RESUMEN
Self-renewing, damage-repair and differentiation of mammalian stratified squamous epithelia are subject to tissue homeostasis, but the regulation mechanisms remain elusive. Here, we investigate the esophageal squamous epithelial tissue homeostasis in vitro and in vivo. We establish a rat esophageal organoid (rEO) in vitro system and show that the landscapes of rEO formation, development and maturation trajectories can mimic those of rat esophageal epithelia in vivo. Single-cell RNA sequencing (scRNA-seq), snapshot immunostaining and functional analyses of stratified "matured" rEOs define that the epithelial pluripotent stem cell determinants, p63 and Sox2, play crucial but distinctive roles for regulating mammalian esophageal tissue homeostasis. We identify two cell populations, p63+Sox2+ and p63-Sox2+, of which the p63+Sox2+ population presented at the basal layer is the cells of origin required for esophageal epithelial stemness maintenance and proliferation, whereas the p63-Sox2+ population presented at the suprabasal layers is the cells of origin having a dual role for esophageal epithelial differentiation (differentiation-prone fate) and rapid tissue damage-repair responses (proliferation-prone fate). Given the fact that p63 and Sox2 are developmental lineage oncogenes and commonly overexpressed in ESCC tissues, p63-Sox2+ population could not be detected in organoids formed by esophageal squamous cell carcinoma (ESCC) cell lines. Taken together, these findings reveal that the tissue homeostasis is maintained distinctively by p63 and/or Sox2-dependent cell lineage populations required for the tissue renewing, damage-repair and protection of carcinogenesis in mammalian esophagi.
Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Ratas , Animales , Neoplasias Esofágicas/genética , Mamíferos , Homeostasis , CarcinogénesisRESUMEN
Nuclear factor erythroid-2 related factor-2 (NFE2L2 or NRF2) is a frequently mutated gene in esophageal squamous cell carcinoma (ESCC). However, the roles of NFE2L2 alterations in ESCC remain elusive. In order to elucidate this issue, 130 ESCC patients who underwent esophagectomy were enrolled. The majority of tumor tissues were positive for NRF2, which was significantly enriched in the nucleus of the primary tumor tissues compared with the noncancerous mucosae. Primary ESCC tumors positive for NRF2 tended to be positive for NAD(P)H quinone oxidoreductase 1 (NQO1) as the downstream target of NRF2. There was a positive correlation between NRF2 and NQO1 expression level in primary tumors. NQO1 staining in primary tumors with NRF2 nuclear expression was significantly stronger than that with NRF2 cytoplasmic expression. In addition, high concordance for the status of NRF2 expression between primary tumors and corresponding metastatic lesions was observed. Next, we found high expression of nuclear NRF2 (the proportion of nuclear NRF2 expression >20% or nuclear NRF2 immunohistochemistry score >20) predicted shorter overall survival in patients with dual-positive expression of NRF2 and NQO1. Captured-based targeted sequencing revealed that NFE2L2 somatic alterations were observed in 52.8% of ESCC patients with dual-positive expression of NRF2 and NQO1. NFE2L2 amplification and mutations within the DLG/ETGE motifs were seen more frequently in ESCC tumors with nuclear or nucleocytoplasmic expression of NRF2 compared with those with cytoplasmic expression of NRF2. We also found high expression of nuclear NRF2 plus the status of NFE2L2 alteration exhibited high performance in predicting prognosis of ESCC patients. Our study demonstrated that high nuclear NRF2 expression and NFE2L2 alterations were associated with poor prognosis of ESCC patients. These findings suggest that NRF2 signaling pathway might play vital roles in ESCC malignancy and the aberrant activation of NRF2 pathway predicts unfavorable prognosis in ESCC.
Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Factor 2 Relacionado con NF-E2 , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/diagnóstico , Carcinoma de Células Escamosas de Esófago/genética , Humanos , Inmunohistoquímica , Factor 2 Relacionado con NF-E2/genética , PronósticoRESUMEN
BACKGROUND: Cancer stem cells (CSCs) are related to the patient's prognosis, recurrence and therapy resistance in oesophageal squamous cell carcinoma (ESCC). Although increasing evidence suggests that aspirin (acetylsalicylic acid, ASA) could lower the incidence and improve the prognosis of ESCC, the mechanism(s) remains to be fully understood. METHODS: We investigated the role of ASA in chemotherapy/chemoprevention in human ESCC cell lines and an N-nitrosomethylbenzylamine-induced rat ESCC carcinogenesis model. The effects of combined treatment with ASA/cisplatin on ESCC cell lines were examined in vitro and in vivo. Sphere-forming cells enriched with putative CSCs (pCSCs) were used to investigate the effect of ASA in CSCs. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) was performed to determine the alterations in chromatin accessibility caused by ASA in ESCC cells. RESULTS: ASA inhibits the CSC properties and enhances cisplatin treatment in human ESCC cells. ATAC-seq indicates that ASA treatment results in remarkable epigenetic alterations on chromatin in ESCC cells, especially their pCSCs, through the modification of histone acetylation levels. The epigenetic changes activate Bim expression and promote cell death in CSCs of ESCC. Furthermore, ASA prevents the carcinogenesis of NMBzA-induced ESCC in the rat model. CONCLUSIONS: ASA could be a potential chemotherapeutic adjuvant and chemopreventive drug for ESCC treatment.
Asunto(s)
Aspirina/administración & dosificación , Cisplatino/administración & dosificación , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Animales , Aspirina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Dimetilnitrosamina/efectos adversos , Dimetilnitrosamina/análogos & derivados , Sinergismo Farmacológico , Epigénesis Genética/efectos de los fármacos , Neoplasias Esofágicas/inducido químicamente , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/inducido químicamente , Carcinoma de Células Escamosas de Esófago/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Metformin is a widely used antidiabetic drug for the management of type 2 diabetes mellitus. Recently, epidemiological studies demonstrate that metformin has anticancer effects on esophageal squamous cell carcinoma (ESCC) and other cancers. However, the effects and potential mechanisms of metformin on ESCC remain elusive. In this study, we used N-nitroso-N-methylbenzylamine (NMBzA), a special carcinogen for esophagi, to develop a rat ESCC model, in which the carcinogenesis progression of ESCC in rat was induced and promoted. We investigated the effects of metformin on carcinogenesis of ESCC in this model. Our results revealed that metformin significantly decreased the incidence and precancerous lesions of ESCC and inhibited proliferation and promoted apoptosis of esophageal epithelial cells in rat treated with NMBzA. Moreover, metformin also increased apoptosis and inhibited migration, colony formation and tumor sphere formation of human ESCC cells in vitro. Immunohistochemistry and western blotting showed that without interfering the metabolism of NMBzA, metformin inhibited the inflammation of esophagi via reducing the expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6). Treatment of metformin led to activation of AMP-activated protein kinase (AMPK) and attenuated signaling of the downstream molecules such as p-mTOR, p-p70S6K and cyclin D1 expression both in vivo and in vitro. Taken together, our study demonstrated that metformin suppressed the carcinogenesis of ESCC through inhibiting AMPK/mammalian target of the rapamycin (mTOR) signaling pathway, resulting in its chemopreventive effects on the carcinogenesis of ESCC.