RESUMO
Transcription factor EB (TFEB) is a conserved master transcriptional activator of autophagy and lysosomal genes that modulates organismal lifespan regulation and stress resistance. As neurons can coordinate organism-wide processes, we investigated the role of neuronal TFEB in stress resistance and longevity. To this end, the Caenorhabditis elegans TFEB ortholog, hlh-30, was rescued panneuronally in hlh-30 loss of function mutants. While important in the long lifespan of daf-2 animals, neuronal HLH-30/TFEB was not sufficient to restore normal lifespan in short-lived hlh-30 mutants. However, neuronal HLH-30/TFEB rescue mediated robust improvements in the heat stress resistance of wildtype but not daf-2 animals. Notably, these mechanisms can be uncoupled, as neuronal HLH-30/TFEB requires DAF-16/FOXO to regulate longevity but not thermoresistance. Through further transcriptomics profiling and functional analysis, we discovered that neuronal HLH-30/TFEB modulates neurotransmission through the hitherto uncharacterized protein W06A11.1 by inducing peripheral mitochondrial fragmentation and organismal heat stress resistance in a non-cell autonomous manner. Taken together, this study uncovers a novel mechanism of heat stress protection mediated by neuronal HLH-30/TFEB.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição/metabolismo , Longevidade/genética , Neurônios/metabolismo , Fatores de Transcrição Forkhead/metabolismoRESUMO
While the bone marrow (BM) microenvironment is significantly remodelled in acute myeloid leukaemia (AML), molecular insight into AML-specific alterations in the microenvironment has been historically limited by the analysis of liquid marrow aspirates rather than core biopsies that contain solid-phase BM stroma. We assessed the effect of anthracycline- and cytarabine-based induction chemotherapy on both haematopoietic and non-haematopoietic cells directly in core BM biopsies using RNA-seq and histological analysis. We compared matched human core BM biopsies at diagnosis and 2 weeks after cytarabine- and anthracycline-based induction therapy in responders (<5% blasts present after treatment) and non-responders (≥5% blasts present after treatment). Our data indicated enrichment in vimentin (VIM), platelet-derived growth factor receptor beta (PDGFRB) and Snail family transcriptional repressor 2 (SNAI2) transcripts in responders, consistent with the reactivation of the mesenchymal population in the BM stroma. Enrichment of osteoblast maturation-related transcripts of biglycan (BGN), osteopontin (SPP1) and osteonectin (SPARC) was observed in non-responders. To the best of our knowledge, this is the first report demonstrating distinct osteogenic and mesenchymal transcriptome profiles specific to AML response to induction chemotherapy assessed directly in core BM biopsies. Detailing treatment response-specific alterations in the BM stroma may inform optimised therapeutic strategies for AML.
Assuntos
Medula Óssea , Leucemia Mieloide Aguda , Humanos , Medula Óssea/patologia , Transcriptoma , Leucemia Mieloide Aguda/tratamento farmacológico , Citarabina/uso terapêutico , Células da Medula Óssea/patologia , Antraciclinas/uso terapêutico , Biópsia , Microambiente TumoralRESUMO
Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH's ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.
Assuntos
Neoplasias Encefálicas , Glioma , Leucemia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Inibidores Enzimáticos/uso terapêutico , Glioma/tratamento farmacológico , Glioma/genética , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Camundongos , Mutação , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Salicilanilidas , TriazóisRESUMO
Improving CAR-T cell therapy for solid tumors requires a better understanding of CAR design and cellular composition. Here, we compared second-generation (BBζ and 28ζ) with third-generation (28BBζ) carbonic anhydrase IX (CAIX)-targeted CAR constructs and investigated the antitumor effect of CAR-T cells with different CD4/CD8 proportions in vitro and in vivo. The results demonstrated that BBζ exhibited superior efficacy compared with 28ζ and 28BBζ CAR-T cells in a clear-cell renal cell carcinoma (ccRCC) skrc-59 cell bearing NSG-SGM3 mouse model. The mice treated with a single dose of BBζ CD4/CD8 mixture (CAR4/8) showed complete tumor remission and remained tumor-free 72 days after CAR-T cells infusion. In the other CAR-T and control groups, tumor-infiltrating T cells were recovered and profiled. We found that BBζ CAR8 cells upregulated expression of major histocompatibility complex (MHC) class II and cytotoxicity-associated genes, while downregulating inhibitory immune checkpoint receptor genes and diminishing differentiation of regulatory T cells (Treg cells), leading to excellent therapeutic efficacy in vivo. Increased memory phenotype, elevated tumor infiltration, and decreased exhaustion genes were observed in the CD4/8 untransduced T (UNT) cells compared with CD8 alone, indicating that CD4/8 would be the favored cellular composition for CAR-T cell therapy with long-term persistence. In summary, these findings support that BBζ CAR4/8 cells are a highly potent, clinically translatable cell therapy for ccRCC.
