Lysyl Oxidase-like 2 Expression Research Articles

LOXL2 inhibition ameliorates pulmonary artery remodeling in pulmonary hypertension.

Conduit pulmonary arterial stiffening and the resultant increase in pulmonary vascular impedance has emerged as an important underlying driver of pulmonary arterial hypertension (PAH). Given that matrix deposition is central to vascular remodeling, we evaluated the role of the collagen crosslinking enzyme lysyl oxidase like 2 (LOXL2) in this study. Human pulmonary artery smooth muscle cells (PASMCs) subjected to hypoxia showed increased LOXL2 secretion. LOXL2 activity and expression were markedly higher in primary PASMCs isolated from pulmonary arteries of the rat Sugen5416 + hypoxia (SuHx) model of severe PH. Similarly, LOXL2 protein and mRNA levels were increased in pulmonary arteries (PA) and lungs of rats with PH (SuHx and monocrotaline (MCT) models). Pulmonary arteries (PAs) isolated from rats with PH exhibited hypercontractility to phenylephrine and attenuated vasorelaxation elicited by acetylcholine, indicating severe endothelial dysfunction. Tensile testing revealed a a significant increase in PA stiffness in PH. Treatment with PAT-1251, a novel small-molecule LOXL2 inhibitor, improved active and passive properties of the PA ex vivo. There was an improvement in right heart function as measured by right ventricular pressure volume loops in-vivo with PAT-1251. Importantly PAT-1251 treatment ameliorated PH, resulting in improved pulmonary artery pressures, right ventricular remodeling, and survival. Hypoxia induced LOXL2 activation is a causal mechanism in pulmonary artery stiffening in PH, as well as pulmonary artery mechanical and functional decline. LOXL2 inhibition with PAT-1251 could be a promising approach to improve pulmonary artery pressures, right ventricular elastance, cardiac relaxation, and survival in PAH.

Sex Differences and Role of Lysyl Oxidase Like 2 (LOXL2) in Angiotensin II-Induced Hypertension in Mice.

Hypertension, a disease with known sexual dimorphism, accelerates aging associated arterial stiffening, in part due to the activation of matrix remodeling caused by increased biomechanical load. In this study, we tested the effect of biological sex and the role of the matrix remodeling enzyme lysyl oxidase like 2 (LOXL2) in hypertension induced arterial stiffening. Angiotensin II (Ang II) was delivered using osmotic pumps in Loxl2+/- and WT male and female mice. Blood pressure and pulse wave velocity (PWV) were measured noninvasively to assess hypertension and aortic stiffness. Wire myography and uniaxial tensile testing were used to test aortic vasoreactivity and mechanical properties. Aortic wall composition was examined by histology and Western blotting. The effect of biomechanical strain on LOXL2 expression and secretion by vascular smooth muscle (VSMC) and endothelial cells (EC) was evaluated by uniaxial cyclic stretching of cultured cells. The role of LOXL2 catalytic function on VSMC alignment in response to mechanical loading was determined with LOXL2 inhibition and knockout. Ang II infusion induced hypertension in WT and Loxl2+/- mice of both sexes and increased PWV in WT males but not in Loxl2+/- males, WT females, or Loxl2+/- females. LOXL2 depletion protected males from Ang II mediated potentiation of vasoconstriction but worsened in females and improved aortic mechanical properties in both sexes. Histological analysis showed increased aortic wall thickness in hypertensive WT males but not females and increased intralamellar distance in both sexes, that was ameliorated in Loxl2+/- mice. Western blotting revealed increased collagen I, decreased collagen IV, and increased LOXL2 accumulation and processing in hypertensive mice. Hypertensive cyclic strain contributed to LOXL2 upregulation in the cell-derived matrix in VSMCs but not ECs. LOXL2 catalytic function facilitated VSMC alignment in response to biomechanical strain. In males, arterial stiffening in hypertension is driven both by VSMC response and matrix remodeling. Females exhibit a delayed onset of Ang II-induced hypertension with minimal ECM remodeling but with VSMC dysfunction. LOXL2 depletion ameliorates arterial stiffening and preserves functional contractility and aortic structure in male hypertensive mice. LOXL2 depletion improves aortic mechanics but worsens aortic contractility in hypertensive females. VSMCs are the primary source of LOXL2 in the aorta and hypertension increases LOXL2 processing and shifts to collagen I accumulation. Overall, LOXL2 depletion offers protection in young hypertensive males and females.

