RESUMO
Solitary fibrous tumors (SFTs) harbor recurrent NAB2-STAT6 gene fusions, promoting constitutional up-regulation of oncogenic early growth response 1 (EGR1)-dependent gene expression. SFTs with the most common canonical NAB2 exon 4-STAT6 exon 2 fusion variant are often located in the thorax (pleuropulmonary) and are less cellular with abundant collagen. In contrast, SFTs with NAB2 exon 6-STAT6 exon 16/17 fusion variants typically display a cellular round to ovoid cell morphology and are often located in the deep soft tissue of the retroperitoneum and intra-abdominal pelvic region or in the meninges. Here, we employed next-generation sequencing-based gene expression profiling to identify significant differences in gene expression associated with anatomic localization and NAB2-STAT6 gene fusion variants. SFTs with the NAB2 exon 4-STAT6 exon 2 fusion variant showed a transcriptional signature enriched for genes involved in DNA binding, gene transcription, and nuclear localization, whereas SFTs with the NAB2 exon 6-STAT6 exon 16/17 fusion variants were enriched for genes involved in tyrosine kinase signaling, cell proliferation, and cytoplasmic localization. Specific transcription factor binding motifs were enriched among differentially expressed genes in SFTs with different fusion variants, implicating co-transcription factors in the modification of chimeric NGFI-A binding protein 2 (NAB2)-STAT6-dependent deregulation of EGR1-dependent gene expression. In summary, this study establishes a potential molecular biologic basis for clinicopathologic differences in SFTs with distinct NAB2-STAT6 gene fusion variants.
Assuntos
Biomarcadores Tumorais/genética , Proteínas Repressoras/genética , Fator de Transcrição STAT6/genética , Tumores Fibrosos Solitários/genética , Éxons/genética , Feminino , Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Proteínas de Fusão Oncogênica/genética , Proteínas Repressoras/metabolismo , Tumores Fibrosos Solitários/patologiaRESUMO
BACKGROUND: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). Treatment options for TNBC patients are limited and further insights into disease aetiology are needed to develop better therapeutic approaches. microRNAs' ability to regulate multiple targets could hold a promising discovery approach to pathways relevant for TNBC aggressiveness. Thus, we address the role of miRNAs in controlling three signalling pathways relevant to the biology of TNBC, and their downstream phenotypes. METHODS: To identify miRNAs regulating WNT/ß-catenin, c-Met, and integrin signalling pathways, we performed a high-throughput targeted proteomic approach, investigating the effect of 800 miRNAs on the expression of 62 proteins in the MDA-MB-231 TNBC cell line. We then developed a novel network analysis, Pathway Coregulatory (PC) score, to detect miRNAs regulating these three pathways. Using in vitro assays for cell growth, migration, apoptosis, and stem-cell content, we validated the function of candidate miRNAs. Bioinformatic analyses using BC patients' datasets were employed to assess expression of miRNAs as well as their pathological relevance in TNBC patients. RESULTS: We identified six candidate miRNAs coordinately regulating the three signalling pathways. Quantifying cell growth of three TNBC cell lines upon miRNA gain-of-function experiments, we characterised miR-193b as a strong and consistent repressor of proliferation. Importantly, the effects of miR-193b were stronger than chemical inhibition of the individual pathways. We further demonstrated that miR-193b induced apoptosis, repressed migration, and regulated stem-cell markers in MDA-MB-231 cells. Furthermore, miR-193b expression was the lowest in patients classified as TNBC or Basal compared to other subtypes. Gene Set Enrichment Analysis showed that miR-193b expression was significantly associated with reduced activity of WNT/ß-catenin and c-Met signalling pathways in TNBC patients. CONCLUSIONS: Integrating miRNA-mediated effects and protein functions on networks, we show that miRNAs predominantly act in a coordinated fashion to activate or repress connected signalling pathways responsible for metastatic traits in TNBC. We further demonstrate that our top candidate, miR-193b, regulates these phenotypes to an extent stronger than individual pathway inhibition, thus emphasizing that its effect on TNBC aggressiveness is mediated by the coordinated repression of these functionally interconnected pathways.
