LOX-like Research Articles

LOX, but not LOXL2, promotes bone metastasis formation and bone destruction in triple-negative breast cancer

The primary function of the lysyl oxidase (LOX) family, including LOX and its paralogue LOX-like (LOXL)-2, is to catalyze the covalent crosslinking of collagen and elastin in the extracellular matrix. LOX and LOXL2 are also facilitating breast cancer invasion and metastatic spread to visceral organs (lungs, liver) in vivo. Conversely, the contribution of LOX and LOXL2 to breast cancer bone metastasis remains scant. Here, using gene overexpression or silencing strategies, we investigated the role of LOX and LOXL2 on the formation of metastatic osteolytic lesions in animal models of triple negative breast cancer. In vivo, the extent of radiographic metastatic osteolytic lesions in animals injected with LOX-overexpressing [LOX(+)] tumor cells was 3-fold higher than that observed in animals bearing tumors silenced for LOX [LOX(−)]. By contrast, the extent of osteolytic lesions between LOXL2(+) and LOXL2(−) tumor-bearing animals did not differ, and was comparable to that observed with LOX(−) tumor-bearing animals. In situ, TRAP staining of bone tissue sections from the hind limbs of LOX(+) tumor-bearing animals was substantially increased compared to LOX(−), LOXL2(+) and LOXL2(−)-tumor-bearing animals, which was indicative of enhanced active-osteoclast resorption. In vitro, tumor-secreted LOX increased osteoclast differentiation induced by RANKL, whereas LOXL2 seemed to counteract LOX’s pro-osteoclastic activity. Furthermore, LOX (but not LOXL2) overexpression in tumor cells induced a robust production of IL-6, the latter being a pro-osteoclastic cytokine. Based on these findings, we propose a model in which LOX and IL-6 secreted from tumor cells act in concert to enhance osteoclast-mediated bone resorption that, in turn, promotes metastatic bone destruction in vivo.

Abstract P2042: Linking Extracellular Matrix Stiffness With Cardiomyocyte Structure And Function

The composition and organization of the extracellular matrix (ECM) provides cues to myocardial cells that direct normal function and responses to stress. However, little is known about how exactly a change in environmental tension leads to alterations in cardiomyocyte cell size and contractile force. Collagen I comprises the majority of cardiac ECM where it is crosslinked and matured by the lysyl oxidase (LOX) family of enzymes that includes LOX and LOX-like (LOXL) proteins. LOX and LOXLs are frequently upregulated in the heart after injury, and their inhibition has been shown to alleviate increases in tissue stiffness. Cardiac injury models result in activation and recruitment of many cells that secrete growth factors and modify the ECM, which makes it difficult to address specifically how cardiomyocytes are responding to changes in matrix tension alone. Therefore, we utilized LOX or LOXL overexpression as a tool to stiffen the ECM in the absence of pathophysiologic injury and paracrine factors. Transgenic overexpression of LOX increases myocardial stiffness and results in mild cardiac dysfunction by 9 months of age. While younger adult mice do not display a phenotype at baseline, they have an exaggerated response to pressure overload hypertrophy. In a different set of experiments, adeno-associated viruses (AAV9) for LOXL1, -2, or -3 were administered to mouse pups via cardiac injection at perinatal day 7. By 12 weeks of age, LOXL1 overexpression leads to cardiac hypertrophy with preserved ejection fraction, while increased LOXL2 or LOXL3 results in decreased cardiac function (each with differential patterns of myocardial fibrosis). Ongoing studies are assessing how these functional changes are associated with alterations in tissue stiffness and cardiomyocyte behavior. Determining how cardiomyocytes respond to alterations in the physical properties of the ECM will improve our understanding of the etiology of progressive cardiomyopathies and fibrosis.

Lysyl Oxidase-like Protein Recognizes Viral Envelope Proteins and Bacterial Polysaccharides against Pathogen Infection via Induction of Expression of Antimicrobial Peptides.