Splicing factor hnRNPA1 regulates alternative splicing of LOXL2 to enhance the production of LOXL2Δ13

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family and has the ability to catalyze the cross-linking of extracellular matrix collagen and elastin. High expression of LOXL2 is related to tumor cell proliferation, invasion and metastasis. LOXL2 contains 14 exons. Previous studies have found that LOXL2 has abnormal alternative splicing and exon skipping in a variety of tissues and cells, resulting in a new alternatively-spliced isoform denoted LOXL2Δ13. LOXL2Δ13 lacks LOXL2WT exon 13, but its encoded protein has greater ability to induce tumor cell proliferation, invasion and metastasis. However, the molecular events that produce LOXL2Δ13 are still unclear. In this study, we found that overexpression of the splicing factor hnRNPA1 in cells can regulate the alternative splicing of LOXL2 and increase the expression of LOXL2Δ13. The exonic splicing silencer (ESS) exists at the 3′ splice site (3′ SS) and 5′ splice site (5′ SS) of LOXL2 exon 13. HnRNPA1 can bind to the ESS and inhibit the inclusion of exon 13. The RRM domain of hnRNPA1 and phosphorylation of hnRNPA1 at S91 and S95 are important for the regulation of LOXL2 alternative splicing. These results show that hnRNPA1 is a splicing factor that enhances the production of LOXL2Δ13.

#2349 Lysyl oxidase-like 2 inhibitor ameliorates tubulointerstitial fibrosis and albuminuria in cyclosporine induced nephropathy

Abstract Background and Aims Lysyl oxidase-like 2 (LOXL2), an amine oxidase, contributes to fibrotic scarring in the tissue by facilitating collagen cross-linking. Recent data demonstrated upregulation of lysyl oxidase and LOXL2 in the fibrotic kidneys after administration of cylosporine A (CsA). Thus, inhibition of LOXL2 may render therapeutic effects against CsA-induced nephropathy by ameliorating tubulointerstitial fibrosis. Method Low salt fed CD-1 mice were administered saline or CsA (15 mg/kg/day, intraperitoneally) for 8 weeks. At 4 weeks, CsA-treated animals were divided into 2 groups respectively and treated with: (1) vehicle, (2) LOXL2 inhibitor (10 mg/kg/day, gavage feeding) for another 4 weeks up to 8 weeks. We explored the reduction of tubulointerstitial fibrosis as well as albuminuria with administration of LOXL2 inhibitor in CsA nephropathy mouse model. Results CsA administration significantly increased the levels of serum creatinine (10.0 ± 1.2 vs. 28.4 ± 4.3, p = 0.02), tubular injury scores (0.1 ± 0.2 vs. 3.6 ± 1.3, p = 0.03), tubulointerstitial fibrosis (Sirius red positive area, 14 ± 6% vs. 204 ± 43%, p = 0.01), F4/80 positive inflammatory cell infiltration in mouse kidney (2 ± 1 vs. 21 ± 4, p = 0.001). These changes were associated with upregulated mRNA expression of LOXL2, TGF-β1, and monocyte chemoattractant protein-1 (MCP-1). Administration of LOXL2 inhibitor significantly suppressed the expression of alpha-SMA and collagen 1A, intrarenal F4/80 positive inflammatory cell infiltrations (21 ± 4 vs. 14 ± 2, p = 0.001) and improved tubular injury scores (3.6 ± 1.3 vs. 1.6 ± 1.1, p = 0.02). Moreover, LOXL2 inhibitor effectively reduced albuminuria excretion (46 ± 4 vs. 22 ± 6, p = 0.02). Conclusion Our results suggest that LOXL2 inhibitor treatment can attenuate CsA nephropathy progression and LOXL2 may be a potential therapeutic target in CsA nephropathy.

LOXL2 inhibition ameliorates pulmonary artery remodeling in pulmonary hypertension

Background: Conduit pulmonary arterial stiffening and the resultant increase in pulmonary vascular impedance has emerged as an important underlying driver of pulmonary arterial hypertension (PAH). Given that matrix deposition is central to vascular remodeling, we evaluated the role of the collagen crosslinking enzyme lysyl oxidase like 2 (LOXL2) in this study. Methods and Results: Human pulmonary artery smooth muscle cells (PASMCs) subjected to hypoxia showed increased LOXL2 secretion. LOXL2 activity and expression were markedly higher in primary PASMCs isolated from pulmonary arteries of the rat Sugen 5416 + hypoxia (SuHx) model of severe PH. Similarly, LOXL2 protein and mRNA levels were increased in pulmonary arteries (PA) and lungs of rats with PH (SuHx and monocrotaline (MCT) models). Pulmonary arteries (PAs) isolated from rats with PH exhibited hypercontractility to phenylephrine and attenuated vasorelaxation elicited by acetylcholine, indicating severe endothelial dysfunction. Tensile testing revealed a a significant increase in PA stiffness in PH. Treatment with PAT-1251, a novel small-molecule LOXL2 inhibitor, improved active and passive properties of the PA ex vivo. There was an improvement in right heart function as measured by right ventricular pressure volume loops in-vivo with PAT-1251. Importantly PAT-1251 treatment ameliorated PH, resulting in improved pulmonary artery pressures, right ventricular remodeling, and survival. Conclusion: Hypoxia induced LOXL2 activation is a causal mechanism in pulmonary artery stiffening in PH, as well as pulmonary artery mechanical and functional decline. LOXL2 inhibition with PAT-1251 is a promising approach to improve pulmonary artery pressures, right ventricular elastance, cardiac relaxation, and survival in PAH. This work was supported by a NHLBI grant R01HL105296 (to D.E.B.), a NHLBI grant R01HL148112 01 (to L.S.), two Stimulating and Advancing ACCM Research (StAAR) grants from the Department of Anesthesiology and Critical Care Medicine, JHU (to L.S. and J.S.), NHLBI grants R01HL073859 and R01HL126514 (to L.A.S) and a NHLBI K08 grant K08HL145132 (to J.S.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Loss of phosphatase and tensin homolog (PTEN) increases Lysyl oxidase-like 2 (LOXL2) expression enhancing the growth of fallopian tube epithelial cells as three-dimensional spheroids

