RESUMEN
Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis and limited treatment options. This study evaluates the prognostic value of stromal markers in TNBC, focusing on the tumor-stroma ratio (TSR) and overall stroma ratio (OSR) in whole slide images (WSI), as well as the expression of type-I collagen, type-III collagen, and fibrillin-1 on tissue microarrays (TMAs), using both visual assessment and digital image analysis (DIA). A total of 101 female TNBC patients, primarily treated with surgery between 2005 and 2016, were included. We found that high visual OSR correlates with worse overall survival (OS), advanced pN categories, lower stromal tumor-infiltrating lymphocyte count (sTIL), lower mitotic index, and patient age (p < 0.05). TSR showed significant connections to the pN category and mitotic index (p < 0.01). High expression levels of type-I collagen (>45%), type-III collagen (>30%), and fibrillin-1 (>20%) were linked to significantly worse OS (p = 0.004, p = 0.013, and p = 0.005, respectively) and progression-free survival (PFS) (p = 0.028, p = 0.025, and p = 0.002, respectively), validated at the mRNA level. Our results highlight the importance of stromal characteristics in promoting tumor progression and metastasis and that targeting extracellular matrix (ECM) components may offer novel therapeutic strategies. Furthermore, DIA can be more accurate and objective in evaluating TSR, OSR, and immunodetected stromal markers than traditional visual examination.
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Biomarcadores de Tumor , Neoplasias de la Mama Triple Negativas , Humanos , Femenino , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/mortalidad , Pronóstico , Persona de Mediana Edad , Biomarcadores de Tumor/metabolismo , Anciano , Adulto , Células del Estroma/metabolismo , Células del Estroma/patología , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Fibrilina-1/metabolismo , Fibrilina-1/genética , Procesamiento de Imagen Asistido por Computador/métodos , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo III/metabolismo , Colágeno Tipo III/genética , Anciano de 80 o más AñosRESUMEN
INTRODUCTION: Mandibular prognathism (MP) patients present with aesthetic concerns and functional issues, including difficulties in mastication and pronunciation. Studies revealed that mandibular prognathism had definitive Mendelian inheritance patterns. This study aimed to ascertain distinct genetic markers associated with mandibular prognathism in individuals of Indian descent, focusing on exploring the prevalent genetic variations associated with certain genes. This study sought to identify the association of the following gene markers with mandibular prognathism: 1) Matrilin-1 (MATN1) (rs1065755), 2) Bone morphogenic protein 3 (BMP-3) (Tyr67Asn), 3) Homeobox protein hox-A2 (HOXA2) (Val327Ile), 4) Rho-GTPase activating protein (ARHGAP 21) (Gly1121Ser), 5) Myosin 1H (MYO1H) (rs10850110).
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Proteínas de Homeodominio , Prognatismo , Humanos , Masculino , India , Femenino , Prognatismo/genética , Proteínas de Homeodominio/genética , Miosina Tipo I/genética , Adulto , Proteínas Activadoras de GTPasa/genética , Adulto Joven , Adolescente , Proteínas de la Matriz Extracelular/genética , Marcadores Genéticos , Estudios de Casos y ControlesRESUMEN
Collagen posttranslational processing is crucial for its proper assembly and function. Disruption of collagen processing leads to tissue development and structure disorders like osteogenesis imperfecta (OI). OI-related collagen processing machinery includes prolyl 3-hydroxylase 1 (P3H1), peptidyl-prolyl cis-trans isomerase B (PPIB), and cartilage-associated protein (CRTAP), with their structural organization and mechanism unclear. We determine cryo-EM structures of the P3H1/CRTAP/PPIB complex. The active sites of P3H1 and PPIB form a face-to-face bifunctional reaction center, indicating a coupled modification mechanism. The structure of the P3H1/CRTAP/PPIB/collagen peptide complex reveals multiple binding sites, suggesting a substrate interacting zone. Unexpectedly, a dual-ternary complex is observed, and the balance between ternary and dual-ternary states can be altered by mutations in the P3H1/PPIB active site and the addition of PPIB inhibitors. These findings provide insights into the structural basis of collagen processing by P3H1/CRTAP/PPIB and the molecular pathology of collagen-related disorders.