RESUMO
PURPOSE: Dexamethasone, a uniquely potent corticosteroid, is frequently administered to patients with brain tumors to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in patients with glioblastoma (GBM), particularly in the context of immunotherapy. EXPERIMENTAL DESIGN: We evaluated the dose-dependent effects of dexamethasone when administered with programmed cell death 1 (PD-1) blockade and/or radiotherapy in immunocompetent C57BL/6 mice with syngeneic GL261 and CT-2A GBM tumors. Clinically, the effect of dexamethasone on survival was evaluated in 181 patients with isocitrate dehydrogenase (IDH) wild-type GBM treated with PD-(L)1 blockade, with adjustment for relevant prognostic factors. RESULTS: Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti-PD-1 therapy reduced survival in a dose-dependent manner. Concurrent dexamethasone also abrogated survival following anti-PD-1 therapy with or without radiotherapy in immune-resistant CT-2A models. Dexamethasone decreased T-lymphocyte numbers by increasing apoptosis, in addition to decreasing lymphocyte functional capacity. Myeloid and natural killer cell populations were also generally reduced by dexamethasone. Thus, dexamethasone appears to negatively affect both adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive patients with IDH wild-type GBM treated with PD-(L)1 blockade revealed poorer survival among those on baseline dexamethasone. Upon multivariable adjustment with relevant prognostic factors, baseline dexamethasone administration was the strongest predictor of poor survival [reference, no dexamethasone; <2 mg HR, 2.16; 95% confidence interval (CI), 1.30-3.68; P = 0.003 and ≥2 mg HR, 1.97; 95% CI, 1.23-3.16; P = 0.005]. CONCLUSIONS: Our preclinical and clinical data indicate that concurrent dexamethasone therapy may be detrimental to immunotherapeutic approaches for patients with GBM.
Assuntos
Edema Encefálico/tratamento farmacológico , Neoplasias Encefálicas/terapia , Dexametasona/farmacologia , Glioblastoma/terapia , Inibidores de Checkpoint Imunológico/farmacologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Edema Encefálico/etiologia , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral/transplante , Quimiorradioterapia/métodos , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Seguimentos , Glioblastoma/complicações , Glioblastoma/genética , Glioblastoma/mortalidade , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Isocitrato Desidrogenase/genética , Estimativa de Kaplan-Meier , Camundongos , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Estudos Retrospectivos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
More than 90% of small cell lung cancers (SCLCs) harbor loss-of-function mutations in the tumor suppressor gene RB1 The canonical function of the RB1 gene product, pRB, is to repress the E2F transcription factor family, but pRB also functions to regulate cellular differentiation in part through its binding to the histone demethylase KDM5A (also known as RBP2 or JARID1A). We show that KDM5A promotes SCLC proliferation and SCLC's neuroendocrine differentiation phenotype in part by sustaining expression of the neuroendocrine transcription factor ASCL1. Mechanistically, we found that KDM5A sustains ASCL1 levels and neuroendocrine differentiation by repressing NOTCH2 and NOTCH target genes. To test the role of KDM5A in SCLC tumorigenesis in vivo, we developed a CRISPR/Cas9-based mouse model of SCLC by delivering an adenovirus (or an adeno-associated virus [AAV]) that expresses Cre recombinase and sgRNAs targeting Rb1, Tp53, and Rbl2 into the lungs of Lox-Stop-Lox Cas9 mice. Coinclusion of a KDM5A sgRNA decreased SCLC tumorigenesis and metastasis, and the SCLCs that formed despite the absence of KDM5A had higher NOTCH activity compared to KDM5A+/+ SCLCs. This work establishes a role for KDM5A in SCLC tumorigenesis and suggests that KDM5 inhibitors should be explored as treatments for SCLC.
