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INTRODUCTION: Lysyl oxidase-like 2 (LOXL2) expression and function is frequently altered in different cancers but scarcely explored in oral squamous cell carcinoma (OSCC). This prompted us to investigate the clinical relevance of LOXL2 expression pattern in OSCC and also a possible crosstalk with Hippo/YAP1 pathway signaling. METHODS: Immunohistochemical analysis of LOXL2 protein expression was performed in 158 OSCC patient samples, together with Yes-associated protein 1 (YAP1) activation status. Correlations with clinicopathological parameters and patient survival were assessed. RESULTS: Tumor cell-intrinsic LOXL2 expression showed two distinct expression patterns: diffuse cytoplasmic staining (64.6%) and heterogeneous perinuclear staining (35.4%). Remarkably, perinuclear LOXL2 staining was significantly associated with lymph node metastasis, advanced clinical stage and perineural invasion. Moreover, patients harboring tumors with perinuclear LOXL2 expression exhibited significantly poorer disease-specific survival (DSS) rates, and perinuclear LOXL2 positivity gradually increased in relation to YAP1 activation. Patients harboring tumors with concomitant perinuclear LOXL2 and fully active YAP1 exhibited the worst DSS. Multivariate Cox analysis further revealed combined perinuclear LOXL2 and fully active YAP1 as a significant independent predictor of poor DSS. CONCLUSION: Tumor-intrinsic perinuclear LOXL2 emerges as a clinically and biologically relevant feature associated with advanced disease, tumor aggressiveness, and poor prognosis in OSCC. Moreover, this study unprecedentedly uncovers a functional relationship between perinuclear LOXL2 and YAP1 activation with major prognostic implications. Notably, combined perinuclear LOXL2 and fully active YAP1 was revealed as independent predictor of poor prognosis. These findings encourage targeting oncogenic LOXL2 functions for personalized treatment regimens.

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The formation of fibrotic bands in female reproductive system, including the uterus, after abdominal and pelvic surgeries, is an important medical challenge associated with many complications, including infertility and pain. Investigating the role of different molecules involved in fibrosis and adhesion formation may help in the development of new drugs to prevent this disorder. Lysyl oxidase-like 2 (LoxL2) is a copper-dependent enzyme that catalyzes the cross-linking of collagen and elastin fibers in the extracellular matrix (ECM) to stabilize ECM. Dysregulation of LoxL2 activity resulting from tissue hypoxia and inflammation after gynecological surgeries in the female reproductive tract increases collagen fibers cross-linking and promotes fibrosis. It has been shown that targeting LoxL2 by Lox inhibitors may reduce fibrosis. Considering the expression of LoxL2 in female reproductive organs and its dysregulation in hypoxia and inflammation, it is possible that LoxL2 has therapeutic potential as a drug target in the prevention of adhesions. In this review, we discuss the role of LoxL2 in the promotion of fibrotic processes focusing on its link with inflammatory and hypoxic conditions. We also justify the use of anti- LoxL2 agents as a potential therapeutic strategy for the prevention of post-surgical scar formation.

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Lysyl oxidase-like 2 (LOXL2) mediates the crosslinking of extracellular collagen, reflecting qualitative changes in liver fibrosis. This study aimed to validate the utility of serum LOXL2 levels as a predictive biomarker for the development of hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV) infection who achieved a sustained virological response (SVR). This retrospective study included 137 patients with chronic HCV infection without history of HCC development and who achieved SVR via direct-acting antiviral therapy. Median LOXL2 levels decreased significantly after SVR achievement (pre-Tx, 2.33 ng/mL; post-Tx, 1.31 ng/mL, p < 0.001). Post-Tx LOXL2 levels, fibrosis-4 index, platelet counts, Wisteria floribunda agglutinin-positive human Mac-2 binding protein levels, and alpha-fetoprotein (AFP) levels were identified as independent predictive factors for post-SVR HCC development in the univariate analysis. The incidence of post-SVR HCC development was significantly higher in patients with post-Tx LOXL2 levels ≥ 2.08 ng/mL and AFP levels ≥ 5.0 ng/mL than in patients with elevated levels of either marker or with lower marker levels. Serum LOXL2 levels can serve as a predictive biomarker for HCC development after achieving SVR. The combination of serum LOXL2 and AFP levels provides robust risk stratification for HCC development after SVR, suggesting an enhanced surveillance strategy.