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Colágeno , Microscopía por Crioelectrón , Ciclofilinas , Proteínas de la Matriz Extracelular , Humanos , Colágeno/metabolismo , Colágeno/química , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Ciclofilinas/metabolismo , Ciclofilinas/química , Ciclofilinas/genética , Dominio Catalítico , Isomerasa de Peptidilprolil/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/genética , Procesamiento Proteico-Postraduccional , Sitios de Unión , Unión Proteica , Autoantígenos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Modelos Moleculares , Mutación , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/química , Glicoproteínas de Membrana , Proteoglicanos , Chaperonas Moleculares , Prolil HidroxilasasRESUMEN
Sepsis is a systemic inflammatory response that can result in cardiac insufficiency or heart failure known as septic myocardial injury. A previous study identified OLFM4 as an important gene in sepsis through bioinformatics analysis. However, there is limited research on the regulatory functions of OLFM4 in sepsis-triggered myocardial injury, and the related molecular mechanisms remain unclear. In this study, the protein expression of OLFM4 was found to be significantly elevated in LPS-stimulated H9C2 cells, and its suppression enhanced cell proliferation and reduced cell apoptosis in LPS-triggered H9C2 cells. The inflammatory factors TNF-α, IL-6, and IL-1ß were increased after LPS treatment, and these effects were mitigated after silencing OLFM4. Moreover, it was confirmed that inhibition of OLFM4 attenuated the NF-κB signaling pathway. In conclusion, the knockdown of OLFM4 protected cardiomyocytes from sepsis by inhibiting apoptosis and inflammatory responses via the NF-κB pathway. These findings provide important insights into the regulatory functions of OLFM4 in the progression of septic myocardial injury.
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Apoptosis , Proteínas de la Matriz Extracelular , Glicoproteínas , Lipopolisacáridos , Miocitos Cardíacos , FN-kappa B , Sepsis , Animales , Humanos , Ratas , Apoptosis/inmunología , Línea Celular , Proliferación Celular , Técnicas de Silenciamiento del Gen , Inflamación/inmunología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/inmunología , FN-kappa B/metabolismo , Sepsis/inmunología , Transducción de Señal/inmunología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismoRESUMEN
We present the results of the first study of a large cohort of patients with inherited retinal dystrophies (IRD) performed for the Polish population using whole-exome sequencing (WES) in the years 2016-2019. Moreover, to facilitate such diagnostic analyses and enable future application of gene therapy and genome editing for IRD patients, a Polish genomic reference database (POLGENOM) was created based on whole-genome sequences of healthy Polish Caucasian nonagenarians and centenarians. The newly constructed database served as a control, providing a comparison for variant frequencies in the Polish population. The diagnostic yield for the selected group of IRD patients reached 64.9%. The study uncovered the most common pathogenic variants in ABCA4 and USH2A in the European population, along with several novel causative variants. A significant frequency of the ABCA4 complex haplotype p.(Leu541Pro; Ala1038Val) was observed, as well as that of the p.Gly1961Glu variant. The first VCAN causative variant NM_004385.5:c.4004-2A>G in Poland was found and described. Moreover, one of the first patients with the RPE65 causative variants was identified, and, in consequence, could receive the dedicated gene therapy. The availability of the reference POLGENOM database enabled comprehensive variant characterisation during the NGS data analysis, confirming the utility of a population-specific genomic database for enhancing diagnostic accuracy. Study findings suggest the significance of genetic testing in elder patients with unclear aetiology of eye diseases. The combined approach of NGS and the reference genomic database can improve the diagnosis, management, and future treatment of IRDs.
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Secuenciación del Exoma , Distrofias Retinianas , Población Blanca , Humanos , Distrofias Retinianas/genética , Polonia , Masculino , Población Blanca/genética , Femenino , Secuenciación del Exoma/métodos , Proteínas de la Matriz Extracelular/genética , Bases de Datos Genéticas , Transportadoras de Casetes de Unión a ATP/genética , Anciano de 80 o más Años , cis-trans-Isomerasas/genética , Anciano , Persona de Mediana Edad , Adulto , MutaciónRESUMEN
The main component of human skin is a collagen-rich extracellular matrix (ECM), known as the matrisome. The matrisome is essential for maintaining the structural integrity and mechanical properties of the skin. Recently, we reported notable decreases in matrisome proteins in natural aging and photoaging human skin. This study aims to investigate the mRNA expression of the core matrisome proteins in human skin, comparing young versus aged and sun-protected versus sun-exposed skin by quantitative real-time PCR and immunostaining. Our findings reveal a notable decrease in core matrisome transcription in aged skin. The mRNA expression of the core matrisome, such as collagen 1A1 (COL1A1), decorin, and dermatopontin, is significantly reduced in aged skin compared to its young skin. Yet, the majority of collagen mRNA expression levels of aged sun-exposed skin are similar to those found in young sun-exposed skin. This discrepancy is primarily attributable to a substantial decrease in collagen transcription in young sun-exposed skin, suggesting early molecular changes in matrisome transcription due to sun exposure, which preceded the emergence of clinical signs of photoaging. These findings shed light on the mRNA transcript profile of major matrisome proteins and their alterations in naturally aged and photoaged human skin, offering valuable insights into skin matrisome biology.
