ADAMTS Proteases Research Articles

ECM1 attenuates hepatic fibrosis by interfering with mediators of latent TGF-β1 activation

ObjectiveExtracellular matrix protein 1 (ECM1) serves as a gatekeeper of hepatic fibrosis by maintaining transforming growth factor-β1 (TGF-β1) in its latent form. ECM1 knockout (KO) causes latent (L) TGF-β1 activation,...

Abstract 4647: Potential of TIMP3 regulating the versican status in the colorectal cancer tumor microenvironment

Abstract Background: In colorectal cancer (CRC), versican (VCAN) accumulation has been found to negatively correlate with CD8+ tumor infiltrating lymphocyte (TIL) abundance and its proteolysis into versikine (Vkine) positively correlates with TIL abundance. TIMP3 has demonstrated the ability to inhibit several ADAMTS proteases able to cleave VCAN to Vkine. Here we investigate the correlation of TIMP3 accumulation in CRCs with the VCAN status and CD8+ T cell infiltration. Methods: Matched primary and metastatic CRC samples from 156 patients who had resected oligometastatic CRC were stained for VCAN, Vkine, TIMP3, and CD8+ TILs. VCAN, Vkine, and TIMP3 were scored using an intensity binning system ranging from 0-3+ and T cells in the epithelium were reported per high power field (HPF). Tumors were designated as high (2 or 3+) or low (0 or 1) for both VCAN and Vkine and positive (+; 1-3) or negative (0) for TIMP3. Tumors designated VCAN low and Vkine high are considered VCAN proteolytic predominant (VPP) and all other combinations are considered VCAN proteolytic weak (VPW). Results: Across the whole dataset, 52% of cancers were VCAN high and 24% were VPP. TIMP3 was detected in 33% of cancers. The frequency of TIMP3 was similar between primary and metastatic tumors (39% and 29%, respectively). Of the VCAN high cancers, 42% were TIMP3+ whereas 61% of the TIMP3+ samples are VCAN high. Of the Vkine high cancers, 44% were TIMP3+ and 36% of VPP cancers were TIMP3+. The prevalence of the VPP phenotype was similar between the TIMP3+ and negative cancers. Additionally, among the TIMP3+ samples 68% were Vkine high versus 46% in TIMP3 negative cancers (p=0.001). VCAN proteolysis correlates with higher CD8+ TIL abundance, thus we assessed the number of CD8+ TILs in TIMP3+ and negative tumors. The TIMP3+ cancers had a median of 2 CD8+ TILs/HPF and the TIMP3 negative cancers has a median of 3 CD8+ TILs/HPF. For VPP cancers, those that were also TIMP3+ had a median of 3 CD8+ TILs/HPF compared to TIMP3 negative cancers which had a median of 6 CD8+ TILs/HPF (p=0.2). Conclusions: TIMP3 correlates with higher levels of VCAN accumulation but does not correlate with lower levels of proteolysis. Future studies are needed to better clarify the impact of TIMP3 and other mediators in regulating VCAN accumulation and proteolysis in the tumor microenvironment. Citation Format: Anna Field, Sean Kraus, Kristina Matkowskyj, Dustin Deming, Cheri A. Pasch, Wei Zhang. Potential of TIMP3 regulating the versican status in the colorectal cancer tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4647.

Versican provides the provisional matrix for uterine spiral artery dilation and fetal growth

The extracellular matrix (ECM) in the endometrium plays a crucial role in mammalian pregnancy. We have shown that versican secreted from the endometrial epithelium promotes embryo implantation. Versican is a proteoglycan, a major player in the provisional matrix, and versikine, its N-terminal fragment cleaved by ADAMTS proteinases, serves as a bioactive molecule. Here, since versican expression in the placenta was dynamically altered in humans and mice, we investigated the role of versican in pregnancy using uterine-specific Vcan deletion mice (uKO mice) and ADAMTS-resistant versican expressing mice (V1R mice). uKO mice exhibited insufficient spiral artery dilation, followed by fetal growth restriction and maternal hypertension. Further analysis revealed impaired proliferation of tissue-resident natural killer cells required for spiral artery dilation. V1R mice showed the same results as the control, eliminating the involvement of versikine. Our results provide a new concept that versican, one factor of ECM, contributes to placentation and following fetal growth.

