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1.
Nat Commun ; 11(1): 4660, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938908

RESUMO

Intratumor spatial heterogeneity facilitates therapeutic resistance in glioblastoma (GBM). Nonetheless, understanding of GBM heterogeneity is largely limited to the surgically resectable tumor core lesion while the seeds for recurrence reside in the unresectable tumor edge. In this study, stratification of GBM to core and edge demonstrates clinically relevant surgical sequelae. We establish regionally derived models of GBM edge and core that retain their spatial identity in a cell autonomous manner. Upon xenotransplantation, edge-derived cells show a higher capacity for infiltrative growth, while core cells demonstrate core lesions with greater therapy resistance. Investigation of intercellular signaling between these two tumor populations uncovers the paracrine crosstalk from tumor core that promotes malignancy and therapy resistance of edge cells. These phenotypic alterations are initiated by HDAC1 in GBM core cells which subsequently affect edge cells by secreting the soluble form of CD109 protein. Our data reveal the role of intracellular communication between regionally different populations of GBM cells in tumor recurrence.


Assuntos
Antígenos CD/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Histona Desacetilase 1/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Feminino , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Humanos , Camundongos SCID , Fenilbutiratos/farmacologia , Transdução de Sinais , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Tumour Biol ; 42(9): 1010428320962588, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32996421

RESUMO

A missense mutation of the guanine nucleotide binding protein alpha stimulating activity polypeptide 1 (GNAS) gene, typically Arg201Cys or Arg201His (R201H/R201C), leads to constitutive activation of the Gsα-cyclic AMP (cAMP) signaling pathway that causes several diseases. However, no germline mutations of GNAS have been identified to date, likely due to their lethality, and no robust human cell models have been generated. Therefore, the aim of this study was to generate GNAS-mutated disease-specific induced pluripotent stem cells as a model for these diseases. We then analyzed the functionality of this induced pluripotent stem cell model and differentiated epithelial cells. We generated disease-specific induced pluripotent stem cells by introducing a mutation in GNAS with the clustered regularly interspaced short palindromic repeats (CRISPR) nickase method, which has lower off-target effects than the conventional CRISPR/Cas9 method. We designed the target vector to contain the R201H mutation in GNAS, which was transfected into human control induced pluripotent stem cells (Nips-B2) by electroporation. We confirmed the establishment of GNASR201H-mutated (GNASR201H/+) induced pluripotent stem cells that exhibited a pluripotent stem cell phenotype. We analyzed the effect of the mutation on cAMP production, and further generated teratomas for immunohistochemical analysis of the luminal epithelial structure. GNAS-mutated induced pluripotent stem cells showed significantly higher levels of intracellular cAMP, which remained elevated state for a long time upon hormonal stimulation with parathyroid hormone or adrenocorticotropic hormone. Immunohistochemical analysis revealed that several mucins, including MUC1, 2, and MUC5AC, are expressed in cytokeratin 18 (CK18)-positive epithelial cells. However, we found few CK18-positive cells in mutated induced pluripotent stem cell-derived teratoma tissues, and reduced MUCINs expression in mutated epithelial cells. There was no difference in CDX2 expression; however, mutated epithelial cells were positive for CEA and CA19-9 expression. GNASR201H-mutated induced pluripotent stem cells and GNASR201H-mutated epithelial cells have distinct phenotypic and differentiation characteristics. We successfully established GNASR201H-mutated human induced pluripotent stem cells with increased cAMP production. Considering the differentiation potential of induced pluripotent stem cells, these cells will be useful as a model for elucidating the pathological mechanisms of GNAS-mutated diseases.


