RESUMEN
Genome-wide association studies (GWAS) have identified rs11672691 at 19q13 associated with aggressive prostate cancer (PCa). Here, we independently confirmed the finding in a cohort of 2,738 PCa patients and discovered the biological mechanism underlying this association. We found an association of the aggressive PCa-associated allele G of rs11672691 with elevated transcript levels of two biologically plausible candidate genes, PCAT19 and CEACAM21, implicated in PCa cell growth and tumor progression. Mechanistically, rs11672691 resides in an enhancer element and alters the binding site of HOXA2, a novel oncogenic transcription factor with prognostic potential in PCa. Remarkably, CRISPR/Cas9-mediated single-nucleotide editing showed the direct effect of rs11672691 on PCAT19 and CEACAM21 expression and PCa cellular aggressive phenotype. Clinical data demonstrated synergistic effects of rs11672691 genotype and PCAT19/CEACAM21 gene expression on PCa prognosis. These results provide a plausible mechanism for rs11672691 associated with aggressive PCa and thus lay the ground work for translating this finding to the clinic.
Asunto(s)
Neoplasias de la Próstata/genética , ARN Largo no Codificante/genética , ARN no Traducido/genética , Adulto , Alelos , Línea Celular Tumoral , Cromosomas Humanos Par 19/genética , Estudios de Cohortes , Regulación Neoplásica de la Expresión Génica/genética , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Genotipo , Proteínas de Homeodominio , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , PronósticoRESUMEN
Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature in vitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers.
Asunto(s)
Actinas , Proteínas de Microfilamentos , Seudópodos , Animales , Perros , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Uniones Adherentes/metabolismo , Células Epiteliales/metabolismo , Uniones Intercelulares/metabolismo , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/metabolismo , Seudópodos/metabolismo , Proteínas de Microfilamentos/metabolismoRESUMEN
Integrin-mediated laminin adhesions mediate epithelial cell anchorage to basement membranes and are critical regulators of epithelial cell polarity. Integrins assemble large multiprotein complexes that link to the cytoskeleton and convey signals into the cells. Comprehensive proteomic analyses of actin network-linked focal adhesions (FA) have been performed, but the molecular composition of intermediate filament-linked hemidesmosomes (HD) remains incompletely characterized. Here we have used proximity-dependent biotin identification (BioID) technology to label and characterize the interactome of epithelia-specific ß4-integrin that, as α6ß4-heterodimer, forms the core of HDs. The analysis identified â¼150 proteins that were specifically labeled by BirA-tagged integrin-ß4. In addition to known HDs proteins, the interactome revealed proteins that may indirectly link integrin-ß4 to actin-connected protein complexes, such as FAs and dystrophin/dystroglycan complexes. The specificity of the screening approach was validated by confirming the HD localization of two candidate ß4-interacting proteins, utrophin (UTRN) and ELKS/Rab6-interacting/CAST family member 1 (ERC1). Interestingly, although establishment of functional HDs depends on the formation of α6ß4-heterodimers, the assembly of ß4-interactome was not strictly dependent on α6-integrin expression. Our survey to the HD interactome sets a precedent for future studies and provides novel insight into the mechanisms of HD assembly and function of the ß4-integrin.
Asunto(s)
Integrina beta4/química , Integrina beta4/metabolismo , Proteómica/métodos , Animales , Biotinilación , Cromatografía Liquida , Perros , Células de Riñón Canino Madin Darby , Mapas de Interacción de Proteínas , Multimerización de Proteína , Espectrometría de Masas en TándemRESUMEN
The development of tissue fibrosis is complex and at the present time, not fully understood. Fibrosis, neurodegeneration and cerebral angiomatosis (FINCA disease) have been described in patients with mutations in NHL repeat-containing protein 2 (NHLRC2). However, the molecular functions of NHLRC2 are uncharacterized. Herein, we identified putative interacting partners for NHLRC2 using proximity-labeling mass spectrometry. We also investigated the function of NHLRC2 using immortalized cells cultured from skin biopsies of FINCA patients and normal fibroblasts with NHLRC2 knock-down and NHLRC2 overexpressing gene modifications. Transmission electron microscopy analysis of immortalized cell cultures from three FINCA patients demonstrated multilamellar bodies and distinctly organized vimentin filaments. Additionally, two of three cultures derived from patient skin biopsies contained cells that exhibited features characteristic of myofibroblasts. Altogether, the data presented in this study show for the first time that NHLRC2 is involved in cellular organization through regulation of the cytoskeleton and vesicle transport. We conclude that compound heterozygous p.Asp148Tyr and p.Arg201GlyfsTer6 mutations in NHLRC2 lead to severe tissue fibrosis in humans by enhancing the differentiation of fibroblasts to myofibroblasts.
