Cartilage Oligomeric Matrix Protein promotes epithelial-mesenchymal transition by interacting with Transgelin in Colorectal Cancer.

Background and Purpose: The role of the cartilage oligomeric matrix protein (COMP) in epithelial-mesenchymal transition (EMT) in tumor progression has been studied, but its exact regulatory mechanism remains unknown. Methods: The interaction between COMP and the actin-binding protein transgelin (TAGLN) was identified by interaction protein prediction and co-immunoprecipitation and verified through the stochastic optical reconstruction microscopy (STORM) and duolink experiments. Western blot and immunofluorescence analyses were conducted to detect the changes in EMT-related markers after COMP overexpression and knockdown. Molecular docking and Biacore of the interaction interface of COMP/TAGLN revealed that Chrysin directly targeted COMP. The promotion of COMP and the Chrysin inhibition of EMT were detected through the cell migration, invasion, apoptosis, and xenotransplantation of nude mice. Results: COMP interacts with TAGLN in EMT in colorectal cancer to regulate cytoskeletal remodeling and promote malignant progression. COMP is highly expressed in highly malignant colorectal cancer and positively correlated with TAGLN expression. COMP knockdown can inhibit colorectal cancer metastasis and invasion, whereas COMP overexpression promotes EMT in colorectal cancer. Through virtual screening of the protein interaction interface, Chrysin, a flavonoid compound extracted from Oroxylum indicum, was found to have the highest docking score to the COMP/TAGLN complex. Chrysin inhibited COMP, thereby preventing EMT and the malignant progression of colorectal cancer. Conclusions: This study illustrated the role of COMP in EMT and suggested that COMP/TAGLN may be a potential tumor therapeutic target. Chrysin exhibits obvious antitumor effects. This work provides a preliminary antitumor therapy to target COMP or its interaction protein to inhibit EMT.

A novel COMP mutation in a Chinese family with multiple epiphyseal dysplasia.

BACKGROUND: Multiple epiphyseal dysplasia (MED) is a skeletal disorder characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis. At least 66% of the reported autosomal dominant MED (AD-MED) cases are caused by COMP mutations. METHODS: We recruited a four-generation Chinese family with early-onset hip osteoarthritis, flatfoot, brachydactyly, and mild short stature. An assessment of the family history, detailed physical examinations, and radiographic evaluations were performed on the proband and other family members, followed by the performance of whole-exome sequencing (WES). The pathogenicity of the candidate mutation was also analyzed. RESULTS: An AD-MED family with 10 affected members and 17 unaffected members was recruited. The main radiographic findings were symmetrical changes in the dysplastic acetabulum and femoral heads, irregular contours of the epiphyses, a shortened femoral neck, and flatfoot. Lower bone density was also observed in the ankle joints, wrist joints, and knees, as well as irregular vertebral end plates. In the proband, we identified the missense mutation c.1153G > T (p. Asp385Tyr), located in exon 11 of the COMP gene. This mutation was assessed as 'pathogenic' because of its low allele frequency and its high likelihood of co-segregation with disease in the reported family. Sanger sequencing validated the novel heterozygous mutation c.1153G > T (p. Asp385Tyr) in exon 11 of COMP in all affected individuals in the family. CONCLUSIONS: Our results underlined a key role of the Asp385 amino acid in the protein function of COMP and confirmed the pathogenicity of the COMP (c.1153G > T; p. Asp385Tyr) mutation in AD-MED disease. We have therefore expanded the known mutational spectrum of COMP and revealed new phenotypic information for AD-MED.

Development of a Cartilage Oligomeric Matrix Protein Neo-Epitope Assay for the Detection of Intra-Thecal Tendon Disease.

