RESUMEN
BACKGROUND AND OBJECTIVES: MicroRNAs play a crucial role in the progression of various cancers, and microRNA-221 (miR-221) has been observed to be significantly overexpressed in osteosarcoma (OS) cells. FBXW11, a vital F-box protein of the ubiquitin-proteasome system, mediates the proliferation and survival of cancer cells by targeting multiple substrates for degradation. FBXW11 inhibits OS growth and metastasis by antagonizing the ß-catenin/Wnt signaling pathway. Therefore, we hypothesized that miR-221 targets FBXW11 to mediate Wnt signaling and promote OS proliferation. METHODS: In this study, we demonstrated the increased expression of miR-221 and FBXW11 in OS tissues and cell lines by real-time polymerase chain reaction (RT-PCR). Moreover, to elucidate the regulatory mechanism(s) of miR-221 and FBXW11 in progression, cell viability and apoptosis were analyzed by the MTT assay and flow cytometry, respectively. RESULTS: The results showed that the overexpression of miR-221 in OS cells dramatically promoted cell growth and cell cycle progression, and inhibited apoptosis, whereas miR-221 inhibitors conversely inhibited proliferation and promoted apoptosis in OS cells. The data also showed that FBXW11 directly targeted miR-221 and miR-221 regulated OS cell proliferation and apoptosis by binding to FBXW11. We further confirmed that miR-221 targeted FBXW11 to promote proliferation and inhibit apoptosis in OS cell lines by inhibiting Wnt signaling. INTERPRETATION AND CONCLUSIONS: Overall, our study revealed a functional mechanism for miR-221 in OS. Further studies will elucidate its role in the progression of OS and inhibiting miR-221 may represent a useful treatment strategy.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Neoplasias Óseas/metabolismo , MicroARNs/metabolismo , Osteosarcoma/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización Wnt , Proteínas con Repetición de beta-Transducina/metabolismo , Adolescente , Adulto , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Niño , Progresión de la Enfermedad , Femenino , Humanos , Masculino , MicroARNs/biosíntesis , MicroARNs/genética , Persona de Mediana Edad , Osteosarcoma/genética , Osteosarcoma/patología , Transfección , Ubiquitina-Proteína Ligasas/biosíntesis , Ubiquitina-Proteína Ligasas/genética , Adulto Joven , Proteínas con Repetición de beta-Transducina/biosíntesis , Proteínas con Repetición de beta-Transducina/genéticaRESUMEN
Epigenetic changes frequently occur during tumorigenesis and DNA hypermethylation may account for the inactivation of tumor suppressor genes in cancer cells. Studies in Multiple Myeloma (MM) have shown variable DNA methylation patterns with focal hypermethylation changes in clinically aggressive subtypes. We studied global methylation patterns in patients with relapsed/refractory MM and found that the majority of methylation peaks were located in the intronic and intragenic regions in MM samples. Therefore, we investigated the effect of methylation on miRNA regulation in MM. To date, the mechanism by which global miRNA suppression occurs in MM has not been fully described. In this study, we report hypermethylation of miRNAs in MM and perform confirmation in MM cell lines using bisulfite sequencing and methylation-specific PCR (MSP) in the presence or absence of the DNA demethylating agent 5-aza-2'-deoxycytidine. We further characterized the hypermethylation-dependent inhibition of miR-152, -10b-5p and -34c-3p which was shown to exert a putative tumor suppressive role in MM. These findings were corroborated by the demonstration that the same miRNAs were down-regulated in MM patients compared to healthy individuals, alongside enrichment of miR-152-, -10b-5p, and miR-34c-3p-predicted targets, as shown at the mRNA level in primary MM cells. Demethylation or gain of function studies of these specific miRNAs led to induction of apoptosis and inhibition of proliferation as well as down-regulation of putative oncogene targets of these miRNAs such as DNMT1, E2F3, BTRC and MYCBP. These findings provide the rationale for epigenetic therapeutic approaches in subgroups of MM.