Lysyl oxidases (LOXs) are copper-dependent monoamine oxidases, and they play critical roles in extracellular matrix (ECM) remodeling. The LOX and LOX-like (LOXL) proteins also have a variety of biological functions, such as development and growth regulation, tumor suppression, and cellular senescence. However, the functions of LOXLs containing repeated scavenger receptor cysteine-rich (SRCR) domains in immunity are rarely reported. In this study, we characterized the antiviral and antibacterial functions of a lysyl oxidase-like (LOXL) protein containing tandem SRCR domains in Marsupenaeus japonicus. The mRNA level of LoxL was significantly upregulated in the hemocytes and intestines of shrimp challenged using white spot syndrome virus (WSSV) or bacteria. After the knockdown of LoxL via RNA interference, WSSV replication and bacterial loads were apparently increased, and the survival rate of the shrimp decreased significantly, suggesting that LOXL functions against pathogen infection in shrimp. Mechanistically, LOXL interacted with the envelope proteins of WSSV or with lipopolysaccharide and peptidoglycan from bacteria in shrimp challenged using WSSV or bacteria, and it promoted the expression of a battery of antimicrobial peptides (AMPs) via the induction of Dorsal nuclear translocation against viral and bacterial infection. Moreover, LOXL expression was also positively regulated by Dorsal in the shrimp challenged by pathogens. These results indicate that, by acting as a pattern recognition receptor, LOXL plays vital roles in antiviral and antibacterial innate immunity by enhancing the expression of AMPs in shrimp.

Systematic Analysis of Expression and Prognostic Values of Lysyl Oxidase Family in Gastric Cancer.

Background: Gastric cancer (GC) remains the fifth most commonly diagnosed malignancy worldwide, with a poor prognosis. The lysyl oxidase (LOX) family, a type of secreted copper-dependent amine oxidases, is comprised of LOX and four LOX-like (LOXL) 1–4 isoforms and has been reported to be dysregulated in a number of different type cancers. However, the diverse expression patterns and prognostic values of LOX family in GC have yet to be systematically analyzed. Methods: ONCOMINE, GEPIA, UALCAN, Kaplan–Meier Plotter, LOGpc, cBioPortal, GeneMANIA and Metascape databases were utilized in this study to analyze the expression, prognostic values, mutations and functional networks of LOX family in GC. Results: The mRNA expression levels of LOX, LOXL1 and LOXL2 were significantly higher in GC, the expression level of LOXL3 was contrary in different databases, while the expression level of LOXL4 made no difference; the expression levels of LOX, LOXL1 and LOXL3 were higher in stages 2–4 than that of normal tissues and stage 1, while the mRNA level of LOXL2 in stage 1–4 was higher than normal tissues; patients with high expression of LOX and LOXL 2-4 had poor OS; the genes correlated with LOX and LOXL2 were enriched in extracellular matrix organization, vasculature development and skeletal system development. Conclusion: Our results indicated that the LOX family, especially LOX and LOXL2, might play an important role in GC oncogenesis, and they may become biomarkers for predicting tumor prognosis and potential targets for tumor therapy.