Assuntos
Diferenciação Celular/genética , Células Neuroendócrinas/citologia , Receptores Notch/fisiologia , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Transdução de Sinais/genética , Carcinoma de Pequenas Células do Pulmão/enzimologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linhagem Celular , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Histona Desmetilases/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Células Neuroendócrinas/patologia , Carcinoma de Pequenas Células do Pulmão/fisiopatologiaRESUMO
H3K27M mutations resulting in epigenetic dysfunction are frequently observed in diffuse intrinsic pontine glioma (DIPGs), an incurable pediatric cancer. We conduct a CRISPR screen revealing that knockout of KDM1A encoding lysine-specific demethylase 1 (LSD1) sensitizes DIPG cells to histone deacetylase (HDAC) inhibitors. Consistently, Corin, a bifunctional inhibitor of HDACs and LSD1, potently inhibits DIPG growth in vitro and in xenografts. Mechanistically, Corin increases H3K27me3 levels suppressed by H3K27M histones, and simultaneously increases HDAC-targeted H3K27ac and LSD1-targeted H3K4me1 at differentiation-associated genes. Corin treatment induces cell death, cell-cycle arrest, and a cellular differentiation phenotype and drives transcriptional changes correlating with increased survival time in DIPG patients. These data suggest a strategy for treating DIPG by simultaneously inhibiting LSD1 and HDACs.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Tronco Encefálico/tratamento farmacológico , Glioma/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Histona Desmetilases/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/mortalidade , Neoplasias do Tronco Encefálico/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glioma/genética , Glioma/mortalidade , Glioma/patologia , Código das Histonas/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Mutação , Ponte/patologia , RNA-Seq , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Myoepithelial cells play key roles in normal mammary gland development and in limiting pre-invasive to invasive breast tumor progression, yet their differentiation and perturbation in ductal carcinoma in situ (DCIS) are poorly understood. Here, we investigated myoepithelial cells in normal breast tissues of BRCA1 and BRCA2 germline mutation carriers and in non-carrier controls, and in sporadic DCIS. We found that in the normal breast of non-carriers, myoepithelial cells frequently co-express the p63 and TCF7 transcription factors and that p63 and TCF7 show overlapping chromatin peaks associated with differentiated myoepithelium-specific genes. In contrast, in normal breast tissues of BRCA1 mutation carriers the frequency of p63+TCF7+ myoepithelial cells is significantly decreased and p63 and TCF7 chromatin peaks do not overlap. These myoepithelial perturbations in normal breast tissues of BRCA1 germline mutation carriers may play a role in their higher risk of breast cancer. The fraction of p63+TCF7+ myoepithelial cells is also significantly decreased in DCIS, which may be associated with invasive progression.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Carcinoma Ductal de Mama/metabolismo , Mutação/genética , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Ductal de Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Feminino , Imunofluorescência , Mutação em Linhagem Germinativa/genética , Humanos , Imuno-Histoquímica , Camundongos , Fator 1 de Transcrição de Linfócitos T/genética , Fator 1 de Transcrição de Linfócitos T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti-PD-1 alone was ineffective, the erdafitinib and anti-PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti-PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti-PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Imunidade/efeitos dos fármacos , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Imunofenotipagem , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Prognóstico , Receptor de Morte Celular Programada 1/genética , Pirazóis/farmacologia , Quinoxalinas/farmacologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Resultado do Tratamento , Microambiente TumoralRESUMO
