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1.
J Immunother Cancer ; 10(7)2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35793873

RESUMO

BACKGROUND: Despite the prominent role of innate immunity in the antitumor response, little is known about the myeloid composition of human non-small cell lung cancer (NSCLC) with respect to histology and molecular subtype. We used multiplexed quantitative immunofluorescence (QIF) to measure the distribution and clinical significance of major myeloid cell subsets in large retrospective NSCLC collections. METHODS: We established a QIF panel to map major myeloid cell subsets in fixed human NSCLC including 4',6-Diamidino-2-Phenylindole for all cells, pancytokeratin for tumor-epithelial cells, CD68 for M1-like macrophages; and CD11b plus HLA-DR to interrogate mature and immature myeloid cell populations such as myeloid derived suppressor cells (MDSCs). We interrogated 793 NSCLCs represented in four tissue microarray-based cohorts: #1 (Yale, n=379) and #2 (Greece, n=230) with diverse NSCLC subtypes; #3 (Yale, n=138) with molecularly annotated lung adenocarcinomas (ADC); and #4 (Yale, n=46) with patient-matched NSCLC and morphologically-normal lung tissue. We examined associations between marker levels, myeloid cell profiles, clinicopathologic/molecular variables and survival. RESULTS: The levels of CD68+ M1 like macrophages were significantly lower and the fraction of CD11b+/HLA-DR- MDSC-like cells was prominently higher in tumor than in matched non-tumor lung tissues. HLA-DR was consistently higher in myeloid cells from tumors with elevated CD68 expression. Stromal CD11b was significantly higher in squamous cell carcinomas (SCC) than in ADC across the cohorts and EGFR-mutated lung ADCs displayed lower CD11b levels than KRAS-mutant tumors. Increased stromal CD68- and HLA-DR-expressing cells was associated with better survival in ADCs from two independent NSCLC cohorts. In SCC, increased stromal CD11b or HLA-DR expression was associated with a trend towards shorter 5-year survival. CONCLUSIONS: NSCLCs display an unfavorable myeloid immune contexture relative to non-tumor lung and exhibit distinct myeloid-cell profiles across histologies and presence of major oncogenic driver-mutations. Elevated M1-like stromal proinflammatory myeloid cells are prognostic in lung ADC, but not in SCC.


Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/patologia , Carcinoma de Células Escamosas/patologia , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Células Mieloides , Estudos Retrospectivos
2.
J Immunother Cancer ; 10(7)2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35868661

RESUMO

BACKGROUND: The expression of SYK in cancer cells has been associated with both tumor promoting and tumor suppressive effects. Despite being proposed as anticancer therapeutic target, the possible role of SYK in modulating local adaptive antitumor immune responses remains uncertain. Using detailed analysis of primary human tumors and in vitro models, we reveal the immunomodulatory effect of SYK protein in human solid cancer. METHODS: We spatially mapped SYK kinase in tumor cells, stromal cells and tumor-infiltrating leukocytes (TILs) in 808 primary non-small cell lung carcinomas (NSCLCs) from two cohorts and in 374 breast carcinomas (BCs) from two independent cohorts. We established the associations of localized SYK with clinicopathologic variables and outcomes. The immunomodulatory role of SYK on tumor cells was assessed using in vitro cytokine stimulation, transcriptomic analysis and selective SYK blockade using a small molecule inhibitor. Functional responses were assessed using cocultures of tumor cells with peripheral blood lymphocytes. T cell responses in baseline and post-treatment biopsies from patients with BC treated with a SYK inhibitor in a phase I clinical trial were also studied. RESULTS: Elevated tumor cell or leukocyte SYK expression was associated with high CD4+ and CD8+ TILs and better outcome in both NSCLC and BC. Tumor cell SYK was associated with oncogenic driver mutations in EGFR or KRAS in lung adenocarcinomas and with triple negative phenotype in BC. In cultured tumor cells, SYK was upregulated by TNFα and required for the TNFα-induced proinflammatory responses and T cell activation. SYK blockade after nivolumab in a phase I clinical trial including three patients with advanced triple negative BC reduced TILs and T cell proliferation. Our work establishes the proinflammatory function of tumor cell SYK in lung and breast cancer. SYK signaling in cultured tumor cells is required for T cell activation and SYK blockade limits adaptive antitumor immune responses and tumor rejection in patients with cancer. CONCLUSIONS: Together, our results establish the immunomodulatory role of SYK expression in human solid tumors. This information could be used to develop novel biomarkers and/or therapeutic strategies.


