RESUMEN
Few effective treatments are available for small cell lung cancer (SCLC), indicating the need to explore new therapeutic options. Here, we focus on an antibody-drug conjugate (ADC) targeting the L1 cell adhesion molecule (L1CAM). Several publicly available databases reveal that (1) L1CAM is expressed at higher levels in SCLC cell lines and tissues than in those of lung adenocarcinoma and (2) the expression levels of L1CAM are slightly higher in SCLC tissues than in adjacent normal tissues. We conducted a series of in vitro experiments using an anti-L1CAM monoclonal antibody (termed HSL175, developed in-house) and the recombinant protein DT3C, which consists of diphtheria toxin lacking the receptor-binding domain but containing the C1, C2, and C3 domains of streptococcal protein G. Our HSL175-DT3C conjugates theoretically kill cells only when the conjugates are internalized by the target (L1CAM-positive) cells through antigen-antibody interaction. The conjugates (an ADC analog) were effective against two SCLC-N (NEUROD1 dominant) cell lines, Lu-135 and STC-1, resulting in decreased viability. In addition, L1CAM silencing rendered the two cell lines resistant to HSL175-DT3C conjugates. These findings suggest that an ADC consisting of a humanized monoclonal antibody based on HSL175 and a potent anticancer drug would be effective against SCLC-N cells.
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Anticuerpos Monoclonales , Inmunoconjugados , Neoplasias Pulmonares , Molécula L1 de Adhesión de Célula Nerviosa , Carcinoma Pulmonar de Células Pequeñas , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/inmunología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/inmunología , Línea Celular Tumoral , Anticuerpos Monoclonales/farmacología , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/inmunología , Inmunoconjugados/farmacología , Antineoplásicos Inmunológicos/farmacologíaRESUMEN
OBJECTIVE: Prognostic stratification of endometrial cancer involves the assessment of stage, uterine risk factors, and molecular classification. This process can be further refined through annotation of prognostic biomarkers, notably L1 cell adhesion molecule (L1CAM) and hormonal receptors. Loss of asparaginase-like protein 1 (ASRGL1) has been shown to correlate with poor outcome in endometrial cancer. Our objective was to assess prognostication of endometrial cancer by ASRGL1 in conjunction with other available methodologies. STUDY DESIGN: This was a retrospective study of patients who underwent primary treatment at a single tertiary center. Tumors were molecularly classified by the Proactive Molecular Risk Classifier for Endometrial Cancer. Expression of ASRGL1, L1CAM, estrogen receptor, and progesterone receptor was determined by immunohistochemistry. ASRGL1 expression intensity was scored into four classes. RESULTS: In a cohort of 775 patients, monitored for a median time of 81 months, ASRGL1 expression intensity was related to improved disease-specific survival in a dose-dependent manner (P < 0.001). Low expression levels were associated with stage II-IV disease and presence of uterine factors, i.e. high grade, lymphovascular space invasion, and deep myometrial invasion (P < 0.001 for all). Among the molecular subgroups, low expression was most prevalent in p53 abnormal carcinomas (P < 0.001). Low ASRGL1 was associated with positive L1CAM expression and negative estrogen and progesterone receptor expression (P < 0.001 for all). After adjustment for stage and uterine factors, strong ASRGL1 staining intensity was associated with a lower risk for cancer-related deaths (hazard ratio 0.56, 95 % confidence interval 0.32-0.97; P = 0.038). ASRGL1 was not associated with the outcome when adjusted for stage, molecular subgroups, L1CAM, and hormonal receptors. When analyzed separately within the different molecular subgroups, ASRGL1 showed an association with disease-specific survival specifically in "no specific molecular profile" subtype carcinomas (P < 0.001). However, this association became nonsignificant upon controlling for confounders. CONCLUSIONS: Low ASRGL1 expression intensity correlates with poor survival in endometrial cancer. ASRGL1 contributes to more accurate prognostication when controlled for stage and uterine factors. However, when adjusted for stage and other biomarkers, including molecular subgroups, ASRGL1 does not improve prognostic stratification.