Small cell lung cancer (SCLC) accounts for 15% of lung cancers and is almost always linked to inactivating RB1 and TP53 mutations. SCLC frequently responds, albeit briefly, to chemotherapy. The canonical function of the RB1 gene product RB1 is to repress the E2F transcription factor family. RB1 also plays both E2F-dependent and E2F-independent mitotic roles. We performed a synthetic lethal CRISPR/Cas9 screen in an RB1 -/- SCLC cell line that conditionally expresses RB1 to identify dependencies that are caused by RB1 loss and discovered that RB1 -/- SCLC cell lines are hyperdependent on multiple proteins linked to chromosomal segregation, including Aurora B kinase. Moreover, we show that an Aurora B kinase inhibitor is efficacious in multiple preclinical SCLC models at concentrations that are well tolerated in mice. These results suggest that RB1 loss is a predictive biomarker for sensitivity to Aurora B kinase inhibitors in SCLC and perhaps other RB1 -/- cancers. SIGNIFICANCE: SCLC is rarely associated with actionable protooncogene mutations. We did a CRISPR/Cas9-based screen that showed that RB1 -/- SCLC are hyperdependent on AURKB, likely because both genes control mitotic fidelity, and confirmed that Aurora B kinase inhibitors are efficacious against RB1 -/- SCLC tumors in mice at nontoxic doses.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.
Assuntos
Aurora Quinase B/metabolismo , Proliferação de Células , Genes Supressores de Tumor , Neoplasias Pulmonares/patologia , Mutação , Proteínas de Ligação a Retinoblastoma/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Aurora Quinase B/genética , Sistemas CRISPR-Cas , Segregação de Cromossomos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Proteínas de Ligação a Retinoblastoma/antagonistas & inibidores , Proteínas de Ligação a Retinoblastoma/genética , Transdução de Sinais , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/metabolismo , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Magnetic Resonance Imaging (MRI) relies on optimal scanning parameters to achieve maximal signal-to-noise ratio (SNR) and high contrast-to-noise ratio (CNR) between tissues resulting in high quality images. The optimization of such parameters is often laborious, time consuming, and user-dependent, making harmonization of imaging parameters a difficult task. In this report, we aim to develop and validate a computer simulation technique that can reliably provide "optimal in vivo scanning parameters" ready to be used for in vivo evaluation of disease models. METHODS: A glioblastoma murine model was investigated using several MRI imaging methods. Such MRI methods underwent a simulated and an in vivo scanning parameter optimization in pre- and post-contrast conditions that involved the investigation of tumor, brain parenchyma and cerebrospinal fluid (CSF) CNR values in addition to the time relaxation values of the related tissues. The CNR tissues information were analyzed and the derived scanning parameters compared in order to validate the simulated methodology as a reliable technique for "optimal in vivo scanning parameters" estimation. RESULTS: The CNRs and the related scanning parameters were better correlated when spin-echo-based sequences were used rather than the gradient-echo-based sequences due to augmented inhomogeneity artifacts affecting the latter methods. "Optimal in vivo scanning parameters" were generated successfully by the simulations after initial scanning parameter adjustments that conformed to some of the parameters derived from the in vivo experiment. CONCLUSION: Scanning parameter optimization using the computer simulation was shown to be a valid surrogate to the in vivo approach in a glioblastoma murine model yielding in a better delineation and differentiation of the tumor from the contralateral hemisphere. In addition to drastically reducing the time invested in choosing optimal scanning parameters when compared to an in vivo approach, this simulation program could also be used to harmonize MRI acquisition parameters across scanners from different vendors.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Simulação por Computador , Glioblastoma/diagnóstico por imagem , Aumento da Imagem/métodos , Imageamento por Ressonância Magnética/métodos , Animais , Encéfalo/diagnóstico por imagem , Linhagem Celular Tumoral , Líquido Cefalorraquidiano/diagnóstico por imagem , Meios de Contraste/administração & dosagem , Modelos Animais de Doenças , Feminino , Gadolínio DTPA/administração & dosagem , Humanos , Camundongos , Razão Sinal-RuídoRESUMO
Gliomas with histone H3 lysine27-to-methionine mutations (H3K27M-glioma) arise primarily in the midline of the central nervous system of young children, suggesting a cooperation between genetics and cellular context in tumorigenesis. Although the genetics of H3K27M-glioma are well characterized, their cellular architecture remains uncharted. We performed single-cell RNA sequencing in 3321 cells from six primary H3K27M-glioma and matched models. We found that H3K27M-glioma primarily contain cells that resemble oligodendrocyte precursor cells (OPC-like), whereas more differentiated malignant cells are a minority. OPC-like cells exhibit greater proliferation and tumor-propagating potential than their more differentiated counterparts and are at least in part sustained by PDGFRA signaling. Our study characterizes oncogenic and developmental programs in H3K27M-glioma at single-cell resolution and across genetic subclones, suggesting potential therapeutic targets in this disease.
