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1.
Cell ; 143(1): 84-98, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20887894

RESUMO

Ca(2+) is an essential and ubiquitous second messenger. Changes in cytosolic Ca(2+) trigger events critical for tumorigenesis, such as cellular motility, proliferation, and apoptosis. We show that an isoform of Secretory Pathway Ca(2+)-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuates basal Ca(2+) levels and tumorigenicity. Contrary to its conventional role in Golgi Ca(2+) sequestration, expression of SPCA2 increased Ca(2+) influx by a mechanism dependent on the store-operated Ca(2+) channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca(2+) stores or STIM1 and STIM2 sensors and uncoupled from Ca(2+)-ATPase activity of SPCA2. Binding of the SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca(2+) influx. Our findings reveal a signaling pathway in which the Orai1-SPCA2 complex elicits constitutive store-independent Ca(2+) signaling that promotes tumorigenesis.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , ATPases Transportadoras de Cálcio/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Dados de Sequência Molecular , Transplante de Neoplasias , Proteína ORAI1 , Ratos , Alinhamento de Sequência , Transplante Heterólogo
2.
Plant Biotechnol J ; 22(8): 2248-2266, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38516995

RESUMO

The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian-based production systems is increasing. Plant-based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state-of-the-art mass spectrometry-based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.


Assuntos
Agrobacterium tumefaciens , Reatores Biológicos , Nicotiana , Proteômica , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologia , Nicotiana/crescimento & desenvolvimento , Reatores Biológicos/microbiologia , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Agricultura Molecular/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteoma/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
3.
Brain Behav Immun ; 122: 471-482, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39163911

RESUMO

Increasing rates of child neurodevelopmental vulnerability are a significant public health challenge. The adverse effect of socioeconomic adversity on offspring cognition may be mediated through elevated prenatal maternal systemic inflammation, but the role of modifiable antecedents such as maternal nutrition has not yet been clarified. This study aimed to examine (1) whether prenatal factors, with an emphasis on maternal nutrition, were associated with prenatal maternal systemic inflammation at 28 weeks' gestation, including the metabolomic marker glycoprotein acetyls (GlycA); (2) the extent to which the association between prenatal maternal nutrition and child cognition and language at age two years was mediated by elevated maternal inflammation in pregnancy; (3) the extent to which the associations between prenatal socioeconomic adversity and child neurodevelopment were mediated through prenatal maternal nutrition and GlycA levels. We used a prospective population-derived pre-birth longitudinal cohort study, the Barwon Infant Study (Barwon region of Victoria, Australia), where 1074 mother-child pairs were recruited by 28 weeks' gestation using an unselected sampling frame. Exposures included prenatal factors such as maternal diet measured by a validated food frequency questionnaire at 28 weeks' gestation and dietary patterns determined by principal component analysis. The main outcome measures were maternal inflammatory biomarkers (GlycA and hsCRP levels) at 28 weeks' gestation, and offspring Bayley-III cognition and language scores at age two years. Results showed that the 'modern wholefoods' and 'processed' maternal dietary patterns were independently associated with reduced and elevated maternal inflammation respectively (GlycA or hsCRP p < 0.001), and also with higher and reduced offspring Bayley-III scores respectively (cognition p ≤ 0.004, language p ≤ 0.009). Associations between dietary patterns and offspring cognition and language were partially mediated by higher maternal GlycA (indirect effect: cognition p ≤ 0.036, language p ≤ 0.05), but were less evident for hsCRP. The maternal dietary patterns mediated 22 % of the association between socioeconomic adversity (lower maternal education and/or lower household income vs otherwise) and poorer offspring cognition (indirect effect p = 0.001). Variation in prenatal GlycA levels that were independent of these dietary measures appeared less important. In conclusion, modifiable prenatal maternal dietary patterns were associated with adverse child neurocognitive outcomes through their effect on maternal inflammation (GlycA). Maternal diet may partially explain the association between socioeconomic adversity and child neurocognitive vulnerability. Maternal diet-by-inflammation pathways are an attractive target for future intervention studies.


