RESUMEN
Epithelial cells have an ability termed 'cell competition', which is an immune surveillance-like function that extrudes precancerous cells from the epithelial layer, leading to apoptosis and clearance. However, it remains unclear how epithelial cells recognize and extrude transformed cells. Here, we discovered that a PirB family protein, leukocyte immunoglobulin-like receptor B3 (LILRB3), which is expressed on non-transformed epithelial cells, recognizes major histocompatibility complex class I (MHC class I) that is highly expressed on transformed cells. MHC class I interaction with LILRB3 expressed on normal epithelial cells triggers an SHP2-ROCK2 pathway that generates a mechanical force to extrude transformed cells. Removal of transformed cells occurs independently of natural killer (NK) cell or CD8+ cytotoxic T cell-mediated activity. This is a new mechanism in that the immunological ligand-receptor system generates a mechanical force in non-immune epithelial cells to extrude precancerous cells in the same epithelial layer.
Asunto(s)
Antígenos CD/metabolismo , Apoptosis , Competencia Celular , Células Epiteliales/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias Pulmonares/metabolismo , Lesiones Precancerosas/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Antígenos CD/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Perros , Células Epiteliales/inmunología , Células Epiteliales/patología , Células HaCaT , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Células de Riñón Canino Madin Darby , Mecanotransducción Celular , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Lesiones Precancerosas/genética , Lesiones Precancerosas/inmunología , Lesiones Precancerosas/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Células RAW 264.7 , Receptores Inmunológicos/genética , Estrés Mecánico , Quinasas Asociadas a rho/metabolismoRESUMEN
At the initial stage of carcinogenesis, oncogenic transformation occurs in single cells within epithelial layers. However, the behavior and fate of the newly emerging transformed cells remain enigmatic. Here, using originally established mouse models, we investigate the fate of RasV12-transformed cells that appear in a mosaic manner within epithelial tissues. In the lung bronchial epithelium, most majority of RasV12-transformed cells are apically extruded, whereas noneliminated RasV12 cells are often basally delaminated leading to various noncell-autonomous changes in surrounding environments; macrophages and activated fibroblasts are accumulated, and normal epithelial cells overlying RasV12 cells overproliferate and form a convex multilayer, which is termed a 'dome-like structure'. In addition, basally extruded RasV12 cells acquire certain features of epithelial-mesenchymal transition (EMT). Furthermore, the expression of COX-2 is profoundly elevated in RasV12 cells in dome-like structures, and treatment with the COX inhibitor ibuprofen suppresses the recruitment of activated fibroblasts and moderately diminishes the formation of dome-like structures. Therefore, basal extrusion of single-oncogenic mutant cells can induce a tumor microenvironment and EMT and generate characteristic precancerous lesions, providing molecular insights into the earlier steps of cancer development.
Asunto(s)
Transformación Celular Neoplásica , Células Epiteliales , Perros , Ratones , Animales , Células de Riñón Canino Madin Darby , Células Epiteliales/patología , Transformación Celular Neoplásica/metabolismo , Epitelio/metabolismo , Oncogenes , Microambiente TumoralRESUMEN
The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak, and their priming toward primitive streak fates. How these different events are coordinated remains unknown. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that captures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this system in combination with microfabrication and in vivo experiments, we found that proliferation-induced crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that the right cell types become specified at the right place during embryonic development.
Asunto(s)
Diferenciación Celular , Gastrulación , Estratos Germinativos , Animales , Ratones , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Proteínas de Dominio T Box/metabolismo , Proteínas de Dominio T Box/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Línea Primitiva/citología , Línea Primitiva/metabolismo , Proteínas Fetales/metabolismo , Proteínas Fetales/genética , Vía de Señalización Wnt , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismoRESUMEN
For the maintenance of epithelial homeostasis, various aberrant or dysfunctional cells are actively eliminated from epithelial layers. This cell extrusion process mainly falls into two modes: cell-competition-mediated extrusion and apoptotic extrusion. However, it is not clearly understood whether and how these processes are governed by common molecular mechanisms. In this study, we demonstrate that the reactive oxygen species (ROS) levels are elevated within a wide range of epithelial layers around extruding transformed or apoptotic cells. The downregulation of ROS suppresses the extrusion process. Furthermore, ATP is extracellularly secreted from extruding cells, which promotes the ROS level and cell extrusion. Moreover, the extracellular ATP and ROS pathways positively regulate the polarized movements of surrounding cells toward extruding cells in both cell-competition-mediated and apoptotic extrusion. Hence, extracellular ATP acts as an "extrude me" signal and plays a prevalent role in cell extrusion, thereby sustaining epithelial homeostasis and preventing pathological conditions or disorders.
