RESUMO
Enteric glia are a distinct population of peripheral glial cells in the enteric nervous system that regulate intestinal homeostasis, epithelial barrier integrity, and gut defense. Given these unique attributes, we investigated the impact of enteric glia depletion on tumor development in azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice, a classical model of colorectal cancer (CRC). Depleting GFAP+ enteric glia resulted in a profoundly reduced tumor burden in AOM/DSS mice and additionally reduced adenomas in the ApcMin /+ mouse model of familial adenomatous polyposis, suggesting a tumor-promoting role for these cells at an early premalignant stage. This was confirmed in further studies of AOM/DSS mice, as enteric glia depletion did not affect the properties of established malignant tumors but did result in a marked reduction in the development of precancerous dysplastic lesions. Surprisingly, the protective effect of enteric glia depletion was not dependent on modulation of anti-tumor immunity or intestinal inflammation. These findings reveal that GFAP+ enteric glia play a critical pro-tumorigenic role during early CRC development and identify these cells as a potential target for CRC prevention.
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Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental non-malignant cells for survival. We compared the transcriptomes of primary CLL cells cocultured or not with protective bone marrow stromal cells (BMSCs) and found that oxidative phosphorylation, mitochondrial function, and hypoxic signaling undergo most significant dysregulation in non-protected CLL cells, with the changes peaking at 6-8 h, directly before induction of apoptosis. A subset of CLL patients displayed a gene expression signature resembling that of cocultured CLL cells and had significantly worse progression-free and overall survival. To identify drugs blocking BMSC-mediated support, we compared the relevant transcriptomic changes to the Connectivity Map database. Correlation was found with the transcriptomic signatures of the cardiac glycoside ouabain and of the ipecac alkaloids emetine and cephaeline. These compounds were highly active against protected primary CLL cells (relative IC50's 287, 190, and 35 nM, respectively) and acted by repressing HIF-1α and disturbing intracellular redox homeostasis. We tested emetine in a murine model of CLL and observed decreased CLL cells in peripheral blood, spleen, and bone marrow, recovery of hematological parameters and doubling of median survival (31.5 vs. 15 days, P = 0.0001). Pathways regulating redox homeostasis are thus therapeutically targetable mediators of microenvironmental support in CLL cells.
Assuntos
Leucemia Linfocítica Crônica de Células B/patologia , Estresse Oxidativo/fisiologia , Microambiente Tumoral/fisiologia , Animais , Técnicas de Cocultura , Emetina/farmacologia , Xenoenxertos , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Pancreatic ductal adenocarcinoma (PDAC) after complete surgical resection is often followed by distant metastatic relapse for reasons that remain unclear. In this study, we investigated how the immune response at secondary sites affects tumor spread in murine models of metastatic PDAC. Early metastases were associated with dense networks of CD11b+CD11c+MHC-II+CD24+CD64lowF4/80low dendritic cells (DC), which developed from monocytes in response to tumor-released GM-CSF. These cells uniquely expressed MGL2 and PD-L2 in the metastatic microenvironment and preferentially induced the expansion of T regulatory cells (Treg) in vitro and in vivo Targeted depletion of this DC population in Mgl2DTR hosts activated cytotoxic lymphocytes, reduced Tregs, and inhibited metastasis development. Moreover, blocking PD-L2 selectively activated CD8 T cells at secondary sites and suppressed metastasis, suggesting that the DCs use this particular pathway to inhibit CD8 T-cell-mediated tumor immunity. Phenotypically similar DCs accumulated at primary and secondary sites in other models and in human PDAC. These studies suggest that a discrete DC subset both expands Tregs and suppresses CD8 T cells to establish an immunosuppressive microenvironment conducive to metastasis formation. Therapeutic strategies to block the accumulation and immunosuppressive activity of such cells may help prevent PDAC progression and metastatic relapse after surgical resection. Cancer Res; 77(15); 4158-70. ©2017 AACR.
Assuntos
Carcinoma Ductal Pancreático/patologia , Células Dendríticas/imunologia , Invasividade Neoplásica/imunologia , Neoplasias Pancreáticas/patologia , Evasão Tumoral/imunologia , Animais , Carcinoma Ductal Pancreático/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Camundongos , Invasividade Neoplásica/patologia , Neoplasias Pancreáticas/imunologiaRESUMO
BM-derived DC (BMDC) are powerful antigen-presenting cells. When loaded with immune complexes (IC), consisting of tumor antigens bound to antitumor antibody, BMDC induce powerful antitumor immunity in mice. However, attempts to employ this strategy clinically with either tumor-associated DC (TADC) or monocyte-derived DC (MoDC) have been disappointing. To investigate the basis for this phenomenon, we compared the response of BMDC, TADC, and MoDC to tumor IgG-IC. Our findings revealed, in both mice and humans, that upon exposure to IgG-IC, BMDC internalized the IC, increased costimulatory molecule expression, and stimulated autologous T cells. In contrast, TADC and, surprisingly, MoDC remained inert upon contact with IC due to dysfunctional signaling following engagement of Fcγ receptors. Such dysfunction is associated with elevated levels of the Src homology region 2 domain-containing phosphatase-1 (SHP-1) and phosphatases regulating Akt activation. Indeed, concomitant inhibition of both SHP-1 and phosphatases that regulate Akt activation conferred upon TADC and MoDC the capacity to take up and process IC and induce antitumor immunity in vivo. This work identifies the molecular checkpoints that govern activation of MoDC and TADC and their capacity to elicit T cell immunity.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Antígenos de Neoplasias/imunologia , Células Dendríticas/imunologia , Neoplasias Pulmonares/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Ativação Linfocitária , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Recidiva Local de Neoplasia , Neoplasias Experimentais/imunologia , Linfócitos T/imunologia , Microambiente TumoralRESUMO
Chronic intestinal inflammation accompanies familial adenomatous polyposis (FAP) and is a major risk factor for colorectal cancer in patients with this disease, but the cause of such inflammation is unknown. Because retinoic acid (RA) plays a critical role in maintaining immune homeostasis in the intestine, we hypothesized that altered RA metabolism contributes to inflammation and tumorigenesis in FAP. To assess this hypothesis, we analyzed RA metabolism in the intestines of patients with FAP as well as APCMin/+ mice, a model that recapitulates FAP in most respects. We also investigated the impact of intestinal RA repletion and depletion on tumorigenesis and inflammation in APCMin/+ mice. Tumors from both FAP patients and APCMin/+ mice displayed striking alterations in RA metabolism that resulted in reduced intestinal RA. APCMin/+ mice placed on a vitamin A-deficient diet exhibited further reductions in intestinal RA with concomitant increases in inflammation and tumor burden. Conversely, restoration of RA by pharmacologic blockade of the RA-catabolizing enzyme CYP26A1 attenuated inflammation and diminished tumor burden. To investigate the effect of RA deficiency on the gut immune system, we studied lamina propria dendritic cells (LPDC) because these cells play a central role in promoting tolerance. APCMin/+ LPDCs preferentially induced Th17 cells, but reverted to inducing Tregs following restoration of intestinal RA in vivo or direct treatment of LPDCs with RA in vitro These findings demonstrate the importance of intestinal RA deficiency in tumorigenesis and suggest that pharmacologic repletion of RA could reduce tumorigenesis in FAP patients. Cancer Immunol Res; 4(11); 917-26. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Enterocolite/genética , Genes APC , Tretinoína/farmacologia , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Polipose Adenomatosa do Colo/complicações , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Animais , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Enterocolite/tratamento farmacológico , Enterocolite/metabolismo , Enterocolite/patologia , Humanos , Camundongos , Fenótipo , Células Th17/imunologia , Células Th17/metabolismo , Tretinoína/metabolismo , Carga Tumoral , Vitamina A/metabolismo , Deficiência de Vitamina A/metabolismoRESUMO
Although all-trans-retinoic acid (atRA) is a key regulator of intestinal immunity, its role in colorectal cancer (CRC) is unknown. We found that mice with colitis-associated CRC had a marked deficiency in colonic atRA due to alterations in atRA metabolism mediated by microbiota-induced intestinal inflammation. Human ulcerative colitis (UC), UC-associated CRC, and sporadic CRC specimens have similar alterations in atRA metabolic enzymes, consistent with reduced colonic atRA. Inhibition of atRA signaling promoted tumorigenesis, whereas atRA supplementation reduced tumor burden. The benefit of atRA treatment was mediated by cytotoxic CD8(+) T cells, which were activated due to MHCI upregulation on tumor cells. Consistent with these findings, increased colonic expression of the atRA-catabolizing enzyme, CYP26A1, correlated with reduced frequencies of tumoral cytotoxic CD8(+) T cells and with worse disease prognosis in human CRC. These results reveal a mechanism by which microbiota drive colon carcinogenesis and highlight atRA metabolism as a therapeutic target for CRC.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias Colorretais/imunologia , Microbiota/imunologia , Tretinoína/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Carcinogênese/imunologia , Colo/imunologia , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ácido Retinoico 4 Hidroxilase/metabolismo , Transdução de Sinais/imunologia , Regulação para Cima/imunologiaRESUMO
Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.