Assuntos
Neoplasias da Mama , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Neoplasias da Mama/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Linfócitos do Interstício Tumoral , Quinase Syk/genética , Quinase Syk/metabolismo , Microambiente Tumoral , Fator de Necrose Tumoral alfa/metabolismo
3.
Cancer Discov ; 11(7): 1700-1715, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33658301

RESUMO

Specific mechanisms by which tumor-infiltrating lymphocytes (TIL) become dysfunctional remain poorly understood. Here, we employed a two-pronged approach using single-cell mass cytometry and tissue imaging technologies to dissect TILs from 25 patients with resectable and 35 patients with advanced non-small cell lung cancer (NSCLC). We identified a burned-out CD8+ TIL subset (Ebo) that specifically accumulated within the tumor microenvironment (TME) but not in adjacent nontumoral tissues. Ebo showed the highest expression of proliferation and activation markers but produced the lowest amount of IFNγ and were the most apoptotic CD8+ TIL subset. Using a humanized patient-derived tumor xenograft model, we demonstrated that Ebo expansion occurred within the TME in a PD-1/B7-H1 pathway-dependent manner. Ebo abundance in baseline tumor tissues was associated with resistance to anti-PD therapy in patients with NSCLC. Our study identifies a dysfunctional TIL subset, with distinct features from previously described exhausted T cells, and implies strategies to overcome immunotherapy resistance. SIGNIFICANCE: We identified a highly proliferative, overactivated, and apoptotic dysfunctional CD8+ tumor-infiltrating subpopulation that is functionally distinct from previously described exhausted T cells. This population is expanded in lung cancer tissues in a PD-1/B7-H1-dependent manner, and its abundance is associated with resistance to cancer immunotherapy, thus becoming a potential tissue biomarker.This article is highlighted in the In This Issue feature, p. 1601.


Assuntos
Linfócitos T CD8-Positivos , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Microambiente Tumoral , Idoso , Animais , Carcinoma Pulmonar de Células não Pequenas/terapia , Feminino , Humanos , Imunoterapia , Neoplasias Pulmonares/terapia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Estudos Prospectivos
4.
PLoS Comput Biol ; 17(3): e1008741, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33780435

RESUMO

Imaging Mass Cytometry (IMC) combines laser ablation and mass spectrometry to quantitate metal-conjugated primary antibodies incubated in intact tumor tissue slides. This strategy allows spatially-resolved multiplexing of dozens of simultaneous protein targets with 1µm resolution. Each slide is a spatial assay consisting of high-dimensional multivariate observations (m-dimensional feature space) collected at different spatial positions and capturing data from a single biological sample or even representative spots from multiple samples when using tissue microarrays. Often, each of these spatial assays could be characterized by several regions of interest (ROIs). To extract meaningful information from the multi-dimensional observations recorded at different ROIs across different assays, we propose to analyze such datasets using a two-step graph-based approach. We first construct for each ROI a graph representing the interactions between the m covariates and compute an m dimensional vector characterizing the steady state distribution among features. We then use all these m-dimensional vectors to construct a graph between the ROIs from all assays. This second graph is subjected to a nonlinear dimension reduction analysis, retrieving the intrinsic geometric representation of the ROIs. Such a representation provides the foundation for efficient and accurate organization of the different ROIs that correlates with their phenotypes. Theoretically, we show that when the ROIs have a particular bi-modal distribution, the new representation gives rise to a better distinction between the two modalities compared to the maximum a posteriori (MAP) estimator. We applied our method to predict the sensitivity to PD-1 axis blockers treatment of lung cancer subjects based on IMC data, achieving 97.3% average accuracy on two IMC datasets. This serves as empirical evidence that the graph of graphs approach enables us to integrate multiple ROIs and the intra-relationships between the features at each ROI, giving rise to an informative representation that is strongly associated with the phenotypic state of the entire image.