Assuntos
Cromograninas/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Mutação , Teratoma/patologia , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Cromograninas/antagonistas & inibidores , Subunidades alfa Gs de Proteínas de Ligação ao GTP/antagonistas & inibidores , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos SCID , Teratoma/genética
3.
Nat Commun ; 11(1): 4166, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820173

RESUMO

T cells engineered to express chimeric antigen receptors (CAR-T cells) have shown impressive clinical efficacy in the treatment of B cell malignancies. However, the development of CAR-T cell therapies for solid tumors is hampered by the lack of truly tumor-specific antigens and poor control over T cell activity. Here we present an avidity-controlled CAR (AvidCAR) platform with inducible and logic control functions. The key is the combination of (i) an improved CAR design which enables controlled CAR dimerization and (ii) a significant reduction of antigen-binding affinities to introduce dependence on bivalent interaction, i.e. avidity. The potential and versatility of the AvidCAR platform is exemplified by designing ON-switch CARs, which can be regulated with a clinically applied drug, and AND-gate CARs specifically recognizing combinations of two antigens. Thus, we expect that AvidCARs will be a highly valuable platform for the development of controllable CAR therapies with improved tumor specificity.


Assuntos
Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , Humanos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo
4.
Nat Commun ; 11(1): 3811, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732914

RESUMO

Intratumoral genomic heterogeneity in glioblastoma (GBM) is a barrier to overcoming therapy resistance. Treatments that are effective independent of genotype are urgently needed. By correlating intracellular metabolite levels with radiation resistance across dozens of genomically-distinct models of GBM, we find that purine metabolites, especially guanylates, strongly correlate with radiation resistance. Inhibiting GTP synthesis radiosensitizes GBM cells and patient-derived neurospheres by impairing DNA repair. Likewise, administration of exogenous purine nucleosides protects sensitive GBM models from radiation by promoting DNA repair. Neither modulating pyrimidine metabolism nor purine salvage has similar effects. An FDA-approved inhibitor of GTP synthesis potentiates the effects of radiation in flank and orthotopic patient-derived xenograft models of GBM. High expression of the rate-limiting enzyme of de novo GTP synthesis is associated with shorter survival in GBM patients. These findings indicate that inhibiting purine synthesis may be a promising strategy to overcome therapy resistance in this genomically heterogeneous disease.


Assuntos
Neoplasias Encefálicas/radioterapia , Reparo do DNA/genética , Glioblastoma/radioterapia , Guanosina Monofosfato/metabolismo , Tolerância a Radiação/genética , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Feminino , Glioblastoma/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , Nucleosídeos de Purina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nat Commun ; 11(1): 3806, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732922

RESUMO

Most triple-negative breast cancer (TNBC) patients fail to respond to T cell-mediated immunotherapies. Unfortunately, the molecular determinants are still poorly understood. Breast cancer is the disease genetically linked to a deficiency in autophagy. Here, we show that autophagy defects in TNBC cells inhibit T cell-mediated tumour killing in vitro and in vivo. Mechanistically, we identify Tenascin-C as a candidate for autophagy deficiency-mediated immunosuppression, in which Tenascin-C is Lys63-ubiquitinated by Skp2, particularly at Lys942 and Lys1882, thus promoting its recognition by p62 and leading to its selective autophagic degradation. High Tenascin-C expression is associated with poor prognosis and inversely correlated with LC3B expression and CD8+ T cells in TNBC patients. More importantly, inhibition of Tenascin-C in autophagy-impaired TNBC cells sensitizes T cell-mediated tumour killing and improves antitumour effects of single anti-PD1/PDL1 therapy. Our results provide a potential strategy for targeting TNBC with the combination of Tenascin-C blockade and immune checkpoint inhibitors.


Assuntos
Autofagia/imunologia , Linfócitos T CD8-Positivos/imunologia , Tenascina/metabolismo , Neoplasias de Mama Triplo Negativas/imunologia , Evasão Tumoral/imunologia , Animais , Antineoplásicos Imunológicos/farmacologia , Autofagia/genética , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Imunoterapia Adotiva , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/terapia , Evasão Tumoral/genética
6.
Nat Commun ; 11(1): 4116, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807793

RESUMO

Glioblastoma contains a rare population of self-renewing brain tumor stem cells (BTSCs) which are endowed with properties to proliferate, spur the growth of new tumors, and at the same time, evade ionizing radiation (IR) and chemotherapy. However, the drivers of BTSC resistance to therapy remain unknown. The cytokine receptor for oncostatin M (OSMR) regulates BTSC proliferation and glioblastoma tumorigenesis. Here, we report our discovery of a mitochondrial OSMR that confers resistance to IR via regulation of oxidative phosphorylation, independent of its role in cell proliferation. Mechanistically, OSMR is targeted to the mitochondrial matrix via the presequence translocase-associated motor complex components, mtHSP70 and TIM44. OSMR interacts with NADH ubiquinone oxidoreductase 1/2 (NDUFS1/2) of complex I and promotes mitochondrial respiration. Deletion of OSMR impairs spare respiratory capacity, increases reactive oxygen species, and sensitizes BTSCs to IR-induced cell death. Importantly, suppression of OSMR improves glioblastoma response to IR and prolongs lifespan.