BackgroundHigh-grade serous ovarian cancer (HGSOC) accounts for 70–80% of all ovarian cancer-related deaths. Multiple studies have suggested that the fallopian tube epithelium (FTE) serves as the cell of origin of HGSOC. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and its loss is sufficient to induce numerous tumorigenic changes in FTE, including increased migration, formation of multicellular tumor spheroids (MTSs), and ovarian colonization. In murine oviductal epithelial (MOE) cells (the equivalent of human FTE) loss of PTEN results in the upregulation of transcripts associated with the extracellular matrix, with a specific focus on the elevation of lysyl oxidase-like 2 (LOXL2). Although LOXL2 is known to drive transformation and invasion in solid tumors and is associated with a poor prognosis in ovarian cancer, its specific role in the tumorigenesis of ovarian cancer originating from FTE remains unclear. Therefore, we aim to investigate whether LOXL2 mediates tumorigenesis from the fallopian tube epithelium. MethodsIn this study, we utilized clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CAS9) technology to delete LOXL2 in PTEN-deficient MOE cells to understand its role in mediating the oncogenic effects of PTEN loss. In addition, CRISPR-CAS9 was used to delete LOXL2 in OVCAR8 ovarian cancer cells. We monitored the changes in tumorigenic properties, such as migration, invasion, and growth of three-dimensional (3D) spheroids, to assess whether the loss of LOXL2 resulted in any changes. ResultsWe found that a reduction in LOXL2 expression did not significantly change the migration or invasive capabilities of PTEN-depleted MOE or human ovarian cancer cells. However, we found that a reduction in LOXL2 expression resulted in a significant reduction in 3D MTS formation and survival in both lines. ConclusionsThese results reveal for the first time that PTEN loss in FTE cells increases LOXL2 expression through downregulation of Pax2, and LOXL2 deletion blocks 3D spheroid formation.

RNA-binding protein RPS7 promotes hepatocellular carcinoma progression via LOXL2-dependent activation of ITGB1/FAK/SRC signaling

BackgroundMetastasis is one of the leading cause contributes to treatment failure and poor prognosis of hepatocellular carcinoma (HCC) patients. The underlying mechanism of HCC metastasis remains to be determined. Although several RNA binding proteins (RBPs) have been found to participate in tumorigenesis and progression of liver cancer, the role of RBPs in HCC patients with extrahepatic metastases is poorly understood.MethodsBy performing RNA-seq of primary HCC tissues (including HCC with extrahepatic metastasis and those did not develop metastasis), we identified a set of HCC metastasis-associated RBPs candidates. Among which, ribosomal protein S7 (RPS7) was found to be remarkably increased in HCC tissues and be strongly related to HCC poor survival. Overexpression or CRISPR-Cas9–mediated knockout were applied to investigate the role of RPS7 on the metastasis-associated phenotypes of HCC cells. RNA sequencing, RIP, RNA-pull down, dual luciferase reporter assay, nascent RNA capture assay, and RNA decay and so on, were applied to reveal the underlying mechanism of RPS7 induced HCC metastasis.ResultsGain- and loss- of function analyses revealed that RPS7 promoted HCC cells adhesion, migration and invasion capabilities, as well as lung metastasis. Mechanistically, we uncovered that lysyl oxidase-like 2 (LOXL2) was a critical downstream target of RPS7. RPS7 could stabilize LOXL2 mRNA by binding to AUUUA motifs in the 3155–3375 region of the 3’UTR of LOXL2 mRNA, thus increased LOXL2 expression via elevating LOXL2 mRNA abundance. Further research revealed that LOXL2 could accelerate focal adhesion formation through maintaining the protein stability of ITGB1 and activating ITGB1-mediated FAK/SRC signaling pathway, and thereby contribute to the pro-metastasis effect of RPS7.ConclusionsTaken together, our data reveal a novel function of RPS7 in HCC metastasis, also reveal the critical roles of the RPS7/LOXL2/ITGB1 axis in HCC metastasis and shed new light on the exploration of molecular drugs against HCC.

LOXL2 in Cancer: A Two-Decade Perspective.

