ABSTRACT
BACKGROUND: Bone tissue homeostasis relies on the coordinated activity of the bone-forming osteoblasts and bone-resorbing osteoclasts. Osteomesopyknosis is considered a distinctive rare sclerosing skeletal disorder of unelucidated pathophysiology and presumably autosomal dominant transmission. However, the causal genes are unknown. METHODS: We present a case report encompassing clinical assessments, imaging studies, and whole-exome sequencing analysis, complemented by functional in vitro experiments. RESULTS: This new case of osteomesopyknosis was associated with a missense ALOX5 variant predicted to induce protein misfolding and proteasomal degradation. Transfection experiments demonstrated that the variant was associated with reduced protein levels restored by proteasomal inhibition with bortezomib. Likewise, gene expression analysis showed that the mutated gene was associated with a decreased RANKL/OPG ratio, which is a critical driver of osteoclast precursor differentiation. CONCLUSION: Our data indicate impaired bone resorption as the underlying mechanism of this rare osteosclerosis, implicating ALOX5 pathogenic variants as potential etiological factors.
Subject(s)
Arachidonate 5-Lipoxygenase , Mutation, Missense , RANK Ligand , Female , Humans , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Osteoclasts/metabolism , Osteoclasts/pathology , Osteosclerosis/genetics , Osteosclerosis/pathology , Osteosclerosis/metabolism , RANK Ligand/metabolism , RANK Ligand/genetics , Signal Transduction , Middle AgedABSTRACT
We have previously shown that the transmembrane protein ODZ1 promotes cytoskeletal remodeling of glioblastoma (GBM) cells and invasion of the surrounding parenchyma through the activation of a RhoA-ROCK pathway. We also described that GBM cells can control the expression of ODZ1 through transcriptional mechanisms triggered by the binding of IL-6 to its receptor and a hypoxic environment. Epidermal growth factor (EGF) plays a key role in the invasive capacity of GBM. However, the molecular mechanisms that enable tumor cells to acquire the morphological changes to migrate out from the tumor core have not been fully characterized. Here, we show that EGF is able to induce the expression of ODZ1 in primary GBM cells. We analyzed the levels of the EGF receptor (EGFR) in 20 GBM primary cell lines and found expression in 19 of them by flow cytometry. We selected two cell lines that do or do not express the EGFR and found that EGFR-expressing cells responded to the EGF ligand by increasing ODZ1 at the mRNA and protein levels. Moreover, blockade of EGF-EGFR binding by Cetuximab, inhibition of the p38 MAPK pathway, or Additionally, the siRNA-mediated knockdown of MAPK11 (p38ß MAPK) reduced the induction of ODZ1 in response to EGF. Overall, we show that EGF may activate an EGFR-mediated signaling pathway through p38ß MAPK, to upregulate the invasion factor ODZ1, which may initiate morphological changes for tumor cells to invade the surrounding parenchyma. These data identify a new candidate of the EGF-EGFR pathway for novel therapeutic approaches.
Subject(s)
Epidermal Growth Factor , ErbB Receptors , Glioblastoma , Tenascin , Up-Regulation , Humans , Cell Line, Tumor , Epidermal Growth Factor/pharmacology , Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Glioblastoma/pathology , Glioblastoma/metabolism , Glioblastoma/genetics , Neoplasm Invasiveness , Signal Transduction/drug effects , Up-Regulation/drug effects , Up-Regulation/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Tenascin/genetics , Tenascin/metabolismABSTRACT
In glioblastoma (GBM) patients, maximal safe resection remains a challenge today due to its invasiveness and diffuse parenchymal infiltration. In this context, plasmonic biosensors could potentially help to discriminate tumor tissue from peritumoral parenchyma based on differences in their optical properties. A nanostructured gold biosensor was used ex vivo to identify tumor tissue in a prospective series of 35 GBM patients who underwent surgical treatment. For each patient, two paired samples, tumor and peritumoral tissue, were extracted. Then, the imprint left by each sample on the surface of the biosensor was individually analyzed, obtaining the difference between their refractive indices. The tumor and non-tumor origins of each tissue were assessed by histopathological analysis. The refractive index (RI) values obtained by analyzing the imprint of the tissue were significantly lower (p = 0.0047) in the peritumoral samples (1.341, Interquartile Range (IQR) 1.339-1.349) compared with the tumor samples (1.350, IQR 1.344-1.363). The ROC (receiver operating characteristic) curve showed the capacity of the biosensor to discriminate between both tissues (area under the curve, 0.8779, p < 0.0001). The Youden index provided an optimal RI cut-off point of 0.003. The sensitivity and specificity of the biosensor were 81% and 80%, respectively. Overall, the plasmonic-based nanostructured biosensor is a label-free system with the potential to be used for real-time intraoperative discrimination between tumor and peritumoral tissue in patients with GBM.
