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MOTIVATION: A major challenge in analyzing cancer patient transcriptomes is that the tumors are inherently heterogeneous and evolving. We analyzed 214 bulk RNA samples of a longitudinal, prospective ovarian cancer cohort and found that the sample composition changes systematically due to chemotherapy and between the anatomical sites, preventing direct comparison of treatment-naive and treated samples. RESULTS: To overcome this, we developed PRISM, a latent statistical framework to simultaneously extract the sample composition and cell-type-specific whole-transcriptome profiles adapted to each individual sample. Our results indicate that the PRISM-derived composition-free transcriptomic profiles and signatures derived from them predict the patient response better than the composite raw bulk data. We validated our findings in independent ovarian cancer and melanoma cohorts, and verified that PRISM accurately estimates the composition and cell-type-specific expression through whole-genome sequencing and RNA in situ hybridization experiments. AVAILABILITYAND IMPLEMENTATION: https://bitbucket.org/anthakki/prism. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Neoplasias Ovarianas , Transcriptoma , Feminino , Humanos , RNA-Seq , Estudos Prospectivos , Análise de Sequência de RNA/métodos , RNA/genética , Perfilação da Expressão Gênica , SoftwareRESUMO
MOTIVATION: Single-cell proteomics technologies, such as mass cytometry, have enabled characterization of cell-to-cell variation and cell populations at a single-cell resolution. These large amounts of data, require dedicated, interactive tools for translating the data into knowledge. RESULTS: We present a comprehensive, interactive method called Cyto to streamline analysis of large-scale cytometry data. Cyto is a workflow-based open-source solution that automates the use of state-of-the-art single-cell analysis methods with interactive visualization. We show the utility of Cyto by applying it to mass cytometry data from peripheral blood and high-grade serous ovarian cancer (HGSOC) samples. Our results show that Cyto is able to reliably capture the immune cell sub-populations from peripheral blood and cellular compositions of unique immune- and cancer cell subpopulations in HGSOC tumor and ascites samples. AVAILABILITYAND IMPLEMENTATION: The method is available as a Docker container at https://hub.docker.com/r/anduril/cyto and the user guide and source code are available at https://bitbucket.org/anduril-dev/cyto. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Proteômica , Software , Interpretação Estatística de Dados , Fluxo de TrabalhoRESUMO
MOTIVATION: Non-parametric dimensionality reduction techniques, such as t-distributed stochastic neighbor embedding (t-SNE), are the most frequently used methods in the exploratory analysis of single-cell datasets. Current implementations scale poorly to massive datasets and often require downsampling or interpolative approximations, which can leave less-frequent populations undiscovered and much information unexploited. RESULTS: We implemented a fast t-SNE package, qSNE, which uses a quasi-Newton optimizer, allowing quadratic convergence rate and automatic perplexity (level of detail) optimizer. Our results show that these improvements make qSNE significantly faster than regular t-SNE packages and enables full analysis of large datasets, such as mass cytometry data, without downsampling. AVAILABILITY AND IMPLEMENTATION: Source code and documentation are openly available at https://bitbucket.org/anthakki/qsne/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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SoftwareRESUMO
Poor chemotherapy response remains a major treatment challenge for high-grade serous ovarian cancer (HGSC). Cancer stem cells are the major contributors to relapse and treatment failure as they can survive conventional therapy. Our objectives were to characterise stemness features in primary patient-derived cell lines, correlate stemness markers with clinical outcome and test the response of our cells to both conventional and exploratory drugs. Tissue and ascites samples, treatment-naive and/or after neoadjuvant chemotherapy, were prospectively collected. Primary cancer cells, cultured under conditions favouring either adherent or spheroid growth, were tested for stemness markers; the same markers were analysed in tissue and correlated with chemotherapy response and survival. Drug sensitivity and resistance testing was performed with 306 oncology compounds. Spheroid growth condition HGSC cells showed increased stemness marker expression (including aldehyde dehydrogenase isoform I; ALDH1A1) as compared with adherent growth condition cells, and increased resistance to platinum and taxane. A set of eight stemness markers separated treatment-naive tumours into two clusters and identified a distinct subgroup of HGSC with enriched stemness features. Expression of ALDH1A1, but not most other stemness markers, was increased after neoadjuvant chemotherapy and its expression in treatment-naive tumours correlated with chemoresistance and reduced survival. In drug sensitivity and resistance testing, five compounds, including two PI3K-mTOR inhibitors, demonstrated significant activity in both cell culture conditions. Thirteen compounds, including EGFR, PI3K-mTOR and aurora kinase inhibitors, were more toxic to spheroid cells than adherent cells. Our results identify stemness markers in HGSC that are associated with a decreased response to conventional chemotherapy and reduced survival if expressed by treatment-naive tumours. EGFR, mTOR-PI3K and aurora kinase inhibitors are candidates for targeting this cell population. