RESUMO
Self-organizing neural organoids grown from pluripotent stem cells1-3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando-a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.
Assuntos
Encéfalo , Linhagem da Célula , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Organoides , Humanos , Encéfalo/citologia , Encéfalo/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/genética , Organoides/citologia , Organoides/metabolismo , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
The persistence of undetectable disseminated tumour cells (DTCs) after primary tumour resection poses a major challenge to effective cancer treatment1-3. These enduring dormant DTCs are seeds of future metastases, and the mechanisms that switch them from dormancy to outgrowth require definition. Because cancer dormancy provides a unique therapeutic window for preventing metastatic disease, a comprehensive understanding of the distribution, composition and dynamics of reservoirs of dormant DTCs is imperative. Here we show that different tissue-specific microenvironments restrain or allow the progression of breast cancer in the liver-a frequent site of metastasis4 that is often associated with a poor prognosis5. Using mouse models, we show that there is a selective increase in natural killer (NK) cells in the dormant milieu. Adjuvant interleukin-15-based immunotherapy ensures an abundant pool of NK cells that sustains dormancy through interferon-γ signalling, thereby preventing hepatic metastases and prolonging survival. Exit from dormancy follows a marked contraction of the NK cell compartment and the concurrent accumulation of activated hepatic stellate cells (aHSCs). Our proteomics studies on liver co-cultures implicate the aHSC-secreted chemokine CXCL12 in the induction of NK cell quiescence through its cognate receptor CXCR4. CXCL12 expression and aHSC abundance are closely correlated in patients with liver metastases. Our data identify the interplay between NK cells and aHSCs as a master switch of cancer dormancy, and suggest that therapies aimed at normalizing the NK cell pool might succeed in preventing metastatic outgrowth.
Assuntos
Neoplasias da Mama/patologia , Células Estreladas do Fígado/citologia , Células Matadoras Naturais/citologia , Animais , Linhagem Celular Tumoral , Quimiocina CXCL12/metabolismo , Técnicas de Cocultura , Feminino , Humanos , Imunoterapia , Interferon gama , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Neoplasias Experimentais/patologia , Proteômica , Transcriptoma , Microambiente TumoralRESUMO
Diversity within or between tumours and metastases (known as intra-patient tumour heterogeneity) that develops during disease progression is a serious hurdle for therapy1-3. Metastasis is the fatal hallmark of cancer and the mechanisms of colonization, the most complex step in the metastatic cascade4, remain poorly defined. A clearer understanding of the cellular and molecular processes that underlie both intra-patient tumour heterogeneity and metastasis is crucial for the success of personalized cancer therapy. Here, using transcriptional profiling of tumours and matched metastases in patient-derived xenograft models in mice, we show cancer-site-specific phenotypes and increased glucocorticoid receptor activity in distant metastases. The glucocorticoid receptor mediates the effects of stress hormones, and of synthetic derivatives of these hormones that are used widely in the clinic as anti-inflammatory and immunosuppressive agents. We show that the increase in stress hormones during breast cancer progression results in the activation of the glucocorticoid receptor at distant metastatic sites, increased colonization and reduced survival. Our transcriptomics, proteomics and phospho-proteomics studies implicate the glucocorticoid receptor in the activation of multiple processes in metastasis and in the increased expression of kinase ROR1, both of which correlate with reduced survival. The ablation of ROR1 reduced metastatic outgrowth and prolonged survival in preclinical models. Our results indicate that the activation of the glucocorticoid receptor increases heterogeneity and metastasis, which suggests that caution is needed when using glucocorticoids to treat patients with breast cancer who have developed cancer-related complications.