Subject(s)
Biosensing Techniques , Brain Neoplasms , Glioblastoma , Humans , Glioblastoma/diagnosis , Glioblastoma/pathology , Brain Neoplasms/diagnosis , Sensitivity and Specificity , ROC CurveABSTRACT
Two key features of cancer cells are sustained proliferation and invasion, which is preceded by a modification of the adhesion properties to the extracellular matrix. Currently, fluorescence-based techniques are mainly used to detect these processes, including flow cytometry and fluorescence resonance energy transfer (FRET) microscopy. We have previously described a simple, fast and label-free method based on a gold nanohole array biosensor to detect the spectral response of single cells, which is highly dependent on the actin cortex. Here we used this biosensor to study two cellular processes where configuration of the actin cortex plays an essential role: cell cycle and cell-matrix adhesion. Colorectal cancer cells were maintained in culture under different conditions to obtain cells stopped either in G0/G1 (resting cells/cells at the initial steps of cell growth) or G2 (cells undergoing division) phases of the cell cycle. Data from the nanohole array biosensor showed an ability to discriminate between both cell populations. Additionally, cancer cells were monitored with the biosensor during the first 60 min after cells were deposited onto a biosensor coated with fibronectin, an extracellular matrix protein. Spectral changes were detected in the first 20 min and increased over time as the cell-biosensor contact surface increased. Our data show that the nanohole array biosensor provides a label-free and real-time procedure to detect cells undergoing division or changes in cell-matrix interaction in both clinical and research settings.
Subject(s)
Biosensing Techniques , Neoplasms , Actins , Biosensing Techniques/methods , Cell Division , Fibronectins , GoldABSTRACT
We present a family with a rare mutation of the LRP6 gene and for the first time provide evidence for its association with low bone mineral density. INTRODUCTION: The Wnt pathway plays a critical role in bone homeostasis. Pathogenic variants of the Wnt co-receptor LRP6 have been associated with abnormal skeletal phenotypes or increased risk of cardiovascular events. PATIENT AND METHODS: Here we report an index premenopausal patient and her family carrying a rare missense LRP6 pathogenic variant (rs141212743; 0.0002 frequency among Europeans). This variant has been previously associated with metabolic syndrome and atherosclerosis, in the presence of normal bone mineral density. However, the LRP6 variant was associated with low bone mineral density in this family, without evidence for association with serum lipid levels or cardiovascular events. CONCLUSION: Thus, this novel association shows that LRP6 pathogenic variants may be involved in some cases of early-onset osteoporosis, but the predominant effect, either skeletal or cardiovascular, may vary depending on the genetic background or other acquired factors.