SUN-169 A COPPER CHELATING DRUG INHIBITS RENAL FIBROSIS BY INHIBITING LYSYL OXIDASE AND LOSS OF PERITUBULAR CAPILLARIES

Chronic kidney disease (CKD) is one of the important global health burdens which involves interstitial fibrosis as an underlying pathological process associated with compromised renal function irrespective of etiological cause of the injury. Lysyl oxidase (LOX) is a copper-dependent enzyme that mediates cross linking of extracellular matrix (ECM) proteins including collagen and elastin. The proper LOX activity is essential for a physiologically functional ECM, while the aberrant expression of LOX and related proteins has been found in conditions with excessively deposited ECM such as organ fibrosis. We hypothesized that a copper chelator, disulfiram (DSF) which is used for alcohol deaddiction, could be repurposed to reduce the activation of LOX proteins and subsequent reduction in collagen deposition in fibrotic conditions. We used Male Swiss albino mice for unilateral ureteral obstruction (UUO)-induced animal model of renal fibrosis and investigated the effects of DSF (10 and 30 mg/kg) on LOX and LOX-like (LOXL) proteins as a strategy to treat/prevent renal fibrosis. We used a pan-LOX inhibitor, β-aminopropionitrile (BAPN, 100 mg/kg) as a standard LOX inhibitor for comparison. We performed histopathology, hydroxyproline assay, western blot, ELISA and immunofluorescence studies to assess the effect of DSF on renal injury and fibrosis. The levels of different isoforms of LOX were found to be higher in the fibrotic kidney as compared to sham/contralateral kidney control. The treatment of animals with DSF led to reduced expression of LOX and related proteins. Interestingly, the reduction in expression of LOX proteins with DSF was found to be better than BAPN. Further, the DSF reduced UUO-induced expression of fibrotic proteins (collagen I, fibronectin, TGF-β). Furthermore, DSF reduced the phenotypic conversion of epithelial cells to mesenchymal type by reducing the epithelial-mesenchymal transition (EMT) program as evident by reduced expression of mesenchymal markers such as α-SMA and vimentin along with improved expression of epithelial marker, E-cadherin. DSF prevented renal injury, reduced tubulointerstitial fibrosis and collagen deposition in fibrotic kidney. In addition to that, DSF also prevented peritubular capillary (PTC) loss in the obstructed kidneys of animals which is a hallmark of clinical and preclinical conditions of renal fibrosis. DSF, an FDA approved drug, can be repurposed for fibrotic disorders due to its modulating effects on the expression of LOX and LOXL proteins. The DSF holds a great potential to prevent organ fibrosis and it could be used as a successful strategy for amelioration of different fibrotic conditions, if translated further.

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.

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.

Lysyl oxidase-like 2 (LOXL2)-mediated cross-linking of tropoelastin.

Lysyl oxidases (LOXs) play a central role in extracellular matrix remodeling during development and tumor growth and fibrosis through cross-linking of collagens and elastin. We have limited knowledge of the structure and substrate specificity of these secreted enzymes. LOXs share a conserved C-terminal catalytic domain but differ in their N-terminal region, which is composed of 4 repeats of scavenger receptor cysteine-rich (SRCR) domains in LOX-like (LOXL) 2. We investigated by X-ray scattering and electron microscopy the low-resolution structure of the full-length enzyme and the structure of a shorter form lacking the catalytic domain. Our data demonstrate that LOXL2 has a rod-like structure with a stalk composed of the SRCR domains and the catalytic domain at its tip. We detected direct interaction between LOXL2 and tropoelastin (TE) and also LOXL2-mediated deamination of TE. Using proteomics, we identified several allysines together with cross-linked TE peptides. The elastin-like material generated was resistant to trypsin proteolysis and displayed mechanical properties similar to mature elastin. Finally, we detected the codistribution of LOXL2 and elastin in the vascular wall. Altogether, these data suggest that LOXL2 could participate in elastogenesis in vivo and could be used as a means of cross-linking TE in vitro for biomimetic and cell-compatible tissue engineering purposes.—Schmelzer, C. E. H., Heinz, A., Troilo, H., Lockhart-Cairns, M.-P., Jowitt, T. A., Marchand, M. F., Bidault, L., Bignon, M., Hedtke, T., Barret, A., McConnell, J. C., Sherratt, M. J., Germain, S., Hulmes, D. J. S., Baldock, C., Muller, L. Lysyl oxidase–like 2 (LOXL2)–mediated cross-linking of tropoelastin.