Lysyl Oxidase Like 2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises five lysine tyrosylquinone (LTQ)-dependent copper amine oxidases in humans. In 2003, LOXL2 was first identified as a promoter of tumour progression and, over the course of two decades, numerous studies have firmly established its involvement in multiple cancers. Extensive research with large cohorts of human tumour samples has demonstrated that dysregulated LOXL2 expression is strongly associated with poor prognosis in patients. Moreover, investigations have revealed the association of LOXL2 with various targets affecting diverse aspects of tumour progression. Additionally, the discovery of a complex network of signalling factors acting at the transcriptional, post-transcriptional, and post-translational levels has provided insights into the mechanisms underlying the aberrant expression of LOXL2 in tumours. Furthermore, the development of genetically modified mouse models with silenced or overexpressed LOXL2 has enabled in-depth exploration of its in vivo role in various cancer models. Given the significant role of LOXL2 in numerous cancers, extensive efforts are underway to identify specific inhibitors that could potentially improve patient prognosis. In this review, we aim to provide a comprehensive overview of two decades of research on the role of LOXL2 in cancer.

Lysyl oxidase-like 2 promotes stemness and enhances antitumor effects of gefitinib in head and neck cancer via IFIT1 and IFIT3.

Lysyl oxidase-like 2 (LOXL2) is a matrix-remodeling enzyme that has recently been identified as an important regulator of tumor progression and metastasis. This study discovered that LOXL2 expression in oral squamous cell carcinoma (OSCC) tissues was significantly associated with tumor clinical stage, lymph node metastasis and patients' overall survival time. LOXL2-overexpressing human buccal SCC TW2.6 (TW2.6/LOXL2) and hypopharyngeal SCC FaDu (FaDu/LOXL2) cells exhibited enhanced migration, invasion, epithelial-mesenchymal transition (EMT), and cancer stem cell (CSC) phenotypes, independently of its enzymatic activity. Moreover, TW2.6/LOXL2 significantly increased tumor-initiating frequency in SCID mice. We further demonstrated that LOXL2 increased the levels of interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) and IFIT3 in TW2.6/LOXL2 and FaDu/LOXL2 cells. We also identified IFIT1 and IFIT3 as key downstream components of LOXL2 action in migration, invasion, EMT, and CSC phenotypes in TW2.6 and FaDu cells. Furthermore, a significant positive correlation between LOXL2 expression and IFIT1 and IFIT3 overexpression in human OSCC tissues was observed. In addition, TW2.6/LOXL2 and FaDu/LOXL2 cells were 3.3- to 3.6-fold more susceptible to the epidermal growth factor receptor (EGFR) inhibitor gefitinib than were their respective control cells. The antitumor effect of gefitinib on orthotopic TW2.6/LOXL2 xenograft tumor was fourfold higher than that on controls. Our results indicate that LOXL2 expression is a strong prognostic factor for OSCC and may be used as a marker to identify patients most likely to respond to EGFR-targeted therapy.

Lysyl oxidase like-2 in fibrosis and cardiovascular disease.

Fibrosis is an important and essential reparative response to injury that, if left uncontrolled, results in the excessive synthesis, deposition, remodeling, and stiffening of the extracellular matrix, which is deleterious to organ function. Thus, the sustained activation of enzymes that catalyze matrix remodeling and cross linking is a fundamental step in the pathology of fibrotic diseases. Recent studies have implicated the amine oxidase lysyl oxidase like-2 (LOXL2) in this process and established significantly elevated expression of LOXL2 as a key component of profibrotic conditions in several organ systems. Understanding the relationship between LOXL2 and fibrosis as well as the mechanisms behind these relationships can offer significant insights for developing novel therapies. Here, we summarize the key findings that demonstrate the link between LOXL2 and fibrosis and inflammation, examine current therapeutics targeting LOXL2 for the treatment of fibrosis, and discuss future directions for experiments and biomedical engineering.

Mesenchymal stem cell-derived exosomal miR-27b-3p alleviates liver fibrosis via downregulating YAP/LOXL2 pathway