Accumulation of versican and lack of versikine ameliorate acute colitis

Versican is a large chondroitin sulfate/dermatan sulfate proteoglycan that plays a key role in the formation of the provisional matrix. Here, we generated dextran sulfate sodium-induced colitis in knockin-mice, R/R, expressing ADAMTS-resistant versican, and investigated the impact of accumulating versican and its turnover in the inflammatory colon mucosa. Histologically, R/R colon showed decreased levels of tissue destruction and an increased number of myofibroblasts and macrophages. Characterization of inflammatory cells revealed an increase in F4/80+ macrophages in R/R colon, compared with wildtype, without a clear shift between M1 and M2 populations. Intestinal stroma exhibited a higher number of myofibroblasts in R/R, suggesting increased levels of tissue regeneration. Coculture of macrophages and stromal fibroblasts obtained from inflammatory colon showed that wild-type macrophages inhibited myofibroblastic differentiation of R/R fibroblasts but not wild-type. This inhibitory effect was due to an increased level of versikine, a cleaved fragment of versican by ADAMTS proteinases. Taken together, our results demonstrate versikine as the direct regulator that inhibits repair of inflamed tissue.

Linalool and eugenol exhibit apoptotic potential on hela and caco-2 cells through the modulation of src kinases and ADAMTS proteases while only eugenol displays anti-angiogenic features on HeLa cells

Linalool and eugenol exhibit apoptotic potential on hela and caco-2 cells through the modulation of src kinases and ADAMTS proteases while only eugenol displays anti-angiogenic features on HeLa cells

Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling.

Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin β1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of β-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that β1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with β-blockers had a distinct cardiac ECM profile. Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that β-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

Isolation and Purification of Versican and Analysis of Versican Proteolysis.

Versican is a widely distributed chondroitin sulfate proteoglycan that forms large complexes with the glycosaminoglycan hyaluronan (HA). As a consequence of HA binding to its receptor CD44 and interactions of the versican C-terminal globular (G3) domain with a variety of extracellular matrix proteins, versican is a key component of well-defined networks in pericellular matrix and extracellular matrix. Versican is crucial for several developmental processes in the embryo ranging from cardiac development to digit separation, and there is an increasing interest in its roles in cancer and inflammation. Versican proteolysis by ADAMTS proteases is highly regulated, occurs at specific peptide bonds, and is relevant to several physiological and disease mechanisms. In this chapter, methods are described for the isolation and detection of intact and cleaved versican in tissues using morphologic and biochemical techniques. These, together with the methodologies for purification and analysis of recombinant versican and an N-terminal versican fragment named versikine that are provided here, are likely to facilitate further progress on the biology of versican and its proteolysis.

Expression gradient of metalloproteinases and their inhibitors from proximal to distal segments of abdominal aortic aneurysm

Abdominal aortic aneurysm refers to abnormal, asymmetric distension of the infrarenal aortic wall due to pathological remodelling of the extracellular matrix. The distribution of enzymes remodelling the extracellular matrix and their expression patterns in the affected tissue are largely unknown. The goal of this work was to investigate the expression profiles of 20 selected genes coding for metalloproteinases and their inhibitors in the proximal to the distal direction of the abdominal aortic aneurysm. RNA samples were purified from four lengthwise fragments of aneurysm and border tissue obtained from 29 patients. The quantities of selected mRNAs were determined by real-time PCR to reveal the expression patterns. The genes of interest encode collagenases (MMP1, MMP8, MMP13), gelatinases (MMP2, MMP9), stromelysins (MMP3, MMP7, MMP10, MMP11, MMP12), membrane-type MMPs (MMP14, MMP15, MMP16), tissue inhibitors of metalloproteinases (TIMP1, TIMP2, TIMP3, TIMP4), and ADAMTS proteinases (ADAMTS1, ADAMTS8, and ADAMTS13). It was found that MMP, TIMP, and ADAMTS are expressed in all parts of the aneurysm with different patterns. A developed aneurysm has such a disturbed expression of the main participants in extracellular matrix remodelling that it is difficult to infer the causes of the disorder development. MMP12 secreted by macrophages at the onset of inflammation may initiate extracellular matrix remodelling, which, if not controlled, initiates a feedback loop leading to aneurysm formation.