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Aminoácido Oxidorreductasas , Carcinoma Hepatocelular , Hepatitis C Crónica , Neoplasias Hepáticas , Respuesta Virológica Sostenida , Femenino , Humanos , Masculino , alfa-Fetoproteínas/metabolismo , alfa-Fetoproteínas/análisis , Aminoácido Oxidorreductasas/sangre , Antivirales/uso terapéutico , Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/virología , Hepacivirus , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/sangre , Hepatitis C Crónica/virología , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/virología , Estudios Retrospectivos

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Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies. A specific mechanism of its metastasis has not been established. In this study, we investigated whether Neural Wiskott-Aldrich syndrome protein (N-WASP) plays a role in distant metastasis of PDAC. We found that N-WASP is markedly expressed in clinical patients with PDAC. Clinical analysis showed a notably more distant metastatic pattern in the N-WASP-high group compared to the N-WASP-low group. N-WASP was noted to be a novel mediator of epithelial-mesenchymal transition (EMT) via gene expression profile studies. Knockdown of N-WASP in pancreatic cancer cells significantly inhibited cell invasion, migration, and EMT. We also observed positive association of lysyl oxidase-like 2 (LOXL2) and focal adhesion kinase (FAK) with the N-WASP-mediated response, wherein EMT and invadopodia function were modulated. Both N-WASP and LOXL2 depletion significantly reduced the incidence of liver and lung metastatic lesions in orthotopic mouse models of pancreatic cancer. These results elucidate a novel role for N-WASP signaling associated with LOXL2 in EMT and invadopodia function, with respect to regulation of intercellular communication in tumor cells for promoting pancreatic cancer metastasis. These findings may aid in the development of therapeutic strategies against pancreatic cancer.

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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Neoplasias Pancreáticas/patología , Transducción de Señal , Proteína del Síndrome de Wiskott-Aldrich/metabolismo

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BACKGROUND/AIM: "Stromal high expression" of Nicotinamide N-methyltransferase (NNMT), previously reported as a poor prognostic factor of gastric cancer, was based on immunohistochemical H-score. However, this could simply indicate an increase in cancer-associated fibroblasts (CAFs) because NNMT is positive for fibroblasts. To verify this, the proportion and staining intensity of stromal NNMT-positive stellate/spindle cells were evaluated separately and examined for its association with related proteins (H3K4me3, H3K27me3, and LOXL2). PATIENTS AND METHODS: Immunohistochemistry for NNMT, H3K4me3, H3K27me3, and LOXL2 was performed on 521 tissue microarrays of gastric cancer. Cancer stromal stellate/spindle cells were evaluated according to morphology, proportion, and stain intensity of NNMT, loss of H3K4me3 and H3K27me3, and stain intensity of LOXL2. Their associations with clinicopathological characteristics and overall survival were examined. RESULTS: Higher staining intensity of NNMT was not related to a poorer prognosis. However, higher proportion of NNMT-positive stellate/spindle cells indirectly contributed to a poor prognosis. It was associated with CAF-like morphology and a global decrease in H3K4me3/H3K27me3, which were both associated with high LOXL2 expression. These three factors were independent poor prognostic factors. In addition, in the LOXL2-high group, prognosis significantly deteriorated with the presence of a global decrease in H3K4me3/H3K27me3. CONCLUSION: The higher proportion of NNMT-positive cancer stromal cells in gastric cancer serves as an indicator for identifying unfavorable prognostic CAFs that show a global decrease in H3K4me3/H3K27me3. This facilitates research on the nature of these cells and their characteristics.