Assuntos
MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Via de Sinalização Wnt/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Metástase Neoplásica , TransfecçãoRESUMO
Neoplasms with a myopericytomatous pattern represent a morphological spectrum of lesions encompassing myopericytoma of the skin and soft tissue, angioleiomyoma, myofibromatosis/infantile haemangiopericytoma and putative neoplasms reported as malignant myopericytoma. Lack of reproducible phenotypic and genetic features of malignant myopericytic neoplasms have prevented the establishment of myopericytic sarcoma as an acceptable diagnostic category. Following detection of a LMNA-NTRK1 gene fusion in an index case of paediatric haemangiopericytoma-like sarcoma by combined whole-genome and RNA sequencing, we identified three additional sarcomas harbouring NTRK1 gene fusions, termed 'spindle cell sarcoma, NOS with myo/haemangiopericytic growth pattern'. The patients were two children aged 11 months and 2 years and two adults aged 51 and 80 years. While the tumours of the adults were strikingly myopericytoma-like, but with clear-cut atypical features, the paediatric cases were more akin to infantile myofibromatosis/haemangiopericytoma. All cases contained numerous thick-walled dysplastic-like vessels with segmental or diffuse nodular myxohyaline myo-intimal proliferations of smooth muscle actin-positive cells, occasionally associated with thrombosis. Immunohistochemistry showed variable expression of smooth muscle actin and CD34, but other mesenchymal markers, including STAT6, were negative. This study showed a novel variant of myo/haemangiopericytic sarcoma with recurrent NTRK1 gene fusions. Given the recent introduction of a novel therapeutic approach targeting NTRK fusion-positive neoplasms, recognition of this rare but likely under-reported sarcoma variant is strongly encouraged.
Assuntos
Biomarcadores Tumorais/genética , Fusão Gênica , Hemangiopericitoma/genética , Receptor trkA/genética , Sarcoma/genética , Neoplasias de Tecidos Moles/genética , Fatores Etários , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Células Cultivadas , Pré-Escolar , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Predisposição Genética para Doença , Hemangiopericitoma/metabolismo , Hemangiopericitoma/patologia , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Lactente , Lamina Tipo A/genética , Masculino , Pessoa de Meia-Idade , Pericitos/metabolismo , Pericitos/patologia , Fenótipo , Receptor trkA/metabolismo , Sarcoma/metabolismo , Sarcoma/patologia , Análise de Sequência de DNA , Neoplasias de Tecidos Moles/metabolismo , Neoplasias de Tecidos Moles/patologia , Transfecção , Tropomiosina/genéticaRESUMO
RATIONALE: The emerging role of the ubiquitin-proteasome system in cardiomyocyte function and homeostasis implies the necessity of tight regulation of protein degradation. However, little is known about cardiac components of this machinery. OBJECTIVE: We sought to determine whether molecules exist that control turnover of cardiac-specific proteins. METHODS AND RESULTS: Using a bioinformatic approach to identify novel cardiac-enriched sarcomere proteins, we identified F-box and leucine-rich repeat protein 22 (Fbxl22). Tissue-specific expression was confirmed by multiple tissue Northern and Western Blot analyses as well as quantitative reverse-transcriptase polymerase chain reaction on a human cDNA library. Immunocolocalization experiments in neonatal and adult rat ventricular cardiomyocytes as well as murine heart tissue located Fbxl22 to the sarcomeric z-disc. To detect cardiac protein interaction partners, we performed a yeast 2-hybrid screen using Fbxl22 as bait. Coimmunoprecipitation confirmed the identified interactions of Fbxl22 with S-phase kinase-associated protein 1 and Cullin1, 2 critical components of SCF (Skp1/Cul1/F-box) E3- ligases. Moreover, we identified several potential substrates, including the z-disc proteins α-actinin and filamin C. Consistently, in vitro overexpression of Fbxl22-mediated degradation of both substrates in a dose-dependent fashion, whereas proteasome inhibition with MG-132 markedly attenuated degradation of both α-actinin and filamin C. Finally, targeted knockdown of Fbxl22 in rat cardiomyocytes as well as zebrafish embryos results in the accumulation of α-actinin associated with severely impaired contractile function and cardiomyopathy in vivo. CONCLUSIONS: These findings reveal the previously uncharacterized cardiac-specific F-box protein Fbxl22 as a component of a novel cardiac E3 ligase. Fbxl22 promotes the proteasome-dependent degradation of key sarcomeric proteins, such as α-actinin and filamin C, and is essential for maintenance of normal contractile function in vivo.
