RESUMO
Dysregulation of extracellular matrix (ECM) deposition and cellular metabolism promotes tumor aggressiveness by sustaining the activity of key growth, invasion, and survival pathways. Yet mechanisms by which biophysical properties of ECM relate to metabolic processes and tumor progression remain undefined. In both cancer cells and carcinoma-associated fibroblasts (CAFs), we found that ECM stiffening mechanoactivates glycolysis and glutamine metabolism and thus coordinates non-essential amino acid flux within the tumor niche. Specifically, we demonstrate a metabolic crosstalk between CAF and cancer cells in which CAF-derived aspartate sustains cancer cell proliferation, while cancer cell-derived glutamate balances the redox state of CAFs to promote ECM remodeling. Collectively, our findings link mechanical stimuli to dysregulated tumor metabolism and thereby highlight a new metabolic network within tumors in which diverse fuel sources are used to promote growth and aggressiveness. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in cancer.
Assuntos
Ácido Aspártico/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Carcinoma/metabolismo , Ácido Glutâmico/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Fibroblastos Associados a Câncer/patologia , Linhagem Celular , Matriz Extracelular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
In squamous cell carcinoma (SCC), tissue invasion by collectively invading cells requires physical forces applied by tumor cells on their surrounding extracellular matrix (ECM). Cancer-related ECM is composed of thick collagen bundles organized by carcinoma-associated fibroblasts (CAF) within the tumor stroma. Here, we show that SCC cell collective invasion is driven by the matrix-dependent mechano-sensitization of EGF signaling in cancer cells. Calcium (Ca2+) was a potent intracellular second messenger that drove actomyosin contractility. Tumor-derived matrix stiffness and EGFR signaling triggered increased intracellular Ca2+ through CaV1.1 expression in SCC cells. Blocking L-type calcium channel expression or activity using Ca2+ channel blockers verapamil and diltiazem reduced SCC cell collective invasion both in vitro and in vivo These results identify verapamil and diltiazem, two drugs long used in medical care, as novel therapeutic strategies to block the tumor-promoting activity of the tumor niche.Significance: This work demonstrates that calcium channels blockers verapamil and diltiazem inhibit mechano-sensitization of EGF-dependent cancer cell collective invasion, introducing potential clinical strategies against stromal-dependent collective invasion.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5229/F1.large.jpg Cancer Res; 78(18); 5229-42. ©2018 AACR.
Assuntos
Sinalização do Cálcio , Carcinoma de Células Escamosas/patologia , Matriz Extracelular/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Actomiosina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colágeno/metabolismo , Diltiazem/farmacologia , Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Invasividade Neoplásica , Esferoides Celulares , Verapamil/farmacologiaRESUMO
Acto-myosin contractility in carcinoma-associated fibroblasts leads to assembly of the tumor extracellular matrix. The pro-inflammatory cytokine LIF governs fibroblast activation in cancer by regulating the myosin light chain 2 activity. So far, however, how LIF mediates cytoskeleton contractility remains unknown. Using phenotypic screening assays based on knock-down of LIF-dependent genes in fibroblasts, we identified the glycoprotein ICAM-1 as a crucial regulator of stroma fibroblast proinvasive matrix remodeling. We demonstrate that the membrane-bound ICAM-1 isoform is necessary and sufficient to promote inflammation-dependent extracellular matrix contraction, which favors cancer cell invasion. Indeed, ICAM-1 mediates generation of acto-myosin contractility downstream of the Src kinases in stromal fibroblasts. Moreover, acto-myosin contractility regulates ICAM-1 expression by establishing a positive feedback signaling. Thus, targeting stromal ICAM-1 might constitute a possible therapeutic mean to counteract tumor cell invasion and dissemination.