Assuntos
Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Radiação Ionizante , Receptores de Oncostatina M/metabolismo , Animais , Morte Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Masculino , Camundongos , Camundongos SCID , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Células-Tronco Neoplásicas/efeitos da radiação , Oncostatina M/metabolismo , Estresse Oxidativo/efeitos da radiação , Receptores de Oncostatina M/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos da radiação
7.
Nat Commun ; 11(1): 4153, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814769

RESUMO

The histone methyltransferase DOT1L methylates lysine 79 (K79) on histone H3 and is involved in Mixed Lineage Leukemia (MLL) fusion leukemogenesis; however, its role in prostate cancer (PCa) is undefined. Here we show that DOT1L is overexpressed in PCa and is associated with poor outcome. Genetic and chemical inhibition of DOT1L selectively impaired the viability of androgen receptor (AR)-positive PCa cells and organoids, including castration-resistant and enzalutamide-resistant cells. The sensitivity of AR-positive cells is due to a distal K79 methylation-marked enhancer in the MYC gene bound by AR and DOT1L not present in AR-negative cells. DOT1L inhibition leads to reduced MYC expression and upregulation of MYC-regulated E3 ubiquitin ligases HECTD4 and MYCBP2, which promote AR and MYC degradation. This leads to further repression of MYC in a negative feed forward manner. Thus DOT1L selectively regulates the tumorigenicity of AR-positive prostate cancer cells and is a promising therapeutic target for PCa.


Assuntos
Histona-Lisina N-Metiltransferase/genética , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-myc/genética , Receptores Androgênicos/genética , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Linhagem Celular Tumoral , Intervalo Livre de Doença , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Compostos de Fenilureia/farmacologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Terapêutica com RNAi/métodos , Receptores Androgênicos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
8.
PLoS One ; 15(7): e0237106, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735605

RESUMO

Animal models are vital to the study of transfusion and development of new blood products. Post-transfusion recovery of human blood components can be studied in mice, however, there is a need to identify strains that can best tolerate xenogeneic transfusions, as well as to optimize such protocols. Specifically, the importance of using immunodeficient mice, such as NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, to study human transfusion has been questioned. In this study, strains of wild-type and NSG mice were compared as hosts for human transfusions with outcomes quantified by flow cytometric analyses of CD235a+ erythrocytes, CD45+ leukocytes, and CD41+CD42b+ platelets. Complete blood counts were evaluated as well as serum cytokines by multiplexing methods. Circulating human blood cells were maintained better in NSG than in wild-type mice. Lethargy and hemoglobinuria were observed in the first hours in wild-type mice along with increased pro-inflammatory cytokines/chemokines such as monocyte chemoattractant protein-1, tumor necrosis factor α, keratinocyte-derived chemokine (KC or CXCL1), and interleukin-6, whereas NSG mice were less severely affected. Whole blood transfusion resulted in rapid sequestration and then release of human cells back into the circulation within several hours. This rebound effect diminished when only erythrocytes were transfused. Nonetheless, human erythrocytes were found in excess of mouse erythrocytes in the liver and lungs and had a shorter half-life in circulation. Variables affecting the outcomes of transfused erythrocytes were cell dose and mouse weight; recipient sex did not affect outcomes. The sensitivity and utility of this xenogeneic model were shown by measuring the effects of erythrocyte damage due to exposure to the oxidizer diamide on post-transfusion recovery. Overall, immunodeficient mice are superior models for xenotransfusion as they maintain improved post-transfusion recovery with negligible immune-associated side effects.