Assuntos
Proteínas F-Box/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Sarcômeros/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Células Cultivadas , Células HEK293 , Humanos , Dados de Sequência Molecular , Miócitos Cardíacos/fisiologia , Transporte Proteico/fisiologia , Ratos , Sarcômeros/fisiologiaRESUMO
BACKGROUND: Neuroligin 4 X-linked (NLGN4X) harbors a human leukocyte antigen (HLA)-A*02-restricted tumor-associated antigen, overexpressed in human gliomas, that was found to induce specific cytotoxic T cell responses following multi-peptide vaccination in patients with newly diagnosed glioblastoma. METHODS: T cell receptor (TCR) discovery was performed using droplet-based single-cell TCR sequencing of NLGN4X-tetramer-sorted T cells postvaccination. The identified TCR was delivered to Jurkat T cells and primary human T cells (NLGN4X-TCR-T). Functional profiling of NLGN4X-TCR-T was performed by flow cytometry and cytotoxicity assays. Therapeutic efficacy of intracerebroventricular NLGN4X-TCR-T was assessed in NOD scid gamma (NSG) major histocompatibility complex (MHC) I/II knockout (KO) (NSG MHC I/II KO) mice bearing NLGN4X-expressing experimental gliomas. RESULTS: An HLA-A*02-restricted vaccine-induced T cell receptor specifically binding NLGN4X131-139 was applied for preclinical therapeutic use. Reactivity, cytotoxicity, and polyfunctionality of this NLGN4X-specific TCR are demonstrated in various cellular models. Intracerebroventricular administration of NLGN4X-TCR-T prolongs survival and leads to an objective response rate of 44.4% in experimental glioma-bearing NSG MHC I/II KO mice compared to 0.0% in control groups. CONCLUSION: NLGN4X-TCR-T demonstrate efficacy in a preclinical glioblastoma model. On a global scale, we provide the first evidence for the therapeutic retrieval of vaccine-induced human TCRs for the off-the-shelf treatment of glioblastoma patients.Keywords cell therapy | glioblastoma | T cell receptor | tumor antigen.
Assuntos
Vacinas Anticâncer , Glioblastoma , Camundongos , Animais , Humanos , Glioblastoma/genética , Glioblastoma/terapia , Vacinas Anticâncer/uso terapêutico , Vacinas de Subunidades Antigênicas , Receptores de Antígenos de Linfócitos T , Linfócitos T , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular NeuronaisRESUMO
Tumor microtubes (TMs) connect glioma cells to a network with considerable relevance for tumor progression and therapy resistance. However, the determination of TM-interconnectivity in individual tumors is challenging and the impact on patient survival unresolved. Here, we establish a connectivity signature from single-cell RNA-sequenced (scRNA-Seq) xenografted primary glioblastoma (GB) cells using a dye uptake methodology, and validate it with recording of cellular calcium epochs and clinical correlations. Astrocyte-like and mesenchymal-like GB cells have the highest connectivity signature scores in scRNA-sequenced patient-derived xenografts and patient samples. In large GB cohorts, TM-network connectivity correlates with the mesenchymal subtype and dismal patient survival. CHI3L1 gene expression serves as a robust molecular marker of connectivity and functionally influences TM networks. The connectivity signature allows insights into brain tumor biology, provides a proof-of-principle that tumor cell TM-connectivity is relevant for patients' prognosis, and serves as a robust prognostic biomarker.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Glioblastoma/genética , Glioma/genética , Neoplasias Encefálicas/genética , Proteína 1 Semelhante à Quitinase-3RESUMO
Plakophilin (PKP1) 1 is a member of the arm-repeat family of catenins and acts as a structural component of desmosomes, which are important stabilizers of cell-cell adhesion. Besides this, PKP1 also occurs in a non-junctional, cytoplasmic form contributing to post-transcriptional regulation of gene expression. Moreover, PKP1 is expressed in the prostate epithelium but its expression is frequently downregulated in prostate cancers with a more aggressive phenotype. This observation may imply a tumour-suppressive role of PKP1. We found that, in prostatic adenocarcinomas with PKP1 deficiency, the occurrence of T-cells, B-cells, macrophages and neutrophils were significantly increased. In a PKP1-deficient prostatic cancer cell line expressing IL8, these levels were statistically meaningfully reduced upon PKP1 re-expression. When analysing prostatic PKP1 knockdown cell lines, the mRNA and protein levels of additional cytokines, namely CXCL1 and IL6, were upregulated. The effect was rescued upon re-expression of a PKP1 RNAi-resistant form. The corresponding mRNAs were co-precipitated with cytoplasmic PKP1, indicating that they are components of PKP1-containing mRNA ribonucleoprotein particles. Moreover, the mRNA half-lives of CXCL1, IL8 and IL6 were significantly increased in PKP1-deficient cells, showing that these mRNAs were stabilized by PKP1. In an in vitro migration assay, the higher cytokine concentrations led to higher migration rates of THP1 and PBMC cells. This finding implies that PKP1 loss of expression in vivo correlates with the recruitment of immune cells into the tumour area to set up a tumour-specific environment. One may speculate that this newly established tumour environment has tumour-suppressive characteristics and thereby accelerates tumour progression and metastasis.