Asunto(s)
Apoptosis , Competencia Celular , Adenosina Trifosfato/metabolismo , Células Epiteliales/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Dupuytren's Disease (DD) is a common fibroproliferative disease of the palmar fascia. We previously identified a causal association with a non-synonymous variant (rs1042704, p.D273N) in MMP14 (encoding MT1-MMP). In this study, we investigated the functional consequences of this variant, and demonstrated that the variant MT1-MMP (MT1-N273) exhibits only 17% of cell surface collagenolytic activity compared to the ancestral enzyme (MT1-D273). Cells expressing both MT1-D273 and MT1-N273 in a 1:1 ratio, mimicking the heterozygous state, possess 38% of the collagenolytic activity compared to the cells expressing MT1-D273, suggesting that MT1-N273 acts in a dominant negative manner. Consistent with the above observation, patient-derived DD myofibroblasts with the alternate allele demonstrated around 30% of full collagenolytic activity detected in ancestral G/G genotype cells, regardless of the heterozygous (G/A) or homozygous (A/A) state. Small angle X-ray scattering analysis of purified soluble Fc-fusion enzymes allowed us to construct a 3D-molecular envelope of MT1-D273 and MT1-N273, and demonstrate altered flexibility and conformation of the ectodomains due to D273 to N substitution. Taking together, rs1042704 significantly reduces collagen catabolism in tissue, which tips the balance of homeostasis of collagen in tissue, contributing to the fibrotic phenotype of DD. Since around 30% of the worldwide population have at least one copy of the low collagenolytic alternate allele, further investigation of rs1042704 across multiple pathologies is needed.
Asunto(s)
Colágeno/metabolismo , Contractura de Dupuytren/genética , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Polimorfismo de Nucleótido Simple , Animales , Células COS , Chlorocebus aethiops , Contractura de Dupuytren/metabolismo , Humanos , Metaloproteinasa 14 de la Matriz/química , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.
Asunto(s)
Transformación Celular Neoplásica , Epitelio/crecimiento & desarrollo , Receptores de Hialuranos/metabolismo , Colágenos no Fibrilares/metabolismo , Animales , Línea Celular , Transformación Celular Neoplásica/genética , Perros , Ferroptosis , Humanos , Células de Riñón Canino Madin Darby , Potencial de la Membrana Mitocondrial , Ratones , Especies Reactivas de OxígenoRESUMEN
At the initial stage of carcinogenesis, when RasV12-transformed cells are surrounded by normal epithelial cells, RasV12 cells are apically extruded from epithelia through cell competition with the surrounding normal cells. In this study, we demonstrate that expression of cyclooxygenase (COX)-2 is upregulated in normal cells surrounding RasV12-transformed cells. Addition of COX inhibitor or COX-2-knockout promotes apical extrusion of RasV12 cells. Furthermore, production of Prostaglandin (PG) E2, a downstream prostanoid of COX-2, is elevated in normal cells surrounding RasV12 cells, and addition of PGE2 suppresses apical extrusion of RasV12 cells. In a cell competition mouse model, expression of COX-2 is elevated in pancreatic epithelia harbouring RasV12-exressing cells, and the COX inhibitor ibuprofen promotes apical extrusion of RasV12 cells. Moreover, caerulein-induced chronic inflammation substantially suppresses apical elimination of RasV12 cells. These results indicate that intrinsically or extrinsically mediated inflammation can promote tumour initiation by diminishing cell competition between normal and transformed cells.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Células Epiteliales/enzimología , Genes ras , Pancreatitis/enzimología , Animales , Anticarcinógenos/farmacología , Línea Celular Transformada , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Ceruletida , Ciclooxigenasa 2/genética , Inhibidores de la Ciclooxigenasa/farmacología , Modelos Animales de Enfermedad , Perros , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Femenino , Ibuprofeno/farmacología , Células de Riñón Canino Madin Darby , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Pancreatitis/inducido químicamente , Pancreatitis/genética , Pancreatitis/patología , Transducción de SeñalRESUMEN
Previous studies have revealed that, at the initial step of carcinogenesis, transformed cells are often eliminated from epithelia via cell competition with the surrounding normal cells. In this study, we performed cell competition-based high-throughput screening for chemical compounds using cultured epithelial cells and confocal microscopy. PLX4720 was identified as a hit compound that promoted apical extrusion of RasV12-transformed cells surrounded by normal epithelial cells. Knockdown/knockout of ZAK, a target of PLX4720, substantially enhanced the apical elimination of RasV12 cells in vitro and in vivo. ZAK negatively modulated the accumulation or activation of multiple cell competition regulators. Moreover, PLX4720 treatment promoted apical elimination of RasV12-transformed cells in vivo and suppressed the formation of potentially precancerous tumors. This is the first report demonstrating that a cell competition-promoting chemical drug facilitates apical elimination of transformed cells in vivo, providing a new dimension in cancer preventive medicine.