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Família Aldeído Desidrogenase 1/metabolismo , Antineoplásicos/farmacologia , Cistadenocarcinoma Seroso/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/patologia , Retinal Desidrogenase/metabolismo , Aurora Quinases/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Quimioterapia Adjuvante/métodos , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/metabolismo , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Terapia de Alvo Molecular/métodos , Gradação de Tumores , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Prognóstico , Esferoides Celulares/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Motivation: DNA methylation aberrations are common in many cancer types. A major challenge hindering comparison of patient-derived samples is that they comprise of heterogeneous collection of cancer and microenvironment cells. We present a computational method that allows comparing cancer methylomes in two or more heterogeneous tumor samples featuring differing, unknown fraction of cancer cells. The method is unique in that it allows comparison also in the absence of normal cell control samples and without prior tumor purity estimates, as these are often unavailable or unreliable in clinical samples. Results: We use simulations and next-generation methylome, RNA and whole-genome sequencing data from two cancer types to demonstrate that the method is accurate and outperforms alternatives. The results show that our method adapts well to various cancer types and to a wide range of tumor content, and works robustly without a control or with controls derived from various sources. Availability and implementation: The method is freely available at https://bitbucket.org/anthakki/dmml. Supplementary information: Supplementary data are available at Bioinformatics online.
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Metilação de DNA , Neoplasias/genética , Humanos , Neoplasias/metabolismoRESUMO
Platinum-resistance is a major limitation to effective chemotherapy regimens in high-grade serous ovarian cancer (HGSOC). To better understand the mechanisms involved we characterized the proteome and phosphoproteome in cisplatin sensitive and resistant HGSOC primary cells using a mass spectrometry-based proteomic strategy. PCA analysis identified a distinctive phosphoproteomic signature between cisplatin sensitive and resistant cell lines. The most phosphorylated protein in cisplatin resistant cells was sequestosome-1 (p62/SQSTM1). Changes in expression of apoptosis and autophagy related proteins Caspase-3 and SQSTM1, respectively, were validated by Western blot analysis. A significant increase in apoptosis in the presence of cisplatin was observed in only the sensitive cell line while SQSTM1 revealed increased expression in the resistant cell line relative to sensitive cell line. Furthermore, site-specific phosphorylation on 20 amino acid residues of SQSTM1 was detected indicating a hyper-phosphorylation phenotype. This elevated hyper-phosphorylation of SQSTM1 in resistant HGSOC cell lines was validated with Western blot analysis. Immunofluoresence staining of s28-pSQSTM1 showed inducible localization to autophagosomes upon cisplatin treatment in the sensitive cell line while being constitutively expressed to autophagosomes in the resistant cell. Furthermore, SQSTM1 expression was localized in cancer cells of clinical high-grade serous tumors. Here, we propose hyper-phosphorylation of SQSTM1 as a marker and a key proteomic change in cisplatin resistance development in ovarian cancers by activating the autophagy pathway and influencing down-regulation of apoptosis.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteína Sequestossoma-1/metabolismo , Autofagossomos/metabolismo , Caspase 1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Espectrometria de Massas , Gradação de Tumores , Fosforilação , Estudos Prospectivos , Proteômica/métodos , Proteína Sequestossoma-1/químicaRESUMO
The functional properties of actin-regulating formin proteins are diverse and in many cases cell-type specific. FHOD1, a formin expressed predominantly in cells of mesenchymal lineage, bundles actin filaments and participates in maintenance of cell shape, migration and cellular protrusions. FHOD1 participates in cancer-associated epithelial to mesenchymal transition (EMT) in oral squamous cell carcinoma and breast cancer. The role of FHOD1 in melanomas has not been characterized. Here, we show that FHOD1 expression is typically strong in cutaneous melanomas and cultured melanoma cells while the expression is low or absent in benign nevi. By using shRNA to knockdown FHOD1 in melanoma cells, we discovered that FHOD1 depleted cells are larger, rounder and have smaller focal adhesions and inferior migratory capacity as compared to control cells. Importantly, we found FHOD1 depleted cells to have reduced colony-forming capacity and attenuated tumor growth in vivo, a finding best explained by the reduced proliferation rate caused by cell cycle arrest. Unexpectedly, FHOD1 depletion did not prevent invasive growth at the tumor margins. These results suggest that FHOD1 participates in key cellular processes that are dysregulated in malignancy, but may not be essential for melanoma cell invasion.