Assuntos
Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Animais , Medula Óssea/imunologia , Ritmo Circadiano/imunologia , Citometria de Fluxo , Variação Genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fenótipo , Padrões de ReferênciaRESUMO
Whereas cancers grow within host tissues and evade host immunity through immune-editing and immunosuppression, tumours are rarely transmissible between individuals. Much like transplanted allogeneic organs, allogeneic tumours are reliably rejected by host T cells, even when the tumour and host share the same major histocompatibility complex alleles, the most potent determinants of transplant rejection. How such tumour-eradicating immunity is initiated remains unknown, although elucidating this process could provide the basis for inducing similar responses against naturally arising tumours. Here we find that allogeneic tumour rejection is initiated in mice by naturally occurring tumour-binding IgG antibodies, which enable dendritic cells (DCs) to internalize tumour antigens and subsequently activate tumour-reactive T cells. We exploited this mechanism to treat autologous and autochthonous tumours successfully. Either systemic administration of DCs loaded with allogeneic-IgG-coated tumour cells or intratumoral injection of allogeneic IgG in combination with DC stimuli induced potent T-cell-mediated antitumour immune responses, resulting in tumour eradication in mouse models of melanoma, pancreas, lung and breast cancer. Moreover, this strategy led to eradication of distant tumours and metastases, as well as the injected primary tumours. To assess the clinical relevance of these findings, we studied antibodies and cells from patients with lung cancer. T cells from these patients responded vigorously to autologous tumour antigens after culture with allogeneic-IgG-loaded DCs, recapitulating our findings in mice. These results reveal that tumour-binding allogeneic IgG can induce powerful antitumour immunity that can be exploited for cancer immunotherapy.
Assuntos
Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/imunologia , Células Dendríticas/imunologia , Imunoglobulina G/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Antineoplásicos/administração & dosagem , Antígenos CD40/metabolismo , Modelos Animais de Doenças , Feminino , Imunoglobulina G/administração & dosagem , Isoanticorpos/administração & dosagem , Isoanticorpos/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Metástase Neoplásica , Transplante de Neoplasias/imunologia , Neoplasias/patologia , Receptores de IgG/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Early detection of colonic polyps can prevent up to 90% of colorectal cancer deaths. Conventional colonoscopy readily detects the majority of premalignant lesions, which exhibit raised morphology. However, lesions that are flat and depressed are often undetected using this method. Therefore, there is a need for molecular-based contrast agents to improve detection rates over conventional colonoscopy. We evaluated a quenched fluorescent activity-based probe (qABP; BMV109) that targets multiple cysteine cathepsins that are overexpressed in intestinal dysplasia in a genetic model of spontaneous intestinal polyp formation and in a chemically induced model of colorectal carcinoma. We found that the qABP selectively targets cysteine cathepsins, resulting in high sensitivity and specificity for intestinal tumors in mice and humans. Additionally, the qABP can be administered by either intravenous injection or by local delivery to the colon, making it a highly valuable tool for improved detection of colorectal lesions using fluorescence-guided colonoscopy.