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Carcinogénesis/efectos de los fármacos , Dimetilnitrosamina/análogos & derivados , Neoplasias Esofágicas/prevención & control , Carcinoma de Células Escamosas de Esófago/prevención & control , Metformina/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Carcinógenos/toxicidad , Proliferación Celular , Dimetilnitrosamina/toxicidad , Neoplasias Esofágicas/inducido químicamente , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/inducido químicamente , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Hipoglucemiantes/farmacología , Masculino , Ratas , Ratas Endogámicas F344 , Serina-Treonina Quinasas TOR/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: Mutations in DNA damage response factors BRCA1 and BRCA2 confer sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors in breast and ovarian cancers. BRCA1/BRCA2-defective tumors can exhibit resistance to PARP inhibitors via multiple mechanisms, one of which involves loss of 53BP1. Deficiency in the DNA damage response factor ataxia-telangiectasia mutated (ATM) can also sensitize tumors to PARP inhibitors, raising the question of whether the presence or absence of 53BP1 can predict sensitivity of ATM-deficient breast cancer to these inhibitors. METHODS: Cytotoxicity of PARP inhibitor and ATM inhibitor in breast cancer cell lines was assessed by MTS, colony formation and apoptosis assays. ShRNA lentiviral vectors were used to knockdown 53BP1 expression in breast cancer cell lines. Phospho-ATM and 53BP1 protein expressions were determined in human breast cancer tissues by immunohistochemistry (IHC). RESULTS: We show that inhibiting ATM increased cytotoxicity of PARP inhibitor in triple-negative and non-triple-negative breast cancer cell lines, and depleting the cells of 53BP1 reduced this cytotoxicity. Inhibiting ATM abrogated homologous recombination induced by PARP inhibitor, and down-regulating 53BP1 partially reversed this effect. Further, overall survival was significantly better in triple-negative breast cancer patients with lower levels of phospho-ATM and tended to be better in patients with negative 53BP1. CONCLUSION: These results suggest that 53BP1 may be a predictor of PARP inhibitor resistance in patients with ATM-deficient tumors.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos , Morfolinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Pironas/farmacología , Proteína 1 de Unión al Supresor Tumoral P53/genética , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Células MCF-7 , Fosforilación , Ftalazinas/farmacología , Piperazinas/farmacología , Pronóstico , Análisis de Supervivencia , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
OBJECTIVE: To evaluate the feasibility of detecting macrophage content on atherosclerotic plaques by optical coherence tomography (OCT) technique. METHODS: Thirty New Zealand white rabbits were equally divided into 3 groups at random: Control group (fed normal rabbit chow, n = 10); lipid diet group (fed regular chow supplemented with cholesterol, n = 10) and balloon injury+ lipid diet group (balloon catheter injury of the common carotid artery after 2 weeks lipid diet, n = 10). After 12 weeks, all rabbits underwent pharmacological triggering with Chinese Russell's viper venom (CRVV, 15 mg/kg, i.p.) and histamine (0.02 mg/kg, i.v.). Common carotid arteries were detected with OCT and the Movat pentachrome stain respectively. OCT and histological examination results were compared and the correlation was analyzed. RESULTS: The intra thickness measured by Movat pentachrome stain and by the OCT was (15.2 ± 0.9) µm and (20.2 ± 7.6) µm, the medial thickness was (434.2 ± 86.5) µm and (453.8 ± 87.2) µm, the plaque thickness was (330.2 ± 87.1) µm and (392.2 ± 134.5) µm, the fibrous cap thickness was (58.3 ± 5.6) µm and (61.2 ± 4.9) µm, respectively (all P > 0.05). The normalized standard deviation of the OCT signal (NSD) was compared with immunohistochemical detection. The OCT signal within the cap is relatively homogeneous for low macrophage density in high lipid diet group. For the raw OCT data, a correlation of r = 0.846 (P < 0.01) was found between OCT NSD and a CD68 area<10%, whereas for the base 10 logarithm OCT data, a correlation of r = 0.646 (P < 0.05) was found between OCT NSD and a CD68 area<10%. In balloon injury + high lipid diet group, the OCT signal within the cap was relatively heterogeneous for high macrophage content. For the raw OCT data, a correlation of r = 0.906 (P < 0.01) was found between OCT NSD and a CD68 area >10%, whereas for the base 10 logarithm OCT data, a correlation of r = 0.593 (P < 0.05) was found between OCT NSD and a CD68 area >10%. For the raw OCT signal NSD, a range of NSDs (7.12% to 7.35%) demonstrated 100% sensitivity and specificity (Kappa value 1.0) for differentiating caps containing >10% CD68 staining. For the base 10 logarithm OCT signal, NSD values ranging from 7.81% to 7.92% provided 70% sensitivity and 75% specificity (value 0.44) for identifying caps containing >10% CD68 staining. CONCLUSIONS: OCT is an effective tool to determine macrophage content in this model. OCT imaging can clearly visualize different types of atherosclerotic plaques and provide detailed information on plaque characteristics.