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Asparaginasa , Biomarcadores de Tumor , Neoplasias Endometriales , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Persona de Mediana Edad , Asparaginasa/análisis , Autoantígenos , Biomarcadores de Tumor/metabolismo , Neoplasias Endometriales/patología , Neoplasias Endometriales/mortalidad , Neoplasias Endometriales/metabolismo , Proteínas de Neoplasias/metabolismo , Estadificación de Neoplasias , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/análisis , Pronóstico , Receptores de Progesterona/metabolismo , Estudios RetrospectivosRESUMEN
OBJECTIVE: To assess the risk stratification of clinicopathologically and molecularly classified endometrial cancer based on estrogen receptor (ER) and L1 cell adhesion molecule (L1CAM) expression. METHODS: This was a retrospective study of patients who underwent primary treatment at a single tertiary center. Carcinomas were classified into 5 clinicopathological risk groups, as per European guidelines. Immunohistochemistry and polymerase-ϵ sequencing were conducted for molecular classification and determination of ER and L1CAM expression. RESULTS: Data from 1044 patients were analyzed. The median follow-up was 67.5 months. In univariable analyses, ER expression correlated with improved disease-specific survival (DSS) in the "no specific molecular profile" (NSMP) (P < 0.001) and mismatch repair deficient (MMRd) (P = 0.002) subgroups. Negative L1CAM expression was associated with enhanced DSS in the NSMP subgroup alone (P < 0.001). ER (hazard ratio [HR] 0.18), but not L1CAM, exhibited prognostic significance within NSMP when controlling for parameters available at the time of diagnosis (tumor histotype, grade, age). ER and L1CAM were not independently associated with DSS within NSMP when controlling for parameters available after surgery (clinicopathological risk groups, age, adjuvant therapy). However, in high-risk-advanced-metastatic cases, both ER (HR 0.26) and L1CAM (HR 3.9) independently correlated with DSS. Similarly, within MMRd, ER was associated with improved DSS in high-risk-advanced-metastatic carcinomas (HR 0.42). CONCLUSION: The prognostic significance of ER and L1CAM varies across clinicopathological risk groups and molecular subgroups of endometrial cancer. Notably, risk assessment for high-risk-advanced-metastatic NSMP and MMRd subtype carcinomas can be refined by ER status.
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Neoplasias Endometriales , Molécula L1 de Adhesión de Célula Nerviosa , Receptores de Estrógenos , Humanos , Femenino , Neoplasias Endometriales/patología , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/genética , Molécula L1 de Adhesión de Célula Nerviosa/biosíntesis , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Estudios Retrospectivos , Persona de Mediana Edad , Receptores de Estrógenos/metabolismo , Receptores de Estrógenos/biosíntesis , Anciano , Adulto , Pronóstico , Anciano de 80 o más Años , InmunohistoquímicaRESUMEN
Polysialylated neural cell adhesion molecule (PSA-NCAM) is expressed in the developing central nervous system (CNS) and plays an important role in neurogenesis. Organophosphorus (OP) toxins, including diazinon (DZN), cause oxidative stress (OS) and damage the CNS. Resveratrol (RV), with its antioxidant effect, leads to the reduction of OS. Therefore, this research was conducted with the aim of the effect of RVon the expression of PSA-NCAM in the hippocampus (HPC) of rat fetuses treated with DZN. In this study, 24 female Wistar rats were divided into 4 groups (n = 6): Control, DZN (40 mg/kg), RV(10 mg/kg), and DZN + RV(40 mg/kg + 10 mg/kg) after confirming they were pregnant. On the 21st day of pregnancy, the mother mice were anesthetized with ketamine and xylazine, and the fetuses were removed; after anesthesia, their brains were removed for immunohistochemistry and western blot (WB) technique. The results of the study showed that in the group receiving DZN, the level of PSA-NCAM protein expression decreased significantly compared to the control group, and the group receiving RV with its antioxidant property increased the expression of PSA-NCAM protein compared to the DZN group. All in all, the exposure of pregnant mice to DZN causes disorders in the CNS, especially the level of PSA-NCAM protein expression in the HPC of fetuses, and the use of RV as an antioxidant by pregnant mothers neutralizes the effects of DZN in the HPC of their fetuses.