Assuntos
Cognição , Inflamação , Fenômenos Fisiológicos da Nutrição Materna , Efeitos Tardios da Exposição Pré-Natal , Humanos , Feminino , Gravidez , Cognição/fisiologia , Pré-Escolar , Masculino , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , Adulto , Estudos Prospectivos , Fatores Socioeconômicos , Desenvolvimento Infantil , Estudos Longitudinais , Idioma , Desenvolvimento da Linguagem , Biomarcadores/sangue
4.
Mol Psychiatry ; 28(9): 3760-3768, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37845496

RESUMO

Childhood mental disorders, including emotional and behavioural problems (EBP) are increasingly prevalent. Higher maternal oxidative stress (OS) during pregnancy (matOSpreg) is linked to offspring mental disorders. Environmental factors contribute to matOSpreg. However, the role of matOSpreg in childhood EBP is unclear. We investigated the associations between (i) matOSpreg and offspring EBP; (ii) social and prenatal environmental factors and matOSpreg; and (iii) social and prenatal factors and childhood EBP and evaluated whether matOSpreg mediated these associations. Maternal urinary OS biomarkers, 8-hydroxyguanosine (8-OHGua; an oxidative RNA damage marker) and 8-hydroxy-2'-deoxyguanosine (8-OHdG; an oxidative DNA damage marker), at 36 weeks of pregnancy were quantified by liquid chromatography-mass spectrometry in a population-derived birth cohort, Barwon Infant Study (n = 1074 mother-infant pairs). Social and prenatal environmental factors were collected by mother-reported questionnaires. Offspring total EBP was measured by Child Behavior Checklist Total Problems T-scores at age two (n = 675) and Strengths and Difficulties Questionnaire Total Difficulties score at age four (n = 791). Prospective associations were examined by multivariable regression analyses adjusted for covariates. Mediation effects were evaluated using counterfactual-based mediation analysis. Higher maternal urinary 8-OHGua at 36 weeks (mat8-OHGua36w) was associated with greater offspring total EBP at age four (ß = 0.38, 95% CI (0.07, 0.69), P = 0.02) and age two (ß = 0.62, 95% CI (-0.06, 1.30), P = 0.07). Weaker evidence of association was detected for 8-OHdG. Five early-life factors were associated with both mat8-OHGua36w and childhood EBP (P-range < 0.001-0.05), including lower maternal education, socioeconomic disadvantage and prenatal tobacco smoking. These risk factor-childhood EBP associations were partly mediated by higher mat8-OHGua36w (P-range = 0.01-0.05). Higher matOSpreg, particularly oxidant RNA damage, is associated with later offspring EBP. Effects of some social and prenatal lifestyle factors on childhood EBP were partly mediated by matOSpreg. Future studies are warranted to further elucidate the role of early-life oxidant damage in childhood EBP.


Assuntos
Efeitos Tardios da Exposição Pré-Natal , Comportamento Problema , Gravidez , Feminino , Lactente , Humanos , Pré-Escolar , Comportamento Problema/psicologia , Mães/psicologia , Oxidantes , RNA
5.
EMBO Rep ; 23(11): e55399, 2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36194675