Assuntos
Catepsinas/química , Corantes Fluorescentes/química , Neoplasias Intestinais/diagnóstico , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Carbocianinas/química , Domínio Catalítico , Catepsinas/metabolismo , Colonoscopia , Modelos Animais de Doenças , Corantes Fluorescentes/metabolismo , Humanos , Imuno-Histoquímica , Neoplasias Intestinais/química , Neoplasias Intestinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pólipos/química , Pólipos/diagnóstico , Pólipos/patologia , Sensibilidade e EspecificidadeRESUMO
Chronic lymphocytic leukemia (CLL) cells fail to enter apoptosis in vivo as opposed to their non-malignant B-lymphocyte counterparts. The ability of CLL cells to escape apoptosis is highly dependent on their microenvironment. Compared to non-malignant B cells, CLL cells are more responsive to complex stimuli that can be reproduced in vitro by the addition of cytokines. To understand the molecular mechanism of the environment-dependent anti-apoptotic signaling circuitry of CLL cells, we quantified the effect of the SDF-1, BAFF, APRIL, anti-IgM, interleukin-4 (IL4) and secreted CD40L (sCD40L) on the survival of in vitro cultured CLL cells and found IL4 and sCD40L to be most efficient in rescuing CLL cells from apoptosis. In quantitative dose-response experiments using cell survival as readout, the binding affinity of IL4 to its receptor was similar between malignant and non-malignant cells. However, the downstream signaling in terms of the amount of STAT6 and its degree of phosphorylation was highly stimulated in CLL cells. In contrast, the response to sCD40L showed a loss of cooperative binding in CLL cells but displayed a largely increased ligand binding affinity. Although a high-throughput microscopy analysis did not reveal a significant difference in the spatial CD40 receptor organization, the downstream signaling showed an enhanced activation of the NF-kB pathway in the malignant cells. Thus, we propose that the anti-apoptotic phenotype of CLL involves a sensitized response for IL4 dependent STAT6 phosphorylation, and an activation of NF-kB signaling due to an increased affinity of sCD40L to its receptor.
Assuntos
Ligante de CD40/metabolismo , Sobrevivência Celular , Interleucina-4/fisiologia , NF-kappa B/metabolismo , Fator de Transcrição STAT6/metabolismo , Apoptose , Linfócitos B/fisiologia , Ligante de CD40/fisiologia , Estudos de Casos e Controles , Humanos , Leucemia Linfocítica Crônica de Células B , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de SinaisRESUMO
BACKGROUND: Thalidomide may represent a novel therapeutic strategy in the treatment of chronic lymphocytic leukemia (CLL). Since the activation of nuclear factor kappa B (NF-κB) causes not only malignant transformation and tumor progression, but also allows tumor cells to evade immune surveillance, NF-κB signaling components might constitute a potential target for future therapy in CLL. OBJECTIVES: The current study is an attempt to characterize proteins regulated by thalidomide. Thalidomide's influence on NF-κB proteins and on regulatory T cells (Treg) in CLL was investigated. MATERIAL AND METHODS: A total of 15 patients with CLL were treated with a combined thalidomide/fludarabine regimen. Peripheral blood mononuclear cells were separated by Ficoll density gradient centrifugation. To evaluate glucocorticoid induced tumour-necrosis-factor-receptor-related protein (GITR) expression in regulatory T cells, cells incubated with anti-CD3, ani-CD4 and anti-CD25 were permeabilized and then stained with anti-FOXP3 and analyzed using flow cytometry. Human TNF enzyme-linked immunosorbent assay (ELISA) was used to determine the tumor necrosis factor (TNF) levels in the serum. To evaluate NF-κB activity, chemiluminescent oligonucleotide-based ELISA was performed. RESULTS: It was found that thalidomide regulates NF-κB activity differentially, and the activity of certain NF-κB components correlated with TNF levels and T regulatory cell (CD4 + CD25 high GITR + ). CONCLUSIONS: These results might indicate that thalidomide not only regulates TNF but also directly interferes with NF-κB components.
