RESUMEN
Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA-mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.
Asunto(s)
Metilación de ADN/genética , Leucemia , ARN Largo no Codificante , Proteínas Supresoras de Tumor , Adulto , Anciano , Anciano de 80 o más Años , Transformación Celular Neoplásica , Cromatina/genética , Cromosomas Humanos Par 13/genética , Regulación hacia Abajo , Epigénesis Genética/genética , Femenino , Células HEK293 , Humanos , Leucemia/sangre , Leucemia/genética , Leucemia/fisiopatología , Masculino , Persona de Mediana Edad , Mutación , FN-kappa B/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Sitio de Iniciación de la Transcripción , Transferasas , Proteínas Supresoras de Tumor/sangre , Proteínas Supresoras de Tumor/genética , Regulación hacia ArribaRESUMEN
MicroRNAs post-transcriptionally regulate gene expression and thereby contribute to the modulation of numerous complex and disease-relevant cellular phenotypes, including cell proliferation, cell motility, apoptosis, and stress response. In breast cancer cell systems, miR-31 has been shown to inhibit cell migration, invasion, and metastasis. Here, we link enhanced expression of miR-31 to the inhibition of the oncogenic NF-κB pathway, thus supporting the tumor-suppressive function of this microRNA. We identified protein kinase C epsilon (PKCε encoded by the PRKCE gene) as a novel direct target of miR-31 and show that down-regulation of PKCε results in impaired NF-κB signaling, enhanced apoptosis, and increased sensitivity of MCF10A breast epithelial and MDA-MB-231 triple-negative breast cancer cells toward ionizing radiation as well as treatment with chemotherapeutics. Mechanistically, we attribute this sensitization to anti-cancer treatments to the PRKCE-mediated down-regulation of the anti-apoptotic factor BCL2. In clinical breast cancer samples, high BCL2 expression was associated with poor prognosis. Furthermore, we found an inverse correlation between miR-31 and BCL2 expression, highlighting the functional relevance of the indirect down-regulation of BCL2 via direct targeting of PRKCE by miR-31.
Asunto(s)
Apoptosis , Neoplasias de la Mama/metabolismo , MicroARNs/fisiología , Proteína Quinasa C-epsilon/genética , Interferencia de ARN , Regiones no Traducidas 3' , Secuencia de Bases , Neoplasias de la Mama/patología , Supervivencia Celular , Resistencia a Antineoplásicos , Femenino , Genes Reporteros , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Células MCF-7 , MicroARNs/genética , MicroARNs/metabolismo , FN-kappa B/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tolerancia a Radiación , Estaurosporina/farmacología , Proteína de Unión al GTP rhoA/metabolismo , Quinasa de Factor Nuclear kappa BRESUMEN
Immune checkpoint blockade (ICB) with PD-1 or PD-L1 antibodies has been approved for the treatment of non-small cell lung cancer (NSCLC). However, only a minority of patients respond, and sustained remissions are rare. Both chemotherapy and antiangiogenic drugs may improve the efficacy of ICB in mouse tumor models and patients with cancer. Here, we used genetically engineered mouse models of Kras G12D/+;p53 -/- NSCLC, including a mismatch repair-deficient variant (Kras G12D/+;p53 -/-;Msh2 -/-) with higher mutational burden, and longitudinal imaging to study tumor response and resistance to combinations of ICB, antiangiogenic therapy, and chemotherapy. Antiangiogenic blockade of vascular endothelial growth factor A and angiopoietin-2 markedly slowed progression of autochthonous lung tumors, but contrary to findings in other cancer types, addition of a PD-1 or PD-L1 antibody was not beneficial and even accelerated progression of a fraction of the tumors. We found that antiangiogenic treatment facilitated tumor infiltration by PD-1+ regulatory T cells (Tregs), which were more efficiently targeted by the PD-1 antibody than CD8+ T cells. Both tumor-associated macrophages (TAMs) of monocyte origin, which are colony-stimulating factor 1 receptor (CSF1R) dependent, and TAMs of alveolar origin, which are sensitive to cisplatin, contributed to establish a transforming growth factor-ß-rich tumor microenvironment that supported PD-1+ Tregs Dual TAM targeting with a combination of a CSF1R inhibitor and cisplatin abated Tregs, redirected the PD-1 antibody to CD8+ T cells, and improved the efficacy of antiangiogenic immunotherapy, achieving regression of most tumors.