RESUMEN

Exosomal microRNAs (miRNAs) from cancer cells play a key role in mediating the oral squamous cell carcinoma (OSCC) microenvironment. The objective of this study was to investigate how the long non-coding RNA (lncRNA) MEG3 affects OSCC angiogenesis through exosomal miR-421. Global miRNA microarray analysis and quantitative real-time PCR (qRT-PCR) were performed to determine the level of miRNAs in OSCC cell-derived exosomes. Cell migration, invasion, tube formation, immunohistochemistry, and hemoglobin concentrations were used to study the effects of exosomal miR-421 in angiogenesis. Western blotting was used to determine the expression level of HS2ST1 and VEGFR2-related downstream proteins. MiRNA array and qRT-PCR identified the upregulation of miR-421 in OSCC cell-derived exosomes. Furthermore, exosomal miR-421 can be taken up by human umbilical vein endothelial cells (HUVECs) and then target HS2ST1 through VEGF-mediated ERK and AKT phosphorylation, thereby promoting HUVEC migration, invasion, and tube formation. Additionally, forced expression of the lncRNA MEG3 in OSCC cells reduced exosomal miR-421 levels and then increased HS2ST1 expression, thereby reducing the VEGF/VEGFR2 pathway in HUVECs. Our results demonstrate a novel mechanism by which lncRNA MEG3 can act as a tumor suppressor and regulate endothelial angiogenesis through the exosomal miR-421/HS2ST1 axis, which provides a potential therapeutic strategy for OSCC angiogenesis.

Asunto(s)

Carcinoma de Células Escamosas , Movimiento Celular , Exosomas , Regulación Neoplásica de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , MicroARNs , Neoplasias de la Boca , Neovascularización Patológica , ARN Largo no Codificante , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Exosomas/metabolismo , Exosomas/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Movimiento Celular/genética , Línea Celular Tumoral , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Angiogénesis

RESUMEN

The cytokine-inducible Src homology 2-containing protein (CISH) is an endogenous suppressors of signal transduction and activator of transcription (STAT) and acts as a key negative regulator of inflammatory cytokine responses. Downregulation of CISH has been reported to associate with increased activation of STAT and enhanced inflammatory pathways. However, whether microRNAs (miRNAs) play a crucial role in CISH/STAT regulation in oral squamous cell carcinoma (OSCC) remains unknown. The expression of CISH on OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Specific targeting by miRNAs was determined by software prediction, luciferase reporter assay, and correlation with target protein expression. The functions of miR-944 and CISH were accessed by transwell migration and invasion analyses using gain- and loss-of-function approaches. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR were used to evaluate the pro-inflammation cytokines expression under the miR-944, CISH, NNK or combinations treatment. We found that the CISH protein, which modulates STAT3 activity, as a direct target of miR-944. CISH protein was significantly down-regulated in OSCC patients and cell lines and its level was inversely correlated with miR-944 expression. The miR-944-induced STAT3 phosphorylation, pro-inflammation cytokines secretion, migration and invasion were abolished by CISH restoration, suggesting that the oncogenic activity of miR-944 is CISH dependent. Furthermore, tobacco extract (NNK) may contribute to miR-944 induction and STAT3 activation. Antagomir-mediated inactivation of miR-944 prevented the NNK-induced STAT3 phosphorylation and pro-inflammation cytokines secretion. Altogether, these data demonstrate that NNK-induced miR944 expression plays an important role in CISH/STAT3-mediated inflammatory response and activation of tumor malignancy.