Assuntos
Actomiosina/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Molécula 1 de Adesão Intercelular/metabolismo , Actomiosina/genética , Células Cultivadas , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Neoplasias de Cabeça e Pescoço/genética , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Transdução de Sinais , Células Estromais/metabolismo , Células Estromais/patologia , Microambiente TumoralRESUMO
Epidermolysis bullosa with pyloric atresia (EB-PA) is a rare autosomal recessive hereditary disease with a variable prognosis from lethal to very mild. EB-PA is classified into Simplex form (EBS-PA: OMIM #612138) and Junctional form (JEB-PA: OMIM #226730), and it is caused by mutations in ITGA6, ITGB4 and PLEC genes. We report the analysis of six patients with EB-PA, including two dizygotic twins. Skin immunofluorescence epitope mapping was performed followed by PCR and direct sequencing of the ITGB4 gene. Two of the patients presented with non-lethal EB-PA associated with missense ITGB4 gene mutations. For the other four, early postnatal demise was associated with complete lack of ß4 integrin due to a variety of ITGB4 novel mutations (2 large deletions, 1 splice-site mutation and 3 missense mutations). One of the deletions spanned 278 bp, being one of the largest reported to date for this gene. Remarkably, we also found for the first time a founder effect for one novel mutation in the ITGB4 gene. We have identified 6 novel mutations in the ITGB4 gene to be added to the mutation database. Our results reveal genotype-phenotype correlations that contribute to the molecular understanding of this heterogeneous disease, a pivotal issue for prognosis and for the development of novel evidence-based therapeutic options for EB management.
Assuntos
Displasia Ectodérmica/genética , Integrina beta4/genética , Deleção de Sequência , Biópsia , Pré-Escolar , Análise Mutacional de DNA , Displasia Ectodérmica/diagnóstico , Mapeamento de Epitopos , Epitopos/química , Feminino , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Queratinócitos/citologia , Masculino , Repetições de Microssatélites/genética , Microscopia de Fluorescência , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase , Prognóstico , Análise de Sequência de DNA , Gêmeos DizigóticosRESUMO
Carcinoma-associated fibroblasts (CAF) mediate the onset of a proinvasive tumour microenvironment. The proinflammatory cytokine LIF reprograms fibroblasts into a proinvasive phenotype, which promotes extracellular matrix remodelling and collective invasion of cancer cells. Here we unveil that exposure to LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signalling, which results in sustained proinvasive activity of CAF. Mechanistically, p300-histone acetyltransferase acetylates STAT3, which, in turn, upregulates and activates the DNMT3b DNA methyltransferase. DNMT3b methylates CpG sites of the SHP-1 phosphatase promoter, which abrogates SHP-1 expression, and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signalling is maintained by DNA methyltransferase DNMT1. Consistently, in human lung and head and neck carcinomas, STAT3 acetylation and phosphorylation are inversely correlated with SHP-1 expression. Combined inhibition of DNMT activities and JAK signalling, in vitro and in vivo, results in long-term reversion of CAF-associated proinvasive activity and restoration of the wild-type fibroblast phenotype.
Assuntos
Carcinogênese/metabolismo , Epigenômica , Fibroblastos/fisiologia , Neoplasias/metabolismo , Animais , Anticorpos Neutralizantes , Carcinogênese/genética , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/metabolismo , Interferência de RNARESUMO
Cellular and molecular crosstalks between cancer and non-cancer tumor-associated cells result in tumor growth and metastatic spreading. During carcinoma development, tumor cells secrete signaling molecules that influence the surrounding non-cancer cells, which, in return, favor tumor cell growth, survival, migration and metastasis. Carcinoma-associated fibroblasts (CAF) are the most abundant population of non-cancer cells found in tumors, and their presence is often associated with poor clinical prognosis. Here, we summarize the pro-carcinogenic roles of CAF cells during carcinogenesis, with a specific focus on their abilities to drive cancer cell-dependent pro-invasive extracellular matrix remodeling.
Assuntos
Fibroblastos , Invasividade Neoplásica/patologia , Neoplasias/patologia , Microambiente Tumoral , HumanosRESUMO
Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF) as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA) expression. We demonstrate that a pulse of transforming growth factor ß (TGF-ß) establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-ß-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor) counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.
Assuntos
Carcinogênese/metabolismo , Carcinoma/metabolismo , Fibroblastos/metabolismo , Fator Inibidor de Leucemia/metabolismo , Melanoma/metabolismo , Microambiente Tumoral , Actinas/genética , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Carcinogênese/patologia , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Janus Quinases/antagonistas & inibidores , Fator Inibidor de Leucemia/genética , Melanoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Nitrilas , Pirazóis/farmacologia , Pirimidinas , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia , Regulação para CimaRESUMO
Incidence of cutaneous squamous cell carcinomas (cSCCs) constantly increases in the Caucasian population. Developing preferentially on precancerous lesions such as actinic keratoses due to chronic sunlight exposure, cSCCs result from the malignant transformation of keratinocytes. Although a resection of the primary tumor is usually curative, a subset of aggressive cSCCs shows a high risk of recurrence and metastases. The characterization of the molecular dysfunctions involved in cSCC development should help to identify new relevant targets against these aggressive cSCCs. In that context, we have used small RNA sequencing to identify 100 microRNAs (miRNAs) whose expression was altered during chemically induced mouse skin tumorigenesis. The decreased expression of the miR-193b/365a cluster during tumor progression suggests a tumor suppressor role. Ectopic expression of these miRNAs in tumor cells indeed inhibited their proliferation, clonogenic potential and migration, which were stimulated in normal keratinocytes when these miRNAs were blocked with antisense oligonucleotides. A combination of in silico predictions and transcriptome analyses identified several target genes of interest. We validated KRAS and MAX as direct targets of miR-193b and miR-365a. Repression of these targets using siRNAs mimicked the effects of miR-193b and miR-365a, suggesting that these genes might mediate, at least in part, the tumor-suppressive action of these miRNAs.