Assuntos
Transfusão de Componentes Sanguíneos/métodos , Modelos Animais , Animais , Transfusão de Eritrócitos , Xenoenxertos , Humanos , Transfusão de Leucócitos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transfusão de Plaquetas
9.
PLoS One ; 15(8): e0235319, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810173

RESUMO

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Organoides/efeitos dos fármacos , Tanquirases/antagonistas & inibidores , Adulto , Animais , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/patologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Organoides/patologia
10.
Nat Commun ; 11(1): 3965, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770022

RESUMO

Dysregulated Wnt/ß-catenin activation plays a critical role in cancer progression, metastasis, and drug resistance. Genotoxic agents such as radiation and chemotherapeutics have been shown to activate the Wnt/ß-catenin signaling although the underlying mechanism remains incompletely understood. Here, we show that genotoxic agent-activated Wnt/ß-catenin signaling is independent of the FZD/LRP heterodimeric receptors and Wnt ligands. OTULIN, a linear linkage-specific deubiquitinase, is essential for the DNA damage-induced ß-catenin activation. OTULIN inhibits linear ubiquitination of ß-catenin, which attenuates its Lys48-linked ubiquitination and proteasomal degradation upon DNA damage. The association with ß-catenin is enhanced by OTULIN Tyr56 phosphorylation, which depends on genotoxic stress-activated ABL1/c-Abl. Inhibiting OTULIN or Wnt/ß-catenin sensitizes triple-negative breast cancer xenograft tumors to chemotherapeutics and reduces metastasis. Increased OTULIN levels are associated with aggressive molecular subtypes and poor survival in breast cancer patients. Thus, OTULIN-mediated Wnt/ß-catenin activation upon genotoxic treatments promotes drug resistance and metastasis in breast cancers.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Endopeptidases/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Metástase Neoplásica , Fosforilação , Fosfotirosina/metabolismo , Ubiquitinação , beta Catenina/metabolismo
11.
Nat Commun ; 11(1): 3726, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709856

RESUMO

Ovarian cancer (OVCA) inevitably acquires resistance to platinum chemotherapy and PARP inhibitors (PARPi). We show that acquisition of PARPi-resistance is accompanied by increased ATR-CHK1 activity and sensitivity to ATR inhibition (ATRi). However, PARPi-resistant cells are remarkably more sensitive to ATRi when combined with PARPi (PARPi-ATRi). Sensitivity to PARPi-ATRi in diverse PARPi and platinum-resistant models, including BRCA1/2 reversion and CCNE1-amplified models, correlate with synergistic increases in replication fork stalling, double-strand breaks, and apoptosis. Surprisingly, BRCA reversion mutations and an ability to form RAD51 foci are frequently not observed in models of acquired PARPi-resistance, suggesting the existence of alternative resistance mechanisms. However, regardless of the mechanisms of resistance, complete and durable therapeutic responses to PARPi-ATRi that significantly increase survival are observed in clinically relevant platinum and acquired PARPi-resistant patient-derived xenografts (PDXs) models. These findings indicate that PARPi-ATRi is a highly promising strategy for OVCAs that acquire resistance to PARPi and platinum.


Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Platina/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Ciclinas/metabolismo , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Técnicas de Inativação de Genes , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/genética , Rad51 Recombinase/metabolismo , Células-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Nat Commun ; 11(1): 3701, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709883

RESUMO

Despite its importance in human cancers, including colorectal cancers (CRC), oncogenic KRAS has been extremely challenging to target therapeutically. To identify potential vulnerabilities in KRAS-mutated CRC, we characterize the impact of oncogenic KRAS on the cell surface of intestinal epithelial cells. Here we show that oncogenic KRAS alters the expression of a myriad of cell-surface proteins implicated in diverse biological functions, and identify many potential surface-accessible therapeutic targets. Cell surface-based loss-of-function screens reveal that ATP7A, a copper-exporter upregulated by mutant KRAS, is essential for neoplastic growth. ATP7A is upregulated at the surface of KRAS-mutated CRC, and protects cells from excess copper-ion toxicity. We find that KRAS-mutated cells acquire copper via a non-canonical mechanism involving macropinocytosis, which appears to be required to support their growth. Together, these results indicate that copper bioavailability is a KRAS-selective vulnerability that could be exploited for the treatment of KRAS-mutated neoplasms.