Assuntos
Placofilinas , Neoplasias da Próstata , Humanos , Masculino , Citocinas/genética , Citocinas/metabolismo , Interleucina-6/genética , Interleucina-8/genética , Interleucina-8/metabolismo , Leucócitos Mononucleares/metabolismo , Placofilinas/genética , Placofilinas/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Regulação para CimaRESUMO
BACKGROUND: The regulatory functions of microRNAs (miRNAs) in anti-tumour immunity have been mainly described in immune effector cells. Since little is known about miRNA effects on the susceptibility of target cells during T cell-target cell interaction, this study focused on the identification of miRNAs expressed in tumour cells controlling their susceptibility to CD8+ T cell-mediated cytotoxicity. METHODS: Luciferase expressing B16F10 melanoma (B16F10 Luci+ ) cells transfected with individual miRNAs covering a comprehensive murine miRNA library were screened for their susceptibility to lysis by an established cytotoxic T lymphocyte (CTL) line (5a, clone Nß) specific for the melanoma-associated antigen tyrosinase-related protein 2. miRNAs with the most pronounced effects on T cell-mediated lysis were validated and stably expressed in B16F10 cells. In silico analyses identified common targets of miRNA sets determined by the screen, which were further confirmed by small interfering RNA (siRNA)-mediated silencing experiments modulating immune surveillance. The Ingenuity Pathway Analysis (IPA) software and RNA sequencing (RNA-seq) data from miRNA-overexpressing cell lines were applied to investigate the underlying mechanisms. The Cancer Genome Atlas (TCGA)-derived miRNA sequencing data were used to assess the correlation of miRNA expression with melanoma patients' survival. RESULTS: The miRNA screen resulted in the selection of seven miRNAs enhancing CTL-mediated melanoma cell killing in vitro. Upon stable overexpression of selected miRNAs, hsa-miR-320a-3p, mmu-miR-7037-5p and mmu-miR-666-3p were determined as most effective in enhancing susceptibility to CTL lysis. In silico analyses and subsequent siRNA-mediated silencing experiments identified Psmc3 and Ndufa1 as common miRNA targets possibly involved in the functional effects observed. The analyses of RNA-seq data with IPA showed pathways, networks, biological functions and key molecules potentially involved in the miRNA-mediated functional effects. Finally, based on TCGA data analysis, a positive correlation of the conserved miRNAs among the panel of the seven identified miRNAs with overall survival of melanoma patients was determined. CONCLUSIONS: For the first time, this study uncovered miRNA species that affect the susceptibility of melanoma cells to T cell-mediated killing. These miRNAs might represent attractive candidates for novel therapy approaches against melanoma and other tumour entities.
Assuntos
Melanoma , MicroRNAs , Humanos , Animais , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Melanoma/genética , RNA Interferente Pequeno , Linfócitos T CD8-Positivos/metabolismoRESUMO
Hyper-angiogenesis is a typical feature of glioblastoma (GBM), the most aggressive brain tumor. We have reported the expression of aldehyde dehydrogenase 1A3 (ALDH1A3) in proliferating vasculature in GBM patients. We hypothesized that ALDH1A3 may act as an angiogenesis promoter in GBM. Two GBM cell lines were lentivirally transduced with either ALDH1A3 (ox) or an empty vector (ev). The angiogenesis phenotype was studied in indirect and direct co-culture of endothelial cells (ECs) with oxGBM cells (oxGBMs) and in an angiogenesis model in vivo. Angiogenesis array was performed in oxGBMs. RT2-PCR, Western blot, and double-immunofluorescence staining were performed to confirm the expression of targets identified from the array. A significantly activated angiogenesis phenotype was observed in ECs indirectly and directly co-cultured with oxGBMs and in vivo. Overexpression of ALDH1A3 (oxALDH1A3) led to a marked upregulation of PAI-1 and IL-8 mRNA and protein and a consequential increased release of both proteins. Moreover, oxALDH1A3-induced angiogenesis was abolished by the treatment of the specific inhibitors, respectively, of PAI-1 and IL-8 receptors, CXCR1/2. This study defined ALDH1A3 as a novel angiogenesis promoter. oxALDH1A3 in GBM cells stimulated EC angiogenesis via paracrine upregulation of PAI-1 and IL-8, suggesting ALDH1A3-PAI-1/IL-8 as a novel signaling for future anti-angiogenesis therapy in GBM.