Assuntos
Carcinoma de Células Escamosas/genética , MicroRNAs/genética , Família Multigênica , Neoplasias Cutâneas/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes ras , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , MicroRNAs/metabolismo , Estadiamento de Neoplasias , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
The aim of this study was to identify the molecular signals produced in human endothelial cells (ECs) by the interaction of α5ß1 integrin with soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) present in the extracellular matrix. We generated a gene expression profile of ECs adhering to sVEGFR-1 or to fibronectin, the classic extracellular matrix ligand for α5ß1 integrin or in a nonadhering condition. Several biological pathways were differently modulated, 3 protein kinase C substrates [adducin, myristoylated alanine-rich protein kinase C substrate (MARCKS), and radixin] were differently expressed and phosphorylated when cells adhering to sVEGFR-1 were compared with those adhering to fibronectin. Rac1 activation and Gα13 protein involvement through the interaction with radixin were also detected after attachment to sVEGFR-1, and these responses depended on active VEGFR-2 signaling. On sVEGFR-1, ECs exhibited a motile phenotype that was consistent with the abundant presence of MARCKS, a stabilizer of dynamic adhesions. Moreover, ECs silenced for radixin expression no longer responded to the proangiogenic VEGFR-1-derived peptide 12. We propose that the presence of sVEGFR-1 in the EC microenvironment directs α5ß1 integrin signaling to generate a dynamic, motile phenotype. Our findings also provide new insights into the mechanism of action of proangiogenic peptide 12, relevant to a therapeutic perspective.
Assuntos
Adesão Celular/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Western Blotting , Movimento Celular/genética , Movimento Celular/fisiologia , Células Cultivadas , Fibronectinas/metabolismo , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologiaRESUMO
The frequent alteration of miRNA expression in many cancers, together with our recent reports showing a robust accumulation of miR-483-3p at the final stage of skin wound healing, and targeting of CDC25A leading to an arrest of keratinocyte proliferation, led us to hypothesize that miR-483-3p could also be endowed with antitumoral properties. We tested that hypothesis by documenting the in vitro and in vivo impacts of miR-483-3p in squamous cell carcinoma (SCC) cells. miR-483-3p sensitized SCC cells to serum deprivation- and drug-induced apoptosis, thus exerting potent tumor suppressor activities. Its pro-apoptotic activity was mediated by a direct targeting of several anti-apoptotic genes, such as API5, BIRC5, and RAN. Interestingly, an in vivo delivery of miR-483-3p into subcutaneous SCC xenografts significantly hampered tumor growth. This effect was explained by an inhibition of cell proliferation and an increase of apoptosis. This argues for its further use as an adjuvant in the many instances of cancers characterized by a downregulation of miR-483-3p.
Assuntos
Apoptose/genética , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Bucais/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Injeções Intralesionais , Camundongos , MicroRNAs/administração & dosagem , MicroRNAs/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Transplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais , Pele , Survivina , Transplante Heterólogo , Transplante Heterotópico , Carga Tumoral/genética , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Cancer cell invasion and dissemination from primary tumors are complex multistep mechanisms which remain poorly understood. It is now clear that cancer cells can adapt their mode of invasion to the signalling provided by the surrounding stroma. Single and collective cancer cell invasion are the two invasion features most currently observed and described by pathologists. Here we describe a three-dimensional organotypic assay that allows the study of squamous cell carcinoma cell collective invasion induced by the carcinoma associated fibroblasts. This model preserves the relationship between epithelial and mesenchymal cells, which are observed in vivo, and allows to decipher the molecular and cellular mechanisms involving the tumor and its stromal microenvironment. This three-dimensional model of invasion provides an invaluable tool to gain major insights in the understanding of tumor cell dissemination.