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Fibroblastos Asociados al Cáncer , Neoplasias Gástricas , Humanos , Histonas , Nicotinamida N-Metiltransferasa/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Pronóstico , Células del Estroma/metabolismo

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OBJECTIVE: Although there have been brilliant advancements in the practical application of therapies targeting immune checkpoints, achieving success in targeting the microenvironment remains elusive. In this study, we aimed to address this gap by focusing on Na+ / H+ exchanger 1 (NHE1) and Lysyl Oxidase Like 2 (LOXL2), which are upregulated in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: The malignancy of a metastatic human HNSCC cell line was assessed in a mouse tongue cancer xenograft model by knocking down (KD) NHE1, responsible for regulating intracellular pH, and LOXL2, responsible for extracellular matrix (ECM) reorganization via cross-linking of ECM proteins. In addition to assessing changes in PD-L1 levels and collagen accumulation following knockdown, the functional status of the PD-L1 / PD-1 immune checkpoint was examined through co-culture with NK92MI, a PD-1 positive phagocytic human Natural Killer (NK) cell line. RESULTS: The tumorigenic potential of each single KD cell line was similar to that of the control cells, whereas the potential was attenuated in cells with simultaneous KD of both factors (double knockdown [dKD]). Additionally, we observed decreased PD-L1 levels in NHE1 KD cells and compromised collagen accumulation in LOXL2 KD and dKD cells. NK92MI cells exhibited phagocytic activity toward HNSCC cells in co-culture, and the number of remaining dKD cells after co-culture was the lowest in comparison to the control and single KD cells. CONCLUSION: This study demonstrated the possibility of achieving efficient anti-tumor effects by simultaneously disturbing multiple factors involved in the modification of the tumor microenvironment.

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Aminoácido Oxidorreductasas , Neoplasias de Cabeza y Cuello , Intercambiador 1 de Sodio-Hidrógeno , Carcinoma de Células Escamosas de Cabeza y Cuello , Neoplasias de la Lengua , Intercambiador 1 de Sodio-Hidrógeno/genética , Intercambiador 1 de Sodio-Hidrógeno/metabolismo , Animales , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Ratones , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Humanos , Línea Celular Tumoral , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Neoplasias de la Lengua/genética , Neoplasias de la Lengua/patología , Neoplasias de la Lengua/metabolismo , Microambiente Tumoral , Técnicas de Silenciamiento del Gen , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Carcinogénesis/genética , Colágeno/metabolismo , Células Asesinas Naturales , Receptor de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/genética

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BACKGROUND: Metastasis 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. METHODS: By 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. RESULTS: Gain- 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. CONCLUSIONS: Taken 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.

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Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas Ribosómicas , Humanos , Aminoácido Oxidorreductasas/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas Ribosómicas/metabolismo , ARN , ARN Mensajero , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal

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Lung carcinoma (LC) is a complicated and highly heterogeneous disease with high morbidity and mortality. Both lysyl oxidase-like (LOXL) 2 and 3 act in cancer progression. This work endeavors to illustrate the influence of LOXL2/LOXL3 on LC progression and the underlying mechanisms. LOXL family genes and CCAAT enhancer binding protein A (CEBPA) were analyzed in the TCGA database for their expression patterns in LC patients and their correlations with the patient's prognosis. CEBPA, LOXL2, and LOXL3 expression levels were determined in LC cells. Gain- and loss-of-function assays were conducted, followed by assays for cell proliferation, epithelial-mesenchymal transition (EMT), apoptosis, invasion, and migration. The binding of CEBPA or B cell lymphoma protein (BCL)-2 to LOXL2/LOXL3 was verified. The ubiquitination level of BCL-2 and histone acetylation level of LOXL2/LOXL3 in LC cells were analyzed. Database analyses revealed that LC patients had high CEBPA, LOXL2, and LOXL3 expression, which were related to poor prognosis. LC cells also exhibited high CEBPA, LOXL2, and LOXL3 levels. LOXL2/LOXL3 knockdown subdued EMT, proliferation, migration, and invasion while enhancing the apoptosis of LC cells. LOXL2/LOXL3 could bind to CEBPA and BCL-2. LOXL2/LOXL3 knockdown upregulated BCL-2 ubiquitination level and diminished BCL-2 expression in LC cells. CEBPA recruited Tip60 to enhance histone acetylation and transcription of LOXL2/LOXL3 in LC cells. BCL-2 overexpression abolished the impacts of LOXL2/LOXL3 knockdown on LC cells. In conclusion, CEBPA boosts LOXL2 and LOXL3 transcription to facilitate BCL-2 stability by recruiting Tip60 and thus contributes to LC cell growth and metastasis.