ADAMTS1 Supports Endothelial Plasticity of Glioblastoma Cells with Relevance for Glioma Progression.

Gliomas in general and the more advanced glioblastomas (GBM) in particular are the most usual tumors of the central nervous system with poor prognosis. GBM patients develop resistance to distinct therapies, in part due to the existence of tumor cell subpopulations with stem-like properties that participate in trans-differentiation events. Within the complex tumor microenvironment, the involvement of extracellular proteases remains poorly understood. The extracellular protease ADAMTS1 has already been reported to contribute to the plasticity of cancer cells. Accordingly, this basic knowledge and the current availability of massive sequencing data from human gliomas, reinforced the development of this work. We first performed an in silico study of ADAMTS1 and endothelial markers in human gliomas, providing the basis to further assess these molecules in several primary glioblastoma-initiating cells and established GBM cells with the ability to acquire an endothelial-like phenotype. Using a co-culture approach of endothelial and GBM cells, we noticed a relevant function of ADAMTS1 in GBM cells leading the organization of endothelial-like networks and, even more significantly, we found a blockade of the formation of tumor-spheres and a deficient response to hypoxia in the absence of ADAMTS1. Our data support a chief role of this protease modulating the phenotypic plasticity of GBM.

Abstract 3294: PD-L1 status and versican proteolysis predict CD8+ T cell infiltration in esophageal cancer

Abstract Background: Esophageal cancer is a highly lethal cancer with a 20% 5-year overall survival despite standard of care treatment.. Pembrolizumab (anti-PD1, Merck) is FDA-approved for PD-L1 positive esophageal cancer, which comprises roughly 40% of all patients. PD-L1 positive (+) esophageal cancers have previously been shown to demonstrate elevated CD8+ T cell infiltration (CD8+ TILs). Versican (VCAN) is an immunosuppressive extracellular matrix proteoglycan which produces an immunostimulatory fragment, versikine (Vkine), upon proteolytic cleavage by ADAMTS proteases. Previously, VCAN proteolysis has been shown to correlate with enhanced CD8+ TILs in colorectal cancer (CRC), indicating potential for use as an immune biomarker. Methods: A human esophageal cancer tissue microarray (TMA) was developed consisting of multiple samples across 106 representative esophageal cancer patients. Immunohistochemistry (IHC) was used to evaluate VCAN and Vkine expression, and a binning system was used to quantify these stains based on stromal intensity (0: 0-10%, 1: 10-25%, 2:25-50%, 3: 50-100%). IHC was also conducted for CD8, and the number of CD8+ tumor-infiltrating cells per 400x field of view was counted. PD-L1 IHC 22C3 pharmDx was performed and quantified per CPS guidelines for GEJ cancers to quantify PD-L1 status. The VCAN proteolytic predominant (VPP) phenotype was defined as both low VCAN (0 or 1) and high Vkine (2 or 3), and VCAN proteolytic weak phenotype (VPW) was defined as either high VCAN (2 or 3) or low Vkine (0 or 1). Results: Overall, 42.7% of all samples were scored as PD-L1+ (CPS≥1). PD-L1+ samples demonstrated greater CD8+ TILs than PD-L1- samples (44.5±71 vs 14±22; p<0.001). The presence of immunosuppressive VCAN negatively correlated with CD8+ TILs (16.8±27 in VCAN+ samples vs 39.4±67 in VCAN- samples; p<0.01). The VPP phenotype was observed in 47.8% of the cancer samples. VPP samples also demonstrated a trend towards greater CD8+ TILs compared to VPW samples (35.9±63 vs 22.7±46; p=0.09); however, this was not statistically significant. Of all samples, 33.9% were both PD-L1+ and VCAN-. These dual PD-L1+ and VCAN- samples demonstrated even greater CD8+ TILs compared to either PD-L1- or VCAN+ samples (49.5±79 vs 18.1±29; p<0.001). Discussion: Our TMA demonstrates enhanced CD8+ TILs in PD-L1+ samples, as previously reported. Additionally, we discovered the presence of VCAN and its proteolysis are independent predictors of enhanced CD8+ TILs in esophageal cancers. These data indicate the potential importance of the tumor matrix in establishing a permissive tumor microenvironment. These data support the potential for combining VCAN testing with PD-L1 testing to further select appropriate patients for immunotherapeutic trials in esophageal cancer. Citation Format: Phillip B. Emmerich, Jacob Witt, Darya Buehler, Mitchell Depke, Nathaniel Verhagen, Linda Clipson, Kristina Matkowskyj, Nataliya Uboha, Andrew Baschnagel, Dustin Deming. PD-L1 status and versican proteolysis predict CD8+ T cell infiltration in esophageal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3294.