Significance of the Lysyl Oxidase Members Lysyl Oxidase Like 1, 3, and 4 in Gastric Cancer

Background/Aims: Lysyl oxidase (LOX) family members play a key role in modifying the primary tumor microenvironment by crosslinking collagens and elastin in the extracellular matrix. The aim of this study was to analyze the LOX-like (LOXL)1, LOXL3, and LOXL4 expressions in gastric cancer tissue by immunohistochemical staining. Methods: The correlations between the clinicopathological features of 597 primary gastric carcinomas and LOX family members – LOXL1, LOXL3, and LOXL4 – were investigated by immunohistochemical studies. The effect of the transforming growth ­factor β1 (TGFβ1) on the expressions of LOXL1, LOXL3, and LOXL4 in gastric cancer was examined using diffuse-type gastric cancer cell lines in vitro. Results: The expressions of LOXL1, LOXL3, and LOXL4 were correlated with T invasion, lymph node metastasis, and lymphatic and venous invasion. LOXL1 expression was associated with histological intestinal-type and expanding growth patterns. The overall survival of patients with LOXL1-, LOXL3-, or LOXL4-positive cancer was poorer than those with negative cancer. LOXL3 and LOXL4 mRNA expressions were significantly high in diffuse-type gastric cancer cells with high invasion ability. TGFβ decreased the LOXL1 expression and increased LOXL3 and LOXL4 expression. Conclusion: LOXL1, LOXL3, and LOXL4 expressions are associated with distant metastasis of gastric cancer.

Lysyl Oxidases: Functions and Disorders.

Lysyl oxidases (LOX) are copper-dependent enzymes that oxidize lysyl and hydroxylysyl residues in collagen and elastin, as a first step in the stabilization of these extracellular matrix proteins through the formation of covalent cross-linkages, an essential process for connective tissue maturation. Five different LOX enzymes have been identified in mammals, LOX and LOX-like (LOXL) 1 to 4, being genetically different protein products with a high degree of homology in the catalytic carboxy terminal end and a more variable amino terminal proregion. Intensive investigation in the last years has delineated the main biological functions of these enzymes and their involvement in several pathologies including fibrosis, cancer, and ocular disorders. This review article summarizes the major findings on the role of LOX isoforms, with particular focus on their contribution to the development and progression of human disorders.

Abstract B44: Lysyl oxidases suppress pancreatic cancer progression and inhibit FAK and ERK signaling

Abstract Pancreatic cancer carries a dismal prognosis, as most patients present with locally advanced and metastatic disease at time of diagnosis. High levels of type I collagen extracellular matrix in pancreatic tumor specimens are associated with poor survival and resistance to therapy. Furthermore, the expression of lysyl oxidases (LOXs), enzymes that mediate cross-linking and linearization of collagen type I, correlate with poor prognosis in breast and pancreatic cancer. However, a clinical trial targeting LOX-like (LOXL) 2 in pancreatic cancer did not show any survival benefits, pointing to a limited understanding of the context in which these enzymes function in cancer progression. To understand how collagen structure influences cancer cell behavior, we used host mice that expressed GFPtopaz fused to collagen type I and performed intravital microscopy after orthotopic injection of fluorescently labeled cancer cells (derived from the KPC mouse models of pancreatic cancer). Strikingly, we observed that invasive cancer cells migrated out of the tumors on linear type I collagen. Surprisingly, inhibition of LOX and LOXL2, using a pharmacologic inhibitor or shRNA-mediated knockdown, had the opposite results of what we expected with increased metastasis, collagen linearization, and focal adhesion kinase (FAK) activity. Indicating a mechanism independently of collagen crosslinking, LOX inhibition promoted invasion and increased FAK and Ras/ERK-MAPK signaling in vitro, when cells were grown on plastic. We demonstrated that the FAK activity was required for the increased metastasis induced by LOX inhibition, using a clinical stage pharmacologic inhibitor. Our data highlight that LOXs in pancreatic cancer play an important role in suppression of the ERK and FAK signaling pathways, and metastasis. Indeed, the earliest report of lysyl oxidase function in cancer was as a tumor suppressor in Ras transformed cells. Citation Format: Mario Shields, Pascal Maguin, Christopher Giuliano, Sarah Dallas, John Erby Wilkinson, Mikala Egeblad. Lysyl oxidases suppress pancreatic cancer progression and inhibit FAK and ERK signaling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B44.