Lysyl oxidase-like 2 (LOXL2) is an extracellular copper-dependent enzyme that plays a central role in fibrosis by catalyzing the crosslinking and deposition of collagen. Therapeutic LOXL2 inhibition has been shown to suppress liver fibrosis progression and promote its reversal. This study investigates the efficacy and underlying mechanisms of human umbilical cord-derived exosomes (MSC-ex) in LOXL2 inhibition of liver fibrosis. MSC-ex, nonselective LOX inhibitor β-aminopropionitrile (BAPN), or PBS were administered into carbon tetrachloride (CCl4)-induced fibrotic livers. Serum LOXL2 and collagen crosslinking were assessed histologically and biochemically. MSC-ex’s mechanisms on LOXL2 regulation were investigated in human hepatic stellate cell line LX-2. We found that systemic administration of MSC-ex significantly reduced LOXL2 expression and collagen crosslinking, delaying the progression of CCl4-induced liver fibrosis. Mechanically, RNA-sequencing and fluorescence in situ hybridization (FISH) indicated that miR-27b-3p was enriched in MSC-ex and exosomal miR-27b-3p repressed Yes-associated protein (YAP) expression by targeting its 3ʹ untranslated region in LX-2. LOXL2 was identified as a novel downstream target gene of YAP, and YAP bound to the LOXL2 promoter to positively regulate transcription. Additionally, the miR-27b-3p inhibitor abrogated the anti-LOXL2 abilities of MSC-ex and diminished the antifibrotic efficacy. miR-27b-3p overexpression promoted MSC-ex mediated YAP/LOXL2 inhibition. Thus, MSC-ex may suppress LOXL2 expression through exosomal miR-27b-3p mediated YAP down-regulation. The findings here may improve our understanding of MSC-ex in liver fibrosis alleviation and provide new opportunities for clinical treatment.

LOXL2 serves as a prognostic biomarker for hepatocellular carcinoma by mediating immune infiltration and vasculogenic mimicry.

The development of human hepatocellular carcinoma (HCC) is a multistep process that is accompanied by progressive changes in the liver microenvironment, including immune evasion and angiogenesis. Lysyl oxidase-like 2 (LOXL2) has been suggested to contribute to tumour progression and metastasis; however, the underlying mechanism remains unclear. The purpose of the present study was to explore the relationship between LOXL2 and immune infiltration and vasculogenic mimicry (VM) and to identify the role of LOXL2 in HCC diagnosis prognosis evaluation. The Cancer Genome Atlas (TCGA), UALCAN, GEPIA and Kaplan-Meier plotter databases were used to analyse LOXL2 expression and perform survival analysis. The Tumour Immune Estimation Resource (TIMER) was used to analyse immune cell infiltration, immune cell biomarkers and immune checkpoints. Immunohistochemistry (IHC) of 201 HCC samples was used to confirm the expression of LOXL2 and its relationship with VM. Coimmunoprecipitation (co-IP) and gain- and loss-of-function studies were performed to confirm the molecular mechanism of LOXL2 in VM. The expression of LOXL2 in HCC was higher than that in normal tissues at both the mRNA and protein levels. High expression of LOXL2 was associated with a poorer prognosis of HCC. The genetic alteration rate of LOXL2 was 5%. LOXL2 was positively related to immune cell infiltration and immune checkpoints (PD-1 and CTLA-4) in HCC. Co-IP showed that LOXL2 can interact directly with IQGAP1. Both gain- and loss-of-function studies showed that LOXL2 significantly induced cell migration, invasion and VM formation when IQGAP1 was upregulated. LOXL2 is involved in immune cell infiltration and promotes VM by upregulating IQGAP1. LOXL2 can be used as a novel biomarker for HCC diagnosis and prognosis prediction.

Abstract 3645: OSM-induced LOXL2 expression hinges on upregulation of c-Myc and promotes tumor growth and metastasis, IL-1beta plays synergistic role in LOXL2 expression

Abstract Ductal carcinoma is the most commonly diagnosed breast cancer in women, the presence of metastases drops patient five-year survival from 95% to an abysmal 27%. This makes studying the mechanisms that promote metastasis of critical importance. Research shows that the extracellular matrix (ECM) of the tumor microenvironment (TME) plays an important role in invasive ductal carcinoma (IDC) progression and metastasis. Specifically; the density, stiffness, and orientation of collagen I fibers in the stroma all impact IDC motility, invasion, and metastasis. Lysyl oxidase-like 2 (LOXL2) is a secreted enzymatic protein that is implicit in ECM remodeling. By catalyzing an oxidative reaction in peptidyl lysine/hydroxylysine present on collagen I fibers, LOXL2 initiates crosslinking of the fibers. Our previous publication demonstrates that interleukin-6-related pro-inflammatory cytokine signaling, specifically oncostatin M (OSM), led to the overexpression and secretion of the LOXL2 enzyme. This increased the metastatic potential of IDC cells by promoting the crosslinking and alignment of collagen I fibers, which in turn led to increased invasion in 3D collagen I matrices. Thus, we hypothesize that OSM-induced LOXL2 expression, secretion, and subsequent collagen I fiber crosslinking and alignment, leads to an increase in IDC metastasis. In vivo studies utilizing nude athymic mice injected with either MCF7-Luc-EV control or MCF7-Luc-OSM overexpressing cells were performed in conjunction with LOXL2 inhibition using specially designed chow containing either LOXL2 SMI or control. Further analysis of OSM signaling pathways was performed in MCF7 and MDA-MB-468 IDC cells to determine mechanism for OSM induction of LOXL2. Our studies demonstrate that canonical OSM signaling pathways (pERK, pSTAT3, and pAKT) all contribute to LOXL2 expression in MCF7 cells suggesting a possible mediator protein we identified as c-Myc. Furthermore, OSM-induced LOXL2 played a significant role in OSM promoted tumor growth and metastasis as LOXL2 inhibition led to decreased tumor size and far less metastatic lesions. We also analyzed and confirmed a synergistic interaction between OSM and interleukin-1beta (IL-1β) in LOXL2 expression. These results confirm the significance of LOXL2 expression in OSM promoted metastasis and suggests LOXL2 would be an important target for future breast cancer therapies, specifically in patients with elevated levels of OSM. Furthermore, the synergistic interaction between OSM and IL-1β in LOXL2 expression suggests that patients with high levels of both cytokines are at even greater risk of metastasis, making these great markers for determining at risk IDC patients. Citation Format: Simion Dinca, Cody Wolf, Laura Bond, Cheryl Jorcyk. OSM-induced LOXL2 expression hinges on upregulation of c-Myc and promotes tumor growth and metastasis, IL-1beta plays synergistic role in LOXL2 expression. [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 3645.