RESUMEN
Epithelial cells form sheets and tubules in various epithelial organs and establish apicobasal polarity and asymmetric vesicle transport to provide functionality in these structures. However, the molecular mechanisms that allow epithelial cells to establish polarity are not clearly understood. Here, we present evidence that the kinase activity of the receptor tyrosine kinase for collagen, discoidin domain receptor 1 (DDR1), is required for efficient establishment of epithelial polarity, proper asymmetric protein secretion, and execution of morphogenic programs. Lack of DDR1 protein or inhibition of DDR1 kinase activity disturbed tubulogenesis, cystogenesis, and the establishment of epithelial polarity and caused defects in the polarized localization of membrane-type 1 matrix metalloproteinase (MT1-MMP), GP135, primary cilia, laminin, and the Golgi apparatus. Disturbed epithelial polarity and cystogenesis upon DDR1 inhibition was caused by excess ROCK (rho-associated, coiled-coil-containing protein kinase)-driven actomyosin contractility, and pharmacological inhibition of ROCK was sufficient to correct these defects. Our data indicate that a DDR1-ROCK signaling axis is essential for the efficient establishment of epithelial polarity.
Asunto(s)
Actomiosina/metabolismo , Polaridad Celular/fisiología , Receptor con Dominio Discoidina 1/metabolismo , Células Epiteliales/metabolismo , Animales , Células CACO-2 , Cilios/metabolismo , Contactina 1/metabolismo , Perros , Femenino , Aparato de Golgi/metabolismo , Humanos , Laminina/metabolismo , Células de Riñón Canino Madin Darby , Masculino , Metaloproteinasa 14 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Organoides , Quinasas Asociadas a rho/metabolismoRESUMEN
Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelia via cell competition with the surrounding normal epithelial cells. However, it remains unknown whether and how soluble factors are involved in this cancer preventive phenomenon. By performing stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometric analyses, we have identified ADAM-like Decysin-1 (ADAMDEC1) as a soluble protein whose expression is upregulated in the mix culture of normal and RasV12-transformed epithelial cells. Expression of ADAMDEC1 is elevated in normal epithelial cells co-cultured with RasV12 cells. Knockdown of ADAMDEC1 in the surrounding normal cells substantially suppresses apical extrusion of RasV12 cells, suggesting that ADAMDEC1 secreted by normal cells positively regulate the elimination of the neighboring transformed cells. In addition, we show that the metalloproteinase activity of ADAMDEC1 is dispensable for the regulation of apical extrusion. Furthermore, ADAMDEC1 facilitates the accumulation of filamin, a crucial regulator of Epithelial Defense Against Cancer (EDAC), in normal cells at the interface with RasV12 cells. This is the first report demonstrating that an epithelial intrinsic soluble factor is involved in cell competition in mammals.
Asunto(s)
Proteínas ADAM/metabolismo , Transformación Celular Neoplásica , Células Epiteliales/patología , Proteínas ADAM/deficiencia , Proteínas ADAM/genética , Animales , Técnicas de Cocultivo , Perros , Filaminas/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Células de Riñón Canino Madin Darby , FN-kappa B/metabolismoRESUMEN
p53 is a tumor suppressor protein, and its missense mutations are frequently found in human cancers. During the multi-step progression of cancer, p53 mutations generally accumulate at the mid or late stage, but not in the early stage, and the underlying mechanism is still unclear. In this study, using mammalian cell culture and mouse ex vivo systems, we demonstrate that when p53R273H- or p53R175H-expressing cells are surrounded by normal epithelial cells, mutant p53 cells undergo necroptosis and are basally extruded from the epithelial monolayer. When mutant p53 cells alone are present, cell death does not occur, indicating that necroptosis results from cell competition with the surrounding normal cells. Furthermore, when p53R273H mutation occurs within RasV12-transformed epithelia, cell death is strongly suppressed and most of the p53R273H-expressing cells remain intact. These results suggest that the order of oncogenic mutations in cancer development could be dictated by cell competition.