Assuntos
Neoplasias Encefálicas/patologia , Carcinogênese/genética , Glioma/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Oncogenes , Neoplasias Encefálicas/genética , Proliferação de Células , Glioma/genética , Histonas/metabolismo , Humanos , Proteína Quinase 7 Ativada por Mitógeno/genética , Mutação , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodosRESUMO
Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.
Assuntos
Inativação Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias Ovarianas/metabolismo , Ubiquitina C/biossíntese , Ubiquitina/biossíntese , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Ubiquitina/genética , Ubiquitina C/genéticaRESUMO
Reduced expression of both classical and desmosomal cadherins has been associated with different types of carcinomas, including prostate cancer. This study aims to provide a comprehensive view of the role and regulation of cell-cell adhesion in prostate cancer aggressiveness by examining the functional implications of both E-cadherin and Desmoglein 2 (DSG2). E-cadherin expression was first examined using immunofluorescence in 50 normal prostate tissues and in a cohort of 414 prostate cancer patients. Correlation and survival analyses were performed to assess its clinical significance. In primary prostate cancer patients, reduced expression of both E-cadherin and DSG2 is significantly associated with an earlier biochemical recurrence. Transgenic DU145 E-cadherin knockdown and constitutively active AKT overexpression lines were generated. Functional implications of such genetic alterations were analyzed in vitro and in vivo, the latter by using tumorigenesis as well as extravasation and metastatic tumor formation assays. We observed that loss of E-cadherin leads to impaired primary and metastatic tumor formation in vivo, suggesting a tumor promoter role for E-cadherin in addition to its known role as a tumor suppressor. Activation of AKT leads to a significant reduction in E-cadherin expression and nuclear localization of Snail, suggesting a role for the PI3K/AKT signaling pathway in the transient repression of E-cadherin. This reduced expression may be regulated by separate mechanisms as neither the loss of E-cadherin nor activation of AKT significantly affected DSG2 expression. In conclusion, these findings illustrate the critical role of cell-cell adhesion in the progression to aggressive prostate cancer, through regulation by the PI3K pathway.