Dihydroartemisinin Potentiates VEGFR-TKIs Antitumorigenic Effect on Osteosarcoma by Regulating Loxl2/VEGFA Expression and Lipid Metabolism Pathway.

Anti-angiogenesis therapy has shown significant anti-tumor effects against a variety of cancers. But resistance to antiangiogenic drugs, intrinsic and evasive, is frequently found in patients during treatment. Here, we report that dihydroartemisinin (DHA), a derivative of the Chinese medicine artemisinin, enhances antiangiogenic drug-induced cytotoxicity in osteosarcoma (OS) cells. Proteomics analysis revealed that DHA treatment significantly affected the activity of the collagen-modifying enzyme lysyl oxidase-like 2 (LOXL2), a regulatory gene associated with poor prognosis of OS. Furthermore, we found that DHA reduced the expression of vascular endothelial growth factor (VEGFA) by downregulating LOXL2. This mechanism was confirmed by QRT-PCR, western blot, and ELISA assays. Correspondingly, vector-enforced expression of LOXL2 markedly reduced VEGFA secretion. Untargeted metabolomic analysis revealed that the lipid metabolism that confers antiangiogenic drug resistance, was also interfered with by DHA. Thus, DHA not only exerts antitumor effects in OS cells directly but also synergizes with the antiangiogenic drug by regulating vascular endothelial growth factor A (VEGFA) expression and lipid metabolism.

LOXL2 reduces 5-FU sensitivity through the Hedgehog/BCL2 signaling pathway in colorectal cancer.

Elevated expression of lysyl oxidase-like 2 (LOXL2) contributes to the malignant tumor progression in multiple cancers. However, the role of LOXL2 in the 5-fluorouracil (5-FU) resistance of colorectal cancer (CRC) remains unclear. This study aimed to explore the effects of LOXL2 on 5-FU sensitivity in CRC. The mRNA and protein levels of LOXL2 were explored in public databases by bioinformatics, validated in clinical tissues using immunohistochemistry, and detected in 5-FU treated cell lines. The 50% inhibitory concentrations (IC50) values were quantified based on the cell viability at different concentrations of 5-FU with CCK-8 assays. Colony formation and flow cytometry assays were performed to measure the proliferation and apoptosis rates. Gene set enrichment and correlation analyses were conducted to identify the probable mechanism of LOXL2 in TCGA samples. Critical molecules of the Hedgehog signaling pathway and anti-apoptotic BCL2 in protein levels were detected with Western blotting. It concluded that LOXL2 was up-regulated and positively linked to the unfavorable prognosis of CRC patients. The LOXL2 expression increased with the rising 5-FU concentrations, especially at 20 and 40 μM. Elevated LOXL2 promoted the resistance to 5-FU, augmented the proliferation, and inhibited 5-FU-induced apoptosis of CRC cells. LOXL2 activated the Hedgehog signaling pathway by promoting the expression of SMO, GLI1, and GLI2, leading to the upregulation of downstream target gene BCL2 in CRC cells. Moreover, the Hedgehog signaling pathway inhibitor cyclopamine blocked the BCL2 upregulation mediated by LOXL2. This study has demonstrated that LOXL2 can reduce 5-FU sensitivity through the Hedgehog/BCL2 signaling pathway in CRC.

Inhibition of lysyl oxidase‑like 2 ameliorates folic acid‑induced renal tubulointerstitial fibrosis.