Asunto(s)
Angiomatosis/patología , Encefalopatías/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Miofibroblastos/patología , Degeneración Nerviosa/genética , Actinas/genética , Angiomatosis/genética , Encefalopatías/genética , Diferenciación Celular/genética , Células Cultivadas , Fibrosis , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación/genética , Miofibroblastos/metabolismo , Piel/metabolismo , Piel/patologíaRESUMEN
Hypoxia and loss of cell polarity are common features of malignant carcinomas. Hypoxia-inducible factor 1 (HIF1) is the major regulator of cellular hypoxia response and mediates the activation of â¼300 genes. Increased HIF1 signaling is known to be associated with epithelial-mesenchymal transformation. Here, we report that hypoxia disrupts polarized epithelial morphogenesis of MDCK cells in a HIF1α-dependent manner by modulating the transforming growth factor-ß (TGFß) signaling pathway. Analysis of potential HIF1 targets in the TGFß pathway identified the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a transmembrane glycoprotein related to the type I receptors of the TGFß family, whose expression was essentially lost in HIF1-depleted cells. Similar to what was observed in HIF1-deficient cells, BAMBI-depleted cells failed to efficiently activate TGFß signaling and retained epithelial polarity during hypoxia. Taken together, we show that hypoxic conditions promote TGFß signaling in a HIF1-dependent manner and BAMBI is identified in this pathway as a novel HIF1-regulated gene that contributes to hypoxia-induced loss of epithelial polarity.
Asunto(s)
Polaridad Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Proteínas de la Membrana/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Perros , Humanos , Hipoxia/genética , Hipoxia/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/genética , Transducción de SeñalRESUMEN
There has been a growing interest toward mitochondrial fatty acid synthesis (mtFAS) since the recent discovery of a neurodegenerative human disorder termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration), which is caused by mutations in the mitochondrial enoyl-CoA/ACP (acyl carrier protein) reductase (MECR) carrying out the last step of mtFAS. We show here that MECR protein is highly expressed in mouse Purkinje cells (PCs). To elucidate mtFAS function in neural tissue, here, we generated a mouse line with a PC-specific knock-out (KO) of Mecr, leading to inactivation of mtFAS confined to this cell type. Both sexes were studied. The mitochondria in KO PCs displayed abnormal morphology, loss of protein lipoylation, and reduced respiratory chain enzymatic activities by the time these mice were 6 months of age, followed by nearly complete loss of PCs by 9 months of age. These animals exhibited balancing difficulties â¼7 months of age and ataxic symptoms were evident from 8-9 months of age on. Our data show that impairment of mtFAS results in functional and ultrastructural changes in mitochondria followed by death of PCs, mimicking aspects of the clinical phenotype. This KO mouse represents a new model for impaired mitochondrial lipid metabolism and cerebellar ataxia with a distinct and well trackable cellular phenotype. This mouse model will allow the future investigation of the feasibility of metabolite supplementation approaches toward the prevention of neurodegeneration due to dysfunctional mtFAS.SIGNIFICANCE STATEMENT We have recently reported a novel neurodegenerative disorder in humans termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration) (Heimer et al., 2016). The cause of neuron degeneration in MEPAN patients is the dysfunction of the highly conserved mitochondrial fatty acid synthesis (mtFAS) pathway due to mutations in MECR, encoding mitochondrial 2-enoyl-CoA/ACP reductase. The report presented here describes the analysis of the first mouse model suffering from mtFAS-defect-induced neurodegenerative changes due to specific disruption of the Mecr gene in Purkinje cells. Our work sheds a light on the mechanisms of neurodegeneration caused by mtFAS deficiency and provides a test bed for future treatment approaches.