Assuntos
Citometria por Imagem , Interpretação de Imagem Assistida por Computador/métodos , Aprendizado de Máquina , Espectrometria de Massas , Algoritmos , Antineoplásicos/uso terapêutico , Bases de Dados Factuais , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Imagem Molecular
5.
Mol Genet Genomics ; 296(3): 501-511, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33743061

RESUMO

Coronavirus disease 2019 (COVID-19), a recent viral pandemic that first began in December 2019, in Hunan wildlife market, Wuhan, China. The infection is caused by a coronavirus, SARS-CoV-2 and clinically characterized by common symptoms including fever, dry cough, loss of taste/smell, myalgia and pneumonia in severe cases. With overwhelming spikes in infection and death, its pathogenesis yet remains elusive. Since the infection spread rapidly, its healthcare demands are overwhelming with uncontrollable emergencies. Although laboratory testing and analysis are developing at an enormous pace, the high momentum of severe cases demand more rapid strategies for initial screening and patient stratification. Several molecular biomarkers like C-reactive protein, interleukin-6 (IL6), eosinophils and cytokines, and artificial intelligence (AI) based screening approaches have been developed by various studies to assist this vast medical demand. This review is an attempt to collate the outcomes of such studies, thus highlighting the utility of AI in rapid screening of molecular markers along with chest X-rays and other COVID-19 symptoms to enable faster diagnosis and patient stratification. By doing so, we also found that molecular markers such as C-reactive protein, IL-6 eosinophils, etc. showed significant differences between severe and non-severe cases of COVID-19 patients. CT findings in the lungs also showed different patterns like lung consolidation significantly higher in patients with poor recovery and lung lesions and fibrosis being higher in patients with good recovery. Thus, from these evidences we perceive that an initial rapid screening using integrated AI approach could be a way forward in efficient patient stratification.


Assuntos
Inteligência Artificial , Proteína C-Reativa/análise , Teste para COVID-19/métodos , COVID-19/diagnóstico , Interleucina-6/sangue , Programas de Rastreamento/métodos , Antivirais/uso terapêutico , Biomarcadores/análise , Biomarcadores/sangue , Citocinas/sangue , Eosinófilos/citologia , Humanos , Pulmão/patologia , Pulmão/virologia , Técnicas de Diagnóstico Molecular , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Tratamento Farmacológico da COVID-19
6.
Clin Cancer Res ; 27(10): 2837-2847, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33602682

RESUMO

PURPOSE: To analyze the distribution, associated immune contexture, and clinical significance of human leukocyte antigen (HLA) class-I and HLA class-II subunits in non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Using spatially resolved and quantitative multiplexed immunofluorescence we studied the tumor/stromal tissue distribution, cancer cell-specific defects, and clinicopathologic/survival associations of ß2 microglobulin (ß2M), HLA-A, and HLA-B,-C heavy chains, as well as HLA class-II ß chain in >700 immunotherapy-naïve NSCLCs from four independent cohorts. Genomic analysis of HLA genes in NSCLC was performed using two publicly available cohorts. RESULTS: Cancer cell-specific downregulation of HLA markers was identified in 30.4% of cases. ß2M was downregulated in 9.8% (70/714), HLA-A in 9% (65/722), HLA-B,-C in 12.1% (87/719), and HLA class-II in 17.7% (127/717) of evaluable samples. Concurrent downregulation of ß2M, HLA-B,-C, and HLA class-II was commonly identified. Deleterious mutations in HLA genes were detected in <5% of lung malignancies. Tumors with cancer cell-specific ß2M downregulation displayed reduced T cells and increased natural killer (NK)-cell infiltration. Samples with cancer cell HLA-A downregulation displayed modest increase in CD8+ T cells and NK-cell infiltration. Samples with cancer cell-selective HLA-B,-C or HLA class-II downregulation displayed reduced T cells and NK-cell infiltration. There was limited association of the markers with clinicopathologic variables and KRAS/EGFR mutations. Cancer cell-selective downregulation of the HLA subunits was associated with shorter overall survival. CONCLUSIONS: Our results reveal frequent and differential defects in HLA class-I and HLA class-II protein subunit expression in immunotherapy-naïve NSCLCs associated with distinct tumor microenvironment composition and patient survival.