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Envejecimiento de la Piel , Piel , Humanos , Envejecimiento de la Piel/genética , Envejecimiento de la Piel/efectos de la radiación , Piel/metabolismo , Piel/efectos de la radiación , Adulto , Anciano , Persona de Mediana Edad , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/genética , Regulación de la Expresión Génica/efectos de la radiación , Masculino , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Adulto Joven , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I/genética , Luz SolarRESUMEN
Biallelic variants in USH2A are associated with retinitis pigmentosa (RP) and Type 2 Usher Syndrome (USH2), leading to impaired vision and, additionally, hearing loss in the latter. Although the introduction of next-generation sequencing into clinical diagnostics has led to a significant uplift in molecular diagnostic rates, many patients remain molecularly unsolved. It is thought that non-coding variants or variants of uncertain significance contribute significantly to this diagnostic gap. This study aims to demonstrate the clinical utility of the reverse transcription-polymerase chain reaction (RT-PCR)-Oxford Nanopore Technology (ONT) sequencing of USH2A mRNA transcripts from nasal epithelial cells to determine the splice-altering effect of candidate variants. Five affected individuals with USH2 or non-syndromic RP who had undergone whole genome sequencing were recruited for further investigation. All individuals had uncertain genotypes in USH2A, including deep intronic rare variants, c.8682-654C>G, c.9055+389G>A, and c.9959-2971C>T; a synonymous variant of uncertain significance, c.2139C>T; p.(Gly713=); and a predicted loss of function duplication spanning an intron/exon boundary, c.3812-3_3837dup p.(Met1280Ter). In silico assessment using SpliceAI provided splice-altering predictions for all candidate variants which were investigated using ONT sequencing. All predictions were found to be accurate; however, in the case of c.3812-3_3837dup, the outcome was a complex cryptic splicing pattern with predominant in-frame exon 18 skipping and a low level of exon 18 inclusion leading to the predicted stop gain. This study detected and functionally characterised simple and complex mis-splicing patterns in USH2A arising from previously unknown deep intronic variants and previously reported variants of uncertain significance, confirming the pathogenicity of the variants.
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Proteínas de la Matriz Extracelular , Empalme del ARN , Síndromes de Usher , Humanos , Proteínas de la Matriz Extracelular/genética , Síndromes de Usher/genética , Femenino , Masculino , Empalme del ARN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Exones/genética , Mutación/genética , Retinitis Pigmentosa/genética , Adulto , ARN Mensajero/genética , ARN Mensajero/metabolismo , Intrones/genética , Persona de Mediana EdadRESUMEN
OBJECTIVES: The aim of this study was to investigate the role and molecular mechanism of proline/arginine-rich end leucine-rich repeat protein (PRELP), a secreted protein in extracellular matrix, in oral squamous cell carcinoma (OSCC) progression. DESIGN: PRELP expression in OSCC was analyzed in the Gene Set Enrichment (GSE) 138206, GSE37991, and GSE23558 datasets as well as cell lines. Also, PRELP expression and its relationship with prognosis and immune infiltration in head and neck squamous cell carcinoma (HNSCC) were confirmed by bioinformatics analysis. The proliferation, apoptosis, invasion, epithelial-to-mesenchymal transition (EMT) and NF-κB activation were detected after alteration of PRELP expression in OSCC cells using CCK-8, EdU, flow cytometry, Transwell, real-time PCR, immunofluorescence and Western blot. Additionally, an NF-κB inhibitor PDTC was used to confirm the regulation mechanism of PRELP. RESULTS: The expression of PRELP in OSCC tissues, cells and in HNSCC samples was low. HNSCC patients with higher PRELP expression was associated with longer overall survival. A positive correlation between PRELP expression and immune cell infiltration was found in HNSCC. Upregulation of PRELP inhibited, whereas PRELP silencing promoted, the proliferation, invasion and EMT of OSCC cells. Also, overexpression of PRELP promoted cell apoptosis. Mechanistically, PRELP suppressed p65 phosphorylation and nuclear translocation. And PDTC treatment partially reversed the influences of PRELP knockdown on the malignant behaviors in OSCC cells. CONCLUSION: PRELP suppressed OSCC progression via inactivation of the NF-κB pathway. Targeting PRELP may be a potential approach for OSCC treatment.