Assuntos
Neoplasias Colorretais/metabolismo , Cobre/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Disponibilidade Biológica , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , ATPases Transportadoras de Cobre/metabolismo , Feminino , Humanos , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Mutação
13.
Nat Commun ; 11(1): 3327, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620863

RESUMO

Gaucher disease is a lysosomal storage disorder caused by insufficient glucocerebrosidase activity. Its hallmark manifestations are attributed to infiltration and inflammation by macrophages. Current therapies for Gaucher disease include life-long intravenous administration of recombinant glucocerebrosidase and orally-available glucosylceramide synthase inhibitors. An alternative approach is to engineer the patient's own hematopoietic system to restore glucocerebrosidase expression, thereby replacing the affected cells, and constituting a potential one-time therapy for this disease. Here, we report an efficient CRISPR/Cas9-based approach that targets glucocerebrosidase expression cassettes with a monocyte/macrophage-specific element to the CCR5 safe-harbor locus in human hematopoietic stem and progenitor cells. The targeted cells generate glucocerebrosidase-expressing macrophages and maintain long-term repopulation and multi-lineage differentiation potential with serial transplantation. The combination of a safe-harbor and a lineage-specific promoter establishes a universal correction strategy and circumvents potential toxicity of ectopic glucocerebrosidase in the stem cells. Furthermore, it constitutes an adaptable platform for other lysosomal enzyme deficiencies.


Assuntos
Edição de Genes/métodos , Glucosilceramidase/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/enzimologia , Macrófagos/enzimologia , Monócitos/enzimologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Doença de Gaucher/genética , Doença de Gaucher/terapia , Glucosilceramidase/genética , Células HEK293 , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Macrófagos/metabolismo , Engenharia Metabólica , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Monócitos/metabolismo , Transplante Autólogo
14.
PLoS One ; 15(7): e0219632, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32706829

RESUMO

INTRODUCTION: Surgical resection and systemic chemotherapy with temozolomide remain the mainstay for treatment of glioblastoma. However, many patients are not candidates for surgical resection given inaccessible tumor location or poor health status. Furthermore, despite being first line treatment, temozolomide has only limited efficacy. METHODS: The development of injectable hydrogel-based carrier systems allows for the delivery of a wide range of chemotherapeutics that can achieve high local concentrations, thus potentially avoiding systemic side effects and wide-spread neurotoxicity. To test this modality in a realistic environment, we developed a diblock copolypeptide hydrogel (DCH) capable of carrying and releasing paclitaxel, a compound that we found to be highly potent against primary gliomasphere cells. RESULTS: The DCH produced minimal tissue reactivity and was well tolerated in the immune-competent mouse brain. Paclitaxel-loaded hydrogel induced less tissue damage, cellular inflammation and reactive astrocytes than cremaphor-taxol (typical taxol-carrier) or hydrogel alone. In a deep subcortical xenograft model of glioblastoma in immunodeficient mice, injection of paclitaxel-loaded hydrogel led to local tumor control and improved survival. However, the tumor cells were highly migratory and were able to eventually escape the area of treatment. CONCLUSIONS: These findings suggest this technology may be ultimately applicable to patients with deep-seated inoperable tumors, but as currently formulated, complete tumor eradication would be highly unlikely. Future studies should focus on targeting the migratory potential of surviving cells.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Glioblastoma/tratamento farmacológico , Hidrogéis/química , Paclitaxel/uso terapêutico , Peptídeos/química , Animais , Antineoplásicos Fitogênicos/química , Linhagem Celular Tumoral , Sistema Nervoso Central/patologia , Portadores de Fármacos/química , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Paclitaxel/química , Taxa de Sobrevida , Temozolomida/química , Temozolomida/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
15.
PLoS One ; 15(7): e0236101, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678829

