RESUMEN
Following tissue damage, epithelial stem cells (SCs) are mobilized to enter the wound, where they confront harsh inflammatory environments that can impede their ability to repair the injury. Here, we investigated the mechanisms that protect skin SCs within this inflammatory environment. Characterization of gene expression profiles of hair follicle SCs (HFSCs) that migrated into the wound site revealed activation of an immune-modulatory program, including expression of CD80, major histocompatibility complex class II (MHCII), and CXC motif chemokine ligand 5 (CXCL5). Deletion of CD80 in HFSCs impaired re-epithelialization, reduced accumulation of peripherally generated Treg (pTreg) cells, and increased infiltration of neutrophils in wounded skin. Importantly, similar wound healing defects were also observed in mice lacking pTreg cells. Our findings suggest that upon skin injury, HFSCs establish a temporary protective network by promoting local expansion of Treg cells, thereby enabling re-epithelialization while still kindling inflammation outside this niche until the barrier is restored.
Asunto(s)
Antígeno B7-1 , Folículo Piloso , Inflamación , Piel , Células Madre , Linfocitos T Reguladores , Cicatrización de Heridas , Animales , Linfocitos T Reguladores/inmunología , Ratones , Cicatrización de Heridas/inmunología , Piel/inmunología , Piel/lesiones , Piel/patología , Células Madre/inmunología , Células Madre/metabolismo , Inflamación/inmunología , Folículo Piloso/inmunología , Antígeno B7-1/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Repitelización/inmunología , Movimiento Celular/inmunología , Proliferación CelularRESUMEN
During development, progenitors simultaneously activate one lineage while silencing another, a feature highly regulated in adult stem cells but derailed in cancers. Equipped to bind cognate motifs in closed chromatin, pioneer factors operate at these crossroads, but how they perform fate switching remains elusive. Here we tackle this question with SOX9, a master regulator that diverts embryonic epidermal stem cells (EpdSCs) into becoming hair follicle stem cells. By engineering mice to re-activate SOX9 in adult EpdSCs, we trigger fate switching. Combining epigenetic, proteomic and functional analyses, we interrogate the ensuing chromatin and transcriptional dynamics, slowed temporally by the mature EpdSC niche microenvironment. We show that as SOX9 binds and opens key hair follicle enhancers de novo in EpdSCs, it simultaneously recruits co-factors away from epidermal enhancers, which are silenced. Unhinged from its normal regulation, sustained SOX9 subsequently activates oncogenic transcriptional regulators that chart the path to cancers typified by constitutive SOX9 expression.
Asunto(s)
Células Madre Adultas , Proteómica , Animales , Ratones , Células Madre Adultas/metabolismo , Diferenciación Celular , Cromatina/genética , Epigénesis Genética , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismoRESUMEN
Macrophages and dendritic cells have long been appreciated for their ability to migrate to and engulf dying cells and debris, including some of the billions of cells that are naturally eliminated from our body daily. However, a substantial number of these dying cells are cleared by 'non-professional phagocytes', local epithelial cells that are critical to organismal fitness. How non-professional phagocytes sense and digest nearby apoptotic corpses while still performing their normal tissue functions is unclear. Here, we explore the molecular mechanisms underlying their multifunctionality. Exploiting the cyclical bouts of tissue regeneration and degeneration during the hair cycle, we show that stem cells can transiently become non-professional phagocytes when confronted with dying cells. Adoption of this phagocytic state requires both local lipids produced by apoptotic corpses to activate RXRα, and tissue-specific retinoids for RARγ activation. This dual factor dependency enables tight regulation of the genes requisite to activate phagocytic apoptotic clearance. The tunable phagocytic program we describe here offers an effective mechanism to offset phagocytic duties against the primary stem cell function of replenishing differentiated cells to preserve tissue integrity during homeostasis. Our findings have broad implications for other non-motile stem or progenitor cells which experience cell death in an immune-privileged niche.
