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INTRODUCTION: Variants of uncertain significance (VUS) surged with affordable genetic testing, posing challenges for determining pathogenicity. We examine the pathogenicity of a novel VUS P93S in Annexin A11 (ANXA11) - an amyotrophic lateral sclerosis/frontotemporal dementia-associated gene - in a corticobasal syndrome kindred. Established ANXA11 mutations cause ANXA11 aggregation, altered lysosomal-RNA granule co-trafficking, and transactive response DNA binding protein of 43 kDa (TDP-43) mis-localization. METHODS: We described the clinical presentation and explored the phenotypic diversity of ANXA11 variants. P93S's effect on ANXA11 function and TDP-43 biology was characterized in induced pluripotent stem cell-derived neurons alongside multiomic neuronal and microglial profiling. RESULTS: ANXA11 mutations were linked to corticobasal syndrome cases. P93S led to decreased lysosome colocalization, neuritic RNA, and nuclear TDP-43 with cryptic exon expression. Multiomic microglial signatures implicated immune dysregulation and interferon signaling pathways. DISCUSSION: This study establishes ANXA11 P93S pathogenicity, broadens the phenotypic spectrum of ANXA11 mutations, underscores neuronal and microglial dysfunction in ANXA11 pathophysiology, and demonstrates the potential of cellular models to determine variant pathogenicity. HIGHLIGHTS: ANXA11 P93S is a pathogenic variant. Corticobasal syndrome is part of the ANXA11 phenotypic spectrum. Hybridization chain reaction fluorescence in situ hybridization (HCR FISH) is a new tool for the detection of cryptic exons due to TDP-43-related loss of splicing regulation. Microglial ANXA11 and related immune pathways are important drivers of disease. Cellular models are powerful tools for adjudicating variants of uncertain significance.
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Modifier genes are believed to account for the clinical variability observed in many Mendelian disorders, but their identification remains challenging due to the limited availability of genomics data from large patient cohorts. Here, we present GENDULF (GENetic moDULators identiFication), one of the first methods to facilitate prediction of disease modifiers using healthy and diseased tissue gene expression data. GENDULF is designed for monogenic diseases in which the mechanism is loss of function leading to reduced expression of the mutated gene. When applied to cystic fibrosis, GENDULF successfully identifies multiple, previously established disease modifiers, including EHF, SLC6A14, and CLCA1. It is then utilized in spinal muscular atrophy (SMA) and predicts U2AF1 as a modifier whose low expression correlates with higher SMN2 pre-mRNA exon 7 retention. Indeed, knockdown of U2AF1 in SMA patient-derived cells leads to increased full-length SMN2 transcript and SMN protein expression. Taking advantage of the increasing availability of transcriptomic data, GENDULF is a novel addition to existing strategies for prediction of genetic disease modifiers, providing insights into disease pathogenesis and uncovering novel therapeutic targets.
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Algoritmos , Minería de Datos , Enfermedad/genética , Genes Modificadores , Transcriptoma/genética , Estudios de Asociación Genética , Ligamiento Genético , Células HEK293 , Humanos , Reproducibilidad de los ResultadosRESUMEN
Rapid eye movement (REM) sleep is an important component of the natural sleep/wake cycle, yet the mechanisms that regulate REM sleep remain incompletely understood. Cholinergic neurons in the mesopontine tegmentum have been implicated in REM sleep regulation, but lesions of this area have had varying effects on REM sleep. Therefore, this study aimed to clarify the role of cholinergic neurons in the pedunculopontine tegmentum (PPT) and laterodorsal tegmentum (LDT) in REM sleep generation. Selective optogenetic activation of cholinergic neurons in the PPT or LDT during non-REM (NREM) sleep increased the number of REM sleep episodes and did not change REM sleep episode duration. Activation of cholinergic neurons in the PPT or LDT during NREM sleep was sufficient to induce REM sleep.