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Carcinoma de Células Escamosas , Transición Epitelial-Mesenquimal , Proteínas de la Matriz Extracelular , Glicoproteínas , Neoplasias de la Boca , FN-kappa B , Humanos , Apoptosis , Western Blotting , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Citometría de Flujo , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/genética , FN-kappa B/metabolismo , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Glicoproteínas/genética , Glicoproteínas/metabolismoRESUMEN
Paclitaxel is among the most active chemotherapy drugs for the aggressive triple negative breast cancer (TNBC). Unfortunately, it often induces painful peripheral neuropathy (CIPN), a major debilitating side effect. Here we demonstrate that in naive and breast tumor-bearing immunocompetent mice, a clinically relevant dose of FTY720/Fingolimod that targets sphingosine-1-phosphate receptor 1 (S1PR1), alleviated paclitaxel-induced neuropathic pain. FTY720 also significantly attenuated paclitaxel-stimulated glial fibrillary acidic protein (GFAP), a marker for activated astrocytes, and expression of the astrocyte-secreted synaptogenic protein Sparcl1/Hevin, a key regulator of synapse formation. Notably, the formation of excitatory synapses containing VGluT2 in the spinal cord dorsal horn induced by paclitaxel was also inhibited by FTY720 treatment, supporting the involvement of astrocytes and Sparcl1 in CIPN. Furthermore, in this TNBC mouse model that mimics human breast cancer, FTY720 administration also enhanced the anti-tumor effects of paclitaxel, leading to reduced tumor progression and lung metastasis. Taken together, our findings suggest that targeting the S1P/S1PR1 axis with FTY720 is a multipronged approach that holds promise as a therapeutic strategy for alleviating both CIPN and enhancing the efficacy of chemotherapy in TNBC treatment.
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Clorhidrato de Fingolimod , Neuralgia , Paclitaxel , Animales , Clorhidrato de Fingolimod/farmacología , Paclitaxel/farmacología , Neuralgia/inducido químicamente , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Neuralgia/patología , Ratones , Femenino , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Línea Celular Tumoral , Receptores de Esfingosina-1-Fosfato/metabolismo , Humanos , Progresión de la Enfermedad , Antineoplásicos Fitogénicos/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteína Ácida Fibrilar de la Glía/genéticaRESUMEN
Serine proteinase inhibitors (serpins) are a family of structurally similar proteins which regulate many diverse biological processes from blood coagulation to extracellular matrix (ECM) remodelling. Chondrogenesis involves the condensation and differentiation of mesenchymal stem cells (MSCs) into chondrocytes which occurs during early development. Here, and for the first time, we demonstrate that one serpin, SERPINA3 (gene name SERPINA3, protein also known as alpha-1 antichymotrypsin), plays a critical role in chondrogenic differentiation. We observed that SERPINA3 expression was markedly induced at early time points during in vitro chondrogenesis. We examined the expression of SERPINA3 in human cartilage development, identifying significant enrichment of SERPINA3 in developing cartilage compared to total limb, which correlated with well-described markers of cartilage differentiation. When SERPINA3 was silenced using siRNA, cartilage pellets were smaller and contained lower proteoglycan as determined by dimethyl methylene blue assay (DMMB) and safranin-O staining. Consistent with this, RNA sequencing revealed significant downregulation of genes associated with cartilage ECM formation perturbing chondrogenesis. Conversely, SERPINA3 silencing had a negligible effect on the gene expression profile during osteogenesis suggesting the role of SERPINA3 is specific to chondrocyte differentiation. The global effect on cartilage formation led us to investigate the effect of SERPINA3 silencing on the master transcriptional regulator of chondrogenesis, SOX9. Indeed, we observed that SOX9 protein levels were markedly reduced at early time points suggesting a role for SERPINA3 in regulating SOX9 expression and activity. In summary, our data support a non-redundant role for SERPINA3 in enabling chondrogenesis via regulation of SOX9 levels.