RESUMEN
Immune and tissue stem cells retain an epigenetic memory of inflammation that intensifies sensitivity to future encounters. We investigated whether and to what consequence stem cells possess and accumulate memories of diverse experiences. Monitoring a choreographed response to wounds, we found that as hair follicle stem cells leave their niche, migrate to repair damaged epidermis, and take up long-term foreign residence there, they accumulate long-lasting epigenetic memories of each experience, culminating in post-repair epigenetic adaptations that sustain the epidermal transcriptional program and surface barrier. Each memory is distinct, separable, and has its own physiological impact, collectively endowing these stem cells with heightened regenerative ability to heal wounds and broadening their tissue-regenerating tasks relative to their naïve counterparts.
Asunto(s)
Células Epidérmicas/citología , Epigénesis Genética , Folículo Piloso/citología , Células Madre/fisiología , Adaptación Fisiológica , Animales , Movimiento Celular , Cromatina/metabolismo , Células Epidérmicas/fisiología , Homeostasis , Inflamación , Ratones , Regeneración , Nicho de Células Madre , Transcriptoma , Cicatrización de HeridasRESUMEN
Through recurrent bouts synchronous with the hair cycle, quiescent melanocyte stem cells (McSCs) become activated to generate proliferative progeny that differentiate into pigment-producing melanocytes. The signaling factors orchestrating these events remain incompletely understood. Here, we use single-cell RNA sequencing with comparative gene expression analysis to elucidate the transcriptional dynamics of McSCs through quiescence, activation, and melanocyte maturation. Unearthing converging signs of increased WNT and BMP signaling along this progression, we endeavored to understand how these pathways are integrated. Employing conditional lineage-specific genetic ablation studies in mice, we found that loss of BMP signaling in the lineage leads to hair graying due to a block in melanocyte maturation. We show that interestingly, BMP signaling functions downstream from activated McSCs and maintains WNT effector, transcription factor LEF1. Employing pseudotime analysis, genetics, and chromatin landscaping, we show that following WNT-mediated activation of McSCs, BMP and WNT pathways collaborate to trigger the commitment of proliferative progeny by fueling LEF1- and MITF-dependent differentiation. Our findings shed light upon the signaling interplay and timing of cues that orchestrate melanocyte lineage progression in the hair follicle and underscore a key role for BMP signaling in driving complete differentiation.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Diferenciación Celular/genética , Melanocitos/citología , Transducción de Señal/genética , Células Madre/citología , Animales , Linaje de la Célula/genética , Perfilación de la Expresión Génica , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , Factor de Transcripción Asociado a Microftalmía/metabolismo , Análisis de la Célula IndividualRESUMEN
Tissue homeostasis and regeneration rely on resident stem cells (SCs), whose behaviour is regulated through niche-dependent crosstalk. The mechanisms underlying SC identity are still unfolding. Here, using spatiotemporal gene ablation in murine hair follicles, we uncover a critical role for the transcription factors (TFs) nuclear factor IB (NFIB) and IX (NFIX) in maintaining SC identity. Without NFI TFs, SCs lose their hair-regenerating capability, and produce skin bearing striking resemblance to irreversible human alopecia, which also displays reduced NFIs. Through single-cell transcriptomics, ATAC-Seq and ChIP-Seq profiling, we expose a key role for NFIB and NFIX in governing super-enhancer maintenance of the key hair follicle SC-specific TF genes. When NFIB and NFIX are genetically removed, the stemness epigenetic landscape is lost. Super-enhancers driving SC identity are decommissioned, while unwanted lineages are de-repressed ectopically. Together, our findings expose NFIB and NFIX as crucial rheostats of tissue homeostasis, functioning to safeguard the SC epigenome from a breach in lineage confinement that otherwise triggers irreversible tissue degeneration.