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Antioxidantes , Diazinón , Hipocampo , Molécula L1 de Adhesión de Célula Nerviosa , Ratas Wistar , Resveratrol , Ácidos Siálicos , Animales , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Femenino , Diazinón/toxicidad , Embarazo , Resveratrol/farmacología , Antioxidantes/farmacología , Antioxidantes/metabolismo , Ácidos Siálicos/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Ratas , Feto/efectos de los fármacos , Feto/metabolismo , Estrés Oxidativo/efectos de los fármacos , Insecticidas/toxicidadRESUMEN
Nephrogenic adenoma (NA) is a benign, reactive lesion seen predominantly in the urinary bladder and often associated with antecedent inflammation, instrumentation, or an operative history. Its histopathologic diversity can create diagnostic dilemmas and pathologists use morphologic evaluation along with available immunohistochemical (IHC) markers to navigate these challenges. IHC assays currently do not designate or specify NA's potential putative cell of origin. Leveraging single-cell RNA-sequencing technology, we nominated a principal (P) cell-collecting duct marker, L1 cell adhesion molecule (L1CAM), as a potential biomarker for NA. IHC characterization revealed L1CAM to be positive in all 35 (100%) patient samples of NA; negative expression was seen in the benign urothelium, benign prostatic glands, urothelial carcinoma (UCA) in situ, prostatic adenocarcinoma, majority of high-grade UCA, and metastatic UCA. In the study, we also used single-cell RNA sequencing to nominate a novel compendium of biomarkers specific for the proximal tubule, loop of Henle, and distal tubule (DT) (including P and intercalated cells), which can be used to perform nephronal mapping using RNA in situ hybridization and IHC technology. Employing this technique on NA we found enrichment of both the P-cell marker L1CAM and, the proximal tubule type-A and -B cell markers, PDZKI1P1 and PIGR, respectively. The cell-type markers for the intercalated cell of DTs (LINC01187 and FOXI1), and the loop of Henle (UMOD and IRX5), were found to be uniformly absent in NA. Overall, our findings show that based on cell type-specific implications of L1CAM expression, the shared expression pattern of L1CAM between DT P cells and NA. L1CAM expression will be of potential value in assisting surgical pathologists toward a diagnosis of NA in challenging patient samples.
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Adenoma , Biomarcadores de Tumor , Molécula L1 de Adhesión de Célula Nerviosa , Humanos , Molécula L1 de Adhesión de Célula Nerviosa/análisis , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/biosíntesis , Adenoma/patología , Adenoma/metabolismo , Masculino , Biomarcadores de Tumor/análisis , Femenino , Anciano , Persona de Mediana Edad , Inmunohistoquímica , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Nefronas/patología , Nefronas/metabolismo , AdultoRESUMEN
L1CAM-positive extracellular vesicles (L1EV) are an emerging biomarker that may better reflect ongoing neuronal damage than other blood-based biomarkers. The physiological roles and regulation of L1EVs and their small RNA cargoes following stroke is unknown. We sought to characterize L1EV small RNAs following stroke and assess L1EV RNA signatures for diagnosing stroke using weighted gene co-expression network analysis and random forest (RF) machine learning algorithms. Interestingly, small RNA sequencing of plasma L1EVs from patients with stroke and control patients (n = 28) identified micro(mi)RNAs known to be enriched in the brain. Weighted gene co-expression network analysis (WGCNA) revealed small RNA transcript modules correlated to diagnosis, initial NIH stroke scale, and age. L1EV RNA signatures associated with the diagnosis of AIS were derived from WGCNA and RF classification. These small RNA signatures demonstrated a high degree of accuracy in the diagnosis of AIS with an area under the curve (AUC) of the signatures ranging from 0.833 to 0.932. Further work is necessary to understand the role of small RNA L1EV cargoes in the response to brain injury, however, this study supports the utility of L1EV small RNA signatures as a biomarker of stroke.
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Biomarcadores , Vesículas Extracelulares , Accidente Cerebrovascular Isquémico , Molécula L1 de Adhesión de Célula Nerviosa , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Masculino , Accidente Cerebrovascular Isquémico/genética , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/sangre , Accidente Cerebrovascular Isquémico/diagnóstico , Molécula L1 de Adhesión de Célula Nerviosa/genética , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Femenino , Anciano , Biomarcadores/sangre , Persona de Mediana Edad , Aprendizaje Automático , MicroARNs/genética , MicroARNs/sangre , MicroARNs/metabolismoRESUMEN
Isolation of neuron-derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)-specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single-EV techniques to establish the neuronal origin and determine the abundance of L1CAM-positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co-expressed on single-EVs with the neuronal proteins ß-III-tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM-positive EVs. Levels of L1CAM-positive EVs carrying the neuronal proteins VAMP2 and ß-III-tubulin range from 30% to 63%, in contrast to 0.8%-3.9% of L1CAM-negative EVs. Plasma fluid-phase L1CAM does not bind to single-EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function.