Abstract 219: NAD Redox Imbalance Accelerates Diabetic Cardiomyopathy via Protein Acetylation and Oxidative Stress

Diabetes is long linked to lowered NAD/NADH ratio, aka NAD redox imbalance, but its causal role to diabetic cardiomyopathy is not established. We used mouse models with latent decrease in cardiac NAD/NADH ratio (cardiac-specific Ndufs4-KO, cKO) and elevated cardiac NAD levels to directly test whether cardiac NAD redox imbalance accelerates diabetic cardiomyopathy. Control and cKO mice were subjected to 8-week T1D stress, and longitudinal cardiac function was measured by echocardiography. Accelerated declines in systolic and diastolic function were observed in T1D cKO mice. Insulin depletion and hyperglycemia were similar in T1D control and T1D cKO mice, and serum metabolomic analyses showed unchanged aqueous and lipid metabolite levels. These metabolite results suggested that T1D control and cKO hearts were stressed under similar diabetic conditions. Importantly, elevation of cardiac NAD levels to attenuate NAD redox imbalance mitigated the accelerated functional declines in T1D cKO hearts. The data from mouse models with manipulated NAD redox states suggested that NAD redox imbalance accelerates diabetic cardiomyopathy. Cardiac fibrosis levels were not different in T1D control and cKO hearts, while transcript levels of fibrotic genes, including Adamts proteinases, integrins, laminins, matrix metalloproteinases and collagens, also showed no difference. Therefore, the accelerated functional declines in T1D cKO hearts are not due to altered extracellular matrix environment, but are rather due to cardiomyocyte dysfunction. We next determined whether the accelerated cardiac dysfunction is mediated via protein acetylation and oxidative stress. NAD-dependent global protein acetylation and inhibitory acetylation of superoxide dismutase 2 were elevated in T1D cKO hearts. Inhibition of SOD2 concomitantly promoted elevation of protein oxidation levels in T1D cKO hearts. The results suggested that NAD redox balance-dependent protein acetylation regulates oxidative stress to promote diabetic cardiomyopathy.