Asunto(s)

Fumar Cigarrillos/efectos adversos , MicroARNs/genética , Neoplasias de la Boca/etiología , Neoplasias de la Boca/metabolismo , Interferencia de ARN , Factor de Transcripción STAT3/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/genética , Regiones no Traducidas 3' , Biomarcadores , Línea Celular Tumoral , Susceptibilidad a Enfermedades , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Humanos , Inmunohistoquímica , Neoplasias de la Boca/patología , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal

RESUMEN

BACKGROUND: Epigenetic silencing of retinoic acid (RA) signaling-related genes have been linked with the pathogenesis and clinical outcome in oral squamous cell carcinoma (OSCC) carcinogenesis. However, the precise mechanisms underlying the abnormal silencing of RA signaling-related genes in OSCC have not been well investigated. METHODS: Using combined analysis of genome-wide gene expression and methylation profile from 40 matched normal-tumor pairs of OSCC specimens, we found a set of retinoid signaling related genes are frequently hypermethylated and downregulated in OSCC patient samples, including alcohol dehydrogenase, iron containing 1 (ADHFE1) and aldehyde dehydrogenase 1 family, member A2 (ALDH1A2), which are the important rate-limiting enzymes in synthesis of RA. The expression of ADHFE1 and ALDH1A2 in OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. The binding sites of miR-30a and miR-379 with DNA methyltransferase 3B (DNMT3B) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and Western blot analyses. The functions of miR-30a, miR-379, and DNMT3B were accessed by growth and colony formation analyses using gain- and loss-of-function approaches. Chromatin immunoprecipitation (ChIP) was performed to explore the molecular mechanisms by arecoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment. RESULTS: We demonstrated that deregulated miR-30a and miR-379 could represent a mechanism for the silencing of ADHFE1 and ALDH1A2 in OSCC through targeting DNMT3B. Ectopic expression of miR-30a and miR-379 could induce re-expression of methylation-silenced ADHFE1 and ALDH1A2, and lead to growth inhibition in oral cancer cells. Furthermore, the dysregulation of the miRNAs and DNMT-3B may result from exposure to tobacco smoking and betel quid chewing. CONCLUSIONS: Our results demonstrate that tobacco smoking and betel quid chewing could repress miR-30a and miR-379, which upregulate the DNMT3B expression, in turn, lead to the hypermethylation of ADHFE1 and ALDH1A genes, consequently, promote the oncogenic activity. These findings highlight the potential use of retinoids in combination with epigenetic modifiers for the prevention or treatment of oral cancer.

Asunto(s)

Carcinoma de Células Escamosas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Silenciador del Gen , MicroARNs/genética , Neoplasias de la Boca/genética , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Familia de Aldehído Deshidrogenasa 1/genética , Familia de Aldehído Deshidrogenasa 1/metabolismo , Arecolina/química , Carcinogénesis/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Redes y Vías Metabólicas , MicroARNs/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Nitrosaminas/química , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismo , Tretinoina/metabolismo , ADN Metiltransferasa 3B

RESUMEN

BACKGROUND: Discoidin domain receptor-1 (DDR1) tyrosine kinase is highly expressed in a variety of human cancers and involved in various steps of tumorigenesis. However, the precise mechanisms underlying the abnormal expression of DDR1 in oral squamous cell carcinoma (OSCC) has not been well investigated. METHODS: The expression of DDR1 on OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Specific targeting by miRNAs was determined by software prediction, luciferase reporter assay, and correlation with target protein expression. The functions of miR-486-3p and DDR1 were accessed by MTT and Annexin V analyses using gain- and loss-of-function approaches. Chromatin immunoprecipitation (ChIP) and methylation specific PCR (MSP) were performed to explore the molecular mechanisms by arecoline treatment. RESULTS: Here, we reported that DDR1 was significantly upregulated in OSCC tissues and its levels were inversely correlated with miR-486-3p expression. The experimental results in vitro confirmed that miR-486-3p decreased DDR1 expression by targeting the 3'-UTR of DDR1 mRNA. Overexpression of miR-486-3p led to growth inhibition and apoptosis induction with a similar function by knockdown of DDR1. Aberrant methylation of ANK1 promoter was a highly prevalent in OSCC and contributes to oral carcinogenesis by epigenetic silencing of ANK1 and miR-486-3p. We found that miR-486-3p can be transcriptionally co-regulated with its host gene ANK1 through epigenetic repression. DNA methylation inhibitor treatment re-expressed ANK1 and miR-486-3p. Importantly, arecoline, a major betel nut alkaloid, recruited DNMT3B binding to ANK1 promoter for DNA methylation and then attenuated the expression of miR-486-3p in OSCC. CONCLUSION: This study was the first to demonstrate that betel nut alkaloid may recruit DNMT3B to regulate miR-486-3p/DDR1 axis in oral cancer andmiR-486-3p and DDR1 may serve as potential therapeutic targets of oral cancer.