RESUMO
Epithelial membrane protein 3 (EMP3) is an N-glycosylated tetraspanin with a putative trafficking function. It is highly expressed in isocitrate dehydrogenase-wild-type glioblastoma (IDH-wt GBM), and its high expression correlates with poor survival. However, the exact trafficking role of EMP3 and how it promotes oncogenic signaling in GBM remain unclear. Here, we show that EMP3 promotes EGFR/CDK2 signaling by regulating the trafficking and enhancing the stability of EGFR. BioID2-based proximity labeling revealed that EMP3 interacts with endocytic proteins involved in the vesicular transport of EGFR. EMP3 knockout (KO) enhances epidermal growth factor (EGF)-induced shuttling of EGFR into RAB7 + late endosomes, thereby promoting EGFR degradation. Increased EGFR degradation is rescued by the RAB7 negative regulator and novel EMP3 interactor TBC1D5. Phosphoproteomic and transcriptomic analyses further showed that EMP3 KO converges into the inhibition of the cyclin-dependent kinase CDK2 and the repression of EGFR-dependent and cell cycle transcriptional programs. Phenotypically, EMP3 KO cells exhibit reduced proliferation rates, blunted mitogenic response to EGF, and increased sensitivity to the pan-kinase inhibitor staurosporine and the EGFR inhibitor osimertinib. Furthermore, EGFR-dependent patient-derived glioblastoma stem cells display a transcriptomic signature consistent with reduced CDK2 activity, as well as increased susceptibility to CDK2 inhibition upon EMP3 knockdown. Lastly, using TCGA data, we showed that GBM tumors with high EMP3 expression have increased total and phosphorylated EGFR levels. Collectively, our findings demonstrate a novel EMP3-dependent mechanism by which EGFR/CDK2 activity is sustained in GBM. Consequently, EMP3's stabilizing effect provides an additional layer of tumor cell resistance against targeted kinase inhibition.
Assuntos
Fator de Crescimento Epidérmico , Glioblastoma , Humanos , Fator de Crescimento Epidérmico/farmacologia , Glioblastoma/patologia , Transdução de Sinais , Receptores ErbB/metabolismo , Proliferação de Células , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Ativadoras de GTPaseRESUMO
Intratumoral heterogeneity impacts the success or failure of anti-cancer therapies. Here, we investigated the evolution and mechanistic heterogeneity in clonal populations of cell models for estrogen receptor positive breast cancer. To this end, we established barcoded models of luminal breast cancer and rendered them resistant to commonly applied first line endocrine therapies. By isolating single clones from the resistant cell pools and characterizing replicates of individual clones we observed inter- (between cell lines) and intra-tumor (between different clones from the same cell line) heterogeneity. Molecular characterization at RNA and phospho-proteomic levels revealed private clonal activation of the unfolded protein response and respective sensitivity to inhibition of the proteasome, and potentially shared sensitivities for repression of protein kinase C. Our in vitro findings are consistent with tumor-heterogeneity that is observed in breast cancer patients thus highlighting the need to uncover heterogeneity at an individual patient level and to adjust therapies accordingly.
RESUMO
Primary tumors and distant site metastases form a bidirectionally communicating system. Yet, the molecular mechanisms of this crosstalk are poorly understood. Here, we identified the proteolytically cleaved fragments of angiopoietin-like 4 (ANGPTL4) as contextually active protumorigenic and antitumorigenic contributors in this communication ecosystem. Preclinical studies in multiple tumor models revealed that the C-terminal fragment (cANGPTL4) promoted tumor growth and metastasis. In contrast, the N-terminal fragment of ANGPTL4 (nANGPTL4) inhibited metastasis and enhanced overall survival in a postsurgical metastasis model by inhibiting WNT signaling and reducing vascularity at the metastatic site. Tracing ANGPTL4 and its fragments in tumor patients detected full-length ANGPTL4 primarily in tumor tissues, whereas nANGPTL4 predominated in systemic circulation and correlated inversely with disease progression. The study highlights the spatial context of the proteolytic cleavage-dependent pro- and antitumorigenic functions of ANGPTL4 and identifies and validates nANGPTL4 as a novel biomarker of tumor progression and antimetastatic therapeutic agent.