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Diferenciación Celular , Condrocitos , Condrogénesis , Matriz Extracelular , Células Madre Mesenquimatosas , Serpinas , Condrogénesis/genética , Humanos , Condrocitos/metabolismo , Condrocitos/citología , Matriz Extracelular/metabolismo , Matriz Extracelular/genética , Serpinas/genética , Serpinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Cartílago/metabolismo , Cartílago/crecimiento & desarrollo , Cartílago/citología , Regulación del Desarrollo de la Expresión Génica , Biomarcadores/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Células CultivadasRESUMEN
Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration-like (AMD-like) retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that enabled simple, sensitive, and high-throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix, reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium-derived factor). In vivo, treatment of 8-month-old R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is an important demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of retinal degenerative diseases, including potentially AMD itself.
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Proteínas de la Matriz Extracelular , Matriz Extracelular , Degeneración Macular , Epitelio Pigmentado de la Retina , Animales , Ratones , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Degeneración Macular/patología , Degeneración Macular/genética , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Humanos , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Modelos Animales de Enfermedad , Distrofias Retinianas/metabolismo , Distrofias Retinianas/patología , Distrofias Retinianas/genética , Drusas del Disco Óptico/congénitoRESUMEN
This article recounts my journey as a scientist in the early days of extracellular matrix research through the discovery of fibronectin, the RGD sequence as a key recognition motif in fibronectin and other adhesion proteins, and isolation and cloning of integrins. I also discuss more recent work on identification of molecular "zip codes" by in vivo screening of peptide libraries expressed on phage, which led us right back to RGD and integrins. Many disease-specific zip codes have turned out to be based on altered expression of extracellular matrix molecules and integrins. Homing peptides and antibodies recognizing zip code molecules are being used in drug delivery applications, some of which have advanced into clinical trials.
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Matriz Extracelular , Fibronectinas , Integrinas , Oligopéptidos , Matriz Extracelular/metabolismo , Humanos , Integrinas/metabolismo , Integrinas/genética , Fibronectinas/metabolismo , Fibronectinas/genética , Fibronectinas/química , Historia del Siglo XXI , Historia del Siglo XX , Oligopéptidos/metabolismo , Oligopéptidos/genética , Oligopéptidos/química , Biblioteca de Péptidos , Animales , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genéticaRESUMEN
Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine. We previously identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene-set -enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular-matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, concurrent with these perturbations in gene and protein expression, changes in cell morphology and phenotype. We also identified that EWS-FLI1 dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insights into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular-matrix components by EWS-FLI1 and AP-1.
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Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Sarcoma de Ewing , Factor de Transcripción AP-1 , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Sarcoma de Ewing/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Humanos , Proteína EWS de Unión a ARN/metabolismo , Proteína EWS de Unión a ARN/genética , Factor de Transcripción AP-1/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Fusión Oncogénica/genética , Línea Celular Tumoral , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Perfilación de la Expresión GénicaRESUMEN
Cerebral arteriovenous malformations represent the most common form of vascular malformations and can cause recurrent bleeding and hemorrhagic stroke. The current issue of the JCI features an article by Zhao et al. describing a mouse model of cerebral arteriovenous malformations. Endothelial cells lacking matrix Gla protein, a BMP inhibitor, underwent epigenetic changes characteristic of an endothelial-to-mesenchymal fate transition. The authors uncovered a two-step process for this transition controlled by the epigenetic regulator histone deacetylase 2 (HDAC2), which controls endothelial cell differentiation, and by enhancer of zeste homolog 1 (EZH1), which suppressed mesenchymal fate. This discovery provides a promising entry point for preventive pharmacological interventions.