The diagnosis of tendon injury relies on clinical signs and diagnostic imaging but imaging is subjective and does not always correlate with clinical signs. A molecular marker would potentially offer a sensitive and specific diagnostic tool that could also provide objective assessment of healing for the comparison of different treatments. Cartilage Oligomeric Matrix Protein (COMP) has been used as a molecular marker for osteoarthritis in humans and horses but assays for the protein in tendon sheath synovial fluids have shown overlap between horses affected by tendinopathy and controls. We hypothesized that quantifying a COMP neoepitope would be more discriminatory of injury. COMP fragments were purified from synovial fluids of horses with intra-thecal tendon injuries and media from equine tendon explants, and mass spectrometry of a consistent and abundant fragment revealed a ~100 kDa COMP fragment with a new N-terminus at the 78th amino-acid (NH2-TPRVSVRP) located just outside the junctional region of the protein. A competitive inhibition ELISA based on a polyclonal antibody raised to this sequence yielded more than a 10-fold rise in the mean neoepitope levels for tendinopathy cases compared to controls (5.3 ± 1.3 µg/mL (n = 7) versus 58.8 ± 64.3 µg/mL (n = 13); p = 0.002). However, there was some cross-reactivity of the neoepitope polyclonal antiserum with intact COMP, which could be blocked by a peptide spanning the neoepitope. The modified assay demonstrated a lower concentration but a significant > 500-fold average rise with tendon injury (2.5 ± 2.2 ng/mL (n = 6) versus 1029.8 ± 2188.8 ng/ml (n = 14); p = 0.013). This neo-epitope assay therefore offers a potentially useful marker for clinical use.

Label-Free and Direct Visualization of Multivalent Binding of Bone Morphogenetic Protein-2 with Cartilage Oligomeric Matrix Protein.

This work presents the first direct evidence of multivalent binding between bone morphogenetic protein-2 (BMP-2) and cartilage oligomeric matrix protein (COMP) using high-resolution atomic force microscopy (AFM) imaging. AFM topographic images reveal the molecular morphology of COMP, a pentameric protein whose five identical monomer units bundle together at N-termini, extending out with flexible chains to C-termini. Upon addition of BMP-2, COMP molecules undergo conformational changes at the C-termini to enable binding with BMP-2 molecules. AFM enables local structural changes of COMP to be revealed upon binding various numbers, 1-5, of BMP-2 molecules. These BMP-2/COMP complexes exhibit very different morphologies from those of COMP: much more compact and thus less flexible. These molecular-level insights deepen current understanding of the mechanism of how the BMP-2/COMP complex enhances osteogenesis among osteoprogenitor cells, i.e., multivalent presentation of BMP-2 via the stable and relatively rigid BMP-2/COMP complex could form a lattice of interaction between multiple BMP-2 and BMP-2 receptors. These ligand-receptor clusters lead to fast initiation and sustained activation of the Smad signaling pathway, resulting in enhanced osteogenesis. This work is also of translational importance as the outcome may enable use of lower BMP-2 dosage for bone repair and regeneration.

Design and Characterization of Fibers and Bionanocomposites Using the Coiled-Coil Domain of Cartilage Oligomeric Matrix Protein.

Tremendous effort has been dedicated to the design and assembly of bioinspired protein-based architectures with potential applications in drug delivery, tissue engineering, biosensing, and bioimaging. Here, we describe our strategy to generate fibers and bionanocomposites using the coiled-coil domain of cartilage oligomeric matrix protein (COMPcc). Our construct, Q, engineered by swapping particular regions of COMPcc to optimize surface charge, self-assembles to form nanofibers. The Q protein nanofibers can efficiently bind curcumin to form robust mesofibers that can be potentially used for drug delivery and biomedical applications. In addition, using the same Q protein, we describe the biotemplation of gold nanoparticles (AuNP) in the presence and absence of the hexahistidine tag (His-tag). The Q bearing His-tag·AuNP (Q·AuNP) readily deposits on electrode surfaces, while Q without His-tag·AuNP (Qx·AuNP) stabilizes the soluble protein·gold bionanocomposites for several days without aggregating.

Protein Engineered Triblock Polymers Composed of Two SADs: Enhanced Mechanical Properties and Binding Abilities.

Recombinant methods have been used to engineer artificial protein triblock polymers composed of two different self-assembling domains (SADs) bearing one elastin (E) flanked by two cartilage oligomeric matrix protein coiled-coil (C) domains to generate CEC. To understand how the two C domains improve small molecule recognition and the mechanical integrity of CEC, we have constructed CL44AECL44A, which bears an impaired CL44A domain that is unstructured as a negative control. The CEC triblock polymer demonstrates increased small molecule binding and ideal elastic behavior for hydrogel formation. The negative control CL44AECL44A does not exhibit binding to small molecule and is inelastic at lower temperatures, affirming the favorable role of C domain and its helical conformation. While both CEC and CL44AECL44A assemble into micelles, CEC is more densely packed with C domains on the surface enabling the development of networks leading to hydrogel formation. Such protein engineered triblock copolymers capable of forming robust hydrogels hold tremendous promise for biomedical applications in drug delivery and tissue engineering.

Cartilage oligomeric matrix protein: COMPopathies and beyond.