Abstract 220: Emerging Roles of Altered NAD Metabolism in Diabetic Cardiomyopathy

Diabetes is linked to altered NAD metabolism whose causal roles in cardiac dysfunction are poorly understood. Synthesis, consumption and redox balance of NAD dictate NAD metabolism and its homeostasis, and we here explored their roles in diabetic cardiomyopathy. 16 week of Type 1 diabetes (T1D) to C57BL6N mice was associated with lowered cardiac NAD/NADH, mild systolic and more severe diastolic dysfunction. We next used mouse models with altered NAD metabolism to determine how NAD redox balance impacts diabetic cardiomyopathy. Using cardiac-specific Ndufs4-KO mice (cKO) as a model of latent decrease in cardiac NAD/NADH, we observed accelerated systolic and diastolic dysfunction (lowered fractional shortening, E’/A’ ratio, and increased e/E’ ratio) in male cKO mice stressed with chronic T1D. Cardiac hypertrophy (heart weight/tibia length), insulin depletion and hyperglycemia levels were similar in these mice. Serum metabolomic analyses (~240 metabolites) also showed unchanged aqueous and lipid metabolite levels, suggesting that the diabetic stresses on these hearts were similar. The accelerated dysfunction of T1D cKO hearts was also observed in another female cohort. Importantly, elevation of cardiac NAD levels to attenuate NAD redox imbalance mitigated the accelerated dysfunction of T1D cKO hearts. In another cohort, control and cKO mice were stressed by 16-week high fat diet feeding. Accelerated systolic and diastolic dysfunction, and increased hypertrophy were observed in T2D cKO mice with similar glycemic levels to control mice. The data suggested that NAD redox imbalance is a positive regulator of cardiac dysfunction induced by T1D or T2D. However, NAD-dependent pathogenic mechanism induced by T1D or T2D (e.g. hypertrophy) can be different, and is under further investigation. Tissue fibrosis and mRNA levels of pro-fibrotic genes, including Adamts proteinases, integrins, laminins, matrix metalloproteinases and collagens were unchanged in T1D cKO hearts. Therefore, the accelerated dysfunction of T1D cKO hearts is due to cardiomyocyte dysfunction. We examined how NAD metabolism, other than the redox balance, may alter cardiomyocyte function. Levels of metabolite and mRNA regulating NAD synthesis and consumption pathways were measured. Of 32 transcripts assayed, Nmrk2 levels were uniquely up-regulated in T1D cKO hearts, and lowered by elevation of cardiac NAD levels. NMRK product levels were concomitantly decreased in T1D cKO hearts. NAD-dependent global acetylation and inhibitory superoxide dismutase 2 acetylation were increased in T1D cKO hearts. Protein oxidation levels were concomitantly raised. Hyperacetylation in proteins like CaM kinase was associated with increased phosphorylation in troponin I, not in MyBPC or PLN in T1D cKO hearts. These data support the emerging, multifaceted roles of altered NAD metabolism in the progression of diabetic cardiomyopathy.

IL-17A induction of ADAMTS-5 in differentiated THP-1 cells is modulated by the ERK signaling pathway.

Atherosclerosis is initiated when lipoproteins are trapped by proteoglycans in the arterial intima. Macrophages play a vital role in this disease, especially in the formation of foam cells and the regulation of pro-inflammatory responses. They also participate in plaque stabilization through the secretion of matrix metalloproteinases. Studies have reported the role of ADAMTS proteases in osteoarthritis and atherosclerotic lesions.In the present study, we have studied the effect of interleukin-17A (IL-17A) on the expression of ADAMTS-5 in the macrophage cell line THP-1. The results show that the mRNA and protein expression levels of ADAMTS-5 were significantly upregulated when differentiated THP-1 cells were treated with 100 ng/mL of IL-17A for 24 h with maximum ADAMTS-5 mRNA expression levels obtained at 8 h of stimulation. Subsequent inhibition studies showed that IL-17A upregulation of ADAMTS-5 was mediated through ERK and JNK pathways in THP-1 cells. Phosphorylation studies revealed that the expression of ADAMTS-5 transcripts was upregulated by IL-17A through the activation of p-c-Raf (S338), p-MEK1/2 (Ser217/221), p-p44/42 MAPK (Thr202/Tyr204), and p-Elk1 (Ser383). ERK1/2 siRNA transfection further confirmed that the ERK pathway is involved in the expression of ADAMTS-5 in IL-17A-stimulated THP-1 cells.