Asunto(s)
Placa Aterosclerótica , Tomografía de Coherencia Óptica , Animales , Arteria Carótida Común , Colesterol , Fibrosis , Macrófagos , Conejos , Sensibilidad y EspecificidadRESUMEN
Macrophage inhibitory factor 1 (MIC1) is frequently altered in various cancers. The aim of this study was to investigate the clinical significance of MIC1 for esophageal squamous cell carcinoma (ESCC). Serum MIC1 of 286 ESCC and 250 healthy subjects was detected, the diagnostic performance was assessed and compared with SCC, CEA, CA199 and CA724, and the value as a prognostic indicator was also evaluated. The expression of MIC1 in ESCC cell lines, tissues were detected, and the inhibition of MIC1 antibody on ESCC was carried out in vitro and in vivo. The results showed that the serum MIC1 of ESCC was significantly higher than normal groups (P < 0.001), and was positively associated with tumor invasion (P = 0.030) as well as lymph node metastasis (P = 0.007). The sensitivity of MIC1 was significantly better than SCC, CEA, CA199 and CA724, especially for stage I ESCC. Patients with higher serum MIC1 also had a poorer prognosis in relapse-free (P = 0.050) and tumor-specific survival (P = 0.005). In vitro studies showed that the expression of MIC1 was upregulated in 37.5% (3/8) ESCC cell lines and 45% (18/40) tissues, and the transcription of MIC1 in tumor tissues was significantly higher than paired adjacent normal tissues (P = 0.001). The antibody of MIC1 inhibited the tumor growth (P < 0.001), and showing preference for tumor tissues in xenograft model. The decreased formation of neovascularization lumen may be involved in the mechanism. We conclude that MIC1 plays an important role in the progression of ESCC and can serve as a potential biomarker and therapeutic target for ESCC.
Asunto(s)
Anticuerpos/administración & dosificación , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/inmunología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Neoplasias Esofágicas/metabolismo , Factor 15 de Diferenciación de Crecimiento/inmunología , Animales , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/patología , Línea Celular , Línea Celular Tumoral , Progresión de la Enfermedad , Neoplasias Esofágicas/sangre , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/inmunología , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/terapia , Carcinoma de Células Escamosas de Esófago , Femenino , Factor 15 de Diferenciación de Crecimiento/sangre , Factor 15 de Diferenciación de Crecimiento/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Metástasis Linfática , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Recurrencia Local de Neoplasia/sangre , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Neovascularización Patológica/sangre , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Pronóstico , Distribución Aleatoria , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Artesunate (ART), an effective antimalarial semisynthetic derivative of artemisinin, exhibits antitumour properties, but the mechanism(s) involved remain elusive. In this study, we investigated the antitumour effects of ART on human oesophageal squamous cell carcinoma (ESCC) cell lines. Treatment of ESCC cell lines with ART resulted in the production of excessive reactive oxygen species (ROS) that induced DNA damage, reduced cell proliferation and inhibited clonogenicity via G1-S cell cycle arrest and/or apoptosis in vitro. The administration of ART to nude mice with ESCC cell xenografts inhibited tumour formation in vivo. However, the cytotoxicity of ART strongly differed among the ESCC cell lines tested. Transcriptomic profiling revealed that although the expression of large numbers of genes in ESCC cell lines was affected by ART treatment, these genes could be functionally clustered into pathways involved in regulating cell cycle progression, DNA metabolism and apoptosis. We revealed that p53 and Cdk4/6-p16-Rb cell cycle checkpoint controls were critical determinants required for mediating ART cytotoxicity in ESCC cell lines. Specifically, KYSE30 cells with p53Mut/p16Mut were the most sensitive to ART, KYSE150 and KYSE180 cells with p53Mut/p16Nor exhibited intermediate responses to ART, and Eca109 cells with p53Nor/p16Nor exhibited the most resistance to ATR. Consistently, perturbation of p53 expression using RNA interference (RNAi) and/or Cdk4/6 activity using the inhibitor palbociclib altered ART cytotoxicity in KYSE30 cells. Given that the p53 and Cdk4/6-cyclin D1-p16-Rb genes are commonly mutated in ESCC, our results potentially shed new light on neoadjuvant chemotherapy strategies for ESCC.