Assuntos
Neoplasias da Mama/patologia , Glucocorticoides/efeitos adversos , Glucocorticoides/metabolismo , Metástase Neoplásica/patologia , Animais , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Dexametasona/efeitos adversos , Dexametasona/metabolismo , Progressão da Doença , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Taxa de SobrevidaRESUMO
Glioblastoma comprises 54% of all the gliomas derived from glial cells and are lethally malignant tumors of the central nervous system (CNS). Glioma cells disrupt the blood-brain barrier, leading to access of circulating immune cells to the CNS. Blocking the interaction between programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) enhances T-cell responses against tumor cells, and inhibition of the PD-1/PD-L1 pathway is used as immunotherapy for cancer, including glioblastoma. Nitric oxide (NO) has multiple physiological roles, such as immune modulation and neural transmission in the CNS. Moreover, it has both tumor-promoting and tumor-suppressive functions. We examined the effects of NOC-18, an NO donor, on the expression of PD-L1 in A172 glioblastoma cells. NOC-18 increased PD-L1 expression in A172 glioblastoma cells. Moreover, this increase is regulated via the c-Jun N-terminal kinase pathway.
Assuntos
Antígeno B7-H1/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , Compostos Nitrosos/farmacologia , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , HumanosRESUMO
We have synthesized 39 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] analogs having two side chains attached to carbon-20 (Gemini) with various modifications and compared their anticancer activities. Five structure-function rules emerged to identify analogs with enhanced anticancer activity. One of these active analogs, BXL-01-0126, was more potent than 1,25(OH)2D3 in mediating 50% clonal inhibition of cancer cell growth. Murine studies found that BXL-01-0126 and 1,25(OH)2D3 had nearly the same potency to raise serum calcium levels. Taken together, BXL-01-0126 when compared to 1,25(OH)2D3 has greater anticancer potency, but similar toxicity causing hypercalcemia. We focused on the effect of these compounds on the stimulation of expression of human cathelicidin antimicrobial peptide (CAMP) whose gene has a vitamin D response element in its promoter. Expression of CAMP mRNA and protein increased in a dose-response fashion after exposure of acute myeloid leukemia (AML) cells to the Gemini analog, BXL-01-126, in vitro. A xenograft model of AML was developed using U937 AML cells injected into NSG-immunodeficient mice. Administration of vitamin D3 compounds to these mice resulted in substantial levels of CAMP in the systemic circulation. This suggests a unique prophylactic treatment at diagnosis or during induction chemotherapy for AML patients to provide them with protection against various microbial infections through CAMP induction.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Calcitriol/análogos & derivados , Catelicidinas/biossíntese , Colecalciferol/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos , Antineoplásicos/química , Calcitriol/síntese química , Calcitriol/química , Calcitriol/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colecalciferol/análogos & derivados , Colecalciferol/síntese química , Citometria de Fluxo , Xenoenxertos , Humanos , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Relação Estrutura-AtividadeRESUMO
Cells assemble fibronectin, the major extracellular matrix (ECM) protein, into fibrillar matrices, which serve as 3D architectural scaffolds to provide, together with other ECM proteins tissue-specific environments. Although recent approaches enable to bioengineer 3D fibrillar fibronectin matrices in vitro, it remains elusive how fibronectin can be co-assembled with other ECM proteins into complex 3D fibrillar matrices that recapitulate tissue-specific compositions and cellular responses. Here, we introduce the engineering of fibrillar fibronectin-templated 3D matrices that can be complemented with other ECM proteins, including vitronectin, collagen, and laminin to resemble ECM architectures observed in vivo. For the co-assembly of different ECM proteins, we employed their innate fibrillogenic mechanisms including shear forces, pH-dependent electrostatic interactions, or specific binding domains. Through recapitulating various tissue-specific ECM compositions and morphologies, the large scale multi-composite 3D fibrillar ECM matrices can guide fibroblast adhesion, 3D fibroblast tissue formation, or tissue morphogenesis of epithelial cells. In other examples, we customize multi-composite 3D fibrillar matrices to support the growth of signal propagating neuronal networks and of human brain organoids. We envision that these 3D fibrillar ECM matrices can be tailored in scale and composition to modulate tissue-specific responses across various biological length scales and systems, and thus to advance manyfold studies of cell biological systems.