VCAN accumulation and proteolysis as predictors of T lymphocyte-excluded and permissive tumor microenvironments.

3127 Background: Immune checkpoint inhibitors (ICIs) represent a major advance for treating solid tumors. However, only a minority of patients (pts) benefit from these therapies and markers that predict response have been elusive. Versican (VCAN) is an immunosuppressive proteoglycan in the tumor microenvironment (TME), which releases an immunostimulatory N-terminal fragment versikine (Vkine) when cleaved by ADAMTS proteases. We have demonstrated in colorectal cancers (CRC) that a low VCAN/high Vkine (VCAN proteolytic predominant [VPP]) phenotype correlates with increased tumor-infiltrating CD8+ T lymphocytes (TILs). Here we examine the accumulation of VCAN as a marker of immune exclusion and its proteolysis as a marker of an immune-permissive TME. Methods: Immunohistochemistry for VCAN, Vkine and CD8+ was performed on samples from 1662 pts across breast (BC), CRC, endometrial cancer, pancreatic adenocarcinoma (PDAC), esophageal cancers and neuroendocrine tumors (NETs), across stages of disease (I-IV) and with diverse prior treatments. Stromal intensities of VCAN and Vkine staining quantified in collaboration with blinded surgical pathologists using a 0-3+ scale. 0/1+ were considered “low” for both VCAN and Vkine, whereas 2/3+ were considered “high”. The number of CD8+ TILs were counted using 400x magnification, the equivalent of a high power field (hpf). Results: Across the entire cohort VCAN phenotypes were diverse (VCAN high/Vkine low, 21%; VCAN high/Vkine high, 23%; VCAN low/Vkine low, 29%; VCAN low/Vkine high (VPP), 27%). Consistent with VCAN accumulation as a marker of T cell exclusion, VCAN low cancers had increased TILs compared to VCAN high (4.8 vs 18.3 TILs/hpf, p < 0.001). Low VCAN was identified in 85% esophageal, 79% NET (including small cell lung cancer [SCLC]) 72% endometrial, 47% MSI-H CRCs, 28% triple-negative BC and only 22% MSS CRC, 18% PDAC, 17% ER+ BCs. The VPP subgroup had the highest TILs per hpf across tumors. VPP was identified in 47% of esophageal, 45% endometrial, 41% NETs (including SCLC), 24% MSI-H CRCs, and only 9% MSS CRC, 7% ER+ BCs, 3% triple-negative BCs, and 0% of PDAC (n = 131 PDAC pts). Conclusions: VCAN accumulation correlates with T lymphocyte exclusion, while VCAN proteolysis predicts an immune permissive phenotype. VCAN accumulation and proteolysis are now incorporated into ICI clinical trials as a potential marker of response. Future studies will clarify the role of these biomarkers in predicting benefits of immuno-oncology treatment strategies.

Extracellular Protease ADAMTS1 Is Required at Early Stages of Human Uveal Melanoma Development by Inducing Stemness and Endothelial-Like Features on Tumor Cells.