Assuntos
Alelos , Carcinoma Pulmonar de Células não Pequenas/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Biologia Computacional/métodos , Análise Mutacional de DNA , Imunofluorescência/métodos , Imunofluorescência/normas , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/mortalidade , Mutação , Prognóstico
7.
Mol Psychiatry ; 26(10): 5592-5607, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33144711

RESUMO

Although APP metabolism is being intensively investigated, a large fraction of its modulators is yet to be characterized. In this context, we combined two genome-wide high-content screenings to assess the functional impact of miRNAs and genes on APP metabolism and the signaling pathways involved. This approach highlighted the involvement of FERMT2 (or Kindlin-2), a genetic risk factor of Alzheimer's disease (AD), as a potential key modulator of axon guidance, a neuronal process that depends on the regulation of APP metabolism. We found that FERMT2 directly interacts with APP to modulate its metabolism, and that FERMT2 underexpression impacts axonal growth, synaptic connectivity, and long-term potentiation in an APP-dependent manner. Last, the rs7143400-T allele, which is associated with an increased AD risk and localized within the 3'UTR of FERMT2, induced a downregulation of FERMT2 expression through binding of miR-4504 among others. This miRNA is mainly expressed in neurons and significantly overexpressed in AD brains compared to controls. Altogether, our data provide strong evidence for a detrimental effect of FERMT2 underexpression in neurons and insight into how this may influence AD pathogenesis.


Assuntos
Doença de Alzheimer , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Humanos , Proteínas de Membrana , Proteínas de Neoplasias , Plasticidade Neuronal/genética , Neurônios , Fatores de Risco
8.
Acta Neuropathol ; 138(4): 631-652, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31065832

RESUMO

The bridging integrator 1 gene (BIN1) is a major genetic risk factor for Alzheimer's disease (AD). In this report, we investigated how BIN1-dependent pathophysiological processes might be associated with Tau. We first generated a cohort of control and transgenic mice either overexpressing human MAPT (TgMAPT) or both human MAPT and BIN1 (TgMAPT;TgBIN1), which we followed-up from 3 to 15 months. In TgMAPT;TgBIN1 mice short-term memory deficits appeared earlier than in TgMAPT mice; however-unlike TgMAPT mice-TgMAPT;TgBIN1 mice did not exhibit any long-term or spatial memory deficits for at least 15 months. After killing the cohort at 18 months, immunohistochemistry revealed that BIN1 overexpression prevents both Tau mislocalization and somatic inclusion in the hippocampus, where an increase in BIN1-Tau interaction was also observed. We then sought mechanisms controlling the BIN1-Tau interaction. We developed a high-content screening approach to characterize modulators of the BIN1-Tau interaction in an agnostic way (1,126 compounds targeting multiple pathways), and we identified-among others-an inhibitor of calcineurin, a Ser/Thr phosphatase. We determined that calcineurin dephosphorylates BIN1 on a cyclin-dependent kinase phosphorylation site at T348, promoting the open conformation of the neuronal BIN1 isoform. Phosphorylation of this site increases the availability of the BIN1 SH3 domain for Tau interaction, as demonstrated by nuclear magnetic resonance experiments and in primary neurons. Finally, we observed that although the levels of the neuronal BIN1 isoform were unchanged in AD brains, phospho-BIN1(T348):BIN1 ratio was increased, suggesting a compensatory mechanism. In conclusion, our data support the idea that BIN1 modulates the AD risk through an intricate regulation of its interaction with Tau. Alteration in BIN1 expression or activity may disrupt this regulatory balance with Tau and have direct effects on learning and memory.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transtornos da Memória/metabolismo , Memória de Longo Prazo/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Tauopatias/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas tau/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Transtornos da Memória/genética , Transtornos da Memória/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Memória Espacial/fisiologia , Tauopatias/genética , Tauopatias/patologia , Proteínas Supressoras de Tumor/genética
9.
J Cyst Fibros ; 16(3): 327-334, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28438500

RESUMO

BACKGROUND: AP2 is a clathrin-based endocytic adaptor complex comprising α, ß2, µ2 and σ2 subunits. µ2 regulates CFTR endocytosis. The α subunit interacts with CFTR in the intestine but its physiologic significance is unclear. METHODS: CFTR short circuit current was measured in intestinal T84 cells following shRNA knock down of AP2α (AP2αKD). Clathrin-coated structures (CCS) were immunolabeled and quantified in AP2αKD intestinal Caco2BBe (C2BBe) cells. GST tagged human AP2α appendage domain was cloned and its interaction with CFTR determined by GST pull down assay. RESULT: AP2αKD in T84 cells resulted in higher CFTR current (57%) compared to control, consistent with increased functional CFTR and delayed endocytosis. Depletion of AP2α reduced CCS in C2BBe cells. Pull down assays revealed an interaction between human AP2α appendage domain and CFTR. CONCLUSION: AP2 α interacts with and modulates CFTR function in the intestine by participating in clathrin assembly and recruitment of CFTR to CCS.


Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades do Complexo de Proteínas Adaptadoras/metabolismo , Fibrose Cística/metabolismo , Mucosa Intestinal/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Endocitose/fisiologia , Células HEK293 , Humanos , Transporte de Íons/fisiologia
10.
Acta Neuropathol ; 133(6): 955-966, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27933404

RESUMO

Genome-wide association studies (GWASs) have identified 19 susceptibility loci for Alzheimer's disease (AD). However, understanding how these genes are involved in the pathophysiology of AD is one of the main challenges of the "post-GWAS" era. At least 123 genes are located within the 19 susceptibility loci; hence, a conventional approach (studying the genes one by one) would not be time- and cost-effective. We therefore developed a genome-wide, high-content siRNA screening approach and used it to assess the functional impact of gene under-expression on APP metabolism. We found that 832 genes modulated APP metabolism. Eight of these genes were located within AD susceptibility loci. Only FERMT2 (a ß3-integrin co-activator) was also significantly associated with a variation in cerebrospinal fluid Aß peptide levels in 2886 AD cases. Lastly, we showed that the under-expression of FERMT2 increases Aß peptide production by raising levels of mature APP at the cell surface and facilitating its recycling. Taken as a whole, our data suggest that FERMT2 modulates the AD risk by regulating APP metabolism and Aß peptide production.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Interferente Pequeno/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Biomarcadores/líquido cefalorraquidiano , Membrana Celular/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Neurônios/metabolismo , Neurônios/patologia , Interferência de RNA , Ratos
11.
Mol Vis ; 22: 332-41, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27122964

RESUMO

PURPOSE: Optic neuritis affects most patients with multiple sclerosis (MS), and current treatments are unreliable. The purpose of this study was to characterize the contribution of Th1 and Th17 cells to the development of optic neuritis. METHODS: Mice were passively transferred myelin-specific Th1 or Th17 cells to induce experimental autoimmune encephalomyelitis (EAE), a model of neuroautoimmunity. Visual acuity was assessed daily with optokinetic tracking, and 1, 2, and 3 weeks post-induction, optic nerves and retinas were harvested for immunohistochemical analyses. RESULTS: Passive transfer experimental autoimmune encephalomyelitis elicits acute episodes of asymmetric visual deficits and is exacerbated in Th17-EAE relative to Th1-EAE. The Th17-EAE optic nerves contained more inflammatory infiltrates and an increased neutrophil to macrophage ratio. Significant geographic degeneration of the retinal ganglion cells accompanied Th17-EAE but not Th1. CONCLUSIONS: Th17-induced transfer EAE recapitulates pathologies observed in MS-associated optic neuritis, namely, monocular episodes of vision loss, optic nerve inflammation, and geographic retinal ganglion cell (RGC) degeneration.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Bainha de Mielina/imunologia , Neurite Óptica/imunologia , Células Ganglionares da Retina/patologia , Células Th17/imunologia , Animais , Apoptose/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Feminino , Imunização Passiva , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Neutrófilos/imunologia , Neurite Óptica/patologia , Células Th1/imunologia , Acuidade Visual/fisiologia
12.
J Biol Chem ; 290(42): 25595-608, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26342078

RESUMO

Pancreatic islet ß-cells that lack the MEN1-encoded protein menin develop into tumors. Such tumors express the phosphorylated isoform of the ß-cell differentiation transcription factor HLXB9. It is not known how phospho-HLXB9 acts as an oncogenic factor in insulin-secreting ß-cell tumors (insulinomas). In this study we investigated the binding partners and target genes of phospho-HLXB9 in mouse insulinoma MIN6 ß-cells. Co-immunoprecipitation coupled with mass spectrometry showed a significant association of phospho-HLXB9 with the survival factor p54nrb/Nono (54-kDa nuclear RNA-binding protein, non-POU-domain-containing octamer). Endogenous phospho-HLXB9 co-localized with endogenous Nono in the nucleus. Overexpression of HLXB9 decreased the level of overexpressed Nono but not endogenous Nono. Anti-phospho-HLXB9 chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) identified the c-Met inhibitor, Cblb, as a direct phospho-HLXB9 target gene. Phospho-HLXB9 occupied the promoter of Cblb and reduced the expression of Cblb mRNA. Cblb overexpression or HLXB9 knockdown decreased c-Met protein and reduced cell migration. Also, increased phospho-HLXB9 coincided with reduced Cblb and increased c-Met in insulinomas of two mouse models of menin loss. These data provide mechanistic insights into the role of phospho-HLXB9 as a pro-oncogenic factor by interacting with a survival factor and by promoting the oncogenic c-Met pathway. These mechanisms have therapeutic implications for reducing ß-cell proliferation in insulinomas by inhibiting phospho-HLXB9 or its interaction with Nono and modulating the expression of its direct (Cblb) or indirect (c-Met) targets. Our data also implicate the use of pro-oncogenic activities of phospho-HLXB9 in ß-cell expansion strategies to alleviate ß-cell loss in diabetes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Proteínas de Homeodomínio/fisiologia , Insulinoma/metabolismo , Oncogenes , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Fatores de Transcrição/fisiologia , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Insulinoma/patologia , Camundongos , Ligação Proteica , Proteínas de Ligação a RNA , Fatores de Transcrição/metabolismo
13.
Int J Endocrinol ; 2015: 149826, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26229531

