Alexandra Naba

The Matrisome: In Silico Definition and In Vivo Characterization by Proteomics of Normal and Tumor Extracellular Matrices

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins providing both biophysical and biochemical cues that are important regulators of cell proliferation, survival, differentiation, and migration. We present here a proteomic strategy developed to characterize the in vivo ECM composition of normal tissues and tumors using enrichment of protein extracts for ECM components and subsequent analysis by mass spectrometry. In parallel, we have developed a bioinformatic approach to predict the in silico “matrisome” defined as the ensemble of ECM proteins and associated factors. We report the characterization of the extracellular matrices of murine lung and colon, each comprising more than 100 ECM proteins and each presenting a characteristic signature. Moreover, using human tumor xenografts in mice, we show that both tumor cells and stromal cells contribute to the production of the tumor matrix and that tumors of differing metastatic potential differ in both the tumor- and the stroma-derived ECM components. The strategy we describe and illustrate here can be broadly applied and, to facilitate application of these methods by others, we provide resources including laboratory protocols, inventories of ECM domains and proteins, and instructions for bioinformatically deriving the human and mouse matrisome.

Extracellular matrix signatures of human mammary carcinoma identify novel metastasis promoters

The extracellular matrix (ECM) is a major component of tumors and a significant contributor to cancer progression. In this study, we use proteomics to investigate the ECM of human mammary carcinoma xenografts and show that primary tumors of differing metastatic potential differ in ECM composition. Both tumor cells and stromal cells contribute to the tumor matrix and tumors of differing metastatic ability differ in both tumor- and stroma-derived ECM components. We define ECM signatures of poorly and highly metastatic mammary carcinomas and these signatures reveal up-regulation of signaling pathways including TGFβ and VEGF. We further demonstrate that several proteins characteristic of highly metastatic tumors (LTBP3, SNED1, EGLN1, and S100A2) play causal roles in metastasis, albeit at different steps. Finally we show that high expression of LTBP3 and SNED1 correlates with poor outcome for ER−/PR−breast cancer patients. This study thus identifies novel biomarkers that may serve as prognostic and diagnostic tools. DOI: http://dx.doi.org/10.7554/eLife.01308.001

Characterization of the Extracellular Matrix of Normal and Diseased Tissues Using Proteomics

The extracellular matrix (ECM) is a complex meshwork of insoluble fibrillar proteins and signaling factors interacting together to provide architectural and instructional cues to the surrounding cells. Alterations in ECM organization or composition and excessive ECM deposition have been observed in diseases such as fibrosis, cardiovascular diseases, and cancer. We provide here optimized protocols to solubilize ECM proteins from normal or tumor tissues, digest the proteins into peptides, analyze ECM peptides by mass spectrometry, and interpret the mass spectrometric data. In addition, we present here two novel R-script-based web tools allowing rapid annotation and relative quantification of ECM proteins, peptides, and intensity/abundance in mass spectrometric data output files. We illustrate this protocol with ECMs obtained from two pairs of tissues, which differ in ECM content and cellularity: triple-negative breast cancer and adjacent mammary tissue, and omental metastasis from high-grade serous ovarian cancer and normal omentum. The complete proteomics data set generated in this study has been deposited to the public repository ProteomeXchange with the data set identifier: PXD005554.

Proteomic characterization of human multiple myeloma bone marrow extracellular matrix

The extracellular matrix (ECM) is a major component of the tumor microenvironment, contributing to the regulation of cell survival, proliferation, differentiation and metastasis. In multiple myeloma (MM), interactions between MM cells and the bone marrow (BM) microenvironment, including the BM ECM, are critical to the pathogenesis of the disease and the development of drug resistance. Nevertheless, composition of the ECM in MM and its role in supporting MM pathogenesis has not been reported. We have applied a novel proteomic-based strategy and defined the BM ECM composition in patients with monoclonal gammopathy of undetermined significance (MGUS), newly diagnosed and relapsed MM compared with healthy donor-derived BM ECM. In this study, we show that the tumor ECM is remodeled at the mRNA and protein levels in MGUS and MM to allow development of a permissive microenvironment. We further demonstrate that two ECM-affiliated proteins, ANXA2 and LGALS1, are more abundant in MM and high expression is associated with a decreased overall survival. This study points to the importance of ECM remodeling in MM and provides a novel proteomic pipeline for interrogating the role of the ECM in cancers with BM tropism.