PKCε Increases Extracellular Elastin and Fibulin-5/DANCE in Dermal Fibroblasts

Background/Aims: In the earlier study, the selective PKCε activator DCP-LA increased elastic fibres in the dermis of HR-1 hairless mice. As a process of elastic fibre formation, tropoelastin, an elastin monomer, is secreted into the extracellular space. Secreted tropoelastin is delivered to the microfibrils by fibulin-5/developmental arteries and neural crest epidermal growth factor-like (DANCE) and undergoes self-association. Then, tropoelastin assembles around the microfibrils, growing into elastin and elastic fibres by lysyl oxidase (LOX)- or LOX-like (LOXL)-mediated cross-linking. The present study was conducted to understand the mechanism underlying DCP-LA-induced increase in elastin/elastic fibre. Methods: Western blotting, immunocytochemistory, and real-time reverse transcription-polymerase chain reaction (RT-PCR) were carried out in cultured human dermal fibroblasts. PKCε, mammalian target of rapamycin complex (mTOR), and p70 S6 kinase (S6K) were knocked-down by transfecting each siRNA. Results: DCP-LA increased elastin and fibulin-5/DANCE in a treatment time (6-24 h)- and a bell-shaped concentration (1 nM-1 µM)-dependent manner in the culture medium of human dermal fibroblasts. DCP-LA markedly increased elastic fibres in the extracellular space of cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was abolished by a PKC inhibitor or knocking-down PKCε. DCP-LA did not affect expression of mRNAs for tropoelastin and fiblin-5/DANCE in cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was not inhibited by the protein synthesis inhibitor cycloheximide or by knocking-down mTOR and S6K. DCP-LA never increased extracellular elastin in the presence of elastase, that breaks down elastin. An inhibitor of matrix metalloproteinase 9, that degrades multiple extracellular matrix components including elastin, had no effect on the basal levels and the DCP-LA-induced increase levels of extracellular elastin. Conclusion: The results of the present study indicate that PKCε, activated by DCP-LA, increases elastin and fibulin-5/DANCE in the extracellular space of cultured fibroblasts by the mechanism independent of transcriptional and translational modulation or inhibition of elastolysis.

Comparative analysis of lysyl oxidase (like) family members in pulmonary fibrosis

Extracellular matrix (ECM) composition and stiffness are major driving forces for the development and persistence of fibrotic diseases. Lysyl oxidase (LOX) and LOX-like (LOXL) proteins play crucial roles in ECM remodeling due to their collagen crosslinking and intracellular functions. Here, we systematically investigated LOX/L expression in primary fibroblasts and epithelial cells under fibrotic conditions, Bleomycin (BLM) induced lung fibrosis and in human IPF tissue. Basal expression of all LOX/L family members was detected in epithelial cells and at higher levels in fibroblasts. Various pro-fibrotic stimuli broadly induced LOX/L expression in fibroblasts, whereas specific induction of LOXL2 and partially LOX was observed in epithelial cells. Immunohistochemical analysis of lung tissue from 14 IPF patients and healthy donors revealed strong induction of LOX and LOXL2 in bronchial and alveolar epithelium as well as fibroblastic foci. Using siRNA experiments we observed that LOXL2 and LOXL3 were crucial for fibroblast-to-myofibroblast transition (FMT). As FMT could only be reconstituted with an enzymatically active LOXL2 variant, we conclude that LOXL2 enzymatic function is crucial for fibroblast transdifferentiation. In summary, our study provides a comprehensive analysis of the LOX/L family in fibrotic lung disease and indicates prominent roles for LOXL2/3 in fibroblast activation and LOX/LOXL2 in IPF.