Asunto(s)
Cerebelo/metabolismo , Ácidos Grasos/biosíntesis , Mitocondrias/metabolismo , Degeneración Nerviosa/metabolismo , Animales , Animales Recién Nacidos , Cerebelo/patología , Ácidos Grasos/genética , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/patología , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/biosíntesis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genéticaRESUMEN
Deep artificial neural network learning is an emerging tool in image analysis. We demonstrate its potential in the field of digital holographic microscopy by addressing the challenging problem of determining the in-focus reconstruction depth of Madin-Darby canine kidney cell clusters encoded in digital holograms. A deep convolutional neural network learns the in-focus depths from half a million hologram amplitude images. The trained network correctly determines the in-focus depth of new holograms with high probability, without performing numerical propagation. This paper reports on extensions to preliminary work published earlier as one of the first applications of deep learning in the field of digital holographic microscopy.
RESUMEN
A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis.
Asunto(s)
Angiomatosis/genética , Encefalopatías/genética , Variación Genética , Péptidos y Proteínas de Señalización Intracelular/genética , Enfermedades Neurodegenerativas/genética , Fibrosis Pulmonar/genética , Angiomatosis/patología , Angiomatosis/fisiopatología , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Encéfalo/patología , Encefalopatías/patología , Encefalopatías/fisiopatología , Células Cultivadas , Familia , Resultado Fatal , Humanos , Lactante , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hepatopatías/genética , Hepatopatías/patología , Hepatopatías/fisiopatología , Masculino , Ratones Endogámicos C57BL , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/fisiopatología , Estudios Prospectivos , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/fisiopatología , Síndrome , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Individual cells in multicellular organisms constantly explore their microenvironment, or niche, to obtain spatial information that is used to regulate cell behavior to maintain tissue integrity. The extracellular matrix (ECM) is an important source of such spatial information. Binding of the integrin family receptors to the ECM triggers formation of integrin adhesion complexes (IACs) that link the ECM network to cellular cytoskeleton via remarkably large multiprotein complexes collectively referred to as the integrin adhesome. Recent advances in proteomics have enabled researchers to study the IAC composition in detail. Various biochemical IAC isolation methods and culture conditions have been employed to study the composition and dynamics of integrin-mediated adhesions mainly in fibroblasts and lymphoblasts. These studies have led to identification of daunting lists of potential IAC components. This review focuses on the current status of proteomics-driven research seeking to understand integrin functions by comprehensive analysis of IAC components. These systems level approaches have revealed the complexity of biochemical and biomechanical signals that are processed at IACs and provide a novel insight into how these signals are conveyed to regulate cellular behavior.
Asunto(s)
Adhesión Celular/fisiología , Integrinas/química , Integrinas/metabolismo , Proteómica/métodos , Animales , Matriz Extracelular/metabolismo , HumanosRESUMEN
Epithelial cells are important building blocks of most tissues and the corner stone of tissue architectures that allow directional transport of nutrients, ions and waste products in and out of the body. In tissues composed of millions of cells every individual cell needs to make right decisions when to differentiate, migrate, divide or die. Tight control of such fundamental cell-level processes ensures proper tissue morphogenesis, homeostasis and function. Cellular decisions are guided by biochemical and mechanical cues from their immediate microenvironment that consists of the extracellular matrix (ECM), neighboring cells and soluble factors. Generation of two distinct surfaces one facing the outside world (the apical domain) and the other contacting the neighboring cells and basal ECM (basolateral domain) is the most fundamental property of epithelial cells. The cues from the ECM are of particular importance in this process and communication between the cells and the ECM is largely mediated by transmembrane ECM receptors. Integrins constitute the largest family of such receptors binding to the ECM. Integrins have been shown to be essential for the establishment of initial polarity cues that define the position of the basal domain and thereby govern the orientation of the forming apico-basal axis. In this review I will discuss the multifaceted roles of integrins in epithelial cells with a particular focus on recent developments unveiling the specific functions of the different integrin heterodimers in regulating epithelial cell polarization and morphogenesis.