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Carcinoma , Neoplasias Pulmonares , Humanos , Histonas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Pulmón/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Aminoácido Oxidorreductasas/genética , Proteínas Potenciadoras de Unión a CCAAT

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Objective To investigate the correlation between the expression of the activator of HSP90 ATPase-1(AHA1),lysyl oxidase like-2 protein(LOXL2)in osteosarcoma tissues with mRNA expression of invasion and metastasis genes and their clinical significance.Methods A total of 90 osteosarcoma patients diagnosed and treated in North China Medical and Health Group Fengfeng General Hospital from February 2016 to March 2017 were selected as the research object.The expression of AHA1 mRNA,LOXL2 mRNA and invasion and metastasis genes Wnt family member 9A(Wnt9a)mRNA,zinc finger E-box binding homologous box 1(ZEB1)mRNA,zinc finger E-box binding homologous box 1(ZEB2)mRNA,N-cadherin(N-cad)mRNA,and vimentin(Vim)mRNA in tissues were detected by real-time fluorescence quantitative PCR.Pearson correlation analysis was used for correlation analysis.The differences in expression of AHA1 mRNA and LOXL2 mRNA in osteosarcoma patients among different clinical characteristics were compared.Kaplan-Meier survival analysis was used to analyze the effect of AHA1 mRNA and LOXL2 mRNA on the prognosis of osteosarcoma patients.The prognostic factors of osteosarcoma patients were analyzed by univariate and multivariate COX regression.Results The expressions of AHA1 mRNA(3.16±0.59),LOXL2 mRNA(2.84±0.44)and invasion and metastasis genes[Wnt9a mRNA(3.23±0.42),ZEB1 mRNA(2.73±0.39),ZEB2 mRNA(2.52±0.56),N-cad mRNA(2.71±0.65)and Vim mRNA(2.81±0.73)]in osteosarcoma tissues were higher than those in paracancerous tissues(1.10±0.21,0.95±0.18,0.79±0.15,0.64±0.11,0.98±0.19,0.68±0.14,0.72±0.15),and the differences were statissically significant(t=31.206,37.716,51.903,48.931,24.706,28.964,26.605,all P<0.05).There was a significant positive correlation between AHA1 mRNA and LOXL2 mRNA expression in osteosarcoma(r=0.712,P<0.05).The expressions of AHA1 mRNA and LOXL2 mRNA were significantly positively correlated with the expressions of invasion and metastasis genes(Wnt9a,ZEB1,ZEB2,N-cad,and Vim mRNA)in tumor tissue of osteosarcoma group(r=0.504～0.720,all P<0.05).The expressions of AHA1 mRNA and LOXL2 mRNA in osteosarcoma tissues with Eneeking stage Ⅲ,soft tissue infiltration,and lung metastasis were higher than those in patients with Eneeking stage Ⅰ～Ⅱ,no soft tissue infiltration,and no lung metastasis,with significant differences(t=14.122～171.054,all P<0.05).The 5-year survival rates of patients in the AHA1 mRNA high expression group and low expression group were 36.36%(16/44)and 78.26%(36/46),respectively.The 5-year cumulative survival rate of patients in the AHA1 mRNA high expression group was significantly lower than that in the low expression group(Log-rank χ2=16.081,P<0.05).The 5-year survival rates of patients with high and low expression of LOXL2 mRNA were 34.88%(15/43)and 78.72%(37/47),respectively.The 5-year cumulative survival rate of patients in the LOXL2 mRNA high expression group was significantly lower than that in the low expression group(Log-rank χ2=15.880,P<0.05).Lung metastasis(OR=1.921,P<0.05),Eneeking stage Ⅲ(OR=1.906,P<0.05),AHA1 mRNA high expression(OR=1.405,P<0.05),and LOXL2 mRNA high expression(OR=1.733,P<0.05)were independent risk factors affecting the poor survival prognosis of osteosarcoma patients.Conclusion The expressions of AHA1 mRNA and LOXL2 mRNA in osteosarcoma were increased,and they were correlated with the expression of invasion and metastasis genes,indicating they may be independent risk factors affecting the poor survival and prognosis of osteosarcoma patients.