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Biomarcadores , Vesículas Extracelulares , Molécula L1 de Adhesión de Célula Nerviosa , Neuronas , Proteína 2 de Membrana Asociada a Vesículas , Humanos , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Vesículas Extracelulares/metabolismo , Biomarcadores/metabolismo , Biomarcadores/sangre , Neuronas/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Extracellular vesicles (EVs) carry disease-specific molecular profiles, demonstrating massive potential in biomarker discovery. In this study, we developed an integrated biochip platform, termed EVID-biochip (EVs identification and detection biochip), which integrates in situ electrochemical protein detection with on-chip antifouling-immunomagnetic beads modified with CD81 antibodies and zwitterion molecules, enabling efficient isolation and detection of neuronal EVs. The capability of the EVID-biochip to isolate common EVs and detect neuronal EVs associated with Parkinson's disease in human serum is successfully demonstrated, using the transmembrane protein L1-cell adhesion molecule (L1CAM) as a target biomarker. The EVID-biochip exhibited high efficiency and specificity for the detection of L1CAM with a sensitivity of 1 pg/mL. Based on the validation of 76 human serum samples, for the first time, this study discovered that the level of L1CAM/neuronal EV particles in serum could serve as a reliable indicator to distinguish Parkinson's disease from control groups with AUC = 0.973. EVID-biochip represents a reliable and rapid liquid biopsy platform for the analysis of complex biofluids offering EVs isolation and detection in a single chip, requiring a small sample volume (300 µL) and an assay time of 1.5 h. This approach has the potential to advance the diagnosis and biomarker discovery of various neurological disorders and other diseases.
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Biomarcadores , Vesículas Extracelulares , Molécula L1 de Adhesión de Célula Nerviosa , Enfermedad de Parkinson , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/sangre , Enfermedad de Parkinson/diagnóstico , Humanos , Vesículas Extracelulares/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Biomarcadores/sangre , Masculino , Femenino , Biopsia Líquida/métodos , Anciano , Persona de Mediana EdadRESUMEN
Krüppel-like factor 12 (KLF12) has been characterized as a transcriptional repressor, and previous studies have unveiled its roles in angiogenesis, neural tube defect, and natural killer (NK) cell proliferation. However, the contribution of KLF12 to cancer treatment remains undefined. Here, we show that KLF12 is downregulated in various cancer types, and KLF12 downregulation promotes cisplatin resistance and cancer metastasis in esophageal squamous cell carcinoma (ESCC). Mechanistically, KLF12 binds to the promoters of L1 Cell Adhesion Molecule (L1CAM) and represses its expression. Depletion of L1CAM abrogates cisplatin resistance and cancer metastasis caused by KLF12 loss. Moreover, the E3 ubiquitin ligase tripartite motif-containing 27 (TRIM27) binds to the N-terminal region of KLF12 and ubiquitinates KLF12 at K326 via K33-linked polyubiquitination. Notably, TRIM27 depletion enhances the transcriptional activity of KLF12 and consequently inhibits L1CAM expression. Overall, our study elucidated a novel regulatory mechanism involving TRIM27, KLF12 and L1CAM, which plays a substantial role in cisplatin resistance and cancer metastasis in ESCC. Targeting these genes could be a promising approach for ESCC treatment.
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Cisplatino , Resistencia a Antineoplásicos , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel , Molécula L1 de Adhesión de Célula Nerviosa , Humanos , Cisplatino/farmacología , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/patología , Carcinoma de Células Escamosas de Esófago/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/genética , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/genética , Línea Celular Tumoral , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Metástasis de la Neoplasia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/efectos de los fármacos , Ratones , Proliferación Celular/efectos de los fármacos , Proteínas de Motivos Tripartitos , Proteínas de Unión al ADN , Proteínas NuclearesRESUMEN
L1 syndrome, a neurological disorder with an X-linked inheritance pattern, mainly results from mutations occurring in the L1 cell adhesion molecule (L1CAM) gene. The L1CAM molecule, belonging to the immunoglobulin (Ig) superfamily of neurocyte adhesion molecules, plays a pivotal role in facilitating intercellular signal transmission across membranes and is indispensable for proper neuronal development and function. This study identified a rare missense variant (c.1759G>C; p.G587R) in the L1CAM gene within a male fetus presenting with hydrocephalus. Due to a lack of functional analysis, the significance of the L1CAM mutation c.1759G>C (p.G587R) remains unknown. We aimed to perform further verification for its pathogenicity. Blood samples were obtained from the proband and his parents for trio clinical exome sequencing and mutation analysis. Expression level analysis was conducted using western blot techniques. Immunofluorescence was employed to investigate L1CAM subcellular localization, while cell aggregation and cell scratch assays were utilized to assess protein function. The study showed that the mutation (c.1759G>C; p.G587R) affected posttranslational glycosylation modification and induced alterations in the subcellular localization of L1-G587R in the cells. It resulted in the diminished expression of L1CAM on the cell surface and accumulation in the endoplasmic reticulum. The p.G587R altered the function of L1CAM protein and reduced homophilic adhesion capacity of proteins, leading to impaired adhesion and migration of proteins between cells. Our findings provide first biological evidence for the association between the missense mutation (c.1759G>c; p.G587R) in the L1CAM gene and L1 syndrome, confirming the pathogenicity of this missense mutation.