Asunto(s)
Polaridad Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Transducción de Señal , Animales , HumanosRESUMEN
The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor-treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting.
Asunto(s)
Marcación de Gen , Técnicas de Transferencia de Gen , Riñón/embriología , Mesodermo/fisiología , Células Madre/fisiología , Animales , Proteína Morfogenética Ósea 7 , Factor 2 de Crecimiento de Fibroblastos , Factores de Transcripción Forkhead/metabolismo , RatonesRESUMEN
BACKGROUND: The growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear. RESULTS: Here we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and ß1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. ß1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures. CONCLUSIONS: In the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of ß1-, α6- and αV-integrins.
Asunto(s)
Integrina alfa6beta1/metabolismo , Integrina alfaV/metabolismo , Animales , Adhesión Celular , Diferenciación Celular , Línea Celular , Proliferación Celular , Supervivencia Celular , Colágeno/química , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Matriz Extracelular/metabolismo , Integrina alfa6beta1/antagonistas & inhibidores , Integrina alfa6beta1/genética , Integrina alfaV/química , Integrina alfaV/genética , Laminina/química , Factor Inhibidor de Leucemia/deficiencia , Ratones , Microscopía Fluorescente , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The extracellular matrix (ECM) undergoes substantial changes during prostate cancer (PCa) progression, thereby regulating PCa growth and invasion. Herein, a meta-analysis of multiple PCa cohorts is performed which revealed that downregulation or genomic loss of ITGA1 and ITGA2 integrin genes is associated with tumor progression and worse prognosis. Genomic deletion of both ITGA1 and ITGA2 activated epithelial-to-mesenchymal transition (EMT) in benign prostate epithelial cells, thereby enhancing their invasive potential in vitro and converting them into tumorigenic cells in vivo. Mechanistically, EMT is induced by enhanced secretion and autocrine activation of TGFß1 and nuclear targeting of YAP1. An unbiased genome-wide co-expression analysis of large PCa cohort datasets identified the transcription factor TEAD1 as a key regulator of ITGA1 and ITGA2 expression in PCa cells while TEAD1 loss phenocopied the dual loss of α1- and α2-integrins in vitro and in vivo. Remarkably, clinical data analysis revealed that TEAD1 downregulation or genomic loss is associated with aggressive PCa and together with low ITGA1 and ITGA2 expression synergistically impacted PCa prognosis and progression. This study thus demonstrated that loss of α1- and α2-integrins, either via deletion/inactivation of the ITGA1/ITGA2 locus or via loss of TEAD1, contributes to PCa progression by inducing TGFß1-driven EMT.
Asunto(s)
Próstata , Neoplasias de la Próstata , Masculino , Humanos , Próstata/metabolismo , Próstata/patología , Línea Celular Tumoral , Neoplasias de la Próstata/genética , Transducción de Señal/genética , Integrina alfa2/genética , Integrina alfa2/metabolismo , Factores de Transcripción de Dominio TEARESUMEN
ErbB4 receptor tyrosine kinase contributes to the development of the heart, the central nervous system, and the lactating mammary gland, but whether it has a role in the development of the kidney epithelium is unknown. Here, we found that expression of Erbb4 isoforms JM-a CYT-1 and JM-a CYT-2 was first detectable around embryonic day 13 in the mouse, mainly in the collecting ducts and both the proximal and distal tubules. In vitro, overexpression of a relevant ErbB4 isoform promoted proliferation and disturbed polarization of kidney epithelial cells when cultured as three-dimensional structures. We examined ErbB4 function in developing kidney tubules in vivo with Pax8-Cre-mediated conditional overexpression of Rosa26 locus-targeted ERBB4 and with conditional Erbb4 knock-out mice. The Pax8-Cre-driven ERBB4 overexpression enhanced proliferation in the collecting ducts, reduced the size of epithelial duct lumens, and promoted formation of cortical tubular cysts. These defects were associated with changes in the subcellular distribution of markers of epithelial cell polarity. Similarly, the Pax8-Cre-mediated Erbb4 knock-out mice manifested dysfunctional kidneys with larger duct lumens and epithelial cell mispolarization. Taken together, these data suggest that ErbB4 signaling modulates proliferation and polarization, cellular functions critical for the development of epithelial ducts in the kidney.
