RESUMO
Acute myeloid leukemia (AML) is characterized by the presence of leukemia stem cells (LSCs), and failure to fully eradicate this population contributes to disease persistence/relapse. Prior studies have characterized metabolic vulnerabilities of LSCs, which demonstrate preferential reliance on oxidative phosphorylation (OXPHOS) for energy metabolism and survival. In the present study, using both genetic and pharmacologic strategies in primary human AML specimens, we show that signal transducer and activator of transcription 3 (STAT3) mediates OXPHOS in LSCs. STAT3 regulates AML-specific expression of MYC, which in turn controls transcription of the neutral amino acid transporter gene SLC1A5. We show that genetic inhibition of MYC or SLC1A5 acts to phenocopy the impairment of OXPHOS observed with STAT3 inhibition, thereby establishing this axis as a regulatory mechanism linking STAT3 to energy metabolism. Inhibition of SLC1A5 reduces intracellular levels of glutamine, glutathione, and multiple tricarboxylic acid (TCA) cycle metabolites, leading to reduced TCA cycle activity and inhibition of OXPHOS. Based on these findings, we used a novel small molecule STAT3 inhibitor, which binds STAT3 and disrupts STAT3-DNA, to evaluate the biological role of STAT3. We show that STAT3 inhibition selectively leads to cell death in AML stem and progenitor cells derived from newly diagnosed patients and patients who have experienced relapse while sparing normal hematopoietic cells. Together, these findings establish a STAT3-mediated mechanism that controls energy metabolism and survival in primitive AML cells.
Assuntos
Sistema ASC de Transporte de Aminoácidos/metabolismo , Leucemia Mieloide Aguda/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Sobrevivência Celular , Humanos , Células-Tronco Neoplásicas/citologia , Fosforilação Oxidativa , Células Tumorais CultivadasRESUMO
Tumor heterogeneity is the concept that different tumor cells provide distinct biomorphological lesions, gene expressions, proliferation, microenvironment and graduated capacity of metastatic lesions. Brain tumor heterogeneity has been recently discussed about the interesting interaction of chronic inflammation, microenvironment, epigenetics and glioma steam cells. Brain tumors remain a challenge with regards to medication and disease, due to the lack of treatment options and unsatisfactory results. These results might be the result of the brain tumor heterogeneity and its multiple resistance mechanisms to chemo and radiotherapy.
Assuntos
Células-Tronco Neoplásicas/citologia , Neoplasias Encefálicas/genética , Heterogeneidade Genética , Perfilação da Expressão Gênica , Glioma/genética , Receptores Proteína Tirosina Quinases/genética , Resistencia a Medicamentos Antineoplásicos/genética , Nicho de Células-Tronco/genética , Microambiente Tumoral , Evolução Clonal/genética , Microambiente Celular/genética , RNA-SeqRESUMO
Breast cancer is an aggressive disease with a high incidence in women worldwide. Two decades ago, a controversial hypothesis was proposed that cancer arises from a subpopulation of "tumor initiating cells" or "cancer stem cells-like" (CSC). Today, CSC are defined as small subset of somatic cancer cells within a tumor with self-renewal properties driven by the aberrant expression of genes involved in the maintenance of a stemness-like phenotype. The understanding of the underlying cellular and molecular mechanisms involved in the maintenance of CSC subpopulation are fundamental in the development and persistence of breast cancer. Nowadays, the hypothesis suggests that genetic and epigenetic alterations give rise to breast cancer stem cells (bCSC), which are responsible for self-renewal, tumor growth, chemoresistance, poor prognosis and low survival in patients. However, the prominence of bCSC, as well as the molecular mechanisms that regulates and promotes the malignant phenotypes, are still poorly understood. The role of non-coding RNAs (ncRNAs), such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) acting as oncogenes or tumor suppressor genes has been recently highlighted by a plethora of studies in breast cancer. These ncRNAs positively or negatively impact on different signaling pathways that govern the cancer hallmarks associated with bCSC, making them attractive targets for therapy. In this review, we present a current summary of the studies on the pivotal roles of lncRNAs and microRNAs in the regulation of genes associated to stemness of bCSC.
Assuntos
Neoplasias da Mama/genética , MicroRNAs/genética , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células-Tronco Neoplásicas/patologiaRESUMO
PURPOSE: Immune cells in the immune microenvironment of lung cancer have a great impact on the development of lung cancer. Our purpose was to analyze the immune cell infiltration features and related marker genes for lung cancer. METHODS: Single cell RNA sequencing data of 11,485 lung cancer cells were retrieved from the Gene Expression Omnibus. After quality control and data normalization, cell clustering was performed using the Seurat package. Based on the marker genes of each cell type from the CellMarker database, each cell was divided into G1, G2M, and S phases. Then, differential expression and functional enrichment analyses were performed. CIBERSORT was used to reconstruct immune cell types. RESULTS: Following cell filtering, highly variable genes were identified for all cells. 14 cell types were clustered. Among them, CD4 + T cell, B cell, plasma cell, natural killer cell and cancer stem cell were the top five cell types. Up-regulated genes were mainly enriched in immune-related biological processes and pathways. Using CIBERSORT, we identified the significantly higher fractions of naïve B cell, memory CD4 + T cell, T follicular helper cell, T regulatory helper cell and M1 macrophage in lung cancer tissues compared to normal tissues. Furthermore, the fractions of resting NK cell, monocyte, M0 macrophage, resting mast cell, eosinophil and neutrophil were significantly lower in tumor tissues than normal tissues. CONCLUSION: Our findings dissected the immune cell infiltration features and related marker genes for lung cancer, which might provide novel insights for the immunotherapy of lung cancer.