Subject(s)
Bone Diseases, Metabolic , Low Density Lipoprotein Receptor-Related Protein-6 , Bone Density/genetics , Female , Humans , Lipids , Low Density Lipoprotein Receptor-Related Protein-6/genetics , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Mutation , Wnt Signaling PathwayABSTRACT
Glioblastoma (GBM) is one of the most aggressive cancers, with dismal prognosis despite continuous efforts to improve treatment. Poor prognosis is mostly due to the invasive nature of GBM. Thus, most research has focused on studying the molecular players involved in GBM cell migration and invasion of the surrounding parenchyma, trying to identify effective therapeutic targets against this lethal cancer. Our laboratory discovered the implication of TENM1, also known as ODZ1, in GBM cell migration in vitro and in tumor invasion using different in vivo models. Moreover, we investigated the microenvironmental stimuli that promote the expression of TENM1 in GBM cells and found that macrophage-secreted IL-6 and the extracellular matrix component fibronectin upregulated TENM1 through activation of Stat3. We also described that hypoxia, a common feature of GBM tumors, was able to induce TENM1 by both an epigenetic mechanism and a HIF2α-mediated transcriptional pathway. The fact that TENM1 is a convergence point for various cancer-related signaling pathways might give us a new therapeutic opportunity for GBM treatment. Here, we briefly review the findings described so far about the mechanisms that control the expression of the GBM invasion factor TENM1.
ABSTRACT
R-loops are three-stranded nucleic acid structures that accumulate on chromatin in neurological diseases and cancers and contribute to genome instability. Using a proximity-dependent labeling system, we identified distinct classes of proteins that regulate R-loops in vivo through different mechanisms. We show that ATRX suppresses R-loops by interacting with RNAs and preventing R-loop formation. Our proteomics screen also discovered an unexpected enrichment for proteins containing zinc fingers and homeodomains. One of the most consistently enriched proteins was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP resolves R-loops in vitro and that it is necessary to suppress R-loops in vivo at its genomic targets. Furthermore, deletion of the ADNP homeodomain severely diminishes R-loop resolution activity in vitro, results in R-loop accumulation at ADNP targets, and compromises neuronal differentiation. Notably, patient-derived human induced pluripotent stem cells that contain an ADNP syndrome-causing mutation exhibit R-loop and CTCF accumulation at ADNP targets. Our findings point to a specific role for ADNP-mediated R-loop resolution in physiological and pathological neuronal function and, more broadly, to a role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers.
Subject(s)
Proteomics , R-Loop Structures/physiology , RNA/metabolism , Animals , Cell Differentiation , Chromatin , Embryonic Stem Cells , Genomic Instability , HEK293 Cells , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Induced Pluripotent Stem Cells , Mice , Mutation , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/metabolism , R-Loop Structures/genetics , Zinc FingersABSTRACT
Background: Glioblastoma (GBM) remains a major clinical challenge due to its invasive capacity, resistance to treatment, and recurrence. We have previously shown that ODZ1 contributes to glioblastoma invasion and that ODZ1 mRNA levels can be upregulated by epigenetic mechanisms in response to hypoxia. Herein, we have further studied the transcriptional regulation of ODZ1 in GBM stem cells (GSCs) under hypoxic conditions and analyzed whether HIF2α has any role in this regulation. Methods: We performed the experiments in three primary GSC cell lines established from tumor specimens. GSCs were cultured under hypoxia, treated with HIF regulators (DMOG, chetomin), or transfected with specific siRNAs, and the expression levels of ODZ1 and HIF2α were analyzed. In addition, the response of the ODZ1 promoter cloned into a luciferase reporter plasmid to the activation of HIF was also studied. Results: The upregulation of both mRNA and protein levels of HIF2α under hypoxia conditions correlated with the expression of ODZ1 mRNA. Moreover, the knockdown of HIF2α by siRNAs downregulated