Abstract P6-05-05: The expression of lysyl oxidase and fibrotic focus is related to inflammation in breast cancer

Abstract Background: Lysyl oxidase (LOX) is an extracellular matrix enzyme that catalyzes the cross-linking of collagens or elastin. Our hypothesis is that LOX contributes to the formation of a fibrotic focus (FF), which is related to inflammation in breast carcinogenesis. In this study, we analyzed the association between the expression LOXs and FF, and investigated prognostic significance in breast cancer. Methods: Tissue microarrarys were constructed from the specimens of 444 patients with primary invasive breast cancer. Immunohistochemical staining for LOX, LOXlike (LOXL)-, LOXL-2 and LOXL-3 was performed. The status of FF within the tumor was assessed. The number of CD4+ T cells, CD8+ T cells, CD68+ macrophages was counted, and intratumoral and peritumoral lymphocyte infiltration were evaluated. The clinicopathologic characteristics of the patients were analyzed. Result: The percentage of positive FF was 39.2% and positive rate of LOX expression was 50% in primary breast cancer tissues. FF was found to be significantly associated with intratumoral and peritumoral inflammation, lymph node metastasis, high histologic grade, larger tumor size. LOX was associated with intratumoral and peritumoral inflammation, CD8+ T cells and menopausal status. LOXL-3 was significantly associated with positive expression of ER and PR, and molecular subtype. Conclusions: FF and the expression of LOX were associated with inflammation in breast cancer in this study. Our results suggest that LOXs may contribute to the formation of a FF indirectly in relation with inflammation in breast cancer. Further studies are needed to clarify the role of LOXs, FF and inflammation in tumorigenesis and prognostic value of them in breast cancer. Citation Format: Park SH, Jeong YJ, Bong JG, Oh HK. The expression of lysyl oxidase and fibrotic focus is related to inflammation in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-05-05.

Inhibition of Lysyl Oxidase and Lysyl Oxidase-Like Enzymes Has Tumour-Promoting and Tumour-Suppressing Roles in Experimental Prostate Cancer.

Lysyl oxidase (LOX) and LOX-like (LOXL) enzymes are key players in extracellular matrix deposition and maturation. LOX promote tumour progression and metastasis, but it may also have tumour-inhibitory effects. Here we show that orthotopic implantation of rat prostate AT-1 tumour cells increased LOX and LOXLs mRNA expressions in the tumour and in the surrounding non-malignant prostate tissue. Inhibition of LOX enzymes, using Beta-aminopropionitrile (BAPN), initiated before implantation of AT-1 cells, reduced tumour growth. Conversely, treatment that was started after the tumours were established resulted in unaffected or increased tumour growth. Moreover, treatment with BAPN did not suppress the formation of spontaneous lymph node metastases, or lung tumour burden, when tumour cells were injected intravenously. A temporal decrease in collagen fibre content, which is a target for LOX, was observed in tumours and in the tumour-adjacent prostate tissue. This may explain why early BAPN treatment is more effective in inhibiting tumour growth compared to treatment initiated later. Our data suggest that the enzymatic function of the LOX family is context-dependent, with both tumour-suppressing and tumour-promoting properties in prostate cancer. Further investigations are needed to understand the circumstances under which LOX inhibition may be used as a therapeutic target for cancer patients.

The function and mechanisms of action of LOXL2 in cancer (Review).

The lysyl oxidase (LOX) family is comprised of five members, and some members have recently emerged as important regulators of tumor progression. Among these, at present, LOX‑like (LOXL)2 is the prototypical LOX and the most comprehensively studied member. A growing body of evidence has implicated LOXL2 in the promotion of cancer cell invasion, metastasis and angiogenesis, as well as in the malignant transformation of solid tumors. Moreover, a high expression of LOXL2 is associated with a poor prognosis. These data have piqued the interest of a number of researchers and research groups, who have identified LOXL2 as a strong target candidate in the development of inhibitors for use as functional and efficacious tumor therapeutics. In the present study, we summarize the recent progress made regarding LOXL2, mainly focusing on its function and mechanisms of action in tumor progression and metastasis. In this review, we note that LOXL2 promotes tumor progression possibly by activating multiple signal pathways through a variety of mechanisms, both biochemical and biomechanical. The data presented herein may open new avenues for the therapeutic utility of LOXL2.