Asunto(s)

Carcinoma de Células Escamosas/genética , Receptor con Dominio Discoidina 1/genética , Genes Supresores de Tumor , MicroARNs/metabolismo , Neoplasias de la Boca/genética , Regiones no Traducidas 3' , Anciano , Ancirinas/química , Ancirinas/genética , Apoptosis/genética , Arecolina/farmacología , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , Receptor con Dominio Discoidina 1/metabolismo , Células HEK293 , Humanos , MicroARNs/genética , Neoplasias de la Boca/metabolismo , Regiones Promotoras Genéticas , ADN Metiltransferasa 3B

RESUMEN

Epigenetic correlates of the head and neck cancer may illuminate its pathogenic roots. Through a gene set enrichment analysis, we found that the oncogenic transcription factor RUNX2 is widely upregulated in the head and neck squamous cell carcinoma (HNSCC) with lymph node metastasis, where it also predicts poor prognosis in patients with HNSCC. Enforced expression of ectopic RUNX2 promoted the metastatic capabilities of HNSCC, whereas RUNX2 silencing inhibited these features. Mechanistic investigations showed that manipulating levels of activin A (INHBA) could rescue or compromise the RUNX2-mediated metastatic capabilities of HNSCC cells. Furthermore, we found that miR-376c-3p encoded within the 3'-untranslated region of RUNX2 played a pivotal role in regulating RUNX2 expression in highly metastatic HNSCC cells, where it was downregulated commonly. Restoring miR-376c expression in this setting suppressed expression of RUNX2/INHBA axis along with metastatic capability. Clinically, we observed an inverse relationship between miR-376c-3p expression and the RUNX2/INHBA axis in HNSCC specimens. In summary, our results defined a novel pathway in which dysregulation of the RUNX2/INHBA axis due to miR-376c downregulation fosters lymph node metastasis in HNSCC. Cancer Res; 76(24); 7140-50. ©2016 AACR.

Asunto(s)

Carcinoma de Células Escamosas/patología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias de Cabeza y Cuello/patología , Subunidades beta de Inhibinas/metabolismo , MicroARNs/biosíntesis , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Inmunoprecipitación de Cromatina , Regulación hacia Abajo , Femenino , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Hibridación in Situ , Metástasis Linfática , Ratones , Ratones Endogámicos NOD , Ratones SCID , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Transducción de Señal/fisiología , Carcinoma de Células Escamosas de Cabeza y Cuello , Análisis de Matrices Tisulares

RESUMEN

Microtubule inhibitors have been shown to inhibit Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal transduction pathway in various cancer cells. However, little is known of the mechanism by which the microtubule inhibitors inhibit STAT3 activity. In the present study, we examined the effect of a novel small-molecule microtubule inhibitor, MPT0B098, on STAT3 signaling in oral squamous cell carcinoma (OSCC). Treatment of various OSCC cells with MPT0B098 induced growth inhibition, cell cycle arrest and apoptosis, as well as increased the protein level of SOCS3. The accumulation of SOCS3 protein enhanced its binding to JAK2 and TYK2 which facilitated the ubiquitination and degradation of JAK2 and TYK2, resulting in a loss of STAT3 activity. The inhibition of STAT3 activity led to sensitization of OSCC cells to MPT0B098 cytotoxicity, indicating that STAT3 is a key mediator of drug resistance in oral carcinogenesis. Moreover, the combination of MPT0B098 with the clinical drug cisplatin or 5-FU significantly augmented growth inhibition and apoptosis in OSCC cells. Taken together, our results provide a novel mechanism for the action of MPT0B098 in which the JAK2/STAT3 signaling pathway is suppressed through the modulation of SOCS3 protein level. The findings also provide a promising combinational therapy of MPT0B098 for OSCC.