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Antígeno B7-H1 , Linfocitos T CD8-positivos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Ratones , Receptor de Muerte Celular Programada 1 , Microambiente Tumoral , Factor A de Crecimiento Endotelial VascularRESUMEN
In sheep, scrapie susceptibility is so strongly associated with single nucleotide polymorphisms (SNPs) in the gene encoding the prion protein (PrP) that this linkage constitutes the basis for selective breeding strategies directed toward controlling the disease. For goats, in contrast, the association between scrapie susceptibility/resistance and variations in the PrP gene is far weaker, with only a few identified SNPs showing an influence on scrapie susceptibility. A recent survey of PrP genotypes in Cypriot goats, however, revealed the existence of a robust association between polymorphisms at codon 146 of the caprine PrP gene and resistance/susceptibility to natural scrapie. Here we describe here a high-throughput assay, based on homogeneous MassExtend technology coupled with mass spectrometry, for genotyping codon 146 of the caprine PrP gene. Our results demonstrate that this assay exhibits high accuracy, reproducibility, and repeatability, thereby making it suitable for large-scale SNP genotyping, as required for scrapie surveillance programs.
Asunto(s)
Pruebas Genéticas , Cabras/genética , Procedimientos Analíticos en Microchip/veterinaria , Polimorfismo Genético , Priones/genética , Animales , Codón , Chipre , Susceptibilidad a Enfermedades/veterinaria , Genotipo , Enfermedades de las Cabras/genética , Procedimientos Analíticos en Microchip/métodos , Scrapie/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy.
Asunto(s)
Doxorrubicina/uso terapéutico , Vesículas Extracelulares/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Paclitaxel/uso terapéutico , Animales , Anexina A6/metabolismo , Línea Celular Tumoral , Quimiocina CCL2/metabolismo , Vesículas Extracelulares/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Ratones TransgénicosRESUMEN
Resistance to antiangiogenic drugs limits their applicability in cancer therapy. Here, we show that revascularization and progression of pancreatic neuroendocrine tumors (PNETs) under extended vascular-endothelial growth factor A (VEGFA) blockade are dependent on periostin (POSTN), a matricellular protein expressed by stromal cells. Genetic deletion of Postn in RIP1-Tag2 mice blunted tumor rebounds of M2-like macrophages and αSMA+ stromal cells in response to prolonged VEGFA inhibition and suppressed PNET revascularization and progression on therapy. POSTN deficiency also impeded the upregulation of basic fibroblast growth factor (FGF2), an adaptive mechanism previously implicated in PNET evasion from antiangiogenic therapy. Higher POSTN expression correlated with markers of M2-like macrophages in human PNETs, and depleting macrophages with a colony-stimulating factor 1 receptor (CSF1R) antibody inhibited PNET revascularization and progression under VEGFA blockade despite continued POSTN production. These findings suggest a role for POSTN in orchestrating resistance to anti-VEGFA therapy in PNETs.