Lysyl Oxidase Family Research Articles

LOXL1 confers antiapoptosis and promotes gliomagenesis through stabilizing BAG2

The lysyl oxidase (LOX) family is closely related to the progression of glioma. To ensure the clinical significance of LOX family in glioma, The Cancer Genome Atlas (TCGA) database was mined and the analysis indicated that higher LOXL1 expression was correlated with more malignant glioma progression. The functions of LOXL1 in promoting glioma cell survival and inhibiting apoptosis were studied by gain- and loss-of-function experiments in cells and animals. LOXL1 was found to exhibit antiapoptotic activity by interacting with multiple antiapoptosis modulators, especially BAG family molecular chaperone regulator 2 (BAG2). LOXL1-D515 interacted with BAG2-K186 through a hydrogen bond, and its lysyl oxidase activity prevented BAG2 degradation by competing with K186 ubiquitylation. Then, we discovered that LOXL1 expression was specifically upregulated through the VEGFR-Src-CEBPA axis. Clinically, the patients with higher LOXL1 levels in their blood had much more abundant BAG2 protein levels in glioma tissues. Conclusively, LOXL1 functions as an important mediator that increases the antiapoptotic capacity of tumor cells, and approaches targeting LOXL1 represent a potential strategy for treating glioma. In addition, blood LOXL1 levels can be used as a biomarker to monitor glioma progression.

Role of the lysyl oxidase family in organ development (Review).

Lysyl oxidase proteins (LOXs) are amine oxidases, which are mainly located in smooth muscle cells and fibroblasts and serve an important role in the formation of the extracellular matrix (ECM) in a copper-dependent manner. Owing to the ability of LOX proteins to modulate crosslinking between collagens and to promote the deposition of other fibers, they serve crucially in organogenesis and the subsequent organ development, as well as disease initiation and progression. In addition, ECM formation significantly influences organ morphological formation in both cancer- and non-tumor-related diseases, in addition to cellular epigenetic transformation and migration, under the influence of LOXs. A number of different signaling pathways regulate the LOXs expression and their enzymatic activation. The tissue remodeling and transformation process shares some resemblance between oncogenesis and embryogenesis. Additionally the roles that LOXs serve appeared to be stressed during oncogenesis and tumor metastasis. It has also been indicated LOXs have a noteworthy role in non-tumor diseases. Nonetheless, the role of LOXs in systemic or local organ development and disease control remains unknown. In the present study, the essential roles that LOXs play in embryogenesis were unveiled partially, whereas the role of LOXs in organ or systematic development requires further investigations. The present review aimed to discuss the roles of members of the LOX family in the context of the remodeling of organogenesis and organ development. In addition, the consequences of the malfunction of these proteins related to the development of abnormalities and resulting diseases is discussed.

Molecular Insights Into Lysyl Oxidases in Cartilage Regeneration and Rejuvenation.

Articular cartilage remains among the most difficult tissues to regenerate due to its poor self-repair capacity. The lysyl oxidase family (LOX; also termed as protein-lysine 6-oxidase), mainly consists of lysyl oxidase (LO) and lysyl oxidase-like 1-4 (LOXL1-LOXL4), has been traditionally defined as cuproenzymes that are essential for stabilization of extracellular matrix, particularly cross-linking of collagen and elastin. LOX is essential in the musculoskeletal system, particularly cartilage. LOXs-mediated collagen cross-links are essential for the functional integrity of articular cartilage. Appropriate modulation of the expression or activity of certain LOX members selectively may become potential promising strategy for cartilage repair. In the current review, we summarized the advances of LOX in cartilage homeostasis and functioning, as well as copper-mediated activation of LOX through hypoxia-responsive signaling axis during recent decades. Also, the molecular signaling network governing LOX expression has been summarized, indicating that appropriate modulation of hypoxia-responsive-signaling-directed LOX expression through manipulation of bioavailability of copper and oxygen is promising for further clinical implications of cartilage regeneration, which has emerged as a potential therapeutic approach for cartilage rejuvenation in tissue engineering and regenerative medicine. Therefore, targeted regulation of copper-mediated hypoxia-responsive signalling axis for selective modulation of LOX expression may become potential effective therapeutics for enhanced cartilage regeneration and rejuvenation in future clinical implications.