RESUMO

Anticancer T cells acquire a dysfunctional state characterized by poor effector function and expression of inhibitory receptors, such as PD-1. Blockade of PD-1 leads to T cell reinvigoration and is increasingly applied as an effective anticancer treatment. Recent work challenged the commonly held view that the phosphatase PTPN11 (known as SHP-2) is essential for PD-1 signaling in T cells, suggesting functional redundancy with the homologous phosphatase PTPN6 (SHP-1). Therefore, we investigated the effect of concomitant Ptpn6 and Ptpn11 deletion in T cells on their ability to mount antitumour responses. In vivo data show that neither sustained nor acute Ptpn6/11 deletion improves T cell-mediated tumor control. Sustained loss of Ptpn6/11 also impairs the therapeutic effects of anti-PD1 treatment. In vitro results show that Ptpn6/11-deleted CD8+ T cells exhibit impaired expansion due to a survival defect and proteomics analyses reveal substantial alterations, including in apoptosis-related pathways. These data indicate that concomitant ablation of Ptpn6/11 in polyclonal T cells fails to improve their anticancer properties, implying that caution shall be taken when considering their inhibition for immunotherapeutic approaches.


Assuntos
Linfócitos T CD8-Positivos , Receptor de Morte Celular Programada 1 , Linfócitos T CD8-Positivos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transdução de Sinais
6.
Brain ; 146(6): 2298-2315, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-36508327

RESUMO

Huntingtin (HTT)-lowering therapies show great promise in treating Huntington's disease. We have developed a microRNA targeting human HTT that is delivered in an adeno-associated serotype 5 viral vector (AAV5-miHTT), and here use animal behaviour, MRI, non-invasive proton magnetic resonance spectroscopy and striatal RNA sequencing as outcome measures in preclinical mouse studies of AAV5-miHTT. The effects of AAV5-miHTT treatment were evaluated in homozygous Q175FDN mice, a mouse model of Huntington's disease with severe neuropathological and behavioural phenotypes. Homozygous mice were used instead of the more commonly used heterozygous strain, which exhibit milder phenotypes. Three-month-old homozygous Q175FDN mice, which had developed acute phenotypes by the time of treatment, were injected bilaterally into the striatum with either formulation buffer (phosphate-buffered saline + 5% sucrose), low dose (5.2 × 109 genome copies/mouse) or high dose (1.3 × 1011 genome copies/mouse) AAV5-miHTT. Wild-type mice injected with formulation buffer served as controls. Behavioural assessments of cognition, T1-weighted structural MRI and striatal proton magnetic resonance spectroscopy were performed 3 months after injection, and shortly afterwards the animals were sacrificed to collect brain tissue for protein and RNA analysis. Motor coordination was assessed at 1-month intervals beginning at 2 months of age until sacrifice. Dose-dependent changes in AAV5 vector DNA level, miHTT expression and mutant HTT were observed in striatum and cortex of AAV5-miHTT-treated Huntington's disease model mice. This pattern of microRNA expression and mutant HTT lowering rescued weight loss in homozygous Q175FDN mice but did not affect motor or cognitive phenotypes. MRI volumetric analysis detected atrophy in four brain regions in homozygous Q175FDN mice, and treatment with high dose AAV5-miHTT rescued this effect in the hippocampus. Like previous magnetic resonance spectroscopy studies in Huntington's disease patients, decreased total N-acetyl aspartate and increased myo-inositol levels were found in the striatum of homozygous Q175FDN mice. These neurochemical findings were partially reversed with AAV5-miHTT treatment. Striatal transcriptional analysis using RNA sequencing revealed mutant HTT-induced changes that were partially reversed by HTT lowering with AAV5-miHTT. Striatal proton magnetic resonance spectroscopy analysis suggests a restoration of neuronal function, and striatal RNA sequencing analysis shows a reversal of transcriptional dysregulation following AAV5-miHTT in a homozygous Huntington's disease mouse model with severe pathology. The results of this study support the use of magnetic resonance spectroscopy in HTT-lowering clinical trials and strengthen the therapeutic potential of AAV5-miHTT in reversing severe striatal dysfunction in Huntington's disease.