Asunto(s)

Carcinoma de Células Escamosas/patología , Indoles/farmacología , Janus Quinasa 2/metabolismo , Neoplasias de la Boca/patología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Regulación hacia Abajo/efectos de los fármacos , Interacciones Farmacológicas , Retroalimentación Fisiológica/efectos de los fármacos , Fluorouracilo/farmacología , Humanos , Janus Quinasa 2/genética , Multimerización de Proteína/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína , Factor de Transcripción STAT3/genética , TYK2 Quinasa/metabolismo , Tubulina (Proteína)/química , Moduladores de Tubulina/farmacología

RESUMEN

Suppressor of cytokine signaling (SOCS) proteins are negative feedback regulators of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Dysregulation of SOCS protein expression in cancers can be one of the mechanisms that maintain STAT activation, but this mechanism is still poorly understood in oral squamous cell carcinoma (OSCC). Here, we report that SOCS2 protein is significantly downregulated in OSCC patients and its levels are inversely correlated with miR-424-5p expression. We identified the SOCS2 protein, which modulates STAT5 activity, as a direct target of miR-424-5p. The miR-424-5p-induced STAT5 phosphorylation, matrix metalloproteinases (MMPs) expression, and cell migration and invasion were blocked by SOCS2 restoration, suggesting that miR-424-5p exhibits its oncogenic activity through negatively regulating SOCS2 levels. Furthermore, miR-424-5p expression could be induced by the cytokine IL-8 primarily through enhancing STAT5 transcriptional activity rather than NF-κB signaling. Antagomir-mediated inactivation of miR-424-5p prevented the IL-8-induced cell migration and invasion, indicating that miR-424-5p is required for IL-8-induced cellular invasiveness. Taken together, these data indicate that STAT5-dependent expression of miR-424-5p plays an important role in mediating IL-8/STAT5/SOCS2 feedback loop, and scavenging miR-424-5p function using antagomir may have therapeutic potential for the treatment of OSCC.

Asunto(s)

Carcinoma de Células Escamosas/patología , Interleucina-8/metabolismo , MicroARNs/genética , Neoplasias de la Boca/patología , Boca/patología , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Boca/metabolismo , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas Supresoras de la Señalización de Citocinas/genética

RESUMEN

Amyloidogenic processing of amyloid-ß precursor protein (AßPP) is associated with cholesterol- and sphingolipid-rich lipid rafts. Caveolin-1, a raft-residing protein, has been implicated in the pathogenesis of Alzheimer's disease. To determine the role of caveolin-1 in governing γ-secretase-mediated AßPP proteolysis, cellular γ-secretase activity was assessed in response to alteration in caveolin-1 expression. We demonstrated that suppression of caveolin-1 expression by RNA interference resulted in a significant increase in γ-secretase-mediated proteolysis of AßPP, generation of amyloid-ß, and cleavage of Notch. Overexpression of caveolin-1 attenuated γ-secretase-mediated proteolysis of AßPP and Notch, substantiating the negative regulation of γ-secretase by caveolin-1. Furthermore, we found that cells deficient in caveolin-1 exhibited significantly increased co-localization of γ-secretase with clathrin-coated non-caveolar endocytic vesicles, demonstrating that the partitioning of γ-secretase between caveolar and non-caveolar membranes can be modulated by caveolin-1. Our data also showed that JNK activation is essential for caveolin-1-mediated regulation of γ-secretase. Together, our results strongly suggest that caveolin-1 is an important regulator of γ-secretase activity.

Asunto(s)

Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Caveolina 1/metabolismo , Membrana Celular/enzimología , Microdominios de Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Línea Celular Transformada , Membrana Celular/ultraestructura , Ensayo de Inmunoadsorción Enzimática/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , MAP Quinasa Quinasa 4/metabolismo , Microdominios de Membrana/efectos de los fármacos , Microscopía Confocal/métodos , Mutación/genética , ARN Interferente Pequeño/farmacología , Receptores Notch/genética , Receptores Notch/metabolismo , Fracciones Subcelulares , Transfección