RESUMO

Lipoma in patients with the multiple endocrine neoplasia type 1 (MEN1) syndrome is a type of benign fat-cell tumor that has biallelic inactivation of MEN1 that encodes menin and could serve as a model to investigate normal and pathologic fat-cell (adipocyte) proliferation and function. The role of menin and its target genes in adipocytes is not known. We used in vitro differentiation to derive matched normal and menin-deficient adipocytes from wild type (WT) and menin-null (Men1-KO) mouse embryonic stem cells (mESCs), respectively, or 3T3-L1 cells without or with menin knockdown to investigate cell size, lipid content, and gene expression changes. Adipocytes derived from Men1-KO mESCs or after menin knockdown in 3T3-L1 cells showed a 1.5-1.7-fold increase in fat-cell size. Global gene expression analysis of mESC-derived adipocytes showed that lack of menin downregulated the expression of many differentially methylated genes including the tumor suppressor long noncoding RNA Meg3 but upregulated gene expression from the prolactin gene family locus. Our results show that menin deficiency leads to fat-cell hypertrophy and provide model systems that could be used to study the regulation of fat-cell size.

14.
J Biol Chem ; 289(9): 5386-98, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24425879

RESUMO

Insulinomas (pancreatic islet ß cell tumors) are the most common type of functioning pancreatic neuroendocrine tumors that occur sporadically or as a part of the MEN1 syndrome that is caused by germ line mutations in MEN1. Tissue-specific tumor predisposition from germ line mutations in ubiquitously expressed genes such as MEN1 could occur because of functional consequences on tissue-specific factors. We previously reported the proapoptotic ß cell differentiation factor HLXB9 as a downstream target of menin (encoded by MEN1). Here we show that GSK-3ß inactivates the proapoptotic activity of HLXB9 by phosphorylating HLXB9 at Ser-78/Ser-80 (pHLXB9). Although HLXB9 is found in the nucleus and cytoplasm, pHLXB9 is predominantly nuclear. Both pHLXB9 and active GSK-3ß are elevated in ß cells with menin knockdown, in MEN1-associated ß cell tumors (insulinomas), and also in human sporadic insulinomas. Pharmacologic inhibition of GSK-3ß blocked cell proliferation in three different rodent insulinoma cell lines by arresting the cells in G2/M phase and caused apoptosis. Taken together, these data suggest that the combination of GSK-3ß and pHLXB9 forms a therapeutically targetable mechanism of insulinoma pathogenesis. Our results reveal that GSK-3ß and pHLXB9 can serve as novel targets for insulinoma treatment and have implications for understanding the pathways associated with ß cell proliferation.


Assuntos
Proliferação de Células , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/patologia , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Proteínas de Homeodomínio/genética , Humanos , Células Secretoras de Insulina/patologia , Insulinoma/genética , Insulinoma/patologia , Camundongos , Fosforilação/genética , Estabilidade Proteica , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Ratos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
15.
Oncotarget ; 5(18): 8202-10, 2014 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-24077665