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Mutación Missense , Molécula L1 de Adhesión de Célula Nerviosa , Humanos , Masculino , Células HEK293 , Hidrocefalia/genética , Hidrocefalia/metabolismo , Hidrocefalia/patología , Molécula L1 de Adhesión de Célula Nerviosa/genética , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Linaje , Recién NacidoRESUMEN
AIMS: Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs. METHODOLOGY: Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization. RESULTS: PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations. CONCLUSIONS: In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).
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Diferenciación Celular , Pulpa Dental , Citometría de Flujo , Pulpa Dental/citología , Humanos , Células Cultivadas , Células-Madre Neurales , Ácidos Siálicos , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Separación Inmunomagnética , NeuronasRESUMEN
Melanoma, the deadliest type of skin cancer, has a high propensity to metastasize to other organs, including the brain, lymph nodes, lungs, and bones. While progress has been made in managing melanoma with targeted and immune therapies, many patients do not benefit from these current treatment modalities. Tumor cell migration is the initial step for invasion and metastasis. A better understanding of the molecular mechanisms underlying metastasis is crucial for developing therapeutic strategies for metastatic diseases, including melanoma. The cell adhesion molecule L1CAM (CD171, in short L1) is upregulated in many human cancers, enhancing tumor cell migration. Earlier studies showed that the small-molecule antagonistic mimetics of L1 suppress glioblastoma cell migration in vitro. This study aims to evaluate if L1 mimetic antagonists can inhibit melanoma cell migration in vitro and in vivo. We showed that two antagonistic mimetics of L1, anagrelide and 2-hydroxy-5-fluoropyrimidine (2H5F), reduced melanoma cell migration in vitro. In in vivo allograft studies, only 2H5F-treated female mice showed a decrease in tumor volume.
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Movimiento Celular , Melanoma , Molécula L1 de Adhesión de Célula Nerviosa , Animales , Femenino , Humanos , Ratones , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Quinazolinas/farmacología , Quinazolinas/uso terapéuticoRESUMEN
TRPV2 voltage-insensitive, calcium-permeable ion channels play important roles in cancer progression, immune response, and neuronal development. Despite TRPV2's physiological impact, underlying endogenous proteins mediating TRPV2 responses and affected signaling pathways remain elusive. Using quantitative peroxidase-catalyzed (APEX2) proximity proteomics we uncover dynamic changes in the TRPV2-proximal proteome and identify calcium signaling and cell adhesion factors recruited to the molecular channel neighborhood in response to activation. Quantitative TRPV2 proximity proteomics further revealed activation-induced enrichment of protein clusters with biological functions in neural and cellular projection. We demonstrate a functional connection between TRPV2 and the neural immunoglobulin cell adhesion molecules NCAM and L1CAM. NCAM and L1CAM stimulation robustly induces TRPV2 [Ca2+]I flux in neuronal PC12 cells and this TRPV2-specific [Ca2+]I flux requires activation of the protein kinase PKCα. TRPV2 expression directly impacts neurite lengths that are modulated by NCAM or L1CAM stimulation. Hence, TRPV2's calcium signaling plays a previously undescribed, yet vital role in cell adhesion, and TRPV2 calcium flux and neurite development are intricately linked via NCAM and L1CAM cell adhesion proteins.
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Calcio , Molécula L1 de Adhesión de Célula Nerviosa , Moléculas de Adhesión de Célula Nerviosa , Proyección Neuronal , Proteoma , Canales Catiónicos TRPV , Animales , Humanos , Ratas , Calcio/metabolismo , Señalización del Calcio , Adhesión Celular , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuritas/metabolismo , Células PC12 , Proteína Quinasa C-alfa/metabolismo , Proteoma/metabolismo , Canales Catiónicos TRPV/metabolismo , Antígeno CD56/metabolismoRESUMEN
Neural cell adhesion molecule L1 (L1CAM) is a cell-surface glycoprotein involved in cancer occurrence and migration. Up to today, L1CAM-targeted therapy appeared limited efficacy in clinical trials although quite a few attempts by monoclonal antibody (mAb) or chimeric antigen receptor T-cell therapy (CAR-T) have been reported. Therefore, the development of new effective therapies targeting L1CAM is highly desirable. It has been demonstrated that T cell-engaging bispecific antibody (TCE) plays an effective role in cancer immunotherapy by redirecting the cytotoxic activity of CD3+ T cells to tumor cells, resulting in tumor cell death. In this study, we designed and characterized a novel bispecific antibody (CE7-TCE) based on the IgG-(L)-ScFv format, which targets L1CAM and CD3 simultaneously. In vitro, CE7-TCE induced specific killing of L1CAM-positive tumor cells through T cells. In vivo, CE7-TCE inhibited tumor growth in human peripheral blood mononuclear cell/tumor cell co-grafting models. To overcome the adaptive immune resistance (AIR) that impairs the efficacy of TCEs, we conducted a combination therapy of CE7-TCE with Pembrolizumab (anti-PD1 mAb), which enhanced the anti-tumor activity of CE7-TCE. Our results confirmed the feasibility of using L1CAM as a TCE target for the treatment of solid tumors and revealed the therapeutic potential of CE7-TCE combined with immune checkpoint inhibitors.