Assuntos
Matriz Extracelular , Fibroblastos , Fibronectinas , Engenharia Tecidual , Fibronectinas/química , Fibronectinas/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Humanos , Engenharia Tecidual/métodos , Fibroblastos/metabolismo , Fibroblastos/citologia , Animais , Alicerces Teciduais/química , Adesão Celular , Camundongos , Organoides/metabolismo , Organoides/citologiaRESUMO
A genome-wide PiggyBac transposon-mediated screen and a resistance screen in a PIK3CAH1047R-mutated murine tumor model reveal NF1 loss in mammary tumors resistant to the phosphatidylinositol 3-kinase α (PI3Kα)-selective inhibitor alpelisib. Depletion of NF1 in PIK3CAH1047R breast cancer cell lines and a patient-derived organoid model shows that NF1 loss reduces sensitivity to PI3Kα inhibition and correlates with enhanced glycolysis and lower levels of reactive oxygen species (ROS). Unexpectedly, the antioxidant N-acetylcysteine (NAC) sensitizes NF1 knockout cells to PI3Kα inhibition and reverts their glycolytic phenotype. Global phospho-proteomics indicates that combination with NAC enhances the inhibitory effect of alpelisib on mTOR signaling. In public datasets of human breast cancer, we find that NF1 is frequently mutated and that such mutations are enriched in metastases, an indication for which use of PI3Kα inhibitors has been approved. Our results raise the attractive possibility of combining PI3Kα inhibition with NAC supplementation, especially in patients with drug-resistant metastases associated with NF1 loss.
Assuntos
Neoplasias da Mama , Humanos , Camundongos , Animais , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fosfatidilinositol 3-Quinase , Acetilcisteína/farmacologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/genéticaRESUMO
19-nor-14-epi-23-yne-1,25(OH)(2) D(3) (inecalcitol) is a unique vitamin D(3) analog. We evaluated the activity of inecalcitol in a human prostate cancer model system. The analog was 11-fold more potent than 1,25(OH)(2) D(3) in causing 50% clonal growth inhibition of androgen-sensitive human prostate cancer LNCaP cells. Inecalcitol, more than 1,25(OH)(2) D(3) , reduced in a dose-dependent manner the expression levels of the transcription factor ETS variant 1 and the serine/threonine protein kinase Pim-1, both of which are upregulated in prostate cancer. Remarkably, dose challenge experiments revealed that inecalcitol maximal tolerated dose (MTD) by intraperitoneal (i.p.) administration was 30 µg/mouse (1,300 µg/kg) three times per week, while we previously found that the MTD of 1,25(OH)(2) D(3) is 0.0625 µg/mouse; therefore, inecalcitol is 480 times less hypercalcemic than 1,25(OH)(2) D(3) . Pharmacokinetic studies showed that plasma half-life of inecalcitol were 18.3 min in mice. A xenograft model of LNCaP cells was developed in immunodeficient mice treated with inecalcitol. The tumors of the diluent-treated control mice increased in size but those in the inecalcitol treatment group did not grow. Our data suggest that inecalcitol inhibits androgen-responsive prostate cancer growth in vivo and should be examined either alone or with other chemotherapy in clinical trials in individuals with rising serum prostate-specific antigen after receiving either surgery or irradiation therapy with curative intent.
Assuntos
Alcinos/uso terapêutico , Androgênios/fisiologia , Colecalciferol/análogos & derivados , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Colecalciferol/uso terapêutico , Humanos , Masculino , Camundongos , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Plasticity delineates cancer subtypes with more or less favourable outcomes. In breast cancer, the subtype triple-negative lacks expression of major differentiation markers, e.g., estrogen receptor α (ERα), and its high cellular plasticity results in greater aggressiveness and poorer prognosis than other subtypes. Whether plasticity itself represents a potential vulnerability of cancer cells is not clear. However, we show here that cancer cell plasticity can be exploited to differentiate triple-negative breast cancer (TNBC). Using a high-throughput imaging-based reporter drug screen with 9 501 compounds, we have identified three polo-like kinase 1 (PLK1) inhibitors as major inducers of ERα protein expression and downstream activity in TNBC cells. PLK1 inhibition upregulates a cell differentiation program characterized by increased DNA damage, mitotic arrest, and ultimately cell death. Furthermore, cells surviving PLK1 inhibition have decreased tumorigenic potential, and targeting PLK1 in already established tumours reduces tumour growth both in cell line- and patient-derived xenograft models. In addition, the upregulation of genes upon PLK1 inhibition correlates with their expression in normal breast tissue and with better overall survival in breast cancer patients. Our results indicate that differentiation therapy based on PLK1 inhibition is a potential alternative strategy to treat TNBC.