Extracellular matrix remodeling within the tumor microenvironment has been recognized as a relevant dynamic framework during tumor growth. However, research on proteases that trigger this remodeling keeps revealing a wide range of actions including both pro- and anti-tumorigenic. The extracellular protease ADAMTS1 exemplifies this dual role. In this work, we first confirmed a positive correlation of ADAMTS1 with endothelial-like phenotype of human melanoma cells together with the finding of associated signatures, including key genes such as endothelial CDH5. Using a CRISPR-Cas9 approach, we observed that the inhibition of ADAMTS1 in an aggressive uveal melanoma model compromised its endothelial-like properties, and more importantly, caused a robust blockade on the progression of tumor xenografts. Although vasculature emerged affected in ADAMTS1-deficient tumors, the most relevant action implied the downregulation of endothelial CDH5 in tumor cells, in association with stemness markers. Indeed, melanoma sphere assays also revealed a deficient commitment to form spheres in the absence of ADAMTS1, directly correlating with stemness markers and, remarkably, also with CDH5. Finally, taking advantage of advanced bioinformatics tools and available public data of uveal melanomas, we disclosed new prognosis factors, including endothelial elements and ADAMTS proteases. Our findings support the key role of ADAMTS proteases for uveal melanoma development since earlier stages, modulating the complex crosstalk between extracellular matrix and the induction of stemness and endothelial-like features. To our knowledge, this is the first report that supports the development of therapeutic targets on the extracellular matrix to overcome uveal melanoma.

ADAMTS Proteins: Concepts, Challenges, and Prospects.

The ADAMTS superfamily comprises secreted metalloproteases (ADAMTS proteases) as well as structurally related secreted glycoproteins that lack catalytic activity (ADAMTS-like proteins). Members of both families participate in diverse morphogenetic processes during embryonic development, and connective tissue maintenance and hemostasis in the adult. Several ADAMTS proteins are heavily implicated in genetic and acquired human and animal disorders. Despite these indicators of a profound biological and medical importance, detailed knowledge about their molecular structures, substrates, biological pathways, and biochemical mechanisms is significantly limited by unique intrinsic characteristics, which have led to several technical challenges. As a group, they are larger, more heavily modified, and harder to purify than other secreted proteases. In addition, idiosyncratic aspects of individual members are deserving of further investigation but can complicate their analysis. Here, some of the key concepts, challenges, and prospects in ADAMTS research are discussed in the context of the knowledge accumulated over the past two decades. Individual chapters in this volume of Methods in Molecular Biology provide practical solutions for surmounting these challenges. Since the biology of a protease is actually the biology of its substrates, there is considerable emphasis on purification of recombinant ADAMTS proteins, identification of their substrates and assays for their proteolytic activity.

Visualization and Quantification of Pericellular Matrix.

The pericellular matrix (PCM), also known as the pericellular coat or glycocalyx, lies between the plasma membrane and the interstitial extracellular matrix (ECM). It can have a dramatic influence on cell function because of its presence at the interface between the cell and its microenvironment. A common tool used to demonstrate the PCM is the particle exclusion assay in which fixed red blood cells are utilized to outline the boundary of the cell together with its PCM. PCM visualization and quantification provide opportunities to uncover the roles of ADAMTS proteases in PCM remodeling in many cell types and processes.

The "other" 15-40%: The Role of Non-Collagenous Extracellular Matrix Proteins and Minor Collagens in Tendon.

Extracellular matrix (ECM) determines the physiological function of all tissues, including musculoskeletal tissues. In tendon, ECM provides overall tissue architecture, which is tailored to match the biomechanical requirements of their physiological function, that is, force transmission from muscle to bone. Tendon ECM also constitutes the microenvironment that allows tendon-resident cells to maintain their phenotype and that transmits biomechanical forces from the macro-level to the micro-level. The structure and function of adult tendons is largely determined by the hierarchical organization of collagen type I fibrils. However, non-collagenous ECM proteins such as small leucine-rich proteoglycans (SLRPs), ADAMTS proteases, and cross-linking enzymes play critical roles in collagen fibrillogenesis and guide the hierarchical bundling of collagen fibrils into tendon fascicles. Other non-collagenous ECM proteins such as the less abundant collagens, fibrillins, or elastin, contribute to tendon formation or determine some of their biomechanical properties. The interfascicular matrix or endotenon and the outer layer of tendons, the epi- and paratenon, includes collagens and non-collagenous ECM proteins, but their function is less well understood. The ECM proteins in the epi- and paratenon may provide the appropriate microenvironment to maintain the identity of distinct tendon cell populations that are thought to play a role during repair processes after injury. The aim of this review is to provide an overview of the role of non-collagenous ECM proteins and less abundant collagens in tendon development and homeostasis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:23-35, 2020.