Assuntos
Caderinas/genética , Desmogleína 2/genética , Regulação Neoplásica da Expressão Gênica , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Junções Aderentes/metabolismo , Animais , Biomarcadores Tumorais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Desmossomos/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismoRESUMO
Prostatic intraepithelial neoplasia is a precursor to prostate cancer. Herein, deletion of the NAD(+)-dependent histone deacetylase Sirt1 induced histological features of prostatic intraepithelial neoplasia at 7 months of age; these features were associated with increased cell proliferation and enhanced mitophagy. In human prostate cancer, lower Sirt1 expression in the luminal epithelium was associated with poor prognosis. Genetic deletion of Sirt1 increased mitochondrial superoxide dismutase 2 (Sod2) acetylation of lysine residue 68, thereby enhancing reactive oxygen species (ROS) production and reducing SOD2 activity. The PARK2 gene, which has several features of a tumor suppressor, encodes an E3 ubiquitin ligase that participates in removal of damaged mitochondria via mitophagy. Increased ROS in Sirt1(-/-) cells enhanced the recruitment of Park2 to the mitochondria, inducing mitophagy. Sirt1 restoration inhibited PARK2 translocation and ROS production requiring the Sirt1 catalytic domain. Thus, the NAD(+)-dependent inhibition of SOD2 activity and ROS by SIRT1 provides a gatekeeper function to reduce PARK2-mediated mitophagy and aberrant cell survival.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mitocôndrias/metabolismo , Mitofagia , Neoplasia Prostática Intraepitelial/metabolismo , Sirtuína 1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células 3T3 , Animais , Sobrevivência Celular , Genótipo , Histona Desacetilases/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Estresse Oxidativo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
PURPOSE: Several risk factors have been claimed to predict the progression of clinically high grade T1 bladder tumors. However, these factors are not specific enough to define which patients should be treated immediately with radical cystectomy. Therefore, it is critical to identify molecular markers that can help provide individualized, risk stratified decision making. Our main goal was to evaluate the role of total p63, p53 and ΔNp63 expression in cases of clinically high grade T1 bladder cancer progression. MATERIALS AND METHODS: Total p63, p53 and ΔNp63 expression was analyzed by immunohistochemistry in 134 clinically high grade T1 tumors. We assessed clinical progression to muscle invasive disease or radical cystectomy as a patient outcome end point. Survival analysis was done for recurrence-free, progression-free, disease specific and overall survival. RESULTS: A total of 132 patients (98.5%) underwent repeat transurethral resection. Cases of early progression (less than 3 months) were excluded from study to avoid under staging. Of the tumors 90 (67.2%) showed ΔNp63 expression loss. During a median followup of 62.1 months 19 patients (14.2%) progressed to muscle invasive disease. The progression rate was 21.1% in patients with tumors characterized by ΔNp63 loss but no progression was observed in those with tumors with ΔNp63 expression (p <0.001). There was no difference in the number of patients who underwent repeat transurethral resection, had associated carcinoma in situ, showed lymphovascular invasion or received followup intravesical bacillus Calmette-Guérin courses. CONCLUSIONS: ΔNp63 expression is a favorable prognostic factor in clinically high grade T1 bladder cancer. This marker identifies patients at low risk for progression who could benefit from conservative therapy with transurethral bladder tumor resection and bacillus Calmette-Guérin, avoiding over treatment with immediate radical cystectomy.
Assuntos
Biomarcadores Tumorais/biossíntese , Fatores de Transcrição/biossíntese , Proteínas Supressoras de Tumor/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Biomarcadores Tumorais/análise , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Masculino , Gradação de Tumores , Fatores de Proteção , Fatores de Transcrição/análise , Proteínas Supressoras de Tumor/análise , Neoplasias da Bexiga Urinária/químicaRESUMO
PURPOSE: The expression of desmogleins (DSGs), which are known to be crucial for establishing and maintaining the cell-cell adhesion required for tissue integrity, has been well characterized in the epidermis and hair follicle; however, their expression in other epithelial tissues such as prostate is poorly understood. Although downregulation of classical cadherins, such as E-cadherin, has been described in prostate cancer tissue samples, the expression of desmogleins has only been previously reported in prostate cancer cell lines. In this study we characterized desmoglein expression in normal prostate tissues, and further investigated whether Desmoglein 2 (DSG2) expression specifically can serve as a potential clinical prognostic factor for patients diagnosed with primary prostate cancer. EXPERIMENTAL DESIGN: We utilized immunofluorescence to examine DSG2 expression in normal prostate (n = 50) and in a clinically well-characterized cohort of prostate cancer patients (n = 414). Correlation of DSG2 expression with clinico-pathological characteristics and biochemical recurrence was analyzed to assess its clinical significance. RESULTS: These studies revealed that DSG2 and DSG4 were specifically expressed in prostatic luminal cells, whereas basal cells lack their expression. In contrast, DSG1 and DSG3 were not expressed in normal prostate epithelium. Further analyses of DSG2 expression in prostate cancer revealed that reduced levels of this biomarker were a significant independent marker of poor clinical outcome. CONCLUSION: Here we report for the first time that a low DSG2 expression phenotype is a useful prognostic biomarker of tumor aggressiveness and may serve as an aid in identifying patients with clinically significant prostate cancer.