Assuntos
Proteína 4 Semelhante a Angiopoietina , Neoplasias , Humanos , Proteína 4 Semelhante a Angiopoietina/farmacologia , Proteína 4 Semelhante a Angiopoietina/uso terapêutico , Angiopoietinas/farmacologia , Angiopoietinas/uso terapêutico , Biomarcadores Tumorais , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêuticoRESUMO
Excessive stress, e.g. due to biomechanical overload or ischemia/reperfusion is a potent inductor of cardiomyocyte apoptosis, which contributes to maladaptive remodeling. Despite substantial progress in the understanding of the molecular pathophysiology, many components of the signaling pathways underlying remodeling in general and apoptosis in particular still remain unknown. Recent evidence suggests that microRNAs (miRs) play an important role in the heart's response to increased cardiac stress. To identify novel modulators of stress-dependent remodeling, we conducted a genome-wide miR-screen of mechanically stretched neonatal rat cardiomyocytes (NRCM). Out of 351 miRs, eight were significantly regulated by biomechanical stress, including microRNA-20a, which is part of the miR17-92 cluster. Interestingly, further expression analyses also revealed upregulation of microRNA-20a in an in vitro hypoxia/"reperfusion" model. Given the potential apoptosis-modulating properties of the miR17-92 cluster, we subjected NRCM to hypoxia and subsequent reoxygenation. AdmiR-20a significantly inhibited hypoxia-mediated apoptosis in a dose-dependent fashion, while targeted knockdown of miR-20a in NRCM induced cardiomyocyte apoptosis. Mechanistically, the antiapoptotic effect of miR-20a appears to be mediated through direct targeting and subsequent downregulation of the proapoptotic factor Egln3. Thus, miR-20a is upregulated in acute biomechanical stress as well as hypoxia and inhibits apoptosis in cardiomyocytes. These properties reveal miR-20a as a cardioprotective micro-RNA and a potential target for novel therapeutic strategies to prevent cardiac remodeling.
Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/genética , Proteínas Imediatamente Precoces/genética , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Estresse Fisiológico , Animais , Cardiomegalia/genética , Células Cultivadas , Perfilação da Expressão Gênica , Inativação Gênica , Prolina Dioxigenases do Fator Induzível por Hipóxia , MicroRNAs/metabolismo , Miócitos Cardíacos/patologia , RatosRESUMO
Muscle LIM protein (MLP) has been proposed to be a central player in the pathogenesis of heart muscle disease. In line with this notion, the homozygous loss of MLP results in cardiac hypertrophy and dilated cardiomyopathy. Moreover, MLP is induced in several models of cardiac hypertrophy such as aortic banding and myocardial infarction. We thus hypothesized that overexpression of MLP might change the hypertrophic response to cardiac stress. In order to answer the question whether MLP modulates cardiac hypertrophy in vivo, we generated a novel transgenic mouse model with cardiac-specific overexpression of MLP. Three independent transgenic lines did not show a pathological phenotype under baseline conditions. Specifically, contractile function and heart weight to body weight ratios at different ages were normal. Next, the transgenic animals were challenged with pressure overload due to aortic constriction. Surprisingly, transgenic mice developed cardiac hypertrophy to the same extent as their wild-type littermates. Moreover, neither contractile dysfunction nor pathological gene expression in response to pressure overload were differentially affected by MLP overexpression. Finally, in a milder in vivo model of hypertrophy induced by chronic infusion of angiotensin-II, cardiac mass and hypertrophic gene expression were again identical in MLP transgenic mice and controls. Taken together, we provide evidence that cardiac overexpression of MLP does not modulate the heart's response to various forms of pathological stress.
Assuntos
Cardiomegalia/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Remodelação Ventricular , Angiotensina II , Animais , Animais Recém-Nascidos , Aorta/cirurgia , Pressão Sanguínea , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/etiologia , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Genótipo , Proteínas com Domínio LIM/genética , Ligadura , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Proteínas Musculares/genética , Contração Miocárdica , Miocárdio/patologia , Fenótipo , Ratos , Ratos Wistar , Ultrassonografia , Função Ventricular EsquerdaRESUMO
BACKGROUND: Previous experimental studies concluded that stem cells (SC) may exert their beneficial effects on the ischemic heart by paracrine activation of antiapoptotic pathways. In order to identify potential cardioprotective mediators, we performed a systematic analysis of the differential gene expression of hematopoietic SC after coculture with cardiomyocytes (CM). METHODS: After 48 h of coculture with neonatal rat ventricular CM (NRVCM), two consecutive cell sorting steps generated a highly purified population of conditioned murine hematopoietic SC (>99%). Next, a genome-wide microarray analysis of cocultured vs. monocultured hematopoietic SC derived from three independent experiments was performed. The analysis of differentially expressed genes was focused on products that are secretable and/or membrane-bound and potentially involved in antiapoptotic signalling. RESULTS: We found CCL-12, Macrophage Inhibitory Factor, Fibronectin and connexin 40 significantly upregulated in our coculture model. An ELISA of cell culture supernatants was performed to confirm secretion of candidate genes and showed that coculture supernatants revealed markedly higher CCL-12 concentrations. Moreover, we stimulated NRVCM with concentrated coculture supernatants which resulted in a significant reduction of apoptosis compared to monoculture-derived supernatant. Mechanistically, NRVCMs stimulated with coculture supernatants showed a higher level of AKT-phosphorylation, consistent with enhanced antiapoptotic signaling. CONCLUSION: In summary, our results show that the interaction between hematopoietic SC and NRVCM led to a modified gene expression and induction of antiapoptotic pathways. These findings may thus at least in part explain the cardioprotective effects of hematopoietic SC.