Assuntos
Doença de Huntington , MicroRNAs , Humanos , Animais , Camundongos , Lactente , Doença de Huntington/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Corpo Estriado/metabolismo , Encéfalo/patologia , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Modelos Animais de Doenças
7.
J Biol Chem ; 298(12): 102644, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36309093

RESUMO

Idiopathic pulmonary fibrosis is a progressive and normally fatal disease with limited treatment options. The tyrosine kinase inhibitor nintedanib has recently been approved for the treatment of idiopathic pulmonary fibrosis, and its effectiveness has been linked to its ability to inhibit a number of receptor tyrosine kinases including the platelet-derived growth factor, vascular endothelial growth factor, and fibroblast growth factor receptors. We show here that nintedanib also inhibits salt-inducible kinase 2 (SIK2), with a similar IC50 to its reported tyrosine kinase targets. Nintedanib also inhibited the related kinases SIK1 and SIK3, although with 12-fold and 72-fold higher IC50s, respectively. To investigate if the inhibition of SIK2 may contribute to the effectiveness of nintedanib in treating lung fibrosis, mice with kinase-inactive knockin mutations were tested using a model of bleomycin-induced lung fibrosis. We found that loss of SIK2 activity protects against bleomycin-induced fibrosis, as judged by collagen deposition and histological scoring. Loss of both SIK1 and SIK2 activity had a similar effect to loss of SIK2 activity. Total SIK3 knockout mice have a developmental phenotype making them unsuitable for analysis in this model; however, we determined that conditional knockout of SIK3 in the immune system did not affect bleomycin-induced lung fibrosis. Together, these results suggest that SIK2 is a potential drug target for the treatment of lung fibrosis.


Assuntos
Fibrose Pulmonar Idiopática , Lesão Pulmonar , Animais , Camundongos , Bleomicina , Fibrose , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/genética , Pulmão/metabolismo , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/genética , Lesão Pulmonar/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Modelos Animais de Doenças
8.
FASEB J ; 36(1): e22108, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34939697

RESUMO

Excessive rapid increases in cytosolic free Ca2+ have a clear association with the induction of cancer cell death. Whereas, characterizing the Ca2+ signaling events that occur during the progression of the apoptotic cascade over a period of hours or days, has not yet been possible. Now using genetically encoded Ca2+ indicators complemented with automated epifluorescence microscopy we have shown that staurosporine-induced apoptosis in MDA-MB-231 breast cancer cells was associated with delayed development of cytosolic free Ca2+ fluctuations, which were then maintained for 24 h. These cytosolic free Ca2+ fluctuations were dependent on the Ca2+ channel ORAI1. Silencing of ORAI1, but not its canonical activators STIM1 and STIM2, promoted apoptosis in this model. The pathway for this regulation implicates a mechanism previously associated with the migration of cancer cells involving ORAI1, the chaperone protein SigmaR1, and Ca2+ -activated K+ channels.


Assuntos
Apoptose , Neoplasias da Mama/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Molécula 1 de Interação Estromal/genética
9.
Proc Natl Acad Sci U S A ; 117(34): 20836-20847, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32769205

RESUMO

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane-depolarizing activity is alleviated by coproduction of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologs at the tspA locus, suggestive of additional roles in intraspecies competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologs.


Assuntos
Staphylococcus aureus/metabolismo , Sistemas de Secreção Tipo VII/metabolismo , Animais , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Família Multigênica/genética , Transporte Proteico/genética , Proteômica , Infecções Estafilocócicas/microbiologia , Toxinas Biológicas/metabolismo , Sistemas de Secreção Tipo VII/fisiologia , Virulência/genética , Peixe-Zebra/microbiologia
10.
Brain Behav Immun ; 104: 83-94, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35618227