LOX family and ZFPM2 as novel diagnostic biomarkers for malignant pleural mesothelioma

BackgroundMalignant pleural mesothelioma (MPM) is a rare and aggressive cancer that develops in the pleural and outer layer of tissues surrounding the lungs. MPM is primarily caused by occupational exposure to asbestos and results in a poor prognosis. Effective therapeutics as well as early diagnostics for the MPM are still lacking. To identify potential diagnostic biomarkers for MPM, we performed bioinformatics analysis of public database.MethodsUtilizing databases from Cancer Cell Line Encyclopedia (CCLE) and Gene Expression Omnibus (GEO), we identified several potential candidates that could act as MPM biomarkers. We carried out additional molecular analyses of these potential markers using MPM patient tissue samples via quantitative polymerase chain reaction.ResultsWe identified Lysyl oxidase (LOX), Lysyl oxidase homologs 1&2 (LOXL1& LOXL2) Zinc Finger Protein, FOG Family Member 2 (ZFPM2) as potential diagnostic biomarkers for MPM. In this study, we found that the LOX family and ZFPM2 showed comparable diagnostic ability to Fibulin-3 or mesothelin (MSLN) and would be better potential biomarkers than Sulfatase 1 (SULF1), Thrombospondin 2 (THBS2) and Cadherin 11 (CDH11).ConclusionsLOX family and ZPFM2 were identified as novel MPM diagnostic biomarkers which could strengthen MPM clinical diagnostic capabilities.

Identification and characterization of the promoter of cancer-related gene LOXL2

Lysyl oxidase like 2, LOXL2, as a member of the lysyl oxidase (LOX) family, has been shown to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. LOXL2 is also engaged to transcription regulation, cell signaling transduction and cell adhesion regulation. It has been reported that LOXL2 is highly expressed in several types of tumors and promotes cell proliferation and migration in various cancer cells. However, the regulatory mechanism of LOXL2 expression remains largely unknown. To further investigate its transcriptional regulatory mechanism, LOXL2 promoter region has been cloned and identified in the present study. Chromatin state analysis revealed that LOXL2 gene locus contained an active promoter near its first exon. We then constructed five different LOXL2 gene promoter luciferase reporter constructs covering 1.7 kb upstream of LOXL2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all the five constructs showed notable promoter activity, and LOXL2 core promoter was located in a region of 185 bp near the transcription initiation site. Transcriptional factor binding analysis indicated that, LOXL2 promoter lacked classical TATA box, but contained putative binding sites for classic transcriptional factors such as Sp1 and NF-κB. Ectopic overexpression of Sp1 significantly enhanced LOXL2 promoter activity as well as its endogenous expression in cells. In contrast, mithramycin A (a selective Sp1 inhibitor) treatment repressed LOXL2 promoter as well as its endogenous transcription. Site directed mutagenesis assay further confirmed that the Sp1 binding sites were essential for proximal prompter activity of LOXL2 gene. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 bound LOXL2 promoter in vivo. Of note, the expression of Sp1 and LOXL2 are positively correlated, and the higher expression of LOXL2 is associated with poor prognosis in colorectal cancer, strongly suggesting the implication of Sp1-mediated LOXL2 transactivation in the pathogenesis of colorectal cancer.

Targeting the lysyl oxidases in tumour desmoplasia

The extracellular matrix (ECM) is a fundamental component of tissue microenvironments and its dysregulation has been implicated in a number of diseases, in particular cancer. Tumour desmoplasia (fibrosis) accompanies the progression of many solid cancers, and is also often induced as a result of many frontline chemotherapies. This has recently led to an increased interest in targeting the underlying processes. The major structural components of the ECM contributing to desmoplasia are the fibrillar collagens, whose key assembly mechanism is the enzymatic stabilisation of procollagen monomers by the lysyl oxidases. The lysyl oxidase family of copper-dependent amine oxidase enzymes are required for covalent cross-linking of collagen (as well as elastin) molecules into the mature ECM. This key step in the assembly of collagens is of particular interest in the cancer field since it is essential to the tumour desmoplastic response. LOX family members are dysregulated in many cancers and consequently the development of small molecule inhibitors targeting their enzymatic activity has been initiated by many groups. Development of specific small molecule inhibitors however has been hindered by the lack of crystal structures of the active sites, and therefore alternate indirect approaches to target LOX have also been explored. In this review, we introduce the importance of, and assembly steps of the ECM in the tumour desmoplastic response focussing on the role of the lysyl oxidases. We also discuss recent progress in targeting this family of enzymes as a potential therapeutic approach.