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Neuronas Colinérgicas/fisiología , Sueño REM/fisiología , Tegmento Mesencefálico/fisiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Channelrhodopsins , Colina O-Acetiltransferasa/genética , Neuronas Colinérgicas/citología , Tecnología de Fibra Óptica , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Transgénicos , Optogenética , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sueño REM/genética , Tegmento Mesencefálico/anatomía & histología , Vigilia/genética , Vigilia/fisiologíaRESUMEN
The diagnosis and treatment of oral lesions is often challenging due to the clinician's limited exposure to the conditions that may cause the lesions and their similar appearances. While many oral ulcers are the result of chronic trauma, some may indicate an underlying systemic condition such as a gastrointestinal dysfunction, malignancy, immunologic abnormality, or cutaneous disease. Correctly establishing a definitive diagnosis is of major importance to clinicians who manage patients with oral mucosal disease. Some of these diseases are infectious; however, most are chronic, symptomatic, and desquamative. Treatment and management requires an understanding of the immunopathologic nature of the lesion. This review will address how to differentiate and diagnose varying types of oral ulcers and provide a treatment strategy.
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Manejo de la Enfermedad , Úlceras Bucales/diagnóstico , Úlceras Bucales/terapia , Diagnóstico Diferencial , HumanosRESUMEN
Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43-depleted human iPSC-derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/genética , Péptidos , ProteómicaRESUMEN
Squamous cell carcinoma (SCC) accounts for 96 percent of all intraoral malignancies. The five-year survival rate is 50 percent and has not improved in 60 years. During SCC progression, subsets of SCC cells undergo an epithelial-to-mesenchymal transition (EMT) to become highly invasive. The extracellular matrix metalloproteinase inducer (EMMPRIN) contributes to EMT by activating local matrix metalloproteinases (MMPs). In this study, we found that EMMPRIN modulates the invasive phenotype and may be a potential therapeutic target.
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Antígenos de Neoplasias/metabolismo , Basigina/metabolismo , Carcinoma de Células Escamosas/química , Integrina alfaV/metabolismo , Integrinas/metabolismo , Invasividad Neoplásica/fisiopatología , Neoplasias de la Lengua/química , Animales , Técnicas de Cocultivo , Transición Epitelial-Mesenquimal , Fibroblastos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Neovascularización Patológica , Células Tumorales CultivadasRESUMEN
High-dimensional data analysis starts with projecting the data to low dimensions to visualize and understand the underlying data structure. Several methods have been developed for dimensionality reduction, but they are limited to cross-sectional datasets. The recently proposed Aligned-UMAP, an extension of the uniform manifold approximation and projection (UMAP) algorithm, can visualize high-dimensional longitudinal datasets. We demonstrated its utility for researchers to identify exciting patterns and trajectories within enormous datasets in biological sciences. We found that the algorithm parameters also play a crucial role and must be tuned carefully to utilize the algorithm's potential fully. We also discussed key points to remember and directions for future extensions of Aligned-UMAP. Further, we made our code open source to enhance the reproducibility and applicability of our work. We believe our benchmarking study becomes more important as more and more high-dimensional longitudinal data in biomedical research become available.
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As genetic testing has become more accessible and affordable, variants of uncertain significance (VUS) are increasingly identified, and determining whether these variants play causal roles in disease is a major challenge. The known disease-associated Annexin A11 (ANXA11) mutations result in ANXA11 aggregation, alterations in lysosomal-RNA granule co-trafficking, and TDP-43 mis-localization and present as amyotrophic lateral sclerosis or frontotemporal dementia. We identified a novel VUS in ANXA11 (P93S) in a kindred with corticobasal syndrome and unique radiographic features that segregated with disease. We then queried neurodegenerative disorder clinic databases to identify the phenotypic spread of ANXA11 mutations. Multi-modal computational analysis of this variant was performed and the effect of this VUS on ANXA11 function and TDP-43 biology was characterized in iPSC-derived neurons. Single-cell sequencing and proteomic analysis of iPSC-derived neurons and microglia were used to determine the multiomic signature of this VUS. Mutations in ANXA11 were found in association with clinically diagnosed corticobasal syndrome, thereby establishing corticobasal syndrome as part of ANXA11 clinical spectrum. In iPSC-derived neurons expressing mutant ANXA11, we found decreased colocalization of lysosomes and decreased neuritic RNA as well as decreased nuclear TDP-43 and increased formation of cryptic exons compared to controls. Multiomic assessment of the P93S variant in iPSC-derived neurons and microglia indicates that the pathogenic omic signature in neurons is modest compared to microglia. Additionally, omic studies reveal that immune dysregulation and interferon signaling pathways in microglia are central to disease. Collectively, these findings identify a new pathogenic variant in ANXA11, expand the range of clinical syndromes caused by ANXA11 mutations, and implicate both neuronal and microglia dysfunction in ANXA11 pathophysiology. This work illustrates the potential for iPSC-derived cellular models to revolutionize the variant annotation process and provides a generalizable approach to determining causality of novel variants across genes.