Assuntos
Carcinoma de Células Escamosas/patologia , Técnicas de Cocultura/métodos , Fibroblastos/patologia , Queratinócitos/patologia , Invasividade Neoplásica/patologia , Pele/patologia , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura/instrumentação , Humanos , Modelos Biológicos , Técnicas de Cultura de Tecidos/instrumentação , Técnicas de Cultura de Tecidos/métodos , Microambiente TumoralRESUMO
Skin aging is linked to reduced epidermal proliferation and general extracellular matrix atrophy. This involves factors such as the cell adhesion receptors integrins and amino acid transporters. CD98hc (SLC3A2), a heterodimeric amino acid transporter, modulates integrin signaling in vitro. We unravel CD98hc functions in vivo in skin. We report that CD98hc invalidation has no appreciable effect on cell adhesion, clearly showing that CD98hc disruption phenocopies neither CD98hc knockdown in cultured keratinocytes nor epidermal ß1 integrin loss in vivo. Instead, we show that CD98hc deletion in murine epidermis results in improper skin homeostasis and epidermal wound healing. These defects resemble aged skin alterations and correlate with reduction of CD98hc expression observed in elderly mice. We also demonstrate that CD98hc absence in vivo induces defects as early as integrin-dependent Src activation. We decipher the molecular mechanisms involved in vivo by revealing a crucial role of the CD98hc/integrins/Rho guanine nucleotide exchange factor (GEF) leukemia-associated RhoGEF (LARG)/RhoA pathway in skin homeostasis. Finally, we demonstrate that the deregulation of RhoA activation in the absence of CD98hc is also a result of impaired CD98hc-dependent amino acid transports.
Assuntos
Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Queratinócitos/metabolismo , Pele/metabolismo , Cicatrização/fisiologia , Fatores Etários , Animais , Proteína Tirosina Quinase CSK , Adesão Celular/genética , Movimento Celular/genética , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Folículo Piloso/metabolismo , Homeostase , Integrinas/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Fenômenos Fisiológicos da Pele , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Fatores de Transcrição/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP , Quinases da Família src/metabolismoRESUMO
In culture, cell confluence generates signals that commit actively growing keratinocytes to exit the cell cycle and differentiate to form a stratified epithelium. Using a comparative proteomic approach, we studied this 'confluence switch' and identified a new pathway triggered by cell confluence that regulates basement membrane (BM) protein composition by suppressing the uPA-uPAR-plasmin pathway. Indeed, confluence triggers adherens junction maturation and enhances TGF-ß and activin A activity, resulting in increased deposition of PAI-1 and perlecan in the BM. Extracellular matrix (ECM)-accumulated PAI-1 suppresses the uPA-uPAR-plasmin pathway and further enhances perlecan deposition by inhibiting its plasmin-dependent proteolysis. We show that perlecan deposition in the ECM strengthens cell adhesion, inhibits keratinocyte motility and promotes additional accumulation of PAI-1 in the ECM at confluence. In agreement, during wound-healing, perlecan concentrates at the wound-margin, where BM matures to stabilize keratinocyte adhesion. Our results demonstrate that confluence-dependent signaling orchestrates not only growth inhibition and differentiation, but also controls ECM proteolysis and BM formation. These data suggest that uncontrolled integration of confluence-dependent signaling, might favor skin disorders, including tumorigenesis, not only by promoting cell hyperproliferation, but also by altering protease activity and deposition of ECM components.
Assuntos
Matriz Extracelular/metabolismo , Fibrinolisina/metabolismo , Queratinócitos/metabolismo , Proteólise , Transdução de Sinais , Ativinas/metabolismo , Junções Aderentes/metabolismo , Animais , Membrana Basal/metabolismo , Adesão Celular , Diferenciação Celular , Movimento Celular , Proliferação de Células , Regulação para Baixo , Retroalimentação Fisiológica , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Queratinócitos/patologia , Camundongos , Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ligação Proteica , Proteômica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , CicatrizaçãoRESUMO
Proinflammatory cytokines are frequently observed in the tumor microenvironment, and chronic inflammation is involved in cancer initiation and progression. We show that cytokine signaling through the receptor subunit GP130-IL6ST and the kinase JAK1 generates actomyosin contractility through Rho-kinase dependent signaling. This pathway generates contractile force in stromal fibroblasts to remodel the extracellular matrix to create tracks for collective migration of squamous carcinoma cells and provides the high levels of actomyosin contractility required for migration of individual melanoma cells in the rounded, "amoeboid" mode. Thus, cytokine signaling can generate actomyosin contractility in both stroma and tumor cells. Strikingly, actomyosin contractility itself positively modulates activity of the transcription factor STAT3 downstream of JAK1, demonstrating positive feedback within the signaling network.