Asunto(s)
Metilación de ADN/genética , Epigénesis Genética , MicroARNs/genética , Mieloma Múltiple/genética , Apoptosis/genética , Azacitidina/administración & dosificación , Azacitidina/análogos & derivados , Línea Celular Tumoral , Proliferación Celular/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Decitabina , Factor de Transcripción E2F3/biosíntesis , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/biosíntesis , Mieloma Múltiple/patología , ARN Mensajero/biosíntesis , Factores de Transcripción/biosíntesis , Proteínas con Repetición de beta-Transducina/biosíntesisRESUMEN
This study investigated the mechanism by which the transcription factor Sp1 is degraded in prostate cancer cells. We recently developed a thiazolidinedione derivative, (Z)-5-(4-hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexyl)-thiazolidine-2,4-dione (OSU-CG12), that induces Sp1 degradation in a manner paralleling that of glucose starvation. Based on our finding that thiazolidinediones suppress beta-catenin and cyclin D1 by up-regulating the E3 ligase SCF(beta-TrCP), we hypothesized that beta-transducin repeat-containing protein (beta-TrCP) targets Sp1 for proteasomal degradation in response to glucose starvation or OSU-CG12. Here we show that either treatment of LNCaP cells increased specific binding of Sp1 with beta-TrCP. This direct binding was confirmed by in vitro pull-down analysis with bacterially expressed beta-TrCP. Although ectopic expression of beta-TrCP enhanced the ability of OSU-CG12 to facilitate Sp1 degradation, suppression of endogenous beta-TrCP function by a dominant-negative mutant or small interfering RNA-mediated knockdown blocked OSU-CG12-facilitated Sp1 ubiquitination and/or degradation. Sp1 contains a C-terminal conventional DSG destruction box ((727)DSGAGS(732)) that mediates beta-TrCP recognition and encompasses a glycogen synthase kinase 3beta (GSK3beta) phosphorylation motif (SXXXS). Pharmacological and molecular genetic approaches and mutational analyses indicate that extracellular signal-regulated kinase-mediated phosphorylation of Thr739 and GSK3beta-mediated phosphorylation of Ser728 and Ser732 were critical for Sp1 degradation. The ability of OSU-CG12 to mimic glucose starvation to activate beta-TrCP-mediated Sp1 degradation has translational potential to foster novel strategies for cancer therapy.
Asunto(s)
Glucosa/deficiencia , Factor de Transcripción Sp1/metabolismo , Tiazolidinedionas/farmacología , Proteínas con Repetición de beta-Transducina/biosíntesis , Línea Celular Tumoral , Femenino , Glucógeno Sintasa Quinasa 3/fisiología , Glucógeno Sintasa Quinasa 3 beta , Humanos , MAP Quinasa Quinasa 1/fisiología , Masculino , Fosforilación , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/genética , Regulación hacia ArribaRESUMEN
OBJECTIVE: To identify the expression of antagonist beta-TrCP protein in Sonic hedgehog signal transduction pathway and Wnt signal transduction pathway in hair follicle tissues. METHODS: The heads of day 18 embryo, and one day and six-days-old postnatal mice were acquired and treated with 40 g/L paraformaldehyde fixation for 48 h and paraffin embedding. The expression of beta-TrCP proteins was examined using LsAB (labelled streptavidin-biotin) method. RESULTS: beta-TrCP proteins were expressed in the cytoplasm of the hair stems of hair follicle, hair cuticle, cuticle of root sheath, Huxley's layer of internal root sheath cells, external root sheath and mesenchymal tissues, but not in connective tissue sheath and Henle's layer of internal root sheath. CONCLUSION: TrCP express in the developmental hair follicle tissues, which implicates that beta-TrCP regulate the developmental hair follicle by mediating the signal transduction pathways.
Asunto(s)
Folículo Piloso/metabolismo , Transducción de Señal , Proteínas con Repetición de beta-Transducina/biosíntesis , Proteínas con Repetición de beta-Transducina/fisiología , Animales , Animales Recién Nacidos , Folículo Piloso/embriología , Folículo Piloso/crecimiento & desarrollo , Inmunohistoquímica , Ratones , Factores de TiempoRESUMEN
The permanent activation of the transcription factor nuclear factor-kappaB (NF-kappaB) in pancreatic cancer cells is associated with a profound resistance towards chemotherapy. In the present study, we show that chemoresistant pancreatic cancer cell lines exhibiting constitutive NF-kappaB activity (i.e., PancTu-1, BxPc3, and Capan-1) express significantly elevated levels of the E3-ubiquitin ligase receptor subunit betaTRCP1, compared with pancreatic carcinoma cell lines lacking constitutive NF-kappaB activity and chemoresistance (i.e., PT45-P1 and T3M4). If transfected with betaTRCP1, PT45-P1 cells exhibit an elevated NF-kappaB activity and become less sensitive towards anticancer drug treatment (i.e., etoposide). Conversely, blockade of betaTRCP1 expression in PancTu-1 cells by transfection with a vector-expressed small interfering RNA reduces NF-kappaB activation and chemoresistance. In PancTu-1 cells, betaTRCP1 expression is inhibited, at least in part, by the interleukin-1 (IL-1) receptor(I) antagonist, whereas stimulation of PT45-P1 cells with IL-1beta resulted in an increased expression of betaTRCP1, and transfection of this cell line with betaTRCP1 induced IL-1beta secretion in a NF-kappaB-dependent fashion. Thus, via its close and mutual link to IL-1beta secretion, betaTRCP1 expression might substantially contribute to the persistent, IL-1beta-dependent activation of NF-kappaB in pancreatic carcinoma cells. In support of this, betaTRCP1 expression is detectable at considerable levels in a great number of pancreatic ductal adenocarcinoma specimens, along with an intense staining for activated NF-kappaB. Altogether, our findings of the elevated betaTRCP1 expression in pancreatic carcinoma cells pinpoint to another important mediator of constitutive NF-kappaB activation and thereby of chemoresistance.