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Citoesqueleto de Actina/metabolismo , Proteínas Fetais/metabolismo , Melanoma/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Cutâneas/metabolismo , Fibras de Estresse/metabolismo , Actinas/metabolismo , Animais , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal/fisiologia , Forminas , Humanos , Camundongos , Ativação Transcricional/fisiologia , Regulação para Cima , Melanoma Maligno CutâneoRESUMO
OBJECTIVE: The melanocortin 1 receptor (MC1-R) is expressed by vascular endothelial cells and shown to enhance nitric oxide (NO) availability and vasodilator function on pharmacological stimulation. However, the physiological role of MC1-R in the endothelium and its contribution to vascular homeostasis remain unresolved. We investigated whether a lack of functional MC1-R signaling carries a phenotype with predisposition to vascular abnormalities. APPROACH AND RESULTS: Recessive yellow mice (MC1R(e/e)), deficient in MC1-R signaling, and their wild-type littermates were studied for morphology and functional characteristics of the aorta. MC1R(e/e) mice showed increased collagen deposition and arterial stiffness accompanied by an elevation in pulse pressure. Contractile capacity and NO-dependent vasodilatation were impaired in the aorta of MC1R(e/e) mice supported by findings of decreased NO availability. These mice also displayed elevated levels of systemic and local cytokines. Exposing the mice to high-sodium diet or acute endotoxemia revealed increased susceptibility to inflammation-driven vascular dysfunction. Finally, we investigated whether a similar phenotype can be found in healthy human subjects carrying variant MC1-R alleles known to attenuate receptor function. In a longitudinal analysis of 2001 subjects with genotype and ultrasound data (The Cardiovascular Risk in Young Finns Study), weak MC1-R function was associated with lower flow-mediated dilatation response of the brachial artery and increased carotid artery stiffness. CONCLUSIONS: The present study demonstrates that deficiency in MC1-R signaling is associated with increased arterial stiffness and impairment in endothelium-dependent vasodilatation, suggesting a physiological role for MC1-R in the regulation of arterial tone.
Assuntos
Endotélio Vascular/metabolismo , Receptor Tipo 1 de Melanocortina/metabolismo , Rigidez Vascular , Animais , Aorta/metabolismo , Pressão Sanguínea , Colágeno/metabolismo , Humanos , Inflamação/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Transdução de Sinais , Vasoconstrição , VasodilataçãoRESUMO
The broad research use of organoids from high-grade serous ovarian cancer (HGSC) has been hampered by low culture success rates and limited availability of fresh tumor material. Here, we describe a method for generation and long-term expansion of HGSC organoids with efficacy markedly improved over previous reports (53% vs. 23%-38%). We established organoids from cryopreserved material, demonstrating the feasibility of using viably biobanked tissue for HGSC organoid derivation. Genomic, histologic, and single-cell transcriptomic analyses revealed that organoids recapitulated genetic and phenotypic features of original tumors. Organoid drug responses correlated with clinical treatment outcomes, although in a culture conditions-dependent manner and only in organoids maintained in human plasma-like medium (HPLM). Organoids from consenting patients are available to the research community through a public biobank and organoid genomic data are explorable through an interactive online tool. Taken together, this resource facilitates the application of HGSC organoids in basic and translational ovarian cancer research.