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Epigénesis Genética , Histona Desacetilasa 2 , Animales , Ratones , Humanos , Histona Desacetilasa 2/metabolismo , Histona Desacetilasa 2/genética , Células Endoteliales/metabolismo , Células Endoteliales/patología , Malformaciones Arteriovenosas Intracraneales/genética , Malformaciones Arteriovenosas Intracraneales/metabolismo , Malformaciones Arteriovenosas Intracraneales/patología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Modelos Animales de Enfermedad , Diferenciación Celular , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genéticaRESUMEN
AIMS: The ELFN1, discovered in 2007, is a single-pass transmembrane protein. Studies conducted thus far to elucidate the function of the Elfn1 have been limited only to animal studies. These studies have reported that ELFN1 is a universal binding partner of metabotropic glutamate receptors (mGluRs) in the central nervous system and its functional deficiency has been associated with the pathogenesis of neurological and neuropsychiatric diseases. In 2021, we described the first disease-associated human ELFN1 pathogenic gene mutation. Severe joint laxity, which was the most striking finding of this new disease and was clearly seen in the patients since early infancy, showed that the ELFN1 may have a possible function in the connective tissue besides the nervous system. Here, we present the first experimental evidence of the extracellular matrix (ECM)-related function of the ELFN1. MATERIALS AND METHODS: Primary skin fibroblasts were isolated from the skin biopsies of ELFN1 mutated patients and healthy foreskin donors. For the clinical trial in a dish, in vitro ECM and DEM (decellularized ECM) models were created from skin fibroblasts. All the in vitro models were comparatively characterized and analyzed. KEY FINDINGS: The mutation in the ELFN1 signal peptide region of patients resulted in a severe lack of ELFN1 expression and dramatically altered the characteristic morphology and behavior (growth, proliferation, and motility) of fibroblasts. SIGNIFICANCE: We propose that ELFN1 is involved in the cell-ECM attachment, and its deficiency is critical enough to cause a loss of cell motility and soft ECM stiffness.
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Proteínas de la Matriz Extracelular , Matriz Extracelular , Fibroblastos , Humanos , Masculino , Movimiento Celular , Proliferación Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Fibroblastos/metabolismo , Mutación , Piel/metabolismo , Piel/patologíaRESUMEN
Reelin (RELN) is a secreted glycoprotein essential for cerebral cortex development. In humans, recessive RELN variants cause cortical and cerebellar malformations, while heterozygous variants were associated with epilepsy, autism, and mild cortical abnormalities. However, the functional effects of RELN variants remain unknown. We identified inherited and de novo RELN missense variants in heterozygous patients with neuronal migration disorders (NMDs) as diverse as pachygyria and polymicrogyria. We investigated in culture and in the developing mouse cerebral cortex how different variants impacted RELN function. Polymicrogyria-associated variants behaved as gain-of-function, showing an enhanced ability to induce neuronal aggregation, while those linked to pachygyria behaved as loss-of-function, leading to defective neuronal aggregation/migration. The pachygyria-associated de novo heterozygous RELN variants acted as dominant-negative by preventing WT RELN secretion in culture, animal models, and patients, thereby causing dominant NMDs. We demonstrated how mutant RELN proteins in vitro and in vivo predict cortical malformation phenotypes, providing valuable insights into the pathogenesis of such disorders.
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Moléculas de Adhesión Celular Neuronal , Movimiento Celular , Proteínas de la Matriz Extracelular , Mutación Missense , Proteínas del Tejido Nervioso , Proteína Reelina , Serina Endopeptidasas , Humanos , Animales , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Ratones , Femenino , Masculino , Movimiento Celular/genética , Neuronas/metabolismo , Neuronas/patología , Polimicrogiria/genética , Polimicrogiria/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Heterocigoto , Lisencefalia/genética , Lisencefalia/patología , AlelosRESUMEN
BACKGROUND: Our previous study has demonstrated that Nischarin (NISCH) exerts its antitumor effects in breast cancer (BC) by suppressing cell migration and invasion. This study aims to explore the underlying mechanism through which NISCH functions in BC. METHODS AND RESULTS: The relevance between EGF Like Repeats and Discoidin Domains 3 (EDIL3) mRNA expression and the overall survival of tumor patients was depicted by the Kaplan-Meier curve. The findings revealed that overexpressed NISCH attenuated cell motility and colony-forming capacities of Hs578T cells, yet silenced NISCH in MDA-MB-231 cells led to contrasting results. Western blot (WB) analysis indicated that overexpression of NISCH significantly down-regulated the Vimentin and Slug expression, and inactivated the FAK/ERK signaling pathway. RNA sequencing (RNA-seq) was performed in NISCH-overexpressed Hs578T cells and the control cells to analyze differentially expressed genes (DeGs), and the results showed a significant down-regulation of EDIL3 mRNA level upon overexpression of NISCH. Subsequent functional analyses demonstrated that overexpression of EDIL3 attenuated the inhibitory effect of NISCH on cell migration, invasion, colony formation, and tube formation. CONCLUSION: In summary, our finding preliminarily revealed that NISCH inhibits the epithelial-mesenchymal transition (EMT) process and angiogenesis in BC cells by down-regulating EDIL3 to inactivate the FAK/ERK signaling pathway, thereby suppressing the progression of BC. Our results hold promise for contributing to the deep understanding of BC pathogenesis and identifying new therapeutic strategies for clinical application.