RESUMO

Dysregulation of histone demethylase Jumonji-C domain-containing protein 5 (JMJD5) has been identified as a great effect on tumorigenesis. Silibinin is a commonly used anti-hepatotoxic drug and exhibits anticancer effect in various cancers. However, the antitumor mechanism between silibinin and JMJD5 in oral squamous cell carcinoma (OSCC) remains unclear. In this study, the clinical significance of JMJD5 on OSCC patients was assessed through tissue microarray. Furthermore, mice bearing patient-derived tumor xenografts (PDTXs) and tongue cancer cell lines were treated with silibinin and evaluated for tumor growth and JMJD5 expression. High expression of JMJD5 in oral cancer was significantly associated with tumor size (P = 0.0241), cervical node metastasis (P = 0.0001) and clinical stage (P = 0.0002), was associated with worse survival rate compared with that of the total cohort (P = 0.0002). Collectively the data indicate that JMJD5 expression may be suitable for detection of unfavorable prognosis in OSCC patients, based in part on its apparent role as a marker of metastasis. In addition, silibinin inhibits cancer growth in vitro and in PDTX models. Furthermore, metastasis-associated protein 1 (MTA1) could regulate the expression for JMJD5 and had a positive correlation with JMJD5. Moreover, silibinin could downregulate JMJD5 and MTA1 in oral cancer. Present study thus identifies that JMJD5 might be an essential prognostic indicator and therapeutic target against OSCC progression. In addition, silibinin is a potential candidate among novel chemotherapeutic agents or adjuvants for modulating JMJD5 in OSCC, through a mechanism likely involving MTA1/JMJD5 axis.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Histona Desmetilases/metabolismo , Neoplasias Bucais/patologia , Proteínas Repressoras/metabolismo , Silibina/farmacologia , Transativadores/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Prognóstico , Proteínas Repressoras/genética , Taxa de Sobrevida , Transativadores/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Nat Commun ; 11(1): 3303, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620742

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) lethality is due to metastatic dissemination. Characterization of rare, heterogeneous circulating tumor cells (CTCs) can provide insight into metastasis and guide development of novel therapies. Using the CTC-iChip to purify CTCs from PDAC patients for RNA-seq characterization, we identify three major correlated gene sets, with stemness genes LIN28B/KLF4, WNT5A, and LGALS3 enriched in each correlated gene set; only LIN28B CTC expression was prognostic. CRISPR knockout of LIN28B-an oncofetal RNA-binding protein exerting diverse effects via negative regulation of let-7 miRNAs and other RNA targets-in cell and animal models confers a less aggressive/metastatic phenotype. This correlates with de-repression of let-7 miRNAs and is mimicked by silencing of downstream let-7 target HMGA2 or chemical inhibition of LIN28B/let-7 binding. Molecular characterization of CTCs provides a unique opportunity to correlated gene set metastatic profiles, identify drivers of dissemination, and develop therapies targeting the "seeds" of metastasis.


Assuntos
Carcinoma Ductal Pancreático/genética , Proteína HMGA2/genética , MicroRNAs/genética , Células Neoplásicas Circulantes/metabolismo , Neoplasias Pancreáticas/genética , Proteínas de Ligação a RNA/genética , Adulto , Idoso , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Proteína HMGA2/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteínas de Ligação a RNA/metabolismo
17.
Nat Commun ; 11(1): 3369, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632153

RESUMO

Induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons are an expected source for cell-based therapies for Parkinson's disease (PD). The regulatory criteria for the clinical application of these therapies, however, have not been established. Here we show the results of our pre-clinical study, in which we evaluate the safety and efficacy of dopaminergic progenitors (DAPs) derived from a clinical-grade human iPSC line. We confirm the characteristics of DAPs by in vitro analyses. We also verify that the DAP population include no residual undifferentiated iPSCs or early neural stem cells and have no genetic aberration in cancer-related genes. Furthermore, in vivo studies using immunodeficient mice reveal no tumorigenicity or toxicity of the cells. When the DAPs are transplanted into the striatum of 6-OHDA-lesioned rats, the animals show behavioral improvement. Based on these results, we started a clinical trial to treat PD patients in 2018.