RESUMO

BACKGROUND: Emotional and behavioral problems (EBP) are common in children. Environmental factors like socioeconomic disadvantage influence EBP pathogenesis and can trigger inflammation. However, the link between early inflammation-EBP in children is unclear. We investigated the associations between i) infant inflammatory biomarkers and subsequent EBP and ii) early life environmental factors and EBP and assessed whether infant inflammation mediated these associations. METHODS: Inflammatory biomarkers glycoprotein acetyls (GlycA) and high-sensitivity C-reactive protein (hsCRP) were quantified at birth and 12 months in a population-derived birth cohort, the Barwon Infant Study. Early life factors including demographic, prenatal, and perinatal factors were collected from antenatal to the two-year period. Internalizing and externalizing problems at age two were measured by the Child Behavior Checklist. Prospective associations were examined by multivariable regression analyses adjusted for potential confounders. Indirect effects of early life factors on EBP through inflammation were identified using mediation analyses. RESULTS: Elevated GlycA levels at birth (GlycAbirth) were associated with greater internalizing problems at age two (ß = 1.32 per SD increase in GlycA; P = 0.001). Inflammation at birth had a stronger magnitude of effect with later EBP than at 12 months. GlycAbirth partially mediated the associations between lower household income (6%), multiparity (12%) and greater number of older siblings (13%) and EBP. Patterns were less evident for hsCRP or externalizing problems. CONCLUSIONS: GlycAbirth was positively associated with EBP at age two and partially mediated the association between several indicators of socioeconomic disadvantage and EBP. Prenatal and perinatal inflammation may be relevant to early neurodevelopment and emotional health.

11.
Brain Behav Immun ; 100: 211-218, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34896180

RESUMO

Poor cognitive outcomes in early childhood predict poor educational outcomes and diminished health over the life course. We sought to investigate (i) whether maternal metabolites predict child cognition, and (ii) if maternal metabolomic profile mediates the relationship between environmental exposures and child cognition. Metabolites were measured using nuclear magnetic resonance-based metabolomics in pregnant women from a population-derived birth cohort. Child cognition was measured at age 2 years. In 662 mother-child pairs, elevated inflammatory markers (ß = -2.62; 95% CI -4.10, -1.15; P = 0.0005) and lower omega-3 fatty acid-related metabolites (ß = 0.49; 95% CI 0.09, 0.88; P = 0.02) in the mother were associated with lower child cognition and partially mediated the association between lower child cognition and multiple risk factors common to socioeconomic disadvantage. Modifying maternal prenatal metabolic pathways related to inflammation and omega-3 fatty acids may offset the adverse associations between prenatal risk factors related to socioeconomic disadvantage and low child cognition.


Assuntos
Ácidos Graxos Ômega-3 , Pré-Escolar , Cognição , Feminino , Humanos , Mães , Gravidez , Fatores Socioeconômicos
12.
Mol Pharm ; 19(6): 1669-1686, 2022 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-35594500

RESUMO

Gene editing mediated by CRISPR/Cas9 systems is due to become a beneficial therapeutic option for treating genetic diseases and some cancers. However, there are challenges in delivering CRISPR components which necessitate sophisticated delivery systems for safe and effective genome editing. Lipid nanoparticles (LNPs) have become an attractive nonviral delivery platform for CRISPR-mediated genome editing due to their low immunogenicity and application flexibility. In this review, we provide a background of CRISPR-mediated gene therapy, as well as LNPs and their applicable characteristics for delivering CRISPR components. We then highlight the challenges of CRISPR delivery, which have driven the significant development of new, safe, and optimized LNP formulations in the past decade. Finally, we discuss considerations for using LNPs to deliver CRISPR and future perspectives on clinical translation of LNP-CRISPR gene editing.


Assuntos
Edição de Genes , Nanopartículas , Sistemas CRISPR-Cas/genética , Técnicas de Transferência de Genes , Lipídeos , Lipossomos
13.
Int J Mol Sci ; 23(11)2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35682546