The Role of Lysyl Oxidase Enzymes in Cardiac Function and Remodeling.

Lysyl oxidase (LOX) proteins comprise a family of five copper-dependent enzymes (LOX and four LOX-like isoenzymes (LOXL1–4)) critical for extracellular matrix (ECM) homeostasis and remodeling. The primary role of LOX enzymes is to oxidize lysyl and hydroxylysyl residues from collagen and elastin chains into highly reactive aldehydes, which spontaneously react with surrounding amino groups and other aldehydes to form inter- and intra-catenary covalent cross-linkages. Therefore, they are essential for the synthesis of a mature ECM and assure matrix integrity. ECM modulates cellular phenotype and function, and strikingly influences the mechanical properties of tissues. This explains the critical role of these enzymes in tissue homeostasis, and in tissue repair and remodeling. Cardiac ECM is mainly composed of fibrillar collagens which form a complex network that provides structural and biochemical support to cardiac cells and regulates cell signaling pathways. It is now becoming apparent that cardiac performance is affected by the structure and composition of the ECM and that any disturbance of the ECM contributes to cardiac disease progression. This review article compiles the major findings on the contribution of the LOX family to the development and progression of myocardial disorders.

LOXL2 Upregulates Phosphorylation of Ezrin to Promote Cytoskeletal Reorganization and Tumor Cell Invasion.

Lysyl oxidase-like 2 (LOXL2), a copper-dependent enzyme of the lysyl oxidase family and its nonsecreted, catalytically dead spliced isoform L2Δ13, enhance cell migration and invasion, stimulate filopodia formation, modulate the expression of cytoskeletal genes, and promote tumor development and metastasis in vivo. We previously showed that LOXL2 reorganizes the actin cytoskeleton in esophageal squamous cell carcinoma (ESCC) cells, however, the underlying molecular mechanisms were not identified. Here, using interactome analysis, we identified ezrin (EZR), fascin (FSCN1), heat shock protein beta-1 (HSPB1), and tropomodulin-3 (TMOD3) as actin-binding proteins that associate with cytoplasmic LOXL2, as well as with its L2Δ13 variant. High levels of LOXL2 and L2Δ13 and their cytoskeletal partners correlated with poor clinical outcome in patients with ESCC. To better understand the significance of these interactions, we focused on the interaction of LOXL2 with ezrin. Phosphorylation of ezrin at T567 was greatly reduced following depletion of LOXL2 and was enhanced following LOXL2/L2Δ13 reexpression. Furthermore, LOXL2 depletion inhibited the ability of ezrin to promote tumor progression. These results suggest that LOXL2-induced ezrin phosphorylation, which also requires PKCα, is critical for LOXL2-induced cytoskeletal reorganization that subsequently promotes tumor cell invasion and metastasis in ESCC. In summary, we have characterized a novel molecular mechanism that mediates, in part, the protumorigenic activity of LOXL2. These findings may enable the future development of therapeutic agents targeting cytoplasmic LOXL2. SIGNIFICANCE: LOXL2 and its spliced isoform L2Δ13 promote cytoskeletal reorganization and invasion of esophageal cancer cells by interacting with cytoplasmic actin-binding proteins such as ezrin.

Lysyl oxidases: from enzyme activity to extracellular matrix cross-links.