Asunto(s)
Apoptosis , Artesunato , Puntos de Control del Ciclo Celular , Proliferación Celular , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Artesunato/farmacología , Artesunato/uso terapéutico , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Animales , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Carcinoma de Células Escamosas de Esófago/genética , Ratones , Línea Celular Tumoral , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ratones Desnudos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Daño del ADN/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Artemisininas/farmacología , Artemisininas/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Antineoplásicos/farmacologíaRESUMEN
Rat model of N-nitrosomethylbenzylamine (NMBzA)-induced esophageal squamous cell carcinoma (ESCC) is routinely used to study ESCC initiation, progression and new therapeutic strategies. However, the model is time-consuming and malignant tumor incidences are low. Here, we report the usage of multi-kinase inhibitor sorafenib as a tumor promoter to establish an efficient two-stage NMBzA-induced rat ESCC carcinogenesis model, resulting in increments of tumor incidences and shortened tumor formation times. By establishing the model and applying whole-genome sequencing, we discover that benign papillomas and malignant ESCCs harbor most of the "driver" events found in rat ESCCs (e.g. recurrent mutations in Ras family, the Hippo and Notch pathways and histone modifier genes) and the mutational landscapes of rat and human ESCCs overlap extensively. We generate tumor cell lines derived from NMBzA-induced papillomas and ESCCs, showing that papilloma cells retain more characteristics of normal epithelial cells than carcinoma cells, especially their exhibitions of normal rat cell karyotypes and inabilities of forming tumors in immunodeficient mice. Three-dimensional (3-D) organoid cultures and single cell RNA sequencing (scRNA-seq) indicate that, when compared to control- and papilloma-organoids, ESCC-organoids display salient abnormalities at tissue and single-cell levels. Multi-omic analyses indicate that NMBzA-induced rat ESCCs are accompanied by progressive hyperactivations of the FAT-Hippo-YAP1 axis and siRNA or inhibitors of YAP1 block the growth of rat ESCCs. Taken together, these studies provide a framework of using an effective rat ESCC model to investigate multilevel functional genomics of ESCC carcinogenesis, which justify targeting YAP1 as a therapeutic strategy for ESCC.
Asunto(s)
Carcinoma de Células Escamosas , Dimetilnitrosamina/análogos & derivados , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Papiloma , Humanos , Ratas , Ratones , Animales , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Neoplasias Esofágicas/inducido químicamente , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , CarcinogénesisRESUMEN
Objectives: The majority of esophageal squamous dysplasia (ESD) patients progress slowly, while a subset of patients can undergo recurrence rapidly or progress to invasive cancer even after proper treatment. However, the molecular mechanisms underlying these clinical observations are still largely unknown. Methods: By sequencing the genomic data of 160 clinical samples from 49 tumor-free ESD patients and 88 esophageal squamous cell carcinoma (ESCC) patients, we demonstrated lower somatic mutation and copy number alteration (CNA) burden in ESD compared with ESCC. Results: Cross-species screening and functional assays identified ACSM5 as a novel driver gene for ESD progression. Furthermore, we revealed that miR-4292 promoted ESD progression and could serve as a non-invasive diagnostic marker for ESD. Conclusions: These findings largely expanded our understanding of ESD genetics and tumorigenesis, which possessed promising significance for improving early diagnosis, reducing overtreatment, and identifying high-risk ESD patients.
RESUMEN
A short tandem repeat (STR) polymorphism in the 3'UTR region of esophageal cancer-related gene 2 (ECRG2, also known as SPINK7) has been widely reported to be associated with the incidence and the prognosis of esophageal squamous cell carcinoma (ESCC). This study explores how the microRNA binding to the STR region affects ECRG2 expression in ESCC. Dual-luciferase reporter assays were used to verify the effects of the four microRNAs (miR-580, miR-1182, miR-1272, and miR-1322) predicted to bind the STR region of the ECRG2 3' untranslated region (UTR). The expression of identified effective microRNA was then analyzed in 44 paired ESCC and adjacent normal tissues and 402 case-controlled serum samples (divided into a discovery group and an independent validation group) by real-time RT-PCR assay. We found that only miR-1322 could significantly down-regulate the ECRG2 with TCA3 allele (P < 0.01), but it could not down-regulate the ECRG2 with TCA4 allele significantly (P > 0.05). MiR-1322 was also expressed significantly higher in ESCC tissue and serum samples than in controls (both P < 0.01). Additionally, serum levels of miR-1322 yielded an under receiver operating characteristic (ROC) curve area of 0.847 (95% CI, 0.795-0.890) for discriminating ESCCs from healthy controls in the discovery group and a similar result was obtained in the validation group (under ROC area is 0.845; 95%CI, 0.780-0.897). We conclude that miR-1322 can regulate ECRG2 in an allele-specific manner and that serum levels of miR-1322 can serve as a potential diagnostic biomarker for patients with ESCC.