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Anticuerpos Biespecíficos , Molécula L1 de Adhesión de Célula Nerviosa , Linfocitos T , Animales , Femenino , Humanos , Ratones , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/inmunología , Antineoplásicos Inmunológicos/farmacología , Complejo CD3/inmunología , Línea Celular Tumoral , Inmunoterapia/métodos , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Molécula L1 de Adhesión de Célula Nerviosa/inmunología , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The X-chromosome-linked cell adhesion molecule L1 (L1CAM), a glycoprotein mainly expressed by neurons in the central and peripheral nervous systems, has been implicated in many neural processes, including neuronal migration and survival, neuritogenesis, synapse formation, synaptic plasticity and regeneration. L1 consists of extracellular, transmembrane and cytoplasmic domains. Proteolytic cleavage of L1's extracellular and transmembrane domains by different proteases generates several L1 fragments with different functions. We found that myelin basic protein (MBP) cleaves L1's extracellular domain, leading to enhanced neuritogenesis and neuronal survival in vitro. To investigate in vivo the importance of the MBP-generated 70 kDa fragment (L1-70), we generated mice with an arginine to alanine substitution at position 687 (L1/687), thereby disrupting L1's MBP cleavage site and obliterating L1-70. Young adult L1/687 males showed normal anxiety and circadian rhythm activities but enhanced locomotion, while females showed altered social interactions. Older L1/687 males were impaired in motor coordination. Furthermore, L1/687 male and female mice had a larger hippocampus, with more neurons in the dentate gyrus and more proliferating cells in the subgranular layer, while the thickness of the corpus callosum and the size of lateral ventricles were normal. In summary, subtle mutant morphological changes result in subtle behavioral changes.
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Encéfalo , Molécula L1 de Adhesión de Célula Nerviosa , Animales , Molécula L1 de Adhesión de Célula Nerviosa/genética , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Ratones , Masculino , Femenino , Encéfalo/metabolismo , Fibronectinas/metabolismo , Fibronectinas/genética , Mutación , Conducta Animal , Dominios Proteicos , Neuronas/metabolismo , Hipocampo/metabolismo , Ratones Endogámicos C57BLRESUMEN
Renal low-grade oncocytic tumor (LOT) is a recently recognized renal cell neoplasm designated within the "other oncocytic tumors" category in the 2022 World Health Organization classification system. Although the clinicopathologic, immunohistochemical, and molecular features reported for LOT have been largely consistent, the data are relatively limited. The morphologic overlap between LOT and other low-grade oncocytic neoplasms, particularly eosinophilic chromophobe renal cell carcinoma (E-chRCC), remains a controversial area in renal tumor classification. To address this uncertainty, we characterized and compared large cohorts of LOT (n = 67) and E-chRCC (n = 69) and revealed notable differences between the 2 entities. Clinically, LOT predominantly affected women, whereas E-chRCC showed a male predilection. Histologically, although almost all LOTs were dominated by a small-nested pattern, E-chRCC mainly showed solid and tubular architectures. Molecular analysis revealed that 87% of LOT cases harbored mutations in the tuberous sclerosis complex (TSC)-mTOR complex 1 (mTORC1) pathway, most frequently in MTOR and RHEB genes; a subset of LOT cases had chromosomal 7 and 19q gains. In contrast, E-chRCC lacked mTORC1 mutations, and 60% of cases displayed chromosomal losses characteristic of chRCC. We also explored the cell of origin for LOT and identified L1 cell adhesion molecule (L1CAM), a collecting duct and connecting tubule principal cell marker, as a highly sensitive and specific ancillary test for differentiating LOT from E-chRCC. This distinctive L1CAM immunohistochemical labeling suggests the principal cells as the cell of origin for LOT, unlike the intercalated cell origin of E-chRCC and oncocytoma. The ultrastructural analysis of LOT showed normal-appearing mitochondria and intracytoplasmic lumina with microvilli, different from what has been described for chRCC. Our study further supports LOT as a unique entity with a benign clinical course. Based on the likely cell of origin and its clinicopathologic characteristics, we propose that changing the nomenclature of LOT to "Oncocytic Principal Cell Adenoma of the Kidney" may be a better way to define and describe this entity.