The lysyl oxidase family comprises five members in mammals, lysyl oxidase (LOX) and four lysyl oxidase like proteins (LOXL1-4). They are copper amine oxidases with a highly conserved catalytic domain, a lysine tyrosylquinone cofactor, and a conserved copper-binding site. They catalyze the first step of the covalent cross-linking of the extracellular matrix (ECM) proteins collagens and elastin, which contribute to ECM stiffness and mechanical properties. The role of LOX and LOXL2 in fibrosis, tumorigenesis, and metastasis, including changes in their expression level and their regulation of cell signaling pathways, have been extensively reviewed, and both enzymes have been identified as therapeutic targets. We review here the molecular features and three-dimensional structure/models of LOX and LOXLs, their role in ECM cross-linking, and the regulation of their cross-linking activity by ECM proteins, proteoglycans, and by inhibitors. We also make an overview of the major ECM cross-links, because they are the ultimate molecular readouts of LOX/LOXL activity in tissues. The recent 3D model of LOX, which recapitulates its known structural and biochemical features, will be useful to decipher the molecular mechanisms of LOX interaction with its various substrates, and to design substrate-specific inhibitors, which are potential antifibrotic and antitumor drugs.

Role of the lysyl oxidase enzyme family in cardiac function and disease

Heart diseases are a major cause of morbidity and mortality world-wide. Lysyl oxidase (LOX) and related LOX-like (LOXL) isoforms play a vital role in remodelling the extracellular matrix (ECM). The LOX family controls ECM formation by cross-linking collagen and elastin chains. LOX/LOXL proteins are copper-dependent amine oxidases that catalyse the oxidation of lysine, causing cross-linking between the lysine moieties of lysine-rich proteins. Dynamic changes in LOX and LOXL protein-expression occur in a variety of cardiac pathologies; these changes are believed to be central to the associated tissue-fibrosis. An awareness of the potential pathophysiological importance of LOX has led to the evaluation of interventions that target LOX/LOXL proteins for heart-disease therapy. The purposes of this review article are: (i) to summarize the basic biochemistry and enzyme function of LOX and LOXL proteins; (ii) to consider their tissue and species distribution; and (iii) to review the results of experimental studies of the roles of LOX and LOXL proteins in heart disease, addressing involvement in the mechanisms, pathophysiology and therapeutic responses based on observations in patient samples and relevant animal models. Therapeutic targeting of LOX family enzymes has shown promising results in animal models, but small-molecule approaches have been limited by non-specificity and off-target effects. Biological approaches show potential promise but are in their infancy. While there is strong evidence for LOX-family protein participation in heart failure, myocardial infarction, cardiac hypertrophy, dilated cardiomyopathy, atrial fibrillation and hypertension, as well as potential interest as therapeutic targets, the precise involvement of LOX-family proteins in heart disease requires further investigation.

2-Aminomethylene-5-sulfonylthiazole Inhibitors of Lysyl Oxidase (LOX) and LOXL2 Show Significant Efficacy in Delaying Tumor Growth.

The lysyl oxidase (LOX) family of extracellular proteins plays a vital role in catalyzing the formation of cross-links in fibrillar elastin and collagens leading to extracellular matrix (ECM) stabilization. These enzymes have also been implicated in tumor progression and metastatic disease and have thus become an attractive therapeutic target for many types of invasive cancers. Following our recently published work on the discovery of aminomethylenethiophenes (AMTs) as potent, orally bioavailable LOX/LOXL2 inhibitors, we report herein the discovery of a series of dual LOX/LOXL2 inhibitors, as well as a subseries of LOXL2-selective inhibitors, bearing an aminomethylenethiazole (AMTz) scaffold. Incorporation of a thiazole core leads to improved potency toward LOXL2 inhibition via an irreversible binding mode of inhibition. SAR studies have enabled the discovery of a predictive 3DQSAR model. Lead AMTz inhibitors exhibit improved pharmacokinetic properties and excellent antitumor efficacy, with significantly reduced tumor growth in a spontaneous breast cancer genetically engineered mouse model.