ADAM and ADAMTS Proteases in Hepatic Disorders

Proteolysis is an irreversible post-translational modification that regulates protein function and signal transduction. This includes remodelling of the extracellular matrix, release of membrane-bound cytokines and receptor ectodomains, as well as the initiation of intracellular signalling cues. Members of the adamalysin protease subfamily, in particular the ADAM (a disintegrin and metalloprotease) and ADAMTS (the ADAM containing thrombospondin motif) families, are involved in these processes. This review presents an overview of how ADAM and ADAMTS proteins are involved in liver physiology and pathophysiology.

Abstract 1322: A meta-analysis of genome-wide association studies identifies novel loci that influence breast cancer prognosis

Abstract Familial studies were among the first to indicate that breast cancer prognosis has a heritable component. Subsequently many variants associated with prognosis have been identified using a range of techniques including genome-wide association studies (GWAs). Despite these advances, much of the heritability remains unexplained. In young women, breast cancer is characterised by a higher incidence of adverse pathological features, unique tumour gene expression profiles and worse survival. In addition, the association of risk with conventional epidemiological exposures is less clear in women with early onset. We hypothesise that some of these difference between early and late onset could be influenced by germline variation. To identify additional variants that influence breast cancer prognosis we conducted a two stage meta-analysis of four GWAs consisting of 6,042 patients from the UK (POSH), Finland (HEBCS), Germany (SUCCESS-A) and Australia (ABCFS). Cox-regression analyses were used to investigate overall survival (OS, n=1,101 events) and disease-free survival (DFS, n=1,316 events) with correction for oestrogen status (ER). These survival analyses were repeated in a subset of patients with early onset (aged ≤40 at diagnosis, n=2,315 patients, OS n=604 events, DFS n=716 events). Meta-analysis identified two intronic SNPs in ADAMTSL1 that were associated exclusively with early onset DFS, rs715212 (Pmeta=3.54x10-5) and rs10963755 (Pmeta=3.91x10-4) without heterogeneity between cohorts. Multivariable Cox-regression demonstrated that the effect of these SNPs were independent of the classical prognostic factors. Most importantly, rs715212 reached genome-wide significance (Pmultivariate=5.37x10-8) in the multivariate model. ADAMTSL1 encodes a glycoprotein that forms part of the extracellular matrix (ECM) and may function in cell-cell or cell-matrix interactions or may regulate other ADAMTS proteases. Previous studies have shown that ADAMTSL1 is hypermethylated in ER positive breast cancer tumours. Using GTEx to perform eQTL analysis, we found that rs715212 is associated with the expression of several biologically relevant genes AREG (P=0.035), TNF (P=0.015), FASLG (P=0.0031) and EGF (P=0.0018) in breast mammary tissue. Interestingly, separate studies have shown that AREG is overexpressed in ER-positive breast tumours from pre-menopausal women versus post-menopausal women. Furthermore, AREG is differentially expressed between parous and non-parous mammary glands and is persistently downregulated by parity, which suggests it may contribute to the susceptibility of the nulliparous gland to breast cancer. We conclude that rs715212 is associated with an increased risk of disease progression in patients with early onset and speculate that this could be due to an interaction with the expression of AREG. Note: This abstract was not presented at the meeting. Citation Format: Latha Kadalayil, Sofia Khan, Heli Nevanlinna, Peter A. Fasching, Fergus J. Couch, John Hopper, Jianjun Liu, Tom Maishman, Lorraine Durcan, Carl Bloomqvist, Andy Collins, Dianna Eccles, William Tapper. A meta-analysis of genome-wide association studies identifies novel loci that influence breast cancer prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1322. doi:10.1158/1538-7445.AM2017-1322