RESUMO

A remodeling of calcium homeostasis, including calcium influx via store-operated calcium entry (SOCE), is a feature of breast cancers. SOCE is critical to maintain calcium balance in the endoplasmic reticulum calcium store and is an important mechanism for calcium signaling in a variety of cell types, including breast cancer cells. The canonical mechanism of SOCE is stromal interacting molecule 1 (STIM1)-mediated activation of ORAI. Elevated ORAI1 expression is a feature of basal breast cancer cells. However, the role of ORAI1 in the regulation of transcription in breast cancer cells of the basal molecular subtype is still unclear. Using CRISPR-Cas9 gene editing, ORAI1 protein expression was disrupted in MDA-MB-231 and MDA-MB-468 basal breast cancer cells. The ORAI1 wild-type and mutants were reintroduced into ORAI1 knockout cells to study the role of ORAI1 in gene transcriptional regulation. In the absence of calcium store depletion, ORAI1 regulated PTGS2 in MDA-MB-231 cells, and this was dependent on ORAI1 pore function and STIM1 binding. The activation of SOCE by thapsigargin resulted in ORAI1-dependent increases in IL6 transcription in MDA-MB-468 cells; this was also dependent on ORAI1 pore function and STIM1 binding and was associated with the translocation of NFAT1. Given the upregulation of ORAI1 in basal breast cancer cells, our results provide further evidence that ORAI1 may contribute to cancer progression through regulation of gene expression.


Assuntos
Neoplasias da Mama , Cálcio , Neoplasias da Mama/genética , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio da Dieta , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Fatores de Transcrição/metabolismo
14.
Semin Cell Dev Biol ; 94: 74-83, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30439562

RESUMO

The past two decades have seen the identification of important roles for calcium signalling in many of the hallmarks of cancer. One of the cancer types that has been a particular focus of such studies is breast cancer. The breast is intrinsically linked to the calcium ion due to the importance of milk calcium in neonatal growth and development. Indeed, some of the calcium channels and pumps involved in transporting calcium ions into milk also have altered expression in some breast cancers. However, altered expression is not confined to channels and pumps important in lactation, other calcium channels and pumps may also be modulated and may even be specific to breast cancer molecular subtypes. This review considers calcium signalling in the context of breast cancer and provides an overview of the roles that have been attributed to specific regulators of cellular calcium levels in processes relevant to breast cancer progression. Emerging areas in the study of calcium signalling in breast cancer are considered, such as the intersection between calcium signalling, the tumour microenvironment and breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Animais , Neoplasias da Mama/patologia , Feminino , Humanos , Microambiente Tumoral
15.
Int J Mol Sci ; 22(21)2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34768796

RESUMO

Tumors exist in a complex milieu where interaction with their associated microenvironment significantly contributes to disease progression. Cancer-associated fibroblasts (CAFs) are the primary component of the tumor microenvironment and participate in complex bidirectional communication with tumor cells. CAFs support the development of various hallmarks of cancer through diverse processes, including direct cell-cell contact, paracrine signaling, and remodeling and deposition of the extracellular matrix. Calcium signaling is a key second messenger in intra- and inter-cellular signaling pathways that contributes to cancer progression; however, the links between calcium signaling and CAFs are less well-explored. In this review, we put into context the role of calcium signaling in interactions between cancer cells and CAFs, with a focus on migration, proliferation, chemoresistance, and genetic instability.


Assuntos
Sinalização do Cálcio , Fibroblastos/metabolismo , Neoplasias/fisiopatologia , Microambiente Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos
16.
Lab Invest ; 100(2): 224-233, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31243341