Abstract 5175: Expression profile and role of LOXL3 in astrocytomas

Abstract Astrocytomas are the most common primary central nervous system tumors in adults. The WHO classification based on histologial characteristics according consider four malignant grades, glioblastoma (GBM) being the most malignant (grade IV). Molecular classification of GBM is based on the integration of somatic mutations, DNA methylation and specific transcript/protein expression and consider four subtypes: proneural, classic, mesenchymal and neural. Lysyl oxidase family is composed of five members (LOX, LOXL1, LOXL2, LOXL3 and LOXL4) which are enzymes responsible for catalyzing lysine-derived cross-links of collagen and elastin. The aim of the present work was to analyze the gene expression of LOXL3 in astrocytomas of different grades and different GBM molecular subtypes, the impact of LOXL3 expression level on overall survival in GBM cases of our cohort. These data were also analyzed in the TCGA database to validate our findings. LOXL3expression increased with astrocytoma malignant grades, and proved to be a prognostic factor, as GBM patients with lower LOXL3 expression presented longer survival time than those with higher expression (p<0.041). Analogous data was obtained in the TCGA database, when considering temozolamide-treated GBM patients (p=0.049). Additionally, LOXL3 expression was higher in mesenchymal GBM subtype than in proneural and classic GBM subtypes in TCGA cohort (p<0.001). To further understand LOXL3 functional role in gliomagenesis, LOXL3 was silenced with two distinct siRNA sequences in U87MG, a mesenchymal subtype of GBM cell line. LOXL3 down regulation was confirmed at gene and protein expression levels. Functional assays with silenced LOXL3 demonstrated a decrease in the cell proliferation (p<0.05), and an increase in the apoptosis associated to temozolamide treatment (p<0.05). Immunofluorescence analysis in U87MG cells showed LOXL3 colocalized with mitochondria. Interestingly, such colocalization decreased when LOXL3 was downregulated, associated to a clear morphological alteration in mitochondria shape. A rough increase in mitochondria volume was observed, suggesting fused mitochondria. In addition, such condition was associated to a 23% decrease in mitochondrial DNA copy number. Altogether, our findings suggest that LOXL3 is upregulated in GBM, with impact in prognosis and might play a role in mitochondria dynamics and/or mitophagy, modulating apoptosis. Transcriptome analysis, currently in progress, will further clarify the associated players in this signaling pathway. Citation Format: Talita de Souza Laurentinho, Roseli da Silva Soares, Suely Kazue Marie, Sueli Mieko Oba-Shinjo. Expression profile and role of LOXL3 in astrocytomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5175.

Abstract 4594: Cytoplasmic LOXL2 regulates actin cytoskeletal organization in esophageal squamous cell carcinoma progression

Abstract Background: Lysyl oxidase-like 2 (LOXL2), a copper-dependent enzyme of the lysyl oxidase family, promotes tumor progression and metastasis. We previously revealed that the tumor-promoting role of LOXL2 is mediated by perturbing the architecture of the actin cytoskeleton in esophageal squamous cell carcinoma (ESCC). However, the molecular mechanisms underlying LOXL2-driven ESCC progression remain elusive, and the cytoskeletal proteins that directly interact with LOXL2 remain uncharacterized. Methods: Knockdown LOXL2 in ESCC cells was used by a lentivirus shRNA targeting LOXL2 and analyzed by RNA-sequencing and bioinformatics analysis. Proteomic analysis following by co-immunoprecipitation and immunofluorescence were applied for identification and validation of interacting partners of LOXL2 and its splicing isoform L2Δ13. Cell migration and invasion were measured by wound healing and transwell assays. By risk score calculations, immunohistochemistry of tissue microarrays were adopted to evaluate the prognosis of ESCC patients. Results: We found that knockdown of LOXL2 in ESCC cells suppresses cell migration and invasion, whereas re-expressions of LOXL2 and its non-enzymatic splicing isoform L2Δ13 rescue these cell behaviors. Silencing of LOXL2 inhibits filopodia formation, and induces changes in the expression of cytoskeleton-associated genes. Our interactome analysis identified four actin-binding proteins, i.e. ezrin (EZR), facsin (FSCN1), heat shock protein beta-1 (HSPB1) and tropomodulin-3 (TMOD3), as novel interactors of LOXL2 and L2Δ13. These novel LOXL2/L2Δ13 interaction networks carry important prognostic values in ESCC, as molecular signatures of combinations of LOXL2/L2Δ13 and their cytoskeletal interactors are associated with clinical outcome in ESCC patients. Mechanistically, the ezrin-LOXL2 interaction promotes PKCα-stimulated phosphorylation of ezrin at Thr 567 (T567), an essential residue for ezrin activation, which enhances cell motility and cell invasion in ESCC. Correlating with this, double-high expression of LOXL2 and phosphorylated ezrin-T567 predicts significantly shorter overall survival in ESCC patients. Conclusion: In conclusion, our findings have uncovered a novel molecular mechanism underlying the tumor-promoting role of cytoplasmic LOXL2, which may open new avenues for the therapeutic targeting of LOXL2 in ESCC. (This study is supported by the Natural Science Foundation of China (No. 81472613) and Li Ka Shing Foundation). Citation Format: Xiuhui Zhan, Jiwei Jiao, Haifeng Zhang, Jianzhong He, Runliu Li, Haiying Zou, Zhiyong Wu, Shaohong Wang, Xiue Xu, Jianyi Wu, Liandi Liao, Yinwei Cheng, Kai Zhang, Gera Neufeld, Liyan Xu, Enmin Li. Cytoplasmic LOXL2 regulates actin cytoskeletal organization in esophageal squamous cell carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4594.