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Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development.
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Mucocutaneous melanoma has a five-year survival rate of less than 10 percent. The alphavbeta3 integrin promotes invasion, which requires actin reorganization by cofilin. The authors previously showed that cofilin and alphavbeta3 promote invasion. K1735 melanoma has several clones, each with different levels of alphavbeta3. The authors found that expression of alphavbeta3 suppresses activation of RhoA thus inhibiting LIMK1 phosphorylation of cofilin. This indicates that alphavbeta3 integrin suppresses the RhoA/ ROCK/LIMK1 pathway.
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Integrina alfaVbeta3/fisiología , Quinasas Lim/fisiología , Melanoma/patología , Transducción de Señal/fisiología , Proteínas de Unión al GTP rho/fisiología , Factores Despolimerizantes de la Actina/metabolismo , Factores Despolimerizantes de la Actina/fisiología , Amidas/farmacología , Animales , Western Blotting , Línea Celular Tumoral , Membrana Celular/ultraestructura , Núcleo Celular/ultraestructura , Citoesqueleto/ultraestructura , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Vectores Genéticos/genética , Humanos , Inmunoprecipitación , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Quinasas Lim/metabolismo , Ratones , Microscopía Fluorescente , Invasividad Neoplásica , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Fosforilación , Piridinas/farmacología , Transfección , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal non-specific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ARN Guía de Sistemas CRISPR-Cas , Línea Celular , Sistemas CRISPR-CasRESUMEN
Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.
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Células Madre Pluripotentes Inducidas , Humanos , Diferenciación Celular , Edición Génica , BioensayoRESUMEN
The iPSC Neurodegenerative Disease Initiative (iNDI) is the largest-ever iPSC genome engineering project. iNDI will model more than 100 mutations associated with Alzheimer's disease and related dementias (ADRD) in isogenic iPSC lines. Resulting cell lines and phenotypic datasets will be broadly shared.
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Ingeniería Genética/métodos , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/terapia , Investigación con Células Madre , Trasplante de Células Madre , Enfermedad de Alzheimer/terapia , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Células-Madre Neurales/trasplanteRESUMEN
Gene replacement and pre-mRNA splicing modifier therapies represent breakthrough gene targeting treatments for the neuromuscular disease spinal muscular atrophy (SMA), but mechanisms underlying variable efficacy of treatment are incompletely understood. Our examination of severe infantile onset human SMA tissues obtained at expedited autopsy revealed persistence of developmentally immature motor neuron axons, many of which are actively degenerating. We identified similar features in a mouse model of severe SMA, in which impaired radial growth and Schwann cell ensheathment of motor axons began during embryogenesis and resulted in reduced acquisition of myelinated axons that impeded motor axon function neonatally. Axons that failed to ensheath degenerated rapidly postnatally, specifically releasing neurofilament light chain protein into the blood. Genetic restoration of survival motor neuron protein (SMN) expression in mouse motor neurons, but not in Schwann cells or muscle, improved SMA motor axon development and maintenance. Treatment with small-molecule SMN2 splice modifiers beginning immediately after birth in mice increased radial growth of the already myelinated axons, but in utero treatment was required to restore axonal growth and associated maturation, prevent subsequent neonatal axon degeneration, and enhance motor axon function. Together, these data reveal a cellular basis for the fulminant neonatal worsening of patients with infantile onset SMA and identify a temporal window for more effective treatment. These findings suggest that minimizing treatment delay is critical to achieve optimal therapeutic efficacy.