RESUMO

The M2 isoform of pyruvate kinase (PKM2) plays an important role in aerobic glycolysis and is a mediator of the Warburg effect in tumors. It was previously thought that tumor cells switch expression of PKM from normal tissue-expressed PKM1 to tumor-specific PKM2 via an alternative splicing mechanism. This view was challenged by a recent report demonstrating that PKM2 is already the major PKM isoform expressed in many differentiated normal tissues. Here, through analyses on sixteen tumor types using the cancer genome atlas RNA-Seq and exon array datasets, we confirmed that isoform switch from PKM1 to PKM2 occurred in glioblastomas but not in other tumor types examined. Despite lacking of isoform switches, PKM2 expression was found to be increased in all cancer types examined, and correlated strongly to poor prognosis in head and neck cancers. We further demonstrated that elevated PKM2 expression correlated well with the hypomethylation status of intron 1 of the PKM gene in multiple cancer types, suggesting epigenetic regulation by DNA methylation as a major mechanism in controlling PKM transcription in tumors. Our study suggests that isoform switch of PKM1 to PKM2 in cancers is tissue-specific and targeting PKM2 activity in tumors remains a promising approach for clinical intervention of multiple cancer types.


Assuntos
Biomarcadores Tumorais/metabolismo , Metilação de DNA , Epigênese Genética , Neoplasias/enzimologia , Piruvato Quinase/metabolismo , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Análise por Conglomerados , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Glioblastoma/enzimologia , Glioblastoma/genética , Humanos , Íntrons , Isoenzimas , Estimativa de Kaplan-Meier , Neoplasias/genética , Neoplasias/mortalidade , Neoplasias/patologia , Prognóstico , Regiões Promotoras Genéticas , Modelos de Riscos Proporcionais , Piruvato Quinase/genética , Fatores de Tempo , Transcrição Gênica
16.
J Biol Chem ; 288(13): 9165-76, 2013 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-23386620

RESUMO

Heat shock factor 1 (HSF1), a master regulator of heat shock responses, plays an important role in tumorigenesis. In this study we demonstrated that HSF1 is required for chemotherapeutic agent-induced cytoprotective autophagy through transcriptional up-regulation of autophagy-related gene ATG7. Interestingly, this is independent of the HSF1 heat shock response function. Treatment of cancer cells with the FDA-approved chemotherapeutic agent carboplatin induced autophagy and growth inhibition, which were significantly increased upon knockdown of HSF1. Mechanistic studies revealed that HSF1 regulates autophagy by directly binding to ATG7 promoter and transcriptionally up-regulating its expression. Significantly, breast cancer patient sample study revealed that a higher ATG7 expression level is associated with poor patient survival. This novel finding was further confirmed by analysis of two independent patient databases, demonstrating a prognostic value of ATG7. Furthermore, a strong positive correlation was observed between levels of HSF1 and ATG7 in triple-negative breast cancer patient samples, thus validating our in vitro findings. This is the first study identifying a critical role for HSF1 in controlling cytoprotective autophagy through regulation of ATG7, which is distinct from the HSF1 function in the heat shock response. This is also the first study demonstrating a prognostic value of ATG7 in breast cancer patients. These findings strongly argue that combining chemotherapeutic agents with autophagy inhibition by repressing HSF1/ATG7 axis represents a promising strategy for future cancer treatment.


Assuntos
Autofagia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Proteína 7 Relacionada à Autofagia , Carboplatina/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Citometria de Fluxo/métodos , Fatores de Transcrição de Choque Térmico , Humanos , Luciferases/metabolismo , Microscopia de Fluorescência/métodos , Prognóstico , RNA Interferente Pequeno/metabolismo , Transcrição Gênica
17.
Endocr Relat Cancer ; 20(1): 111-22, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23419452

RESUMO

The multiple endocrine neoplasia type 1 (MEN1) syndrome is caused by germline mutations in the MEN1 gene encoding menin, with tissue-specific tumors of the parathyroids, anterior pituitary, and enteropancreatic endocrine tissues. Also, 30-40% of sporadic pancreatic endocrine tumors show somatic MEN1 gene inactivation. Although menin is expressed in all cell types of the pancreas, mouse models with loss of menin in either pancreatic α-cells, or ß-cells, or total pancreas develop ß-cell-specific endocrine tumors (insulinomas). Loss of widely expressed tumor suppressor genes may produce tissue-specific tumors by reactivating one or more embryonic-specific differentiation factors. Therefore, we determined the effect of menin overexpression or knockdown on the expression of ß-cell differentiation factors in a mouse ß-cell line (MIN6). We show that the ß-cell differentiation factor Hlxb9 is posttranscriptionally upregulated upon menin knockdown, and it interacts with menin. Hlxb9 reduces cell proliferation and causes apoptosis in the presence of menin, and it regulates genes that modulate insulin level. Thus, upon menin loss or from other causes, dysregulation of Hlxb9 predicts a possible combined mechanism for ß-cell proliferation and insulin production in insulinomas. These observations help to understand how a ubiquitously expressed protein such as menin might control tissue-specific tumorigenesis. Also, our findings identify Hlxb9 as an important factor for ß-cell proliferation and insulin regulation.


