RESUMEN
BACKGROUND: Nutrient deprivation, hypoxia, radiotherapy and chemotherapy induce endoplasmic reticulum (ER) stress, which activates the so-called unfolded protein response (UPR). Extensive and acute ER stress directs the UPR towards activation of death-triggering pathways. Cancer cells are selected to resist mild and prolonged ER stress by activating pro-survival UPR. We recently found that drug-resistant tumor cells are simultaneously resistant to ER stress-triggered cell death. It is not known if cancer cells adapted to ER stressing conditions acquire a chemoresistant phenotype. METHODS: To investigate this issue, we generated human cancer cells clones with acquired resistance to ER stress from ER stress-sensitive and chemosensitive cells. RESULTS: ER stress-resistant cells were cross-resistant to multiple chemotherapeutic drugs: such multidrug resistance (MDR) was due to the overexpression of the plasma-membrane transporter MDR related protein 1 (MRP1). Gene profiling analysis unveiled that cells with acquired resistance to ER stress and chemotherapy share higher expression of the UPR sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK), which mediated the erythroid-derived 2-like 2 (Nrf2)-driven transcription of MRP1. Disrupting PERK/Nrf2 axis reversed at the same time resistance to ER stress and chemotherapy. The inducible silencing of PERK reduced tumor growth and restored chemosensitivity in resistant tumor xenografts. CONCLUSIONS: Our work demonstrates for the first time that the adaptation to ER stress in cancer cells produces a MDR phenotype. The PERK/Nrf2/MRP1 axis is responsible for the resistance to ER stress and chemotherapy, and may represent a good therapeutic target in aggressive and resistant tumors.
Asunto(s)
Neoplasias del Colon/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Factor 2 Relacionado con NF-E2/genética , eIF-2 Quinasa/genética , Animales , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Doxorrubicina/administración & dosificación , Resistencia a Antineoplásicos/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células HT29 , Humanos , Ratones , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , Ensayos Antitumor por Modelo de Xenoinjerto , eIF-2 Quinasa/antagonistas & inhibidoresRESUMEN
Regulation of hematopoiesis during human development remains poorly defined. Here we applied single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to over 8,000 human immunophenotypic blood cells from fetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream of hematopoietic stem cells (HSCs)/multipotent progenitors (MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSCs/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSCs/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental hematopoiesis in the context of blood pathologies and regenerative medicine.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Hematopoyesis , Linaje de la Célula/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas , Humanos , RNA-Seq , Análisis de la Célula IndividualRESUMEN
BACKGROUND: Chemotherapy triggers endoplasmic reticulum (ER) stress, which in turn regulates levels of the active (LAP) and the natural dominant-negative (LIP) forms of the transcription factor C/EBP-ß. LAP upregulates and LIP downregulates the multidrug resistance (MDR) protein P-glycoprotein (Pgp), but it is not known how critical is their role in establishing MDR. METHODS: Cell viability was quantitated by crystal violet staining and measuring absorbance at 540nm. Expression of various proteins was determined by immunoblotting. mRNA levels were determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). LIP and LAP were overexpressed using expression plasmids followed by selection with blasticidin. Tumor cells expressing doxycycline-inducible LIP were orthotopically implanted in mice (n = 15 mice per group), and tumor size was measured daily by caliper. Tumor sections were stained with hematoxylin and eosin and immunostained for Pgp, proliferation, and ER stress markers. RESULTS: MDR cells do not express basal, chemotherapy-triggered, or ER stress-triggered LIP and fail to activate the CHOP-caspase-3 death-triggering axis upon ER stress or chemotherapy challenge. Overexpression of LIP reversed the MDR phenotype in vitro and in tumors implanted in mice. LIP was undetectable in MDR cells, probably due to its ubiquitination, which was 3.56-fold higher, resulting in lysosomal and proteasomal degradation of LIP. CONCLUSIONS: Spontaneous and drug-selected MDR cells lack LIP, which is eliminated by ubiquitin-mediated degradation. Loss of LIP drives MDR not only by increasing Pgp expression but also by a two-fold attenuation of ER stress-triggered cell death.