Asunto(s)
Alelos , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , MicroARNs/genética , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Regiones no Traducidas 3' , Adulto , Anciano , Anciano de 80 o más Años , Emparejamiento Base , Secuencia de Bases , Biomarcadores de Tumor , Carcinoma de Células Escamosas/metabolismo , Estudios de Casos y Controles , Línea Celular Tumoral , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago , Femenino , Regulación Neoplásica de la Expresión Génica , Orden Génico , Humanos , Masculino , MicroARNs/metabolismo , Repeticiones de Microsatélite , Persona de Mediana Edad , Conformación de Ácido Nucleico , Polimorfismo Genético , Proteínas Inhibidoras de Proteinasas Secretoras/química , Interferencia de ARN , Inhibidores de Serinpeptidasas Tipo KazalRESUMEN
Cyclin-dependent kinase 12 (CDK12), a transcription-related cyclin dependent kinase (CDK), plays a momentous part in multitudinous biological functions, such as replication, transcription initiation to elongation and termination, precursor mRNA (pre-mRNA) splicing, intron polyadenylation (IPA), and translation. CDK12 can act as a tumour suppressor or oncogene in disparate cellular environments, and its dysregulation likely provokes tumorigenesis. A comprehensive understanding of CDK12 will tremendously facilitate the exploitation of novel tactics for the treatment and precaution of cancer. Currently, CDK12 inhibitors are nonspecific and nonselective, which profoundly hinders the pharmacological target validation and drug exploitation process. Herein, we summarize the newly comprehension of the biological functions of CDK12 with a focus on recently emerged advancements of CDK12-associated therapeutic approaches in cancers.
Asunto(s)
Quinasas Ciclina-Dependientes , Neoplasias , Humanos , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/genética , CarcinogénesisRESUMEN
Despite its remarkable potential for transforming low-resolution images, deep learning faces significant challenges in achieving high-quality superresolution microscopy imaging from wide-field (conventional) microscopy. Here, we present X-Microscopy, a computational tool comprising two deep learning subnets, UR-Net-8 and X-Net, which enables STORM-like superresolution microscopy image reconstruction from wide-field images with input-size flexibility. X-Microscopy was trained using samples of various subcellular structures, including cytoskeletal filaments, dot-like, beehive-like, and nanocluster-like structures, to generate prediction models capable of producing images of comparable quality to STORM-like images. In addition to enabling multicolour superresolution image reconstructions, X-Microscopy also facilitates superresolution image reconstruction from different conventional microscopic systems. The capabilities of X-Microscopy offer promising prospects for making superresolution microscopy accessible to a broader range of users, going beyond the confines of well-equipped laboratories.
RESUMEN
Somatic stem cells are essential for the maintenance of tissue homeostasis. Despite its importance, how the esophageal stratified squamous epithelium executes its self-renewal and maintenance remains elusive. In this study, using 5-bromo-2'-deoxyuridine label-chase in rats in vivo and rat esophageal organoids in vitro together with genome-wide DNA methylation and single-cell RNA sequencing, we identified a slow-cycling/quiescent stem cell population that contained high levels of hemidesmosomes (HDs) and low levels of Wnt signaling localized spatially and randomly at the basal layer of the esophageal epithelium. Pseudotime cell trajectory analysis indicated that tissue cells originated from quiescent basal stem cells in the basal layer. Perturbations of HD component expression and/or Wnt signaling reduced the stem cell population in the basal layer of esophageal keratinocyte organoids, resulting in alterations in the organoid formation rate, size, morphogenesis, and proliferation-differentiation homeostasis. Furthermore, not only high levels of HDs and low levels of Wnt signaling but also an interplay between HD and Wnt signaling defined the stem cells of the basal layer. Hence, HDs and Wnt signaling are critical determinants for defining the stem cells of the basal layer required for tissue homeostasis in mammalian esophagi.
Asunto(s)
Carcinoma de Células Escamosas , Células Madre , Ratas , Animales , Células Madre/metabolismo , Epitelio/metabolismo , Esófago/metabolismo , Diferenciación Celular , Carcinoma de Células Escamosas/metabolismo , Vía de Señalización Wnt , Proliferación Celular , MamíferosRESUMEN
OBJECTIVE: To investigate the mechanism of loss of human esophageal cancer-related gene 4 (ECRG4) expression in esophageal squamous cell carcinoma (ESCC.) METHODS: PCR-SSCP and DNA sequencing analysis were used to detect the mutation of ECRG4 exons in esophageal cancer and matched adjacent normal tissues of 80 patients. DNA bisulfite-modifying ssPCR sequencing assay was used to examine the methylation status of ECRG4 promoter in human esophageal squamous cell carcinoma EC9706 cells. The re-expression of ECRG4 mRNA was examined by RT-PCR in EC9706 cells, after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. RESULTS: No mutation in the four ECRG4 exons was found in all the ESCC and matched normal adjacent tissues. RT-PCR showed that 11 of 16 CpG islands of ECRG4 promoter were hypermethylated, while ECRG4 mRNA expression level was undetectable in the EC9706 cells. The ECRG4 mRNA was re-expressed after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. CONCLUSION: The epigenetic mechanism of methylation is a reason of loss of ECRG4 gene expression in the ESCC cell line EC9706.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Islas de CpG/genética , Metilación de ADN , Neoplasias Esofágicas/metabolismo , Proteínas de Neoplasias/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Trióxido de Arsénico , Arsenicales/farmacología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Decitabina , Epigénesis Genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Exones , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mutación , Proteínas de Neoplasias/genética , Óxidos/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteínas Supresoras de TumorRESUMEN
BACKGROUND: Cancer stem cells (CSCs) are considered the main cause of cancer recurrence and metastasis, and DNA methylation is involved in the maintenance of CSCs. However, the methylation profile of esophageal CSCs remains unknown. METHODS: Side population (SP) cells were isolated from esophageal squamous cell carcinoma (ESCC) cell lines KYSE150 and EC109. Sphere-forming cells were collected from human primary esophageal cancer cells. SP cells and sphere-forming cells were used as substitutes for cancer stem-like cells. We investigated the genome-wide DNA methylation profile in esophageal cancer stem-like cells using reduced representation bisulfite sequencing (RRBS). RESULTS: Methylated cytosine (mC) was found mostly in CpG dinucleotides, located mostly in the intronic, intergenic, and exonic regions. Forty intersected differentially methylated regions (DMRs) were identified in these 3 groups of samples. Thirteen differentially methylated genes with the same alteration trend were detected; these included OTX1, SPACA1, CD163L1, ST8SIA2, TECR, CADM3, GRM1, LRRK1, CHSY1, PROKR2, LINC00658, LOC100506688, and NKD2. DMRs covering ST8SIA2 and GRM1 were located in exons. These differentially methylated genes were involved in 10 categories of biological processes and 3 cell signaling pathways. CONCLUSIONS: When compared to non-CSCs, cancer stem-like cells have a differential methylation status, which provides an important biological base for understanding esophageal CSCs and developing therapeutic targets for esophageal cancer.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Neoplasias Esofágicas/genética , Regulación Neoplásica de la Expresión Génica , Estudio de Asociación del Genoma Completo , Células Madre Neoplásicas/metabolismo , Biomarcadores de Tumor , Línea Celular Tumoral , Biología Computacional/métodos , Islas de CpG , Bases de Datos Genéticas , Epigenómica/métodos , Neoplasias Esofágicas/mortalidad , Neoplasias Esofágicas/patología , Citometría de Flujo , Humanos , Células Madre Neoplásicas/patología , PronósticoRESUMEN
The clinical outcome of neuroblastoma (NB) has significantly improved in the last 30 years for patients with localized disease; however, the overall survival (OS) for patients with metastasis remains poor. Apatinib, a selective inhibitor of the vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase, which was discovered to be highly associated with metastasis, has been reported to exert antitumor effects in numerous types of cancer. However, the effect of apatinib in NB remains relatively unknown. The present study aimed to investigate the antitumor effects of apatinib in NB cells in vitro. The results revealed that apatinib inhibited cell viability and colony formation, whilst inducing cell cycle arrest and the apoptosis of NB cells. Additionally, apatinib inhibited the migration and invasion of NB cells, in addition to promoting the autophagy of NB cells. Western blotting demonstrated that the protein expression levels of phosphorylated (p)-AKT, p-mTOR and p-P70S6K, and downstream molecules associated with the cell cycle and apoptosis, such as cyclin D1 and the Bcl-2/Bax ratio of NB cells, were significantly decreased following treatment with apatinib. In addition, western blotting and immunofluorescence assays identified that the expression level of microtubule-associated protein 1A/1B-light chain 3-II, which is expressed in autophagosomes, was upregulated following apatinib treatment. In conclusion, the findings of the present study suggested that apatinib may induce apoptosis and autophagy via the PI3K/AKT/mTOR and mitogen-activated protein kinase/ERK signaling pathways in NB cells. Thus, apatinib may be a potential antitumor agent for the clinical treatment of NB.