RESUMO

The Ca2+ signal is essential in both hypoxia- and epidermal growth factor (EGF)-mediated epithelial to mesenchymal transition (EMT) in MDA-MB-468 breast cancer cells. This finding suggests that Ca2+-permeable ion channels participate in the induction of expression of some mesenchymal markers such as vimentin. However, the ion channels involved in vimentin expression induction have not been fully characterized. This work sought to define how differential modulation of the calcium signal effects the induction of vimentin and the Ca2+ influx pathways involved. We identified that the intracellular Ca2+ chelator EGTA-AM, cytochalasin D (a modulator of cytoskeletal dynamics and cell morphology), and the sarco/endoplasmic reticulum ATPase inhibitor thapsigargin are all inducers of vimentin in MDA-MB-468 breast cancer cells. EGTA-AM- and thapsigargin-mediated induction of vimentin expression in MDA-MB-468 cells involves store-operated Ca2+ entry, as evidenced by sensitivity to silencing of the molecular components of this pathway, STIM1 and ORAI1. In stark contrast, cytochalasin D-mediated vimentin induction was insensitive to silencing of ORAI1, despite sensitivity to silencing of its canonical activator the endoplasmic reticulum Ca2+ sensor STIM1. Cytochalasin D-mediated vimentin induction was, however, sensitive to silencing of another reported STIM1 target, TRPC1. Subsequent studies identified that EGTA-AM-induced vimentin expression also partially involved a TRPC1-dependent pathway. These studies define a complex interplay between vimentin expression in this model and the specific Ca2+-permeable ion channels involved. The complexity in the engagement of different Ca2+ influx pathways that regulate vimentin induction are opportunities but also potential challenges in targeting Ca2+ signaling to block EMT in cancer cells. Our findings further highlight the need to identify potential indispensable ion channels that can regulate induction of specific mesenchymal markers via different stimuli.


Assuntos
Sinalização do Cálcio/fisiologia , Proteína ORAI1/metabolismo , Canais de Cátion TRPC/metabolismo , Vimentina/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Citocalasina D/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Tapsigargina/farmacologia
17.
Biochem Biophys Res Commun ; 522(2): 532-538, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31780263

RESUMO

Triple-negative breast cancers (TNBC) are often associated with high relapse rates, despite treatment with chemotherapy agents such as doxorubicin. A better understanding of the signaling and molecular changes associated with doxorubicin may provide novel insights into strategies to enhance treatment efficacy. Calcium signaling is involved in many pathways influencing the efficacy of chemotherapy agents such as proliferation and cell death. However, there are a limited number of studies exploring the effect of doxorubicin on calcium signaling in TNBC. In this study, MDA-MB-231 triple-negative, basal breast cancer cells stably expressing the genetically-encoded calcium indicator GCaMP6m (GCaMP6m-MDA-MB-231) were used to define alterations in calcium signaling. The effects of doxorubicin in GCaMP6m-MDA-MB-231 cells were determined using live cell imaging and fluorescence microscopy. Changes in mRNA levels of specific calcium regulating proteins as a result of doxorubicin treatment were also assessed using real time qPCR. Doxorubicin (1 µM) produced alterations in intracellular calcium signaling, including enhancing the sensitivity of MDA-MB-231 cells to ATP stimulation and prolonging the recovery time after store-operated calcium entry. Upregulation in mRNA levels of ORAI1, TRPC1, SERCA1, IP3R2 and PMCA2 with doxorubicin 1 µM treatment was also observed. Doxorubicin treatment is associated with specific remodeling in calcium signaling in MDA-MB-231 cells, with associated changes in mRNA levels of specific calcium-regulating proteins.


Assuntos
Neoplasias da Mama/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Doxorrubicina/farmacologia , Proteínas de Neoplasias/metabolismo , Trifosfato de Adenosina/farmacologia , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Feminino , Homeostase/efeitos dos fármacos , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos
18.
Proc Natl Acad Sci U S A ; 114(21): E4223-E4232, 2017 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-28484017