Assuntos
Neoplasias de Mama Triplo Negativas , Mama/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Receptor alfa de Estrogênio , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Afadin is an actin-filament-binding protein that binds to nectin, an immunoglobulin-like cell-cell adhesion molecule, and plays an important role in the formation of adherens junctions. Here, we show that afadin, which did not bind to nectin and was localized at the leading edge of moving cells, has another role: enhancement of the directional, but not random, cell movement. When NIH3T3 cells were stimulated with platelet-derived growth factor (PDGF), afadin colocalized with PDGF receptor, alphavbeta3 integrin and nectin-like molecule-5 at the leading edge and facilitated the formation of leading-edge structures and directional cell movement in the direction of PDGF stimulation. However, these phenotypes were markedly perturbed by knockdown of afadin, and were dependent on the binding of afadin to active Rap1. Binding of Rap1 to afadin was necessary for the recruitment of afadin and the tyrosine phosphatase SHP-2 to the leading edge. SHP-2 was previously reported to tightly regulate the activation of PDGF receptor and its downstream signaling pathway for the formation of the leading edge. These results indicate that afadin has a novel role in PDGF-induced directional cell movement, presumably in cooperation with active Rap1 and SHP-2.
Assuntos
Movimento Celular/efeitos dos fármacos , Proteínas dos Microfilamentos/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Western Blotting , Bovinos , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Imunoprecipitação , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Células NIH 3T3 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Acute promyelocytic leukemia (APL) is a hematopoietic malignant disease characterized by the chromosomal translocation t(15;17), resulting in the formation of the PML-RARA gene. Here, 47 t(15;17) APL samples were analyzed with high-density single-nucleotide polymorphism microarray (50-K and 250-K SNP-chips) using the new algorithm AsCNAR (allele-specific copy-number analysis using anonymous references). Copy-number-neutral loss of heterozygosity (CNN-LOH) was identified at chromosomes 10q (3 cases), 11p (3 cases), and 19q (1 case). Twenty-eight samples (60%) did not have an obvious alteration (normal-copy-number [NC] group). Nineteen samples (40%) showed either one or more genomic abnormalities: 8 samples (17%) had trisomy 8 either with or without an additional duplication, deletion, or CNN-LOH (+8 group); and 11 samples (23%) had genomic abnormalities without trisomy 8 (other abnormalities group). These chromosomal abnormalities were acquired somatic mutations. Interestingly, FLT3-ITD mutations (11/47 cases) occurred only in the group with no genomic alteration (NC group). Taken together, these results suggest that the pathway of development of APL differs in each group: FLT3-ITD, trisomy 8, and other genomic changes. Here, we showed for the first time hidden abnormalities and novel disease-related genomic changes in t(15;17) APL.
Assuntos
Cromossomos Humanos Par 15 , Cromossomos Humanos Par 17 , Leucemia Promielocítica Aguda/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Translocação Genética , Adolescente , Adulto , Idoso , Criança , Feminino , Dosagem de Genes , Regulação Leucêmica da Expressão Gênica , Genômica , Humanos , Leucemia Promielocítica Aguda/patologia , Perda de Heterozigosidade/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Células Tumorais Cultivadas , Adulto Jovem , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
BACKGROUND: Differences in survival have been reported between pediatric and adult acute lymphoblastic leukemia. The inferior prognosis in adult acute lymphoblastic leukemia is not fully understood but could be attributed, in part, to differences in genomic alterations found in adult as compared to in pediatric acute lymphoblastic leukemia. DESIGN AND METHODS: We compared two different sets of high-density single nucleotide polymorphism array genotyping data from 75 new diagnostic adult and 399 previously published diagnostic pediatric acute lymphoblastic leukemia samples. The patients' samples were randomly acquired from among Caucasian and Asian populations and hybridized to either Affymetrix 50K or 250K single nucleotide polymorphism arrays. The array data were investigated with Copy Number Analysis for GeneChips (CNAG) software for allele-specific copy number analysis. RESULTS: The high density