Assuntos
Apoptose , Técnicas de Cocultura/métodos , Células-Tronco Hematopoéticas/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , Comunicação Parácrina , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/genética , Separação Celular , Citoproteção , Ativação Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Ventrículos do Coração/citologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Frações Subcelulares/metabolismoRESUMO
RATIONALE AND OBJECTIVE: The M-band represents a transverse structure in the center of the sarcomeric A-band and provides an anchor for the myosin-containing thick filaments. In contrast to other sarcomeric structures, eg, the Z-disc, only few M-band-specific proteins have been identified to date, and its exact molecular composition remains unclear. METHODS AND RESULTS: Using a bioinformatic approach to identify novel heart- and muscle-specific genes, we found a leucine rich protein, myomasp (Myosin-interacting, M-band-associated stress-responsive protein)/LRRC39. RT-PCR and Northern and Western blot analyses confirmed a cardiac-enriched expression pattern, and immunolocalization of myomasp revealed a strong and specific signal at the sarcomeric M-band. Yeast 2-hybrid screens, as well as coimmunoprecipitation experiments, identified the C terminus of myosin heavy chain (MYH)7 as an interaction partner for myomasp. Knockdown of myomasp in neonatal rat ventricular myocytes (NRVCMs) led to a significant upregulation of the stretch-sensitive genes GDF-15 and BNP. Conversely, the expression of MYH7 and the M-band proteins myomesin-1 and -2 was found to be markedly reduced. Mechanistically, knockdown of myomasp in NRVCM led to a dose-dependent suppression of serum response factor-dependent gene expression, consistent with earlier observations linking the M-band to serum response factor-mediated signaling. Finally, downregulation of myomasp/LRRC39 in spontaneously beating engineered heart tissue constructs resulted in significantly lower force generation and reduced fractional shortening. Likewise, knockdown of the myomasp/LRRC39 ortholog in zebrafish resulted in severely impaired heart function and cardiomyopathy in vivo. CONCLUSIONS: These findings reveal myomasp as a previously unrecognized component of an M-band-associated signaling pathway that regulates cardiomyocyte gene expression in response to biomechanical stress.
Assuntos
Proteínas de Transporte/metabolismo , Mecanotransdução Celular , Proteínas Musculares/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Proteínas/metabolismo , Sarcômeros/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Northern Blotting , Western Blotting , Miosinas Cardíacas/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Proteínas de Transporte/genética , Células Cultivadas , Clonagem Molecular , Conectina , Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Proteínas de Repetições Ricas em Leucina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteínas/genética , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Resposta Sérica/metabolismo , Estresse Mecânico , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Peixe-ZebraRESUMO
RATIONALE: The intercalated disc (ID) is a highly specialized cell-cell contact structure that ensures mechanical and electric coupling of contracting cardiomyocytes. Recently, the ID has been recognized to be a hot spot of cardiac disease, in particular inherited cardiomyopathy. OBJECTIVE: Given its complex structure and function we hypothesized that important molecular constituents of the ID still remain unknown. METHODS AND RESULTS: Using a bioinformatics screen, we discovered and cloned a previously uncharacterized 54 kDa cardiac protein which we termed Myozap (Myocardium-enriched zonula occludens-1-associated protein). Myozap is strongly expressed in the heart and lung. In cardiac tissue it localized to the ID and directly binds to desmoplakin and zonula occludens-1. In a yeast 2-hybrid screen for additional binding partners of Myozap we identified myosin phosphatase-RhoA interacting protein (MRIP), a negative regulator of Rho activity. Myozap, in turn, strongly activates SRF-dependent transcription through its ERM (Ezrin/radixin/moesin)-like domain in a Rho-dependent fashion. Finally, in vivo knockdown of the Myozap ortholog in zebrafish led to severe contractile dysfunction and cardiomyopathy. CONCLUSIONS: Taken together, these findings reveal Myozap as a previously unrecognized component of a Rho-dependent signaling pathway that links the intercalated disc to cardiac gene regulation. Moreover, its subcellular localization and the observation of a severe cardiac phenotype in zebrafish, implicate Myozap in the pathogenesis of cardiomyopathy.