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Tumores Neuroendocrinos/metabolismo , Neoplasias Pancreáticas/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Macrófagos/metabolismo , Ratones Transgénicos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Tumores Neuroendocrinos/irrigación sanguínea , Tumores Neuroendocrinos/tratamiento farmacológico , Tumores Neuroendocrinos/genética , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Nuclear Factor kappa B (NF-κB) signaling is frequently deregulated in a variety of cancers and is constitutively active in estrogen receptor negative (ER-) breast cancer subtypes. These molecular subtypes of breast cancer are associated with poor overall survival. We focused on mechanisms of NF-κB regulation by microRNAs (miRNAs), which regulate eukaryotic gene expression at the post-transcriptional level. In a previous genome-wide miRNA screen, we had identified miR-30c-2-3p as one of the strongest negative regulators of NF-κB signaling. Here we have uncovered the underlying molecular mechanisms and its consequences in breast cancer. In vitro results show that miR-30c-2-3p directly targets both TNFRSF1A-associated via death domain (TRADD), an adaptor protein of the TNFR/NF-κB signaling pathway, and the cell cycle protein Cyclin E1 (CCNE1). Ectopic expression of miR-30c-2-3p downregulated essential cytokines IL8, IL6, CXCL1, and reduced cell proliferation as well as invasion in MDA-MB-231 breast cancer cells. RNA interference (RNAi) induced silencing of TRADD phenocopied the effects on invasion and cytokine expression caused by miR-30c-2-3p, while inhibition of CCNE1 phenocopied the effects on cell proliferation. We further confirmed the tumor suppressive role of this miRNA using a dataset of 781 breast tumors, where higher expression was associated with better survival in breast cancer patients. In summary we have elucidated the mechanism by which miR-30c-2-3p negatively regulates NF-κB signaling and cell cycle progression in breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Ciclo Celular , Ciclina E/metabolismo , MicroARNs/metabolismo , FN-kappa B/metabolismo , Proteínas Oncogénicas/metabolismo , ARN Neoplásico/metabolismo , Transducción de Señal , Proteína de Dominio de Muerte Asociada a Receptor de TNF/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Ciclina E/genética , Femenino , Humanos , MicroARNs/genética , FN-kappa B/genética , Proteínas Oncogénicas/genética , ARN Neoplásico/genética , Proteína de Dominio de Muerte Asociada a Receptor de TNF/genéticaRESUMEN
Tumor relapse after chemotherapy-induced regression is a major clinical problem, because it often involves inoperable metastatic disease. Tumor-associated macrophages (TAM) are known to limit the cytotoxic effects of chemotherapy in preclinical models of cancer. Here, we report that an alternatively activated (M2) subpopulation of TAMs (MRC1(+)TIE2(Hi)CXCR4(Hi)) accumulate around blood vessels in tumors after chemotherapy, where they promote tumor revascularization and relapse, in part, via VEGF-A release. A similar perivascular, M2-related TAM subset was present in human breast carcinomas and bone metastases after chemotherapy. Although a small proportion of M2 TAMs were also present in hypoxic tumor areas, when we genetically ablated their ability to respond to hypoxia via hypoxia-inducible factors 1 and 2, tumor relapse was unaffected. TAMs were the predominant cells expressing immunoreactive CXCR4 in chemotherapy-treated mouse tumors, with the highest levels expressed by MRC1(+) TAMs clustering around the tumor vasculature. Furthermore, the primary CXCR4 ligand, CXCL12, was upregulated in these perivascular sites after chemotherapy, where it was selectively chemotactic for MRC1(+) TAMs. Interestingly, HMOX-1, a marker of oxidative stress, was also upregulated in perivascular areas after chemotherapy. This enzyme generates carbon monoxide from the breakdown of heme, a gas known to upregulate CXCL12. Finally, pharmacologic blockade of CXCR4 selectively reduced M2-related TAMs after chemotherapy, especially those in direct contact with blood vessels, thereby reducing tumor revascularization and regrowth. Our studies rationalize a strategy to leverage chemotherapeutic efficacy by selectively targeting this perivascular, relapse-promoting M2-related TAM cell population.