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Neoplasias Ovarianas , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Organoides/patologia , GenômicaRESUMO
The time between the last cycle of chemotherapy and recurrence, the platinum-free interval (PFI), predicts overall survival in high-grade serous ovarian cancer (HGSOC). To identify secreted proteins associated with a shorter PFI, we utilized machine learning to predict the PFI from ascites composition. Ascites from stage III/IV HGSOC patients treated with neoadjuvant chemotherapy (NACT) or primary debulking surgery (PDS) were screened for secreted proteins and Lasso regression models were built to predict the PFI. Through regularization techniques, the number of analytes used in each model was reduced; to minimize overfitting, we utilized an analysis of model robustness. This resulted in models with 26 analytes and a root-mean-square error (RMSE) of 19 days for the NACT cohort and 16 analytes and an RMSE of 7 days for the PDS cohort. High concentrations of MMP-2 and EMMPRIN correlated with a shorter PFI in the NACT patients, whereas high concentrations of uPA Urokinase and MMP-3 correlated with a shorter PFI in PDS patients. Our results suggest that the analysis of ascites may be useful for outcome prediction and identified factors in the tumor microenvironment that may lead to worse outcomes. Our approach to tuning for model stability, rather than only model accuracy, may be applicable to other biomarker discovery tasks.
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Identification of ovarian cancer patient subpopulations with increased sensitivity to targeted therapies could offer significant clinical benefit. We report that 22% of the high-grade ovarian cancer tumors at diagnosis express CIP2A oncoprotein at low levels. Furthermore, regardless of their significantly lower likelihood of disease relapse after standard chemotherapy, a portion of relapsed tumors retain their CIP2A-deficient phenotype. Through a screen for therapeutics that would preferentially kill CIP2A-deficient ovarian cancer cells, we identified reactive oxygen species inducer APR-246, tested previously in ovarian cancer clinical trials. Consistent with CIP2A-deficient ovarian cancer subtype in humans, CIP2A is dispensable for development of MISIIR-Tag-driven mouse ovarian cancer tumors. Nevertheless, CIP2A-null ovarian cancer tumor cells from MISIIR-Tag mice displayed APR-246 hypersensitivity both in vitro and in vivo. Mechanistically, the lack of CIP2A expression hypersensitizes the ovarian cancer cells to APR-246 by inhibition of NF-κB activity. Accordingly, combination of APR-246 and NF-κB inhibitor compounds strongly synergized in killing of CIP2A-positive ovarian cancer cells. Collectively, the results warrant consideration of clinical testing of APR-246 for CIP2A-deficient ovarian cancer tumor subtype patients. Results also reveal CIP2A as a candidate APR-246 combination therapy target for ovarian cancer.
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NF-kappa B , Neoplasias Ovarianas , Animais , Autoantígenos/genética , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , QuinuclidinasRESUMO
Objective: A major challenge in the treatment of platinum-resistant high-grade serous ovarian cancer (HGSOC) is lack of effective therapies. Much of ongoing research on drug candidates relies on HGSOC cell lines that are poorly documented. The goal of this study was to screen for effective, state-of-the-art drug candidates using primary HGSOC cells. In addition, our aim was to dissect the inhibitory activities of Wee1 inhibitor adavosertib on primary and conventional HGSOC cell lines. Methods: A comprehensive drug sensitivity and resistance testing (DSRT) on 306 drug compounds was performed on three patient-derived genetically unique HGSOC cell lines and two commonly used ovarian cancer cell lines. The effect of adavosertib on the cell lines was tested in several assays, including cell-cycle analysis, apoptosis induction, proliferation, wound healing, DNA damage, and effect on nuclear integrity. Results: Several compounds exerted cytotoxic activity toward all cell lines, when tested in both adherent and spheroid conditions. In further cytotoxicity tests, adavosertib exerted the most consistent cytotoxic activity. Adavosertib affected cell-cycle control in patient-derived and conventional HGSOC cells, inducing G2/M accumulation and reducing cyclin B1 levels. It induced apoptosis and inhibited proliferation and migration in all cell lines. Furthermore, the DNA damage marker γH2AX and the number of abnormal cell nuclei were clearly increased following adavosertib treatment. Based on the homologous recombination (HR) signature and functional HR assays of the cell lines, the effects of adavosertib were independent of the cells' HR status. Conclusion: Our study indicates that Wee1 inhibitor adavosertib affects several critical functions related to proliferation, cell cycle and division, apoptosis, and invasion. Importantly, the effects are consistent in all tested cell lines, including primary HGSOC cells, and independent of the HR status of the cells. Wee1 inhibition may thus provide treatment opportunities especially for patients, whose cancer has acquired resistance to platinum-based chemotherapy or PARP inhibitors.