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Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , NF-kappa B/fisiologia , Linfócitos T Reguladores/imunologia , Talidomida/farmacologia , ADP-Ribosil Ciclase 1/análise , Idoso , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Proteína-Tirosina Quinase ZAP-70/análiseRESUMO
CONTEXT: Talactoferrin alfa (TLF) is a unique recombinant form of human lactoferrin. The hypothesized mechanism of action involves TLF binding to the intestinal endothelium inducing dendritic cell maturation and cytokine release leading to infiltration of tumor with monocytes and T-lymphocytes and inhibition of tumor growth. OBJECTIVE: Based on promising phase II trial results, this correlative study was undertaken to examine immune mechanism of action of TLF in metastatic non-small cell lung cancer (NSCLC) patients. METHODS: Talactoferrin was administered orally at 1.5 g bid weeks 1-12 with 2 weeks off on a 14-week cycle. Enrolled patients had a pathologic diagnosis of NSCLC previously treated with at least two lines of systemic treatment. Patients had core biopsy of tumor before initiation of talactoferrin and at week 7 on TLF. Flow cytometry and quantitative immunohistochemistry for immune correlates were performed on the biopsied specimens. RESULTS: Four patients with metastatic NSCLC were enrolled. The trial was halted pre-maturely in light of negative phase III trial results. For the two patients who had repeat on-treatment tumor biopsies, a consistent increase in monocytes as a percentage of total immune cells was observed. Otherwise, no clear trend of increase or decrease was observed in any other immune cell parameters compared to matched patient pre-treatment biopsies. CONCLUSION: Repeat biopsies for immune correlates by flow cytometry and quantitative immunohistochemistry in NSCLC patients are feasible. In the few patients sampled before trial closure, increased monocytes as a total percentage of the immune cell population within tumor was observed in response to TLF.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Lactoferrina/imunologia , Neoplasias Pulmonares/imunologia , Recidiva Local de Neoplasia/imunologia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Dendritic cells (DCs) are important mediators of anti-tumor immune responses. We hypothesized that an in-depth analysis of dendritic cells and their spatial relationships to each other as well as to other immune cells within tumor draining lymph nodes (TDLNs) could provide a better understanding of immune function and dysregulation in cancer. METHODS: We analyzed immune cells within TDLNs from 59 breast cancer patients with at least 5 years of clinical follow-up using immunohistochemical staining with a novel quantitative image analysis system. We developed algorithms to analyze spatial distribution patterns of immune cells in cancer versus healthy intra-mammary lymph nodes (HLNs) to derive information about possible mechanisms underlying immune-dysregulation in breast cancer. We used the non-parametric Mann-Whitney test for inter-group comparisons, Wilcoxon Matched-Pairs Signed Ranks test for intra-group comparisons and log-rank (Mantel-Cox) test for Kaplan Maier analyses. RESULTS: Degree of clustering of DCs (in terms of spatial proximity of the cells to each other) was reduced in TDLNs compared to HLNs. While there were more numerous DC clusters in TDLNs compared to HLNs,DC clusters within TDLNs tended to have fewer member DCs and also consisted of fewer cells displaying the DC maturity marker CD83. The average number of T cells within a standardized radius of a clustered DC was increased compared to that of an unclustered DC, suggesting that DC clustering was associated with T cell interaction. Furthermore, the number of T cells within the radius of a clustered DC was reduced in tumor-positive TDLNs compared to HLNs. Importantly, clinical outcome analysis revealed that DC clustering in tumor-positive TDLNs correlated with the duration of disease-free survival in breast cancer patients. CONCLUSIONS: These findings are the first to describe the spatial organization of DCs within TDLNs and their association with survival outcome. In addition, we characterized specific changes in number, size, maturity, and T cell co-localization of such clusters. Strategies to enhance DC function in-vivo, including maturation and clustering, may provide additional tools for developing more efficacious DC cancer vaccines.
Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Células Dendríticas/imunologia , Linfonodos/imunologia , Linfonodos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Mama/patologia , Estudos de Casos e Controles , Agregação Celular , Contagem de Células , Diferenciação Celular , Análise por Conglomerados , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Linfócitos T/imunologia , Resultado do TratamentoRESUMO
Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA-mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.
Assuntos
Metilação de DNA/genética , Leucemia , RNA Longo não Codificante , Proteínas Supressoras de Tumor , Adulto , Idoso , Idoso de 80 Anos ou mais , Transformação Celular Neoplásica , Cromatina/genética , Cromossomos Humanos Par 13/genética , Regulação para Baixo , Epigênese Genética/genética , Feminino , Células HEK293 , Humanos , Leucemia/sangue , Leucemia/genética , Leucemia/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mutação , NF-kappa B/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Sítio de Iniciação de Transcrição , Transferases , Proteínas Supressoras de Tumor/sangue , Proteínas Supressoras de Tumor/genética , Regulação para CimaRESUMO