RESUMEN
Bone metastasis is common in late-stage breast cancer patients and leads to skeletal-related events that affect the quality of life and decrease survival. Numerous miRNAs have been confirmed to be involved in metastatic breast cancer, such as the miR200 family. Our previous study identified microRNA-429 (miR-429) as a regulatory molecule in breast cancer bone metastasis. However, the effects of miR-429 and its regulatory axis in the metastatic breast cancer bone microenvironment have not been thoroughly investigated. We observed a positive correlation between miR-429 expression in clinical tissues and the bone metastasis-free interval and a negative correlation between miR-429 expression and the degree of bone metastasis. We cultured bone metastatic MDA-MB-231 cells and used conditioned medium (CM) to detect the effect of miR-429 on osteoblast and osteoclast cells in vitro. We constructed an orthotopic bone destruction model and a left ventricle implantation model to examine the effect of miR-429 on the metastatic bone environment in vivo. The transfection experiments showed that the expression levels of V-crk sarcoma virus CT10 oncogene homolog-like (CrkL) and MMP-9 were negatively regulated by miR-429. The in vitro coculture experiments showed that miR-429 promoted osteoblast differentiation and that CrkL promoted osteoclast differentiation. The two animal models showed that miR-429 diminished local bone destruction and distant bone metastasis but CrkL enhanced these effects. Furthermore, CrkL and MMP-9 expression decreased simultaneously in response to increased miR-429 expression. These findings further reveal the possible mechanism and effect of the miR-429/CrkL/MMP-9 regulatory axis in the bone microenvironment in breast cancer bone metastasis.
Asunto(s)
Neoplasias Óseas , Neoplasias de la Mama , MicroARNs , Animales , Neoplasias Óseas/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metaloproteinasa 9 de la Matriz/genética , MicroARNs/genética , Metástasis de la Neoplasia , Calidad de Vida , Microambiente TumoralRESUMEN
The ECRG4 gene was initially identified and cloned in our laboratory from human normal esophageal epithelium (GenBank accession no. AF325503). We revealed the expression of ECRG4 protein was downregulated in 68.5% (89/130) ESCC samples using tissue microarray. The low ECRG4 protein expression was significantly associated with regional lymph node metastasis, primary tumor size, and tumor stage in ESCC (p < 0.05). ECRG4 mRNA expression was downregulated in ESCC due to the hypermethylation in the gene promoter. The treatment with 5-aza-2'-deoxycytidine, which is a DNA methyltransferase inhibitor restored ECRG4 mRNA expression in ESCC cells. The result indicated that promoter hypermethylation may be 1 main mechanism leading to the silencing of ECRG4. The high expression of ECRG4 in patients with ESCC was associated with longer survival compared with those with low ECRG4 expression by Kaplan-Meier survival analysis (p < 0.05). ECRG4 protein was an independent prognostic factor for ESCC by multivariable Cox proportional hazards regression analysis (p < 0.05). The restoration of ECRG4 expression in ESCC cells inhibited cell proliferation, colony formation, anchorage-independent growth, cell cycle progression and tumor growth in vivo (p < 0.05). The transfection of ECRG4 gene in ESCC cells inhibited the expression of NF-kappaB and nuclear translocation, in addition to the expression of COX-2, a NF-kappaB target gene, was attenuated. Taken together, ECRG4 is a novel candidate tumor suppressor gene in ESCC, and ECRG4 protein is a candidate prognostic marker for ESCC.