Cartilage oligomeric matrix protein (COMP) is a large pentameric glycoprotein that interacts with multiple extracellular matrix proteins in cartilage and other tissues. While, COMP is known to play a role in collagen secretion and fibrillogenesis, chondrocyte proliferation and mechanical strength of tendons, the complete range of COMP functions remains to be defined. COMPopathies describe pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), two skeletal dysplasias caused by autosomal dominant COMP mutations. The majority of the mutations are in the calcium binding domains and compromise protein folding. COMPopathies are ER storage disorders in which the retention of COMP in the chondrocyte ER stimulates overwhelming cellular stress. The retention causes oxidative and inflammation processes leading to chondrocyte death and loss of long bone growth. In contrast, dysregulation of wild-type COMP expression is found in numerous diseases including: fibrosis, cardiomyopathy and breast and prostate cancers. The most exciting clinical application is the use of COMP as a biomarker for idiopathic pulmonary fibrosis and cartilage degeneration associated osteoarthritis and rheumatoid and, as a prognostic marker for joint injury. The ever expanding roles of COMP in single gene disorders and multifactorial diseases will lead to a better understanding of its functions in ECM and tissue homeostasis towards the goal of developing new therapeutic avenues.

Decreased Plasma COMP and Increased Plasma CTX-II Levels in a Chinese Pseudoachondroplasia Family with Novel COMP Mutation.

Pseudoachondroplasia (PSACH) is an autosomal dominant osteochondrodysplasia caused by mutations in the gene encoding cartilage oligomeric matrix protein (COMP). Accurate clinical diagnosis of PSACH is sometimes difficult. Here, we identified a novel COMP mutation (c.1675G>A, p.Glu559Lys) in a Chinese PSACH family. We detected the plasma levels of COMP and type II collagen (CTX-II) in the four affected individuals. The results showed the levels of plasma COMP significantly decreased and plasma CTX-II significantly increased in the three PSACH patients with COMP mutation. However, both plasma levels of COMP and CTX-II were not to have found significant difference between the presymptomatic carrier and the age-matched subjects. In vitro analysis and immunofluorescence displayed wild type COMP homogenously expressed in cytoplasm, but mutant proteins were irregularly accumulated inside the HEK-293 cells. Western blot revealed that the quantity of the mutant COMP was more compared to wild type COMP in cells after transfection for 12 hours and 24 hours. Subsequently, 3D structural analysis showed three changes have taken place in secondary structure of the mutant COMP. In conclusion, the novel mutation of COMP may result in intracellular accumulation of the mutant protein. Decreased plasma COMP and increased plasma CTX-II may potentially serve as diagnostic markers of PSACH but may not be applicable in the presymptomatic carrier.

A B-Z junction induced by an A A mismatch in GAC repeats in the gene for cartilage oligomeric matrix protein promotes binding with the hZαADAR1 protein.

GAC repeat expansion from five to seven in the exonic region of the gene for cartilage oligomeric matrix protein (COMP) leads to pseudoachondroplasia, a skeletal abnormality. However, the molecular mechanism by which GAC expansions in the COMP gene lead to skeletal dysplasias is poorly understood. Here we used molecular dynamics simulations, which indicate that an A … A mismatch in a d(GAC)6·d(GAC)6 duplex induces negative supercoiling, leading to a local B-to-Z DNA transition. This transition facilitates the binding of d(GAC)7·d(GAC)7 with the Zα-binding domain of human adenosine deaminase acting on RNA 1 (ADAR1, hZαADAR1), as confirmed by CD, NMR, and microscale thermophoresis studies. The CD results indicated that hZαADAR1 recognizes the zigzag backbone of d(GAC)7·d(GAC)7 at the B-Z junction and subsequently converts it into Z-DNA via the so-called passive mechanism. Molecular dynamics simulations carried out for the modeled hZαADAR1-d(GAC)6d(GAC)6 complex confirmed the retention of previously reported important interactions between the two molecules. These findings suggest that hZαADAR1 binding with the GAC hairpin stem in COMP can lead to a non-genetic, RNA editing-mediated substitution in COMP that may then play a crucial role in the development of pseudoachondroplasia.

Cartilage Oligomeric Matrix Protein: Matricellular and Matricrine Signaling in Cardiovascular Homeostasis and Disease.