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Aims and objectives: Salidroside (SAL), an active component isolated from the Chinese plant Rose Rhodiola, has anti-inflammatory, antioxidant, anti-cancer, neuroprotective, and renal protective properties. Atrial fibrosis developed due to angiotensin II (Ang II) plays a crucial function in developing atrial fibrillation (AF). This research investigates the involvement of SAL in AF, its vulnerability to AF, and Ang II-induced inflammatory atrial fibrosis. Methods: Ang II (2 mg/kg/day) was infused underneath the skin into male C57BL/6 mice (8-10 weeks old, n = 40) for four weeks to create the AF model. SAL (50 mg/kg/day) was given intraperitoneally once per day for 28 days. Analyses of morphology, histology, and biochemical were carried out. Transesophageal burst pacing was used in vivo to induce AF. Results: Ang II injection increased mice's heart rate and systolic blood pressure (SBP), whereas SAL treatment was significantly reduced. Ang II infusion increased left atrial diameter (LAD) in mice, which was attenuated after SAL treatment. SAL alone did not affect AF inducibility, but SAL therapy markedly decreased Ang II-induced AF inducibility. Additionally, the expression levels of interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were inhibited with SAL therapy in mice. Compared to the Ang II group, Ang II infusion raised malondialdehyde (MDA) levels and reduced superoxide dismutase (SOD) and catalase (CAT) activity, but SAL therapy altered all of these effects. SAL treatment significantly reduced LOXL2, TGF-ß1, p-Smad2 and p-Smad3 protein expression than the Ang II group mice. Conclusion: SAL inhibits atrial fibrosis and potentially attenuates increased susceptibility to AF by suppressing the LOXL2-TGF-ß1-Smad2/3 pathway.

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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.

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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Animales , Ratones , Humanos , Gefitinib/farmacología , Carcinoma de Células Escamosas/patología , Proteína-Lisina 6-Oxidasa , Ratones SCID , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Proteínas de Unión al ARN/genética , Receptores ErbB , Regulación Neoplásica de la Expresión Génica , Transición Epitelial-Mesenquimal , Línea Celular Tumoral , Péptidos y Proteínas de Señalización Intracelular

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Noninvasive imaging of idiopathic pulmonary fibrosis (IPF) remains a challenge. The aim of this study was to develop an antibody-based radiotracer targeting Lysyl Oxidase-like 2 (LOXL2), an enzyme involved in the fibrogenesis process, for SPECT/CT imaging of pulmonary fibrosis. The bifunctional chelator DOTAGA-PEG4-NH2 was chemoenzymatically conjugated to the murine antibody AB0023 using microbial transglutaminase, resulting in a degree of labeling (number of chelators per antibody) of 2.3. Biolayer interferometry confirmed that the binding affinity of DOTAGA-AB0023 to LOXL2 was preserved with a dissociation constant of 2.45 ± 0.04 nM. DOTAGA-AB0023 was then labeled with 111In and in vivo experiments were carried out in a mice model of progressive pulmonary fibrosis induced by intratracheal administration of bleomycin. [111In]In-DOTAGA-AB0023 was injected in three groups of mice (control, fibrotic, and treated with nintedanib). SPECT/CT images were recorded over 4 days p.i. and an ex vivo biodistribution study was performed by gamma counting. A significant accumulation of the tracer in the lungs of the fibrotic mice was observed at D18 post-bleomycin. Interestingly, the tracer uptake was found selectively upregulated in fibrotic lesions observed on CT scans. Images of mice that received the antifibrotic drug nintedanib from D8 up to D18 showed a decrease in [111In]In-DOTAGA-AB0023 lung uptake associated with a decrease in pulmonary fibrosis measured by CT scan. In conclusion, we report the first radioimmunotracer targeting the protein LOXL2 for nuclear imaging of IPF. The tracer showed promising results in a preclinical model of bleomycin-induced pulmonary fibrosis, with high lung uptake in fibrotic areas, and accounted for the antifibrotic activity of nintedanib.

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Fibrosis Pulmonar Idiopática , Proteína-Lisina 6-Oxidasa , Animales , Ratones , Proteína-Lisina 6-Oxidasa/metabolismo , Distribución Tisular , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/diagnóstico por imagen , Pulmón/metabolismo , Fibrosis , Tomografía Computarizada de Emisión de Fotón Único , Bleomicina , Anticuerpos/metabolismo

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Kidney fibrosis is closely associated with the progression of chronic kidney disease (CKD). Furthermore, copper-containing secretory amine oxidases, such as lysyl oxidase (LOX) and LOX-like 1-4 (LOXL1-4), play pivotal roles in the regulation of extracellular components and facilitate fibrosis. In this study, we investigated the regulation of LOX enzymes in human tubular epithelial HK2 cells to help clarify the role of LOX enzymes in kidney fibrosis. Among 5 LOX enzymes, LOXL2 expression is abundantly expressed in HK2 cells. LOX enzymes inhibitor, ß-aminopropionitrile, suppressed transforming growth factor-ß1 (TGF-ß1)-promoted epithelial-to-mesenchymal transition processes in HK2 cells, indicating that LOX enzymes are involved in TGF-ß1-mediated fibrotic processes. Recent studies suggest that LOX enzymes are secreted into the extracellular spaces by exosomes and promote fibrotic processes. Similar to the previous reports, we observed that exosomes secreted from HK2 cells carry LOXL2 into the extracellular spaces. Furthermore, we determined that N-glycosylation on the asparagine residues plays a key role in LOXL2 secretion. Amino acid mutations in three asparagine residues, which can be glycosylated, suppressed the secretion of mutated LOXL2. Moreover, N-acetylglucosaminyltransferase 5, an enzyme used for the biosynthesis of ß1,6N-acetylglucosamine-branched N-glycans, participated in LOXL2 secretion, and the N-glycosylation inhibitor, glucosamine hydrochloride (GS), inhibited TGF-ß1-mediated LOXL2 secretion and fibrotic processes. Overall, TGF-ß1 promotes LOXL2 secretion and may participate in kidney fibrosis. Our results provide novel insight into the antifibrotic properties of GS that contribute to the inhibition of CKD progression.

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Insuficiencia Renal Crónica , Factor de Crecimiento Transformador beta1 , Humanos , Factor de Crecimiento Transformador beta1/metabolismo , Glicosilación , Glucosamina , Asparagina , Fibrosis , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo

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Hypoxia was proved to enhance the angiogenesis of stem cells. However, the mechanism of the angiogenic potential in hypoxia-pretreated dental pulp stem cells (DPSCs) is poorly understood. We previously confirmed that hypoxia enhances the angiogenic potential of DPSC-derived exosomes with upregulation of lysyl oxidase-like 2 (LOXL2). Therefore, our study aimed to illuminate whether these exosomes promote angiogenesis via transfer of LOXL2. Exosomes were generated from hypoxia-pretreated DPSCs (Hypo-Exos) stably silencing LOXL2 after lentiviral transfection and characterized with transmission electron microscopy, nanosight and Western blot. The efficiency of silencing was verified using quantitative real-time PCR (qRT-PCR) and Western blot. CCK-8, scratch and transwell assays were conducted to explore the effects of LOXL2 silencing on DPSCs proliferation and migration. Human umbilical vein endothelial cells (HUVECs) were co-incubated with exosomes to assess the migration and angiogenic capacity through transwell and matrigel tube formation assays. The relative expression of angiogenesis-associated genes was characterized by qRT-PCR and Western blot. LOXL2 was successfully silenced in DPSCs and inhibited DPSC proliferation and migration. LOXL2 silencing in Hypo-Exos partially reduced promotion of HUVEC migration and tube formation and inhibited the expression of angiogenesis-associated genes. Thus, LOXL2 is one of various factors mediating the angiogenic effects of Hypo-Exos.

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Exosomas , Humanos , Exosomas/metabolismo , Proliferación Celular/genética , Neovascularización Fisiológica/genética , Células Endoteliales de la Vena Umbilical Humana , Células Madre , Aminoácido Oxidorreductasas/genética

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The ductus arteriosus (DA) connects the aorta to the pulmonary artery (PA), directing placentally oxygenated blood away from the developing lungs. High pulmonary vascular resistance and low systemic vascular resistance facilitate shunting of blood in utero from the pulmonary to the systemic circulation through the widely patent DA, thereby optimizing fetal oxygen (O2) delivery. With the transition from fetal (hypoxia) to neonatal (normoxia) oxygen conditions, the DA constricts while the PA dilates. This process often fails in prematurity, promoting congenital heart disease. Impaired O2-responsivness in the DA promotes persistent ductus arteriosus (PDA), the most common form of congenital heart disease. Knowledge of DA oxygen sensing has greatly advanced in the past few decades, however we still lack a complete understanding of the sensing mechanism. The genomic revolution of the past two decades has facilitated unprecedented discovery in every biological system. This review will demonstrate how multiomic integration of data generated from the DA can breathe new life into our understanding of the DA's oxygen response.

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Conducto Arterioso Permeable , Conducto Arterial , Cardiopatías Congénitas , Recién Nacido , Humanos , Conducto Arterial/fisiología , Oxígeno , Recien Nacido Prematuro

RESUMEN

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Asunto(s)

Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Pronóstico , Biomarcadores , Línea Celular Tumoral , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Microambiente Tumoral , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo

RESUMEN

Lysyl oxidase-like 2 (LOXL2) has been recognized as an attractive drug target for anti-fibrotic and anti-tumor therapies. However, the structure-based drug design of LOXL2 has been very challenging due to the lack of structural information of the catalytically-competent LOXL2. In this study; we generated a 3D-predicted structure of the C-terminal amine oxidase domain of LOXL2 containing the lysine tyrosylquinone (LTQ) cofactor from the 2.4Å crystal structure of the Zn2+-bound precursor (lacking LTQ; PDB:5ZE3); this was achieved by molecular modeling and molecular dynamics simulation based on our solution studies of a mature LOXL2 that is inhibited by 2-hydrazinopyridine. The overall structures of the 3D-modeled mature LOXL2 and the Zn2+-bound precursor are very similar (RMSD = 1.070Å), and disulfide bonds are conserved. The major difference of the mature and the precursor LOXL2 is the secondary structure of the pentapeptide (His652-Lys653-Ala654-Ser655-Phe656) containing Lys653 (the precursor residue of the LTQ cofactor). We anticipate that this peptide is flexible in solution to accommodate the conformation that enables the LTQ cofactor formation as opposed to the ß-sheet observed in 5ZE3. We discuss the active site environment surrounding LTQ and Cu2+ of the 3D-predicted structure.