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Atrofia Muscular Espinal , Animales , Axones , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Neuronas Motoras , Atrofia Muscular Espinal/terapia , Proteína 1 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
Oral squamous cell carcinoma (SCC) is composed of a heterogeneous population of cells which range anywhere from epithelial to mesenchymal in phenotype. Several oral cancer specimens with antibodies to TRA160, a marker of pluripotent cells, were screened. Compared with the well differentiated lesions, pluripotent cells were more numerous in specimens from poorly differentiated tumors. In vitro, the expression of TRA160 was much greater in invasive oral SCC9beta6 cells compared with the poorly invasive SCC9SN or SCC9beta6D1 cells, which express a truncated beta6. In vitro, pluripotent cells were instrumental in aggressively closing an experimental wound assay. Lastly, TRA-1-60+/beta6+ tumor cells which formed vascular-like structures in vivo were identified. SCC9beta6 cells formed interconnecting channels, whereas SCC9SN cells did not in an in vitro Matrigel angiogenesis assay. The results of this study clearly demonstrated the differential distribution of pluripotent stem cells in oral SCC and that the beta6 integrin may be an important regulatory component of the pluripotent phenotype.
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Antígenos de Neoplasias/metabolismo , Carcinoma de Células Escamosas/metabolismo , Integrinas/metabolismo , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Células Madre Neoplásicas/metabolismo , Antígenos de Superficie/metabolismo , Western Blotting , Carcinoma de Células Escamosas/patología , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Células Madre Neoplásicas/patología , Proteoglicanos/metabolismo , Células Tumorales Cultivadas , Cicatrización de HeridasRESUMEN
In this study, we evaluated whether the forced expression of beta6 integrin would modulate the epithelial to mesenchymal transition (EMT). When the full length beta6 integrin was expressed in poorly invasive squamous cell carcinoma SCC9 cells, the resulting SCC9/6 cells acquired a fibroblast-like morphology, increased expression of the mesenchymal marker vimentin and reduced expression of the epithelial markers keratin and E-cadherin. SCC9beta6D1 cells, which express a truncated form of beta6 subunit lacking the C-terminal 11 amino acids (AA), retained their epithelial morphology and did not alter vimentin or E-cadherin expression. This suggests that the full-length beta6 subunit can induce EMT in oral SCC cells. We previously showed that expression of beta6 increases both MMP-3 activation and tenascin-C expression and we now show that both molecules are MEK dependent. These results also demonstrate that the terminal 11 AA of beta6 contain information important for establishing an epithelial to mesenchymal transition.
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Antígenos de Neoplasias/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Integrinas/metabolismo , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Antígenos de Neoplasias/biosíntesis , Cadherinas/biosíntesis , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Activación Enzimática , Células Epiteliales/patología , Humanos , Integrinas/antagonistas & inhibidores , Integrinas/biosíntesis , Queratinas/biosíntesis , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Mesodermo/patología , Neoplasias de la Boca/enzimología , Tenascina/metabolismo , Vimentina/biosíntesisRESUMEN
Oral cancer is the sixth most frequent cancer worldwide. Prognosis for these patients remains poor. Recently, the epidermal growth factor receptor has been targeted as an adjunct to radiotherapy and surgery with limited success. The authors now present data suggesting that the alphanubeta6 integrin, which is a marker for aggressive oral cancer, may regulate epidermal growth factor receptor expression. The authors suggest perhaps targeting both alphanubeta6 and EGFR may provide additional benefits.