Could the “Stiff Rim Sign” Be an Indicator of Lysyl Oxidase Activity in Breast Cancer?

Background: Shear wave elastography (SWE) is a non-invasive and easily applicable imaging modality, which can provide quantitative information of tissue stiffness. Peritumoral high SWE elasticity values (stiff rim sign) has been reported in many studies. Lysyl oxidase (LOX) enzyme is implicated in the formation of peri-tumoral stiffness. Objectives: The aim of the study was to investigate the correlation between SWE measures with LOX gene expression levels in breast cancer patients. Patients and Methods: Forty seven women were included in the study. The lesions evaluated by SWE and ultrasound guided tru-cut biopsies were performed from both of the central and peripheral parts. SWE values, LOX family gene expression levels, histopathological features of the lesions, as well as axillary and distant metastasis statuses were evaluated statistically. Results: Thirty of the patients had breast cancer (BC) (the patient group) and 17 of them had fibroadenoma (the control group). Lysyl oxidase like1 (LOXL1) expression level in BC samples (central parts) were found to be significantly higher than the control group (P = 0.022). Stiff rim sign was present in all BC lesions and none of the control group. The elastography values of the patient group were significantly higher than the control group statistically (P 0.05) both for patient and control groups. Conclusion: Although there were no significant correlations between LOX expressions and SWE parameters in our study, axillary and distant metastasis were found to be correlated with SWE features, which emphasized the prognostic potential of SWE.

Interplay Between LOX Enzymes and Integrins in the Tumor Microenvironment.

Members of the lysyl oxidase (LOX) family are secreted copper-dependent amine oxidases that catalyze the covalent crosslinking of collagens and elastin in the extracellular matrix (ECM), an essential process for the structural integrity of all tissues. LOX enzymes can also remodel the tumor microenvironment and have been implicated in all stages of tumor initiation and progression of many cancer types. Changes in the ECM can influence several cancer cell phenotypes. Integrin adhesion complexes (IACs) physically connect cells with their microenvironment. This review article summarizes the main findings on the role of LOX proteins in modulating the tumor microenvironment, with a particular focus on how ECM changes are integrated by IACs to modulate cells behavior. Finally, we discuss how the development of selective LOX inhibitors may lead to novel and effective therapies in cancer treatment.

Lysyl Oxidase Like 2 Processing by Factor Xa Modulates its Activity and Localization