the expression of ODZ1. We found, in the ODZ1 promoter, a HIF consensus binding site (GCGTG) 1358 bp from the transcription start site (TSS) and a HIF-like site (CCGTG) 826 bp from the TSS. Luciferase assays revealed that the stabilization of HIF by DMOG resulted in the increased activity of the ODZ1 promoter. Conclusions: Our data indicate that the HIF2α-mediated upregulation of ODZ1 helps strengthen the transcriptional control of this migration factor under hypoxia in glioblastoma stem cells. The discovery of this novel transcriptional pathway identifies new targets to develop strategies that may avoid GBM tumor invasion and recurrence.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic , Glioblastoma/etiology , Glioblastoma/metabolism , Neoplastic Stem Cells/metabolism , Nerve Tissue Proteins/genetics , Tenascin/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Hypoxia , Cell Line, Tumor , Gene Knockdown Techniques , Glioblastoma/pathology , Humans , Neoplastic Stem Cells/pathology , Nerve Tissue Proteins/metabolism , RNA, Messenger/genetics , Tenascin/metabolismABSTRACT
We have previously shown that the transmembrane protein ODZ1 serves for glioblastoma (GBM) cells to invade the surrounding tissue through activation of RhoA/ROCK pathway. However, the transcriptional machinery used by GBM cells to regulate the expression of ODZ1 is unknown. Here we show that interaction with tumor microenvironment elements, mainly activated monocytes through IL-6 secretion, and the extracellular matrix protein fibronectin, induces the Stat3 transcriptional pathway and upregulates ODZ1 which results in GBM cell migration. This signaling route is abrogated by blocking the IL-6 receptor, inhibiting Jak kinases or knocking down Stat3. Furthermore, we have identified a Stat3 responsive element in the ODZ1 gene promoter, about 1 kb from the transcription start site. Luciferase-reporter assays confirmed that the promoter responds to the presence of monocytic cells and this activation is greatly reduced when the Stat3 site is mutated or following treatment with a neutralizing anti-IL-6 receptor antibody or transfecting GBM cells with a dominant negative variant of Stat3. Overall, we show that monocyte-secreted IL-6 and the extracellular matrix protein fibronectin activate the axis Stat3-ODZ1 and promote migration of GBM cells. This is the first described transcriptional mechanism used by tumor cells to promote the expression of the invasion factor ODZ1.
Subject(s)
Gene Expression Regulation, Neoplastic , Glioblastoma/pathology , Interleukin-6/metabolism , Nerve Tissue Proteins/metabolism , STAT3 Transcription Factor/metabolism , Tenascin/metabolism , Transcriptional Activation , Tumor Microenvironment , Cell Movement , Glioblastoma/genetics , Glioblastoma/immunology , Glioblastoma/metabolism , Humans , Nerve Tissue Proteins/genetics , Promoter Regions, Genetic , Receptors, Interleukin-6/metabolism , STAT3 Transcription Factor/genetics , Signal Transduction , Tenascin/genetics , Tumor Cells, CulturedABSTRACT
The transmembrane protein ODZ1 has been associated with the invasive capacity of glioblastoma (GBM) cells through upregulation of RhoA/ROCK signaling, but the mechanisms triggering the ODZ1 pathway remain elusive. In addition, it is widely accepted that hypoxia is one of the main biological hallmarks of the GBM microenvironment and it is associated with treatment resistance and poor prognosis. Here we show that hypoxic tumor regions express higher levels of ODZ1 and that hypoxia induces ODZ1 expression in GBM cells by regulating the methylation status of the ODZ1 promoter. Hypoxia-induced upregulation of ODZ1 correlates with higher migration capacity of GBM cells that is drastically reduced by knocking down ODZ1. In vitro methylation of the promoter decreases its transactivation activity and we found a functionally active CpG site at the 3'end of the promoter. This site is hypermethylated in somatic neural cells and mainly hypomethylated in GBM cells. Mutagenesis of this CpG site reduces the promoter activity in response to hypoxia. Overall, we identify hypoxia as the first extracellular activator of ODZ1 expression and describe that hypoxia controls the levels of this migration-inducer, at least in part, by regulating the methylation status of the ODZ1 gene promoter.
