RESUMEN
Diffuse-type gastric cancer (DGC) is a highly invasive subtype of gastric adenocarcinoma that frequently exhibits scattered peritoneal metastasis. Previous studies have shown that the genes of receptor tyrosine kinases (RTKs), such as fibroblast growth factor receptor 2 (FGFR2) or Met, are amplified in some DGC cell lines, leading to the constitutive activation of corresponding RTKs. In these cell lines, the survival of cancer cells appears to be dependent on the activation of RTKs. To gain novel insights into the downstream signaling pathways of RTKs specific to DGC, phosphotyrosine-containing proteins associated with activated FGFR2 were purified through two sequential rounds of immunoprecipitation from the lysates of two DGC cell lines. As a result, transferrin receptor 1 (TfR1) was identified as the binding partner of FGFR2. Biochemical analysis confirmed that TfR1 protein binds to FGFR2 and is phosphorylated at tyrosine 20 (Tyr20) in an FGFR2 kinase activity-dependent manner. The knockdown of TfR1 and treatment with an inhibitor of FGFR2 caused significant impairment in iron uptake and suppression of cellular proliferation in vitro. Moreover, the suppression of expression levels of TfR1 in the DGC cells significantly reduced their tumorigenicity and potency of peritoneal dissemination. It was indicated that TfR1, when phosphorylated by the binding partner FGFR2 in DGC cells, promotes proliferation and tumorigenicity of these cancer cells. These results suggest that the control of TfR1 function may serve as a therapeutic target in DGC with activated FGFR2.
Asunto(s)
Neoplasias Gástricas , Línea Celular Tumoral , Humanos , Fosforilación , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Neoplasias Gástricas/patología , Tirosina/metabolismoRESUMEN
Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MßCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MßCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MßCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism.
Asunto(s)
Caspasa 8/metabolismo , Colesterol/metabolismo , Dacarbazina/análogos & derivados , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Microdominios de Membrana/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Antineoplásicos Alquilantes/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Dacarbazina/administración & dosificación , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Glioblastoma/patología , Humanos , Líquido Intracelular/metabolismo , Microdominios de Membrana/efectos de los fármacos , TemozolomidaRESUMEN
The epithelial-mesenchymal transition (EMT) is associated with tumor progression. We reported previously that expression of the δEF1 family proteins (δEF1/ZEB1 and SIP1/ZEB2), key regulators of the EMT, is positively correlated with EMT phenotypes and aggressiveness of breast cancer. Here, we show that the expression levels of regulator of G-protein signaling 16 (RGS16) are negatively correlated with those of the δEF1 family proteins. On the basis of the results of gain- and loss-of-function analyses, we suggest that δEF1 family proteins promote cell motility of breast cancer cells directly or indirectly through repressing expression of RGS16.
Asunto(s)
Movimiento Celular/fisiología , Proteínas de Homeodominio/fisiología , Proteínas RGS/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/fisiología , Humanos , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Temozolomide (TMZ) is one of the few effective anticancer agents against gliomas. However, acquisition of TMZ resistance or adaptation by gliomas is currently a crucial problem, especially increased invasiveness which is critical for the determination of clinical prognosis. This study investigated the molecular regulatory mechanisms of TMZ resistance in gliomas involved in invasiveness, particularly invadopodia formation, a molecular complex formed at the invasive front to cause extracellular matrix degradation during cellular local invasion. The TMZ-resistant clone of the U343 MG human glioma cell line (U343-R cells) was established. U343-R cells demonstrated higher invadopodia formation compared with U343 cells without TMZ resistance (U343-Con cells). Immunoblot analysis of DNA damage-related mitogen-activated protein kinase signals found increased phosphorylation of c-Jun terminal kinase (JNK) and higher activation of its downstream signaling in U343-R cells compared with U343-Con cells. Treatment of U343-R cells with specific inhibitors of JNK or siRNA targeting JNK suppressed up-regulation of invadopodia formation. In addition, paxillin, one of the known JNK effectors which is phosphorylated and affects cell migration, was phosphorylated at serine 178 in JNK activity-dependent manner. Expression of paxillin with mutation of the serine 178 phosphorylation site in U343-R cells blocked invadopodia formation. The present findings suggest that increased formation of invadopodia in U343-R cells is mediated by hyperactivation of JNK-paxillin signaling, and both JNK and paxillin might become targets of novel therapies against TMZ-resistant gliomas.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Dacarbazina/análogos & derivados , Resistencia a Antineoplásicos , Glioma/tratamiento farmacológico , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Paxillin/metabolismo , Podosomas/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Dacarbazina/farmacología , Glioma/metabolismo , Glioma/patología , Humanos , Fosforilación/efectos de los fármacos , Podosomas/metabolismo , Podosomas/patología , Transducción de Señal/efectos de los fármacos , TemozolomidaRESUMEN
Epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Transforming growth factor (TGF)-ß induces EMT in mouse epithelial cells. During prolonged treatment, TGF-ß successively induces myofibroblastic differentiation with increased expression of myofibroblast marker proteins, including smooth muscle α actin and calponin. We recently showed that fibroblast growth factor-2 prevented myofibroblastic differentiation induced by TGF-ß, and transdifferentiated the cells to those with much more aggressive characteristics (enhanced EMT). To identify the molecular markers specifically expressed in cells undergoing enhanced EMT induced by the combination of TGF-ß and fibroblast growth factor-2, we carried out a microarray-based analysis and found that integrin α3 (ITGA3) and Ret were upregulated. Intriguingly, ITGA3 was also overexpressed in breast cancer cells with aggressive phenotypes and its expression was correlated with that of δEF-1, a key regulator of EMT. Moreover, the expression of both genes was downregulated by U0126, a MEK 1/2 inhibitor. Therefore, ITGA3 is a potential marker protein for cells undergoing enhanced EMT and for cancer cells with aggressive phenotypes, which is positively regulated by δEF-1 and the MEK-ERK pathway.
Asunto(s)
Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Integrina alfa3/genética , Integrina alfa3/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Células Cultivadas , Regulación hacia Abajo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Perfilación de la Expresión Génica/métodos , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Miofibroblastos/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia ArribaRESUMEN
Bone morphogenetic proteins (BMPs) regulate various biological processes, mostly mediated by cells of mesenchymal origin. However, the roles of BMPs in epithelial cells are poorly understood. Here, we demonstrate that, in polarized epithelial cells, BMP signals are transmitted from BMP receptor complexes exclusively localized at the basolateral surface of the cell membrane. In addition, basolateral stimulation with BMP increased expression of components of tight junctions and enhanced the transepithelial resistance (TER), counteracting reduction of TER by treatment with TGF-ß or an anti-tumor drug. We conclude that BMPs maintain epithelial polarity via intracellular signaling from basolaterally localized BMP receptors.
Asunto(s)
Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 6/genética , Receptores de Proteínas Morfogenéticas Óseas/genética , Células Epiteliales/efectos de los fármacos , Factor 2 de Diferenciación de Crecimiento/genética , Transducción de Señal , Animales , Proteína Morfogenética Ósea 4/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Proteína Morfogenética Ósea 6/metabolismo , Proteína Morfogenética Ósea 6/farmacología , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular , Polaridad Celular/efectos de los fármacos , Cisplatino/farmacología , Perros , Células Epiteliales/citología , Células Epiteliales/metabolismo , Expresión Génica , Factor 2 de Diferenciación de Crecimiento/metabolismo , Factor 2 de Diferenciación de Crecimiento/farmacología , Ratones , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/genética , Uniones Estrechas/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Genetic alterations and deregulation of the miRNA biogenesis pathway components have been reported in human tumors. Tissue-specific deletion of the Dicer gene, which encodes an essential miRNA processing enzyme, promotes carcinogenesis in animal models. These features indicate that aberrant miRNA biogenesis components are directly associated with cancer. For the present study, we conducted quantitative RT-PCR of 14 genes that are related to the miRNA biogenesis pathway in 47 paired samples of primary hepatocellular carcinoma (HCC) and matched non-cancerous liver. Expression of seven genes (Dgcr8, p68, p72, Dicer, Ago3, Ago4 and Piwil4) was significantly decreased in primary HCC, especially in non-viral HCC subtypes, compared to the non-cancerous liver. Combinations of decreased expression of the miRNA biogenesis components in non-cancerous liver were related to cigarette smoking, alcohol intake and diabetes, which are known to be risk factors for HCC, and were also associated with the occurrence of multicentric tumors. Reduction of two of these genes (Dicer and p68) in HCC was associated with poor prognosis. Trimethylation of histone H3 lysine 27 in the promoters is implicated in the deregulation of these miRNA-biogenesis-related genes in non-HBV genome integrated HCC cell lines. In conclusion, deregulation of the miRNA biogenesis pathway components is frequently observed in non-viral-associated HCC and is linked to etiological risk factors and poor prognosis. Our study further showed that epigenetic regulation could be implicated in the deregulation of these genes during hepatocarcinogenesis.