Assuntos
Actomiosina/metabolismo , Janus Quinase 1/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Quinases Associadas a rho/metabolismo , Movimento Celular , Humanos , Melanoma/metabolismo , Melanoma/patologia , Neoplasias/patologia , Fator de Transcrição STAT3/metabolismo , Células Estromais/patologiaRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering condition caused by mutations in the gene coding for collagen type VII. Genetically engineered RDEB dog keratinocytes were used to generate autologous epidermal sheets subsequently grafted on two RDEB dogs carrying a homozygous missense mutation in the col7a1 gene and expressing baseline amounts of the aberrant protein. Transplanted cells regenerated a differentiated and vascularized auto-renewing epidermis progressively repopulated by dendritic cells and melanocytes. No adverse immune reaction was detected in either dog. In dog 1, the grafted epidermis firmly adhered to the dermis throughout the 24-month follow-up, which correlated with efficient transduction (100%) of highly clonogenic epithelial cells and sustained transgene expression. In dog 2, less efficient (65%) transduction of primary keratinocytes resulted in a loss of the transplanted epidermis and graft blistering 5 months after transplantation. These data provide the proof of principle for ex vivo gene therapy of RDEB patients with missense mutations in collagen type VII by engraftment of the reconstructed epidermis, and demonstrate that highly efficient transduction of epidermal stem cells is crucial for successful gene therapy of inherited skin diseases in which correction of the genetic defect confers no major selective advantage in cell culture.
Assuntos
Epidermólise Bolhosa Distrófica/terapia , Transplante de Pele/métodos , Animais , Colágeno Tipo VII/metabolismo , Cães , Terapia Genética/métodos , Homozigoto , Microscopia de Fluorescência/métodos , Modelos Genéticos , Mutação , Mutação de Sentido Incorreto , Retroviridae/genética , Pele/patologia , Transgenes , Transplante AutólogoRESUMO
The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up-regulation of miR-483-3p in 2 distinct models of wound healing: scratch-injured cultures of human keratinocytes and wounded skin in mice. miR-483-3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of "healing" progression. Using an in vitro wound-healing model, videomicroscopy, and 5-bromo-2'-uridine incorporation, we observed that overexpression of miR-483-3p inhibits keratinocyte migration and proliferation, whereas delivery of anti-miR-483-3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti-miR treatment. Expression profiling of keratinocytes transfected with miR-483-3p identified 39 transcripts that were both predicted targets of miR-483-3p and down-regulated after miR-483-3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR-483-3p that control keratinocyte proliferation. miR-483-3p-mediated down-regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.
Assuntos
Proliferação de Células , Queratinócitos/metabolismo , MicroRNAs/metabolismo , Ferimentos e Lesões/metabolismo , Animais , Anticorpos , Células Epiteliais , Inativação Gênica , Humanos , Queratinócitos/citologia , Camundongos , MicroRNAs/genética , Oligonucleotídeos , Pele/metabolismo , Fatores de TempoRESUMO
RATIONALE: Integrins play a crucial role in controlling endothelial cell proliferation and migration during angiogenesis. The Delta-like 4 (Dll4)/Notch pathway establishes an adequate ratio between stalk and tip cell populations by restricting tip cell formation through "lateral inhibition" in response to a vascular endothelial growth factor gradient. Because angiogenesis requires a tight coordination of these cellular processes, we hypothesized that adhesion, vascular endothelial growth factor, and Notch signaling pathways are interconnected. OBJECTIVE: This study was aimed at characterizing the cross-talk between integrin and Notch signaling in endothelial cells. METHODS AND RESULTS: Adhesion of primary human endothelial cells to laminin-111 triggers Dll4 expression, leading to subsequent Notch pathway activation. SiRNA-mediated knockdown of α2ß1 and α6ß1 integrins abolishes Dll4 induction, which discloses a selective integrin signaling acting upstream of Notch pathway. The increase in Foxc2 transcription, triggered by α2ß1 binding to laminin, is required but not sufficient per se for Dll4 expression. Furthermore, vascular endothelial growth factor stimulates laminin γ1 deposition, which leads to integrin signaling and Dll4 induction. Interestingly, loss of integrins α2 or α6 mimics the effects of Dll4 silencing and induces excessive network branching in an in vitro sprouting angiogenesis assay on three-dimensional matrigel. CONCLUSIONS: We show that, in endothelial cells, ligation of α2ß1 and α6ß1 integrins induces the Notch pathway, and we disclose a novel role of basement membrane proteins in the processes controlling tip vs stalk cell selection.