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Neoplasias de la Mama , Movimiento Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Neovascularización Patológica , Humanos , Transición Epitelial-Mesenquimal/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Línea Celular Tumoral , Movimiento Celular/genética , Sistema de Señalización de MAP Quinasas/genética , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Proliferación Celular/genética , Vimentina/metabolismo , Vimentina/genética , Transducción de Señal , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Factores de Transcripción de la Familia Snail/metabolismo , Factores de Transcripción de la Familia Snail/genética , Angiogénesis , Proteínas de Unión al Calcio , Moléculas de Adhesión CelularRESUMEN
BACKGROUND: Usher syndrome (USH) encompasses a group of disorders characterized by congenital sensorineural hearing loss (SNHL) and retinitis pigmentosa (RP). We described the clinical findings, natural history, and molecular analyses of USH patients identified during a large-scale screening to identify quantitative traits related to ocular disorders in the SardiNIA project cohort. METHODS: We identified 3 USH-affected families out of a cohort of 6,148 healthy subjects. 9 subjects presented a pathological phenotype, with SNHL and RP. All patients and their family members underwent a complete ophthalmic examination including best-corrected visual acuity, slit-lamp biomicroscopy, fundoscopy, fundus autofluorescence, spectral-domain optical coherence tomography, and electrophysiological testing. Audiological evaluation was performed with a clinical audiometer. Genotyping was performed using several arrays integrated with whole genome sequence data providing approximately 22 million markers equally distributed for each subject analyzed. Molecular diagnostics focused on analysis of the following candidate genes: MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31, CLRN1, and PDZD7. RESULTS: A single missense causal variant in USH2A gene was identified in homozygous status in all patients and in heterozygous status in unaffected parents. The presence of multiple homozygous patients with the same phenotypic severity of the syndromic form suggests that the Sardinian USH phenotype is the result of a founder effect on a specific pathogenic variant related haplotype. The frequency of heterozygotes in general Sardinian population is 1.89. Additionally, to provide new insights into the structure of usherin and the pathological mechanisms caused by small pathogenic in-frame variants, like p.Pro3272Leu, molecular dynamics simulations of native and mutant protein-protein and protein-ligand complexes were performed that predicted a destabilization of the protein with a decrease in the free energy change. CONCLUSIONS: Our results suggest that our approach is effective for the genetic diagnosis of USH. Based on the heterozygous frequency, targeted screening of this variant in the general population and in families at risk or with familial USH can be suggested. This can lead to more accurate molecular diagnosis, better genetic counseling, and improved molecular epidemiology data that are critical for future intervention plans. TRIAL REGISTRATION: We did not perform any health-related interventions for the participants.
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Linaje , Síndromes de Usher , Humanos , Síndromes de Usher/genética , Síndromes de Usher/diagnóstico , Italia/epidemiología , Masculino , Femenino , Adulto , Persona de Mediana Edad , Proteínas de la Matriz Extracelular/genética , Análisis Mutacional de ADN , Tomografía de Coherencia Óptica , Fenotipo , Efecto Fundador , Mutación Missense , Electrorretinografía , Adulto Joven , Adolescente , Agudeza Visual , Pruebas Genéticas/métodosRESUMEN
Fibulin-2 is a multidomain, disulfide-rich, homodimeric protein which belongs to a broader extracellular matrix family. It plays an important role in the development of elastic fiber structures. Malfunction of fibulin due to mutation or poor expression can result in a variety of diseases including synpolydactyly, limb abnormalities, eye disorders leading to blindness, cardiovascular diseases and cancer. Traditionally, fibulins have either been produced in mammalian cell systems or were isolated from the extracellular matrix, a procedure that results in poor availability for structural and functional studies. Here, we produced seven fibulin-2 constructs covering 62% of the mature protein (749 out of 1195 residues) using a prokaryotic expression system. Biophysical studies confirm that the purified constructs are folded and that the presence of disulfide bonds within the constructs makes them extremely thermostable. In addition, we solved the first crystal structure for any fibulin isoform, a structure corresponding to the previously suggested three motifs related to anaphylatoxin. The structure reveals that the three anaphylatoxins moieties form a single-domain structure.