IntroductionLysyl oxidase like 2 (LOXL2) is a member of the lysyl oxidase (LOX) family of amine oxidases that catalyzes the oxidative deamination of lysine residues to crosslink extracellular matrix (ECM) proteins, thereby forming a stable and highly organized matrix. LOXL2 is shown to be involved in a diverse set of diseases including hepatic fibrosis and cancer metastasis, due to its critical role in tubulogenesis, matrix assembly, and angiogenesis. Despite the significant advances made in linking LOXL2 to fibrosis initiation and disease progression, the mechanisms of LOXL2 regulation are still largely unknown.Prior studies have shown that LOXL2 is processed extracellularly and LOXL2 processing putatively regulates its catalytic function. However, the exact identity of the protease that processes LOXL2 remains unknown. Specifically, LOXL2 is composed of a C‐terminal prototypic LOX domain that has the enzymatic function to catalyze ECM protein crosslinking, and four N‐terminal scavenger receptor cysteine rich (SRCR) domains, which are typically involved in protein‐protein recognition and signaling. Extracellular processing releases an N‐terminus fragment of 317 amino acids containing SRCR domains 1–2 and a C‐terminus fragment of SRCR domains 3–4 along with the LOX domain.Methods and ResultsIn silico digest of LOXL2 using the ExPASy peptide cutter showed Factor Xa as the potential protease that cleaves LOXL2 at position 317. Therefore, we first tested processing of exogenously expressed LOXL2 in cell culture media in vitro. Factor Xa dose dependently increased LOXL2 processing, and this was inhibited using aprotinin (non‐specific inhibitor) and rivaroxaban (specific inhibitor). We next determined the effect of LOXL2 processing on its sub‐cellular localization. Comparing the full‐length and processed LOXL2 content in the conditioned cell‐culture media and ECM showed that full length LOXL2 is predominantly found in the ECM and the processed form is found predominantly in the media. Next, we investigated whether LOXL2 processing regulates its catalytic activity. To this end, we measured the in‐situ assembly of FITC‐labeled collagen I or elastin, the main substrates of LOXL2, by processed and full length LOXL2. The incorporation of collagen I/elastin and the deposition of LOXL2 were visualized by confocal epifluorescence microscopy. Our findings indicate that LOXL2 processing markedly reduces collagen I/elastin incorporation when compared to that by full‐length LOXL2.ConclusionsFactor Xa is the protease responsible for LOXL2 processing in vitro. LOXL2 processing inhibits its ability to utilize collagen I and elastin as substrates. Future studies will continue to 1) investigate whether LOXL2 processing regulates its substrate specificity by shifting LOXL2 catalysis to Collagen IV, 2) determine the functions of the cleaved SRCR1–2 propeptide, 3) identify the specific mechanism by which processed and full length LOXL2 are trafficked to the cell‐derived ECM, and 4) evaluate the in vivo significance of this processing in the vascular compartment.Support or Funding InformationFunded by JHMI ACCM StAAR Grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ATP7A delivers copper to the lysyl oxidase family of enzymes and promotes tumorigenesis and metastasis

Lysyl oxidase (LOX) and LOX-like (LOXL) proteins are copper-dependent metalloenzymes with well-documented roles in tumor metastasis and fibrotic diseases. The mechanism by which copper is delivered to these enzymes is poorly understood. In this study, we demonstrate that the copper transporter ATP7A is necessary for the activity of LOX and LOXL enzymes. Silencing of ATP7A inhibited LOX activity in the 4T1 mammary carcinoma cell line, resulting in a loss of LOX-dependent mechanisms of metastasis, including the phosphorylation of focal adhesion kinase and myeloid cell recruitment to the lungs, in an orthotopic mouse model of breast cancer. ATP7A silencing was also found to attenuate LOX activity and metastasis of Lewis lung carcinoma cells in mice. Meta-analysis of breast cancer patients found that high ATP7A expression was significantly correlated with reduced survival. Taken together, these results identify ATP7A as a therapeutic target for blocking LOX- and LOXL-dependent malignancies.

Divergent roles of lysyl oxidase family members in ornithine decarboxylase- and RAS-transformed mouse fibroblasts and human melanoma cells.