Asunto(s)
Neoplasias de la Mama/genética , Macrófagos/patología , Recurrencia Local de Neoplasia/genética , Neovascularización Patológica/genética , Receptores CXCR4/biosíntesis , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patología , Quimiocina CXCL12/biosíntesis , Quimiocina CXCL12/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Macrófagos/metabolismo , Ratones , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Neovascularización Patológica/tratamiento farmacológico , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/genética , Transducción de Señal/efectos de los fármacos , Tamoxifeno/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Angiopoietin-2 (ANG2/ANGPT2) is a context-dependent TIE2 receptor agonist/antagonist and proangiogenic factor. Although ANG2 neutralization improves tumor angiogenesis and growth inhibition by vascular endothelial growth factor (VEGF)-A signaling blockade, the mechanistic underpinnings of such therapeutic benefits remain poorly explored. We employed late-stage RIP1-Tag2 pancreatic neuroendocrine tumors (PNETs) and MMTV-PyMT mammary adenocarcinomas, which develop resistance to VEGF receptor 2 (VEGFR2) blockade. We found that VEGFR2 inhibition upregulated ANG2 and vascular TIE2 and enhanced infiltration by TIE2-expressing macrophages in the PNETs. Dual ANG2/VEGFR2 blockade suppressed revascularization and progression in most of the PNETs, whereas it had only minor additive effects in the mammary tumors, which did not upregulate ANG2 upon VEGFR2 inhibition. ANG2/VEGFR2 blockade did not elicit increased PNET invasion and metastasis, although it exacerbated tumor hypoxia and hematopoietic cell infiltration. These findings suggest that evasive tumor resistance to anti-VEGFA therapy may involve the adaptive enforcement of ANG2-TIE2 signaling, which can be reversed by ANG2 neutralization.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos , Neoplasias Pancreáticas/metabolismo , Ribonucleasa Pancreática/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Adenocarcinoma/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/tratamiento farmacológico , Ribonucleasa Pancreática/antagonistas & inhibidores , Ribonucleasa Pancreática/inmunología , Transducción de Señal , Regulación hacia Arriba , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
MicroRNA-200c (miR-200c) has been shown to suppress epithelial-mesenchymal transition (EMT), which is attributed mainly to targeting of ZEB1/ZEB2, repressors of the cell-cell contact protein E-cadherin. Here we demonstrated that modulation of miR-200c in breast cancer cells regulates cell migration, cell elongation, and transforming growth factor ß (TGF-ß)-induced stress fiber formation by impacting the reorganization of cytoskeleton that is independent of the ZEB/E-cadherin axis. We identified FHOD1 and PPM1F, direct regulators of the actin cytoskeleton, as novel targets of miR-200c. Remarkably, expression levels of FHOD1 and PPM1F were inversely correlated with the level of miR-200c in breast cancer cell lines, breast cancer patient samples, and 58 cancer cell lines of various origins. Furthermore, individual knockdown/overexpression of these target genes phenocopied the effects of miR-200c overexpression/inhibition on cell elongation, stress fiber formation, migration, and invasion. Mechanistically, targeting of FHOD1 by miR-200c resulted in decreased expression and transcriptional activity of serum response factor (SRF), mediated by interference with the translocation of the SRF coactivator mycocardin-related transcription factor A (MRTF-A). This finally led to downregulation of the expression and phosphorylation of the SRF target myosin light chain 2 (MLC2) gene, required for stress fiber formation and contractility. Thus, miR-200c impacts on metastasis by regulating several EMT-related processes, including a novel mechanism involving the direct targeting of actin-regulatory proteins.
Asunto(s)
Neoplasias de la Mama/patología , Proteínas Fetales/metabolismo , MicroARNs/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Neoplasias de la Mama/metabolismo , Miosinas Cardíacas/biosíntesis , Línea Celular Tumoral , Movimiento Celular , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Femenino , Forminas , Regulación Neoplásica de la Expresión Génica , Humanos , Cadenas Ligeras de Miosina/biosíntesis , Invasividad Neoplásica , Proteínas de Fusión Oncogénica/metabolismo , Factor de Respuesta Sérica/biosíntesis , Fibras de Estrés/metabolismo , Transactivadores , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Analysis of biological processes is frequently performed with the help of phenotypic assays where data is mostly acquired in single end-point analysis. Alternative phenotypic profiling techniques are desired where time-series information is essential to the biological question, for instance to differentiate early and late regulators of cell proliferation in loss-of-function studies. So far there is no study addressing this question despite of high unmet interests, mostly due to the limitation of conventional end-point assaying technologies. We present the first human kinome screen with a real-time cell analysis system (RTCA) to capture dynamic RNAi phenotypes, employing time-resolved monitoring of cell proliferation via electrical impedance. RTCA allowed us to investigate the dynamics of phenotypes of cell proliferation instead of using conventional end-point analysis. By introducing data transformation with first-order derivative, i.e. the cell-index growth rate, we demonstrate this system suitable for high-throughput screenings (HTS). The screen validated previously identified inhibitor genes and, additionally, identified activators of cell proliferation. With the information of time kinetics available, we could establish a network of mitotic-event related genes to be among the first displaying inhibiting effects after RNAi knockdown. The time-resolved screen captured kinetics of cell proliferation caused by RNAi targeting human kinome, serving as a resource for researchers. Our work establishes RTCA technology as a novel robust tool with biological and pharmacological relevance amenable for high-throughput screening.