Assuntos
Diferenciação Celular , Proliferação de Células , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/patologia , Insulina/metabolismo , Insulinoma/patologia , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Insulina/genética , Células Secretoras de Insulina/metabolismo , Insulinoma/genética , Insulinoma/metabolismo , Rim/citologia , Rim/metabolismo , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
18.
J Basic Microbiol ; 53(6): 518-31, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22961447

RESUMO

Deinococcus radiodurans is known for its extraordinary resistance to various DNA damaging agents including γ-radiation and desiccation. The pqqE:cat and Δdr2518 mutants making these cells devoid of pyrroloquinoline quinone (PQQ) and a PQQ inducible Ser/Thr protein kinase, respectively, became sensitive to γ-radiation. Transcriptome analysis of these mutants showed differential expression of the genes including those play roles in oxidative stress tolerance and (DSB) repair in D. radiodurans and in genome maintenance and stress response in other bacteria. Escherichia coli cells expressing DR2518 and PQQ showed improved resistance to γ-radiation, which increased further when both DR2518 and PQQ were present together. Although, profiles of genes getting affected in these mutants were different, there were still a few common genes showing similar expression trends in both the mutants and some others as reported earlier in oxyR and pprI mutant of this bacterium. These results suggested that PQQ and DR2518 have independent roles in γ-radiation resistance of D. radiodurans but their co-existence improves radioresistance further, possibly by regulating differential expression of the genes important for bacterial response to oxidative stress and DNA damage.


Assuntos
Deinococcus/fisiologia , Deinococcus/efeitos da radiação , Cofator PQQ/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA , Reparo do DNA , DNA Bacteriano/genética , DNA Bacteriano/efeitos da radiação , Deinococcus/genética , Deinococcus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Raios gama , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Mutação , Estresse Oxidativo/genética , Cofator PQQ/genética , Proteínas Quinases/genética , Tolerância a Radiação/fisiologia
19.
J Biol Chem ; 288(6): 4334-45, 2013 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-23255607

RESUMO

Chemoresistance is a major obstacle in cancer treatment. Our previous studies have shown that miR-125b plays an important role in chemoresistance. Here we report a novel mechanism that up-regulation of miR-125b through Wnt signaling by Snail enriches cancer stem cells. Overexpression of Snail dramatically increases the expression of miR-125b through the Snail-activated Wnt/ß-catenin/TCF4 axis. Snail confers chemoresistance by repressing Bak1 through up-regulation of miR-125b. Restoring the expression of Bak1 or depleting miR-125b re-sensitizes Snail-expressing cancer cells to Taxol, indicating that miR-125b is critical in Snail-induced chemoresistance. Moreover, overexpression of miR-125b significantly increases the cancer stem cell population (CD24-CD44+), while depletion of miR-125b or rescue of the expression of Bak1 increases the non-stem cell population (CD24+CD44+) in Snail-overexpressing cells. These findings strongly support that miR-125b functions as a key mediator in Snail-induced cancer stem cell enrichment and chemoresistance. This novel mechanism for Snail-induced stem cell propagation and chemoresistance may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.


Assuntos
Resistencia a Medicamentos Antineoplásicos , MicroRNAs/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , RNA Neoplásico/biossíntese , Fatores de Transcrição/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Humanos , MicroRNAs/genética , Proteínas de Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Paclitaxel/farmacologia , RNA Neoplásico/genética , Fatores de Transcrição da Família Snail , Fator de Transcrição 4 , Fatores de Transcrição/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
20.
Nat Commun ; 3: 1271, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23232401

RESUMO

It is well known that ErbB2, a receptor tyrosine kinase, localizes to the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and cellular ATP levels were also decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to the ErbB2-targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 has an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics.


Assuntos
Mitocôndrias/fisiologia , Receptor ErbB-2/fisiologia , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Respiração Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Transporte de Elétrons/fisiologia , Feminino , Glicólise/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Humanos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Transporte Proteico , Receptor ErbB-2/metabolismo , Trastuzumab
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