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Adenoma Oxifílico , Biomarcadores de Tumor , Carcinoma de Células Renales , Neoplasias Renales , Molécula L1 de Adhesión de Célula Nerviosa , Humanos , Neoplasias Renales/patología , Neoplasias Renales/genética , Neoplasias Renales/química , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/química , Femenino , Masculino , Persona de Mediana Edad , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Molécula L1 de Adhesión de Célula Nerviosa/genética , Molécula L1 de Adhesión de Célula Nerviosa/análisis , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Anciano , Adulto , Adenoma Oxifílico/patología , Adenoma Oxifílico/genética , Diagnóstico Diferencial , Anciano de 80 o más Años , Inmunohistoquímica , Clasificación del Tumor , MutaciónRESUMEN
OBJECTIVE: We investigate the prognostic role of ß-catenin and L1 neuronal cell-adhesion molecule (L1CAM) according to risk groups in endometrial carcinomas (EC). METHODS: A total of 335 EC patients were classified according to the Proactive Molecular Risk Classifier for Endometrial Cancer. We evaluated the expression of ß-catenin and L1CAM using immunohistochemistry, and their association with clinicopathological characteristics and survival. RESULTS: The expressions of ß-catenin and L1CAM were observed in 10.4% of all patients, respectively, and showed mutually exclusive pattern. While ß-catenin expression was associated with endometrioid histology (p = 0.035) and low tumor grade (p = 0.045), L1CAM expression was associated with non-endometrioid histology (p < 0.001), high tumor grade (p < 0.001), lymphovascular space invasion (p = 0.006), and advanced International Federation of Gynecology and Obstetrics (FIGO) stage (p = 0.001). ß-catenin expression was most frequent in the no specific molecular (NSMP) group (26/35, 74.3%), followed by the DNA polymerase-ε-mutated (POLE-mut) (6/35, 17.1%), and mismatch repair-deficiency (dMMR) (3/35, 8.6%). L1CAM expression was most frequent in the p53-abnormal group (22/35, 62.9%), followed by the NSMP (6/35, 17.1%), dMMR (4/35, 11.4%), and POLE-mut (3/35, 8.6%). Although both markers did not show statistical significance in multivariate analysis for both progression-free survival (PFS) and overall survival in entire cohort, ß-catenin positivity was identified as the sole factor associated with worse PFS in the high-intermediate risk subgroup (p = 0.001). CONCLUSION: The expression of nuclear ß-catenin may serve as a potential biomarker for predicting recurrence and guiding therapeutic strategies in high-intermediate risk EC patients.
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Neoplasias Endometriales , Molécula L1 de Adhesión de Célula Nerviosa , beta Catenina , Humanos , Femenino , Neoplasias Endometriales/patología , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/genética , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/biosíntesis , Molécula L1 de Adhesión de Célula Nerviosa/genética , beta Catenina/metabolismo , beta Catenina/biosíntesis , beta Catenina/genética , Persona de Mediana Edad , Anciano , Pronóstico , Adulto , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Anciano de 80 o más Años , Carcinoma Endometrioide/patología , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/genética , Clasificación del Tumor , Estadificación de NeoplasiasRESUMEN
Melanoma incidence and mortality rates are historically higher for men than women. Although emerging studies have highlighted tumorigenic roles for the male sex hormone androgen and its receptor (AR) in melanoma, cellular and molecular mechanisms underlying these sex-associated discrepancies are poorly defined. Here, we delineate a previously undisclosed mechanism by which androgen-activated AR transcriptionally upregulates fucosyltransferase 4 (FUT4) expression, which drives melanoma invasiveness by interfering with adherens junctions (AJs). Global phosphoproteomic and fucoproteomic profiling, coupled with in vitro and in vivo functional validation, further reveal that AR-induced FUT4 fucosylates L1 cell adhesion molecule (L1CAM), which is required for FUT4-increased metastatic capacity. Tumor microarray and gene expression analyses demonstrate that AR-FUT4-L1CAM-AJs signaling correlates with pathological staging in melanoma patients. By delineating key androgen-triggered signaling that enhances metastatic aggressiveness, our findings help explain sex-associated clinical outcome disparities and highlight AR/FUT4 and its effectors as potential prognostic biomarkers and therapeutic targets in melanoma.