Asunto(s)
Polaridad Celular , Receptores ErbB/metabolismo , Túbulos Renales/embriología , Animales , Proliferación Celular , Perros , Células Epiteliales/fisiología , Receptores ErbB/genética , Humanos , Isoenzimas/metabolismo , Túbulos Renales/citología , Túbulos Renales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Organogénesis , Receptor ErbB-4RESUMEN
Collagen XVIII (ColXVIII) is a component of the extracellular matrix implicated in embryogenesis and control of tissue homoeostasis. We now provide evidence that ColXVIII has a specific role in renal branching morphogenesis as observed in analyses of total and isoform-specific knockout embryos and mice. The expression of the short and the two longer isoforms differ temporally and spatially during renal development. The lack of ColXVIII or its specific isoforms lead to congenital defects in the 3D patterning of the ureteric tree where the short isoform plays a prominent role. Moreover, the ex vivo data suggests that ColXVIII is involved in the kidney epithelial tree patterning via its N-terminal domains, and especially the Thrombospondin-1-like domain common to all isoforms. This morphogenetic function likely involves integrins expressed in the ureteric epithelium. Altogether, the results point to an important role for ColXVIII in the matrix-integrin-mediated functions regulating renal development.
Asunto(s)
Colágeno Tipo XVIII , Riñón , Isoformas de Proteínas , Animales , Ratones , Colágeno Tipo XVIII/genética , Colágeno Tipo XVIII/metabolismo , Integrinas , Riñón/embriología , Riñón/metabolismo , Morfogénesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Uréter/embriología , Uréter/metabolismoRESUMEN
BACKGROUND: Aberrant somatic genomic alteration including copy number amplification is a hallmark of cancer genomes. We previously profiled genomic landscapes of prostate cancer (PCa), yet the underlying causal genes with prognostic potential has not been defined. It remains unclear how a somatic genomic event cooperates with inherited germline variants contribute to cancer predisposition and progression. METHODS: We applied integrated genomic and clinical data, experimental models and bioinformatic analysis to identify GATA2 as a highly prevalent metastasis-associated genomic amplification in PCa. Biological roles of GATA2 in PCa metastasis was determined in vitro and in vivo. Global chromatin co-occupancy and co-regulation of GATA2 and SMAD4 was investigated by coimmunoprecipitation, ChIP-seq and RNA-seq assays. Tumor cellular assays, qRT-PCR, western blot, ChIP, luciferase assays and CRISPR-Cas9 editing methods were performed to mechanistically understand the cooperation of GATA2 with SMAD4 in promoting TGFß1 and AR signaling and mediating inherited PCa risk and progression. RESULTS: In this study, by integrated genomics and experimental analysis, we identified GATA2 as a prevalent metastasis-associated genomic amplification to transcriptionally augment its own expression in PCa. Functional experiments demonstrated that GATA2 physically interacted and cooperated with SMAD4 for genome-wide chromatin co-occupancy and co-regulation of PCa genes and metastasis pathways like TGFß signaling. Mechanistically, GATA2 was cooperative with SMAD4 to enhance TGFß and AR signaling pathways, and activated the expression of TGFß1 via directly binding to a distal enhancer of TGFß1. Strinkingly, GATA2 and SMAD4 globally mediated inherited PCa risk and formed a transcriptional complex with HOXB13 at the PCa risk-associated rs339331/6q22 enhancer, leading to increased expression of the PCa susceptibility gene RFX6. CONCLUSIONS: Our study prioritizes causal genomic amplification genes with prognostic values in PCa and reveals the pivotal roles of GATA2 in transcriptionally activating the expression of its own and TGFß1, thereby co-opting to TGFß1/SMAD4 signaling and RFX6 at 6q22 to modulate PCa predisposition and progression.