single nucleotide polymorphism array analysis of 75 samples of adult acute lymphoblastic leukemia led to the identification of numerous cryptic and submicroscopic genomic lesions with a mean of 7.6 genomic alterations per sample. The patterns and frequencies of lesions detected in the adult samples largely reproduced known genomic hallmarks detected in previous single nucleotide polymorphism-array studies of pediatric acute lymphoblastic leukemia, such as common deletions of 3p14.2 (FHIT), 5q33.3 (EBF), 6q, 9p21.3 (CDKN2A/B), 9p13.2 (PAX5), 13q14.2 (RB1) and 17q11.2 (NF1). Some differences between adult and pediatric acute lymphoblastic leukemia were identified when the pediatric data set was partitioned into hyperdiploid and non-hyperdiploid cases and then compared to the nearly exclusively non-hyperdiploid adult samples. In this analysis, adult samples had a higher rate of deletions of chromosome 17p (TP53) and duplication of 17q. CONCLUSIONS: Our analysis of adult acute lymphoblastic leukemia cases led to the identification of new potential target lesions relevant for the pathogenesis of acute lymphoblastic leukemia. However, no unequivocal pattern of submicroscopic genomic alterations was found to separate adult acute lymphoblastic leukemia from pediatric acute lymphoblastic leukemia. Therefore, apart from different therapy regimen, differences of prognosis between adult and pediatric acute lymphoblastic leukemia are probably based on genetic subgroups according to cytogenetically detectable lesions but not focal genomic copy number microlesions.
Assuntos
Perfilação da Expressão Gênica/métodos , Genoma Humano , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Criança , Dosagem de Genes , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Deleção de Sequência , Adulto JovemRESUMO
The active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], modulates proliferation and induces differentiation of many cancer cells. A new class of analogs of vitamin D(3) has been synthesized, having two side-chains attached to carbon-20 (Gemini) and deuterium substituted on one side-chain. We have examined six of these analogs for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP, PC-3, DU145), lung (H520), colon (HT-29), and breast (MCF-7) cancer cell lines. Dose-response clonogenic studies showed that all six analogs had greater antiproliferative activities against cancer cells than 1,25(OH)(2)D(3). Although they had similar potency, the most active of these analogs was BXL-01-0120. BXL-01-0120 was 529-fold more potent than 1,25(OH)(2)D(3) in causing 50% clonal growth inhibition (ED(50)) of HL-60 cells. Pulse-exposure studies demonstrated that exposure to BXL-01-120 (10(-9)M, 48h) resulted in 85% clonal inhibition of HL-60 growth. BXL-01-0120 (10(-11)M, 4 days) induced the differentiation marker, CD11b. Also, morphologically differentiation was more prominent compared to 1,25(OH)(2)D(3). Annexin V assay showed that BXL-01-0120 (10(-10)M, 4 days) induced significantly (p<0.05) more apoptosis than 1,25(OH)(2)D(3). In summary, these analogs have a unique structure resulting in extremely potent inhibition of clonal proliferation of various types of cancer cells, especially HL-60 cells.
Assuntos
Antineoplásicos/farmacologia , Colecalciferol/análogos & derivados , Colecalciferol/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , HumanosRESUMO
Necl-5/Tage4/poliovirus receptor/CD155 is the poliovirus receptor and upregulated in rodent and human carcinoma. We have recently shown that mouse Necl-5 is upregulated by an oncogenic Ki-Ras (V12Ki-Ras) in NIH3T3 cells and enhances cell movement induced by growth factors, including platelet-derived growth factor and fibroblast growth factor (FGF), in an integrin alpha(v)beta(3)-dependent manner in wild type and V12Ki-Ras-transformed NIH3T3 cells. In addition, it enhances the growth factor-induced cell proliferation. We examined here how mouse Necl-5 was upregulated by V12Ki-Ras in NIH3T3 cells. Expression of the luciferase reporter gene fused to the Necl-5 promoter was induced by V12Ki-Ras in NIH3T3 cells. This induction was mediated through the Raf-MEK-ERK pathway. The Necl-5 promoter has an AP-1-binding site and this site was required for the V12Ki-Ras-induced activation of the Necl-5 promoter. Expression of the luciferase reporter gene fused to the Necl-5 promoter was also induced by FGF through the Raf-MEK-ERK-AP-1 pathway in NIH3T3 cells. These results indicate that the expression of mouse Necl-5 is induced by FGF or V12Ki-Ras through the Raf-MEK-ERK-AP-1 pathway.