We have previously shown that proto-oncoprotein c-Jun is activated in ornithine decarboxylase (ODC)- and RAS-transformed mouse fibroblasts, and that the transformed morphology of these cells can be reversed by expressing the transactivation domain deletion mutant of c-Jun (TAM67). Here, we found that lysyl oxidase (Lox), encoding an extracellular matrix-modifying enzyme, is downregulated in a c-Jun-dependent manner in ODC-transformed fibroblasts (Odc cells). In addition to Lox, the Lox family members Lox-like 1 and 3 (Loxl1 and Loxl3) were found to be downregulated in Odc as well as in RAS-transformed fibroblasts (E4), whereas Lox-like 4 (Loxl4) was upregulated in Odc and downregulated in E4 cells compared to normal N1 fibroblasts. Tetracycline-regulatable LOX re-expression in Odc cells led to inhibition of cell growth and invasion in three-dimensional Matrigel in an activity-independent manner. On the contrary, LOX and especially LOXL2, LOXL3, and LOXL4 were found to be upregulated in several human melanoma cell lines, and LOX inhibitor B-aminopropionitrile inhibited the invasive growth of these cells particularly when co-cultured with fibroblasts in Matrigel. Knocking down the expression of LOX and especially LOXL2 in melanoma cells almost completely abrogated the invasive growth capability. Further, LOXL2 was significantly upregulated in clinical human primary melanomas compared to benign nevi, and high expression of LOXL2 in primary melanomas was associated with formation of metastases and shorter survival of patients. Thus, our studies reveal that inactive pro-LOX (together with Lox propeptide) functions as a tumor suppressor in ODC- and RAS-transformed murine fibroblasts by inhibiting cell growth and invasion, and active LOX and LOXL2 as tumor promoters in human melanoma cells by promoting their invasive growth.

LOXL3 novel mutation causing a rare form of autosomal recessive Stickler syndrome.

Stickler syndrome is a collagenopathy that is typically inherited as autosomal dominant disease caused by monoallelic mutations in COL2A1, COL11A2, and COL11A1. Rarely, biallelic mutations in COL9A1, COL9A2, and COL9A3 cause an autosomal recessive Stickler syndrome. One previous report described two siblings with Stickler syndrome and a homozygous mutation in LOXL3, suggesting that biallelic mutations in LOXL3 can also cause autosomal recessive Stickler syndrome. LOXL3 is a member of the lysyl oxidase family of genes which encode enzymes oxidizing the side chain of peptidyl lysine permitting the covalent crosslinking of collagen and elastin chains. Therefore, LOXL3 deficiency is expected to result in collagen defect. Furthermore, Loxl3 deficient mouse model demonstrated features overlapping with Stickler syndrome. In this report, we describe a child and his father who had clinical features consistent with Stickler syndrome and found to have a homozygous novel mutation c.1036C>T (p.Arg346Trp) in LOXL3. This report not only supports that biallelic LOXL3 mutations cause autosomal recessive Stickler syndrome, but also further delineates the phenotype associated with LOXL3 mutations. In addition, the family described here shows an interesting example for pseudodominance, which can be observed in recessive diseases when one parent is affected and the other is heterozygous carrier.

117 - Prdx1 regulation of LOX-dependent ECM remodeling in breast cancer

Breast cancer progression and metastasis includes not only cell-autonomous properties of cancer epithelial cells, but also the influence of the neighboring tumor stromal cells. In breast cancer, almost 80% of stromal associated fibroblasts (SAFs) acquire a cancer associated fibroblast (CAF)-like “activated phenotype”. This CAF activated phenotype is associated with elevated levels of reactive oxygen species (ROS) that are linked with tumor remodeling and spreading. Members of the lysyl oxidase (LOX) family of enzymes participate in tumor remodeling through the promotion of collagen crosslinking and collagen fibril production. We hypothesize that stromal Prdx1 regulates ROS dependent metastasis/migration of cancer cells through effects on LOX activity. Our preliminary data reveal that Prdx1 prevents CAF-induced malignant phenotypes in breast cancer (epithelial) cells in an H2O2-dependent manner. When compared to wild-type mice, Prdx1-/- SAFs show a marked increase in CAF-specific characteristics, including increased expression of CAF-specific markers, motility and invasiveness of SAFs and SAF-induced chemotactic migration and invasion by breast cancer epithelial cells in vitro. Lack of Prdx1 in mammary SAFs results in the upregulation of markers of the activated phenotype, such as collagen, vimentin and α-SMA leading to an increase in co-migration and invasion. As shPrdx1 SAFs show CAF-like mesenchymal properties in vitro, we tested in vivo if Prdx1 suppresses migration of breast cancer cells by generating a syngeneic mouse model to image BALB/c derived SAFs shPrdx1/SAFs pLKO1 (expressing iRFP).