Asunto(s)
Neoplasias de la Próstata , Masculino , Humanos , Neoplasias de la Próstata/patología , Próstata/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Cromatina , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Proteína Smad4/genética , Proteína Smad4/metabolismo , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismoRESUMEN
The tumor extracellular matrix (ECM) critically regulates cancer progression and treatment response. Expression of the basement membrane component collagen XVIII (ColXVIII) is induced in solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII was markedly upregulated in human breast cancer (BC) and was closely associated with a poor prognosis in high-grade BCs. We discovered a role for ColXVIII as a modulator of epidermal growth factor receptor tyrosine kinase (ErbB) signaling and show that it forms a complex with ErbB1 and -2 (also known as EGFR and human epidermal growth factor receptor 2 [HER2]) and α6-integrin to promote cancer cell proliferation in a pathway involving its N-terminal portion and the MAPK/ERK1/2 and PI3K/AKT cascades. Studies using Col18a1 mouse models crossed with the mouse mammary tumor virus-polyoma virus middle T antigen (MMTV-PyMT) mammary carcinogenesis model showed that ColXVIII promoted BC growth and metastasis in a tumor cell-autonomous manner. Moreover, the number of mammary cancer stem cells was significantly reduced in the MMTV-PyMT and human cell models upon ColXVIII inhibition. Finally, ablation of ColXVIII substantially improved the efficacy of ErbB-targeting therapies in both preclinical models. In summary, ColXVIII was found to sustain the stemness properties of BC cells and tumor progression and metastasis through ErbB signaling, suggesting that targeting ColXVIII in the tumor milieu may have important therapeutic potential.
Asunto(s)
Neoplasias de la Mama , Colágeno Tipo XVIII , Ratones , Animales , Humanos , Femenino , Colágeno Tipo XVIII/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor ErbB-2/metabolismo , Transformación Celular Neoplásica , Transducción de SeñalRESUMEN
The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell-cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell-cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell-cell and cell-ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.
Asunto(s)
Polaridad Celular/fisiología , Células Epiteliales/metabolismo , Glucuronosiltransferasa/metabolismo , Ácido Hialurónico/biosíntesis , Huso Acromático/metabolismo , Animales , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula , Línea Celular , Perros , Epitelio/metabolismo , Glucuronosiltransferasa/genética , Hialuronano Sintasas , Riñón/citología , Morfogénesis/fisiologíaRESUMEN
Hemidesmosomes (HDs) are adhesive structures that ensure stable anchorage of cells to the basement membrane. They are formed by α6ß4-integrin heterodimers and linked to intermediate filaments via plectin. It has been reported that one of the most common events during the pathogenesis of prostate cancer (PCa) is the loss of HD organization. While the expression levels of ß4-integrins are strongly reduced, the expression levels of α6-integrins and plectin are maintained or even elevated, and seem to promote tumorigenic properties of PCa cells, such as proliferation, invasion, metastasis, apoptosis- and drug-resistance. In this review, we discuss the potential mechanisms of how HD components might contribute to various cellular signaling pathways to promote prostate carcinogenesis. Moreover, we summarize the current knowledge on the involvement of α6ß4-integrins and plectin in PCa initiation and progression.
RESUMEN
Epithelial cell adhesion is mediated by actin cytoskeleton-linked focal adhesions (FAs) and intermediate filament-associated hemidesmosomes (HDs). HDs are formed by α6ß4-integrins and mediate stable anchoring to the extracellular matrix (ECM) while FAs containing ß1-integrins regulate cell migration. Loss of HDs has been reported in various cancers such as prostate cancer where it correlates with increased invasive migration. Here we have studied cell migration properties and FA dynamics in genetically engineered prostate epithelial cell lines with intact or disrupted HDs. Disruption of HDs by depleting α6- or ß4-integrin expression promoted collective cell migration and modulated migratory activity. Dynamic analysis of fluorescent protein-tagged FA marker proteins revealed faster FA assembly and disassembly kinetics in HD-depleted cells. FRAP analysis showed that loss of HDs correlated with faster diffusion rates of focal adhesion kinase (FAK) and vinculin in and out of FAs. These data suggest that loss of α6ß4-mediated HDs promote cell migration and FA assembly dynamics by influencing the molecular diffusion rates of FAK.