Assuntos
Desmogleínas/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Adulto , Idoso , Linhagem Celular Tumoral , Estudos de Coortes , Desmogleína 2/genética , Desmogleína 2/metabolismo , Desmogleínas/genética , Progressão da Doença , Seguimentos , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Avaliação de Resultados da Assistência ao Paciente , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidadeRESUMO
The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.
Assuntos
Técnicas Citológicas/métodos , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/patologia , Citometria de Fluxo/métodos , Humanos , Masculino , Células-Tronco Neoplásicas/química , Neoplasias da Próstata/química , Neoplasias de Próstata Resistentes à Castração/química , Neoplasias de Próstata Resistentes à Castração/patologia , Coloração e Rotulagem/métodosRESUMO
UNLABELLED: p53 mutations and downregulation of promyelocytic leukemia (PML) are common genetic alterations in human cancers. In healthy cells these two key tumor suppressors exist in a positive regulatory loop, promoting cell death and cellular senescence. However, the influence of their interplay on tumorigenesis has not been explored directly in vivo. The contribution of PML to mutant p53 driven cancer was evaluated in a mouse model harboring a p53 mutation (p53 (wild-type/R172H) ) that recapitulates a frequent p53 mutation (p53 (R175H) ) in human sporadic and Li-Fraumeni cancers. These mice with PML displayed perturbation of the hematopoietic compartment, manifested either as lymphoma or extramedullary hematopoiesis (EMH). EMH was associated with peripheral blood leucocytosis and macrocytic anemia, suggestive of myeloproliferative- myelodysplastic overlap. In contrast, a complete loss of PML from these mice resulted in a marked alteration in tumor profile. While the incidence of lymphomas was unaltered, EMH was not detected and the majority of mice succumbed to sarcomas. Further, males lacking PML exhibited a high incidence of soft tissue sarcomas and reduced survival, while females largely developed osteosarcomas, without impact on survival. Together, these findings demonstrate that PML is an important tumor suppressor dictating disease development in a pertinent mouse model of human cancer. KEY POINTS: (1) A mutant p53 allele disrupts hematopoiesis in mice, by promoting lymphomas and myeloproliferative / myelodysplastic overlap. (2) Coincidental p53 allele mutation and PML loss shifts the tumor profile toward sarcoma formation, which is paralleled in human leiomyosarcomas (indicated by immunohistochemistry; IHC).
Assuntos
Leucemia Promielocítica Aguda/patologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Neoplasias Ósseas/etiologia , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Humanos , Estimativa de Kaplan-Meier , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas Nucleares/genética , Osteossarcoma/etiologia , Proteína da Leucemia Promielocítica , Fatores Sexuais , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
miRNAs are small noncoding RNAs with critical roles in a large variety of biological processes such as development and tumorigenesis. miRNA expression profiling has been reported to be a powerful tool to classify tissue samples, including cancers, based on their developmental lineage. In this study, we have profiled the expression of miRNAs in bladder carcinoma in situ (CIS) and distinct cell compartments of the normal bladder, namely umbrella and basal-intermediate urothelial cells, as well as the muscularis propria. We identified several miRNAs differentially expressed between umbrella and basal-intermediate cells (miR-133a, miR-139-3p, miR-142-3p, miR-199b-5p, and miR-221). In situ hybridization confirmed the expression of miR-133a and miR-139-3p in umbrella cells, and miR-142-3p in basal-intermediate cells. Strikingly, miRNA expression levels of CIS most closely resembled the miRNA profile of umbrella cells. Finally, we examined well-established umbrella and basal-intermediate cell immunohistochemical biomarkers in an independent series of CIS samples. Again, this analysis revealed the significant expression of umbrella-specific markers in CIS when compared to non-CIS lesions. Overall, our studies represent a comprehensive and accurate description of the different miRNAs expressed in CIS tumors and three distinct histological areas of the urinary bladder. Notably, this study provides evidence of the possible origin relationship between CIS and normal umbrella cells.