Asunto(s)
Alopecia/patología , Diferenciación Celular , Cromatina/metabolismo , Folículo Piloso/citología , Factores de Transcripción NFI/fisiología , Células Madre/citología , Alopecia/genética , Alopecia/metabolismo , Animales , Células Cultivadas , Cromatina/genética , Femenino , Folículo Piloso/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regeneración , Células Madre/metabolismoRESUMEN
Sharma investigates vesicular trafficking to lysosomes and how pathogens hijack the endolysosomal system during infection.
Asunto(s)
Biología Celular/historia , Endosomas/metabolismo , Lisosomas/metabolismo , Transporte Biológico Activo , Historia del Siglo XXI , Humanos , Retratos como AsuntoRESUMEN
Wang studies lysosomal degradation pathways using C. elegans as a model system.
Asunto(s)
Autofagia , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Biología Celular/historia , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Femenino , Historia del Siglo XXI , Humanos , Retratos como AsuntoRESUMEN
Mierzwa studies mechanisms of cell division in different cell types and tissue contexts.
Asunto(s)
Arte/historia , Biología Celular/historia , Historia del Siglo XX , Historia del Siglo XXI , Retratos como AsuntoRESUMEN
McLaughlin studies how neurons respond to acute and chronic stress.
Asunto(s)
Neuroprotección , Neurociencias/historia , Estrés Fisiológico , Mujeres Trabajadoras/historia , Femenino , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Neuronas , Retratos como AsuntoRESUMEN
Davis uses microscopy and imaging approaches to study immune cell interactions.
Asunto(s)
Alergia e Inmunología , Células Asesinas Naturales , Historia del Siglo XXI , HumanosRESUMEN
Chipuk studies the interaction between mitochondrial dynamics and cell death in cancer.
Asunto(s)
Mitocondrias , Humanos , New YorkRESUMEN
Auwerx studies the signaling networks that underlie mitochondrial function and metabolism.
Asunto(s)
Investigación Biomédica Traslacional , Animales , Historia del Siglo XX , Historia del Siglo XXI , HumanosRESUMEN
Purpose: Anaplastic lymphoma kinase (ALK) is the most frequently mutated oncogene in the pediatric cancer neuroblastoma. We performed an in vitro screen for synergistic drug combinations that target neuroblastomas with mutations in ALK to determine whether drug combinations could enhance antitumor efficacy.Experimental Design: We screened combinations of eight molecularly targeted agents against 17 comprehensively characterized human neuroblastoma-derived cell lines. We investigated the combination of ceritinib and ribociclib on in vitro proliferation, cell cycle, viability, caspase activation, and the cyclin D/CDK4/CDK6/RB and pALK signaling networks in cell lines with representative ALK status. We performed in vivo trials in CB17 SCID mice bearing conventional and patient-derived xenograft models comparing ceritinib alone, ribociclib alone, and the combination, with plasma pharmacokinetics to evaluate for drug-drug interactions.Results: The combination of ribociclib, a dual inhibitor of cyclin-dependent kinase (CDK) 4 and 6, and the ALK inhibitor ceritinib demonstrated higher cytotoxicity (P = 0.008) and synergy scores (P = 0.006) in cell lines with ALK mutations as compared with cell lines lacking mutations or alterations in ALK Compared with either drug alone, combination therapy enhanced growth inhibition, cell-cycle arrest, and caspase-independent cell death. Combination therapy achieved complete regressions in neuroblastoma xenografts with ALK-F1174L and F1245C de novo resistance mutations and prevented the emergence of resistance. Murine ribociclib and ceritinib plasma concentrations were unaltered by combination therapy.Conclusions: This preclinical combination drug screen with in vivo validation has provided the rationale for a first-in-children trial of combination ceritinib and ribociclib in a molecularly selected pediatric population. Clin Cancer Res; 23(11); 2856-68. ©2016 AACR.