PP.04.09

Objective: Lysyl oxidase (LOX) is a key enzyme in extracellular matrix (ECM) remodelling. Because LOX deficiency is lethal, we have developed a transgenic mouse model to study the impact of LOX overexpression on cardiovascular remodelling. Design and method: A new mouse model that over-expresses human LOX was generated by conventional methods. Transgene expression was determined by real time PCR in 8 different tissues including aorta, heart, kidney, white adipose tissue (WAT), brown adipose tissue, lung, liver and skeletal muscle. We tested the impact of LOX over-expression on cardiovascular remodelling in TgLOX mice and their wild-type (WT) littermates mice after chronic infusion with Ang II (1.4 μg/kg/min) or saline (n = 10 per group). Aortic diameter and cardiac function were evaluated by ultrasonography. Results: The maximum expression of human LOX was found in aorta followed by heart and WAT. Neither the expression of endogenous LOX nor that of LOX-Like (LOXL) isoenzymes was modified by transgene expression in these tissues. Ang II-induced aortic diameter dilation was similar in TgLOX and WT mice after Ang II infusion. However, the mortality rate due to aortic rupture was higher in WT mice (20 %) compared to TgLOX mice (0 %). The quantification of heart function showed that Ang II infusion decreased ejection fraction (EF) and fractional shortening (FS) in TgLOX mice, while they were augmented in WT mice. A stronger hypertrophic response induced by Ang II occurred in TgLOX mice as evidenced by an increased LV mass and left ventricle posterior wall (LVPW) thickness. Accordingly, the left ventricular inner diameter (LVID) was significantly lowered in TgLOX mice. After Ang II infusion, cardiac output, stroke volume and mRNA levels of hypertrophic markers such as ANP and B-HMC remained similar in both, transgenic and WT mice. Finally, LOX overexpression increased the Ang II-induced expression of pro-inflammatory (Emr-1, IL6, Mmp-9) and fibrosis-related (Serpin-1, Col-1a1) markers in cardiac tissue compared with WT mice Conclusions: We have developed a valuable model to improve our knowledge about LOX biology in the cardiovascular system. Our data evidence that LOX over-expression impairs cardiac function under hypertensive conditions.

Origin and evolution of lysyl oxidases.

Lysyl oxidases (LOX) are copper-dependent enzymes that oxidize primary amine substrates to reactive aldehydes. The best-studied role of LOX enzymes is the remodeling of the extracellular matrix (ECM) in animals by cross-linking collagens and elastin, although intracellular functions have been reported as well. Five different LOX enzymes have been identified in mammals, LOX and LOX-like (LOXL) 1 to 4, showing a highly conserved catalytic carboxy terminal domain and more divergence in the rest of the sequence. Here we have surveyed a wide selection of genomes in order to infer the evolutionary history of LOX. We identified LOX proteins not only in animals, but also in many other eukaryotes, as well as in bacteria and archaea – which reveals a pre-metazoan origin for this gene family. LOX genes expanded during metazoan evolution resulting in two superfamilies, LOXL2/L3/L4 and LOX/L1/L5. Considering the current knowledge on the function of mammalian LOX isoforms in ECM remodeling, we propose that LOXL2/L3/L4 members might have preferentially been involved in making cross-linked collagen IV-based basement membrane, whereas the diversification of LOX/L1/L5 forms contributed to chordate/vertebrate-specific ECM innovations, such as elastin and fibronectin. Our work provides a novel view on the evolution of this family of enzymes.

The lysyl oxidase inhibitor β-aminopropionitrile reduces body weight gain and improves the metabolic profile in diet-induced obesity in rats.