Cardiovascular (CV) diseases remain a leading cause of morbidity and mortality in the world. Increasing the understanding of the pathogenesis of various CV diseases may provide novel therapeutic targets to improve their prevention and treatment. Cartilage oligomeric matrix protein (COMP), also known as thrombospondin-5 (TSP-5), is a matricellular protein that is abundantly expressed in both cartilage and the CV system. Our group and others have identified COMP as playing critical roles in maintaining CV homeostasis. COMP, expressed and produced by vascular smooth muscle cells (VSMCs), maintains VSMC contractile phenotypes. COMP deficiency enhances VSMC migration and aggravates VSMC calcification and atherosclerosis. Moreover, a lack of COMP leads to spontaneous dilated cardiomyopathy in mice. COMP is also secreted by platelets in circulating blood and negatively regulates haemostasis and thrombosis. A series of COMP binding proteins, such as integrin α7ß1, integrin ß3, thrombin, and bone morphogenetic protein 2, have been identified in the CV system, and they have been determined to mediate various COMP functions. The matrix metalloproteinase (A Disintegrin and Metalloproteinase with Thrombospondin motifs) ADAMTS-7 is a regulatory enzyme that is responsible for the degradation of COMP in the CV system. ADAMTS-7 expression correlates with atherosclerosis and vascular calcification in both human genome-wide association studies and in vivo mice models via COMP-dependent and COMP-independent mechanisms. In this review, we summarize what is currently known about the matricellular and matricrine signaling of COMP mediated by its respective binding partners as well as its proteolytic regulation by ADAMTS-7 in CV disease.

Molecular synergy in biolubrication: The role of cartilage oligomeric matrix protein (COMP) in surface-structuring of lubricin.

HYPOTHESIS: Synovial surfaces are lubricated by biomolecular aggregates that act in synergy, and lubricin is one key biolubricant. Its molecular structure allows extensive hydration and this is conducive to its lubrication performance. However, in order to fullfil its lubrication function it needs to be anchored and oriented on the surface in a proper way. We suggest that cartilage oligomeric matrix protein (COMP) is one of the biomolecules that promotes anchoring of lubricin in a fashion that facilitates lubrication. EXPERIMENTS: Weakly hydrophobic poly(methyl methacrylate) (PMMA) surfaces were coated by COMP and lubricin, individually and in combinations. Adsorption was investigated using a quartz crystal microbalance, and friction between the biopolymer-coated surfaces was determined by employing the atomic force microscope-colloidal probe technique. FINDINGS: It was found that COMP facilitated firm directed attachment of lubricin in a manner that resulted in low friction forces, significantly lower than what was achieved when lubricin was directly adsorbed to PMMA. Evidently, COMP provides means for lubricin to attach strongly and in a favourable conformation for efficient lubrication of this surface. We suggest that our findings can be extrapolated to cartilage surfaces, where co-localization of COMP and lubricin has been demonstrated.

Intravitreal AAV2.COMP-Ang1 Prevents Neurovascular Degeneration in a Murine Model of Diabetic Retinopathy.

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the U.S. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here, we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable with nondiabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR.

Influence of fluorination on protein-engineered coiled-coil fibers.

We describe the design and characterization of fluorinated coiled-coil proteins able to assemble into robust nano- and microfibers. Fluorination is achieved biosynthetically by residue-specific incorporation of 5,5,5-trifluoroleucine (TFL). The fluorinated proteins C+TFL and Q+TFL are highly α-helical as confirmed via circular dichroism (CD) and more resistant to thermal denaturation compared to their nonfluorinated counterparts, C and Q. The fluorinated proteins demonstrate enhanced fiber assembly at pH 8.0 with higher order structure in contrast to nonfluorinated proteins, which are unable to form fibers under the same conditions. Ionic strength dependent fiber assembly is observed for fluorinated as well as wild-type proteins in which the fluorinated proteins exhibited more stable, thicker fibers. The fluorinated and nonfluorinated proteins reveal metal ion-dependent small molecule recognition and supramolecular assemblies. In the presence of Zn (II), enhanced thermal stability and fiber assembly is observed for the fluorinated proteins and their nonfluorinated counterparts. Whereas Ni (II) promotes aggregation with no fiber assembly, the stabilization of α-helix by Zn (II) results in enhanced binding to curcumin by the fluorinated proteins. Surprisingly, the nonfluorinated proteins exhibit multiple-fold increase in curcumin binding in the presence of Zn (II). In the context of the growing number of protein-based fiber assemblies, these fluorinated coiled-coil proteins introduce a new paradigm in the development of highly stable, robust self-assembling fibers under more physiologically relevant pH conditions that promotes the binding and release of small molecules in response to external cues.