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Neuroblastoma/tratamiento farmacológico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Aminopiridinas/administración & dosificación , Quinasa de Linfoma Anaplásico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D/genética , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/genética , Sinergismo Farmacológico , Humanos , Ratones , Mutación , Neuroblastoma/genética , Neuroblastoma/patología , Purinas/administración & dosificación , Pirimidinas/administración & dosificación , Proteínas Tirosina Quinasas Receptoras/genética , Proteína de Retinoblastoma/genética , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Sulfonas/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The presence of an ALK aberration correlates with inferior survival for patients with high-risk neuroblastoma. The emergence of ALK inhibitors such as crizotinib has provided novel treatment opportunities. However, certain ALK mutations result in de novo crizotinib resistance, and a phase I trial of crizotinib showed a lack of response in patients harboring those ALK mutations. Thus, understanding mechanisms of resistance and defining circumvention strategies for the clinic is critical. EXPERIMENTAL DESIGN: The sensitivity of human neuroblastoma-derived cell lines, cell line-derived, and patient-derived xenograft (PDX) models with varying ALK statuses to crizotinib combined with topotecan and cyclophosphamide (topo/cyclo) was examined. Cultured cells and xenografts were evaluated for effects of these drugs on proliferation, signaling, and cell death, and assessment of synergy. RESULTS: In neuroblastoma murine xenografts harboring the most common ALK mutations, including those mutations associated with resistance to crizotinib (but not in those with wild-type ALK), crizotinib combined with topo/cyclo enhanced tumor responses and mouse event-free survival. Crizotinib + topo/cyclo showed synergistic cytotoxicity and higher caspase-dependent apoptosis than crizotinib or topo/cyclo alone in neuroblastoma cell lines with ALK aberrations (mutation or amplification). CONCLUSIONS: Combining crizotinib with chemotherapeutic agents commonly used in treating newly diagnosed patients with high-risk neuroblastoma restores sensitivity in preclinical models harboring both sensitive ALK aberrations and de novo-resistant ALK mutations. These data support clinical testing of crizotinib and conventional chemotherapy with the goal of integrating ALK inhibition into multiagent therapy for ALK-aberrant neuroblastoma patients.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neuroblastoma/tratamiento farmacológico , Quinasa de Linfoma Anaplásico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Crizotinib , Ciclofosfamida/administración & dosificación , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Humanos , Concentración 50 Inhibidora , Ratones SCID , Mutación , Neuroblastoma/genética , Pirazoles/administración & dosificación , Piridinas/administración & dosificación , Proteínas Tirosina Quinasas Receptoras/genética , Topotecan/administración & dosificación , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: Neuroblastomas harboring activating point mutations in anaplastic lymphoma kinase (ALK) are differentially sensitive to the ALK inhibitor crizotinib, with certain mutations conferring intrinsic crizotinib resistance. To overcome this clinical obstacle, our goal was to identify inhibitors with improved potency that can target intractable ALK variants such as F1174L. We find that PF-06463922 has high potency across ALK variants and inhibits ALK more effectively than crizotinib in vitro. Most importantly, PF-06463922 induces complete tumor regression in both crizotinib-resistant and crizotinib-sensitive xenograft mouse models of neuroblastoma, as well as in patient-derived xenografts harboring the crizotinib-resistant F1174L or F1245C mutations. These studies demonstrate that PF-06463922 has the potential to overcome crizotinib resistance and exerts unprecedented activity as a single targeted agent against F1174L and F1245C ALK-mutated xenograft tumors, while also inducing responses in an R1275Q xenograft model. Taken together, these results provide the rationale to move PF-06463922 into clinical trials for treatment of patients with ALK-mutated neuroblastoma. SIGNIFICANCE: The next-generation ALK/ROS1 inhibitor PF-06463922 exerts unparalleled activity in ALK-driven neuroblastoma models with primary crizotinib resistance. Our biochemical and in vivo data provide the preclinical rationale for fast-tracking the development of this agent in children with relapsed/refractory ALK-mutant neuroblastoma.