ABSTRACTExtracellular matrix (ECM) remodelling of the adipose tissue plays a pivotal role in the pathophysiology of obesity. The lysyl oxidase (LOX) family of amine oxidases, including LOX and LOX-like (LOXL) isoenzymes, controls ECM maturation, and upregulation of LOX activity is essential in fibrosis; however, its involvement in adipose tissue dysfunction in obesity is unclear. In this study, we observed that LOX is the main isoenzyme expressed in human adipose tissue and that its expression is strongly upregulated in samples from obese individuals that had been referred to bariatric surgery. LOX expression was also induced in the adipose tissue from male Wistar rats fed a high-fat diet (HFD). Interestingly, treatment with β-aminopropionitrile (BAPN), a specific and irreversible inhibitor of LOX activity, attenuated the increase in body weight and fat mass that was observed in obese animals and shifted adipocyte size toward smaller adipocytes. BAPN also ameliorated the increase in collagen content that was observed in adipose tissue from obese animals and improved several metabolic parameters – it ameliorated glucose and insulin levels, decreased homeostasis model assessment (HOMA) index and reduced plasma triglyceride levels. Furthermore, in white adipose tissue from obese animals, BAPN prevented the downregulation of adiponectin and glucose transporter 4 (GLUT4), as well as the increase in suppressor of cytokine signaling 3 (SOCS3) and dipeptidyl peptidase 4 (DPP4) levels, triggered by the HFD. Likewise, in the TNFα-induced insulin-resistant 3T3-L1 adipocyte model, BAPN prevented the downregulation of adiponectin and GLUT4 and the increase in SOCS3 levels, and consequently normalised insulin-stimulated glucose uptake. Therefore, our data provide evidence that LOX plays a pathologically relevant role in the metabolic dysfunction induced by obesity and emphasise the interest of novel pharmacological interventions that target adipose tissue fibrosis and LOX activity for the clinical management of this disease.

Abstract 1980: Role of lysyl oxidase and lysyl oxidase-like 2 in gastric carcinoma

Abstract Object: Lysyl oxidase (LOX) family is the extracellular matrix-modifying enzymes comprising of LOX and LOX-like (LOXL) 1-4. These enzymes have intracellular functions involved in the regulation of motility, migration, differentiation, and transcription in various types of cells. LOX is associated with mesenchymal reconstruction that is essential for cancer invasion and metastasis. LOX overexpression is crucial to promoting tumor growth and metastasis in several cancers such as breast cancer and colorectal cancer, however, role of LOX in gastric cancer remain unknown. Here, we investigated the clinicopathological significance of LOX and LOXL2 in gastric cancer. Materials and Methods: A total of 549 patients who had undergone surgery for gastric cancer at Osaka-City University were enrolled. LOX and LOXL2 expressions were evaluated by intensity of staining and percentage of stained tumor cells at the invading tumor front. This study was approved by the Osaka City University ethics committee. Informed consent was obtained from all patients. Results: LOX and LOXL2 expression were positive in 265 (48.3%) and 342 (58.5%) of the 549 gastric tumors. Both LOX and LOXL2 expression in cancer cells were significantly associated with macroscopic type (p<0.01), histological type (p<0.01), depth of invasion (p<0.01), lymph node status (p<0.01), vessel invasion (p<0.01). The prognosis of patients with LOX-positive tumors or LOXL2-positive tumors was significantly (p<0.001 or p<0.001, log-rank) worse than that of patients with LOX-negative or LOXL2-negative tumors, respectively. A multivariate analysis indicated LOX and LOXL2 expression in cancer cells to be an independent prognostic factor (p=0.016 or p<0.001). Conclusions: LOX and LOXL2 expression might be associated with metastatic activity of gastric cancer cells. LOX and LOXL2 might be useful predictive prognostic factors for patients with gastric cancer. Citation Format: Hiroaki Kasashima, Masakazu Yashiro, Yukie Go, Go Masuda, Haruhito Kinoshita, Mao Tokumoto, Tamami Morisaki, Tatsunari Fukuoka, Takahiro Toyokawa, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. Role of lysyl oxidase and lysyl oxidase-like 2 in gastric carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1980. doi:10.1158/1538-7445.AM2014-1980