ABSTRACT
The invasive capacity of GBM is one of the key tumoral features associated with treatment resistance, recurrence, and poor overall survival. The molecular machinery underlying GBM invasiveness comprises an intricate network of signaling pathways and interactions with the extracellular matrix and host cells. Among them, PI3k/Akt, Wnt, Hedgehog, and NFkB play a crucial role in the cellular processes related to invasion. A better understanding of these pathways could potentially help in developing new therapeutic approaches with better outcomes. Nevertheless, despite significant advances made over the last decade on these molecular and cellular mechanisms, they have not been translated into the clinical practice. Moreover, targeting the infiltrative tumor and its significance regarding outcome is still a major clinical challenge. For instance, the pre- and intraoperative methods used to identify the infiltrative tumor are limited when trying to accurately define the tumor boundaries and the burden of tumor cells in the infiltrated parenchyma. Besides, the impact of treating the infiltrative tumor remains unclear. Here we aim to highlight the molecular and clinical hallmarks of invasion in GBM.
ABSTRACT
ADNP syndrome is an intellectual disability associated with Autism spectrum disorder caused by mutations in ADNP. We generated an iPSC line from an ADNP syndrome pediatric patient harboring the mutation p.Trp719* (GENYOi004-A). Peripheral blood mononuclear cells were reprogrammed using a non-transmissible form of Sendai viruses expressing the four Yamanaka factors (Oct3/4, SOX2, KLF4 and c-MYC). Characterization of GENYOi004-A included mutation analysis of ADNP by allele-specific PCR, genetic identity by Short Tandem Repeats polymorphism profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and pluripotency studies in vivo. GENYOi004-A will be useful to evaluate ADNP syndrome alterations at early developmental stages.
Subject(s)
Autism Spectrum Disorder/genetics , Cell Differentiation , Homeodomain Proteins/genetics , Induced Pluripotent Stem Cells/pathology , Leukocytes, Mononuclear/pathology , Mutation , Nerve Tissue Proteins/genetics , Teratoma/etiology , Animals , Autism Spectrum Disorder/pathology , Cells, Cultured , Cellular Reprogramming , Child , Female , Humans , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Leukocytes, Mononuclear/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Phenotype , Teratoma/pathologyABSTRACT
Mutations in ADNP have been recently associated with intellectual disability and autism spectrum disorder. However, the clinical features of patients with this syndrome are not fully identified, and no treatment currently exists for these patients. Here, we extended the ADNP syndrome phenotype describing skin abnormalities in both a patient with ADNP syndrome and an Adnp haploinsufficient mice. The patient displayed thin dermis, hyperkeratotic lesions in periarticular areas and delayed wound healing. Patient-derived skin keratinocytes showed reduced proliferation and increased differentiation. Additionally, detection of cell cycle markers indicated that mutant cells exhibited impaired cell cycle progression. Treatment of ADNP-deficient keratinocytes with the ADNP-derived NAP peptide significantly reduced the expression of differentiation markers. Sonography and immunofluorescence staining of epidermal layers revealed that the dermis was thinner in the patient than in a healthy control. Adnp haploinsufficient mice (Adnp+/-) mimicked the human condition showing reduced dermal thickness. Intranasal administration of NAP significantly increased dermal thickness and normalized the levels of cell cycle and differentiation markers. Our observations provide a novel activity of the autism-linked ADNP in the skin that may serve to define the clinical phenotype of patients with ADNP syndrome and provide an attractive therapeutic option for skin alterations in these patients.