Cholera toxin B subunit-five-stranded α-helical coiled-coil fusion protein: "five-to-five" molecular chimera displays robust physicochemical stability.

To create a physicochemically stable cholera toxin (CT) B subunit (CTB), it was fused to the five-stranded α-helical coiled-coil domain of cartilage oligomeric matrix protein (COMP). The chimeric fusion protein (CTB-COMP) was expressed in Pichia pastoris, predominantly as a pentamer, and retained its affinity for the monosialoganglioside GM1, a natural receptor of CT. The fusion protein displayed thermostability, tolerating the boiling temperature of water for 10min, whereas unfused CTB readily dissociated to its monomers and lost its affinity for GM1. The fusion protein also displayed resistance to strong acid at pHs as low as 0.1, and to the protein denaturant sodium dodecyl sulfate at concentrations up to 10%. Intranasal administration of the fusion protein to mice induced anti-B subunit serum IgG, even after the protein was boiled, whereas unfused CTB showed no thermostable mucosal immunogenicity. This study demonstrates that CTB fused to a pentameric α-helical coiled coil has a novel physicochemical phenotype, which may provide important insight into the molecular design of enterotoxin-B-subunit-based vaccines and vaccine delivery molecules.

Novel cartilage oligomeric matrix protein (COMP) neoepitopes identified in synovial fluids from patients with joint diseases using affinity chromatography and mass spectrometry.

To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDSPAGE followed by in-gel digestion and mass spectrometric identification and characterization.Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided.

Proteomic analysis of tendon extracellular matrix reveals disease stage-specific fragmentation and differential cleavage of COMP (cartilage oligomeric matrix protein).

During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1ß and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1ß enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease.

Bionanocomposites: differential effects of cellulose nanocrystals on protein diblock copolymers.

We investigate the effects of mixing a colloidal suspension of tunicate-derived cellulose nanocrystals (t-CNCs) with aqueous colloidal suspensions of two protein diblock copolymers, EC and CE, which bear two different self-assembling domains (SADs) derived from elastin (E) and the coiled-coil region of cartilage oligomeric matrix protein (C). The resulting aqueous mixtures reveal improved mechanical integrity for the CE+t-CNC mixture, which exhibits an elastic gel network. This is in contrast to EC+t-CNC, which does not form a gel, indicating that block orientation influences the ability to interact with t-CNCs. Surface analysis and interfacial characterization indicate that the differential mechanical properties of the two samples are due to the prevalent display of the E domain by CE, which interacts more with t-CNCs leading to a stronger network with t-CNCs. On the other hand, EC, which is predominantly C-rich on its surface, does not interact as much with t-CNCs. This suggests that the surface characteristics of the protein polymers, due to folding and self-assembly, are important factors for the interactions with t-CNCs, and a significant influence on the overall mechanical properties. These results have interesting implications for the understanding of cellulose hydrophobic interactions, natural biomaterials and the development of artificially assembled bionanocomposites.

Identification, prioritization, and evaluation of glycoproteins for aggressive prostate cancer using quantitative glycoproteomics and antibody-based assays on tissue specimens.

Prostate cancer is highly heterogeneous in nature; while the majority of cases are clinically insignificant, some cases are lethal. Currently, there are no reliable screening methods for aggressive prostate cancer. Since most established serum and urine biomarkers are glycoproteins secreted or leaked from the diseased tissue, the current study seeks to identify glycoprotein markers specific to aggressive prostate cancer using tissue specimens. With LC-MS/MS glycoproteomic analysis, we identified 350 glycopeptides with 17 being altered in aggressive prostate cancer. ELISA assays were developed/purchased to evaluate four candidates, that is, cartilage oligomeric matrix protein (COMP), periostin, membrane primary amine oxidase (VAP-1), and cathepsin L, in independent tissue sets. In agreement with the proteomic analysis, we found that COMP and periostin expressions were significantly increased in aggressive prostate tumors while VAP-1 expression was significantly decreased in aggressive tumor. In addition, the expression of these proteins in prostate metastases also follows the same pattern observed in the proteomic analysis. This study provides a workflow for biomarker discovery, prioritization, and evaluation of aggressive prostate cancer markers using tissue specimens. Our data suggest that increase in COMP and periostin and decrease in VAP-1 expression in the prostate may be associated with aggressive prostate cancer.