Assuntos
Cardiomiopatias/metabolismo , Proteínas Musculares/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Bovinos , Chlorocebus aethiops , Clonagem Molecular , Biologia Computacional , Desmoplaquinas/metabolismo , Cães , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Proteínas Musculares/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Peixe-Zebra , Proteína da Zônula de Oclusão-1 , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Autophagy is an intracellular degradation process that maintains the cellular homeostasis and it is regulated in multiple ways, both in health and disease. Assessment of autophagic flux in cells is an important approach for understanding the function of autophagy in biological contexts. Here, we describe a new tool for the qualitative and quantitative determination of autophagic flux using a dual lentiviral reporter system that generates a fusion HiBiT-GFP-LC3B protein suitable for generating stable cell lines.
Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos , Autofagia/genética , Linhagem Celular , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
BACKGROUND: MicroRNAs (miRNAs) and isomiRs play important roles in tumorigenesis as essential regulators of gene expression. 5'isomiRs exhibit a shifted seed sequence compared to the canonical miRNA, resulting in different target spectra and thereby extending the phenotypic impact of the respective common pre-miRNA. However, for most miRNAs, expression and function of 5'isomiRs have not been studied in detail yet. Therefore, this study aims to investigate the functions of miRNAs and their 5'isomiRs. METHODS: The expression of 5'isomiRs was assessed in The Cancer Genome Atlas (TCGA) breast cancer patient dataset. Phenotypic effects of miR-183 overexpression in triple-negative breast cancer (TNBC) cell lines were investigated in vitro and in vivo by quantifying migration, proliferation, tumor growth and metastasis. Direct targeting of E2F1 by miR-183-5p|+2 was validated with a 3'UTR luciferase assay and linked to the phenotypes of isomiR overexpression. RESULTS: TCGA breast cancer patient data indicated that three variants of miR-183-5p are highly expressed and upregulated, namely miR-183-5p|0, miR-183-5p|+1 and miR-183-5p|+2. However, TNBC cell lines displayed reduced proliferation and invasion upon overexpression of pre-miR-183. While invasion was reduced individually by all three isomiRs, proliferation and cell cycle progression were specifically inhibited by overexpression of miR-183-5p|+2. Proteomic analysis revealed reduced expression of E2F target genes upon overexpression of this isomiR, which could be attributed to direct targeting of E2F1, specifically by miR-183-5p|+2. Knockdown of E2F1 partially phenocopied the effect of miR-183-5p|+2 overexpression on cell proliferation and cell cycle. Gene set enrichment analysis of TCGA and METABRIC patient data indicated that the activity of E2F strongly correlated with the expression of miR-183-5p, suggesting transcriptional regulation of the miRNA by a factor of the E2F family. Indeed, in vitro, expression of miR-183-5p was regulated by E2F1. Hence, miR-183-5p|+2 directly targeting E2F1 appears to be part of a negative feedback loop potentially fine-tuning its activity. CONCLUSIONS: This study demonstrates that 5'isomiRs originating from the same arm of the same pre-miRNA (i.e. pre-miR-183-5p) may exhibit different functions and thereby collectively contribute to the same phenotype. Here, one of three isomiRs was shown to counteract expression of the pre-miRNA by negatively regulating a transcriptional activator (i.e. E2F1). We speculate that this might be part of a regulatory mechanism to prevent uncontrolled cell proliferation, which is disabled during cancer progression.
Assuntos
MicroRNAs , Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Retroalimentação , Humanos , MicroRNAs/metabolismo , Proteômica , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Anticancer therapies have been limited by the emergence of mutations and other adaptations. In bacteria, antibiotics activate the SOS response, which mobilizes error-prone factors that allow for continuous replication at the cost of mutagenesis. We investigated whether the treatment of lung cancer with EGFR inhibitors (EGFRi) similarly engages hypermutators. In cycling drug-tolerant persister (DTP) cells and in EGFRi-treated patients presenting residual disease, we observed upregulation of GAS6, whereas ablation of GAS6's receptor, AXL, eradicated resistance. Reciprocally, AXL overexpression enhanced DTP survival and accelerated the emergence of T790M, an EGFR mutation typical to resistant cells. Mechanistically, AXL induces low-fidelity DNA polymerases and activates their organizer, RAD18, by promoting neddylation. Metabolomics uncovered another hypermutator, AXL-driven activation of MYC, and increased purine synthesis that is unbalanced by pyrimidines. Aligning anti-AXL combination treatments with the transition from DTPs to resistant cells cured patient-derived xenografts. Hence, similar to bacteria, tumors tolerate therapy by engaging pharmacologically targetable endogenous mutators. SIGNIFICANCE: EGFR-mutant lung cancers treated with kinase inhibitors often evolve resistance due to secondary mutations. We report that in similarity to the bacterial SOS response stimulated by antibiotics, endogenous mutators are activated in drug-treated cells, and this heralds tolerance. Blocking the process prevented resistance in xenograft models, which offers new treatment strategies. This article is highlighted in the In This Issue feature, p. 2483.