Assuntos
Células Endoteliais/metabolismo , Integrina alfa2beta1/metabolismo , Integrina alfa6beta1/metabolismo , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Receptores Notch/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Membrana Basal , Proteínas de Ligação ao Cálcio , Adesão Celular , Células Cultivadas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Laminina/metabolismo , Proteínas de Membrana/fisiologia , Neovascularização Fisiológica , Receptor Cross-TalkRESUMO
Patients suffering from recessive dystrophic epidermolysis bullosa (RDEB), a hereditary blistering disease of epithelia, show susceptibility to develop highly aggressive squamous cell carcinoma (SCC). Tumors metastasize early and are associated with mortality in the 30th-40th years of life in this patient group. So far, no adequate therapy is available for RDEB SCC. An approach is suicide gene therapy, in which a cell death-inducing agent is introduced to cancer cells. However, lack of specificity has constrained clinical application of this modality. Therefore, we used spliceosome-mediated RNA trans-splicing technology, capable of replacing a tumor-specific transcript with one encoding a cell death-inducing peptide/toxin, to provide tumor-restricted expression. We designed 3' pre-trans-splicing molecules (PTM) and evaluated their efficiency to trans-splice an RDEB SCC-associated target gene, the matrix metalloproteinase-9 (MMP9), in a fluorescence-based test system. A highly efficient PTM was further adapted to insert the toxin streptolysin O (SLO) of Streptococcus pyogenes into the MMP9 gene. Transfection of RDEB SCC cells with the SLO-PTM resulted in cell death and induction of toxin function restricted to RDEB SCC cells. Thus, RNA trans-splicing is a suicide gene therapy approach with increased specificity to treat highly malignant SCC tumors.
Assuntos
Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/genética , Epidermólise Bolhosa Distrófica/complicações , Epidermólise Bolhosa Distrófica/genética , Técnicas de Transferência de Genes , Genes Transgênicos Suicidas/genética , Metaloproteinase 9 da Matriz/genética , Spliceossomos/metabolismo , Estreptolisinas/genética , Trans-Splicing/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Carcinoma de Células Escamosas/terapia , Linhagem Celular , Sobrevivência Celular/genética , Epidermólise Bolhosa Distrófica/terapia , Citometria de Fluxo , Ordem dos Genes , Células HEK293 , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Estreptolisinas/metabolismoRESUMO
Functional defects in type VII collagen, caused by premature termination codons on both alleles of the COL7A1 gene, are responsible for the severe autosomal recessive types of the skin blistering disease, recessive dystrophic epidermolysis bullosa (RDEB). The full-length COL7A1 complementary DNA (cDNA) is about 9 kb, a size that is hardly accommodated by therapeutically used retroviral vectors. Although there have been successful attempts to produce functional type VII collagen protein in model systems of RDEB, the risk of genetic rearrangements of the large repetitive cDNA sequence may hamper the clinical application of full-length COL7A1 cDNA in the human system. Therefore, we used trans-splicing to reduce the size of the COL7A1 transcript. Retroviral transduction of RDEB keratinocytes with a 3' pre-trans-splicing molecule resulted in correction of full-length type VII collagen expression. Unlike parental RDEB keratinocytes, transduced cells displayed normal morphology and reduced invasive capacity. Moreover, transduced cells showed normal localization of type VII collagen at the basement membrane zone in skin equivalents, where it assembled into anchoring fibril-like structures. Thus, using trans-splicing we achieved correction of an RDEB phenotype in vitro, which marks an important step toward its application in gene therapy in vivo.
Assuntos
Códon sem Sentido/genética , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica , Terapia Genética/métodos , Transdução Genética/métodos , Processamento Alternativo/genética , Biópsia , Células Cultivadas , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Epidermólise Bolhosa Distrófica/terapia , Humanos , Queratinócitos/patologia , Queratinócitos/fisiologia , Técnicas de Cultura de Órgãos , Fenótipo , RNA Mensageiro/genética , Retroviridae/genéticaRESUMO
Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.