ, Birkedal-Hansen B, breast cancer cells. It has previously been shown in human DeCarlo A et al. 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The binding of Mss4 to In summary, our study shows for the ﬁrst time that cancer cell alpha-integrin subunits regulates matrix metalloproteinase activation and ﬁbro- nectin remodeling. FASEB J 2007; 21: 497–510. adhesome and degradome interact in metastatic breast cancer 20 Riikonen T, Westermarck J, Koivisto L, Broberg A, Kahari VM, Heino J. Integrin cells, and are modulated during migration and invasion of these alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1 cancer cells. These results suggest that targeting nodes in the (I) gene expression. J Biol Chem 1995; 270: 13548–13552. adhesome-degradome network of breast cancer may be an 21 Whittaker M, Floyd CD, Brown P. Gearing AJ. Design and therapeutic application effective strategy for treating metastatic breast cancer. of matrix metalloproteinase inhibitors. (Chem. Rev. 1999, 99, 2735-2776. Published on the web september 8, 1999). Chem Rev 2001; 101:2205–2206. 22 Vandenbroucke RE, Libert C. Is there new hope for therapeutic matrix metallo- ACKNOWLEDGMENTS proteinase inhibition? Nat Rev Drug Discov 2014; 13: 904–927. This work was supported by NIH R01 CA154725. 23 Iwasaki M, Nishikawa A, Fujimoto T, Akutagawa N, Manase K, Endo T et al. Anti- invasive effect of MMI-166, a new selective matrix metalloproteinase inhibitor, in cervical carcinoma cell lines. Gynecol Oncol 2002; 85: 103–107. 24 Jacobsen JA, Major Jourden JL, Miller MT, Cohen SM. To bind zinc or not to bind CONTRIBUTIONS zinc: an examination of innovative approaches to improved metalloproteinase AR and KG contributed in conception/design, development of methodology, analysis inhibition. Biochim Biophys Acta 2010; 1803:72–94. and interpretation of the data, and study supervision. AR and MC contributed in 25 Overall CM, Kleifeld O. Towards third generation matrix metalloproteinase inhi- acquisition of the data. AR, ZMB, BK and KG comtributed in writing, review and/or bitors for cancer therapy. Br J Cancer 2006; 94:941–946. revision of the manuscript. KG provided administrative, technical or material support. 26 Van Lint P, Libert C. Chemokine and cytokine processing by matrix metallopro- AR, ZMB, BK and KG contributed in pathological diagnosis, analyzation and teinases and its effect on leukocyte migration and inﬂammation. J Leuk Biol 2007; interpretation of the immunohistochemical data. AR, BK, MC and KG carried out 82: 1375–1381. experiments and analyzed the data. 27 Rizwan A, Bulte C, Kalaichelvan A, Cheng M, Krishnamachary B, Bhujwalla ZM et al. Metastatic breast cancer cells in lymph nodes increase nodal collagen density. Sci Rep 2015; 5: 10002. COMPETING INTERESTS 28 Gyorffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q et al. An online The authors declare no conﬂict of interest. survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat 2009; 123:725–731. 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