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Chemotherapy resistance is a critical contributor to cancer mortality and thus an urgent unmet challenge in oncology. To characterize chemotherapy resistance processes in high-grade serous ovarian cancer, we prospectively collected tissue samples before and after chemotherapy and analyzed their transcriptomic profiles at a single-cell resolution. After removing patient-specific signals by a novel analysis approach, PRIMUS, we found a consistent increase in stress-associated cell state during chemotherapy, which was validated by RNA in situ hybridization and bulk RNA sequencing. The stress-associated state exists before chemotherapy, is subclonally enriched during the treatment, and associates with poor progression-free survival. Co-occurrence with an inflammatory cancer-associated fibroblast subtype in tumors implies that chemotherapy is associated with stress response in both cancer cells and stroma, driving a paracrine feed-forward loop. In summary, we have found a resistant state that integrates stromal signaling and subclonal evolution and offers targets to overcome chemotherapy resistance.
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Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Análise de Sequência de RNA , Transcriptoma , Sequenciamento do ExomaRESUMO
Critical DNA repair pathways become deranged during cancer development. This vulnerability may be exploited with DNA-targeting chemotherapy. Topoisomerase II inhibitors induce double-strand breaks which, if not repaired, are detrimental to the cell. This repair process requires high-fidelity functional homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). If either of these pathways is defective, a compensatory pathway may rescue the cells and induce treatment resistance. Consistently, HR proficiency, either inherent or acquired during the course of the disease, enables tumor cells competent to repair the DNA damage, which is a major problem for chemotherapy in general. In this context, c-Abl is a protein tyrosine kinase that is involved in DNA damage-induced stress. We used a low-dose topoisomerase II inhibitor mitoxantrone to induce DNA damage which caused a transient cell cycle delay but allowed eventual passage through this checkpoint in most cells. We show that the percentage of HR and NHEJ efficient HeLa cells decreased more than 50% by combining c-Abl inhibitor imatinib with mitoxantrone. This inhibition of DNA repair caused more than 87% of cells in G2/M arrest and a significant increase in apoptosis. To validate the effect of the combination treatment, we tested it on commercial and patient-derived cell lines in high-grade serous ovarian cancer (HGSOC), where chemotherapy resistance correlates with HR proficiency and is a major clinical problem. Results obtained with HR-proficient and deficient HGSOC cell lines show a 50-85% increase of sensitivity by the combination treatment. Our data raise the possibility of successful targeting of treatment-resistant HR-proficient cancers.
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Due to its dynamic nature, the evolution of cancer cell-extracellular matrix (ECM) crosstalk, critically affecting metastasis and treatment resistance, remains elusive. Our results show that platinum-chemotherapy itself enhances resistance by progressively changing the cancer cell-intrinsic adhesion signaling and cell-surrounding ECM. Examining ovarian high-grade serous carcinoma (HGSC) transcriptome and histology, we describe the fibrotic ECM heterogeneity at primary tumors and distinct metastatic sites, prior and after chemotherapy. Using cell models from systematic ECM screen to collagen-based 2D and 3D cultures, we demonstrate that both specific ECM substrates and stiffness increase resistance to platinum-mediated, apoptosis-inducing DNA damage via FAK and ß1 integrin-pMLC-YAP signaling. Among such substrates around metastatic HGSCs, COL6 was upregulated by chemotherapy and enhanced the resistance of relapse, but not treatment-naïve, HGSC organoids. These results identify matrix adhesion as an adaptive response, driving HGSC aggressiveness via co-evolving ECM composition and sensing, suggesting stromal and tumor strategies for ECM pathway targeting.