Subject(s)
Autism Spectrum Disorder/genetics , Homeodomain Proteins/genetics , Nerve Tissue Proteins/genetics , Skin Abnormalities/genetics , Animals , Autism Spectrum Disorder/pathology , Cell Cycle/genetics , Cell Differentiation/genetics , Disease Models, Animal , Haploinsufficiency/genetics , Humans , Intellectual Disability/genetics , Intellectual Disability/pathology , Keratinocytes/metabolism , Mice , Mice, Transgenic , Mutation/genetics , Skin Abnormalities/pathology , Wound Healing/geneticsABSTRACT
We have previously described that the NFκB pathway is upregulated during differentiation of glioblastoma stem-like cells (GSCs) which keeps differentiating GSCs in a proliferative astrocytic precursor state. However, extracellular signals and cellular mediators of this pathway are not clear yet. Here, we show that TLR4 is a key factor to promote NFκB activation in differentiating GSCs. TLR4 is upregulated during differentiation of GSCs and promotes transcriptional activation of NFκB as determined by luciferase-reporter assays and expression of NFκB target genes. Downregulation of TLR4 by shRNAs or blockade with anti-TLR4 specific antibodies drastically inhibited NFκB activity which promoted further differentiation and reduced proliferation of GSCs. We found that hyaluronic acid (HA), a main component of brain extracellular matrix, triggers the TLR4-NFκB pathway in differentiating GSCs. Moreover, HA is synthesized and released by GSCs undergoing differentiation and leads to transcriptional activation of NFκB, which is inhibited following downregulation of TLR4 or blockade of HA synthesis. Thus, we have demonstrated that during the process of differentiation, GSCs upregulate TLR4 and release the TLR4 ligand HA, which activates the TLR4-NFκB signaling pathway. This strategy may efficiently be used by differentiating GSCs to maintain their proliferative potential and consequently their tumorigenic capacity.
Subject(s)
NF-kappa B/metabolism , Neoplastic Stem Cells/metabolism , Toll-Like Receptor 4/metabolism , Brain Neoplasms/metabolism , Cell Differentiation/genetics , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic/genetics , Glioblastoma/metabolism , Humans , Hyaluronic Acid/metabolism , Hyaluronic Acid/physiology , Neoplastic Stem Cells/physiology , Signal Transduction/genetics , Toll-Like Receptor 4/physiologyABSTRACT
Rheumatoid arthritis (RA) is a systemic autoimmune disease whose main feature is persistent joint inflammation. Toll-like receptors (TLRs) play critical roles in the activation of innate and adaptive immune responses, and influence the activity of NFκB, a key player in chronic inflammation. We aimed at investigating the association of TLR allelic variants with susceptibility and severity of RA through a systematic, high-throughput, analysis of TLR genes. All coding exons and flanking regions of nine members of the TLR family (TLR1-9) were analyzed in 66 patients with RA and 30 healthy controls by next generation sequencing. We focussed on three single allelic variants, N248S in TLR1, Q11L in TLR7 and M1V in TLR8 based on the allelic frequencies in both patient and control populations, the predicted impact on protein function and the novelty in RA research. Analysis of these selected variants in a larger cohort of 402 patients with RA and in 208 controls revealed no association with susceptibility. However, the M1V allele was associated with a lower need for disease-modifying antirheumatic drugs (DMARDs) (p=0.008) and biologic treatments (p=0.021). Functional studies showed that the M1V variant leads to a reduced production of inflammatory cytokines, IL-1ß, IL-6 and TNFα, in response to TLR8 agonists. Thus, the presence of this variant confers a significant protective effect on disease severity. These results show for the first time the association between the M1V variant of TLR8 and reduced disease severity in RA, which could have prognostic value for these patients.
Subject(s)
Alleles , Gene Frequency , Rheumatic Fever/genetics , Toll-Like Receptors/genetics , Aged , Aged, 80 and over , Antirheumatic Agents/administration & dosage , Cytokines/genetics , Cytokines/immunology , Female , Genetic Markers , Humans , Male , Middle Aged , Rheumatic Fever/drug therapy , Rheumatic Fever/immunology , Toll-Like Receptors/immunologyABSTRACT
BACKGROUND: Toll-like receptor (TLR) family members are key players in inflammation. TLR10 has been poorly studied in chronic inflammatory disorders, and its clinical relevance in rheumatoid arthritis (RA) is as yet unknown. We aimed at identifying TLR10 variants within all coding regions of the gene in patients with RA as well as studying their functional and clinical significance. METHODS: TLR10 gene variants were studied by performing sequencing of 66 patients with RA and 30 control subjects. A selected variant, I473T, was then analyzed in 1654 patients and 1702 healthy control subjects. The capacity of this TLR10 variant to modify the transcriptional activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) was determined by using a luciferase reporter assay and analyzing the expression of NFkB target genes by quantitative polymerase chain reaction. Differences between groups were analyzed by using the Mann-Whitney U test and the unpaired two-tailed Student's t test. RESULTS: We detected ten missense variants in the TLR10 gene and focused on the I473T substitution based on allele frequencies and the predicted functional impact. I473T variant is not associated with susceptibility to RA, but it significantly correlates with erosive disease in patients seropositive for antibodies to citrullinated protein antigens (p = 0.017 in the total cohort and p = 0.0049 in female patients) and with a lower response to infliximab treatment as measured by the change in Disease Activity Score in 28 joints (p = 0.012) and by the European League Against Rheumatism criteria (p = 0.049). Functional studies showed that TLR10 reduced activation of the NFkB inflammatory pathway in hematopoietic cells, whereas the I473T variant lacked this inhibitory capacity. Consistently, after exposure to infliximab, cells expressing the I437T variant showed higher NFkB activity than cells carrying wild-type TLR10. CONCLUSIONS: A TLR10 allelic variant, I473T, has impaired NFkB inhibitory activity and is highly associated with disease severity and low response to infliximab in patients with RA.