Assuntos
Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Genes ras , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Neoplasias/genética , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Quinases raf/metabolismo , Células 3T3 , Animais , Antígenos de Neoplasias/efeitos dos fármacos , Moléculas de Adesão Celular/efeitos dos fármacos , Primers do DNA , Cinética , Camundongos , Proteínas de Neoplasias/efeitos dos fármacos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Somatic mutations in the spliceosome gene ZRSR2-located on the X chromosome-are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3'-splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here we characterize ZRSR2 as an essential component of the minor spliceosome (U12 dependent) assembly. shRNA-mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns and RNA-sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns, while splicing of the U2-type introns remain mostly unaffected. ZRSR2-deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS.
Assuntos
Processamento Alternativo , Mutação , Síndromes Mielodisplásicas/genética , Proteínas Nucleares/genética , RNA Nuclear Pequeno , Ribonucleoproteínas/genética , Animais , Antígenos CD34/metabolismo , Sequência de Bases , Células da Medula Óssea/citologia , Diferenciação Celular , Proliferação de Células , Éxons , Feminino , Genômica , Humanos , Íntrons , Células K562 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Dados de Sequência Molecular , Transplante de Neoplasias , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , SpliceossomosRESUMO
CONTEXT: Anaplastic thyroid cancer (ATC) has no effective treatment, resulting in a high rate of mortality. We established cell lines from a primary ATC and its lymph node metastasis, and investigated the molecular factors and genomic changes associated with tumor growth. OBJECTIVE: The aim of the study was to understand the molecular and genomic changes of highly aggressive ATC and its clonal evolution to develop rational therapies. DESIGN: We established unique cell lines from primary (OGK-P) and metastatic (OGK-M) ATC specimen, as well as primagraft from the metastatic ATC, which was serially xeno-transplanted for more than 1 year in NOD scid gamma mice were established. These cell lines and primagraft were used as tools to examine gene expression, copy number changes, and somatic mutations using RNA array, SNP Chip, and whole exome sequencing. RESULTS: Mice carrying sc (OGK-P and OGK-M) tumors developed splenomegaly and neutrophilia with high expression of cytokines including CSF1, CSF2, CSF3, IL-1ß, and IL-6. Levels of HIF-1α and its targeted genes were also elevated in these tumors. The treatment of tumor carrying mice with Bevacizumab effectively decreased tumor growth, macrophage infiltration, and peripheral WBCs. SNP chip analysis showed homozygous deletion of exons 3-22 of the PARD3 gene in the cells. Forced expression of PARD3 decreased cell proliferation, motility, and invasiveness, restores cell-cell contacts and enhanced cell adhesion. Next generation exome sequencing identified the somatic changes present in the primary, metastatic, and primagraft tumors demonstrating evolution of the mutational signature over the year of passage in vivo. CONCLUSION: To our knowledge, we established the first paired human primary and metastatic ATC cell lines offering unique possibilities for comparative functional investigations in vitro and in vivo. Our exome sequencing also identified novel mutations, as well as clonal evolution in both the metastasis and primagraft.