Tubulointerstitial fibrosis is characterized by accumulation of the extracellular matrix in the interstitium. Lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase family, is known for promoting cancer metastasis, invasion and stromal fibrosis in various organs. Our previous study demonstrated expression of LOXL2 in kidney podocytes and tubular epithelial cells, and the association between elevated LOXL2 and tubulointerstitial fibrosis. The present study evaluated the effect of LOXL2 inhibition using an inhibitory monoclonal antibody (AB0023) on tubulointerstitial fibrosis in a folic acid-induced tubulointerstitial fibrosis mouse model. The association of LOXL2 with epithelial-mesenchymal transformation-related molecules was also evaluated in vitro using HK-2 cells. The present data demonstrated that AB0023 prevented the progression of tubulointerstitial fibrosis significantly, as determined by trichrome and picro-sirius red staining, as well as the total collagen assay. The mean expression of phosphorylated Smad2 and Smad4 was lower in the AB0023-treated group although it was not statistically significant. Following transforming growth factor-β (TGF-β) challenge, LOXL2-deficient HK-2 cells exhibited significantly lower expression of the mesenchymal markers vimentin and fibronectin than control HK-2 cells. In conclusion, LOXL2 inhibition ameliorates renal fibrosis through the TGF-β/Smad signalling pathway.

Theragnostic Implications of MIR29C/LOXL2 Axis in Lung Adenocarcinoma

<p>肺腺癌的早期精準診斷以及其抗藥性的生成一直是臨床上的挑戰。近年來發現 到微核酸 29C 以及類二型賴氨酸氧化酶分別扮演的抑癌以及促癌角色。然而,微核 酸 29C/ 類二型賴氨酸氧化酶軸線的臨床意義以及生物功能尚未釐清。本研究應用 多體學策略探討微核酸 29C/ 類二型賴氨酸氧化酶軸線是否可作為肺腺癌預測模組以 及作為治療標的的潛力。自 cBioPortal 伺服器擷取 7,561 件肺腺癌檢體中,微核酸 29C 的突變光譜展現隨機分布,擴增率僅 3%,且基因套數變化不影響病人存活時 間。相比之下,低表達微核酸 29C 與短存活時間顯著相關,且與類二型賴氨酸氧化 酶呈現負相關。高表達類二型賴氨酸氧化酶與短存活時間顯著相關,並且與 CD8 陽 性 T 細胞呈現負相關,而與癌相關纖維母細胞呈現正相關,顯示出抑制免疫的腫瘤 微環境特徵。此外,微核酸 29C 與臨床分期呈現負向關聯性。TargetScan 生資工具 預測出微核酸 29C 的 3’ 端可標靶類二型賴氨酸氧化酶的兩處 3’ 端未轉譯區,核苷 酸位序 555-561 以及 757-763,表明微核酸 29C 有抑制類二型賴氨酸氧化酶的潛力。 總結本研究發現低表達微核酸 29C 連同高表達類二型賴氨酸氧化酶可預測肺腺癌不 良預後。微核酸 29C/ 類二型賴氨酸氧化酶軸線指出一肺腺癌新生物路徑以及治療 標的。</p> <p> </p><p>The early, precise diagnosis and drug resistance of lung adenocarcinoma (LUAD) remains a challenging issue. The tumor suppression role of microRNA- 29C (MIR29C) and the tumor promotion role of lysyl oxidase-like 2 (LOXL2) have recently been observed. However, the clinical implication and biological function of MIR29C/lysyl oxidase-like 2 (LOXL2) axis remains unclear. In this study, we utilized the multi-omic approach to investigate whether the MIR29C/LOXL2 axis can serve as a prediction panel and determine its potential as therapeutic targets. Using the cBioPortal server, the mutation landscape of MIR29C in LUAD samples showed that the mutation spectrum presented a random distribution and the amplification rate was as low as 3%, while LOXL2 presented as 6%. The copy number alteration of MIR29C had no effect on the overall survival (OS) rate, whereas that of LOXL2 was associated with poor OS. In contrast, the low expression of MIR29C was significantly associated with shorter OS. MIR29C negatively correlated with LOXL2 mRNA level. High expression of LOXL2 was significantly associated with shorter OS, and connected with lower infiltration levels of CD8+T cells and higher infiltrate levels of cancer associated fibroblast, representing hallmarks of an immunosuppressive tumor microenvironment. In addition, MIR29C showed a negative association with advanced stages. Using TargetScan Release 7.2, MIR29C-3p was predicted to target LOXL2 3’UTR at position 555-561 and position 757-763, suggesting its inhibitory potential on LOXL2 expression. In summary, this study revealed that low expression of MIR29C and high expression of LOXL2 predict poor outcomes for LUAD patients. The MIR29C/LOXL2 axis may point out a new biological pathway that could be a potential therapeutic target for LUAD.</p> <p> </p>

LOXL2 Inhibitor Attenuates Angiotensin II-Induced Atrial Fibrosis and Vulnerability to Atrial Fibrillation through Inhibition of Transforming Growth Factor Beta-1 Smad2/3 Pathway