Assuntos
Cistadenocarcinoma Seroso/genética , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Colágeno/genética , Colágeno/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Evolução Molecular , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Recidiva Local de Neoplasia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Diaphanous-related formins govern actin-based processes involved in many cellular functions, such as cell movement and invasion. Possible connections to developmental processes and cellular changes associated with malignant phenotype make them interesting study targets. In spite of this, very little is known of the tissue distribution and cellular location of any mammalian formin. Here we have carried out a comprehensive analysis of the formin family member formin -like 2 (FMNL2) in human tissues. RESULTS: An FMNL2 antibody was raised and characterized. The affinity-purified FMNL2 antibody was validated by Western blotting, Northern blotting, a peptide competition assay and siRNA experiments. Bioinformatics-based mRNA profiling indicated that FMNL2 is widely expressed in human tissues. The highest mRNA levels were seen in central and peripheral nervous systems. Immunohistochemical analysis of 26 different human tissues showed that FMNL2 is widely expressed, in agreement with the mRNA profile. The widest expression was detected in the central nervous system, since both neurons and glial cells expressed FMNL2. Strong expression was also seen in many epithelia. However, the expression in different cell types was not ubiquitous. Many mesenchymal cell types showed weak immunoreactivity and cells lacking expression were seen in many tissues. The subcellular location of FMNL2 was cytoplasmic, and in some tissues a strong perinuclear dot was detected. In cultured cells FMNL2 showed mostly a cytoplasmic localization with perinuclear accumulation consistent with the Golgi apparatus. Furthermore, FMNL2 co-localized with F-actin to the tips of cellular protrusions in WM164 human melanoma cells. This finding is in line with FMNL2's proposed function in the formation of actin filaments in cellular protrusions, during amoeboid cellular migration. CONCLUSION: FMNL2 is expressed in multiple human tissues, not only in the central nervous system. The expression is especially strong in gastrointestinal and mammary epithelia, lymphatic tissues, placenta, and in the reproductive tract. In cultured melanoma cells, FMNL2 co-localizes with F-actin dots at the tips of cellular protrusions.
Assuntos
Sistema Nervoso Central/metabolismo , Epitélio/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteínas/metabolismo , Actinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Extensões da Superfície Celular/metabolismo , Sistema Nervoso Central/patologia , Biologia Computacional , Citoplasma/metabolismo , Epitélio/patologia , Forminas , Perfilação da Expressão Gênica , Humanos , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Células-Tronco Mesenquimais/patologia , Ligação Proteica , Transporte Proteico , Proteínas/genética , Proteínas/imunologiaRESUMO
The melanocortin MC1 and MC3 receptors elicit anti-inflammatory actions in leukocytes and activation of these receptors has been shown to alleviate arterial inflammation in experimental atherosclerosis. Thus, we aimed to investigate whether selective targeting of melanocortin MC3 receptor protects against atherosclerosis. Apolipoprotein E deficient (ApoE-/-) mice were fed high-fat diet for 12 weeks and randomly assigned to receive either vehicle (n = 11) or the selective melanocortin MC3 receptor agonist [D-Trp(8)]-gamma-melanocyte-stimulating hormone ([D-Trp8]-γ-MSH; 15 µg/day, n = 10) for the last 4 weeks. Lesion size as well as macrophage and collagen content in the aortic root plaques were determined. Furthermore, leukocyte counts in the blood and aorta and cytokine mRNA expression levels in the spleen, liver and aorta were quantified. No effect was observed in the body weight development or plasma cholesterol level between the two treatment groups. However, [D-Trp8]-γ-MSH treatment significantly reduced plasma levels of chemokine (C-C motif) ligands 2, 4 and 5. Likewise, cytokine and adhesion molecule expression levels were reduced in the spleen and liver of γ-MSH-treated mice, but not substantially in the aorta. In line with these findings, [D-Trp8]-γ-MSH treatment reduced leukocyte counts in the blood and aorta. Despite reduced inflammation, [D-Trp8]-γ-MSH did not change lesion size, macrophage content or collagen deposition of aortic root plaques. In conclusion, the findings indicate that selective activation of melanocortin MC3 receptor by [D-Trp8]-γ-MSH suppresses systemic and local inflammation and thereby also limits leukocyte accumulation in the aorta. However, the treatment was ineffective in reducing atherosclerotic plaque size.