Assuntos
Linhagem Celular Tumoral/patologia , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Idoso , Animais , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Transplante de Neoplasias , Neutrófilos/patologia , Esplenomegalia/etiologia , Esplenomegalia/patologia , Carcinoma Anaplásico da Tireoide/complicações , Neoplasias da Glândula Tireoide/complicaçõesRESUMO
The use of genome-wide copy-number analysis and massive parallel sequencing has revolutionized the understanding of the clonal architecture of pediatric acute lymphoblastic leukemia (ALL) by demonstrating that this disease is composed of highly variable clonal ancestries following the rules of Darwinian selection. The current study aimed to analyze the molecular composition of childhood ALL biopsies and patient-derived xenografts with particular emphasis on mechanisms associated with acquired chemoresistance. Genomic DNA from seven primary pediatric ALL patient samples, 29 serially passaged xenografts, and six in vivo selected chemoresistant xenografts were analyzed with 250K single-nucleotide polymorphism arrays. Copy-number analysis of non-drug-selected xenografts confirmed a highly variable molecular pattern of variegated subclones. Whereas primary patient samples from initial diagnosis displayed a mean of 5.7 copy-number alterations per sample, serially passaged xenografts contained a mean of 8.2 and chemoresistant xenografts a mean of 10.5 copy-number alterations per sample, respectively. Resistance to cytarabine was explained by a new homozygous deletion of the DCK gene, whereas methotrexate resistance was associated with monoallelic deletion of FPGS and mutation of the remaining allele. This study demonstrates that selecting for chemoresistance in xenografted human ALL cells can reveal novel mechanisms associated with drug resistance.
Assuntos
Antineoplásicos/farmacologia , Células Clonais/patologia , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Animais , Antineoplásicos/uso terapêutico , Biópsia , Citarabina/farmacologia , Citarabina/uso terapêutico , DNA de Neoplasias/genética , Desoxicitidina Quinase/genética , Dexametasona/uso terapêutico , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Dosagem de Genes , Xenoenxertos , Humanos , Masculino , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Peptídeo Sintases/genética , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Quimera por Radiação , Análise de Sequência de DNA , Vincristina/uso terapêuticoRESUMO
Aberrantly activated c-MET signaling occurs in several cancers, promoting the development of c-MET inhibitors. In this study, we found that eight of eight thyroid cancer cell lines (including six anaplastic thyroid cell lines) have prominent expression of c-MET protein. Fifty percent of the thyroid cancer cell lines (four of eight) were growth inhibited by two small molecule c-MET inhibitors (tivantinib and crizotinib) associated with apoptosis and G(2)-M cell-cycle arrest. However, crizotinib did not inhibit 50% proliferation of thyroid cancer cells (SW1736 and TL3) at a concentration at which the drug completely inhibited ligand-stimulated c-MET phosphorylation. However, tivantinib was less potent than crizotinib at inhibiting c-MET phosphorylation, but was more potent than crizotinib at decreasing cell growth. Suppressing c-MET protein expression and phosphorylation using siRNA targeting c-MET did not induce cell-cycle arrest and apoptosis. Taken together, tivantinib and crizotinib have off-target(s) activity, contributing to their antitumor activity. In vivo study showed that crizotinib markedly inhibited the growth of thyroid cancer cells (SW1736) in immunodeficient mice. In summary, c-MET inhibitors (tivantinib and crizotinib) suppress the growth of aggressive thyroid cancer cells, and this potential therapeutic benefit results from their non-MET-targeting effects.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Crizotinibe , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-met/genética , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Pirrolidinonas/administração & dosagem , Quinolinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Glândula Tireoide/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The CCAAT/enhancer binding proteins (C/EBPs) are transcription factors involved in hematopoietic cell development and induction of several inflammatory mediators. C/EBPε is expressed only in myeloid cells including monocytes/macrophages. Atherosclerosis is an inflammatory disorder of the vascular wall and circulating immune cells such as monocytes/macrophages. Mice deficient in the low density lipoprotein (LDL) receptor (Ldlr-/-) fed on a high cholesterol diet (HCD) show elevated blood cholesterol levels and are widely used as models to study human atherosclerosis. In this study, we generated Ldlr and Cebpe double-knockout (llee) mice and compared their atherogenic phenotypes to Ldlr single deficient (llEE) mice after HCD. Macrophages from llee mice have reduced lipid uptake by foam cells and impaired phagokinetic motility in vitro compared to macrophages from llEE mice. Also, compared to llEE mice, llee mice have alterations of lipid metabolism, and reduced atheroma and obesity, particularly the males. Peritoneal macrophages of llee male mice have reduced mRNA expression of FABP4, a fatty acid binding protein implicated in atherosclerosis. Overall, our study suggests that the myeloid specific factor C/EBPε is involved in systemic lipid metabolism and that silencing of C/EBPε could decrease the development of atherosclerosis.