Subject(s)
Arthritis, Rheumatoid/genetics , Drug Resistance/genetics , NF-kappa B/immunology , Toll-Like Receptor 10/genetics , Aged , Antirheumatic Agents/therapeutic use , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/immunology , Female , Gene Expression Regulation/immunology , Genotype , High-Throughput Nucleotide Sequencing , Humans , Infliximab/therapeutic use , Male , Middle Aged , NF-kappa B/biosynthesis , Polymorphism, Single Nucleotide , Prognosis , Real-Time Polymerase Chain Reaction , Toll-Like Receptor 10/immunologyABSTRACT
Loss of the regulatory mechanisms that avoid excessive or constitutive activation of NF-κB may be associated with chronic inflammatory disorders, including rheumatoid arthritis (RA). After massive sequencing of 158 regulators of the NF-κB pathway in RA patients, we focused on a scarcely known gene, ASCC1, and showed that it potently inhibits the expression of NF-κB target genes (TRAIL, TNF-α, cIAP-1, IL8) and blocks activation of a NF-κB-luciferase reporter construct in five different human cell lines. Therefore, ASCC1 may contribute to avoiding a pathologic activation of this transcription factor. A truncated variant of ASCC1 (p.S78*) was found in RA patients and control individuals. Functional in vitro studies revealed that truncation abrogated the NF-κB inhibition capacity of ASCC1. In contrast with full-length protein, truncated ASCC1 did not reduce the transcriptional activation of NF-κB and the secretion of TNF-α in response to inflammatory stimuli. We analyzed the clinical impact of p.S78* variant in 433 patients with RA and found that heterozygous carriers of this variant needed more disease-modifying antirheumatic drugs, and more patients with this genotype needed treatment with corticoids and biologic agents. Moreover, the truncated allele-carrier group had lower rates of remission compared with the full-length variant carriers. Overall, our findings show for the first time, to our knowledge, that ASCC1 inhibits NF-κB activation and that a truncated and inactive variant of ASCC1 is associated with a more severe disease, which could have clinical value for assessing the progression and prognosis of RA.
Subject(s)
Arthritis, Rheumatoid/pathology , Carrier Proteins/physiology , NF-kappa B/antagonists & inhibitors , Transcriptional Activation/genetics , Aged , Antirheumatic Agents/therapeutic use , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/genetics , Base Sequence , Carrier Proteins/genetics , Cell Line, Tumor , Enzyme Activation , Female , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , Inhibitor of Apoptosis Proteins/biosynthesis , Interleukin-8/biosynthesis , MCF-7 Cells , Male , NF-kappa B/metabolism , Sequence Analysis, DNA , Signal Transduction/genetics , TNF-Related Apoptosis-Inducing Ligand/biosynthesis , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
Inhibiting cancer cell migration and infiltration to other tissues makes the difference between life and death. Multiwalled carbon nanotubes (MWCNTs) display intrinsic biomimetic properties with microtubules, severely interfering with the function of these protein filaments during cell proliferation, triggering cell death. Here it is shown MWCNTs disrupt the centrosomal microtubule cytoskeletal organization triggering potent antimigratory effects in different cancer cells.