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Anti-Inflamatórios/uso terapêutico , Hormônios Estimuladores de Melanócitos/uso terapêutico , Placa Aterosclerótica/tratamento farmacológico , Receptor Tipo 3 de Melanocortina/agonistas , Animais , Anti-Inflamatórios/farmacologia , Aorta/efeitos dos fármacos , Aorta/imunologia , Aorta/patologia , Células Cultivadas , Colesterol/sangue , Citocinas/sangue , Citocinas/genética , Dieta Hiperlipídica , Células Endoteliais , Feminino , Inflamação/imunologia , Contagem de Leucócitos , Fígado/efeitos dos fármacos , Fígado/imunologia , Hormônios Estimuladores de Melanócitos/farmacologia , Camundongos Knockout para ApoE , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Receptor Tipo 3 de Melanocortina/imunologia , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
A Leu7Pro change in the signal peptide of preproNPY is a functional substitution, which changes the processing of NPY in cells and associates with several cardiovascular and metabolic conditions in humans. The current study investigates the effect of the P7 allele in endothelial cells, where decreased nitric oxide (NO) production is a promoting factor to endothelial dysfunction. The function of NO system was assessed in the human umbilical vein endothelial cells (HUVECs) with [p.L7]+[p.L7] or [p.L7]+[p.P7] genotype. NPY seems to have a significant influence on NO system in HUVECs, and the responses are time and genotype dependent. HUVECs with [p.L7]+[p.P7] genotype seem to have higher basal production of NO, but after a long term treatment with NPY these cells express less eNOS mRNA and overall eNOS protein levels are lower. These significant differences in the NO bioavailability may explain the association of the L7P polymorphism with several cardiovascular complications.
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Endotélio Vascular/metabolismo , Neuropeptídeo Y/genética , Óxido Nítrico/biossíntese , Polimorfismo Genético , Células Cultivadas , Endotélio Vascular/enzimologia , Humanos , Leucina/genética , Leucina/metabolismo , Óxido Nítrico/genética , Óxido Nítrico Sintase Tipo III/biossíntese , Prolina/genética , Prolina/metabolismo , RNA Mensageiro/biossíntese , Veias Umbilicais/metabolismoRESUMO
Neuropeptide Y (NPY) is a universally expressed neuropeptide involved in the regulation of several physiological functions. The rather common leucine7 to proline7 (L7P) polymorphism in the signal peptide of preproNPY is a functional substitution, which changes the processing and release of NPY in cells. The mutation is associated with altered lipid levels and accelerated atherosclerosis in humans. Based on previous studies, we investigated the effect of the Pro7 allele in endothelial cells, which are known to play a role in the development of atherosclerosis. Cell proliferation and apoptosis were studied in primary cultured, genotyped human umbilical vein endothelial cells (HUVECs). Our results indicate that cells with the [p.L7]+[p.P7] genotype seem to have a tendency to be more sensitive to the growth stimulating effect of NPY and less sensitive to the effect of vascular endothelial growth factor compared to cells with the [p.L7]+[p.L7] genotype. Additionally, cells with the [p.L7]+[p.P7] genotype seem to be more sensitive to apoptosis than [p.L7]+[p.L7] cells. We speculate that the L7P substitution in preproNPY might cause a state of cellular pre-senescence, leading to endothelial dysfunction. This might be one reason for the associations of the L7P polymorphism with atherosclerosis and type II diabetes found in clinical studies.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Neuropeptídeo Y/fisiologia , Polimorfismo Genético/fisiologia , Veias Umbilicais/citologia , Apoptose/genética , Apoptose/fisiologia , Proliferação de Células , Células Cultivadas , Genótipo , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Microscopia Confocal , Neuropeptídeo Y/genética , Polimorfismo Genético/genéticaRESUMO
Preproneuropeptide Y is a precursor peptide to mature neuropeptide Y (NPY), which is a universally expressed peptide in the central and peripheral nervous system. NPY is normally routed to endoplasmic reticulum and secretory vesicles in cells, which secrete NPY. In our previous studies, we found a functional Leucine7 to Proline7 (L7P) polymorphism in the signal peptide sequence of preproNPY. This polymorphism affects the secretion of NPY and causes multiple physiological effects in humans. The sequence of NPY mRNA contains two in frame kozak sequences that allow translation initiation to shift, and translation of two proteins. In addition to mature NPY(1-36) also a putative truncated NPY(17-36) with mitochondrial targeting signal is produced. The purpose of this study was to investigate the protein mobility of the putative mitochondrial fragment and the effect of the L7P polymorphism on the cellular level using GFP tagged constructs. The mobility was studied with fluorescence recovery after photobleaching technique in a neuronal cell line. We found that the mobility of the secretory vesicles with NPY(1-36) in cells with L7P genotype was increased in comparison to vesicle mobility in cells with the more abundant L7L genotype. The mobility in the cells with the putative mitochondrial construct was found to be very low. According to the results of the present study, the mitochondrial truncated peptide stays in the mitochondrion. It can be hypothesized that this could be one of the factors affecting energy balance of the membranes of the mitochondrion.