Chronic lymphocytic leukemia is characterized by the accumulation of B cells that are resistant to apoptosis. This resistance is induced by pro-survival stimuli from the microenvironment. TCL1 and ATM are central to the pathogenesis of the disease and associated with more aggressive disease. Their protein products have recently been shown to physically interact in leukemic cells and to impact on NF-κB signaling, which is a key regulator of apoptosis. In the present study we show that TCL1 and ATM are significantly co-expressed and up-regulated in malignant cells compared to non-malignant B cells, and that expression of TCL1 is partially deregulated by aberrant DNA-methylation. In addition, complex external stimuli induce essentially similar TCL1 and ATM time-course kinetics. In line with a coordinative regulation of NF-κB signaling by TCL1, its knockdown induced apoptosis in primary leukemia cells. These findings suggest that both genes functionally cooperate to modulate similar apoptosis-related cellular pathways.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Idoso , Linhagem Celular Tumoral , Deleção Cromossômica , Cromossomos Humanos Par 11 , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Cancer-mediated immune dysfunction contributes to tumor progression and correlates with patient outcome. Metastasis to tumor draining lymph nodes (TDLNs) is an important step in breast cancer progression and is used to predict patient outcome and survival. Although lymph nodes are important immune organs, the role of immune cells in TDLNs has not been thoroughly investigated. We hypothesized that the host immune response in node negative (NN) patients is more intact and thereby can resist tumor invasion compared to node positive (NP) patients. As such, lymph node metastasis requires breakdown of the host immune response in addition to escape of cancer cells from the tumor. To investigate the immunological differences between NN and NP breast cancer patients, we purified and profiled immune cells from the three major compartments where cancer and immune cells interact: tumor, TDLNs and peripheral blood. Significant down-regulation of genes associated with immune-related pathways and up-regulation of genes associated with tumor-promoting pathways was consistently observed in NP patients' TDLNs compared to NN patients. Importantly, these signatures were seen even in NP patients' tumor-free TDLNs, suggesting that such immune changes are not driven solely by local tumor invasion. Furthermore, similar patterns were also observed in NP patients' tumor and blood immune cells, suggesting that immunological differences between NN and NP patients are systemic. Together, these findings suggest that alterations in overall immune function may underlie risk for LN metastasis in breast cancer patients.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Perfilação da Expressão Gênica , Linfonodos/patologia , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Feminino , Seguimentos , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Biópsia de Linfonodo SentinelaRESUMO
The discovery of immune cells with regulatory effects has created considerable excitement for their potential use in inducing tolerance to transplanted tissues. Despite the fact that these cells possess essential functions in vivo, attempts to translate them into effective clinical therapies has proved challenging due to a number of unanticipated complexities in their behavior. This article provides a broad summary of research done to understand the largest of the regulatory cell subtypes, namely CD4+Foxp3+ Regulatory T cells (T(Regs)). Special attention will be paid to current and future difficulties in using T(Regs) clinically, as well as room for improvement and innovation in this field.
Assuntos
Sobrevivência de Enxerto/imunologia , Tolerância Imunológica/imunologia , Linfócitos T Reguladores/imunologia , Transplantes , Rejeição de Enxerto/imunologia , Humanos , Imunoterapia , Modelos Imunológicos , Linfócitos T Reguladores/citologiaRESUMO
Immune modulatory drugs have been successfully used to treat patients with multiple myeloma (MM), myelodysplastic syndromes displaying loss of 5q (del5q MDS) and chronic lymphocytic leukemia (CLL). Immune modulatory drugs are used in first-line therapy in combination with functionally complementary compounds, but have also shown efficacy in refractory disease. However, their exact mode of action remains unclear. Here we describe the clinical impact of these compounds on MM, del5q MDS and CLL, discuss their mode of action with respect to intracellular targets, focus on the phenotypic changes that immune modulatory compounds induce in the tumor microenvironment and how they modulate the immune response.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Fatores Imunológicos/farmacologia , HumanosRESUMO
Chronic lymphocytic leukaemia (CLL) cells convert CD14(+) cells from patients into 'nurse-like' cells (NLCs). CLL cells can also convert CD14(+) peripheral blood mononuclear cells (PBMCs) from healthy donors into cells with morphological similarities to NLCs (CD14(CLL) -cells). However it is unclear whether only CLL cells induce this conversion process. This study showed that CD14(+) PBMCs from healthy donors could also be converted into differentiated cells (CD14(B) -cells) by non-malignant B-cells. In order to identify changes specifically induced by CLL cells, we compared gene expression profiles of NLCs, CD14(CLL) -cells and CD14(B) -cells. CD14(+) cells cultured with CLL cells were more similar to NLCs than those cultured with non-malignant B-cells. The most significant changes induced by CLL cells were deregulation of the antigen presentation pathway and of genes related to immunity. NLCs had reduced levels of lysozyme activity, CD74 and HLA-DR in-vitro while expression of inhibitory FCGR2B was increased. These findings suggest an impaired immunocompetence of NLCs which, if found in-vivo, could contribute to the immunodeficiency in CLL patients.