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Antígenos de Neoplasias/fisiología , Carcinoma de Células Escamosas/patología , Receptores ErbB/fisiología , Integrinas/fisiología , Neoplasias de la Boca/patología , Anticuerpos Monoclonales , Antígenos de Neoplasias/análisis , Biomarcadores de Tumor/análisis , Western Blotting , Línea Celular Tumoral , Membrana Celular/patología , Receptores ErbB/análisis , Matriz Extracelular/patología , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Humanos , Inmunohistoquímica , Integrinas/análisis , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-fyn/fisiología , Transducción de Señal/fisiologíaRESUMEN
BACKGROUNDSpinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein. New SMN-enhancing therapeutics are associated with variable clinical benefits. Limited knowledge of baseline and drug-induced SMN levels in disease-relevant tissues hinders efforts to optimize these treatments.METHODSSMN mRNA and protein levels were quantified in human tissues isolated during expedited autopsies.RESULTSSMN protein expression varied broadly among prenatal control spinal cord samples, but was restricted at relatively low levels in controls and SMA patients after 3 months of life. A 2.3-fold perinatal decrease in median SMN protein levels was not paralleled by comparable changes in SMN mRNA. In tissues isolated from nusinersen-treated SMA patients, antisense oligonucleotide (ASO) concentration and full-length (exon 7 including) SMN2 (SMN2-FL) mRNA level increases were highest in lumbar and thoracic spinal cord. An increased number of cells showed SMN immunolabeling in spinal cord of treated patients, but was not associated with an increase in whole-tissue SMN protein levels.CONCLUSIONSA normally occurring perinatal decrease in whole-tissue SMN protein levels supports efforts to initiate SMN-inducing therapies as soon after birth as possible. Limited ASO distribution to rostral spinal and brain regions in some patients likely limits clinical response of motor units in these regions for those patients. These results have important implications for optimizing treatment of SMA patients and warrant further investigations to enhance bioavailability of intrathecally administered ASOs.FUNDINGSMA Foundation, SMART, NIH (R01-NS096770, R01-NS062869), Ionis Pharmaceuticals, and PTC Therapeutics. Biogen provided support for absolute real-time RT-PCR.
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Envejecimiento , Neuronas Motoras , Atrofia Muscular Espinal , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Médula Espinal , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Autopsia , Supervivencia Celular , Femenino , Humanos , Masculino , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patología , Médula Espinal/metabolismo , Médula Espinal/patología , Proteína 2 para la Supervivencia de la Neurona Motora/antagonistas & inhibidores , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
The extracellular matrix metalloproteinase inducer (EMMPRIN), found on the surface of many tumor cells, stimulates the production of matrix metalloproteinases (MMPs) by both fibroblasts and the tumor cells themselves. To evaluate its possible role as a tumor promoter, we first overexpressed EMMPRIN, by retroviral transduction, into poorly invasive squamous cell carcinoma (SCC) cells. Secondly, we knocked down its expression using small interfering RNA (siRNA) in invasive SCC cells. The cell lines were then re-evaluated for migration on fibronectin (FN). Overexpression of EMMPRIN, promoted motility, whereas the siRNA decreased migration. The MMP expression by these variant SCC cell lines was also manipulated by EMMPRIN. The expression of MMP-2, -3, and -9 coincided with the expression of EMMPRIN. Cocultures of SCC/peritumor fibroblasts (PTF) were used to investigate tenascin-C (TN-C) matrix deposition. The cocultures overexpressing EMMPRIN, deposited several fold greater levels of TN-C compared to the control cocultures. In addition, the siRNA cocultures deposited minimal amounts of TN-C. In the presence of the broad spectrum MMP inhibitor, GM6001, TN-C deposition by the EMMPRIN overexpressing cocultures was suppressed. Thus EMMPRIN regulates migration, MMP production by SCC cells and deposition of the TN-C matrix.
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Basigina/biosíntesis , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Movimiento Celular/fisiología , Tenascina/metabolismo , Neoplasias de la Lengua/metabolismo , Neoplasias de la Lengua/patología , Basigina/genética , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Humanos , Isoenzimas , Metaloproteinasas de la Matriz/biosíntesis , ARN Interferente Pequeño/genética , Neoplasias de la Lengua/enzimologíaRESUMEN
The 5-year survival rate for patients with oral cancer remains at 50%, in large part due the high rate of post-treatment recurrence. In this study, we transfected epithelial-specific integrin αvß6 and Fyn-kinase, a member of the Src-family kinases, into embryonic murine fibroblasts. In oral cancer, expression of αvß6 is neo-expressed. Using a variety of in vitro assays, including cell migration and multicellular spheroid formation, we determined that these embryonic fibroblasts expressing αvß6 and Fyn-kinase were able to acquire an epithelial phenotype. This is in direct contrast to human oral SCC, where expression of αvß6 with Fyn-kinase promotes epithelial to mesenchymal transition. This demonstrates that signaling pathways can be species-specific.