Subject(s)
Biocompatible Materials/chemistry , Nanotubes, Carbon/chemistry , Biocompatible Materials/toxicity , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , HeLa Cells , Humans , MCF-7 Cells , Microscopy, Confocal , Microtubules , Nanotubes, Carbon/toxicity , Neoplasms/metabolism , Neoplasms/pathology , Spectrum Analysis, RamanABSTRACT
Sunitinib, an inhibitor of kinases, including VEGFR and platelet-derived growth factor receptor (PDGFR), efficiently induces apoptosis in vitro in glioblastoma (GBM) cells, but does not show any survival benefit in vivo. One detrimental aspect of current in vitro models is that they do not take into account the contribution of extrinsic factors to the cellular response to drug treatment. Here, we studied the effects of substrate properties including elasticity, dimensionality, and matrix composition on the response of GBM stem-like cells (GSC) to chemotherapeutic agents. Thirty-seven cell cultures, including GSCs, parenchymal GBM cells, and GBM cell lines, were treated with nine antitumor compounds. Contrary to the expected chemoresistance of GSCs, these cells were more sensitive to most agents than GBM parenchymal cells or GBM cell lines cultured on flat (two-dimensional; 2D) plastic or collagen-coated surfaces. However, GSCs cultured in collagen-based three-dimensional (3D) environments increased their resistance, particularly to receptor tyrosine kinase inhibitors, such as sunitinib, BIBF1120, and imatinib. Differences in substrate rigidity or matrix components did not modify the response of GSCs to the inhibitors. Moreover, the MEK-ERK and PI3K-Akt pathways, but not PDGFR, mediate at least in part, this dimensionality-dependent chemoresistance. These findings suggest that survival of GSCs on 2D substrates, but not in a 3D environment, relies on kinases that can be efficiently targeted by sunitinib-like inhibitors. Overall, our data may help explain the lack of correlation between in vitro and in vivo models used to study the therapeutic potential of kinase inhibitors, and provide a rationale for developing more robust drug screening models.
Subject(s)
Antineoplastic Agents/administration & dosage , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Glioblastoma/drug therapy , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Enzyme Inhibitors/administration & dosage , Glioblastoma/pathology , Humans , In Vitro Techniques , Neoplastic Stem Cells/drug effects , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: The hereditary hemorrhagic telangiectasia syndrome (HHT), also known as the Rendu-Osler-Weber syndrome is a multiorganic vascular disorder inherited as an autosomal dominant trait. Diagnostic clinical criteria include: epistaxis, telangiectases in mucocutaneous and gastrointestinal sites, arteriovenous malformations (AVMs) most commonly found in pulmonary, hepatic and cerebral circulations, and familial inheritance. HHT is transmitted in 90% of the cases as an autosomal dominant condition due to mutations in either endoglin (ENG), or activin receptor-like kinase 1 (ACVRL1/ALK1) genes (HHT type 1 and 2, respectively). METHODS: We have carried out a genetic analysis of four independent Spanish families with HHT clinical criteria, which has permitted the identification of new large deletions in ENG. These mutations were first detected using the MLPA technique and subsequently, the deletion breakpoints were mapped using a customized copy number variation (CNV) microarray. The array was designed to cover the ENG gene and surrounding areas. RESULTS: All tested families carried large deletions ranging from 3-kb to 100-kb, involving the ENG gene promoter, several ENG exons, and the two downstream genes FGSH and CDK9. Interestingly, common breakpoints coincident with Alu repetitive sequences were found among these families. CONCLUSIONS: The systematic hybridization of DNA from HHT families, with deletions or duplications, to custom designed microarrays, could allow the mapping of breakpoints, coincident with repetitive Alu sequences that might act as "hot spots" in the development of chromosomal anomalies.