Asunto(s)

Proteína-Lisina 6-Oxidasa , Quinonas , Proteína-Lisina 6-Oxidasa/química , Modelos Moleculares , Quinonas/química , Monoaminooxidasa , Aminas , Aminoácido Oxidorreductasas/química

RESUMEN

Lysyl oxidase-2 (LOXL2) is a Cu2+ and lysine tyrosylquinone (LTQ)-dependent amine oxidase that catalyzes the oxidative deamination of peptidyl lysine and hydroxylysine residues to promote crosslinking of extracellular matrix proteins. LTQ is post-translationally derived from Lys653 and Tyr689, but its biogenesis mechanism remains still elusive. A 2.4 Å Zn2+-bound precursor structure lacking LTQ (PDB:5ZE3) has become available, where Lys653 and Tyr689 are 16.6 Å apart, thus a substantial conformational rearrangement is expected to take place for LTQ biogenesis. However, we have recently shown that the overall structures of the precursor (no LTQ) and the mature (LTQ-containing) LOXL2s are very similar and disulfide bonds are conserved. In this study, we aim to gain insights into the spatial arrangement of LTQ and the active site Cu2+ in the mature LOXL2 using a recombinant LOXL2 that is inhibited by 2-hydrazinopyridine (2HP). Comparative UV-vis and resonance Raman spectroscopic studies of the 2HP-inhibited LOXL2 and the corresponding model compounds and an EPR study of the latter support that 2HP-modified LTQ serves as a tridentate ligand to the active site Cu2. We propose that LTQ resides within 2.9 Å of the active site of Cu2+ in the mature LOXL2, and both LTQ and Cu2+ are solvent-exposed.

Asunto(s)

Lisina , Proteína-Lisina 6-Oxidasa , Lisina/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Dominio Catalítico , Quinonas/química

RESUMEN

Lysyl-oxidase like-2 (LOXL2) regulates extracellular matrix remodeling and promotes tumor invasion and metastasis. Altered metabolism is a core hallmark of cancer, however, it remains unclear whether and how LOXL2 contributes to tumor metabolism. Here, we found that LOXL2 and its catalytically inactive L2Δ13 splice variant boost glucose metabolism of esophageal tumor cells, facilitate tumor cell proliferation and promote tumor development in vivo. Consistently, integrated transcriptomic and metabolomic analysis of a knock-in mouse model expressing L2Δ13 gene revealed that LOXL2/L2Δ13 overexpression perturbs glucose and lipid metabolism. Mechanistically, we identified aldolase A, glyceraldehyde-3-phosphate dehydrogenase and enolase as glycolytic proteins that interact physically with LOXL2 and L2Δ13. In the case of aldolase A, LOXL2/L2Δ13 stimulated its mobilization from the actin cytoskeleton to enhance aldolase activity during malignant transformation. Using stable isotope labeling of amino acids in cell culture (SILAC) followed by proteomic analysis, we identified LOXL2 and L2Δ13 as novel deacetylases that trigger metabolic reprogramming. Both LOXL2 and L2Δ13 directly catalyzed the deacetylation of aldolase A at K13, resulting in enhanced glycolysis which subsequently reprogramed tumor metabolism and promoted tumor progression. High level expression of LOXL2/L2Δ13 combined with decreased acetylation of aldolase-K13 predicted poor clinical outcome in patients with esophageal cancer. In summary, we have characterized a novel molecular mechanism that mediates the pro-tumorigenic activity of LOXL2 independently of its classical amine oxidase activity. These findings may enable the future development of therapeutic agents targeting the metabolic machinery via LOXL2 or L2Δ13. HIGHLIGHT OF THE STUDY: LOXL2 and its catalytically inactive isoform L2Δ13 function as new deacetylases to promote metabolic reprogramming and tumor progression in esophageal cancer by directly activating glycolytic enzymes such as aldolase A.

RESUMEN

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-ß (TGF-ß) 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-ß/Smad signalling pathway.