Assuntos
Marcadores Genéticos/genética , Imunidade Celular , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , RNA-Seq/métodos , Linfócitos B/citologia , Linfócitos T CD4-Positivos/citologia , Ciclo Celular , Bases de Dados Genéticas , Expressão Gênica , Humanos , Imunidade Celular/genética , Células Matadoras Naturais/citologia , Macrófagos/citologia , Células-Tronco Neoplásicas/citologia , Plasmócitos/citologia , Células T Auxiliares Foliculares/citologia , Linfócitos T Reguladores/citologia , Microambiente Tumoral/imunologia , Regulação para CimaRESUMO
Glioma diagnosis is based on histomorphology and grading; however, such classification does not have predictive clinical outcome after glioblastomas have developed. To date, no bona fide biomarkers that significantly translate into a survival benefit to glioblastoma patients have been identified. We previously reported that the IDH mutant G-CIMP-high subtype would be a predecessor to the G-CIMP-low subtype. Here, we performed a comprehensive DNA methylation longitudinal analysis of diffuse gliomas from 77 patients (200 tumors) to enlighten the epigenome-based malignant transformation of initially lower-grade gliomas. Intra-subtype heterogeneity among G-CIMP-high primary tumors allowed us to identify predictive biomarkers for assessing the risk of malignant recurrence at early stages of disease. G-CIMP-low recurrence appeared in 9.5% of all gliomas, and these resembled IDH-wild-type primary glioblastoma. G-CIMP-low recurrence can be characterized by distinct epigenetic changes at candidate functional tissue enhancers with AP-1/SOX binding elements, mesenchymal stem cell-like epigenomic phenotype, and genomic instability. Molecular abnormalities of longitudinal G-CIMP offer possibilities to defy glioblastoma progression.
Assuntos
Neoplasias Encefálicas/patologia , Metilação de DNA , Glioma/patologia , Recidiva Local de Neoplasia/genética , Adulto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/terapia , Ilhas de CpG , Feminino , Instabilidade Genômica , Glioma/genética , Glioma/mortalidade , Glioma/terapia , Humanos , Isocitrato Desidrogenase/genética , Estimativa de Kaplan-Meier , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Fenótipo , PrognósticoRESUMO
Tumor-initiating cells possess the capacity for self-renewal and to create heterogeneous cell lineages within a tumor. Therefore, the identification and isolation of cancer stem cells is an essential step in the analysis of their biology. The aim of the present study was to determine whether the cell surface protein neuropilin 1 (NRP1) can be used as a biomarker of stem-like cells in lung cancer tumors. For this purpose, NRP1-negative (NRP1-) and NRP1-positive (NRP1+) cell subpopulations from two lung cancer cell lines were sorted by flow cytometry. The NRP1+ cell subpopulation showed an increased expression of pluripotency markers OCT-4, Bmi-1 and NANOG, as well as higher cell migration, clonogenic and self-renewal capacities. NRP1 gene knockdown resulted not only in a decreased expression of stemness markers but also in a decrease in the clonogenic, cell migration and self-renewal potential. In addition, the NRP1+ cell subpopulation exhibited dysregulated expression of epithelial-to-mesenchymal transition-associated genes, including the ΔNp63 isoform protein, a previously reported characteristic of cancer stem cells. Notably, a genome-wide expression analysis of NRP1-knockdown cells revealed a potential new NRP1 pathway involving OLFML3 and genes associated with mitochondrial function. In conclusion, we demonstrated that NRP1+ lung cancer cells have tumor-initiating properties. NRP1 could be a useful biomarker for tumor-initiating cells in lung cancer tumors.
Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/citologia , Neuropilina-1/genética , Neuropilina-1/metabolismo , Células A549 , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Células Tumorais CultivadasRESUMO
The aim of the present study was to demonstrate that ivermectin preferentially inhibited cancer stemlike cells (CSC) in breast cancer cells and downregulated the expression of 'stemness' genes. Computational searching of DrugBank, a database of approved drugs, was performed using the principles of twodimensional similarity searching; the chemical structure of salinomycin was used as a query. Growth inhibition of the breast cancer cell lin e MDAMB231 by ivermectin was investigated in the total cell population, in cell spheroids and in sorted cells that expressed cluster of differentiation (CD)44+/CD24. The effects of ivermectin treatment on the expression of pluripotency and selfrenewal transcription factors, such as homeobox protein nanog (nanog), octamerbinding protein 4 (oct4) and SRYbox 2 (sox2), were evaluated by reverse transcriptionquantitative polymerase chain reaction and western blotting. Ivermectin exhibited a similarity value of 0.78 in reference to salinomycin. Ivermectin demonstrated an inhibitory effect upon the growth of MDAMB231 cells in the range of 0.28 µM. Ivermectin preferentially inhibits the viability of CSCenriched populations (CD44+/CD24 and cells growing in spheroids) compared with the total cell population. The opposite pattern was observed with paclitaxel treatment. Ivermectin exposure reduced the expression of nanog, oct4 and sox2 at the mRNA and protein levels. Ivermectin preferentially inhibited the CSC subpopulation in the MDAMB231 cells and downregulated the expression of genes involved in the maintenance of pluripotency and selfrenewal.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ivermectina/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Praguicidas/farmacologia , Antiparasitários/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Autorrenovação Celular/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
Breast cancer human cells culture as spheroids develop autophagy and apoptosis, which promotes Trastuzumab resistance in HER2 overexpressing cells. Our aim was to study the association of the hostile environment developed in 3D with the breast cancer stem cells population and the HER2 modulation. Human mammary adenocarcinoma cell lines were cultured as spheroids using the hanging drop method. We generated hypoxia conditions by using a hypoxic chamber and CoCl2 treatment. Breast cancer stem cells were measured with mammosphere assays, the analysis of CD44 + CD24low population by flow cytometry and the pluripotent gene expression by RT-qPCR. HER2 expression was evaluated by flow cytometry and Western blot. MTS assays were conducted to study cell viability. Hostil environment developed in spheroids, defined by hypoxia and autophagy, modulated the response to Trastuzumab. In HER2+ cells with acquired resistance, we observed an increase in the breast cancer stem cell population. In BT474 spheroids, Trastuzumab induced the acquisition of resistance, along with an increase in breast cancer stem cells. Also, in 3D culture conditions we determined a modulation in the HER2 expression. Moreover, breast cancer stem cells showed enhanced HER2 expression. Finally, cells without HER2 gene amplification cultured as spheroids were sensitive to Trastuzumab, diminishing HER2 expression and cancer stem cells. Our findings show that 3D architecture is able to modulate breast cancer stem cell population and HER2 distribution, modifying the cell response to Trastuzumab.
Assuntos
Neoplasias da Mama/genética , Técnicas de Cultura de Células/métodos , Resistencia a Medicamentos Antineoplásicos , Células-Tronco Neoplásicas/citologia , Receptor ErbB-2/genética , Trastuzumab/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cobalto/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Receptor ErbB-2/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismoRESUMO
In recent years, it has become evident that intra-tumor heterogeneity of breast cancer is a big challenge for the diagnosis, treatment, and clinical course of tumor-bearing patients. The advances in molecular biology and other technologies have led to the knowledge that a breast cancer tumor is comprised of multiple cellular entities. Here we review the two theories that have been described, trying to explain the origin of intra-tumor heterogeneity: clonal evolution and cancer stem cells. The first one considers that a single cell gives rise to many subpopulations through the accumulation of multiple aberrations, while the cancer stem cells theory foresees a hierarchical tumor evolution where only a few cells with self-renewal capacity give rise to different subpopulations. We also analyze the genetic, epigenetic, and microenvironment contributions to breast cancer intra-tumor heterogeneity. Finally, the clinical and therapeutic impact of intra-tumor heterogeneity on the outcome of breast cancer patients is discussed.
Assuntos
Neoplasias da Mama/patologia , Evolução Clonal/fisiologia , Células-Tronco Neoplásicas/citologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/terapia , Autorrenovação Celular/fisiologia , Epigênese Genética/fisiologia , Feminino , Humanos , Biologia Molecular/métodos , Microambiente Tumoral/fisiologiaRESUMO
PURPOSE: In a previous study the vaccine was effective against bladder cancer in a mouse model. However, a small portion of tumors regrew because the vaccine could not eliminate bladder cancer stem cells (CSCs). In this study, we showed a modified method for the isolation of bladder CSCs using a combination of differential adhesion method and serum-free culture medium (SFM) method. MATERIALS AND METHODS: Trypsin-resistant cells and trypsin-sensitive cells were isolated from MB49, EJ and 5637 cells by a combination of differential adhesion method and SFM method. The CSCs characterizations of trypsin-resistant cells were verified by the flow cytometry, the western blotting, the quantitative polymerase chain reaction, the resistance to chemotherapy assay, the transwell assay, and the tumor xenograft formation assay. RESULTS: Trypsin-resistant cells were isolated and identified in CSCs characters, with high expression of CSCs markers, higher resistance to chemotherapy, greater migration in vitro, and stronger tumorigenicity in vivo. CONCLUSION: Trypsin-resistant cells displayed specific CSCs properties. Our study showed trypsin-resistant cells were isolated successfully with a modified method using a combination of differential adhesion method and SFM method.