Quantitative proteomic profiling of the extracellular matrix of pancreatic islets during the angiogenic switch and insulinoma progression

The angiogenic switch, the time at which a tumor becomes vascularized, is a critical step in tumor progression. Indeed, without blood supply, tumors will fail to grow beyond 1 mm3 and are unlikely to disseminate. The extracellular matrix (ECM), a major component of the tumor microenvironment, is known to undergo significant changes during angiogenesis and tumor progression. However the extent of these changes remains unknown. In this study, we used quantitative proteomics to profile the composition of the ECM of pancreatic islets in a mouse model of insulinoma characterized by a precisely timed angiogenic switch. Out of the 120 ECM proteins quantified, 35 were detected in significantly different abundance as pancreatic islets progressed from being hyperplastic to angiogenic to insulinomas. Among these, the core ECM proteins, EFEMP1, fibrillin 1, and periostin were found in higher abundance, and decorin, Dmbt1, hemicentin, and Vwa5 in lower abundance. The angiogenic switch being a common feature of solid tumors, we propose that some of the proteins identified represent potential novel anti-angiogenic targets. In addition, we report the characterization of the ECM composition of normal pancreatic islets and propose that this could be of interest for the design of tissue-engineering strategies for treatment of diabetes.

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

Quantitative proteomics identify Tenascin-C as a promoter of lung cancer progression and contributor to a signature prognostic of patient survival

Significance Quantitative mass spectrometric profiling of the extracellular matrix composition of normal lung, fibrotic lung, primary lung tumors, and lung metastases to the lymph nodes uncovered specific signatures distinguishing these tissues. CRISPR/Cas9-mediated gene activation of one of the identified factors, Tenascin-C (Tnc), showed that this protein plays a role in mediating lung adenocarcinoma metastasis. Tnc expression is repressed, directly or indirectly, by the transcription factor Nkx2-1. Bioinformatic analysis shows that expression of three matrisome factors (TNC, S100A10, and S100A11) can predict survival in patients with lung adenocarcinoma. These factors could serve as disease markers that could be exploited for better diagnosis of lung cancer, and their future study could be used to inform the design of more potent treatments for patients. The extracellular microenvironment is an integral component of normal and diseased tissues that is poorly understood owing to its complexity. To investigate the contribution of the microenvironment to lung fibrosis and adenocarcinoma progression, two pathologies characterized by excessive stromal expansion, we used mouse models to characterize the extracellular matrix (ECM) composition of normal lung, fibrotic lung, lung tumors, and metastases. Using quantitative proteomics, we identified and assayed the abundance of 113 ECM proteins, which revealed robust ECM protein signatures unique to fibrosis, primary tumors, or metastases. These analyses indicated significantly increased abundance of several S100 proteins, including Fibronectin and Tenascin-C (Tnc), in primary lung tumors and associated lymph node metastases compared with normal tissue. We further showed that Tnc expression is repressed by the transcription factor Nkx2-1, a well-established suppressor of metastatic progression. We found that increasing the levels of Tnc, via CRISPR-mediated transcriptional activation of the endogenous gene, enhanced the metastatic dissemination of lung adenocarcinoma cells. Interrogation of human cancer gene expression data revealed that high TNC expression correlates with worse prognosis for lung adenocarcinoma, and that a three-gene expression signature comprising TNC, S100A10, and S100A11 is a robust predictor of patient survival independent of age, sex, smoking history, and mutational load. Our findings suggest that the poorly understood ECM composition of the fibrotic and tumor microenvironment is an underexplored source of diagnostic markers and potential therapeutic targets for cancer patients.

Abstract 4855: Proteomic characterization of the extracellular matrix in multiple myeloma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: The extracellular matrix is a major component of the tumor microenvironment, contributing to the regulation of cell survival, proliferation, differentiation and metastasis. In multiple myeloma (MM) ECM components, such as integrins, fibronectin and collagens, have been shown are critical to the pathogenesis of MM and the development of drug resistance. To date, despite some knowledge of the composition of the ECM in tumors, detailed profiling of the composition of the ECM in MM has not been carried out. Recent advances in proteomics have led to the characterization of the ECM and ECM-related proteins (“matrisome”) in normal human tissues and tumors in a systematic and comprehensive approach. Methods: Tumor Xenograft models; MM1S-GFP-Luc+ cells (5x106) were injected intravenously into SCID-Bg mice (n=4/group) and animals underwent weekly bioluminescent imaging (BLI). Mice were sacrificed after two weeks in order to mimic early tumor development (luminescence= 1x105 p/sec/cm2/sr). and compared to mice demonstrating high tumor burden (1x108 p/sec/cm2/sr) 5 weeks post injection. Human bone marrow aspirates; Whole bone marrow was obtained from MM patients (newly diagnosed n=9, relapsed n=9) and healthy human donors (n=9) following written informed consent. Sequential extractions of whole pooled bone marrow from mice and individual bone marrow humans was performed using the CNMCS(Cytosol/Nucleus/Membrane/Cytoskeleton) Compartmental Protein Extraction Kit (Cytomol, CA). Following this, proteins underwent reversed-phase high performance liquid chromatography followed by tandem mass spectrometry (MS). Identified peptide spectra were counted as a semi quantitative measure of abundance. Results: We detected a total of 1202, 982 and 329 unique proteins from enriched whole bone marrow samples from relapsed patients, newly diagnosed patients and mice, respectively. Of these, critical ECM components such as laminins, matrix metalloproteinases and collagens were found to be enriched in human MM ECM in comparison to healthy donors with increased abundance apparent with disease progression in mice. Specifically Bone Marrow Proteoglycan and Proteoglycan 3 were amongst the ECM proteins significantly enriched in the ECM of newly diagnosed patients. PRG3 is a p53 responsive gene that has been demonstrated to be upregulated in apoptotic cells in several cancers, including CLL. The ECM protein Elastin, the expression of which is closely associated with the invasive/metastatic potential of various cancer types, was also upregulated in the MM ECM where it may play an important role in the tumor-ECM interaction. Conclusions: We have profiled the ECM in MM using mass spectrometry with a view to determining the specific components that may be important in MM disease biology. Through this approach molecular mechanisms that influence MM development and progression can be uncovered and potential targets for therapy identified. Citation Format: Siobhan V. Glavey, Alexandra Naba, Manuela Gambella, Alberto Rocci, Antonio Sacco, John Asara, Antonio Palumbo, Richard O. Hynes, Aldo M. Roccaro, Irene M. Ghobrial. Proteomic characterization of the extracellular matrix in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4855. doi:10.1158/1538-7445.AM2014-4855

Abstract SY28-01: Multiple changes in tumor extracellular matrices

The extracellular matrix (ECM) is a fundamental and important component of metazoan organisms providing architectural support and anchorage for the cells. The ECM consists of a complex meshwork of highly cross-linked proteins and exists as interstitial forms within organs and as specialized forms, such as basement membranes underlying epithelia, vascular endothelium and surrounding certain other tissues and cell types (e.g., neurons, muscles). Cells adhere to the ECM via transmembrane receptors, among which integrins are the most prominent. These cell-matrix interactions result in the stimulation of various signaling pathways controlling proliferation and survival, differentiation, migration, etc. The composition of the ECM and the repertoire of ECM receptors determine the responses of the cells. The biophysical properties of the ECM (deformability or stiffness) have also been shown to modulate these cellular functions. In addition to core ECM components (fibronectins, collagens, laminins, proteoglycans, etc.) the ECM serves as a reservoir for growth factors and cytokines and ECM-remodeling enzymes (crosslinkers, proteases) that collaborate with ECM proteins to signal to the cells. Hence, the ECM provides not only biophysical cues but also biochemical signals that regulate cell behavior. In addition to being important for normal development, alterations of the ECM have been associated with various pathologies such as fibrosis, skeletal diseases and cancer. The role of the ECM in cancer is of particular interest. Long-standing as well as recent data implicate tumor ECM as a significant contributor to tumor progression. Indeed, the ECM is a major component of the tumor microenvironment and classical pathology has shown that excessive deposition of ECM is a common feature of tumors with poor prognosis. More recently, gene expression screens have revealed that many genes encoding ECM components and ECM receptors are dysregulated during tumor progression. Finally, modifications of the extracellular matrix architecture and biophysical properties have been shown to influence tumor progression. Despite these clear indications that tumor ECM and the interactions of cells with it are very likely to play important roles in tumor progression, we do not have a good picture of ECM composition, origins and functions in tumors. One reason for this lies in the biochemical properties of ECM proteins (large size, insolubility, cross-linking, etc.) that have rendered attempts to characterize systematically the composition of the ECM from tissues and tumors very challenging. Thanks to the completion of the genomes of many species and to previous studies, it is now clear that vertebrate genomes contain hundreds of genes encoding ECM proteins. Specific features of ECM proteins have emerged from these studies, in particular their distinctive structures based on the repetition of conserved domains. During the last few years, several attempts have been made at in silico predictions of the complement of ECM proteins. Furthermore, recent studies have begun to characterize experimentally the composition of the extracellular matrix of specific model systems such as retinal and vascular basement membranes, mammary gland and cartilage. However, there remains a pressing need for a better definition of the number and diversity of ECM proteins and even of what should be included in that definition. Limitations arise also from the lack of experimental reagents and approaches due to the biochemical intractability of ECM and the lack of an adequate library of antibodies or other probes to characterize ECM proteins in situ. Thus, deciphering the complexity of the extracellular matrix in vivo represents an important scientific challenge. We have developed proteomics-based methods coupled with a bioinformatic definition of the “matrisome” (ECM and ECM-associated proteins) to analyze the protein composition of the tissue extracellular matrix. We have applied this strategy to characterize in detail the extracellular matrices both of normal murine tissues (e.g., lung and colon) and of both non-metastatic and metastatic tumors. In each case, these comprise well over 100 proteins. Moreover, we have applied this approach to understand the origins of tumor ECM proteins and have been able to show, using human/mouse xenograft models, that both tumor cells and stromal cells contribute in characteristic ways to the ECM of the tumor microenvironment. Furthermore, we show that both tumor and stromal cells contribute to significant changes in the extracellular matrices of tumors of differing metastatic potential. The strategy we have developed can be broadly applied and we have begun to apply it to human patient material in order to characterize the ECM composition of tumors or varying prognosis with the goal of developing ECM signatures that may be of diagnostic and/or prognostic value. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY28-01. doi:1538-7445.AM2012-SY28-01