RESUMEN

Single-domain antibody (sdAb) holds the promising strategies for diverse research and translational applications. Here, we describe a method for the adaptation of the in situ proximity ligation assay (isPLA) followed by sequencing (isPLA-seq) to facilitate screening of a high-sensitive, high-throughput sdAb library for a given protein at subcellular and single-cell resolution. Based on the sequence of complementarity-determining region 3 (CDR3), the recombinant sdAb can be produced for in vitro and in vivo utilities. This method provides a general means to identify the functional measure of sdAb and its complementary epitopes and its potential applications to investigate cellular processes.

Asunto(s)

Ensayos Analíticos de Alto Rendimiento/métodos , Anticuerpos de Dominio Único/inmunología , Secuencia de Bases , Línea Celular , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Biblioteca de Genes , Humanos , Inmunofenotipificación , Imagen Molecular , Proteína Sequestosoma-1/inmunología , Anticuerpos de Dominio Único/química

RESUMEN

To support cellular homeostasis and mitigate chemotherapeutic stress, cancer cells must gain a series of adaptive intracellular processes. Here we identify that NUPR1, a tamoxifen (Tam)-induced transcriptional coregulator, is necessary for the maintenance of Tam resistance through physical interaction with ESR1 in breast cancers. Mechanistically, NUPR1 binds to the promoter regions of several genes involved in autophagy process and drug resistance such as BECN1, GREB1, RAB31, PGR, CYP1B1, and regulates their transcription. In Tam-resistant ESR1 breast cancer cells, NUPR1 depletion results in premature senescence in vitro and tumor suppression in vivo. Moreover, enforced-autophagic flux augments cytoplasmic vacuolization in NUPR1-depleted Tam resistant cells, which facilitates the transition from autophagic survival to premature senescence. Collectively, these findings suggest a critical role for NUPR1 as a transcriptional coregulator in enabling endocrine persistence of breast cancers, thus providing a vulnerable diagnostic and/or therapeutic target for endocrine resistance.

Asunto(s)

Antineoplásicos Hormonales/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma Ductal de Mama/tratamiento farmacológico , Resistencia a Antineoplásicos , Receptor alfa de Estrógeno/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Tamoxifeno/farmacología , Transcripción Genética , Animales , Autofagia/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Ratones SCID , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas , Transcriptoma , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Nuclear protein 1 (NUPR1)/p8, a transcriptional regulator, has the ability to facilitate lung cancer cell survival. Adeno­associated virus (AAV)­based vectors are efficient vehicles for gene transfer and expression. In this study, an AAV­mediated NUPR1 shRNA vector was constructed that effectively inhibited the expression of NUPR1 in a tumor xenograft model derived from lung adenocarcinoma A549 cells. Trifluoperazine (TFP), which is an antipsychotic drug, has the ability to bind to NUPR1 and mimic NUPR1 deficiency in cancer cells. It was also found that the combination of TFP and AAV­mediated NUPR1 shRNA delivery led to significant tumor growth inhibition in nude mice bearing human lung cancer xenografts. Moreover, AAV­mediated NUPR1 shRNA therapy induced premature senescence in vitro and in vivo. Collectively, the findings of this study suggest a putative role for the combination of AAV­NUPR1 shRNA and TFP in lung cancer therapy.

Asunto(s)

Adenocarcinoma del Pulmón/tratamiento farmacológico , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/antagonistas & inhibidores , ARN Interferente Pequeño/administración & dosificación , Trifluoperazina/administración & dosificación , Células A549 , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Supervivencia Celular/efectos de los fármacos , Dependovirus/genética , Sinergismo Farmacológico , Técnicas de Silenciamiento del Gen , Vectores Genéticos/administración & dosificación , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , ARN Interferente Pequeño/farmacología , Trifluoperazina/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto