RESUMEN
Localization of mRNAs can involve multiple steps, each with its own cis-acting localization signals and transport factors. How is the transition between different steps orchestrated? We show that the initial step in localization of Drosophila oskar mRNA - transport from nurse cells to the oocyte - relies on multiple cis-acting signals. Some of these are binding sites for the translational control factor Bruno, suggesting that Bruno plays an additional role in mRNA transport. Although transport of oskar mRNA is essential and robust, the localization activity of individual transport signals is weak. Notably, increasing the strength of individual transport signals, or adding a strong transport signal, disrupts the later stages of oskar mRNA localization. We propose that the oskar transport signals are weak by necessity; their weakness facilitates transfer of the oskar mRNA from the oocyte transport machinery to the machinery for posterior localization.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Oocitos/metabolismo , Transporte de ARN/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Drosophila/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Mutación/genética , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
BACKGROUND: Worldwide, gastric cancer (GC) is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects. OBJECTIVE: To determine oncogenic mechanisms and novel therapeutic targets specific for GC by identifying commonly dysregulated genes from the tumours of both Asian-Pacific and Caucasian patients. METHODS: We generated transcriptomic profiles of 22 Caucasian GC tumours and their matched non-cancerous samples and performed an integrative analysis across different GC gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signalling pathways by RNAi and/or pharmacological inhibition. RESULTS: Hepatocyte nuclear factor-4α (HNF4α) upregulation was a key signalling event in gastric tumours from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumour cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signalling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest and tumour growth inhibition. HNF4α also regulated WNT signalling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumours. CONCLUSIONS: Our results indicate that HNF4α is a targetable oncoprotein in GC, is regulated by AMPK signalling through AMPKα and resides upstream of WNT signalling. HNF4α may regulate 'metabolic switch' characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signalling cascade represents a potentially targetable pathway for drug development.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 4 del Hepatocito/genética , Proteínas Proto-Oncogénicas/genética , Neoplasias Gástricas/genética , Proteínas Wnt/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Adenocarcinoma/etnología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Pueblo Asiatico , Biomarcadores de Tumor/metabolismo , Western Blotting , Estudios de Casos y Controles , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas/metabolismo , Distribución Aleatoria , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/etnología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Regulación hacia Arriba , Población Blanca , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Proteína Wnt-5aRESUMEN
Although trastuzumab is a successful targeted therapy for breast cancer patients with tumors expressing HER2 (ERBB2), many patients eventually progress to drug resistance. Here, we identified subpathways differentially expressed between trastuzumab-resistant vs. -sensitive breast cancer cells, in conjunction with additional transcriptomic preclinical and clinical gene datasets, to rigorously identify overexpressed, resistance-associated genes. From this approach, we identified 32 genes reproducibly upregulated in trastuzumab resistance. 25 genes were upregulated in drug-resistant JIMT-1 cells, which also downregulated HER2 protein by >80% in the presence of trastuzumab. 24 genes were downregulated in trastuzumab-sensitive SKBR3 cells. Trastuzumab sensitivity was restored by siRNA knockdown of these genes in the resistant cells, and overexpression of 5 of the 25 genes was found in at least one of five refractory HER2 + breast cancer. In summary, our rigorous computational approach, followed by experimental validation, significantly implicate ATF4, CHEK2, ENAH, ICOSLG, and RAD51 as potential biomarkers of trastuzumab resistance. These results provide further proof-of-concept of our methodology for successfully identifying potential biomarkers and druggable signal pathways involved in tumor progression to drug resistance.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Adulto , Anciano , Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trastuzumab , Células Tumorales CultivadasRESUMEN
Asymmetric positioning of proteins within cells is crucial for cell polarization and function. Deployment of Oskar protein at the posterior pole of the Drosophila oocyte relies on localization of the oskar mRNA, repression of its translation prior to localization, and finally activation of translation. Translational repression is mediated by BREs, regulatory elements positioned in two clusters near both ends of the oskar mRNA 3' UTR. Here we show that some BREs are bifunctional: both clusters of BREs contribute to translational repression, and the 3' cluster has an additional role in release from BRE-dependent repression. Remarkably, both BRE functions can be provided in trans by an oskar mRNA with wild-type BREs that is itself unable to encode Oskar protein. Regulation in trans is likely enabled by assembly of oskar transcripts in cytoplasmic RNPs. Concentration of transcripts in such RNPs is common, and trans regulation of mRNAs may therefore be widespread.