Asunto(s)
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , MicroARNs/genética , Adulto , Anciano , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Células Hep G2 , Histonas/genética , Humanos , Masculino , MicroARNs/biosíntesis , Persona de Mediana Edad , Pronóstico , Regiones Promotoras Genéticas , Factores de RiesgoRESUMEN
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. We sequenced and analyzed the whole genomes of 27 HCCs, 25 of which were associated with hepatitis B or C virus infections, including two sets of multicentric tumors. Although no common somatic mutations were identified in the multicentric tumor pairs, their whole-genome substitution patterns were similar, suggesting that these tumors developed from independent mutations, although their shared etiological backgrounds may have strongly influenced their somatic mutation patterns. Statistical and functional analyses yielded a list of recurrently mutated genes. Multiple chromatin regulators, including ARID1A, ARID1B, ARID2, MLL and MLL3, were mutated in â¼50% of the tumors. Hepatitis B virus genome integration in the TERT locus was frequently observed in a high clonal proportion. Our whole-genome sequencing analysis of HCCs identified the influence of etiological background on somatic mutation patterns and subsequent carcinogenesis, as well as recurrent mutations in chromatin regulators in HCCs.
Asunto(s)
Carcinoma Hepatocelular/genética , Cromatina/genética , Neoplasias Hepáticas/genética , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Femenino , Genoma Viral/genética , Hepatitis B/genética , Virus de la Hepatitis B/genética , Hepatitis C/genética , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Telomerasa/genética , Integración Viral/genéticaRESUMEN
Hepatocellular carcinoma, one of the most common virus-associated cancers, is the third most frequent cause of cancer-related death worldwide. By massively parallel sequencing of a primary hepatitis C virus-positive hepatocellular carcinoma (36× coverage) and matched lymphocytes (>28× coverage) from the same individual, we identified more than 11,000 somatic substitutions of the tumor genome that showed predominance of T>C/A>G transition and a decrease of the T>C substitution on the transcribed strand, suggesting preferential DNA repair. Gene annotation enrichment analysis of 63 validated non-synonymous substitutions revealed enrichment of phosphoproteins. We further validated 22 chromosomal rearrangements, generating four fusion transcripts that had altered transcriptional regulation (BCORL1-ELF4) or promoter activity. Whole-exome sequencing at a higher sequence depth (>76× coverage) revealed a TSC1 nonsense substitution in a subpopulation of the tumor cells. This first high-resolution characterization of a virus-associated cancer genome identified previously uncharacterized mutation patterns, intra-chromosomal rearrangements and fusion genes, as well as genetic heterogeneity within the tumor.
Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/virología , Exones , Reordenamiento Génico , Genes Supresores de Tumor , Variación Genética , Biblioteca Genómica , Genómica , Hepacivirus/patogenicidad , Humanos , Mutación INDEL , Neoplasias Hepáticas/virología , Mutación , Oncogenes , Polimorfismo de Nucleótido Simple , Selección GenéticaRESUMEN
The epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Here, we demonstrate that transforming growth factor (TGF)-ß induced EMT and that long-term exposure to TGF-ß elicited the epithelial-myofibroblastic transition (EMyoT) by inactivating the MEK-Erk pathway. During the EMT process, TGF-ß induced isoform switching of fibroblast growth factor (FGF) receptors, causing the cells to become sensitive to FGF-2. Addition of FGF-2 to TGF-ß-treated cells perturbed EMyoT by reactivating the MEK-Erk pathway and subsequently enhanced EMT through the formation of MEK-Erk-dependent complexes of the transcription factor δEF1/ZEB1 with the transcriptional corepressor CtBP1. Consequently, normal epithelial cells that have undergone EMT as a result of combined TGF-ß and FGF-2 stimulation promoted the invasion of cancer cells. Thus, TGF-ß and FGF-2 may cooperate with each other and may regulate EMT of various kinds of cells in cancer microenvironment during cancer progression.