Objectives: Angiotensin II (Ang II)-induced atrial fibrosis plays a vital role in the development of atrial fibrillation (AF). Lysyl oxidase-like 2 (LOXL2) plays an essential role in matrix remodeling and fibrogenesis, indicating it may involve fibrosis-associated diseases. This study aims to elucidate the role of LOXL2 in AF, and its specific inhibitor can suppress Ang II-induced inflammatory atrial fibrosis and attenuate the enhanced vulnerability to AF. Methods: Male mice C57BL/6 were subcutaneously infused with either saline or Ang II (2 mg/kg/day) for 4 weeks. DMSO or LOXL2 inhibitor LOXL2-IN-1 hydrochloride (LOXL2-IN-1) at a dose of 100 μg/kg/day were intraperitoneally injected once daily for 4 weeks. Morphological, histological, and biochemical analyses were performed. AF was induced by transesophageal burst pacing in vivo. Results: Expression of LOXL2 was increased in serum of AF patients and Ang II-treated mice. LOXL2-IN-1 significantly attenuated Ang II-induced AF vulnerability, cardiac hypertrophy, atrial inflammation, and fibrosis. LOXL2-IN-1 suppressed Ang II-induced expression of transforming growth factor beta-1 (TGF-β1) and collagen I and phosphorylation of Smad2/3 in atrial tissue. Conclusions: LOXL2 is a target of AF, and its inhibitor prevents atrial fibrosis and attenuated enhanced vulnerability to AF potentially through the TGF-β/Smad pathway.

LOXL2 attenuates osteoarthritis through inactivating Integrin/FAK signaling

Temporomandibular joint OA (TMJOA) is a common degenerative joint disease, leads to structural damage and ultimately loss of function. Matrix degradation is one of the first pathogenesis during the progression of OA, it was effective to inhibit matrix degradation to block the development of OA. In this study, an in vivo model (compressive mechanical force) and an in vitro model (IL-1β) were used to induce OA-like changes in TMJ cartilage and chondrocytes. We revealed lysyl oxidase like-2 (LOXL2) play a critical role in TMJOA. LOXL2 expression decreased in mechanical stress/IL-β induced TMJOA-like lesions in both in vivo models and in vitro models. Furthermore, recombinant LOXL2 (rhLOXL2) treatment ameliorated the degenerative changes induced by mechanical stress in vivo, including the thinning cartilage, down-expression of collagen II and proteoglycan, and over-expression of TNF-a, while LOXL2 antibody (anti-LOXL2) treatment exacerbated these changes. Mechanistically, the protection of LOXL2 in chondrocytes was induced partly through activation of the Integrin/FAK pathway. The inhibition of the Integrin/FAK pathway could neutralized the effects caused by rhLOXL2. Collectively, our study suggests that the LOXL2 plays a protective role in mechanical stress induced TMJOA-like changes, and the Integrin/FAK pathway may be a key downstream pathway in this process.

Novel mechanism for OSM-promoted extracellular matrix remodeling in breast cancer: LOXL2 upregulation and subsequent ECM alignment

BackgroundInvasive ductal carcinoma (IDC) is a serious problem for patients as it metastasizes, decreasing 5-year patient survival from > 95 to ~ 27%. The breast tumor microenvironment (TME) is often saturated with proinflammatory cytokines, such as oncostatin M (OSM), which promote epithelial-to-mesenchymal transitions (EMT) in IDC and increased metastasis. The extracellular matrix (ECM) also plays an important role in promoting invasive and metastatic potential of IDC. Specifically, the reorganization and alignment of collagen fibers in stromal ECM leads to directed tumor cell motility, which promotes metastasis. Lysyl oxidase like-2 (LOXL2) catalyzes ECM remodeling by crosslinking of collagen I in the ECM. We propose a novel mechanism whereby OSM induces LOXL2 expression, mediating stromal ECM remodeling of the breast TME.MethodsBioinformatics was utilized to determine survival and gene correlation in patients. IDC cell lines were treated with OSM (also IL-6, LIF, and IL-1β) and analyzed for LOXL2 expression by qRT-PCR and immunolabelling techniques. Collagen I contraction assays, 3D invasion assays, and confocal microscopy were performed with and without LOXL2 inhibition to determine the impact of OSM-induced LOXL2 on the ECM.ResultsOur studies demonstrate that IDC patients with high LOXL2 and OSM co-expression had worse rates of metastasis-free survival than those with high levels of either, individually, and LOXL2 expression is positively correlated to OSM/OSM receptor (OSMR) expression in IDC patients. Furthermore, human IDC cells treated with OSM resulted in a significant increase in LOXL2 mRNA, which led to upregulated protein expression of secreted, glycosylated, and enzymatically active LOXL2. The expression of LOXL2 in IDC cells did not affect OSM-promoted EMT, and LOXL2 was localized to the cytoplasm and/or secreted. OSM-induced LOXL2 promoted an increase in ECM collagen I fiber crosslinking, which led to significant fiber alignment between cells and increased IDC cell invasion.ConclusionsAligned collagen fibers in the ECM provide pathways for tumor cells to migrate more easily through the stroma to nearby vasculature and tissue. These results provide a new paradigm through which proinflammatory cytokine OSM promotes tumor progression. Understanding the nuances in IDC metastasis will lead to better potential therapeutics to combat against the possibility.