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Melanoma , Molécula L1 de Adhesión de Célula Nerviosa , Humanos , Masculino , Femenino , Melanoma/metabolismo , Andrógenos , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Antígeno Lewis X/metabolismo , Glicosilación , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Línea Celular Tumoral , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismoRESUMEN
BACKGROUND: Vascular endothelial cells are pivotal in the pathophysiological progression following spinal cord injury (SCI). The UTX (Ubiquitously Transcribed Tetratripeptide Repeat on Chromosome X) serves as a significant regulator of endothelial cell phenotype. The manipulation of endogenous neural stem cells (NSCs) offers a compelling strategy for the amelioration of SCI. METHODS: Two mouse models were used to investigate SCI: NSCs lineage-traced mice and mice with conditional UTX knockout (UTX KO) in endothelial cells. To study the effects of UTX KO on neural differentiation, we harvested extracellular vesicles (EVs) from both UTX KO spinal cord microvascular endothelial cells (SCMECs) and negative control SCMECs. These EVs were then employed to modulate the differentiation trajectory of endogenous NSCs in the SCI model. RESULTS: In our NSCs lineage-traced mice model of SCI, a marked decrease in neurogenesis was observed post-injury. Notably, NSCs in UTX KO SCMECs mice showed enhanced neuronal differentiation compared to controls. RNA sequencing and western blot analyses revealed an upregulation of L1 cell adhesion molecule (L1CAM), a gene associated with neurogenesis, in UTX KO SCMECs and their secreted EVs. This aligns with the observed promotion of neurogenesis in UTX KO conditions. In vivo administration of L1CAM-rich EVs from UTX KO SCMECs (KO EVs) to the mice significantly enhanced neural differentiation. Similarly, in vitro exposure of NSCs to KO EVs resulted in increased activation of the Akt signaling pathway, further promoting neural differentiation. Conversely, inhibiting Akt phosphorylation or knocking down L1CAM negated the beneficial effects of KO EVs on NSC neuronal differentiation. CONCLUSIONS: In conclusion, our findings substantiate that EVs derived from UTX KO SCMECs can act as facilitators of neural differentiation following SCI. This study not only elucidates a novel mechanism but also opens new horizons for therapeutic interventions in the treatment of SCI. Video Abstract.
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Vesículas Extracelulares , Molécula L1 de Adhesión de Célula Nerviosa , Células-Madre Neurales , Traumatismos de la Médula Espinal , Animales , Ratones , Diferenciación Celular , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/farmacología , Células-Madre Neurales/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/terapiaRESUMEN
BACKGROUND: Premature infants may suffer from high levels of bilirubin that could lead to neurotoxicity. Bilirubin has been shown to decrease L1-mediated ERK1/2 signaling, L1 phosphorylation, and L1 tyrosine 1176 dephosphorylation. Furthermore, bilirubin redistributes L1 into lipid rafts (LR) and decreases L1-mediated neurite outgrowth. We demonstrate that choline supplementation improves L1 function and signaling in the presence of bilirubin. METHODS: Cerebellar granule neurons (CGN) were cultured with and without supplemental choline, and the effects on L1 signaling and function were measured in the presence of bilirubin. L1 activation of ERK1/2, L1 phosphorylation and dephosphorylation were measured. L1 distribution in LR was quantified and neurite outgrowth of CGN was determined. RESULTS: Forty µM choline significantly reduced the effect of bilirubin on L1 activation of ERK1/2 by 220% (p = 0.04), and increased L1 triggered changes in tyrosine phosphorylation /dephosphorylation of L1 by 34% (p = 0.026) and 35% (p = 0.02) respectively. Choline ameliorated the redistribution of L1 in lipid rafts by 38% (p = 0.02) and increased L1-mediated mean neurite length by 11% (p = 0.04). CONCLUSION: Choline pretreatment of CGN significantly reduced the disruption of L1 function by bilirubin. The supplementation of pregnant women and preterm infants with choline may increase infant resilience to the effects of bilirubin. IMPACT: This article establishes choline as an intervention for the neurotoxic effects of bilirubin on lipid rafts. This article provides clear evidence toward establishing one intervention for bilirubin neurotoxicity, where little is understood. This article paves the way for future investigation into the mechanism of the ameliorative effect of choline on bilirubin neurotoxicity.