RESUMO

Rational modulation of the immune response with biologics represents one of the most promising and active areas for the realization of new therapeutic strategies. In particular, the use of function blocking monoclonal antibodies targeting checkpoint inhibitors such as CTLA-4 and PD-1 have proven to be highly effective for the systemic activation of the human immune system to treat a wide range of cancers. Ipilimumab is a fully human antibody targeting CTLA-4 that received FDA approval for the treatment of metastatic melanoma in 2011. Ipilimumab is the first-in-class immunotherapeutic for blockade of CTLA-4 and significantly benefits overall survival of patients with metastatic melanoma. Understanding the chemical and physical determinants recognized by these mAbs provides direct insight into the mechanisms of pathway blockade, the organization of the antigen-antibody complexes at the cell surface, and opportunities to further engineer affinity and selectivity. Here, we report the 3.0 Å resolution X-ray crystal structure of the complex formed by ipilimumab with its human CTLA-4 target. This structure reveals that ipilimumab contacts the front ß-sheet of CTLA-4 and intersects with the CTLA-4:Β7 recognition surface, indicating that direct steric overlap between ipilimumab and the B7 ligands is a major mechanistic contributor to ipilimumab function. The crystallographically observed binding interface was confirmed by a comprehensive cell-based binding assay against a library of CTLA-4 mutants and by direct biochemical approaches. This structure also highlights determinants responsible for the selectivity exhibited by ipilimumab toward CTLA-4 relative to the homologous and functionally related CD28.


Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Antineoplásicos Imunológicos/farmacologia , Sítios de Ligação de Anticorpos/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Ipilimumab/farmacologia , Melanoma/tratamento farmacológico , Fatores Biológicos/farmacologia , Antígeno CTLA-4/imunologia , Linhagem Celular , Cristalografia por Raios X , Células HEK293 , Humanos , Imunoterapia/métodos , Ligação Proteica , Estrutura Terciária de Proteína
19.
Int J Mol Sci ; 21(24)2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33322037

RESUMO

Epithelial to mesenchymal transition (EMT) in cancer is important in therapeutic resistance and invasiveness. Calcium signaling is key to the induction of EMT in breast cancer cells. Although inhibition of specific calcium-permeable ion channels regulates the induction of a sub-set of EMT markers in breast cancer cells, it is still unclear if activation of a specific calcium channel can be a driver for the induction of EMT events. In this study, we exploited the availability of a selective pharmacological activator of the calcium-permeable ion channel TRPV4 to assess the direct role of calcium influx in EMT marker induction. Gene association studies revealed a link between TRPV4 and gene-ontologies associated with EMT and poorer relapse-free survival in lymph node-positive basal breast cancers. TRPV4 was an important component of the calcium influx phase induced in MDA-MB-468 breast cancer cells by the EMT inducer epidermal growth factor (EGF). Pharmacological activation of TRPV4 then drove the induction of a variety of EMT markers in breast cancer cells. These studies demonstrate that calcium influx through specific pathways appears to be sufficient to trigger EMT events.


Assuntos
Neoplasias da Mama/genética , Transição Epitelial-Mesenquimal , Canais de Cátion TRPV/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Sulfonamidas/farmacologia , Análise de Sobrevida , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/genética
20.
Evol Comput ; 28(4): 621-641, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101026

RESUMO

Connection patterns among Local Optima Networks (LONs) can inform heuristic design for optimisation. LON research has predominantly required complete enumeration of a fitness landscape, thereby restricting analysis to problems diminutive in size compared to real-life situations. LON sampling algorithms are therefore important. In this article, we study LON construction algorithms for the Quadratic Assignment Problem (QAP). Using machine learning, we use estimated LON features to predict search performance for competitive heuristics used in the QAP domain. The results show that by using random forest regression, LON construction algorithms produce fitness landscape features which can explain almost all search variance. We find that LON samples better relate to search than enumerated LONs do. The importance of fitness levels of sampled LONs in search predictions is crystallised. Features from LONs produced by different algorithms are combined in predictions for the first time, with promising results for this "super-sampling": a model to predict tabu search success explained 99% of variance. Arguments are made for the use-case of each LON algorithm and for combining the exploitative process of one with the exploratory optimisation of the other.


Assuntos
Algoritmos , Heurística , Simulação por Computador , Humanos , Modelos Lineares , Cadeias de Markov , Estudos de Amostragem , Ferramenta de Busca
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