Assuntos
Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/transplante , Doença de Parkinson/terapia , Transplante de Células-Tronco/métodos , Animais , Diferenciação Celular/genética , Linhagem Celular , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Macaca fascicularis , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Avaliação de Resultados em Cuidados de Saúde/métodos , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Ratos Nus , Transplante Heterólogo
18.
Nat Commun ; 11(1): 3406, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641768

RESUMO

Cancer stem cells are critical for cancer initiation, development, and treatment resistance. Our understanding of these processes, and how they relate to glioblastoma heterogeneity, is limited. To overcome these limitations, we performed single-cell RNA sequencing on 53586 adult glioblastoma cells and 22637 normal human fetal brain cells, and compared the lineage hierarchy of the developing human brain to the transcriptome of cancer cells. We find a conserved neural tri-lineage cancer hierarchy centered around glial progenitor-like cells. We also find that this progenitor population contains the majority of the cancer's cycling cells, and, using RNA velocity, is often the originator of the other cell types. Finally, we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development, suggests a possible origin for glioblastoma hierarchy, and helps to identify cancer stem cell-specific targets.


Assuntos
Neoplasias Encefálicas/genética , Encéfalo/metabolismo , Glioblastoma/genética , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Adulto , Animais , Antineoplásicos Alquilantes/farmacologia , Encéfalo/embriologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Feminino , Feto , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Análise de Célula Única/métodos , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
19.
Nat Commun ; 11(1): 3520, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665551

RESUMO

PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Imunoprecipitação da Cromatina , Biologia Computacional , Proteínas de Ligação a DNA/genética , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Linfoma/genética , Linfoma/metabolismo , Camundongos , Camundongos SCID , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Distribuição Aleatória , Fatores de Transcrição/genética , Transcriptoma/genética
20.
Prostate ; 80(13): 1134-1144, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32628304

RESUMO

BACKGROUND: Although androgen deprivation therapy (ADT) is the initial treatment strategy for prostate cancer (PCa), recurrent castration-resistant prostate cancer (CRPC) eventually ensues. In this study, cancer-derived immunoglobulin G (CIgG) is found to be induced after ADT, identifying CIgG as a potential CRPC driver gene. METHODS: The expression of CIgG and its clinical significance in PCa tissue was analyzed by The Cancer Genome Atlas database and immunohistochemistry. Subsequently, the sequence features of prostate cell line VHDJH rearrangements were analyzed. We also assessed the effect of CIgG on the migratory, invasive and proliferative abilities of PCa cells in vitro and vivo. Suspended microsphere, colony formation and drug-resistant assays were performed using PC3 cells with high CIgG expression (CIgGhigh ) and low CIgG expression (CIgG-/low ), and A nonobese diabetic/severe combined immunodeficiency mouse tumor xenograft model was developed for the study of the tumorigenic effects of the different cell populations. The SOX2-CIgG signaling pathway was validated by immunohistochemistry, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, Western blot, luciferase, and chromatin immunoprecipitation assays and bioinformatics analyses. Finally, we investigated the effect of RP215 inhibition on the progression of PCa in vivo using a Babl/c nude mouse xenograft model. RESULTS: CIgG is frequently expressed in PCa and associated with clinicopathological characteristics, moreover, CIgG transcripts with unique patterns of VHDJH rearrangements are found in PCa cells. Functional analyses identified that CIgG was induced by ADT and upregulated by SOX2 (SRY (sex determining region Y)-box 2) in PCa, promoting the development of PCa. In addition, our findings underscore a novel role of CIgG signaling in the maintenance of stemness and the progression of cancer through mitogen activated protein kinase/extracellular-signal-regulated kinase and AKT in PCa. In vivo experiments further demonstrated that depleting CIgG significantly suppressed the growth of PCa cell xenografts. Furthermore, a CIgG monoclonal antibody named RP215 exhibits tumor inhibitory effect as well. CONCLUSION: Our data suggests that CIgG could be a driver of PCa development, and that targeting the SOX2-CIgG axis may therefore inhibit PCa development after ADT.


Assuntos
Imunoglobulina G/imunologia , Neoplasias de Próstata Resistentes à Castração/imunologia , Fatores de Transcrição SOXB1/imunologia , Animais , Células HEK293 , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Células PC-3 , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , Transdução de Sinais/imunologia , Análise Serial de Tecidos
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