Asunto(s)
Empalme Alternativo/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Actinas/genética , Actinas/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Empalme Alternativo/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Humanos , Modelos Biológicos , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/fisiología , Invasividad Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
The epithelial-mesenchymal transition (EMT), a crucial event in cancer progression and embryonic development, is induced by transforming growth factor (TGF)-beta. Expression of E-cadherin, a representative epithelial marker, is repressed through transcriptional reduction by TGF-beta. Here, we show that endocytosis of cell surface E-cadherin during EMT induced by TGF-beta and during scattering induced by hepatocyte growth factor (HGF) can be blocked by inhibiting proteasome with lactacystin and MG132 in normal epithelial cells and in cancer cells. Although loss of cell surface E-cadherin following TGF-beta treatment induced translocation of beta-catenin, an E-cadherin-anchoring molecule, to the nucleus, proteasome inhibition prevented this process and resulted in co-localization of beta-catenin with E-cadherin at the cell surface, leading to establishment of cell-cell adhesion. However, promotion of cell migration by TGF-beta was not significantly affected by proteasome inhibition. Proteasome-dependent events thus appear to be involved in stabilization of cell surface E-cadherin.
Asunto(s)
Cadherinas/metabolismo , Membrana Celular/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Cadherinas/genética , Línea Celular , Endocitosis , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Factor de Crecimiento de Hepatocito , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Inhibidores de Proteasoma , Estabilidad Proteica , Factor de Crecimiento Transformador beta/farmacología , beta Catenina/metabolismoRESUMEN
The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during the progression of epithelial tumors. EMT can be induced by transforming growth factor (TGF)-beta in some tumor cells. Here, we demonstrate the molecular mechanism whereby Snail, a key regulator of EMT, is induced by TGF-beta in tumor cells. Snail induction by TGF-beta was highly dependent on cooperation with active Ras signals, and silencing of Ras abolished Snail induction by TGF-beta in pancreatic cancer Panc-1 cells. Transfection of constitutively active Ras into HeLa cells led to induction of Snail by TGF-beta, while representative direct targets of TGF-beta, including Smad7 and PAI-1, were not affected by Ras signaling. Using mitogen-activated protein kinase inhibitors or Smad3 or Smad2 mutants, we found that phosphorylation at the linker region of Smad2/3 was not required for the induction of Snail by TGF-beta. Taken together, these findings indicate that Ras and TGF-beta-Smad signaling selectively cooperate in the induction of Snail, which occurs in a Smad-dependent manner, but independently of phosphorylation at the linker region of R-Smads by Ras signaling.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Glandulares y Epiteliales/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , Transducción de Señal , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Inducción Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Mutación , Neoplasias Glandulares y Epiteliales/genética , Fosforilación , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal/efectos de los fármacos , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Epithelial-mesenchymal transition (EMT), a crucial event in cancer progression and embryonic development, is induced by transforming growth factor (TGF)-beta in mouse mammary NMuMG epithelial cells. Id proteins have previously been reported to inhibit major features of TGF-beta-induced EMT. In this study, we show that expression of the deltaEF1 family proteins, deltaEF1 (ZEB1) and SIP1, is gradually increased by TGF-beta with expression profiles reciprocal to that of E-cadherin. SIP1 and deltaEF1 each dramatically down-regulated the transcription of E-cadherin in NMuMG cells through direct binding to the E-cadherin promoter. Silencing of the expression of both SIP1 and deltaEF1, but not either alone, completely abolished TGF-beta-induced E-cadherin repression. However, expression of mesenchymal markers, including fibronectin, N-cadherin, and vimentin, was not affected by knockdown of SIP1 and deltaEF1. TGF-beta-induced the expression of Ets1, which in turn activated deltaEF1 promoter activity. Moreover, up-regulation of SIP1 and deltaEF1 expression by TGF-beta was suppressed by knockdown of Ets1 expression. In addition, Id2 suppressed the TGF-beta- and Ets1-induced up-regulation of deltaEF1. Taken together, these findings suggest that the deltaEF1 family proteins, SIP1 and deltaEF1, are necessary, but not sufficient, for TGF-beta-induced EMT and that Ets1 induced by TGF-beta may function as an upstream transcriptional regulator of SIP1 and deltaEF1.